Abstract: In order to prepare the special antiserum of Tomato chlorosis virus (ToCV),the total RNA of tomato leaves infected with ToCV were extracted.According to the coat protein gene of ToCV,specific primers were designed to amplify the coding region of coat protein by RT-PCR,the prokaryotic expression vector pET32a-ToCVCP was constructed and the recombinant protein was expressed in E.coli Rosetta (DE3).The results showed that the ToCVCP gene(NCBI:KT809400)owned 774 bp nucleotides,encoding 257 amino acids.The similarity of nucleotide and predicted protein were 97.2%-99.6% and 97.3%-100.0%,respectively,compared with the CP gene of other ToCV isolates registered in GenBank.The nucleotide sequence of conserved sites accounted for 91.3% of all loci,the amino acid sequence conserved sites accounted for 88.3% of all loci,indicated that the geographical origin of different ToCV CP gene had a relative higher conservative property.The ToCV CP gene was subcloned into the expression vector pET-32a(+) and the fusion protein was expressed in vitro.SDS-PAGE analysis showed that a specific recombinant protein of approximately 33 kDa was produced in the Rosetta (DE3) with the prokaryotic expression vector pET32a-ToCVCP in 37℃ with 1.0 mmol/L IPTG for 6 hours.

Key words: Tomato chlorosis virus, Coat protein gene, Prokaryotic expression, Gene cloning

CLC Number: 

• Q78

• S641.03