Hypericin,the principal bioactive naphthodianthrone from Hypericum perforatum,exhibits broadspectrum antibacterial,antitumor,antidepressant,and antiviral properties.However,its low natural abundance and suboptimal pur...Hypericin,the principal bioactive naphthodianthrone from Hypericum perforatum,exhibits broadspectrum antibacterial,antitumor,antidepressant,and antiviral properties.However,its low natural abundance and suboptimal purity hinder large-scale applications.In this study,two hypericin synthase isoforms,Hyp1A and Hyp1B,were recombinantly expressed in Escherichia coli.Following IPTG induction,Hyp1A accumulated to 94.2µg·g^(-1) wet cell mass,which increased to 1.41 mg·g^(-1) upon fusion with a solubility-enhancing tag.In contrast,Hyp1B achieved 7.53 mg·g^(-1) wet cell mass without modification,highlighting distinct expression phenotypes.Molecular dynamics simulations revealed that Hyp1A harbors a higher proportion of random coil and fewerα-helices relative to Hyp1B,correlating with reduced structural stability.In vitro assays confirmed that Hyp1B catalyzes emodin dimerization to hypericin,albeit with limited activity under current conditions.Collectively,these findings demonstrate the feasibility of emodin-to-hypericin bioconversion and establish a foundation for engineering more efficient biosynthetic processes and advancing the clinical application of hypericin.展开更多
Since its discovery in the 1980s,the insect cell-baculovirus expression vector system(IC-BEVS)has been widely used in biomedical applications,such as recombinant protein expression,drug screening,vaccine development,g...Since its discovery in the 1980s,the insect cell-baculovirus expression vector system(IC-BEVS)has been widely used in biomedical applications,such as recombinant protein expression,drug screening,vaccine development,gene therapy and so on[1].As a eukaryotic system,IC-BEVS has great development prospects due to its advantages such as high safety,simple operation,simultaneous expression of multi-subunit proteins,and suitability for large-scale cultivation[2].展开更多
Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to thei...Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to their physiological significance, only very limited number of eukaryoUc membrane protein structures have been obtained due to the technical challenges in the genera- tion of recombinant proteins. In this review, we examine the major recombinant expression systems for eukaryotic membrane proteins and compare their relative advantages and disadvantages. We also attempted to summarize the recent technical strategies in the advancement of eukaryotic membrane protein purification and crystallization.展开更多
Thymidylate synthase （TS, E.C.2.1.1.45） catalyzes a critical reaction in the only pathway of de novo synthesis of thymidylate （dTMP） in human cells, and is an important target of chemotherapy. To evaluate the inhi...Thymidylate synthase （TS, E.C.2.1.1.45） catalyzes a critical reaction in the only pathway of de novo synthesis of thymidylate （dTMP） in human cells, and is an important target of chemotherapy. To evaluate the inhibitory activities of novel compounds to TS, a convenient method of activity assay using 6x His-tagged recombinant human TS （rhTS） was established and 49 novel synthetic folate analogues were screened to discover potential TS inhibitors. During the process, 4 novel compounds were found to effectively inhibit TS, while the IC 50 of a positive control raltitrexed was 3.4 μM in this assay.展开更多
Antimicrobial peptides （AMPs） are making headlines in science because they demonstrate superior microbicidal characteristics compared to synthetic and semi-synthetic antibiotics.
