樱桃病毒A外壳蛋白基因克隆及其原核表达

Cloning and Prokaryotic Expressing of Coat Protein Gene of Cherry Virus A

为制备樱桃病毒A(Cherry virusA,CVA)抗血清,克隆CVA外壳蛋白基因(CP),构建原核表达载体,优化蛋白表达条件。根据CVA全基因组序列(NC_003689.1)设计特异引物,RT-PCR方法扩增CVA外壳蛋白基因,克隆、测序,构建原核表达载体pET30a-CVACP,转化大肠杆菌BL21(DE3)株系,采用异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达。序列分析显示,该区段长为603bp,编码201个氨基酸,与法国分离物PF(HQ267856.1)的同源性为96.8%,与印度分离物JKSPMi-5(FN669548.1)同源性为86.3%。成功构建了原核表达载体pET30a-CVACP,SDS-PAGE电泳分析显示,转pET30a-CVACP载体的BL21(DE3)菌株表达分子量约24kDa的重组蛋白,该重组蛋白在37℃、1.5mmol/L IPTG、诱导4h条件下表达量最大。

In order to prepare the special antiserum of cherry virus A, the coat protein gene of CVA was cloned, the prokaryotic expression vector was constructed and the expression condition was optimized. According to the complete genome sequence of CVA in Genbank （NC_003689.1）, specific primers were designed to amplify the coding region of coat protein by RT-PCR, the PCR products were cloned and sequenced. The prokaryotic expression vector pET30a-CVACP was constructed and transformed into E.coli BL21 （DE3）. Isopropyl-β-D- thiogalactopyranoside （IPTG） was used to induce expression of recombinant protein. Sequence analysis showed that the amplicons included 603 bp nucleotides, encoding 201 amino acids. There were 96.8% identical with the isolate PF （HQ267856.1） from France and 86.3% identical with the isolate JKSPMi-5 （FN669548.1） from India. The prokaryotic expression vector pET30a-CVACP was constructed successfully, SDS-PAGE analysis showed that a specific recombinant protein of approximately 24 kDa was induced in the BL21 （DE3） which the prokaryotic expression vector pET30a-CVACP was transformed into and the optimized expression condition was 37℃, and 1.5 mmol/L IPTG with 4 hours.