Hepatitis B virus(HBV),a major cause of human liver disease worldwide,encodes three envelope proteins needed for the attachment and entry of the virus into susceptible host cells.A second virus,hepatitis delta virus,w...Hepatitis B virus(HBV),a major cause of human liver disease worldwide,encodes three envelope proteins needed for the attachment and entry of the virus into susceptible host cells.A second virus,hepatitis delta virus,which is known to enhance liver disease in HBV infected patients,diverts the same HBV envelope proteins to achieve its own assembly and infection.In the lab,lentiviral vectors based on human immunodeficiency virus type 1 can be assembled using the HBV envelope proteins,and will similarly infect susceptible cells.This article provides a partial review and some personal reflections of how these three viruses infect and of how recipient cells become susceptible,along with some consideration of questions that remain to be answered.展开更多
Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutin...Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.展开更多
The envelope protein （Env） of lentiviruses such as HIV, SIV, FIV and EIAV is larger than that of other retroviruses. The Chinese EIAV attenuated vaccine is based on Env and has helped to successfully control this vi...The envelope protein （Env） of lentiviruses such as HIV, SIV, FIV and EIAV is larger than that of other retroviruses. The Chinese EIAV attenuated vaccine is based on Env and has helped to successfully control this virus, demonstrating that envelope is crucial for vaccine. We compared Env variation of the four kinds of lentiviruses. Phylogenetic analysis showed that the evolutionary relationship of Env between HIV and SIV was the closest and they appeared to descend from a common ancestor, and the relationship of HIV and EIAV was the furthest. EIAV had the shortest Env length and the least number of potential N-linked glyeosylation sites （PNGS） as well as glyeosylation density compared to various immunodefieiency viruses. However, HIV had the longest Env length and the most PNGS. Moreover, the alignment of HIV and SIV showed that PNGS were primarily distributed within extraeellular membrane protein gp120 rather than transmembrane gp41. It implies that the size difference among these viruses is associated with a lentivirus specific function and also the diversity of env. There arc low levels of modification of glycosylation sites of Env and selection of optimal protective epitopes might be useful for development of an effective vaccine against HIV/AIDS.展开更多
White spot syndrome virus （WSSV） is one of the major shrimp pathogens causing large economic losses to shrimp farming. In an attempt to identify the envelope proteins involved in the virus infection, purified WSSV v...White spot syndrome virus （WSSV） is one of the major shrimp pathogens causing large economic losses to shrimp farming. In an attempt to identify the envelope proteins involved in the virus infection, purified WSSV virions were mixed with three antisera against WSSV envelope proteins （VP39, VP124 and VP187 ）, individually. And then they were injected intramuscularly into crayfish （Procambarus clarkii） to conduct in vivo neutralization assays. The results showed that for groups injected with virions only and groups injected with the mixture of virions and antiserum against VP124, the crayfish mortalities were 100% and 60% on the 8th day postinfection, individually. The virus infection could be delayed or neutralized by antibody against the envelope protein VP124. Quantitative PCR was used to further investigate the influence of three antisera described above on the virus infection. The results showed that the antiserum against VP124 could restrain the propagation of WSSV in crayfish. All of the results suggested that the viral envelope protein VP124 played a role in WSSV infection.展开更多
The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had caused over 382 million cases and over 2.7 million deaths globally as of 23 March 2021. By ...The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had caused over 382 million cases and over 2.7 million deaths globally as of 23 March 2021. By that date, at least 10 SARS-CoV-2 variants had emerged. The transmissibility and lethality of the variants are higher than those of the Wuhan reference strain. Therefore, a universal vaccine for the reference strain and all variants (present and future) is indispensable. The coronavirus envelope (E) protein is an integral membrane protein crucial to the viral lifecycle and the pathogenesis of coronaviruses. The SARS-CoV-2 E protein has a postsynaptic density protein 95/Drosophila disc large tumor suppressor/zonula occludens-1 (PDZ) binding motif (PBM), and its interaction with PDZ-domain-2 of the human tight junction protein may interrupt the integrity of lung epithelium. Furthermore, the SARS-CoV-2 E protein itself is a homopentameric cation channel viroporin, which may be involved in viral release. This protein is thus a potential target for the development of a universal COVID-19 vaccine, because of its highly conserved amino acid sequence. The variant mutations occur mainly in the spike protein, and conservation of E protein remained in most Variants of Concern (VOC). Only one of the extant VOC have mutations in the E protein that P71L mutation occurs in the South African variant 501Y.V2 (B.1.351). If a vaccine is designed to target E protein, two scenarios are possible: 1) SARS-CoV-2 maintains a highly conserved E protein amino acid sequence, rendering the virus consistently or permanently susceptible to the vaccine;or 2) the E protein mutates and new variants evolve accordingly. In scenario 2, the tertiary structure and function of the E protein homopentameric cation channel viroporin, PBM, or other aspects affecting pathogenicity would be attenuated. Either scenario would thus ameliorate the pandemic. I therefore propose that a vaccine targeting the SARS-CoV-2 E protein would be effective against the Wuhan reference strain and all current and future SARS-CoV-2 variants. Efforts to create E protein-based vaccines are ongoing. Further research and clinical trials are needed to realize this universal COVID-19 vaccine.展开更多
AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragm...AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.展开更多
AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E, coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHO...AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E, coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E, coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chrbmatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli, C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni2+-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 giycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.展开更多
To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira b...To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.展开更多
West Nile virus(WNV)is a mosquito-transmitted flavivirus distributed globally for decades and can cause disease in humans and animals.So far,no WNV vaccine has been licensed for human use.Therefore,the development of ...West Nile virus(WNV)is a mosquito-transmitted flavivirus distributed globally for decades and can cause disease in humans and animals.So far,no WNV vaccine has been licensed for human use.Therefore,the development of novel candidate vaccines and the improvement of vaccination strategies is imperative.As the WNV envelope(E)glycoprotein plays an important role in mediating viral binding to cellular receptors and virus-cell membrane fusion,it is a critical target for the host humoral response.Here,we prepared a recombinant truncated envelope protein of WNV(rWNV-80E)and developed a WNV subunit vaccine formulation with a combination of aluminum hydroxide(alum)and a synthetic oligonucleotide CpG as adjuvants.C57BL/6 mice were immunized twice intramuscularly at 28-day intervals with 5μg purified rWNV-80E adjuvanted with Alum/CpG.WNV E-specific IgG was detected by enzyme-linked immunosorbent assay and neutralizing antibodies(nAbs)was detected using single-round infectious particles of WNV.Furthermore,T cell immunity was detected by enzyme-linked immunospot assay and intracellular cytokine staining assay.Notably,rWNV-80E was highly immunogenic and elicited potent humoral and cell immunity,as evidenced by significant levels of IFN-γ and TNF-αsecretion in the T cells of mice.In summary,the Alum/CpG-adjuvanted rWNV-80E subunit vaccine elicited potent and balanced B-and T-cell immunity in mice,and therefore it is a promising candidate vaccine that warrants further investigation for use in human or veterinary applications.展开更多
AIM: To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context se...AIM: To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing. METHODS: HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively.Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products. RESULTS: E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively. Deglycosylation analysis showed that this difference was mainly due to different glycosylation. Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94. Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences. CONCLUSION: The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2. As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER. Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.展开更多
Our recent study reported that ATP1B3 inhibits hepatitis B virus(HBV)replication via inducing NF-κB activation.However,ATP1B3 mutants which were defective in NF-κB activation still maintained the moderate degree of ...Our recent study reported that ATP1B3 inhibits hepatitis B virus(HBV)replication via inducing NF-κB activation.However,ATP1B3 mutants which were defective in NF-κB activation still maintained the moderate degree of suppression on HBV replication,suggesting that another uncharacterized mechanism is also responsible for ATP1B3-mediated HBV suppression.Here,we demonstrated that ATP1B3 reduced the expression of HBV envelope proteins LHBs,MHBs and SHBs,but had no effect on intracellular HBV DNA,RNA levels as well as HBV promoter activities.Further investigation showed that proteasome inhibitor MG132 rescued ATP1B3-mediated envelope proteins degradation,demonstrating that proteasome-dependent pathway is involved in ATP1B3-induced degradation of envelope proteins.Co-IP showed that ATP1B3 interacts with LHBs and MHBs and induces LHBs and MHBs polyubiquitination.Immunofluorescence colocalization analysis confirmed LHBs and MHBs colocalized with ATP1B3 together.Our work provides important information for targeting ATP1B3 as a potential therapeutic molecule for HBV infection.展开更多
The small envelope protein （E） gene of avian infectious bronchitis virus （IBV） M41 strain was cloned, and then it was subcloned into prokaryotic expressing vector pGEX-6P-1. The recombinant plasmid was transformed...The small envelope protein （E） gene of avian infectious bronchitis virus （IBV） M41 strain was cloned, and then it was subcloned into prokaryotic expressing vector pGEX-6P-1. The recombinant plasmid was transformed into E.coli. BL21 and induced by IPTG. SDS-PAGE result showed that when objective protein fused with GST （about 20 ku）, the relative molecular mass of fusion protein was 38 ku. It indicated that objective protein was about 12.4 ku. The result showed that E protein was expressed successfully, it was useful to the subsequent E protein research.展开更多
文摘Hepatitis B virus(HBV),a major cause of human liver disease worldwide,encodes three envelope proteins needed for the attachment and entry of the virus into susceptible host cells.A second virus,hepatitis delta virus,which is known to enhance liver disease in HBV infected patients,diverts the same HBV envelope proteins to achieve its own assembly and infection.In the lab,lentiviral vectors based on human immunodeficiency virus type 1 can be assembled using the HBV envelope proteins,and will similarly infect susceptible cells.This article provides a partial review and some personal reflections of how these three viruses infect and of how recipient cells become susceptible,along with some consideration of questions that remain to be answered.