It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But ...It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But the characteristics of the cell line do not always remain stable and may change upon continuous passage.Recently an alphanodavirus,named Tn5 Cell Line Virus (or TNCL Virus),was identified in High Five cells in particular.Therefore,we established a new cell line,QB-Tn9-4s,from Trichoplusia ni,which was determined to be free of TNCL virus by RT-PCR analysis.In this paper,we describe the development of a novel cell clone,QB-CL-B,from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV,and the level of recombinant protein production.This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate;although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies,there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.展开更多
Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into pl...Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99,pQE60 and pET32c to construct different recombinant prokaryotic expression systems.After selecting,the soluble rhHAPO fusion protein was expressed stably in E.coli BL21(DE3) by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid(NTA) affinity chromatography.After cleavage with enterokinase,the rhHAPO protein was applied to Fast Flow SP sepharose column.Results:The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells.Conclusion:We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application.展开更多
The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen ...The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.展开更多
To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites ...To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.展开更多
The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced （accession no. EU734749）, which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The ge...The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced （accession no. EU734749）, which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The genes encoding pre-pro-fibrinolytic enzyme （including signal peptide, propeptide, and mature peptide） and fibrinolytic enzyme （including mature peptide） were cloned into pET28a vector respectively and then transformed into Escherichia coli BL21 （DE3）. The recombinant ofpre-pro-fibrinolytic enzyme showed enzyme activity of 183 U mL^-1, while no detectable enzyme activity could be found from the recombinant of the mature peptide.展开更多
Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome repli...Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.展开更多
Malolactic enzyme is the function enzyme which catalyses the reaction for L-malate converting to L-lactic during malolactic fermentation （MLF）. In this paper, researches concerning the malolactic enzyme gene mleA cl...Malolactic enzyme is the function enzyme which catalyses the reaction for L-malate converting to L-lactic during malolactic fermentation （MLF）. In this paper, researches concerning the malolactic enzyme gene mleA cloned from a patent strain Oenococcus oeni SD-2a screened in Chinese wine and integrated expressing in Saccharomyces cerevisiae were performed in order to carry out both alcoholic fermentation （AF） and malolactic fermentation （MLF） during winemaking, with a view to achieving a better control of MLF in enology. To construct the expression plasmid named pYILmleA, cloned mleA gene, PGK1 promoter, and ADH1 terminator were ligated and inserted into integrating vector YIp5. Yeast transformants were screened on SD/-Ura and identified by auxotrophic test, mating type test, and colony PCR. Target protein was detected by SDS-PAGE and the targeted gene integrated to the chromosome was detected by dot bloting hybridization. After the transformant was cultured in SD/-Ura adding glucose （10%） and L-malate （5 648 mg L-1） for 4 d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC. 1 278-1 312 mg L-1 L-lactic acids were detected, while the comparative drop rates of L-malate were 20.18-20.85%. L-malate and L-lactic contents of the transformants showed extra significant difference and significant difference with the control ones by t-test respectively. The result indicated that the functional expression was achieved in recombinants S. cerevisiae.展开更多
According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gen...According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.展开更多
African swine fever(ASF) is a lethal hemorrhagic disease that affects wild and domestic swine. The etiological agent of ASF is African swine fever virus(ASFV). Since the first case was described in Kenya in 1921, the ...African swine fever(ASF) is a lethal hemorrhagic disease that affects wild and domestic swine. The etiological agent of ASF is African swine fever virus(ASFV). Since the first case was described in Kenya in 1921, the disease has spread to many other countries. No commercial vaccines are available to prevent ASF. In this study, we generated a recombinant Newcastle disease virus(r NDV) expressing ASFV protein 72(p72) by reverse genetics and evaluated its humoral and cellular immunogenicity in a mouse model. The recombinant virus, r NDV/p72, replicated well in embryonated chicken eggs and was safe to use in chicks and mice. The p72 gene in r NDV/p72 was stably maintained through ten passages. Mice immunized with r NDV/p72 developed high titers of ASFV p72 specific Ig G antibody, and had higher levels of Ig G1 than IgG2 a. Immunization also elicited T-cell proliferation and secretion of IFN-γ and IL-4. Taken together, these results indicate that r NDV expressing ASFV p72 might be a potential vaccine candidate for preventing ASF.展开更多
Objective To study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus （HCMV） infection. Methods Fab monoclonal antibodies to HCMV were recovered by repertoire cloni...Objective To study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus （HCMV） infection. Methods Fab monoclonal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of VH and VL and neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-K-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G. Results SDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibody recognized a conformational HCMV protein. Conclusion IgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 μg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.展开更多
Dear Editor,Infectious bursal disease virus（IBDV）causes infectious bursal disease,a highly contagious immunosuppressive disease that affects young chickens and causes economic losses in the poultry industry worldwid...Dear Editor,Infectious bursal disease virus（IBDV）causes infectious bursal disease,a highly contagious immunosuppressive disease that affects young chickens and causes economic losses in the poultry industry worldwide.IBDV replicates mainly in actively dividing B lymphocytes within the bursa of Fabricius（BF）,leading to immunosuppression in affected flocks（Mahgoub et al.,2012）.Viral protein 2（VP2）,展开更多
In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion prot...In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.展开更多
文摘Hypericin,the principal bioactive naphthodianthrone from Hypericum perforatum,exhibits broadspectrum antibacterial,antitumor,antidepressant,and antiviral properties.However,its low natural abundance and suboptimal purity hinder large-scale applications.In this study,two hypericin synthase isoforms,Hyp1A and Hyp1B,were recombinantly expressed in Escherichia coli.Following IPTG induction,Hyp1A accumulated to 94.2µg·g^(-1) wet cell mass,which increased to 1.41 mg·g^(-1) upon fusion with a solubility-enhancing tag.In contrast,Hyp1B achieved 7.53 mg·g^(-1) wet cell mass without modification,highlighting distinct expression phenotypes.Molecular dynamics simulations revealed that Hyp1A harbors a higher proportion of random coil and fewerα-helices relative to Hyp1B,correlating with reduced structural stability.In vitro assays confirmed that Hyp1B catalyzes emodin dimerization to hypericin,albeit with limited activity under current conditions.Collectively,these findings demonstrate the feasibility of emodin-to-hypericin bioconversion and establish a foundation for engineering more efficient biosynthetic processes and advancing the clinical application of hypericin.
