Recombinant Expression And Antibody Preparation And Identify ORF72 And TKof Koi Herpesvirus Virus (KHV)

There are many reports of the detection method of Koi herpesvirus.They are mostly is PCR detection,but lack of immunological detection methods. The advantages of PCR is high sensitivity,but the results may be false positive.The results often appear to determine the uncertainty.It is difficult to determine whether there is a complete virus particle or only residual viral RNA. Therefore established an effective method for immunological detection is important. To establish an effective immunological detection methods, do the following work:Clone TK (ORF55) and ORF72 geneAccording to NCB1 published sequences (GenBank accession number: DQ 177346.1), design specific primers of TK (ORF55) and ORF72 genes. The size of amplified fragments were 1113bp and 672bp. And cloned it into the prokaryotic expression vector pGEX-4T1 and pET28a then transformed it into E. coli (DH5a), the recombinant plasmid pGEX-4T1-TK and pET28a-ORF72 were be identifited by PCR, restriction enzyme digestion, DNA sequencing.pET28a-ORF72 expression, purification of the expressed protein with anti-KHV pET28a-ORF72 polyclonal antibodyThe plasmid was transformed into E.coli BL21 (DE3), induced by IPTG. By SDS-PAGE analysis, the recombinant protein are highly expressed. The moleculars of proteins are 48kDa and 41kDa, and named pGEX-4T1-TK and pET28a-ORF72. Expression product has a good reactogenicity by Western blot. Use His-Tag fusion protein purification column Ni+-NTA His Bind of MERCK companies to purificate pET28a-ORF72 protein,the protein content is 0.4mg/mL. First, with Freund's complete adjuvant, FCA and the purified protein (1:1), subcutaneous multi-point injection. After 2 weeks, the second with Freund's incomplete adjuvant and the purified protein (1:1), subcutaneous multi-point injection. After 2 weeks,do the third. After 1 weeks,collection blood of Rabbit.Get the polyclonal antibodies by separated serum.Identification of polyclonal antibodyCoated virus suspension, respectively, protein.The titer of Polyclonal antibody is 9000. after The total protein and purification protein transfer to the NC membrane after SDS-PAGE.Dilution ratio of protein to protein, KHV virus suspension to do antigen, SVC virus suspension as control, the enzyme spot test found to produce the immune response.Multiple experiments for the KHV antibody immunological detection of the foundation.