文摘Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.
基金Natural Science Foundation of China(30970162)Tianjin Municipal Science and Technology Foundation(08ZCGHHZ01800)
文摘The envelope protein （Env） of lentiviruses such as HIV, SIV, FIV and EIAV is larger than that of other retroviruses. The Chinese EIAV attenuated vaccine is based on Env and has helped to successfully control this virus, demonstrating that envelope is crucial for vaccine. We compared Env variation of the four kinds of lentiviruses. Phylogenetic analysis showed that the evolutionary relationship of Env between HIV and SIV was the closest and they appeared to descend from a common ancestor, and the relationship of HIV and EIAV was the furthest. EIAV had the shortest Env length and the least number of potential N-linked glyeosylation sites （PNGS） as well as glyeosylation density compared to various immunodefieiency viruses. However, HIV had the longest Env length and the most PNGS. Moreover, the alignment of HIV and SIV showed that PNGS were primarily distributed within extraeellular membrane protein gp120 rather than transmembrane gp41. It implies that the size difference among these viruses is associated with a lentivirus specific function and also the diversity of env. There arc low levels of modification of glycosylation sites of Env and selection of optimal protective epitopes might be useful for development of an effective vaccine against HIV/AIDS.
基金The"863" Program of China under contract No.2003AA626020the Fujian Science Fund of Chima under contract No.2003F001
文摘White spot syndrome virus （WSSV） is one of the major shrimp pathogens causing large economic losses to shrimp farming. In an attempt to identify the envelope proteins involved in the virus infection, purified WSSV virions were mixed with three antisera against WSSV envelope proteins （VP39, VP124 and VP187 ）, individually. And then they were injected intramuscularly into crayfish （Procambarus clarkii） to conduct in vivo neutralization assays. The results showed that for groups injected with virions only and groups injected with the mixture of virions and antiserum against VP124, the crayfish mortalities were 100% and 60% on the 8th day postinfection, individually. The virus infection could be delayed or neutralized by antibody against the envelope protein VP124. Quantitative PCR was used to further investigate the influence of three antisera described above on the virus infection. The results showed that the antiserum against VP124 could restrain the propagation of WSSV in crayfish. All of the results suggested that the viral envelope protein VP124 played a role in WSSV infection.
文摘The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had caused over 382 million cases and over 2.7 million deaths globally as of 23 March 2021. By that date, at least 10 SARS-CoV-2 variants had emerged. The transmissibility and lethality of the variants are higher than those of the Wuhan reference strain. Therefore, a universal vaccine for the reference strain and all variants (present and future) is indispensable. The coronavirus envelope (E) protein is an integral membrane protein crucial to the viral lifecycle and the pathogenesis of coronaviruses. The SARS-CoV-2 E protein has a postsynaptic density protein 95/Drosophila disc large tumor suppressor/zonula occludens-1 (PDZ) binding motif (PBM), and its interaction with PDZ-domain-2 of the human tight junction protein may interrupt the integrity of lung epithelium. Furthermore, the SARS-CoV-2 E protein itself is a homopentameric cation channel viroporin, which may be involved in viral release. This protein is thus a potential target for the development of a universal COVID-19 vaccine, because of its highly conserved amino acid sequence. The variant mutations occur mainly in the spike protein, and conservation of E protein remained in most Variants of Concern (VOC). Only one of the extant VOC have mutations in the E protein that P71L mutation occurs in the South African variant 501Y.V2 (B.1.351). If a vaccine is designed to target E protein, two scenarios are possible: 1) SARS-CoV-2 maintains a highly conserved E protein amino acid sequence, rendering the virus consistently or permanently susceptible to the vaccine;or 2) the E protein mutates and new variants evolve accordingly. In scenario 2, the tertiary structure and function of the E protein homopentameric cation channel viroporin, PBM, or other aspects affecting pathogenicity would be attenuated. Either scenario would thus ameliorate the pandemic. I therefore propose that a vaccine targeting the SARS-CoV-2 E protein would be effective against the Wuhan reference strain and all current and future SARS-CoV-2 variants. Efforts to create E protein-based vaccines are ongoing. Further research and clinical trials are needed to realize this universal COVID-19 vaccine.
