[Objective] The research aimed to induce the expression of FMDV structural protein VP1 in E.coli and purify the protein,then detect the activity.[Method] The fragment coding VP1 was amplified by PCR and doubly digeste...[Objective] The research aimed to induce the expression of FMDV structural protein VP1 in E.coli and purify the protein,then detect the activity.[Method] The fragment coding VP1 was amplified by PCR and doubly digested with BamH Ⅰ and XhoⅠ,then cloned into expression vector pGEX-4T-1 and pPROExHTb respectively to get recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1.The recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1 was transformed into E.coli BL21(DE3)and induced by IPTG,fusion protein was identifie...展开更多
In order to develop an anti-FMDV Asial type monoclonal antibody （mAb）, BABL/c mice were immunized with recombinant FMDV VP1 protein. Three mAbs, 1B8, 5El and 5E2, were then further optimized. The result indicated th...In order to develop an anti-FMDV Asial type monoclonal antibody （mAb）, BABL/c mice were immunized with recombinant FMDV VP1 protein. Three mAbs, 1B8, 5El and 5E2, were then further optimized. The result indicated that prepared anti-FMDV Asial mAbs had no cross-reactivity with Swine vesicular disease （SVD） and FMDV O, A and C type antigen. Their titers in abdomen liquor were 1：5×10^6, 1：2×10^6 and 1：5×10^6, respectively. 1B8 was found to be of IgG1 subtype, 5El and 5E2 belonged to IgG2b subtype. In this study, the prepared mAbs are specific for detecting FMDV type Asia1, and is potentially useful for pen-side diagnosis.展开更多
Enterovirus 71 （EV71） is a member of the Entero-virus genus of the Picomaviridae family and is the major cause of Hand, foot, and mouth disease （HFMD） in children. Different strains from Gansu were cloned and the ...Enterovirus 71 （EV71） is a member of the Entero-virus genus of the Picomaviridae family and is the major cause of Hand, foot, and mouth disease （HFMD） in children. Different strains from Gansu were cloned and the P1 protein was sequenced and analysed. Results indicate that there are three kinds of EV71 infections prevalent in Gansu. The VP 1 protein from one of these strains, 55F, was expressed. The recombinant protein was expressed with high level and reacted specifically with the EV71 patient antibody, the recombinant protein was also applied to raise antiserum in rabbits and after the fourth injection a high titer of antiserum was detected by ELISA assay. These data are useful for further clarification of prevalent EV71 strains in the north of China at the molecular level and provide a basis for EV71 diagnosis.展开更多
The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to...The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.展开更多
VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability...VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.展开更多
Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted...Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.展开更多
The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vect...The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.展开更多
The researches on chlamydia in recent years show that chlamydia bacteriophage may be a potential and effective means to solve the clinical infection of chlamydia trachomatis（Ct）. We investigated the biological effec...The researches on chlamydia in recent years show that chlamydia bacteriophage may be a potential and effective means to solve the clinical infection of chlamydia trachomatis（Ct）. We investigated the biological effect of chlamydiaphage phi CPG1 capsid protein Vp1 on Ct both in Mc Coy cells and genital tract of mice. Different concentrations of Vp1 were co-incubated with Ct E serotype strain in Mc Coy cells. Female BALB/c mice were used to establish Ct E strain-induced urogenital infection model. They were randomly divided into five groups and given different treatments on the fifth day after Ct inoculation. Animals in groups 1 and 2 were given 30 μL different concentrations of Vp1 in the genital tract respectively, those in group 3 were intramuscularly injected with 30 μL Vp1, those in the infected group did not receive any intervention, and those in the control group received 30 μL PBS in the genital tract. The vaginal discharge was collected to identify the live chlamydia by cell culture and gene fragment