旋毛虫膜蛋白新基因Tmp10的免疫筛选及鉴定

目的免疫筛选旋毛虫成虫c DNA文库,寻找抗旋毛虫病的疫苗候选抗原或诊断抗原。方法应用旋毛虫感染的猪血清免疫筛选旋毛虫成虫c DNA文库,获得阳性克隆。将一阳性克隆与原核表达载体p ET-28a(+)构建重组质粒,异丙醇硫代-β-D-半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)诱导后,利用亲和层析柱纯化目的蛋白,并通过酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)和Western blotting鉴定其抗原特异性和免疫原性。结果免疫筛选旋毛虫成虫c DNA文库获得42个阳性克隆,选取一个新基因Tmp10,成功构建了重组质粒p ET-28a(+)/Tmp10,诱导表达后获得的重组蛋白(r Tmp10)的相对分子质量约为40 000。Western blotting检测结果显示,该重组蛋白可被旋毛虫感染小鼠和猪血清所识别,具有抗原特异性。r Tmp10免疫小鼠可诱导产生高滴度的抗体,显示其具有较好的免疫原性。结论免疫筛选旋毛虫成虫c DNA文库获得一个新基因Tmp10,生物信息学分析预测其为膜蛋白,其重组蛋白(r Tmp10)具有较好的抗原特异性和免疫原性。

Immunoscreening and identification of a novel full-length cDNA encoding membrane protein of Trichinella spiralis

Objective In order to obtain vaccine or diagnostic candidate antigens for trichinellosis, the adult cDNA library of T. spiralis was immunoscreened. Methods The adult cDNA library of T. spiralis was screened using the swine sera infected with T. spiralis. The cDNA sequence of the positive clone was analyzed and the recombinant protein was expressed and purified. The antigenicity and immunogenicity of the recombinant protein was analyzed with ELISA and Western blotting. Results Forty-two positive clones were identified by immunscreened. A novel gene Tmp10 of T. spiralis was sequenced and recombinant plasmid pET-28a(+)/Tmp10 was successfully constructed. The recombinant protein(rTmp10) was expressed and the molecular weight was about 40 000. The results of Western blotting showed that the rTmp10 could be recognized by sera from the mice and swine infected with T. spiralis respectively, which showed that the rTmp10 had the specific antigenicity. The rTmp10 could induce a high level of specific anti-Tmp10 IgG antibodies, which showed that the rTmp10 had the immunogenicity. Conclusion The novel gene Tmp10 of T. spiralis was obtained by immunoscreening and predicted to be a membrane protein. The recombinant protein Tmp10 had the high specific antigenicity and immunogenicity.