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Preparation Of Monoclonal Antibodies Against P72Gene Of African Swine Fever Virus And Construction Of The Recombinant Baculovirus

Posted on:2013-03-15Degree:MasterType:Thesis
Country:ChinaCandidate:X H YangFull Text:PDF
GTID:2233330374478897Subject:Prevention of Veterinary Medicine
Abstract/Summary:PDF Full Text Request
African swine fever(ASF) is an acute and fulminating infectious disease caused by African swine fever virus(ASFV) in domestic pigs. The mortality can be100%with symptoms such as fever and hemorrhage. By now, this disease has done great harm to swine industry worldwide, it is declared to be one of the "List A"diseases by OIE. There is risk for incoming of ASF even though there is no ASF in our country. Therefore, it is very important to study on diagnostic methods and vaccine for preventing the disease.The aim of our study was to obtain P72protein of ASFV expressed in prokaryotic expression vector. Then, we prepared monoclonal antibody against P72protein, and constructed the recombinant baculovirus of P72gene. These are very useful for diagnostic methods and prevention of ASF.The main projects are as follows:1. Prokaryotic expression of P72gene and Preparation of Monoclonal Antibodies against P72proteinAccording to published sequence of the P72gene, it was synthesized and then cloned into prokaryotic expression vector pGEX-KG. Recombinant plasmid pKG-P72was transformed into E.coli BL21(DE3). The expression of P72gene was induced by IPTG and the result of SDS-PAGE demonstrated that P72protein was expressed in E.coli efficiently as soluble protein. BALB/c mice were immunized with recombinant P72protein, its spleen cells were used to prepare the monoclonal antibodies(McAbs) by hybridoma technique. Five hybridoma cell strains secreting McAbs stably against P72protein were selected by indirect enzyme linked immunosorbent assay (ELISA), named as1E1、1F3、3F1、3D11、4F10, respectively. And the subtype of these five McAbs were all IgG2a. The results of Western Blot and indirect immunofluorescence experiment indicated that these five McAbs were specific for P72prokaryotic and eukaryotic protein.2. Construction recombinant baculovirus of P72geneFirst, eukaryotic expression plasmid pcDNA-P72was constructed, then it was digested by Bgl Ⅱ and Xbal to get CMV-P72gene, which was inserted into transfer vector pFast-VSV-G. And then pFast-G-P72was transformed into E.coli DH10Bac containing baculovirus shuttle vector (bacmid) and helper vector. P72gene was transposed into the bacmid, colonies of E.coli containing Bacmid-G-P72were collected by blue/white selection. After isolation and purification, sf9cells were transfected by Bacmid-G-P72DNA to yield recombinant baculovirus BV-G-p72. To investigate the expression of P72gene, EPC cells were transduced with recombinant baculovirus BV-G-p72, Western Blot and indirect immunofluorescence assay showed that P72protein expressed in eukaryotic cells.
Keywords/Search Tags:African Swine Fever virus, P72gene, Monoclonal Antibody, Baculovirus
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