建兰花叶病毒TGB1基因的原核表达及多克隆抗体制备

Prokaryotic expression of TGB1 gene of Cymbidium mosaic virus and preparation of polyclonal antibodies against the recombinant TGB1

摘要: 以GenBank中的CyMV-TGB1(GenBank 登录号：HQ681906.1)基因全序列设计1 对特异性引物，采用RT-PCR 从感染建兰花叶病毒的白花刺果曼陀罗叶片总RNA 扩增出该病毒的TGB1 基因。测序结果表明，该TGB1基因全长702 bp，编码233 个氨基酸残基。构建了原核表达载体pET32a(+)-TGB1，将重组质粒转化大肠杆菌BL21(DE3)，在25℃"以1.0 mmol/L IPTG 诱导表达重组蛋白基因。SDS-PAGE 分析表明，重组蛋白分子量约为44 ku，与预测相符。以该重组蛋白为抗原免疫新西兰大白兔，制备的抗体效价达1：25 600。

Abstract: Based on the TGB1 sequence of Cymbidium mosaic virus (CyMV, GenBank Accession No: HQ681906.1),a pair of specific primers was designed for amplifying the TGB1 gene from the total RNA of Datura stramonium leaves infected with CyMV. DNA sequencing showed that the TGB1 gene length was 702 bp and encoded 233 amino acid residues. The TGB1 gene was subcloned into a prokaryotic expression vector pET32a (+). By inducing with 1.0 mmol/L IPTG at 25℃, the recombinant TGB1 gene was expressed in E. coli BL21(DE3) and its product was identified as a specific band of 44 ku by SDS-PAGE. Polyclonal antibodies against recombinant TGB1 protein were obtained by hypodermal injection of rabbits. The titre of antiserum was 1:25 600.