弓形虫主要表面抗原 P30基因的原核融合表达及鉴定

THE PROKARYOTIC EXPERSSION AND IDENTIFICATION OF TOXOPLASMA GONDII MAJOR ANTIGEN P30 FUSED WITH GST

目的在已建 P30克隆载体的基础上,进一步构建表达质粒,诱导以融合蛋白形式表达的能被免疫血清识别的 P30蛋白,并探索最佳表达条件。方法双酶切 P30克隆载体和空白表达质粒 PGEX-4T-1,连接后转化入 DH5α进行 PCR 筛选,酶切鉴定和测序验证。重新将重组质粒转化入表达宿主菌 BL21进行诱导。对比不同诱导时间的菌体蛋白 PAGE 图谱,并进行 Western blot鉴定。改变诱导物浓度和培养温度,优化出最佳表达条件。结果构建成功序列和读框正确的重组质粒并表达出特异性 P30抗原,该抗原能被抗弓形虫蛋白血清识别。表达产量在 IPTG 为0.1mM时较高,而培养温度影响不大。结论表达出的具有免疫原性的重组蛋白,经纯化和切割后,可为研究 P30在虫体入侵,诱发机体免疫反应中的作用及疫苗和诊断试剂的开发提供充足的条件。

Aim Based on the p30 cloning vector of our previous study,to constrcut an expression recombinant and to in- duce GST-fused p30 which could be recognized by immune serum with the expression conditions to be opti- mized Methods The p30 cloning vector and expression plasmid both cut by EocRI and Hind Ⅲ were ligated and transformed to DH5α.Screened by PCR and restriction enzymes digestion,the then sequenced PGEX-4T -1-P30 was again transtormed to host cell BL21.Expression induction by the means of time course was fol- lowed by Western blot to detect the desired p30.By measurement of the yield of p30,expression conditions were optimized Results An expression recombinant with correct sequence and reading frame was constructed. The expressed GST-fused p30 has an appropriate molecular weight adn was recognized by anti-Toxoplasma an- tibodies Conclusions The harvested fusion protein,after pruification and digestion by thrombin,can be used for investigation on invasion,immunity,and development of vaccine and diagnostic reagents