TTV病毒ORF1抗原的基因克隆及原核表达

目的 扩增和克隆TTV-ORF1 DNA,构建原核表达载体并在大肠杆菌表达.方法 从TTV病毒感染者外周血中提取DNA,用PCR从中扩增出TTV-ORF1 DNA,插入载体pGEM-T Easy中;测序正确后,克隆至原核表达载体pGEX-4T-1中,构建重组表达载体pGEX-4T-1-ORF1,转化大肠杆菌BL21株进行表达.结果 成功获得TTV-ORF1编码基因并构建原核表达载体,测序结果与GenBank收录序列相一致.诱导表达得到51kD的TTV-ORF1融合蛋白,占细菌总蛋白量的27.2%.Western blot证实为目的蛋白.结论 成功获得TTV-ORF1 DNA,构建得原核表达载体并获得高效表达.为TTV的ELISA检测和疫苗提供抗原.

Cloning and Expression of TTV-ORF1

Objective To clone TTV-ORF1 DNA and express its recombinant protein in E.coli BL21(DE3).Methods The cDNA of TTV-ORF1 gene was amplified by means of PCR from human blood.After cloning with pGEM-T easy vector and sequencing,the MAGE-1 gene was inserted into the prokaryotic expression vector pGEX-4T-1.BL21(DE3) was transformed with the recombinant and expressed the fusion protein GST-TTV-ORF1.Results The sequence of TTV-ORF1 was identical to the known sequence(GenBank NC-002076).The expression product reached the highest level at 4 h after IPTG induction.SDS-PAGE and scanning analysis of gel density indicated that the expressed protein was about 51kD and account for 27.2% of the total bacterial protein.Conclusion The TTV-ORF1 gene has been successfully cloned and expressed,which provides the immunogen for ELISA and vaccine.