番茄黑环病毒外壳蛋白基因的克隆与原核表达

Cloning and Prokaryotic Expressing of Coat Protein Gene in Tomato Black Ring Virus

利用RT-PCR方法获得了番茄黑环病毒(TBRV)外壳蛋白(coat protein,CP)基因,将CP基因克隆到pMD19-T Simple载体,并进行测序,大小为1 399 bp。与GenBank中已报道的TBRV的CP基因核苷酸相似度达99%以上。将TBRV CP基因定向插入双酶切的pET30a中,成功构建了原核表达载体pET30a-TBRVCP,转化大肠杆菌BL-21,表达重组蛋白。SDS-PAGE电泳分析发现,通过诱导条件的优化,重组蛋白在37℃,1.0 mmol/L IPTG、4 h条件下表达量最高。

The coat protein （CP） gene of tomato balck ring virus （TBRV） was amplified by RT- PCR. The amplified fragment was cloned and confirmed by sequence analysis which consisted of 1 399 bp. TBRV CP gene was cloned into pMD19 - T simple vector. Sequence analysi：s showed that the nucleotide was 99% aligned with TBRV CP gene in GenBank. TBRV CP gene was inserted into pET30a digested by Hind IU /BamH, the prokaryotic expression vector pET30a - TBRV CP was constructed, and transformed into E. coli BL -21. After induction by 1.0 mmol/L IPTG for 4 h in 37 ℃, the recombinant TBRV CP was successively expressed and the the yields were the largest in SDS -PAGE analysis.