The Effect Of Carp HSP70 And HSP90 Eukaryotic Expression Product To The Immune Effect Of KHV Nucleic Acid Vaccine PIRES- ORF81

Heat shock proteins(HSPs) are stress proteins which induce under external environmental stress. HSPs are classified into different families by molecular mass. Every family has different biological characteristics. Among them, heat shock protein 70 and heat shock protein 90 have been reported that with the function of the molecular chaperone. They present antigens to improve immune effect in the body. And they help to complete the correct protein conformation. Hsps play a role to nascent peptide chain by identifying, folding, assembling, transfering, matureing etc. Hsps help cell to resist apoptosis and promote proliferation.Koi Herpesvirus causes significant morbidity disease in common carp and spreads rapidly with highly mortality. KHV disease is listed as a notifiable disease by the World Organisation for Animal Health(Office International des Epizooties OIE).In previous studies, many open reading frame have been cloned and proved that have immunogenicity. But the immune effect has not been a good breakthrough. Therefore, to explore a efficient immune vaccine is imminent.In this research, according to the HSP70 and HSP90 cDNA sequence which had logged in Genebank,we design two pairs of specific primers by Primer Premiers 5.0 software. We acquire 240 bp HSP70 gene and 687 bp HSP90 gene by PCR cloning. Insert two genes respectively to pMD19- T carrier. And then,here identified that the cloned plasmid pMD19-T- HSP70 and pMD19- T- HSP90 were built successfully by PCR and enzyme digestion evaluation. Biotechnology company sequencing results show that the homology was as high as 99% between the gene we cloned and carp HSP70 and HSP90.Two genes were respectively connected to expression vector pEGFP-C1. PCR identification, digestion with double enzyme showed that the recombinant expression plasmids pEGFP C1-HSP70 and pEGFP C1-HSP90 were successfully constructed. Mixing recombinant expression plasmid and Transfection reagents in several immune dose transfected carp muscle cells. The expression of recombinant expression plasmid pEGFP C1-HSP70 and pEGFP C1-HSP90 in carp muscle cells were observed by fluorescent inverted microscope.By using the mixture of recombinant expression plasmid in several immune dose and pIRES- ORF81 injected Carp`s intramuscular and immunized once a week, three times in total. Blood was sampling before injection and serum was collected to detect antibody levels by enzyme-linked immunosorbent assay(ELISA). Two weeks after the third immunization, experiment result showed that antibody titer reach the highest level. Compared with pIRES-ORF81 alone immune, antibody level of combined immunization increased obviously, but no significant difference was observed between them. The result of challenged showed that the protective rate of combined immunization is relatively higher than DNA vaccine alone immune. Observing the pathological tissue sections, pathological change was not found in both DNA vaccine alone immune and combined immunization.