多头带绦虫Tm7基因的表达及间接ELISA检测方法的建立

Prokaryotic Expression ofTm7 Gene ofTaenia multicepsand Establishment of Indirect ELISA Using the Expressed Protein

以多头带绦虫Tm7重组蛋白为抗原,建立动物多头蚴病早期诊断方法。本研究从羊脑多头蚴原头节提取总RNA,采用RT-PCR技术首次扩增出Tm7基因的全序列,该基因的开放阅读框为207bp,编码68个氨基酸。将此基因克隆到pET-32a(+)载体,构建重组表达质粒pET-32a-Tm7,经转化大肠杆菌BL21(DE3)后IPTG诱导表达,用SDS-PAGE和Western blot检测表达产物。以纯化后的表达蛋白作为抗原,建立检测羊脑多头蚴病抗体的重组蛋白间接ELISA方法。研究结果表明,Tm7基因在大肠杆菌中成功表达,表达产物为约27ku的融合表达蛋白,该蛋白能识别羊脑多头蚴病阳性血清。建立的间接ELISA方法,检测敏感性可达93.3%,特异性达94.1%,研究结果表明Tm7重组蛋白可作为脑多头蚴病的诊断抗原。

In order to establish diagnostic method of coenuriasis,total RNA was extracted from protoscoleces of cyst which were recovered from the brain of sheep.The complete sequence of Tm7 was amplified and sequenced.The open reading frame of Tm7 was then amplified,its ORF consisting of 207 bp and encoding 68 amino acids.A recombinant plasmid pET-32a-Tm7 was constructed and transformed into E.coli BL 21 for in vitro expression.SDS-PAGE and Western blot were employed for analyzing the recombinant protein.The purified recombinant protein was used for development of an indirect ELISA for detection of anti-coenuriasis antibodies.The results showed that target fusion protein was expressed in E.coli,it was about 27 kD in molecular weight and could be recognized by coenurosis positive serum.Futhermore,in indirect ELISA this recombinant protein showed 93.3% of sensitivity and 94.1% of sensitivity,indicated it can be used as diagnostic antigen.