Preparation Of Monoclonal Antibodies Against IBDV And Sequence Analysis Of VP2 Gene

In order to identify antigen epitope and develop the new types of diagnostic kits for infectious bursal disease virus(IBDV),we purified the IBDV B87 strain and expressed the viral VP2 in prokaryotic expression system, and produced McAbs against these viral antigens. The details are as follows:Firstly, IBDV B87 strain was proliferated in chick embryo allantois and concentrated by ultracentrifugation. Balb/c mice were immunized with the concentrated virus emulsified with Freund's adjuvant. A fusion was carried out of immunized mice spleen cells with myeloma cell SP2/0. The positive clones were found and selected with an indirect ELISA method by IBDV-coated enzyme label plate. We obtained 4 strains cells secreting antibody against IBDV, named as I8, I13, I25 and I86. The cells were cultured in vivo to produce ascites antibodies.The results of analysis revealed that the titers of McAbs, I8, I13, I25 and I86 in ascites by ELISA were 1:6×10~4,1:1×10~5,1:2×10~6and 1:6×10~4 respectively. The result of analyzing subtype showed that I8, I13, I25 were IgG2b, while I86 was IgG1. The results of specific test confirmed that McAbs in ascites had high specificity because they could only react with IBDV but not with NDV and allantoic fluid. Add ELSIA results showed that I8 and I13 could bind the same epitope, while I8 (I13), I25 and I86 could bind different antigenic sites respectively. I8, I13, I25 and I86 in ascites diluted by 200 times could react with VP2 protein by indirect ELISA but negative ascites could not. This indicated that I8, I13, I25 and I86 in ascites could recognize VP2 protein specifically.Secondly conserved VP2 gene region of IBDV B87 strain was amplified and cloned into the prokaryotic expression vector PET-32a and PGEX4T-1. The expressive vector was constructed and confirmed by PCR, and the tests of restriction enzyme digestion and DNA sequencing analysis were carried out. HIS-VP2 protein was purified by NI purification resin. Then the recombinant protein was concentrated and purified. Mice was immunized with HIS-VP2 protein, then the spleen cells were fused with myeloma cells. An indirect ELISA screening method was established for selection of fusion cells by using HIS-VP2 and GST-VP2 protein coating enzyme label plate respectively. Two positive cell strains were obtained by consecutive subcloning eventually, named as V1 and V38. The ascites were produced in vivo, and their titers, specificity, stability, antigenicity, etc were identified.SDS-PAGE tests showed that VP2 recombinant plasmid had high expression with the form of inclusion bodies. Purified HIS-VP2 protein concentration was 2.62mg/mL and had a high purity, and it could be identified by anti-IBDV polyclonal antibody. McAbs identification results showed that the titers of V1 and V38 in ascites were 1:104 or more, and V1 and V38 could react with IBDV by ELISA, and they had high specific and stability property, but V1 and V38 could bind different epitopes .Finally, in order to explore the IBDV gene mutation in the Anhui province, we designed a pair of primers used for amplification of IBDV VP2 based on nucleotide sequence of IBDV published on the GenBank. Using Anhui IBDV strains (named AH-1, AH-2, AH-7, AH-9)which were diagnosed by colloid gold rapid diagnostic test strips of IBDV as templates, we amplified 567bp of VP2 hypervariable regions by RT-PCR. Then the VP2 genes were connected to the PMD18-T vector, The results of PCR and nucleotide sequence analysis confirmed that four recombinant plasmid of Anhui strain VP2 hypervariable region were successfully constructed. Applying Bioinformatics software, we compared the four Anhui strains VP2 gene and deduced amino acid sequence with other 11 strains of IBDV published on GenBank , and made up phylogenetic trees based on comparison result. Comparison results showed that Anhui IBDV strains were divided into two categories, namely AH-2, AH-9 and CU-1, B87, NB, D78, STC belong to a branch, these showed they had close relationship; and AH-1, AH-7 and OKYM, HK46, G9201, UK661 belongs to another branch. Antigenicity and toxicity analysis showed that Anhui strain antigen had no qualitative change, but they had undergone a certain mutation compared with the classical strains and vaccine strains in nucleotide and amino acid level respectively. AH-1, AH-7 belonged to high virulent strains, AH-2, AH-9 belonged to attenuated strains. This results indicated that there were both very virulent strain and attenuated strain in Anhui, and the high virulent strain was close to Europe,the United States and Japan strain, they might be have one common source.