Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C...Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.展开更多
Sumoylation is an important protein modification discovered recently. SUMO（small ubiquitin-related modifier） pathway regulates the protein stability and transcriptional activity with a 12-kDa small molecular protein...Sumoylation is an important protein modification discovered recently. SUMO（small ubiquitin-related modifier） pathway regulates the protein stability and transcriptional activity with a 12-kDa small molecular protein, SUMO, ligated to the target protein. The purification of SUMO proteins is a key step to reveal their function. The purpose of this study was to construct the recombinant SUMO1 gene cloned to a pGEX-4T-1 vector to express and purify the SUMO1-GST fusion protein in Escherichia coli. First, the full length DNA sequence of SUMO1 gene was amplified by PCR and was ligated to pMD18-T vector. Then the SUMO1 gene was subcloned to pGEX-4T-1 prokaryotic expression vector between BamHI and XhoI sites, and transformed in Escherichia coli DH5α cells. The right colonies were identified by restrictive enzyme digestion and sequencing. The correct rebombinant plasmid of pGEX-4T-1-SUMO1 was transformed in Escherichia coli BL21 cells and then induced by IPTG（isopropyl- β-D-1- thiogalacto-pyranoside） to express the SUMO1-GST fusion protein. The highly purified SUMO1-GST（glutathione S-transferase） fusion protein was obtained by affinity chromatography. Finally, the properties of SUMO1-GST fusion protein were confirmed by Coomassie brilliant blue strain and Western blot analysis. The recombinant plasmid of pGEX-4T-1-SUMO1 was successfully constructed, and SUMO1-GST fusion proteins were successfully expressed.展开更多
Objective: To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRET to study the dimerization and function of CD11 b( Mac-1 α subunit) and CD18(Mac-1 β subunit). Methods: The mammalia...Objective: To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRET to study the dimerization and function of CD11 b( Mac-1 α subunit) and CD18(Mac-1 β subunit). Methods: The mammalian cell expression vector for CD11b fused with CFP at the carboxyl terminal was constructed to create recombinant plasmid of pCD11b-CFP. Then pCD11b-CFP was co-transfected with pYFP-CD18 into CHO cell, a fibroblast like cell line, as a target cell within which there are some signal pathways involved in inflammatory stimulation but without endogenous Mac-1. Then CHO cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were selected by Western blot and laser scanning confocal microscope. Results: The cyan and yellow fluorescence in co-transfected positive CHO cells were observed under a fluorescence microscope. CHO-Mac-1-FP cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were obtained as demonstrated by Western blot successfully. The adhesive activity of CHO-Mac-1-FP cells with CHO-1CAM-1 cells was increased markedly by treatment with PMA, suggesting the translocation of GD11b-CFP and YFP-CD18 to the plasma membrane in CHO-Mac-1-FP cells and dimerization of CD11b-CFP and YFP-CD18 just as the function of the wild type Mac-1. Conclusion: CHO-Mac-1-FP cells with adhesive activity are established successfully, thus CHO-Mac-1-FP cells may be useful for the study of Mac-1 by FRET and for other purposes.展开更多
Objective:To induce potent CD8^+T-cells against glypican-3(GPC3),which is overexpressed in hepatocellular carcinoma(HCC),to suspend tumor development.Methods:Since the chemokine receptor XCR1 is selectively expressed ...Objective:To induce potent CD8^+T-cells against glypican-3(GPC3),which is overexpressed in hepatocellular carcinoma(HCC),to suspend tumor development.Methods:Since the chemokine receptor XCR1 is selectively expressed on professional cross-presenting CD8α^+dendritic cells(DCs).展开更多
