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Expression of Non-Structural Proteins 3AB and 3ABC of EMCV and Prepareration of Moloclonal Antibody Against 3AB Protein

Author: ShenFang
Tutor: JiangPing
School: Nanjing Agricultural College
Course: Preventive Veterinary Medicine
Keywords: Encephalomyocarditis virus Non-structural protein Prokaryotic expression Baculovirus expression system Moloclonal antibody
CLC: S852.65
Type: Master's thesis
Year: 2008
Downloads: 116
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Abstract


Encephalomyocarditis virus （EMCV） is a new kind of zoonoses virus. which causes acute myocarditis and sudden death in preweaning piglets or severe reproductive failure in sows. Most of the methods for serological survey of disease have been developed to detect the antibody to the whole virus or structural proteins but not non-structural protein. FMDV and EMCV were belong to picornaviridae, they have similar genome structure. It has reported that 3AB and 3ABC ELISA could be used to differentiate vaccinated animals from FMDV infected animals. In this study 3AB and 3ABC genes of EMCV were expressed in prokaryotic expression system and baculovirus expression system, respectively, and one monoclonal antibody against 3AB was prepared. The details as follow:1. Expression of EMCV 3AB and 3ABC genes in prokaryotic expression system and prepare polyclonal antibodies against 3AB proteinNSP 3AB and 3ABC genes of Encephalomyocarditis virus （EMCV） was amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and recombinant proteins 3AB and 3ABC were expressed in E. coli with 36kDa and 62kDa sucessfully. And most of recombinant proteins GST-3ABC were cleaved into GST-3AB and 3C, it may due to the effection of 3C as proteinase. Then mices were immunized by purified recombinant 3AB protein of inclusion-body, the serum was collected two weeks after 3 times immunization. The result of Western blot show that polyclonal antibodies against 3AB protein could specificially react with GST-3AB and GST-3ABC proteins.2. Expression and identification of 3AB and 3ABC genes of EMCV in baculovirus expression systemIn this experiment, the 3AB and 3ABC genes of EMCV were cloned into baculovirus expression vector pFastBacTM, and confirmed by enzyme digestion. Then, recombinant plasmid was transformed to DH10 Bac competent cells, and the recombinant Bacmid-3AB and Bacmid-3ABC plasmids were screened by blue-white colony and PCR. After transfection of the purified plasmids into Sf9 cells respectively, the recombinant baculoviruses were obtained. Expression of the 3AB gene of EMCV was confirmed by indirect immunofluorescent assay （IFA）, SDS-PAGE and Western-blot. The expressed 3AB gene product had a molecular weight of 16kDa and could be recognized by the antibody to 3AB protein of EMCV. The results showed that the 3AB protein was efficiently expressed in the recombinant baculovirus. As same as results of prokaryotic expression the recombinant 3ABC protein was also cleaved into 3AB and 3C.3. Preparation and identification of moloclonal antibody against of 3AB protein The Balb/c mices were immunized by purified recombinant 3AB protein which expressed in prokaryotic expression system, and the splenocytes of the immunized mice were fused with murine myeloma cells to produce hybridoma cell line. After subcloning by 3 times, one strain of hybridoma cell line steadily secreting antibodies of 3AB protein was obtained, named 2D12. The McAb belongs to IgG1 /κ. The McAb was confirmed by indirect immunofluorescent assay （IFA） and Western-blot.EMCV recombinant 3AB and 3ABC proteins, and the McAb against 3AB protein were obtained throught genomic technologies. All the research provides a potential value for structural and functional studies of EMCV non-structural protein and early diagnosis of Encephalomyocarditis virus infection.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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