The acetylcholinesterase 2（AChE2）cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115.One transformant with high-level expression of the recombinant AChE（rA...The acetylcholinesterase 2（AChE2）cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115.One transformant with high-level expression of the recombinant AChE（rAChE,23.2 U mL-1in supernatant）was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate.The maximum production of r ACh E reached about 11.8 mg of the enzyme protein per liter of culture.The r ACh E was first precipitated with ammonium sulfate（50%saturation）and then purified with procainamide affinity column chromatography.The enzyme was purified 12.1-fold with a yield of 22.8%and a high specific activity of 448.3 U mg-1.It was sensitive to inhibition by methamidophos and pirimicarb,the calculated 50% inhibitory concentration（IC50）values of the two pesticides were 0.357 and 0.888 mg L-1,respectively,and the calculated 70% inhibitory concentration（IC70）values were 0.521 and 0.839 mg L-1,respectively.The results suggested that it has a potential application in the detection of pesticide residues.展开更多
Using genomic DNA of bolting-tolerant lettuce as a template,flanking fragments of lettuce plastid rpo A gene were amplified and cloned by PCR. Targeting the sites of these two fragments,homologous recombinant fragment...Using genomic DNA of bolting-tolerant lettuce as a template,flanking fragments of lettuce plastid rpo A gene were amplified and cloned by PCR. Targeting the sites of these two fragments,homologous recombinant fragments of exogenous gene were integrated to construct lettuce plastid expression vector p Brpo AGFP,which harbored the expression cassette Prrn-gfp-aad A-Tpsb A. The results showed that the amplified flanking fragments were 1.2 and 1.1 kb in size. After sequencing,restriction digestion,ligation and transformation,lettuce plastid expression vector containing expression cassette Prrn-gfp-aad A-Tpsb A was constructed and confirmed by SDS-PAGE electrophoresis. The results of SDS-PAGE electrophoresis indicated that gfp gene was efficiently expressed under the regulation of plasmid specific promoter Prrn and terminator Tpsb A. GFP accounted for 45. 6% of total soluble proteins; inclusion bodies accounted for 47.5 % of bacterial proteins,which reached relatively high expression levels. The construction of lettuce plastid expression vector p Brpo A-GFP laid a solid foundation for establishment of subsequent lettuce plastid transformation system and genetic improvement of lettuce using various functional genes.展开更多
Objective:To investigate the effect of utilizing a molecular partner on high-level expression of Mxisca domestica(M.domestica) cecropin in Escherichia coli(E.coli) and to identify the expressed products.Methods:The ge...Objective:To investigate the effect of utilizing a molecular partner on high-level expression of Mxisca domestica(M.domestica) cecropin in Escherichia coli(E.coli) and to identify the expressed products.Methods:The genomic sequence of M.domestica cecropin A(MC) and M. domestica ubiquitin(UBI) were searched from Cenbank and amplified by reverse transcriptase polymerase chain reaction(RT-PCR).Two expression plasmids,pET32a-MC and pET32a-UBI-MC, were constructed and transferred into E.coli and were then induced by Isopropylβ-D-1- Thiogalactopyranoside(IPTG).The expression of the fusion proteins Trx-MC and Trx-UBI-MC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Fusion protein Trx-MC was verified by Western blot analysis.The bactericidal activity of the purified MC was quantitatively determined using E.coli BL21(DE3).Results:The result showed that the fusion proteins were successively expressed in E.coli BL21 cells.A band at the expected position of 24 kDa representing the Trx-MC target protein was positivelystained,and the band at 4 kDa representing the hydrolysis of mature MC protein was also observed at the expected position. The expression levels of Trx-UBI-MC were higher than that of Trx-MC in E.coli.MC exhibited antimicrobial activity.Conclusions:With high-level expression of housefly cecropin A in E.coli using a fusion protein,MC exhibited antimicrobial activity.展开更多
Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to pl...Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to play a vital role in the regulation of cell proliferative cycle[1，2]. Recently, many studies showed that CaM is also present in extracellular fluid such as cell culture media and normal body fluid and has been reported to stimulate proliferation in a range of normal and neoplastic cells, apparently acting as an autocrine growth factor[3-11]. In 1988, Crocker et al reported for the first time that addition of extracellular pure pig brain CaM could promote DNA synthesis and cell [7]proliferation in K562 human leukaemic lymphocytes[7].After that, more and more research was done on extracellular CaM and evidences demonstrated that extracellular CaM could also stimulate cell proliferation in normal human umbilical vein endothelial cells[5], keratinocytes[4], suspension-cultured cells of Angelica Dahurica, etc[6]. CaM is a monomeric protein of 148 amino acids that contains four homologous Ca2 + -binding domains. CaM has been highly conserved throughout the evolution. Only 1 out of 148 amino acids of human CaM is different from that of fish CaM. Complementary DNAs encoding rat, eel, chicken, human, and trypanosome CaM have been cloned.展开更多
We have studied the expression of nm23(NDP) in 50 cases nasopharyngeal biopsies with anti-nm23(NDP) antibodies. As a result, the NDP positive rate in nasopharyngeal carcinoma (NPC) (95.54%) markedly increased (P<0....We have studied the expression of nm23(NDP) in 50 cases nasopharyngeal biopsies with anti-nm23(NDP) antibodies. As a result, the NDP positive rate in nasopharyngeal carcinoma (NPC) (95.54%) markedly increased (P<0.05), as compared with that in the normal nasopharyngeal epithelia (50.00-60.00%) and lymphocytes (52.00%). There were cytopfasmic type, nucleus type and mixed cytoplasmonucleus type according to NDP location in a cell. Their positive rates were 64.44%, 15.56% and 20.00% respectively in nasopharyngeal carcinoma. The expression of NDP had no relation with cervical lymphometastases in NPC, and the NDP positive rates had no significance between bilateral cervical lymphometastases and unilateral (P<0.05). But the NDP expression had most relation with the NPC staging. The expression rate and the intensity in Ⅲ or Ⅳ stage patients were markedly higher than that in II stage. It points out that the high-level expression of NDP had relation with the rapid cellular proliferation in NPC, and it may indicate the bad prognoses.展开更多