基金Supported by National 863 Project,No.102-07-02-079th Five-Year Sci-Tech Plan,No.96-906A-03-08
文摘AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.
基金Supported by National High Technology Research and Development Program of China (863 Program), No. 2001AA215171
文摘AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli(E, coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E, coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chrbmatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli, C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni2+-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 giycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.
文摘To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.
基金the National Key Research and Development Program of China(2022YFC2304100,2022YFC2303401,2016YFD0500301).
文摘West Nile virus(WNV)is a mosquito-transmitted flavivirus distributed globally for decades and can cause disease in humans and animals.So far,no WNV vaccine has been licensed for human use.Therefore,the development of novel candidate vaccines and the improvement of vaccination strategies is imperative.As the WNV envelope(E)glycoprotein plays an important role in mediating viral binding to cellular receptors and virus-cell membrane fusion,it is a critical target for the host humoral response.Here,we prepared a recombinant truncated envelope protein of WNV(rWNV-80E)and developed a WNV subunit vaccine formulation with a combination of aluminum hydroxide(alum)and a synthetic oligonucleotide CpG as adjuvants.C57BL/6 mice were immunized twice intramuscularly at 28-day intervals with 5μg purified rWNV-80E adjuvanted with Alum/CpG.WNV E-specific IgG was detected by enzyme-linked immunosorbent assay and neutralizing antibodies(nAbs)was detected using single-round infectious particles of WNV.Furthermore,T cell immunity was detected by enzyme-linked immunospot assay and intracellular cytokine staining assay.Notably,rWNV-80E was highly immunogenic and elicited potent humoral and cell immunity,as evidenced by significant levels of IFN-γ and TNF-αsecretion in the T cells of mice.In summary,the Alum/CpG-adjuvanted rWNV-80E subunit vaccine elicited potent and balanced B-and T-cell immunity in mice,and therefore it is a promising candidate vaccine that warrants further investigation for use in human or veterinary applications.
基金the National 863 High Technology Foundation of China,No.863-102-07-02-02,No.2001AA215171the project CHN 98/112 (WTZ-Internationales Buro des BMBF).
文摘AIM: To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing. METHODS: HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively.Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products. RESULTS: E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively. Deglycosylation analysis showed that this difference was mainly due to different glycosylation. Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94. Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences. CONCLUSION: The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2. As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER. Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.
基金This work was supported by funding from the National Natural Science Foundation of China(81672004 and 31270202)the Chinese Ministry of Science and Technology(2018ZX10302104-001-010)+3 种基金Science and Technology Department of Jilin Province(20160101044JC and 20190101003JH)Health Commission of Jilin Province(2016J065)the Key Laboratory of Molecular Virology,Jilin Province(20102209)Norman Bethune Program of Jilin University(2015331)to Baisong Zheng.
文摘Our recent study reported that ATP1B3 inhibits hepatitis B virus(HBV)replication via inducing NF-κB activation.However,ATP1B3 mutants which were defective in NF-κB activation still maintained the moderate degree of suppression on HBV replication,suggesting that another uncharacterized mechanism is also responsible for ATP1B3-mediated HBV suppression.Here,we demonstrated that ATP1B3 reduced the expression of HBV envelope proteins LHBs,MHBs and SHBs,but had no effect on intracellular HBV DNA,RNA levels as well as HBV promoter activities.Further investigation showed that proteasome inhibitor MG132 rescued ATP1B3-mediated envelope proteins degradation,demonstrating that proteasome-dependent pathway is involved in ATP1B3-induced degradation of envelope proteins.Co-IP showed that ATP1B3 interacts with LHBs and MHBs and induces LHBs and MHBs polyubiquitination.Immunofluorescence colocalization analysis confirmed LHBs and MHBs colocalized with ATP1B3 together.Our work provides important information for targeting ATP1B3 as a potential therapeutic molecule for HBV infection.
文摘The small envelope protein （E） gene of avian infectious bronchitis virus （IBV） M41 strain was cloned, and then it was subcloned into prokaryotic expressing vector pGEX-6P-1. The recombinant plasmid was transformed into E.coli. BL21 and induced by IPTG. SDS-PAGE result showed that when objective protein fused with GST （about 20 ku）, the relative molecular mass of fusion protein was 38 ku. It indicated that objective protein was about 12.4 ku. The result showed that E protein was expressed successfully, it was useful to the subsequent E protein research.
