旋毛虫抗原基因Ts21的原核表达与重组蛋白鉴定

目的 原核表达旋毛虫相对分子质量（Mr）21 000抗原基因（Ts21）并对重组蛋白进行纯化和抗原性鉴定。 方法 将旋毛虫抗原基因Ts21亚克隆入原核表达载体pMAL-c2X，构建重组表达质粒pMAL-c2X-Ts21，经酶切鉴定正确后转化大肠埃希菌TB1，以异丙基-β-D硫代半乳糖苷（IPTG）诱导表达。亲和层析法纯化表达产物。应用十二烷基磺酸钠?鄄聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹（Western blotting）分析鉴定重组蛋白的抗原性。将重组蛋白免疫小鼠制备免疫血清，ELISA检测抗体滴度，免疫荧光检测（IFA）确定Ts21蛋白在肌幼虫体内的分布。 结果 SDS-PAGE结果显示，表达产物为Mr 63 500的重组融合蛋白，IPTG诱导4 h后表达量最大，薄层凝胶光密度扫描显示表达的融合蛋白占菌体蛋白总量的18.2%。Western blotting结果显示该重组蛋白可被旋毛虫、纳氏旋毛虫感染小鼠血清及旋毛虫病患者血清识别，但不能被乡土旋毛虫、布氏旋毛虫及伪旋毛虫感染小鼠血清识别；与钩虫病、囊尾蚴病、日本血吸虫病患者血清无交叉反应，但与并殖吸虫病、华支睾吸虫病及棘球蚴病患者血清有交叉反应。重组蛋白免疫小鼠可产生高滴度的血清抗体，IFA显示Ts21蛋白主要分布于肌幼虫体壁。 结论 成功制备了旋毛虫Ts21基因的重组蛋白，该蛋白具有较好的抗原性。

Prokaryotic Expression of Trichinella spiralis Gene Ts21 and Identification of the Recombinant Protein

Objective To express the antigen gene Ts21 of Trichinella spiralis, purify the recombinant protein and test its antigenicity. Methods T. spiralis gene Ts21 was sub-cloned into the prokaryotic expression vector pMAL-c2X and the recombinant pMAL-c2X-Ts21 was constructed. The recombinant plasmid was transformed into E. coli TB1 strain and induced by IPTG. The expression products were purified by MBP-binding affinity chromatography. The antigenicity of the recombinant protein was examined by SDS-PAGE and Western blotting. Mice were immunized with the recombinant protein, the titer of the immune sera was detected by ELISA. The distribution of Ts21 protein in muscle larvae was observed by IFA. Results The molecular weight of the expressed fusion protein was about Mr 63 500 and the expression level peaked at 4 h post-incubation. The portion of the fusion protein accounted for 18.2% of all the protein by thin-layer gel optical scanning. Western blotting demonstrated that the recombinant protein was recognized by sera from mice infected by T. spiralis (T1) and T. nelsoni (T7) as well as sera of patients with trichinellosis, but not by sera from mice in-fected with T. nativa (T2), T. britovi (T3) and T. pseudospiralis (T4). The recombinant protein did not react with sera from patients with ancylostomiasis, cysticercosis and schistosomiasis, but cross-reacted with sera from patients with parag-onimiasis, clonorchiasis and echinococcosis. High titers of antibodies were produced in mice immunized with the recom-binant protein. IFA showed that the Ts21 protein was mainly distributed in the cuticle of muscle larvae. Conclusion The Ts21 antigen gene of T. spiralis has been expressed and the recombinant protein shows antigenicity.