Low-density lipoprotein receptor-related protein 1(LRP1,also known as CD91),a multifunctional endocytic and cell signaling receptor,is widely expressed on the surface of multiple cell types such as hepatocytes,fibrobl...Low-density lipoprotein receptor-related protein 1(LRP1,also known as CD91),a multifunctional endocytic and cell signaling receptor,is widely expressed on the surface of multiple cell types such as hepatocytes,fibroblasts,neurons,astrocytes,macrophages,smooth muscle cells,and malignant cells.Emerging in vitro and in vivo evidence demonstrates that LRP1 is critically involved in many processes that drive tumorigenesis and tumor progression.For example,LRP1 not only promotes tumor cell migration and invasion by regulating matrix metalloproteinase(MMP)-2and MMP-9 expression and functions but also inhibits cell apoptosis by regulating the insulin receptor,the serine/threonine protein kinase signaling pathway,and the expression of Caspase-3.LRPI-mediated phosphorylation of the extracellular signal-regulated kinase pathway and c-jun N-terminal kinase are also involved in tumor cell proliferation and invasion.In addition,LRP1 has been shown to be down-regulated by microRNA-205 and methylation of LRP1CpG islands.Furthermore,a novel fusion gene,LRP1-SNRNP25,promotes osteosarcoma cell invasion and migration.Only by understanding the mechanisms of these effects can we develop novel diagnostic and therapeutic strategies for cancers mediated by LRP1.展开更多
To enhance the stability of recombinant human collagen α1(I) chains(rhCOL1 A1) in production and purification stages, a gene fragment fusing COL1 A1 and insulin protein coding domains was synthesized and inserted int...To enhance the stability of recombinant human collagen α1(I) chains(rhCOL1 A1) in production and purification stages, a gene fragment fusing COL1 A1 and insulin protein coding domains was synthesized and inserted into the pPIC9 K expression vector. The fusion peptide-expressing Pichia pastoris strain was created by transformation.After optimization of shake flask cultures, the ultimate intracellular expression level of the insulin-collagen α1(I) chain fusion protein(INS-COL1 A1) reached about 300 mg·L^(-1), and no obvious protein degradation was found in the fermentation and purification processes. The His-tagged recombinant fusion protein was detected by western blotting and was effectively purified using Ni^(2+)-chelating chromatography. A prominent improvement in the stability of INS-COL1 A1 was observed compared to rhCOL1 A1 in vitro, and the rhCOL1 A1 released from the fusion protein was studied by LC–MS/MS and in bioassays. The results showed that the purified rhCOL1 A1 was consistent with the native protein in amino acid composition and had a similar biological compatibility. To our knowledge, this is the first study to demonstrate the use of insulin as a fusion protein to improve the stability of easily degradable proteins.展开更多
Lassa virus(LASV)is an enveloped,negative-sense RNA virus that causes Lassa hemorrhagic fever.Successful entry of LASV requires the viral glycoprotein 1(GP1)to undergo a receptor switch from its primary receptor alpha...Lassa virus(LASV)is an enveloped,negative-sense RNA virus that causes Lassa hemorrhagic fever.Successful entry of LASV requires the viral glycoprotein 1(GP1)to undergo a receptor switch from its primary receptor alpha-dystroglycan(α-DG)to its endosomal receptor lysosome-associated membrane protein 1(LAMP1).A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch.To test the hypothesis that other non-conserved residues also contribute to receptor switch,we constructed a series of mutant LASV GP1 proteins and tested them for binding to LAMP1.Four residues,L84,K88,L107,and H170,were identified as critical for receptor switch.Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus(LCMV)residue(L84 N,K88E,L10F,and H170S)reduced the binding affinity of LASV GP1 for LAMP1.Moreover,all mutations caused decreases in glycoprotein precursor(GPC)-mediated membrane fusion at both pH 4.5 and 5.2.The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types,while L107F and H170S had only mild effects on infectivity.Using biolayer light interferometry assay,we found that all four mutants had decreased binding affinity to LAMP1,in the order of binding affinity being L84 N>L107F>K88E>H170S.The four amino acid loci identified for the first time in this study have important reference significance for the in-depth investigation of the mechanism of receptor switching and immune escape of LASV occurrence and the development of reserve anti-LASV infection drugs.展开更多
