番茄褪绿病毒 CP 基因克隆、序列分析及原核表达

摘要

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 de-signed 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 ToCV CP gene(NCBI:KT809400)owned 774 bp nucleotides,encoding 257 amino acids.The similarity of nucleo-tide 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 or-igin 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 ex-pression vector pET32a-ToCVCP in 37 ℃with 1 .0 mmol /L IPTG for 6 hours.%为制备番茄褪绿病毒（Tomato chlorosis virus，ToCV）抗血清，以番茄病叶为试验材料，提取总 RNA，根据ToCV CP 基因设计特异性引物，利用 RT-PCR 方法克隆目的基因，构建原核表达载体 pET32a-ToCVCP，在大肠杆菌Rosetta （DE3）菌株中表达 CP 蛋白。结果表明：ToCV CP 基因（GenBank 登录号：KT809400）全长774 bp，编码257个氨基酸，与 GenBank 中其他地区分离物核苷酸序列同源性为97．2％～99．6％，推导的氨基酸序列同源性为97．3％～100．0％。核苷酸序列保守位点占全部位点的91．3％，氨基酸序列保守位点占全部位点的88．3％，表明不同地理来源的番茄褪绿病毒的 CP 基因保守性较高。将 ToCV CP 基因克隆到原核表达载体 pET-32a（＋）上，并在体外条件下诱导表达出融合蛋白。SDS-PAGE 分析表明，转 pET32a-ToCVCP 载体的大肠杆菌 Rosetta （DE3）菌株表达了分子量约33 kDa的重组蛋白。该重组蛋白在37℃，1．0 mmol ／L IPTG、诱导6 h，表达量最大。