Cloning, Expression And Identification Of Immunogenicity ORF27and ORF146Genes Of Koi Herpesvirus

Koi herpes virus disease (Koi Herpesvirus Diease, KHVD) is composed of Koi herpesvirus(Koi Herpesvirus, KHV) is a highly infectious cause and high fatal diseases, mainly infected Koi,common carp and its variants. In1998, KHVD in Israel was found for the first time, after more thana dozen countries and regions of the world to spread and popular, in2003to enter the China,because the disease is difficult to control and caused great economic losses, so that countries attachgreat importance to. In this experiment, glycoprotein gene and membrane protein KHV gene(ORF27and ORF146) for the purpose of pET-32a gene, prokaryotic expression vector, to constructthe pET32a-ORF27and the pET32a-ORF146two kinds of prokaryotic recombinant expressionplasmid in vitro proof, which can obtain good expression, the mouse immune experiment.According to KHVORF27and KHVORF146GenBank gene sequence has been logged,designed two pairs of specific primers, amplified by PCR and the two target genes; respectivelyinserted into the pMD18-T vector to construct a recombinant plasmid, two, confirmed by DNAsequencing right after the function, structure and application of bioinformatics in the initial analysisof KHVORF27and KHVORF146gene the. Analysis results show that: the ORF27gene is207bp inlength, encoding69amino acids, the theoretical molecular mass of7366.62Da, isoelectric point is4.487, the ORF146gene is999bp in length, is a complete open reading frame, encoded333aminoacids, the theoretical molecular mass of37466.56Da, isoelectric point7.885. Epitope analysisshowed that: two the gene has good antigenicity. The sequencing results in the GenBank nucleicacid BLAST analysis， Koi herpes virus Chinese Jilin strain (KHV-CJ) ORF27gene and ORF146gene sequence identity with American strains (KHV-U) ORF27gene and ORF146gene was100%.The two target genes ORF27and ORF146was inserted into the prokaryotic expression vector ofPET-32a, pET32a-ORF27and pET32a-ORF146to construct prokaryotic expression recombinantplasmid, identified by PCR and restriction enzyme digestion, the recombinant plasmid wastransformed into Escherichia coli BL21, and then the expression, detected by SDS-PAGE methodshowed that ORF27, ORF146target protein expression.Concentration of purified proteins were measured after protein respectively:0.526μ g/μ L and0.213μ g/μ L. The purified protein was induced by injection of immunization in Kunming mice,divided into pET32a-ORF27and pET32a-ORF146two groups, each group at seventh,0,7,14,21,28days of injection, antibodies were detected by ELASA method, analysis of humoral immuneresponse in mouse.ELASA results showed that the expression of specific antibody in mice: injection of blood proteincould be detected in KHV. antibody level reached maximum in21days, then the antibody levelbegan to decrease, while the control group was not detected antibody; under the same dosage, antibody level of protein expression of two recombinant plasmids obtained were better, and thelevel of antibodies in the basic the same, but compared with the control group, antibody levels weresignificantly (P <0.05).