Two GST-IL-1 fusion genes were constructed by inserting different cDNA fragments of human interleukin1 (IL-1) into the 3'-terminus of GST gene in the fusion protein expression vector pGEX-4T. After IPTG induction ...Two GST-IL-1 fusion genes were constructed by inserting different cDNA fragments of human interleukin1 (IL-1) into the 3'-terminus of GST gene in the fusion protein expression vector pGEX-4T. After IPTG induction ,SDS-PAGE was employed to detect the gene expression. No corresponding protein encoded by GST gene fused with the whole-length 816 bp IL-1 cDNA was observed, nor was free GST protein. However, the fusion protein of GST and IL-1 cDNA without the 189 bp at the 5'- terminus was detected, amounting to 30% of the total bacterial protein expressed. This might suggest that the sequence of 1-189 bp of IL-1 cDNA affected the expression of the fusion gene. That is to say, the downstream sequence distant from the translation start codon AUG in the target gene could significantly affect the expression of the fusion gene.展开更多
Objective: To explore the mechanisms of microRNA-150, cyclin B1 and mitochondrial-associated protein 2 in regulating the apoptosis and inhibiting the invasion and migration of Huh-7 cells. Methods: Huh-7 cells were di...Objective: To explore the mechanisms of microRNA-150, cyclin B1 and mitochondrial-associated protein 2 in regulating the apoptosis and inhibiting the invasion and migration of Huh-7 cells. Methods: Huh-7 cells were divided into the control group, the negative control group (NC group) and the miR-150 overexpression group (mimic group). The miR-150 overexpressing cell line was constructed by plasmid transfection. The cell viability and apoptosis were detected by cell counting kit-8 and flow cytometry. The cell migration and invasion capacity were measured by cell wound scratch assay and Transwell. The levels of miRNA and mRNA were detected by real-time quantitative polymerase chain reaction and the relative expression levels of proteins were detected by Western blot. Results: MiR-150 significantly inhibited the cell viability of Huh-7 and promoted its apoptosis (P<0.01). After 24 h of cultivation, the mobility of the control group and the NC group were (83.54±4.66)%and (85.57±4.74)%, respectively. The mobility of the mimic group was (49.63±3.78)%, which was significantly lower than that of the control group and the NC group (P<0.01). After 24 h of cultivation, the invasive rate of the control group and the NC group were (100.56±2.87)%and (101.63±3.74)%, respectively, and the invasive rate of mimic group was (51.63±5.32)%, which was significantly lower than that of the control group and the NC group (P<0.01). The expression levels of cyclin B1 protein and mRNA in the mimic group were significantly lower than those in the control group and the NC group (P<0.01), and the level of mitochondrial-associated protein 2 in the mimic group was significantly higher than that in the control group and the NC group (P<0.01). Conclusions: MiR-150 may inhibit the proliferation, migration, invasion and apoptosis of hepatoma carcinoma cell by regulating cyclin B1 or up-regulating mitochondrial-associated protein 2 levels.展开更多
Feline herpesvirus type 1(FHV-1)is a common and highly contagious pathogen in domestic cats that causes upper respiratory tract infections and ocular diseases.Accurate and rapid diagnosis of FHV-1 infections is essent...Feline herpesvirus type 1(FHV-1)is a common and highly contagious pathogen in domestic cats that causes upper respiratory tract infections and ocular diseases.Accurate and rapid diagnosis of FHV-1 infections is essential for effective disease management and control.In this study,we developed an immunochromatographic lateral flow(ICLF)assay for the rapid and accurate detection of FHV-1-specific antibodies.The assay was founded upon the successful expression and purification of a 26 kDa recombinant glycoprotein B-glycoprotein D(gB-gD)fusion protein,which served as the primary antigen for the test.Rigorous testing for specificity and cross-reactivity confirmed the strip’s ability to exclusively detect FHV-1 antibodies,even in the presence of a variety of other feline viruses.The assay demonstrated excellent precision,reproducibility across dilutions,and long-term stability,retaining efficacy for 24 months during storage.Furthermore,clinical sample analysis revealed exceptional sensitivity(97%)and specificity(100%).In conclusion,the ICLF strip developed in this study represents a reliable,highly specific,and stable diagnostic tool for the rapid detection and management of FHV-1 infections in cats.展开更多
基金Supported by the National Natural Science Foundation of China(Nos.30700827 and 30871301)Jilin Provincial Science & Technology Department of China(Nos.20070719 and 20080731)Northeast Normal University,China(Nos.20070401,NENU-STC07005)
文摘Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.