Objective To try making huZP3a^22-176 and huZP3b^177-348 polypeptides （representing an intact huZP^322-348 protein without its N-terminal signal peptide and C-terminal transmembrane domain ） express in E. coli at a ...Objective To try making huZP3a^22-176 and huZP3b^177-348 polypeptides （representing an intact huZP^322-348 protein without its N-terminal signal peptide and C-terminal transmembrane domain ） express in E. coli at a higher level Methods The cDNAs encoding huZP3a and huZP3b were obtained with PCR method. The pBV221 plasmid was used to construct thermo-inducible recombinant expression vector. Purification of two target expression products employed an improved method of preparative gel polyacrylamide gel electrophoresis. Results Two polypeptides of recombinant huZP3a （rhuZP3a） and recombinant huZP3b （rhuZP3b） were all expressed respectively in an E. coli BL21（DE3）pLysS strain at a higher level, which were recognized by two specific polyclonal antisera in Western blotting test which recognize a linear B cell epitope present in rhuZP3a or rhuZP3b respectively. Using the shake-flask method, approximately 5 mg of rhuZP3a and rhuZP3b with more than 95% relative homogeneity were harvested from 1 L culture respectively. Conclusion The availability of two rhuZP3 polypeptides will help in detecting the immunogenicities of rhuZP3a and rhuZP3b through animal experiments and confirming the function domain of non-glycosylated huZP3 to induce acrosome reaction in vitro.展开更多
Human interferon alpha-8(IFN-α8) is an important cytokine with multiple biological functions.A genetically engineered strain, E. coli XL1-Blue/pBm, was constructed by DNA recombination technology and characterized by...Human interferon alpha-8(IFN-α8) is an important cytokine with multiple biological functions.A genetically engineered strain, E. coli XL1-Blue/pBm, was constructed by DNA recombination technology and characterized by restriction analysis, DNA sequencing.展开更多
In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL 1 was constructed. The recombinant was identified by three restriction endonucleases, then...In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL 1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL 1 was transformed into expression host M15 （pREP4） and induced by isopropyl-a-D-thiogalactoside （IPTG） to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol＇L-1 IPTG induction as the expression temperature declined from 37 to 28℃. The 6-His tag facilitates affinity binding to Ni^2＋-nitrolotriacetic acid （NTA） and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose coupled with Ni^2＋-NTA affinity chromatography. The optimal substrate for Pt4CL 1 was 4-coumarate.展开更多
Humanoid robots hold significant promise for social interaction and emotional companionship.However,their effectiveness hinges on the ability to convey nuanced and authentic emotions.Here,we presented a universal huma...Humanoid robots hold significant promise for social interaction and emotional companionship.However,their effectiveness hinges on the ability to convey nuanced and authentic emotions.Here,we presented a universal humanoid robot head with a facial kinematics model.Using a reinforcement learning framework guided by symmetry assessment,emotion decoupling,and MLLM authenticity evaluation,our system autonomously learns to generate adaptive facial expressions through dynamic landmark adjustments.By transferring the simulation training results to real-world environments,the robot can perform natural and expressive expressions.Another novel feature is the independent regulation of emotion intensity and expression magnitude across emotional categories,which enhances the ability to achieve culturally adaptive and socially resonant robotic expressions significantly.This research advances adaptive humanoid interaction,offering an easier and more efficient pathway toward culturally resonant and psychologically plausible robotic expressions.展开更多
[Objectives]The present study was conducted to investigate the change rule ofβ-fructofuranosidase gene expression and its enzyme activity in the midgut of 5 th instar silkworm(Bombyx mori),in order to provide a refer...[Objectives]The present study was conducted to investigate the change rule ofβ-fructofuranosidase gene expression and its enzyme activity in the midgut of 5 th instar silkworm(Bombyx mori),in order to provide a reference for illustrating the enzymatic mechanism of usingβ-fructofuranosidase to absorb sucrose nutrition from mulberry leaves.[Methods]Real-time fluorescent quantitative PCR was applied to analyze the expression of BmSuc1 and BmSuc2 in midgut of 5 th-instar silkworm larvae,meanwhile the activities ofβ-fructofuranosidase was determined.[Results]BmSuc1 was expressed in the midgut of 5 th-instar silkworm larvae at different developmental stages.Its expression was upregulated at the beginning of the 5 th instar and during the peak feeding period,whereas BmSuc2 expression remained very low throughout the entire 5 th instar.The activity ofβ-fructofuranosidase was relatively high during the peak feeding period of 5 th-instar larvae,showing a trend of increasing first and then decreasing.[Conclusions]The expression pattern of the BmSuc1 gene and the changes inβ-fructofuranosidase activity were generally consistent with the physiological process of sugar nutrient absorption and utilization from mulberry leaves in 5 th-instar silkworms.It suggests that BmSuc1,as a sucrose hydrolase gene,plays a major role in the digestion and absorption of sucrose nutrients from mulberry leaves in the midgut tissue.展开更多
The pathogenesis-related protein PR10 plays a vital role in plant growth,development,and stress responses.This study systematically identified and analyzed PR10 genes in cultivated peanut(Arachis hypogaea L.),examinin...The pathogenesis-related protein PR10 plays a vital role in plant growth,development,and stress responses.This study systematically identified and analyzed PR10 genes in cultivated peanut(Arachis hypogaea L.),examining their phylogenetic relationships,conserved motifs,gene structures,and syntenic relationships.The analysis identified 54 Ah PR10 genes,which were classified into eight groups based on phylogenetic relationships,supported by gene structure and conserved motif characterization.Analysis of chromosomal distribution and synteny demonstrated that segmental duplications played a crucial role in the expansion of the Ah PR10 gene family.The identified Ah PR10 genes exhibited both constitutive and inducible expression patterns.Significantly,Ah PR10-7,Ah PR10-33,and Ah PR10-41 demonstrated potential importance in peanut resistance to Aspergillus flavus.In vitro fungistatic experiments demonstrated that recombinant Ah PR10-33 effectively inhibited A.flavus mycelial growth.These findings provide valuable insights for future investigations into Ah PR10 functions in protecting peanut from A.flavus infection.展开更多
