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Preparation and Characterization of Monoclonal Antibodies against vp1 protein of Foot-and-mouth Disease Virus O/China99

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Virologica Sinica 2009年第6期24卷 566-572页

作者： Shuai SONG Tong LIN Jun-jun SHAO Shan-dian GAO Guo-zheng CONG Jun-zheng DU Hui-yun CHANG National FMD Reference Laboratory Key Laboratory of Animal Virology of Ministry of Agriculture State Key Laboratory of veterinary Etiological Biology Lanzhou Veterinary Research Institute Chinese Academy of Agricutral Science Lanzhou 730046 China Guangdong Open Laboratory of Veterinary Public Healty Institute of Veterinary Medicine Guangdong Academy of Agricultural Science Guangzhou 510640 China

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.

关键词： Foot-and-mouth disease virus (FMDV) Monoclonal antibody Neutralizing activity vp1 protein

维普期刊数据库

Establishment of Indirect ELISA Diagnosis Technique based on the vp1 protein of Foot and Mouth Disease Virus Serotype A

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Animal Husbandry and Feed Science 2014年第6期6卷 300-303,311页

作者： Lu Qingxia Liu Chang Jin Qianyue Guo Guanpeng Xing Guangxu Liu Yunchao Deng Ruiguang Zhang Gaiping Key Laboratory of Animal Immunology of the Ministry of Agriculture/Henan Provincial Key Laboratory of Animal Immunology Henan Academy of Agricultural Sciences College of Veterinary Medicine and Animal Science Henan Agricultural University

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.

关键词： Foot and mouth disease virus serotype A vp1 protein Indirect ELISA

Genetic analysis of the P1 region of human enterovirus 71 strains isolated in Gansu during 2008—2009 and expression of the 55 F strain vp1 protein

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Genetic analysis of the P1 region of human enterovirus 71 st...

Expression,Purification and Activity Detection of Structural protein vp1 of Foot-and-Mouth Disease Virus Serotype A

2011中国生物制品年会暨第十一次全国生物制品学术研讨会

作者： LI Jian-qiang1,LI Wei1,YANG Jun-jie1,FAN Xiu-juan1,ZHAO Xing-wen1,WANG Yu-lin2 (1.Lanzhou Institute of Biological Products,Lanzhou 730046,China 2.National Vaccine and Serum Institute,Beijing 100024,China)

Enterovirus 71(EV71) is a member of the Entero-virus genus of the Picornaviridae family and is the major cause of hand,foot,and mouth disease(HFMD) in *** sequence of different strains from Gansu were cloned and anlysised,it indicated there were three kids of EV71 infections prevalented in Gansu.55F,one of the strains,vp1 protein was *** recombinant protein expressed with high level and reacted specifically with the EV71 patient antibody, the recombinant protein was also applied to raise antiserum in rabbits and the high titer of antiserum was detected after the fouth injection by the ELISA *** data are useful further study molecular prevalent in the north of China and provided basis for EV71 diagnosis.

关键词： EV71 genetic analysis P1 region expression vp1 protein

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Animal Husbandry and Feed Science 2013年第5期5卷 205-209,226页

作者： LU Qing-xia LIU Chang XING Guang-xu HAO Hui-fang JIN Qian-yue GUO Guan-peng WANG Fang-yu YANG Su-zhen YANG Ji-fei LIU Yun-chao DENG Rui-guang ZHANG Gai-ping Key Laboratory of Animal Immunology of the Ministry of Agriculture /Henan Provincial Key Laboratory of Animal Immunology Henan Academyof Agricultural Sciences College of Veterinary Medicine and Animal Science Henan Agricultural University

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.

关键词： Foot-and-mouth disease virus serotype A vp1 protein Prokaryotic expression Purification of protein Activity analysis

Genetic Analysis of the P1 Region of Human Enterovirus 71 Strains and Expression of the 55 F Strainvp1 protein

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Virologica Sinica 2012年第1期27卷 10-18页

作者： Jian-qiang Li Jun-jie Yang Xiu-juan Fan Zhen-peng Sun Yan Sun Huan Li Zi-xin Meng Wei Li Lanzou Institute of Biological Products Lanzhou 730070China

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.