文摘Sumoylation is an important protein modification discovered recently. SUMO（small ubiquitin-related modifier） pathway regulates the protein stability and transcriptional activity with a 12-kDa small molecular protein, SUMO, ligated to the target protein. The purification of SUMO proteins is a key step to reveal their function. The purpose of this study was to construct the recombinant SUMO1 gene cloned to a pGEX-4T-1 vector to express and purify the SUMO1-GST fusion protein in Escherichia coli. First, the full length DNA sequence of SUMO1 gene was amplified by PCR and was ligated to pMD18-T vector. Then the SUMO1 gene was subcloned to pGEX-4T-1 prokaryotic expression vector between BamHI and XhoI sites, and transformed in Escherichia coli DH5α cells. The right colonies were identified by restrictive enzyme digestion and sequencing. The correct rebombinant plasmid of pGEX-4T-1-SUMO1 was transformed in Escherichia coli BL21 cells and then induced by IPTG（isopropyl- β-D-1- thiogalacto-pyranoside） to express the SUMO1-GST fusion protein. The highly purified SUMO1-GST（glutathione S-transferase） fusion protein was obtained by affinity chromatography. Finally, the properties of SUMO1-GST fusion protein were confirmed by Coomassie brilliant blue strain and Western blot analysis. The recombinant plasmid of pGEX-4T-1-SUMO1 was successfully constructed, and SUMO1-GST fusion proteins were successfully expressed.
基金Supported by National Natural Science Foundation of China (No.30000068, 39730210)
文摘Objective: To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRET to study the dimerization and function of CD11 b( Mac-1 α subunit) and CD18(Mac-1 β subunit). Methods: The mammalian cell expression vector for CD11b fused with CFP at the carboxyl terminal was constructed to create recombinant plasmid of pCD11b-CFP. Then pCD11b-CFP was co-transfected with pYFP-CD18 into CHO cell, a fibroblast like cell line, as a target cell within which there are some signal pathways involved in inflammatory stimulation but without endogenous Mac-1. Then CHO cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were selected by Western blot and laser scanning confocal microscope. Results: The cyan and yellow fluorescence in co-transfected positive CHO cells were observed under a fluorescence microscope. CHO-Mac-1-FP cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were obtained as demonstrated by Western blot successfully. The adhesive activity of CHO-Mac-1-FP cells with CHO-1CAM-1 cells was increased markedly by treatment with PMA, suggesting the translocation of GD11b-CFP and YFP-CD18 to the plasma membrane in CHO-Mac-1-FP cells and dimerization of CD11b-CFP and YFP-CD18 just as the function of the wild type Mac-1. Conclusion: CHO-Mac-1-FP cells with adhesive activity are established successfully, thus CHO-Mac-1-FP cells may be useful for the study of Mac-1 by FRET and for other purposes.
文摘Objective:To induce potent CD8^+T-cells against glypican-3(GPC3),which is overexpressed in hepatocellular carcinoma(HCC),to suspend tumor development.Methods:Since the chemokine receptor XCR1 is selectively expressed on professional cross-presenting CD8α^+dendritic cells(DCs).
基金the National Natural Science Foundation of China(81372872 to J.Yang,81402215 to X.Du,and 81320108022 to K.Chen)funds from the University Cancer Foundation via the Sister Institution Network Fund at the Tianjin Medical University Cancer Institute and Hospital,Fudan University Shanghai Cancer Center,and University of Texas MD Anderson Cancer Centersupported by the program for Innovative Research Team in University in China(IRT1076 to K.Chen)
文摘Low-density lipoprotein receptor-related protein 1(LRP1,also known as CD91),a multifunctional endocytic and cell signaling receptor,is widely expressed on the surface of multiple cell types such as hepatocytes,fibroblasts,neurons,astrocytes,macrophages,smooth muscle cells,and malignant cells.Emerging in vitro and in vivo evidence demonstrates that LRP1 is critically involved in many processes that drive tumorigenesis and tumor progression.For example,LRP1 not only promotes tumor cell migration and invasion by regulating matrix metalloproteinase(MMP)-2and MMP-9 expression and functions but also inhibits cell apoptosis by regulating the insulin receptor,the serine/threonine protein kinase signaling pathway,and the expression of Caspase-3.LRPI-mediated phosphorylation of the extracellular signal-regulated kinase pathway and c-jun N-terminal kinase are also involved in tumor cell proliferation and invasion.In addition,LRP1 has been shown to be down-regulated by microRNA-205 and methylation of LRP1CpG islands.Furthermore,a novel fusion gene,LRP1-SNRNP25,promotes osteosarcoma cell invasion and migration.Only by understanding the mechanisms of these effects can we develop novel diagnostic and therapeutic strategies for cancers mediated by LRP1.
