华支睾吸虫特异性富甘氨酸2a类抗原基因的克隆、表达和免疫学诊断价值评价

Cloning and expression of the Clonorchis sinensis-specific glycine rich antigen 2a genes and its immunodiagnosis value

目的采取免疫学方法筛选华支睾吸虫成虫cDNA表达文库,寻找新的抗原基因。方法使用华支睾吸虫病人混合血清以及华支睾吸虫排泄分泌抗原的免疫小鼠血清,分别免疫学筛选华支睾吸虫成虫λZAP cDNA表达文库;将阳性噬菌体克隆、测序以及核苷酸序列比对分析;将目的基因的编码区克隆至原核表达质粒pET28a(+)中,采用制备不带N端融合标签的天然蛋白的策略表达融合蛋白,使用组氨酸标签亲和纯化柱(Ni-NTA树脂)纯化融合蛋白;采用间接ELISA法,检测血清中的特异性抗体,共检测35例华支睾吸虫虫卵粪检阳性血清,36例正常人血清,15例日本血吸虫、15例卫氏并殖吸虫和13例猪囊尾蚴病人血清,评价重组蛋白的免疫学诊断价值。结果发现华支睾吸虫特异性富甘氨酸2a(GRA2a)类抗原基因家族,将其中的Cs4抗原基因植入重组表达质粒pET28a(+)的NcoI位点,成功实现融合蛋白的表达,并进一步纯化得到可溶性重组蛋白。采用间接ELISA法检测血清中的特异性抗体,该ELISA法的敏感性为80.0%,特异性为97.2%,总符合率为88.7%;日本血吸虫、卫氏并殖吸虫和猪囊尾蚴病人血清的假阳性率分别为6.7%、6.7%和7.7%。经核苷酸序列同源性比较,华支睾吸虫GRA2a类抗原是迄今为止尚未进行功能研究的华支睾吸虫特异性抗原。结论发现华支睾吸虫GRA2a类抗原基因家族;成功构建重组表达质粒,并表达、纯化出可溶性重组蛋白;该抗原具有较高的免疫学诊断价值。

To research and identify new specific antigen gene of Clonorchis sinensis,a λ ZAP cDNA library of adult Clonorchis sinensis was immunoscreened with pooled sera of clonorchiasis cases or sera of mice immunized with excretory-secretory antigen of C.sinensis.The positive clones obtained were sequenced and analyzed.The coding sequence of the target gene was subcloned into prokaryotic plasmid pET28a（＋） with a non-fusion expression strategy.The recombinant protein was purified by His-bind affinity column（Ni-NTA） and its immunodiagnosis value was evaluated by indirect ELISA to detect the level of specific antibodies in the sera,including 35 samples of human clonorchiasis sera whose egg were positive by stool examination,36 healthy control sera samples,15 sera samples from patients infected with Schistosoma japonicum,15 sera samples from Paragonimus westermani and 13 sera samples from Taenia solium patients.The sensitivity,specificity and total coincidence rate of ELISA were 80.0%,97.2% and 88.7%,respectively.And false positive rates for sera samples from S.japonicum,P.westermani and T.solium patients were 6.7%,6.7% and 7.7%,respectively.Sequence analysis revealed that these GRA2a antigens were C.Sinensis-specific and their functions have not been identified in literatures.In this study,the GRA2a gene family of C.sinensis was identified.The expression plasmid pET28a-Cs4DSPM was constructed and soluble recombinant protein of pET28a-Cs4DSPM was purified successfully and it indicated high diagnostic value for clinical detection.