Due to the unique microstructure and diverse opsin genes of the trinocular compound eye,stomatopoda possess an extraordinary ability to perceive multiple properties of light.They not only can detect natural light(NL)a...Due to the unique microstructure and diverse opsin genes of the trinocular compound eye,stomatopoda possess an extraordinary ability to perceive multiple properties of light.They not only can detect natural light(NL)and linearly polarized light(LPL),but also are the only animals capable of recognizing circularly polarized light(CPL).Here,we integrated single-cell RNA sequencing,previously published Illumina data,and in-situ hybridization(ISH)to quantify and localize functional opsin genes in Oratosquilla oratoria,a common stomatopoda species in the China Sea.A total of high-quality 31777 cells were captured for the first time in the O.oratoria compound eye,which were classified into 25 cell subpopulations,and hypothesized that cluster 22 is a critical cell subpopulation responsible for light(whether NL,LPL,or CPL)response in O.oratoria.Furthermore,we propose that the long-wavelengthsensitive opsin gene(lws)gene family,retinol dehydrogenase(rdh),voltage-gated ion channel(vgic),arrestin(arr),and myosin(myo)collectively mediate the light response in O.oratoria.Considering that very few vision-related opsin genes show differential expression in right-handed CPL(RCPL)-vs.-dark(DL),which provides additional evidence that stomatopoda cannot recognize RCPL.Meanwhile,we believe that UV-stimulated scaffold protein A(uvssa)and red pigment concentrating hormone(rpch)play special contributions in the left-handed CPL(LCPL)environment response.ISH revealing that 16 lws,6 middle-wavelength-sensitive(mws),and 2 ultraviolet(uv)opsin genes were expressed in the photoreceptors of the O.oratoria compound eye.Although the inability to determine the functional types of cell subpopulations limits the resolution of opsin genes,these findings systematically elucidate the specific expression patterns of opsin genes in O.oratoria and represent a significant step toward refining the visual ecological theory of O.oratoria and other stomatopod species.展开更多
Botrytis cinerea is a major necrotrophic pathogen responsible for significant crop losses worldwide.Alternative strategies to control B.cinerea are urgently needed to reduce dependence on chemical fungicides,which are...Botrytis cinerea is a major necrotrophic pathogen responsible for significant crop losses worldwide.Alternative strategies to control B.cinerea are urgently needed to reduce dependence on chemical fungicides,which are increasingly ineffective due to resistance and pose environmental risks.In this study,we identified two immunogenic epitopes derived from the B.cinerea cell death-inducing protein BcCrh1 and used them to engineer disease-resistant plants through a novel,spatially compartmentalized dual-epitope immune activation strategy.The first epitope is derived from a 35-amino acid intracellular peptide that exhibits both immunogenicity and cell death-inducing activity,which was mutated to separate these two properties.The second peptide represents an immunogenic portion of the protein that activates extracellular plant immunity.Transcriptomic and metabolomic analyses revealed that these epitopes trigger complementary defense pathways,and their co-expression integrates these responses into a robust,multilayered immunity,providing significantly enhanced protection compared with individual expression.Although constitutive expression of two epitopes conferred resistance,it also led to growth penalties.In contrast,pathogen-inducible expression of two epitopes preserved normal plant development while maintaining strong resistance to both B.cinerea and Pseudomonas syringae in Arabidopsis and tomato.This inducible strategy offers a major advantage by minimizing fitness costs while maximizing protection,highlighting the potential of spatially and temporally targeted epitope-based immune activation for durable and sustainable crop protection.展开更多
Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challengin...Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challenging due to its large genome size and high proportion of repetitive sequences.Allele-specific expression(ASE)plays a key role in regulating plant development and evolution,yet research on ASE in coconut is limited(Shao et al.,2019;Li et al.,2021;Zhang et al.,2021;Hu et al.,2022).Among phenotypic traits,fruit color is especially important as an indicator of maturity,guiding harvest timing and post-harvest processes(Kapoor et al.,2022).While prior studies have explored various coconut traits such as salt tolerance,fiber content,and plant height(Wang et al.,2021;Yang et al.,2021),investigations into ASE and fruit color remain scarce.展开更多
The prognostic and therapeutic roles of biological markers in early-stage breast cancer(eBC)warrant further investigation.Non-Breast Cancer(BRCA)genes,along with moderate-and low-penetrance breast cancer risk variant ...The prognostic and therapeutic roles of biological markers in early-stage breast cancer(eBC)warrant further investigation.Non-Breast Cancer(BRCA)genes,along with moderate-and low-penetrance breast cancer risk variant genes,are crucial formaintaining genome stability,yet their prognostic significance in eBCremains unclear.This study aimed to evaluate the impact of non-BRCA genes on clinical outcomes in eBC patients.Significant correlations were observed between the messenger ribonucleic acid(mRNA)expression levels of the genes Ataxia-telangiectasia mutated(ATM),Bloom helicase gene(BLM),and WRN RecQ Like Helicase(WRN)and patient prognosis.High mRNA expression of ATM was associated with longer metastasis-free survival(MFS).Conversely,lower mRNA expression of BLM correlated with favorable outcomes,particularly in triple-negative tumors.Additionally,high levels of WRN mRNA expression were linked to significantly longer MFS compared to low expression levels.This study highlights the prognostic significance of ATM,BLM,and WRN in predicting survival outcomes in eBC patients.Background:The prognostic significance of various biological and non-BRCA genetic in early-stage breast cancer(eBC)remains unclear and warrants further investigation.This study therefore aimed to evaluate the prognostic impact of these genes on clinical outcomes in breast cancer.Methods:Patients included in this study were subdivided into two groups based on low and high messenger ribonucleic acid(mRNA)expression levels.Statistical analysis,including Kaplan-Meier curves,univariable,andmultivariable Cox regression analyses,was performed to assess metastasis-free survival(MFS)of mRNA expression of non-BRCA genes.Subgroup analyses were also conducted among four different molecular subtypes of eBC.Results:Our analysis revealed significant correlations between mRNA-expression levels of Ataxiatelangiectasia mutated(ATM),Bloom helicase gene(BLM),and WRN RecQ Like Helicase(WRN)and patient prognosis.High mRNA expression of ATM correlated with longer MFS in the entire cohort(p=0.022,Log Rank),and in luminal-B-like tumors(p=0.036).Lower mRNA expression of BLM was associated with favorable outcomes(p=0.011,Log Rank),particularly in triple-negative eBC(p=0.030,Log Rank).Finally,high levels of WRN mRNA expression correlated with significantly longerMFS compared to lowmRNA expression levels(p=0.009,Log Rank).Conclusions:This study underscores the prognostic significance of moderate penetrance breast cancer risk variant genes,such as ATM,BLM,and WRN,for survival outcomes in eBC.展开更多