基金Supported by the National Natural Science Foundation of China(21676214,21576160,21506171)Shaanxi Key Laboratory of Degradable Biomedical Materials Program(2014SZS07-K04,2014SZS07-P05,15JS105,15JS106,2014SZS07-Z01,2014SZS07-K03)Shaanxi R&D Center of Biomaterials and Fermentation Engineering Program(2015HBGC-04)
文摘To enhance the stability of recombinant human collagen α1(I) chains(rhCOL1 A1) in production and purification stages, a gene fragment fusing COL1 A1 and insulin protein coding domains was synthesized and inserted into the pPIC9 K expression vector. The fusion peptide-expressing Pichia pastoris strain was created by transformation.After optimization of shake flask cultures, the ultimate intracellular expression level of the insulin-collagen α1(I) chain fusion protein(INS-COL1 A1) reached about 300 mg·L^(-1), and no obvious protein degradation was found in the fermentation and purification processes. The His-tagged recombinant fusion protein was detected by western blotting and was effectively purified using Ni^(2+)-chelating chromatography. A prominent improvement in the stability of INS-COL1 A1 was observed compared to rhCOL1 A1 in vitro, and the rhCOL1 A1 released from the fusion protein was studied by LC–MS/MS and in bioassays. The results showed that the purified rhCOL1 A1 was consistent with the native protein in amino acid composition and had a similar biological compatibility. To our knowledge, this is the first study to demonstrate the use of insulin as a fusion protein to improve the stability of easily degradable proteins.
基金supported by the National Key Research and Development Program of China(2023YFC2605504,2022YFC2303300)the National Natural Sciences Foundation of China(82172273 and 31670165)+3 种基金the Open Research Fund Program of the State Key Laboratory of Virology of China(2023JZZD-01)the Health research project of Shaanxi Province(2022D040)the Science and Technology Planning Project of Shaanxi Provincial Education Department(22JK0545)the Natural Science Basic Research Program of Shaanxi(2024JC-YBQN-0922).
文摘Lassa virus(LASV)is an enveloped,negative-sense RNA virus that causes Lassa hemorrhagic fever.Successful entry of LASV requires the viral glycoprotein 1(GP1)to undergo a receptor switch from its primary receptor alpha-dystroglycan(α-DG)to its endosomal receptor lysosome-associated membrane protein 1(LAMP1).A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch.To test the hypothesis that other non-conserved residues also contribute to receptor switch,we constructed a series of mutant LASV GP1 proteins and tested them for binding to LAMP1.Four residues,L84,K88,L107,and H170,were identified as critical for receptor switch.Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus(LCMV)residue(L84 N,K88E,L10F,and H170S)reduced the binding affinity of LASV GP1 for LAMP1.Moreover,all mutations caused decreases in glycoprotein precursor(GPC)-mediated membrane fusion at both pH 4.5 and 5.2.The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types,while L107F and H170S had only mild effects on infectivity.Using biolayer light interferometry assay,we found that all four mutants had decreased binding affinity to LAMP1,in the order of binding affinity being L84 N>L107F>K88E>H170S.The four amino acid loci identified for the first time in this study have important reference significance for the in-depth investigation of the mechanism of receptor switching and immune escape of LASV occurrence and the development of reserve anti-LASV infection drugs.