文摘Since its discovery in the 1980s,the insect cell-baculovirus expression vector system(IC-BEVS)has been widely used in biomedical applications,such as recombinant protein expression,drug screening,vaccine development,gene therapy and so on[1].As a eukaryotic system,IC-BEVS has great development prospects due to its advantages such as high safety,simple operation,simultaneous expression of multi-subunit proteins,and suitability for large-scale cultivation[2].
基金ACKNOWLEDGEMENTS We apologize to colleagues whose work could not be cited due to the scope of this review. We would like to thank members in Yan laboratory for discussions. We thank Brendan Lehnert, Xinlei Sheng, Quanxiu Li, Dan Ma and Xinhui Zhou for critical reading. This work was supported by funds from the National Basic Research Program （973 Program） （No. 2011CB910501）, the National Natural Science Foundation of China （Grant Nos. 31321062-20131319400, 31125009, and 91017011 ）, and funds from Tsinghua-Peking Center for Life Sciences. The research of N.Y. was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute.
文摘Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to their physiological significance, only very limited number of eukaryoUc membrane protein structures have been obtained due to the technical challenges in the genera- tion of recombinant proteins. In this review, we examine the major recombinant expression systems for eukaryotic membrane proteins and compare their relative advantages and disadvantages. We also attempted to summarize the recent technical strategies in the advancement of eukaryotic membrane protein purification and crystallization.
基金supported by National Natural Science Foundation of China(Grant No.20972011,21042009,21172014)
文摘Thymidylate synthase （TS, E.C.2.1.1.45） catalyzes a critical reaction in the only pathway of de novo synthesis of thymidylate （dTMP） in human cells, and is an important target of chemotherapy. To evaluate the inhibitory activities of novel compounds to TS, a convenient method of activity assay using 6x His-tagged recombinant human TS （rhTS） was established and 49 novel synthetic folate analogues were screened to discover potential TS inhibitors. During the process, 4 novel compounds were found to effectively inhibit TS, while the IC 50 of a positive control raltitrexed was 3.4 μM in this assay.
基金supported by grants from the Project of Jilin province Technology Platform(NO.20070210)the Young Research Fond of Jilin University
文摘Antimicrobial peptides （AMPs） are making headlines in science because they demonstrate superior microbicidal characteristics compared to synthetic and semi-synthetic antibiotics.
基金supported in part by the Chinese National Basic Research Program(973)2009CB118900Chinese National Science Foundation Project30771451Boyce Thompson Institute Project BTI-QAU1-23-2007
文摘It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But the characteristics of the cell line do not always remain stable and may change upon continuous passage.Recently an alphanodavirus,named Tn5 Cell Line Virus (or TNCL Virus),was identified in High Five cells in particular.Therefore,we established a new cell line,QB-Tn9-4s,from Trichoplusia ni,which was determined to be free of TNCL virus by RT-PCR analysis.In this paper,we describe the development of a novel cell clone,QB-CL-B,from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV,and the level of recombinant protein production.This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate;although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies,there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.
基金the Natural Science Foundation of China (30300186)the Grant of 863 projects from the Ministry of Science & Technology of China (2002AA223354)
文摘Objective:To express the soluble recombinant hemangiopoietin protein in E.coli BL21(DE3).Methods:Using human fetal live cDNA as a template,a partial cDNA fragment of HAPO coding N-terminal region was subcloned into plasmids pTrc99,pQE60 and pET32c to construct different recombinant prokaryotic expression systems.After selecting,the soluble rhHAPO fusion protein was expressed stably in E.coli BL21(DE3) by vector pET32c-HAPO and further isolated by nickelnitrilotriacetic acid(NTA) affinity chromatography.After cleavage with enterokinase,the rhHAPO protein was applied to Fast Flow SP sepharose column.Results:The rhHAPO protein had a purity of more than 95% and a good bioactivity based on the cell adhesion assay in ECV304 cells.Conclusion:We have established a protein engineering system to produce rhHAPO which may provide the possibility for clinical application.
文摘The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.
文摘To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.
