华支睾吸虫3个半胱氨酸蛋白酶N端短片段原核表达及其抗原性分析

Prokaryotic expression and antigenicity analysis of three N-terminal short fragments of cysteine proteases from Clonorchis sinensis

目的通过克隆和表达获得华支睾吸虫3个半胱氨酸蛋白酶N端短片段重组蛋白并分析其抗原性。方法以pET-28a-CsCP1-3重组质粒为模板进行目的片段CsCP1a-3a PCR扩增,构建pET28a-CsCP1a-3a表达载体,并转化至BL21(DE3)大肠埃希菌,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物进行SDS-PAGE分析;表达产物经变性、纯化及复性,与华支睾吸虫感染大鼠血清行Western blot分析。结果成功构建pET28a-CsCP1a-3a重组质粒;SDS-PAGE分析显示,重组蛋白CsCP1a-3a以包涵体形式表达;重组蛋白CsCP1a-3a可被华支睾吸虫感染后第4、5周大鼠血清识别。结论重组蛋白CsCP1a-3a具有较好抗原性,可作为华支睾吸虫感染诊断候选分子。

Objective Clone and express three short N-terminal fragments of cysteine proteases from Clonorchis sinensis and analyze their antigenicity.Methods The CsCP1a-3a genes were amplified by PCR using pET-28a-CsCP1-3 plasmid as template.The target gene fragments were ligated into the prokaryotic expression vector pET-28a by Double enzyme digestion and ligation to construct recombinant plasmids pET-28a-CsCP1a-3a.After identification by double enzyme digestion and sequencing,the recombinant plasmids were transformed into competent cell BL21(DE3)for expression induction and the expressed products were obtained by IPTG induction.The expressed products were purified using a Ni-NTA column under denatured conditions and refolded using the gradient dialysis method,characterized by SDSPAGE and analyzed with C.sinensis infected rat serum by Western blot.Results The molecular weight of the target fragments obtained by double enzyme digestion were the same as that amplified by PCR using pET-28a-CsCP1-3 plasmid as template,and consistent with the theoretical molecular weight of the target genes.E.coli BL21-pET-28a-CsCP1a-3a were induced by IPTG,and the recombinant proteins were expressed in the form of inclusion bodies.After denaturation purification and renunciation,0.7-0.8 mg/mL pure soluble protein were obtained.Western blot showed that the recombinant proteins CsCP1a-3a could not recognize the negative serum,but could recognize the positive serum.The earliest positive band was detected at the 4th week of infection,which was consistent with the detection of eggs.Conclusion The pET28a-CsCP1a-3a recombinant plasmids were successfully constructed and pure soluble recombinant proteins were obtained.The recombinant proteins show good antigenicity and can be used as candidate molecules for diagnosis of C.sinensis infection.