The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase （ALAS）, was expressed in Escherichia coil Rosetta （DE3） and the enzymatic properties of the purified recombinant ALAS （RC-ALAS） ...The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase （ALAS）, was expressed in Escherichia coil Rosetta （DE3） and the enzymatic properties of the purified recombinant ALAS （RC-ALAS） were studied. Compared with ALASs encoded by hemA genes from Agrobacterium radiobacter（AR-ALAS） and Rhodobacter sphaeroides （RS-ALAS）, the specific activity of RC-ALAS reached 198.2 U/mg, which was about 31.2% and 69.5% higher than those of AR-ALAS （151.1 U/mg） and RS-ALAS （116.9 U/mg）, respectively. The optimum pH values and temperatures of the three above mentioned enzymes were all pH 7.5 and 37 ℃, respectively. Moreover, RC-ALAS was more sensitive to pH, while the other two were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid （EDTA）, and sodium dodecyl sulfate （SDS） on the three ALASs were also investigated. The results indicate that they had the same effects on the activities of the three ALASs. SDS and metal ions such as Co^2＋, Zn^2＋, and Cu^2＋ strongly inhibited the activities of the ALASs, while Mn^2＋ exerted slight inhibition, and K^＋, Ca^2＋, Ba^2＋, Mg^2＋, or EDTA had no significant effect. The specificity constant of succinyl coenzyme A [（kcatlKm）^S-CoA] of RC-ALAS was 1.4989, which was higher than those of AR-ALAS （0.7456） and RS-ALAS （1.1699）, showing its high catalytic efficiency. The fed-batch fermentation was conducted using the recombinant strain containing the R. capsulatus hemA gene, and the yield of 5-aminolevulinic acid （ALA） achieved was 8.8 g/L （67 mmol/L） under the appropriate conditions.展开更多
By using the recombinant DNA technology, the gene of the bovine mature prion protein bPrPcL) has been cloned into pET30a and the resulting plasmid has been expressed in E.coli BL21(DE3). After solubilizing in 8 mol/L ...By using the recombinant DNA technology, the gene of the bovine mature prion protein bPrPcL) has been cloned into pET30a and the resulting plasmid has been expressed in E.coli BL21(DE3). After solubilizing in 8 mol/L urea, the expression product was purified by cation ion exchange chromatography. The purified product was refolded by dilution and the recovery was about 15%. Analysis of mass spectrum, circular dichroism (CD) spectrum and Fourier transform infrared (FTIR) spectrum demonstrate that the molecular weight of the bPrPcL is 23 630 u, the bPrPcL has a high α-helix content (36.1%) and low (3-sheet content (11.9%).展开更多
Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells...Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA (IFN αA) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10^(10) units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.展开更多
Artificial intelligence,such as deep learning technology,has advanced the study of facial expression recognition since facial expression carries rich emotional information and is significant for many naturalistic situ...Artificial intelligence,such as deep learning technology,has advanced the study of facial expression recognition since facial expression carries rich emotional information and is significant for many naturalistic situations.To pursue a high facial expression recognition accuracy,the network model of deep learning is generally designed to be very deep while the model’s real-time performance is typically constrained and limited.With MobileNetV3,a lightweight model with a good accuracy,a further study is conducted by adding a basic ResNet module to each of its existing modules and an SSH(Single Stage Headless Face Detector)context module to expand the model’s perceptual field.In this article,the enhanced model named Res-MobileNetV3,could alleviate the subpar of real-time performance and compress the size of large network models,which can process information at a rate of up to 33 frames per second.Although the improved model has been verified to be slightly inferior to the current state-of-the-art method in aspect of accuracy rate on the publically available face expression datasets,it can bring a good balance on accuracy,real-time performance,model size and model complexity in practical applications.展开更多
Background Numerous Asian cases of avian influenza virus infection, especially the highly pathogenic strain H5N1, in humans have raised the concern that another influenza pandemic is close. However, there are no effec...Background Numerous Asian cases of avian influenza virus infection, especially the highly pathogenic strain H5N1, in humans have raised the concern that another influenza pandemic is close. However, there are no effective therapeutic drugs or preventative vaccines available. Hemagglutinin is the membrane glycoprotein of avian influenza virus responsible for receptor binding to human cells and the main immunogenic protein that elicits a strong immune response. Although this protein is of great importance to the study of pathogenesis and vaccine development, its expression and purification are difficult due to high levels of glycosylation. Methods In this study, we expressed codon-optimized, full-length hemagglutinin 5 （H5） protein fused with a human IgG Fc tag （H5-Fc） in HEK293 cells. To enhance secretion of this protein, we also deleted the transmembrane domain and the intracellular domain of the H5 protein （H5ATM-Fc）. Purified proteins were obtained using a protein A column. Results ELISA revealed that the yield of soluble H5ATM-Fc protein in the supernatant was about 20 mg/L. Western blotting and fluorescence activated cell sorter （FACS） indicated that the purified H5 protein was correctly folded and biologically active. Conclusion Purification of H5 proteins from mammalian cells could be used for large-scale production of recombinant H5 protein for basic scientific research or the development of vaccines.展开更多
基金supported by a grant from the Public Benefit Research Foundation of China (200903052)the Science and Technology Department of Guangdong Province, China (2009A020101003)
文摘The acetylcholinesterase 2（AChE2）cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115.One transformant with high-level expression of the recombinant AChE（rAChE,23.2 U mL-1in supernatant）was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate.The maximum production of r ACh E reached about 11.8 mg of the enzyme protein per liter of culture.The r ACh E was first precipitated with ammonium sulfate（50%saturation）and then purified with procainamide affinity column chromatography.The enzyme was purified 12.1-fold with a yield of 22.8%and a high specific activity of 448.3 U mg-1.It was sensitive to inhibition by methamidophos and pirimicarb,the calculated 50% inhibitory concentration（IC50）values of the two pesticides were 0.357 and 0.888 mg L-1,respectively,and the calculated 70% inhibitory concentration（IC70）values were 0.521 and 0.839 mg L-1,respectively.The results suggested that it has a potential application in the detection of pesticide residues.
