摘要
目的选取一个华支睾吸虫磷酸甘油酸激酶基因进行克隆表达、纯化,并鉴定该重组蛋白的分子特性及潜在的诊断应用价值。方法根据Genbank中华支睾吸虫磷酸甘油酸激酶基因顺序(L47982)设计引物,从华支睾吸虫成虫cDNA扩增特异性片段,与pTrcHis表达载体连接,重组质粒在大肠埃希菌DH5α中表达,使用亲合层析柱纯化重组蛋白。分别使用SDS-PAGE、Western blot及ELISA鉴定重组蛋白的一般分子特性和抗原活性,以免疫组化染色法探讨该抗原在成虫组织内的分布。结果重组质粒在大肠埃希菌中成功表达,亲合层析获得高纯度的磷酸甘油酸激酶融合蛋白(rCsPGK),分子量约4.7×104。Western blot显示该重组抗原与华支睾吸虫感染阳性血清有较好的结合反应。ELISA结果显示,rCsPGK用于检测特异性抗体在数值上可区分阴阳性血清,受检阳性与混合阴性血清A值之比(P/N)为1.22～1.86,明显低于使用全虫粗抗原的P/N值(4.24～8.21)。免疫组化显示,针对rCsPGK的抗体与成虫体壁的上皮组织、卵黄腺及卵巢内虫卵有明显的染色反应。结论该研究用pTrcHis原核表达系统成功克隆和表达了华支睾吸虫磷酸甘油酸激酶融合蛋白。使用亲合层析能获得高纯度、具有潜在诊断应用价值的rCsPGK抗原,但用于检测特异性抗体的敏感性低于全虫粗抗原。免疫组化表明,华支睾吸虫磷酸甘油酸激酶可能主要分布在成虫体壁的上皮组织、卵黄腺及卵巢内虫卵等组织内。
Objective To choose a Clonorchis sinensis phosphoglycerate kinase （CsPGK） gene for molecular expression and protein purification, and identify the molecular characters of the recombinant protein and its potential applied value to immunodiagnosis. Methods Primers were designed based on the CsPGK gene sequence in Genbank （ L 47982 ） , and the specific fragment amplified from Clonorchis sinensis （ C. sinensis ） adultworm cDNA were ligated with pTrcHis expression vector. The recombinant plasmid was expressed in E. coli DH5α and the fusion protein was purified by affinity chromatography. The general molecular characters and antigen activity of the recombinant protein were identified by SDS - PAGE, Western blot and ELISA, respectively. The distribution of this antigen in aduhworm tissues was explored by immunohistochemistry. Results The recombinant plasmid was successfully expressed in E. coli and the rCsPGK with high purity and a molecular weight of 4.7 × 10^4 was acquired by affinity chromatography. Western blot analysis showed that the recombinant antigen had a good reaction with the positive sera from C. sinensis infected persons. ELISA results showed that rCsPGK, used to detect the specific antibody,could differentiate the positive and negative sera. The A value ratios of the positive sera and the pool negative serum （P/N） detected with the recombinant antigen were 1.22 - 1.86, significantly lower than the P/ N values with the crude aduhworm antigen （4.24 -8.21 ）. In immunohistochemistry, the body wall epithelium, vitelline gland and intrauterine eggs of Clonorchis sinensis adultworm reacted obviously with the anti - rCsPGK sera. Conclusion In this study, a Clonorchis sinensis phosphoglycerate kinase fusion protein was cloned and expressed successfully by pTrcHis prokaryotic expression system. By affinity chroma- tography, the recombinant antigen with high purity and potential applied value to diagnosis can be acquired, but the sensitivity of rCsPGK for the detection of specific antibody lower than the crude aduhworm antigen. Immunohistochemistry suggests that Clonorchis sinensis phosphoglycerate kinase may distribute mainly in the body wall epithelium, vitelline gland and intrauterine eggs of Clonorchis sinensis adultworm.
出处
《华南预防医学》
2008年第6期20-24,共5页
South China Journal of Preventive Medicine
基金
广东省医学科学研究基金项目(No.A2005081)
广东省"十一五"医学重点专科(食品安全监测检测)项目
日中笹川医学研究者制度的部分资助
关键词
华支睾吸虫
磷酸甘油酸激酶
基因表达
Clonorchis sinensis
Phosphoglycerate kinase
Gene expression
作者简介
裴福全（1969-），男，硕士，副主任医师，主要从事人体寄生虫病防治与研究工作
