新疆卡拉库尔羊Fas基因重组蛋白包涵体的纯化及抗原性分析

Purification and Antigenic Analysis of Recombinant Protein Inclusion Bodies of Fas Gene Xinjiang Karakul Sheep

目的:为了探明Fas基因重组蛋白是否保留天然蛋白的抗原性,为下一步大量制备基础诊断、预防、控制、治疗试剂奠定基础。本实验将Fas基因与原核表达载体pET-28a成功构建了重组融合表达质粒pET-28a-Fas然后转化至E.coliBL21(DE3)感受态当中,找到最佳表达条件,初步纯化了重组融合蛋白,通过对家兔免疫后的血清进行琼扩实验与蛋白质印迹实验分析其抗原性。结果表明,纯化后的包涵体有明显的条带,经测定融合蛋白分子量为39.7 KDa。结论:说明纯化效果好,优化融合蛋白表达量占菌体总蛋白的15.6%,并且证明Fas具有免疫原性和反应原性。

The recombinant protein of Fas gene was analyzed to investigate the antigenic activity for the production, diagnosis, prevention, and treatment. The Fas gene was transformed to prokaryotic expression vector pET- 28a to obtain the recombinant vector pET- 28a - Fas. Preliminary recombinant protein was purificated after the recombinant vector pET - 28a - Fas transformed into E. coli BL21 （DE3） competent ceils at the optimized method. After the immunization of rabbits with recombinant protein, agar diffusion test and western blot were carried out to analyze the antigenic activity in the serum. The result revealed that a special band at 39. 7 KDa wasobserved form the purified inclusion body. And the recombinant protein takes 15. 6% in the bacteria. It can be concluded that the purification meets the need and the recombinant protein possesses the immunogenicity and reactionogenicity.