多头蚴病保护性基因的分离与表达

Isolation and expression of the protective gene from Taenia multiceps

目的分离与表达多头绦虫（Taenia multiceps）基因45m，确定其免疫原性及在虫体不同发育阶段的表达。方法通过对绦虫同源基因序列分析设计引物，运用RT—PCR的方法，首次从绵羊多头绦虫的幼虫阶段分离到新的基因45m，将其克隆到pET41b表达载体中，获得了含正确读码框的重组质粒，将表达蛋白初步运用于动物免疫试验。结果45m基因长度为698bp，编码232个氨基酸。表达的重组蛋白分子量约为63kD，表达产量可达100～120mg／L。其蛋白序列与已知抗羊带绦虫（45W）和牛带绦虫保护性抗原的同源性分别为89％和85％。本动物绵羊的免疫接种试验表明该蛋白可有效激起机体的抗原抗体反应，其抗血清可以与多头绦虫的成虫和幼虫抗原总蛋白在63kD处发生特异性反应。结论45m蛋白可能具有较强抗原性，该蛋白在虫体的成虫和幼虫阶段均有表达，推测认为45m可能成为多头蚴病的候选疫苗。

Coenurosis caused by a dog tape worm Taenia multiceps is an imortant disease of sheep and cattle in China, especially in Xinjiang. To isolate and express a protective gene from T. multiceps, termed as 45m, this gene from the larva stage of the worm was amplified through RT-PCR with a pair of primers identical to the T. ovis 45W gene,and the amplified fragment was then inserted into expression vector pET-41b（＋）and expressed in E. coli BL21. It was demonstrated that the length of the 45m gene was 698 bps,coding for 232 amino acids. The molecular mass of the recombinant protein expressed was 63 kDa and the expression system could yield amount of proteins of 100-120 mg/L. In addition, the sequence homology of proteins with the known protective antigens of T. ovis（45W）and T. saginata were 89% and 85 % respectively. As demonstrated by Western blot analysis,the native proteins from larval and adult stages of worms could be recognized by the sheep anti-45M serum. Also, this recombinant fusion protein 45m showed a strong antigenicity. So, it is apparent that this protein can be used as the vaccine candidate to protect sheep and eattles from Coenurus cerebralis infection.