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目的 获取旋毛虫抗原的 c DNA克隆并进行蛋白的原核表达。 方法 应用兔抗旋毛虫成虫可溶性全虫抗原血清对旋毛虫成虫 c DNA文库进行筛选 ,并用兔人工感染旋毛虫血清对强阳性克隆进行再筛选。将编号为 Ts87阳性克隆的基因片段亚克隆入 PET- 2 8a( +)表达载体 ,IPTG诱导表达后用 SDS- PAGE电泳分析表达产物。 结果 免疫筛选获得阳性克隆 Ts87;成功构建重组表达质粒 PET- 2 8a( +) / Ts87。诱导表达该融合蛋白 ,SDS- PAGE电泳表明 ,其能表达一分子质量约为 40 ku的融合蛋白 ,与预测分子质量相符。 结论 筛选到 c DNA克隆 Ts87,与兔抗旋毛虫成虫可溶性全虫抗原血清和兔人工感染旋毛虫血清均产生特异性免疫反应 ;PET原核表达系统所获重组蛋白为蛋白功能研究奠定了基础。
Objective To obtain c DNA clone of Trichinella antigens and prokaryotic expression of the protein. Methods The cDNA library of Trichinella spiralis adults was screened by the serum of rabbit anti-Trichinella spiralis adult soluble insects, and the strong positive clones were screened by artificial infection of Trichinella spiralis. The gene fragment named Ts87 was subcloned into the PET-2 8a (+) expression vector. The expression product was analyzed by SDS-PAGE electrophoresis after IPTG induction. Results The positive clones Ts87 were obtained by immunoscreening. The recombinant expression plasmid PET-2 8a (+) / Ts87 was successfully constructed. The fusion protein was induced and expressed by SDS-PAGE. It showed that it expressed a fusion protein of about 40 ku in molecular mass, which was consistent with the predicted molecular mass. CONCLUSION: The c DNA clone Ts87 was screened for specific immunoreactivity with rabbit anti-Trichinella spiralis soluble parasite antigen serum and rabbit infected Trichinella spiralis serum. The recombinant protein obtained by PET prokaryotic expression system laid the foundation for protein function research.