基金Supported by Natural Science Foundation of Yunnan Province(2011FB049)National Natural Science Foundation of China(31260481,31460516)+2 种基金Fund of Yunnan Education Department(2013Y251)Fund of the Department of Life Science and Technology,Kunming University(GXKM201505)Talent Fund for PhD(YJL11015)
文摘Using genomic DNA of bolting-tolerant lettuce as a template,flanking fragments of lettuce plastid rpo A gene were amplified and cloned by PCR. Targeting the sites of these two fragments,homologous recombinant fragments of exogenous gene were integrated to construct lettuce plastid expression vector p Brpo AGFP,which harbored the expression cassette Prrn-gfp-aad A-Tpsb A. The results showed that the amplified flanking fragments were 1.2 and 1.1 kb in size. After sequencing,restriction digestion,ligation and transformation,lettuce plastid expression vector containing expression cassette Prrn-gfp-aad A-Tpsb A was constructed and confirmed by SDS-PAGE electrophoresis. The results of SDS-PAGE electrophoresis indicated that gfp gene was efficiently expressed under the regulation of plasmid specific promoter Prrn and terminator Tpsb A. GFP accounted for 45. 6% of total soluble proteins; inclusion bodies accounted for 47.5 % of bacterial proteins,which reached relatively high expression levels. The construction of lettuce plastid expression vector p Brpo A-GFP laid a solid foundation for establishment of subsequent lettuce plastid transformation system and genetic improvement of lettuce using various functional genes.
基金supported by grants from National Natural Science Foundation of China(No.30972566,3087198)
文摘Objective:To investigate the effect of utilizing a molecular partner on high-level expression of Mxisca domestica(M.domestica) cecropin in Escherichia coli(E.coli) and to identify the expressed products.Methods:The genomic sequence of M.domestica cecropin A(MC) and M. domestica ubiquitin(UBI) were searched from Cenbank and amplified by reverse transcriptase polymerase chain reaction(RT-PCR).Two expression plasmids,pET32a-MC and pET32a-UBI-MC, were constructed and transferred into E.coli and were then induced by Isopropylβ-D-1- Thiogalactopyranoside(IPTG).The expression of the fusion proteins Trx-MC and Trx-UBI-MC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Fusion protein Trx-MC was verified by Western blot analysis.The bactericidal activity of the purified MC was quantitatively determined using E.coli BL21(DE3).Results:The result showed that the fusion proteins were successively expressed in E.coli BL21 cells.A band at the expected position of 24 kDa representing the Trx-MC target protein was positivelystained,and the band at 4 kDa representing the hydrolysis of mature MC protein was also observed at the expected position. The expression levels of Trx-UBI-MC were higher than that of Trx-MC in E.coli.MC exhibited antimicrobial activity.Conclusions:With high-level expression of housefly cecropin A in E.coli using a fusion protein,MC exhibited antimicrobial activity.
基金the Natural Science Fundation of Jiangsu Province,№BK95141307
文摘Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to play a vital role in the regulation of cell proliferative cycle[1，2]. Recently, many studies showed that CaM is also present in extracellular fluid such as cell culture media and normal body fluid and has been reported to stimulate proliferation in a range of normal and neoplastic cells, apparently acting as an autocrine growth factor[3-11]. In 1988, Crocker et al reported for the first time that addition of extracellular pure pig brain CaM could promote DNA synthesis and cell [7]proliferation in K562 human leukaemic lymphocytes[7].After that, more and more research was done on extracellular CaM and evidences demonstrated that extracellular CaM could also stimulate cell proliferation in normal human umbilical vein endothelial cells[5], keratinocytes[4], suspension-cultured cells of Angelica Dahurica, etc[6]. CaM is a monomeric protein of 148 amino acids that contains four homologous Ca2 + -binding domains. CaM has been highly conserved throughout the evolution. Only 1 out of 148 amino acids of human CaM is different from that of fish CaM. Complementary DNAs encoding rat, eel, chicken, human, and trypanosome CaM have been cloned.
文摘We have studied the expression of nm23(NDP) in 50 cases nasopharyngeal biopsies with anti-nm23(NDP) antibodies. As a result, the NDP positive rate in nasopharyngeal carcinoma (NPC) (95.54%) markedly increased (P<0.05), as compared with that in the normal nasopharyngeal epithelia (50.00-60.00%) and lymphocytes (52.00%). There were cytopfasmic type, nucleus type and mixed cytoplasmonucleus type according to NDP location in a cell. Their positive rates were 64.44%, 15.56% and 20.00% respectively in nasopharyngeal carcinoma. The expression of NDP had no relation with cervical lymphometastases in NPC, and the NDP positive rates had no significance between bilateral cervical lymphometastases and unilateral (P<0.05). But the NDP expression had most relation with the NPC staging. The expression rate and the intensity in Ⅲ or Ⅳ stage patients were markedly higher than that in II stage. It points out that the high-level expression of NDP had relation with the rapid cellular proliferation in NPC, and it may indicate the bad prognoses.