基金funded by the Science and Technology Research Project during the 11th Five-Year Plan period of Hebei Province, China (06220106D)
文摘The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced （accession no. EU734749）, which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The genes encoding pre-pro-fibrinolytic enzyme （including signal peptide, propeptide, and mature peptide） and fibrinolytic enzyme （including mature peptide） were cloned into pET28a vector respectively and then transformed into Escherichia coli BL21 （DE3）. The recombinant ofpre-pro-fibrinolytic enzyme showed enzyme activity of 183 U mL^-1, while no detectable enzyme activity could be found from the recombinant of the mature peptide.
基金National Basic Research Program of China (973 Program, Grant No. 2009CB118701)National Natural Scientific Foundation of China (Grant Nos. 30671615, 30871940)Innovation project of the Chinese Academy of Sciences (Grant No.KSCX2-YW-N-021)
文摘Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.
基金supported by the National High-Tech Research and Development Program of China (863 Program of China, 2007AA10Z314)andthe Earmarked Fund for Modern Agro-Industry Technology Research System, China (Z225020801)
文摘Malolactic enzyme is the function enzyme which catalyses the reaction for L-malate converting to L-lactic during malolactic fermentation （MLF）. In this paper, researches concerning the malolactic enzyme gene mleA cloned from a patent strain Oenococcus oeni SD-2a screened in Chinese wine and integrated expressing in Saccharomyces cerevisiae were performed in order to carry out both alcoholic fermentation （AF） and malolactic fermentation （MLF） during winemaking, with a view to achieving a better control of MLF in enology. To construct the expression plasmid named pYILmleA, cloned mleA gene, PGK1 promoter, and ADH1 terminator were ligated and inserted into integrating vector YIp5. Yeast transformants were screened on SD/-Ura and identified by auxotrophic test, mating type test, and colony PCR. Target protein was detected by SDS-PAGE and the targeted gene integrated to the chromosome was detected by dot bloting hybridization. After the transformant was cultured in SD/-Ura adding glucose （10%） and L-malate （5 648 mg L-1） for 4 d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC. 1 278-1 312 mg L-1 L-lactic acids were detected, while the comparative drop rates of L-malate were 20.18-20.85%. L-malate and L-lactic contents of the transformants showed extra significant difference and significant difference with the control ones by t-test respectively. The result indicated that the functional expression was achieved in recombinants S. cerevisiae.
基金Supported by 863 Projects (2008AA10Z311)National Science and Technology Support Projects (2009BADB9B06)+1 种基金Started Post-doctoral Research Grant of Heilongjiang Province (LBH-Q07023)Harbin Technological Innovation of Special Funds (2007RFQXN020)
文摘According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.
基金financially supported by the National Supporting Program(2013BAD12B05)Natural Science Foundation of China(NSFC)(no.31101839 and no.31302117)+1 种基金the key program of Gansu Province(no.1013JHTA008)Jiangsu Co-innovation Center program for Prevention and Control of Important Animal Infectious Diseases and Zoonoses
文摘African swine fever(ASF) is a lethal hemorrhagic disease that affects wild and domestic swine. The etiological agent of ASF is African swine fever virus(ASFV). Since the first case was described in Kenya in 1921, the disease has spread to many other countries. No commercial vaccines are available to prevent ASF. In this study, we generated a recombinant Newcastle disease virus(r NDV) expressing ASFV protein 72(p72) by reverse genetics and evaluated its humoral and cellular immunogenicity in a mouse model. The recombinant virus, r NDV/p72, replicated well in embryonated chicken eggs and was safe to use in chicks and mice. The p72 gene in r NDV/p72 was stably maintained through ten passages. Mice immunized with r NDV/p72 developed high titers of ASFV p72 specific Ig G antibody, and had higher levels of Ig G1 than IgG2 a. Immunization also elicited T-cell proliferation and secretion of IFN-γ and IL-4. Taken together, these results indicate that r NDV expressing ASFV p72 might be a potential vaccine candidate for preventing ASF.
文摘Objective To study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus （HCMV） infection. Methods Fab monoclonal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of VH and VL and neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-K-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G. Results SDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibody recognized a conformational HCMV protein. Conclusion IgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 μg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.
基金supported by grants AERG 232141/PNBIO 1131032 from the Instituto Nacional de Tecnología Agropecuaria(INTA)PICT 2008-0400 from the Agencia Nacional de Promoción Científica y Tecnológica(ANPCyT),Argentina
文摘Dear Editor,Infectious bursal disease virus（IBDV）causes infectious bursal disease,a highly contagious immunosuppressive disease that affects young chickens and causes economic losses in the poultry industry worldwide.IBDV replicates mainly in actively dividing B lymphocytes within the bursa of Fabricius（BF）,leading to immunosuppression in affected flocks（Mahgoub et al.,2012）.Viral protein 2（VP2）,
文摘In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.