文摘Two GST-IL-1 fusion genes were constructed by inserting different cDNA fragments of human interleukin1 (IL-1) into the 3'-terminus of GST gene in the fusion protein expression vector pGEX-4T. After IPTG induction ,SDS-PAGE was employed to detect the gene expression. No corresponding protein encoded by GST gene fused with the whole-length 816 bp IL-1 cDNA was observed, nor was free GST protein. However, the fusion protein of GST and IL-1 cDNA without the 189 bp at the 5'- terminus was detected, amounting to 30% of the total bacterial protein expressed. This might suggest that the sequence of 1-189 bp of IL-1 cDNA affected the expression of the fusion gene. That is to say, the downstream sequence distant from the translation start codon AUG in the target gene could significantly affect the expression of the fusion gene.
文摘Objective: To explore the mechanisms of microRNA-150, cyclin B1 and mitochondrial-associated protein 2 in regulating the apoptosis and inhibiting the invasion and migration of Huh-7 cells. Methods: Huh-7 cells were divided into the control group, the negative control group (NC group) and the miR-150 overexpression group (mimic group). The miR-150 overexpressing cell line was constructed by plasmid transfection. The cell viability and apoptosis were detected by cell counting kit-8 and flow cytometry. The cell migration and invasion capacity were measured by cell wound scratch assay and Transwell. The levels of miRNA and mRNA were detected by real-time quantitative polymerase chain reaction and the relative expression levels of proteins were detected by Western blot. Results: MiR-150 significantly inhibited the cell viability of Huh-7 and promoted its apoptosis (P<0.01). After 24 h of cultivation, the mobility of the control group and the NC group were (83.54±4.66)%and (85.57±4.74)%, respectively. The mobility of the mimic group was (49.63±3.78)%, which was significantly lower than that of the control group and the NC group (P<0.01). After 24 h of cultivation, the invasive rate of the control group and the NC group were (100.56±2.87)%and (101.63±3.74)%, respectively, and the invasive rate of mimic group was (51.63±5.32)%, which was significantly lower than that of the control group and the NC group (P<0.01). The expression levels of cyclin B1 protein and mRNA in the mimic group were significantly lower than those in the control group and the NC group (P<0.01), and the level of mitochondrial-associated protein 2 in the mimic group was significantly higher than that in the control group and the NC group (P<0.01). Conclusions: MiR-150 may inhibit the proliferation, migration, invasion and apoptosis of hepatoma carcinoma cell by regulating cyclin B1 or up-regulating mitochondrial-associated protein 2 levels.
基金supported by the Fundamental Research Program of Shanxi Province (202403021221077)the Central Funds Guiding the Local Science and Technology Development in Shanxi Province (YDZJSX2021 A034)+3 种基金the Project of Scientific Research for Excellent Doctors,Shanxi Province,China(SXBYKY2021047)the Research Fund (Clinical Diagnosis and Treatment of Pet) for Young College Teachers in Ruipeng Commonwealth Foundation (RPJJ2020021)the Project of Science and Technology Innovation Fund of Shanxi Agricultural University (2021BQ06)the Inner Mongolia Autonomous Region First Class Discipline Research Special Project(YLXKZX-NND-012)
文摘Feline herpesvirus type 1(FHV-1)is a common and highly contagious pathogen in domestic cats that causes upper respiratory tract infections and ocular diseases.Accurate and rapid diagnosis of FHV-1 infections is essential for effective disease management and control.In this study,we developed an immunochromatographic lateral flow(ICLF)assay for the rapid and accurate detection of FHV-1-specific antibodies.The assay was founded upon the successful expression and purification of a 26 kDa recombinant glycoprotein B-glycoprotein D(gB-gD)fusion protein,which served as the primary antigen for the test.Rigorous testing for specificity and cross-reactivity confirmed the strip’s ability to exclusively detect FHV-1 antibodies,even in the presence of a variety of other feline viruses.The assay demonstrated excellent precision,reproducibility across dilutions,and long-term stability,retaining efficacy for 24 months during storage.Furthermore,clinical sample analysis revealed exceptional sensitivity(97%)and specificity(100%).In conclusion,the ICLF strip developed in this study represents a reliable,highly specific,and stable diagnostic tool for the rapid detection and management of FHV-1 infections in cats.