基金This work was supported by grant (No. 03JG05014) from the Population Family Planning Commission ofShanghai. China and the Medical and Health Science Research Foundation of Zhejiang Province(No. 2004A002)
文摘Objective To try making huZP3a^22-176 and huZP3b^177-348 polypeptides （representing an intact huZP^322-348 protein without its N-terminal signal peptide and C-terminal transmembrane domain ） express in E. coli at a higher level Methods The cDNAs encoding huZP3a and huZP3b were obtained with PCR method. The pBV221 plasmid was used to construct thermo-inducible recombinant expression vector. Purification of two target expression products employed an improved method of preparative gel polyacrylamide gel electrophoresis. Results Two polypeptides of recombinant huZP3a （rhuZP3a） and recombinant huZP3b （rhuZP3b） were all expressed respectively in an E. coli BL21（DE3）pLysS strain at a higher level, which were recognized by two specific polyclonal antisera in Western blotting test which recognize a linear B cell epitope present in rhuZP3a or rhuZP3b respectively. Using the shake-flask method, approximately 5 mg of rhuZP3a and rhuZP3b with more than 95% relative homogeneity were harvested from 1 L culture respectively. Conclusion The availability of two rhuZP3 polypeptides will help in detecting the immunogenicities of rhuZP3a and rhuZP3b through animal experiments and confirming the function domain of non-glycosylated huZP3 to induce acrosome reaction in vitro.
文摘Human interferon alpha-8(IFN-α8) is an important cytokine with multiple biological functions.A genetically engineered strain, E. coli XL1-Blue/pBm, was constructed by DNA recombination technology and characterized by restriction analysis, DNA sequencing.
文摘In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL 1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL 1 was transformed into expression host M15 （pREP4） and induced by isopropyl-a-D-thiogalactoside （IPTG） to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol＇L-1 IPTG induction as the expression temperature declined from 37 to 28℃. The 6-His tag facilitates affinity binding to Ni^2＋-nitrolotriacetic acid （NTA） and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose coupled with Ni^2＋-NTA affinity chromatography. The optimal substrate for Pt4CL 1 was 4-coumarate.
基金supported by the National Natural Science Foundation of China(Grant No.52405041)the Major Program of the Zhejiang Provincial Natural Science Foundation of China(Grant No.LD25E050001)the Key R&D Program of Zhejiang Province(Grant No.2025C01186)。
文摘Humanoid robots hold significant promise for social interaction and emotional companionship.However,their effectiveness hinges on the ability to convey nuanced and authentic emotions.Here,we presented a universal humanoid robot head with a facial kinematics model.Using a reinforcement learning framework guided by symmetry assessment,emotion decoupling,and MLLM authenticity evaluation,our system autonomously learns to generate adaptive facial expressions through dynamic landmark adjustments.By transferring the simulation training results to real-world environments,the robot can perform natural and expressive expressions.Another novel feature is the independent regulation of emotion intensity and expression magnitude across emotional categories,which enhances the ability to achieve culturally adaptive and socially resonant robotic expressions significantly.This research advances adaptive humanoid interaction,offering an easier and more efficient pathway toward culturally resonant and psychologically plausible robotic expressions.
基金Supported by General Project of Yunnan Provincial Agricultural Basic Research Joint Special Project(202301BD070001-229)Yunnan Provincial Key R&D Program(202403AK140075)+1 种基金Modern Sericulture Industry Technology System of Yunan Province(KJTX-07)Honghe Comprehensive Test Station of National Sericulture Industry Technology System(CARS-18).
文摘[Objectives]The present study was conducted to investigate the change rule ofβ-fructofuranosidase gene expression and its enzyme activity in the midgut of 5 th instar silkworm(Bombyx mori),in order to provide a reference for illustrating the enzymatic mechanism of usingβ-fructofuranosidase to absorb sucrose nutrition from mulberry leaves.[Methods]Real-time fluorescent quantitative PCR was applied to analyze the expression of BmSuc1 and BmSuc2 in midgut of 5 th-instar silkworm larvae,meanwhile the activities ofβ-fructofuranosidase was determined.[Results]BmSuc1 was expressed in the midgut of 5 th-instar silkworm larvae at different developmental stages.Its expression was upregulated at the beginning of the 5 th instar and during the peak feeding period,whereas BmSuc2 expression remained very low throughout the entire 5 th instar.The activity ofβ-fructofuranosidase was relatively high during the peak feeding period of 5 th-instar larvae,showing a trend of increasing first and then decreasing.[Conclusions]The expression pattern of the BmSuc1 gene and the changes inβ-fructofuranosidase activity were generally consistent with the physiological process of sugar nutrient absorption and utilization from mulberry leaves in 5 th-instar silkworms.It suggests that BmSuc1,as a sucrose hydrolase gene,plays a major role in the digestion and absorption of sucrose nutrients from mulberry leaves in the midgut tissue.
基金supported by the National Key R&D Program of China(2022YFD1200400)the National Natural Science Foundation of China(32301851)。
文摘The pathogenesis-related protein PR10 plays a vital role in plant growth,development,and stress responses.This study systematically identified and analyzed PR10 genes in cultivated peanut(Arachis hypogaea L.),examining their phylogenetic relationships,conserved motifs,gene structures,and syntenic relationships.The analysis identified 54 Ah PR10 genes,which were classified into eight groups based on phylogenetic relationships,supported by gene structure and conserved motif characterization.Analysis of chromosomal distribution and synteny demonstrated that segmental duplications played a crucial role in the expansion of the Ah PR10 gene family.The identified Ah PR10 genes exhibited both constitutive and inducible expression patterns.Significantly,Ah PR10-7,Ah PR10-33,and Ah PR10-41 demonstrated potential importance in peanut resistance to Aspergillus flavus.In vitro fungistatic experiments demonstrated that recombinant Ah PR10-33 effectively inhibited A.flavus mycelial growth.These findings provide valuable insights for future investigations into Ah PR10 functions in protecting peanut from A.flavus infection.
