Preparation Of The Monoclonal Antibodies Against VP2Protein Of Bluetongue Virus Serotype3and Identification Of B-cell Epitopes

Bluetongue, as a non-contagious, insect-transmitted infectious disease, is caused by the bluetongue virus (BTV). Sheep, goats and cattle as well as other domestic and wild ruminants are susceptible to BTV infection. BTV is associated with a variety of clinical signs and symptoms depending on the serotypes infected; continuous high fever, mucosal edema and ulcers are among the most common signs. To date, BT has affected animals in tropical, subtropical and temperate countries, in accordance with the distribution of arthropod vectors, and transmission of BTV is through the bite of Culicoides spp.BTV belongs to the prototype virus of the Orbivirus genus in the Reoviridae family. The virion of BTV is non-enveloped, icosahedron symmetry, and is consisted of10linear dsRNA genome segments, which encod7structual protein (VP1-VP7) and5non-structual protein. The outer capsid of BTV3is composed of a continuous layer of two proteins, VP2and VP5. BTV serotypes have been identified based on the antigenic profile of the major outer capsid protein, VP2.A pair of primers were designed according to the gene L2open reading frame (ORF)(GenBank accession No. AJ585124), and RNA was isolated from BTV3-infected BHK-21cells using trizol, L2gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplified material was cloned into the pMAL-c4X prokaryotic expression vector and the pFAST-Bac plasmid to produce recombinant VP2protein by a baculovirus expression system.For the preparation of monoclonal antibody (MAb) against protein VP2of BTV3, SP2/0myeloma cells were fused with spleen cells from BALB/c mice immunized with purified recombinant VP2of BTV3expressed in E.coli. Two hybridoma cell lines stably secreting MAb against VP2of BTV3was identified by indirect ELISA coated with the purified recombinant VP2expressed by baculovirus expression system, named2F8and4E8.Western-blot and Indirect immunofluorescence assay (IFA) were used to vertify2F8and4E8, respectively.The result of western-blot indicated that2F8and4E8can react with BTV3VP2; the result of IFA indicated that Only BTV3showed strongly idiosyncratic reaction with both of MAb2F8and4E8when challenged them with BTV serotypes (BTV1-BTV24). Through Pep-Scan, overlapping peptides were expressed to identify the antigenic epitopes of2F8and4E8, were found to be466EMIQSVIDDG WDQ478and508SQWFPSYYNKWT520. Alignment of the defined linear epitopes with the corresponding region of other BTV serotypes by Blast, showed that these peptide epitopes are poorly conserved in BTV serotypes other than BTV3.There is no report about the Preparation of the MAb against VP2Protein of BTV3in our country, the two MAbs we produced and the research about their antigenic epitopes afford a approach to better understanding of the structure and function of BTV3VP2protein, as well as it will make contribution to the development of epitope-based vaccines.