Molecular cloning and expression of coat protein gene of an Iranian isolate of sugarcane streak mosaic virus in Escherichia coli

Sugarcane streak mosaic virus (SCSMV) is an important causal agent of sugarcane mosaic disease in Asian countries. It seems that the most appropriate way to control the virus is to use virus-free cuttings. ELISA is the most common laboratory test to detect viruses on a large-scale; therefore, antibody preparation against SCSMV to detect the presence of the virus in sugarcane cuttings is of importance. However, commercial antiserum to detect SCSMV is not available. To provide viral antigens for use in the polyclonal antibody production process, SCSMV-CP was amplified using specific primers for CP gene containing BamHI and HindIII digestion sites at respective ۵′ end of the forward and reverse primers, respectively. An ۸۵۰ bp PCR product was amplified, purified, and ligated into a pTZ۵۷R/T vector to generate pTZ۵۷-SCSMV-CP. It was then transformed into E. coli strain DH۵α. After culturing bacterial cells at ۳۷ ºC overnight, the integrity of proper recombinant pTZ۵۷-SCSMV-CP clones was confirmed by restriction enzyme analysis of extracted recombinant plasmid DNAs and PCR with specific-forward and reverse primers. The CP gene was released from pTZ۵۷-SCSMV-CP by restriction enzymes and subcloned into the expression vector pET۲۸a. The resultant recombinant plasmid, pET۲۸-SCSMV-CP, was transformed into E. coli strain B۲۱ (DE۳) by the heat shock method. Transformed bacterial cells were grown on solid LB medium containing ۲۵ μg/ml kanamycin overnight. The recombinant pE۲۸a expression vector containing CP gene was confirmed by colony PCR, restriction analysis by BamHI and HindIII, and sequencing of the insert DNA. Finally, expression of CP protein was induced by adding ۱ mM IPTG to bacterial cultures containing the recombinant pET۲۸-SCSMV-CP DNA. Sampling was done four hours after induction, and the bacterial protein was extracted. The extracted proteins from the harvested cells were separated by ۱۲% SDS-PAGE, and a strong ۳۵-KDa protein band was revealed in the gel.

کلیدواژه ها:

Antibody ، Coat protein ، Cloning ، Restriction enzyme digestion ، Expression vector