Prokaryotic Expression And Preparation Of Polyclonal Antibody Of Spring Viremia Of Carp Virus Glycoprotein And Matrix Protein

Spring Viremia of Carp (SVC) is an acute, hemorrhagic and contagious disease resulting in high mortality, and it has caused severe economic losses on national carp breeding industry. The causative agent of SVC is spring viremia of carp viruse(SVCV), which is closely belong to the genus Vesiculovirus, family Rhabdoviridae. According to the genome sequence of SVCV available in Genbank (NC₀02803), the open reading frames of Spring Viremia of Carp Virus (SVCV) two main structural protein genes including G protein gene and M protein gene were artificially synthesized, and then cloned into pMD19-T vector. After restriction digestion and sequence verification, the fragments of G protein gene and M protein gene were cloned into the prokaryotic expression vector pET-32a (+). The two recombinant plasmids pET-32a(+)-G and pET-32a(+)-M were then transformed into BL21(D E3) strain and expressed under the induction of 0.1mmol/L IPTG at 37℃for 5 hours, then the G protein and M protein were highly expressed in E.coli and accumulated in inclusion body. The purified G protein and M protein were respectively obtained after gel extraction mini kit and Ni-NTA His-bind Resin purification. The purified proteins were separately used to immunize rabbits to produce rabbit antiserums. The antiserums were titrated by ELISA, showing the positive signal of the anti-G serum down to a 1:2560000 dilution and anti-M serum down to a 1:64000 dilution. Western blot analysis indicated it may be relevant with splicing of the expressed G protein that there were two specific bands when rabbit anti-G serum reacted with SVCV protein produced by cell, and SVCV M protein produced by cell can be specifically identified by rabbit anti-M serum. In conclusion, the experiments provide important theoretical basis for futher study of SVCV immunodiagnosis and the function of the G protein gene and M protein gene.