华支睾吸虫成虫转录辅激活因子基因在原核细胞中的克隆、表达及其生物功能分析

Cloning and Prokaryotic Expression of Transcriptional Co-activator Gene of Clonorchis sinensis and Functional Analysis of the Expressed Protein

目的 构建华支睾吸虫成虫转录辅激活因子 (transcriptionalcoactivator ,TC)基因 (TC基因 )原核重组质粒 ,进行原核表达、鉴定及生物功能分析。 方法 根据TC基因已知序列设计 1对引物 ,从华支睾吸虫成虫cDNA文库中扩增TC基因片段 ;将目的基因进行聚合酶链反应 (PCR) ,其产物和空质粒 pGEX 4T 1、pET3 0a (+ )同时用限制性内切酶BamHⅠ、SalⅠ双酶切 ,纯化回收后连接并转化大肠埃希菌 (E .coliBL2 1)。将构建的重组质粒pGEX 4T 1 TC和 pET3 0a (+ ) TC分别经双酶切、PCR及测序鉴定后在大肠埃希菌BL2 1中诱导表达。用十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和蛋白质印迹法 (Westernblotting)鉴定其表达效果 ,用亲和层析法纯化重组质粒pET3 0a (+ ) TC表达产生的组氨酸重组蛋白 (His TC)。 结果 构建了TC基因原核重组质粒pGEX 4T 1 TC和 pET3 0a (+ ) TC。SDS PAGE结果表明TC基因在大肠埃希菌BL2 1系统获得高效表达 ,其重组蛋白分子量与理论值相符。Westernblotting结果表明重组质粒 pGEX 4T 1 TC表达的谷胱甘肽硫转移酶 (GST)重组蛋白(GST TC)可被华支睾吸虫免疫兔血清识别 ,具有免疫反应性。纯化的TC基因的组氨酸 (His)重组蛋白 (His TC)经SDS PAGE显示单一条带。

Objective To construct prokaryotic recombinant plasmids of transcriptional co-activator (TC) gene of Clonorchis sinensis, express and purify the recombinant protein and analyze its biological function. Methods A pair of primers was designed according to the known sequence of TC gene. The TC gene fragment was amplified by PCR. After purification and digestion with BamHⅠ and SalⅠ , the TC gene was connected to the prokaryotic expression vectors, pGEX-4T-1 and pET30a(+). By cloning target gene into these vectors, pGEX-4T-1 and pET30a(+), prokaryotic recombinant plasmids of TC gene were constructed and transferred into E.coli BL21. The positive expressed recombinants were detected by SDS-PAGE and Western blotting. Immobilized metal (Ni 2+ ) chelation affinity chromatography was used to purify His-TC produced by the expression of the recombinant protein pET30a(+)-TC. Results The recombinant plasmids, pGEX-4T-1-TC and pET30a(+)-TC, were constructed successfully. SDS-PAGE testified that the molecular weight of the recombinant protein was correct. Western blot analysis of GST-TC recombinant protein testified that the recombinant protein could be recognized by immunized rabbit serum, which means the protein is GST-immune active and the clone can express recombinant Clonorchis sinensis antigen. After affinity chromatography of the pET-TC protein, there was only one protein band with expected size on the SDS-PAGE gel. Conclusion The TC gene was screened from cDNA library of adult Clonorchis sinensis, cloned, expressed and purified. The purified protein of TC gene will be of importance for further research on the biological function of the gene.