关键词： EV71 Genetic analysis P1 region Expression vp1 protein

维普期刊数据库

Recombinant Bivalent Vaccine against Foot-and-Mouth Disease Virus Serotype O/A Infection in Guinea Pig

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Acta Biochimica et Biophysica Sinica 2004年第9期36卷 589-596页

作者： Jian-Zhong YI1, Ming-Qiu LIU1, Cai-Zhu ZHU2, Qiang ZHANG1, Zu-Tian SHENG3, Qing-Yun DU3, Wei-Yao YAN1, and Zhao-Xin ZHENG1* State Key Laboratory of Genetics Engineering, Life Science School, Fudan University, Shanghai 200433, China 1The Veterinary Institute of Chinese Academy of Agricultural Sciences, Lanzhou 730046, China 2The Virus Research Institute of Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China The Veterinary Institute of Chinese Academy of Agricultural SciencesLanzhou 730046 China The Virus Research Institute of Zhejiang Academy of Agricultural SciencesHangzhou 310021 China State Key Laboratory of Genetics Engineering Life Science School Fudan UniversityShanghai 200433 China

In this study, two DNA fragments encoding amino acid (141–160)-(21–40)-(141–160) of the vp1 of FMDV (foot-and-mouth disease virus) serotype O and (138–160)-(21–40)-(138–160) of the serotype A FMDV were chemically synthesized. These two tandem-repeat fragments were ligated and transfected into prokaryotic expression vector pTrcHis A to construct pTH-O-A. The other vector called pTH-O-scIgG-A was constructed similarly only that the two tandem-repeat DNA fragments were linked by the bovine- IgG heavy chain coding sequence. Guinea pigs immunized with the two bivalent vaccines pTH-O-A and pTH-O-scIgG-A showed both specific antibody activity and T cell proliferation responses. FMDV challenge tests showed that 85% and 70% of guinea pigs vaccinated twice with 200 μg of the fusion protein of pTH-O-A were protected from FMDV serotype O and serotype A infection respectively. 70% and 57% of the guinea pigs immunized with the fusion protein of pTH-O-scIgG-A were protected from FMDV serotype O and serotype A infection respectively.

关键词： foot-and-mouth disease FMDV serotype O FMDV serotype A vp1 protein bivalent vaccine guinea pig

KIF5B-mediated internalization of FMDV promotes virus infection

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Virologica Sinica 2024年第3期39卷 378-389页

作者： Wei Zhang Fan Yang Yang Yang Weijun Cao Wenhua Shao Jiali Wang Mengyao Huang Zhitong Chen Xiaoyi Zhao Weiwei Li Zixiang Zhu Haixue Zheng State Key Laboratory for Animal Disease Control and Prevention College of Veterinary MedicineLanzhou UniversityLanzhou Veterinary Research InstituteChinese Academy of Agricultural SciencesLanzhou 730000China Gansu Province Research Center for Basic Disciplines of Pathogen Biology Lanzhou 730046China

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 *** this study,we found that kinesin family member 5B(KIF5B)plays a vital role during FMDV ***,we confirmed the interaction between KIF5B and FMDV structural protein vp1 by co-immunoprecipitation(Co-IP)and co-localization in FMDV-infected *** particular,the stalk[amino acids(aa)413–678]domain of KIF5B was indispensable for KIF5B-vp1 ***,overexpression of KIF5B dramatically enhanced FMDV replication;consistently,knockdown or knockout of KIF5B suppressed FMDV ***,we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin ***5B also promotes the transmission of viral particles to early and late endosomes during the early stages of *** conclusion,our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular *** study may provide a new therapeutic target for developing FMDV antiviral drugs.

关键词： FMDV vp1 protein KIF5B Endosome Clathrin