Efficient Expression of Coat Protein Gene of Cucumber Mosaic Virus and Preparation of Viral-specific Antiserum

CP (coat protein, CP) gene of CMV (cucumber mosaic virus, CMV) passionfruit isolate was cloned by reverse transcription-polymorase chain reaction (RT PCR), Then it was subcloned into pET 22b(+) vector at Bam H I/ Sac I sites. The recombinant vector was transformed into BL21 (DE3) host strain of E.coli . The target fusion peptide with molecular weight of 31.8 kDa was expressed under the condition of 37℃ and induced by IPTG at final concentration of 1mmol/L. SDS PAGE showed that the maxim expression amount of it can account for 28.9% of total soluble proteins (TSP) after 6 hours inducing culture. The specificity of expressed peptide was confirmed by western blot with CMV specific antiserum. Afterwards, rabbit was immunized using the expressed target peptide as antigen and antiserum was obtained. Indirect enzyme-linked immunosorbent assay (Id-ELISA) showed the antiserum had titer of 10 -6 with high specificity to CMV.The primary Id-ELISA tests showed that it could detect less than 30ng/ml of purified virus, and 3125 fold. diluted saps prepared from tobacco leaves infected by CMV.;