细粒棘球绦虫亲环素A的重组表达产物的纯化及免疫学鉴定

Cloning and expression of cyclophilin A gene from Echinoccocus granulosus

目的克隆细粒棘球绦虫亲环素A（EgCypA）基因并体外表达纯化该蛋白以评价EgCypA的血清学免疫检测价值。方法利用生物信息学方法分析细粒棘球绦虫cDNA文库中的EgCypA的理化及生物学特性并设计引物，以细粒棘球绦虫cDNA为模板进行PCR扩增得到EgCypA基因，将该基因克隆到原核表达载体pET28a中，将重组载体导入大肠埃希菌，IPTG诱导该重组菌株表达EgCypA，使用聚丙烯酰胺凝胶电泳（SDS—PAGE）明确重组蛋白表达情况；利用Western blot法进行纯化的重组蛋白对细粒棘球绦虫感染血清及其他寄生虫感染血清免疫反应性的鉴定，以此评价该重组蛋白的免疫检测价值。结果SDS—PAGE显示重组菌株在相对分子质量18400～25000表达了大量蛋白，该蛋白的分子量同生物信息学分析的EgCypA的分子量吻合。Western blot结果显示细粒棘球绦虫相关血清与重组蛋白作用后于17000～26000之间出现了特异性的反应条带，其他寄生虫血清则无此反应。结论EgCypA具有良好的抗原性，是很有前景的细粒棘球绦虫特异性诊断抗原，可以作为免疫学检查细粒棘球绦虫早期感染的候选分子。

Objective In order to assess the value of Echinoccocus granulosus cyclophilin A （EgypA） in immune diagnosis, this novel gene was cloned and expressed. Methods By screening the EST library, the coding region of EgCypA was identified, and the PCR primers were designed based on this sequence. Bioinformatic tools were used to deduce the amino acid sequence of EgCypA and analyzed its biological characteristics. EgCypA was amplified from E. granulosus cDNA library by using PCR. Then, it was cloned into the prokaryotic expression vector pET28a and transformed into E. coli BI21. The recombinant protein was expressed in E. eoli BL21 after IPTG induction. The inmmnogenicity of EgCypA was evaluated by Western blot using the Echinoccocus granulosus and other parasites infected animals＇ sera. Results Results of SDS-PAGE electrophoresis show that the recombinant BL21 expressed 18 400 -25 000 protein, which was identical with the molecular weight calculated by bioinformatie analysis. Western blot shows that the recombinant protein only reacted with its immune serum and E. granulosus cystic fluid immune serum, and EgCypA immune serum could react with the excretion and secretion antigen from E. granulosus protoscolexes, and no cross-reaction between EgCypA and sera from other parasites infected animals. Conclusions Cloning and expression of EgCypA are successful. EgCypA has good immunologic activity and could be a candidate molecular for immune diagnosis of echinococcosis in early stage.