日本血吸虫腺苷酸激酶基因的表达及其重组蛋白免疫反应性的评价

目的　将亚克隆入 pET3 2a( +)质粒的日本血吸虫腺苷酸激酶 (adenylatekinase ,AK )基因进行表达及蛋白纯化 ,并对表达产物的免疫反应性进行评价。　方法　将重组表达质粒 pET3 2a( +) AK转入宿主菌大肠埃希菌BL2 1,异丙基 β D 硫代半乳糖苷 (IPTG)诱导表达后超声裂菌 ,离心 ,取上清进行十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE) ;将SDS PAGE后的蛋白转移至聚偏二氟乙烯 (PVDF)膜上 ,分别用 6 组氨酸 ( 6 His)抗体、日本血吸虫尾蚴感染6wk的兔血清及紫外照射减毒尾蚴免疫的兔血清为一抗进行蛋白质印迹试验 (Westernblotting) ;用金属Ni螯合物亲和层析树脂 (Ni NTA)层析柱纯化目标蛋白 ;以纯化后的融合蛋白为包被抗原 ,酶联免疫吸附测定 (ELISA)检测正常兔血清和血吸虫感染兔血清。　结果　重组菌用IPTG诱导表达后进行SDS PAGE ,于相对分子质量 (Mr) 40 0 0 0处见一特异表达带 ,与预期表达的融合蛋白大小相符 ;Western印迹分析显示该重组的融合蛋白带有预期的 6 His基团 ,且能与尾蚴感染兔血清及紫外照射减毒尾蚴免疫的兔血清发生特异性反应 ;ELISA结果显示纯化的重组AK蛋白可以特异识别血吸虫感染兔血清。　结论　AK基因在工程菌中以可溶性融合蛋白的形式得到表达 ,该重组蛋白与血吸虫感染兔血清及紫外照射减毒尾蚴免疫的兔血清具有特异的免疫反应性。

Expression of Adenylate Kinase of Schistosoma japonicum and Evaluation on the Immunoreactivity of the Recombinant Protein

Objective To express and purify the Schistosoma japonicum adenylate kinase(AK) protein of which the cDNA sequence was subcloned into the pET32a( ) soluble expression plasmid, and to evaluate its immunoreactivity. MethodsThe recombinant plasmid was transformed into {E.coli} BL21, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analyzed by SDS_PAGE after boiling and centrifugation. The target protein was purified with the Ni_NTA agarose. The proteins on the gel were transferred to the PVDF film and then the immunoreactivity was tested by Western blotting using anti_6_His antibody, sera from rabbits 6 weeks after infected with cercariae or UV_attenuated cercariae. The purified protein was coated for ELISA to test the cercariae infected rabbit serum and the normal rabbit serum. Results The molecular weight of the target fusion protein was {Mr 40 000} after being induced with IPTG. The fused protein showed a single band when reacted with anti_6_His antibody, the cercariae infected rabbit serum and attenuated cercariae infected rabbit serum. Conclusion The AK protein is expressed as a fused protein with thioredoxin and its molecular weight is about {Mr 40 000}. This protein has a positive immunoreactivity with sera of rabbits infected with cercariae and UV_attenuated cercariae.