烟草环斑病毒外壳蛋白基因的原核表达及抗血清的制备

Cloning and Expression of the Coat Protein Gene of Tobacco Ringspot Virus and Preparation of Virus-specific Antiserum

烟草环斑病毒(Tobacco ringspot virus,TRSV)是我国二类进境检疫危险性有害生物,对农业生产危害较大.本研究依据TRSV外壳蛋白基因cp序列设计合成了2条引物,通过RT PCR扩增得到长约1500bp的目的片段.将目的片段与质粒pET-22b(+)连接,构建了含TRSV cp基因的融合蛋白原核表达载体pETRSV-CP.序列分析表明,TRSV-SD1的cp基因全长1548bp,编码515个氨基酸与GenBank中其它TRSV分离物cp基因相比,核苷酸及推导的氨基酸序列同源性为90.7%～94.6%.将pETRSV-CP转入大肠杆菌,诱导表达.SDS-PAGE结果显示,表达的TRSV CP融合蛋白的相对分子质量约为58kDa.以此融合蛋白制备的抗血清的效价为1/1024,抗血清与TRSV具有良好的特异性反应.

Coat protein （CP） gene of Tobacco ringspot virus （TRSV） Shandong isolate 1 was cloned by reverse transcription-polymerase chain reaction （RT-PCR）, and was subcloned into the pET-22b（＋） prokaryotic expression vector. The recombinant vector was transformed into E. coli strain BL21. Sequence analysis revealed that the cp gene was 1548 nucleotides in length,encodes a coat protein of 515 amino acids, and shares 90.7%-94.6% nucleotides and amino acid homology with TRSV cp genes registered in GenBank. The target fusion peptide with a molecular weight of 58kDa was expressed under the condition of 23-25 C and induced by IPTG at a final concentration of lmmol/L. Rabbit was immunized using the expressed target peptide as antigen, and the antiserum was obtained. The antiserum had a titer of 1/1024 with high specificity to TRSV.