DEVELOPMENT AND IMPACT ASSESSMENT OF RECOMBINANT LACTOCOCCUS LACTIS MICROBIAL AGENT FOR COMMON CARP(CYPRINUS CARPIO)RHBDD3 PROTEIN
Studies have shown that fish rhomboid domain-containing protein 3(Rhbdd3)can inhibit aquatic virus infection.However,there is still lack of related application in practice.The Lactococcus lactis NICE(nisin controlled gene expression system)is a high-efficient,safe,and economic protein expression system.For the development of recombinant Lactococcus lactis expressing Rhbdd3,the rhbdd3 gene of common carp was cloned into pNZ8148 vector.The correctness of recombinant plasmid pNZ8148-rhbdd3 was verified by sequencing before electrotransformed into L.lactis NZ9000 competent cells.The recombinant Rhbdd3 protein was induced in different concentrations of nisin for 1,3,5 h and then analyzed by SDS-PAGE and the Western blotting.Then,the verified recombinant L.lactis were added to the base feed and fed to the common carp daily.The fish were then infected with spring viremia of carp virus(SVCV)two weeks later.The relative expression of immune-related genes and the viral N gene copies were detected by RT-qPCR.Results show that the molecular weight of expressed protein was 39 kDa,which is equal to the expected protein size.The optimal condition of induction was 100 ng/mL nisin for 5 h.The expression of immune-related genes including IFN1,IRF7,Viperin,Mx1,and ISG15 Viperin were all up-regulated compared with the control group at 14 days post feeding.In addition,the replication of SVCV in experimental group was significantly lower than that of the control group,indicating that the recombinant L.lactis enhanced the immunity of the common carp.The present study provided a strategy for the large-scale preparation of Rhbdd3 protein as an effective prevention and control measure against aquatic viral disease.
Rhbdd3 proteinLactococcus lactisCyprinus carpiospring viremia of carp virus(SVCV)microbial preparation
万方数据
维普
