斯氏狸殖吸虫成虫半胱氨酸蛋白酶基因片段的表达及鉴定

Expression and identification of cysteine proteinase from adult worm of Pagumogonimus skrjabini in Escherichia coli

目的 获得表达斯氏狸殖吸虫半胱蛋白酶基因片段编码多肽 ,对其免疫反应性作初步研究 ,在吸虫所致疾病的血清学诊断的应用奠定基础。方法 用PCR法扩增目的基因片段 ,1%琼脂糖凝胶回收纯化后与PinPointT载体相连 ,并导入到大肠杆菌JM10 9菌株中去 ,诱导表达其编码的多肽片段。用裂解液制备抗原标本 ,经SDS PAGE电泳后 ,经考马斯亮蓝和生物素链亲合素碱性磷酸酶染色检测其表达情况 ,Westernblotting鉴定其免疫反应性。结果 转化实验共获得8株阳性重组子 ,经测序鉴定证实仅 1株正向连接。经过诱导表达 ,制备重组抗原标本 ,经SDS PAGE、转膜后生物素 链亲和素 碱性磷酸酶系统染色显示在 3 2× 10 3处有一融合蛋白条带 ,反向连接菌株与之相似 ,也出现一条表达带 ,但分子量远小于预期值 ,仅仅 14× 10 3左右。Western blotting显示仅正向连接菌株在 3 2× 10 3的位置有一阳性染色条带。结论 成功的构建了斯氏狸殖吸虫半胱氨酸蛋白酶的原核表达载体 ,经过诱导后表达的重组抗原 。

Objective To express the recombinant antigen coded by Pagumogonimus skrjabini adult cysteine protease gene fragment and to investigate the immunoreactivity of the fusion protein for further usage on serodiagnosis of the fluke disease. Methods The target gene fragment was amplified by PCR. After purification with gel purification recoverykit, the target gene fragment was ligated with PinPointTM Xa 1 T vector and transduced into E.coli JM109 strain. The expressed fusion protein sample was prepared with alkaline lysis solution, and then analyzed by SDS PAGE. The expression level was determined by Coomassie blue staining and the streptavidin alkaline phosphatase staining. The immunoreactivity of fusion protein was examined by Western blotting. Results A total of 8 positive clones were harvested, and only one had the proper orientation verified by sequencing. The recombinant antigen was obtained after being induced with IPTG (Isopropltio β D galactoside), and a positive band of 32×10 3 was found by streptavidin alkaline phosphatase staining. In the same position, the fusion protein was also detected by Western blotting. Conclusion The expression vector of adult cysteine protease gene PinPointTM Xa 1 T vector was successfully constructed. The recombinant antigen obtained after being induced with IPTG possesses good immunoreactivity.