华支睾吸虫钙结合蛋白Cs16的重组表达及血清诊断应用的初步评价

Recombinant expression of calcium-binding protein from Clonorchis sinensis and preliminary evaluation of its application in immunodiagnosis

目的以原核和真核表达系统克隆、表达华支睾吸虫含有EF手型结构域的钙结合蛋白(Cs16),纯化并初步评价其在华支睾吸虫病免疫诊断中的作用。方法以华支睾吸虫成虫c DNA为模板,PCR扩增含有EF手型结构域的Cs16基因,分别构建原核重组质粒p GEX-4T-1-Cs16和真核重组质粒p PIC9K-Cs16,将酶切和测序鉴定正确的重组质粒分别转入大肠埃希菌BL21和毕赤酵母GS115后,表达并纯化目的蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)验证其纯度及免疫原性。以华支睾吸虫重组蛋白Cs16为包被抗原,采用ELISA检测24例华支睾吸虫病患者血清中相应抗体的反应性及其与日本血吸虫病患者血清的交叉反应性。结果PCR扩增获得Cs16基因片段,大小为515 bp。构建的重组质粒p GEX-4T-1-Cs16和p PIC9K-Cs16经酶切和测序鉴定正确。SDS-PAGE结果显示,2个重组质粒在大肠埃希菌BL21和毕赤酵母GS115中均为可溶性表达,重组蛋白Cs16相对分子质量约为16 000。Western blotting结果显示,2个重组蛋白能分别被抗GST抗体和抗His抗体特异性识别。ELISA检测结果显示,原核表达和真核表达重组蛋白Cs16的敏感性分别为70.8%(17/24)和54.2%(13/24),特异性分别为95.2%(20/21)和100%(21/21),差异均有统计学意义(P<0.05)。与日本血吸虫病患者血清的交叉反应为1/10。结论获得了原核和真核Cs16重组抗原,其在华支睾吸虫病的诊断上具有潜在的应用价值。

Objective To clone, express the calcium-binding EF-hand-domain-containing protein of Clonorchis sinensis（Cs16） by using the prokaryotic and eukaryotic expression systems, and evaluate its application in immunodiagnosis. Methods The Cs16 gene was amplified by PCR using specific primers and c DNA library of Clonorchis sinensis, and sub-cloned into prokaryotic and eukaryotic expression vectors p GEX-4 T-1 and p PIC9 K,respectively. The recombinant vectors p GEX-4 T-1-Cs16 and p PIC9 K-Cs16 were transformed into Escherichia coli BL21 and yeast GS115 for protein expression. The recombinant proteins were purified and analyzed for purity and antigenicity by SDS-PAGE and Western blotting, respectively. The purified recombinant protein was used as the coating antigen for indirect ELISA to evaluate its diagnostic performance against serum samples from 24 clonorchiosis patients and its cross-reactivity to serum samples from schistosomiasis patients. Results PCR resulted in a 515 bp of Cs16 fragment. The recombinant plasmid p GEX-4 t-1-Cs16 and p PIC9 K-Cs16 were verified by enzymatic digestion and sequencing. SDS-PAGE analysis showed that the two recombinant plasmids were solubly expressed in E. coli BL21 and yeast GS115, producing recombinant protein Cs16 with Mrof 16 000. Western blotting results showed that the recombinant proteins were specifically recognized by anti-GST and anti-His. ELISA showed that the sensitivity of recombinant Cs16 proteins expressed in the prokaryotic and eukaryotic expression systems was 70.8%（17/24） and 54.2%（13/24）, respectively, and the specificity was 95.2%（20/21） and 100%（21/21）, respectively, with significant differences between the two systems（ P〈0.05）. The cross-reactivity with sera from schistosomiasis patients was 1/10. Conclusion The recombinant Cs16 can be expressed prokaryotically and eukaryotically, and has potential applications in immunodiagnosis of clonorchiasis.