The Study Of Carp ?-defensin To The Immune Effect Of Koi Herpes Virus Recombinant Plasmid

Defensins are polypeptides that kill microorganisms such as bacteria,fungi and viruses,and are important components of the natural immune system.They have the advantages of rapid reaction time,drug resistance,and broad-spectrum antimicrobial activity.As the first line of defense against the invasion of pathogenic microorganisms,defensins quickly eliminate bacteria,fungi and viruses,and play an important role in defense against external pathogenic microorganisms.Koi Herpesvirus(KHV),also known as herpesvirus 3(CyHV-3),belongs to the herpes virus family and can cause Koi Herpesvirus Diease(KHVD).The disease has a rapid onset and has a high mortality rate.Many open reading frames of the koi herpes virus have been studied,and have certain immunity,but the effect has been poor.Therefore,this study uses the codfish ?-defensin(BD)and the herpes virus recombinant plasmid for co-immunization in order to get the ideal immune effect.In this study,based on the ID JF343439 sequence of the ?-defensins registered in GenBank,a 204 bp ?-defensin gene was cloned and RT-PCR was used to design specific primers for bioinformatics analysis.At the same time,two pairs of specific primers were designed based on the GenBank-encoded ORF56 and ORF150 DNA sequences.The 930 bp ORF56 gene and the 849 bp ORF150 gene were obtained by PCR.The three genes were inserted into the pMD18-T vector and identified by PCR and double enzyme digestion.The recombinant plasmids pMD18-T-BD,pMD18-T-ORF56,and pMD18-T-ORF150 were successfully constructed.After sequencing,the results showed that the target gene fragment was successfully cloned.In this study,the three genes were linked to the eukaryotic expression vector PVAX,and the recombinant plasmids PVAX-BD,PVAX-ORF56,and PVAX-ORF150 were successfully constructed.The three recombinant plasmids were transfected into salmon muscle cells,and the fluorescence was observed through an inverted microscope,indicating that the recombinant plasmid was successfully constructed.The carp was injected intramuscularly with various immunizing doses of recombinant plasmids,immunized once a week,coimmunized three times,and blood was collected from the test fish.The antibody level was detected by enzyme-linked immunosorbent assay(ELISA).The results showed that: PVAX-BD can enhance PVAX-ORF56,PVAX-ORF150 combined immunization effect.