基金Supported by the Natural Science Foundation of Shandong Province(No.ZR2021QD110)the National Natural Science Foundation of China(No.42106128)。
文摘Due to the unique microstructure and diverse opsin genes of the trinocular compound eye,stomatopoda possess an extraordinary ability to perceive multiple properties of light.They not only can detect natural light(NL)and linearly polarized light(LPL),but also are the only animals capable of recognizing circularly polarized light(CPL).Here,we integrated single-cell RNA sequencing,previously published Illumina data,and in-situ hybridization(ISH)to quantify and localize functional opsin genes in Oratosquilla oratoria,a common stomatopoda species in the China Sea.A total of high-quality 31777 cells were captured for the first time in the O.oratoria compound eye,which were classified into 25 cell subpopulations,and hypothesized that cluster 22 is a critical cell subpopulation responsible for light(whether NL,LPL,or CPL)response in O.oratoria.Furthermore,we propose that the long-wavelengthsensitive opsin gene(lws)gene family,retinol dehydrogenase(rdh),voltage-gated ion channel(vgic),arrestin(arr),and myosin(myo)collectively mediate the light response in O.oratoria.Considering that very few vision-related opsin genes show differential expression in right-handed CPL(RCPL)-vs.-dark(DL),which provides additional evidence that stomatopoda cannot recognize RCPL.Meanwhile,we believe that UV-stimulated scaffold protein A(uvssa)and red pigment concentrating hormone(rpch)play special contributions in the left-handed CPL(LCPL)environment response.ISH revealing that 16 lws,6 middle-wavelength-sensitive(mws),and 2 ultraviolet(uv)opsin genes were expressed in the photoreceptors of the O.oratoria compound eye.Although the inability to determine the functional types of cell subpopulations limits the resolution of opsin genes,these findings systematically elucidate the specific expression patterns of opsin genes in O.oratoria and represent a significant step toward refining the visual ecological theory of O.oratoria and other stomatopod species.
基金supported by the National Natural Science Foundation of China(grant no.32372514)the Research and Innovation Initiatives of WHPU(grant no.2024J02)+1 种基金Y.L.(202108280009)was funded by the China Scholarship Councilsupported by BARD(grant no.5261-20C)to A.S and T.M.
文摘Botrytis cinerea is a major necrotrophic pathogen responsible for significant crop losses worldwide.Alternative strategies to control B.cinerea are urgently needed to reduce dependence on chemical fungicides,which are increasingly ineffective due to resistance and pose environmental risks.In this study,we identified two immunogenic epitopes derived from the B.cinerea cell death-inducing protein BcCrh1 and used them to engineer disease-resistant plants through a novel,spatially compartmentalized dual-epitope immune activation strategy.The first epitope is derived from a 35-amino acid intracellular peptide that exhibits both immunogenicity and cell death-inducing activity,which was mutated to separate these two properties.The second peptide represents an immunogenic portion of the protein that activates extracellular plant immunity.Transcriptomic and metabolomic analyses revealed that these epitopes trigger complementary defense pathways,and their co-expression integrates these responses into a robust,multilayered immunity,providing significantly enhanced protection compared with individual expression.Although constitutive expression of two epitopes conferred resistance,it also led to growth penalties.In contrast,pathogen-inducible expression of two epitopes preserved normal plant development while maintaining strong resistance to both B.cinerea and Pseudomonas syringae in Arabidopsis and tomato.This inducible strategy offers a major advantage by minimizing fitness costs while maximizing protection,highlighting the potential of spatially and temporally targeted epitope-based immune activation for durable and sustainable crop protection.
基金supported by Central Public-interest Scientific Institution Basal Research Fund(CATAS-Nos.1630152023007,1630152023011,1630152023012,1630152023013)the National Natural Science Foundation of China(Grant No.32071805).
文摘Coconut(Cocos nucifera L.),a major oil and fruit crop of the Arecaceae family,is extensively cultivated across the Asia—Pacific region.Despite its agricultural importance,genome assembly in coconut remains challenging due to its large genome size and high proportion of repetitive sequences.Allele-specific expression(ASE)plays a key role in regulating plant development and evolution,yet research on ASE in coconut is limited(Shao et al.,2019;Li et al.,2021;Zhang et al.,2021;Hu et al.,2022).Among phenotypic traits,fruit color is especially important as an indicator of maturity,guiding harvest timing and post-harvest processes(Kapoor et al.,2022).While prior studies have explored various coconut traits such as salt tolerance,fiber content,and plant height(Wang et al.,2021;Yang et al.,2021),investigations into ASE and fruit color remain scarce.
文摘The prognostic and therapeutic roles of biological markers in early-stage breast cancer(eBC)warrant further investigation.Non-Breast Cancer(BRCA)genes,along with moderate-and low-penetrance breast cancer risk variant genes,are crucial formaintaining genome stability,yet their prognostic significance in eBCremains unclear.This study aimed to evaluate the impact of non-BRCA genes on clinical outcomes in eBC patients.Significant correlations were observed between the messenger ribonucleic acid(mRNA)expression levels of the genes Ataxia-telangiectasia mutated(ATM),Bloom helicase gene(BLM),and WRN RecQ Like Helicase(WRN)and patient prognosis.High mRNA expression of ATM was associated with longer metastasis-free survival(MFS).Conversely,lower mRNA expression of BLM correlated with favorable outcomes,particularly in triple-negative tumors.Additionally,high levels of WRN mRNA expression were linked to significantly longer MFS compared to low expression levels.This study highlights the prognostic significance of ATM,BLM,and WRN in predicting survival outcomes in eBC patients.Background:The prognostic significance of various biological and non-BRCA genetic in early-stage breast cancer(eBC)remains unclear and warrants further investigation.This study therefore aimed to evaluate the prognostic impact of these genes on clinical outcomes in breast cancer.Methods:Patients included in this study were subdivided into two groups based on low and high messenger ribonucleic acid(mRNA)expression levels.Statistical analysis,including Kaplan-Meier curves,univariable,andmultivariable Cox regression analyses,was performed to assess metastasis-free survival(MFS)of mRNA expression of non-BRCA genes.Subgroup analyses were also conducted among four different molecular subtypes of eBC.Results:Our analysis revealed significant correlations between mRNA-expression levels of Ataxiatelangiectasia mutated(ATM),Bloom helicase gene(BLM),and WRN RecQ Like Helicase(WRN)and patient prognosis.High mRNA expression of ATM correlated with longer MFS in the entire cohort(p=0.022,Log Rank),and in luminal-B-like tumors(p=0.036).Lower mRNA expression of BLM was associated with favorable outcomes(p=0.011,Log Rank),particularly in triple-negative eBC(p=0.030,Log Rank).Finally,high levels of WRN mRNA expression correlated with significantly longerMFS compared to lowmRNA expression levels(p=0.009,Log Rank).Conclusions:This study underscores the prognostic significance of moderate penetrance breast cancer risk variant genes,such as ATM,BLM,and WRN,for survival outcomes in eBC.