by real time PCR different days after infection. Inhibition rate of 100 μg/m L and 50 μg/m L Vp1 proteins against Ct E strain in the Mc Coy cell cultures was 91% and 79% respectively. The number of intracellular Ct inclusion in the Mc Coy cells co-cultured with vaginal discharge of group 1 and group 2 was less than in the infected group, and that in group 1 was less than in group 2, on the 7th day after Ct inoculation. Real-time PCR showed that chlamydia concentration of the vaginal discharge in group 2 was lower than in the infected group, and that in group 1 was lower than in group 2 on the 10 th day. It was suggested that Vp1 capsid proteins had inhibitory effect on the proliferation of Ct serovar E strain in cell culture and mouse genital tract.展开更多
Foot-and-mouth disease(FMD)is a highly contagious and economically important disease,which is caused by the FMD virus(FMDV).Although the cell receptor for FMDV has been identified,the specific mechanism of FMDV intern...Foot-and-mouth disease(FMD)is a highly contagious and economically important disease,which is caused by the FMD virus(FMDV).Although the cell receptor for FMDV has been identified,the specific mechanism of FMDV internalization after infection remains unknown.In this study,we found that kinesin family member 5B(KIF5B)plays a vital role during FMDV internalization.Moreover,we confirmed the interaction between KIF5B and FMDV structural protein VP1 by co-immunoprecipitation(Co-IP)and co-localization in FMDV-infected cells.In particular,the stalk[amino acids(aa)413–678]domain of KIF5B was indispensable for KIF5B-VP1 interaction.Moreover,overexpression of KIF5B dramatically enhanced FMDV replication;consistently,knockdown or knockout of KIF5B suppressed FMDV replication.Furthermore,we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating.KIF5B also promotes the transmission of viral particles to early and late endosomes during the early stages of infection.In conclusion,our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular transport.This study may provide a new therapeutic target for developing FMDV antiviral drugs.展开更多
基金Supported by National Supporting Plan(2006BAD06A14)Transgenic Major Projects(2008ZX08011-004)~~
文摘[Objective] The research aimed to induce the expression of FMDV structural protein VP1 in E.coli and purify the protein,then detect the activity.[Method] The fragment coding VP1 was amplified by PCR and doubly digested with BamH Ⅰ and XhoⅠ,then cloned into expression vector pGEX-4T-1 and pPROExHTb respectively to get recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1.The recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1 was transformed into E.coli BL21(DE3)and induced by IPTG,fusion protein was identifie...
基金The National high Technology Research and Development Program of China (No.2006AA10A204)The National science & Technology Pillar Program (No. 2006BAD06A17)
文摘In order to develop an anti-FMDV Asial type monoclonal antibody （mAb）, BABL/c mice were immunized with recombinant FMDV VP1 protein. Three mAbs, 1B8, 5El and 5E2, were then further optimized. The result indicated that prepared anti-FMDV Asial mAbs had no cross-reactivity with Swine vesicular disease （SVD） and FMDV O, A and C type antigen. Their titers in abdomen liquor were 1：5×10^6, 1：2×10^6 and 1：5×10^6, respectively. 1B8 was found to be of IgG1 subtype, 5El and 5E2 belonged to IgG2b subtype. In this study, the prepared mAbs are specific for detecting FMDV type Asia1, and is potentially useful for pen-side diagnosis.
文摘Enterovirus 71 （EV71） is a member of the Entero-virus genus of the Picomaviridae family and is the major cause of Hand, foot, and mouth disease （HFMD） in children. Different strains from Gansu were cloned and the P1 protein was sequenced and analysed. Results indicate that there are three kinds of EV71 infections prevalent in Gansu. The VP 1 protein from one of these strains, 55F, was expressed. The recombinant protein was expressed with high level and reacted specifically with the EV71 patient antibody, the recombinant protein was also applied to raise antiserum in rabbits and after the fourth injection a high titer of antiserum was detected by ELISA assay. These data are useful for further clarification of prevalent EV71 strains in the north of China at the molecular level and provide a basis for EV71 diagnosis.