基金Project supported by the National Natural Science Foundation of China (No.20306026)the National Basic Research Program (973) of China (No.2007CB707805)
文摘The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase （ALAS）, was expressed in Escherichia coil Rosetta （DE3） and the enzymatic properties of the purified recombinant ALAS （RC-ALAS） were studied. Compared with ALASs encoded by hemA genes from Agrobacterium radiobacter（AR-ALAS） and Rhodobacter sphaeroides （RS-ALAS）, the specific activity of RC-ALAS reached 198.2 U/mg, which was about 31.2% and 69.5% higher than those of AR-ALAS （151.1 U/mg） and RS-ALAS （116.9 U/mg）, respectively. The optimum pH values and temperatures of the three above mentioned enzymes were all pH 7.5 and 37 ℃, respectively. Moreover, RC-ALAS was more sensitive to pH, while the other two were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid （EDTA）, and sodium dodecyl sulfate （SDS） on the three ALASs were also investigated. The results indicate that they had the same effects on the activities of the three ALASs. SDS and metal ions such as Co^2＋, Zn^2＋, and Cu^2＋ strongly inhibited the activities of the ALASs, while Mn^2＋ exerted slight inhibition, and K^＋, Ca^2＋, Ba^2＋, Mg^2＋, or EDTA had no significant effect. The specificity constant of succinyl coenzyme A [（kcatlKm）^S-CoA] of RC-ALAS was 1.4989, which was higher than those of AR-ALAS （0.7456） and RS-ALAS （1.1699）, showing its high catalytic efficiency. The fed-batch fermentation was conducted using the recombinant strain containing the R. capsulatus hemA gene, and the yield of 5-aminolevulinic acid （ALA） achieved was 8.8 g/L （67 mmol/L） under the appropriate conditions.
文摘By using the recombinant DNA technology, the gene of the bovine mature prion protein bPrPcL) has been cloned into pET30a and the resulting plasmid has been expressed in E.coli BL21(DE3). After solubilizing in 8 mol/L urea, the expression product was purified by cation ion exchange chromatography. The purified product was refolded by dilution and the recovery was about 15%. Analysis of mass spectrum, circular dichroism (CD) spectrum and Fourier transform infrared (FTIR) spectrum demonstrate that the molecular weight of the bPrPcL is 23 630 u, the bPrPcL has a high α-helix content (36.1%) and low (3-sheet content (11.9%).
文摘Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA (IFN αA) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10^(10) units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.
基金supported by China Academy of Railway Sciences Corporation Limited(No.2021YJ127).
文摘Artificial intelligence,such as deep learning technology,has advanced the study of facial expression recognition since facial expression carries rich emotional information and is significant for many naturalistic situations.To pursue a high facial expression recognition accuracy,the network model of deep learning is generally designed to be very deep while the model’s real-time performance is typically constrained and limited.With MobileNetV3,a lightweight model with a good accuracy,a further study is conducted by adding a basic ResNet module to each of its existing modules and an SSH(Single Stage Headless Face Detector)context module to expand the model’s perceptual field.In this article,the enhanced model named Res-MobileNetV3,could alleviate the subpar of real-time performance and compress the size of large network models,which can process information at a rate of up to 33 frames per second.Although the improved model has been verified to be slightly inferior to the current state-of-the-art method in aspect of accuracy rate on the publically available face expression datasets,it can bring a good balance on accuracy,real-time performance,model size and model complexity in practical applications.
基金This work was supported by the grants from the National Natural Science Foundation of China （No. 30625013, No. 30623009 and No. 30721063） and the Ministry of Science and Technology of China （No. 2009CB522105 and No. 2005CB523000）.
文摘Background Numerous Asian cases of avian influenza virus infection, especially the highly pathogenic strain H5N1, in humans have raised the concern that another influenza pandemic is close. However, there are no effective therapeutic drugs or preventative vaccines available. Hemagglutinin is the membrane glycoprotein of avian influenza virus responsible for receptor binding to human cells and the main immunogenic protein that elicits a strong immune response. Although this protein is of great importance to the study of pathogenesis and vaccine development, its expression and purification are difficult due to high levels of glycosylation. Methods In this study, we expressed codon-optimized, full-length hemagglutinin 5 （H5） protein fused with a human IgG Fc tag （H5-Fc） in HEK293 cells. To enhance secretion of this protein, we also deleted the transmembrane domain and the intracellular domain of the H5 protein （H5ATM-Fc）. Purified proteins were obtained using a protein A column. Results ELISA revealed that the yield of soluble H5ATM-Fc protein in the supernatant was about 20 mg/L. Western blotting and fluorescence activated cell sorter （FACS） indicated that the purified H5 protein was correctly folded and biologically active. Conclusion Purification of H5 proteins from mammalian cells could be used for large-scale production of recombinant H5 protein for basic scientific research or the development of vaccines.