基金Supported by Joint Funds of the NSFC and Henan Province(U1204327)Henan Provincial Key Laboratory Construction(122300413217)
文摘The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.
文摘VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.
基金The national high technology research and development program of China 863 (2006AA10A204)The national science and technology pillar program (2006BAD06A17)
文摘Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.
基金Supported by NSFC-Joint Personnel Training Fund of Henan Province(U1204327)Special Fund for Construction of Provincial Key Laboratory in Henan Province(122300413217)
文摘The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.
基金supported by National Natural Science Foundation of China(No.31370211)
文摘The researches on chlamydia in recent years show that chlamydia bacteriophage may be a potential and effective means to solve the clinical infection of chlamydia trachomatis（Ct）. We investigated the biological effect of chlamydiaphage phi CPG1 capsid protein Vp1 on Ct both in Mc Coy cells and genital tract of mice. Different concentrations of Vp1 were co-incubated with Ct E serotype strain in Mc Coy cells. Female BALB/c mice were used to establish Ct E strain-induced urogenital infection model. They were randomly divided into five groups and given different treatments on the fifth day after Ct inoculation. Animals in groups 1 and 2 were given 30 μL different concentrations of Vp1 in the genital tract respectively, those in group 3 were intramuscularly injected with 30 μL Vp1, those in the infected group did not receive any intervention, and those in the control group received 30 μL PBS in the genital tract. The vaginal discharge was collected to identify the live chlamydia by cell culture and gene fragment by real time PCR different days after infection. Inhibition rate of 100 μg/m L and 50 μg/m L Vp1 proteins against Ct E strain in the Mc Coy cell cultures was 91% and 79% respectively. The number of intracellular Ct inclusion in the Mc Coy cells co-cultured with vaginal discharge of group 1 and group 2 was less than in the infected group, and that in group 1 was less than in group 2, on the 7th day after Ct inoculation. Real-time PCR showed that chlamydia concentration of the vaginal discharge in group 2 was lower than in the infected group, and that in group 1 was lower than in group 2 on the 10 th day. It was suggested that Vp1 capsid proteins had inhibitory effect on the proliferation of Ct serovar E strain in cell culture and mouse genital tract.
基金supported by the National Natural Sciences Foundation of China(No.32102639 and 32072831)the National Key Research and Development Program of China(No.2021YFD1800300)+5 种基金the Gansu Science Foundation for Distinguished Young Scholars(No.21JR7RA026)the Earmarked Fund for CARS-35,the Strategic Priority Research Program of the National Center of Technology Innovation for Pigs(No.NCTIP-XD/C03)the Science and Technology Major Project of Gansu Province(No.22ZD6NA001)the Natural Science Foundation of Gansu Province(No.22JR5RA034 and 23JRRA549)the open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province(No.2023SDZG02)the Fundamental Research Funds for the Central Universities(No.lzujbky-2022-ey20).
文摘Foot-and-mouth disease(FMD)is a highly contagious and economically important disease,which is caused by the FMD virus(FMDV).Although the cell receptor for FMDV has been identified,the specific mechanism of FMDV internalization after infection remains unknown.In this study,we found that kinesin family member 5B(KIF5B)plays a vital role during FMDV internalization.Moreover,we confirmed the interaction between KIF5B and FMDV structural protein VP1 by co-immunoprecipitation(Co-IP)and co-localization in FMDV-infected cells.In particular,the stalk[amino acids(aa)413–678]domain of KIF5B was indispensable for KIF5B-VP1 interaction.Moreover,overexpression of KIF5B dramatically enhanced FMDV replication;consistently,knockdown or knockout of KIF5B suppressed FMDV replication.Furthermore,we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating.KIF5B also promotes the transmission of viral particles to early and late endosomes during the early stages of infection.In conclusion,our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular transport.This study may provide a new therapeutic target for developing FMDV antiviral drugs.
