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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。
Objective To screen the cDNA expression library of adult Clonorchis sinensis by immunological method and search for new antigenic genes. Methods The sera of clonorchis sinensis mixed serum and Clonorchis sinensis were used to immunopurify the cDNA library of λZAP cDNA of Clonorchis sinensis, and the positive clones were cloned, sequenced and compared with nucleotide sequences The coding region of the target gene was cloned into the prokaryotic expression plasmid pET28a (+). The fusion protein was expressed by the method of preparing native protein without the N-terminal fusion tag. The recombinant protein was purified by using histidine tag affinity purification column (Ni-NTA resin ). The specific antibodies in serum were detected by indirect ELISA. Serum samples from 35 cases of Clonorchis sinensis ovariectomized sera, 36 cases of normal human serum, 15 cases of Schistosoma japonicum and 15 cases of Wechsler Flukes and 13 cases of Cysticercus cellulosae patients serum, evaluate the immunological diagnostic value of recombinant protein. The results showed that the glycine 2a (GRA2a) antigen gene family of Clonorchis sinensis was successfully cloned, and the Cs4 antigen gene was inserted into the NcoI site of the recombinant expression plasmid pET28a (+). The fusion protein was successfully expressed and further purified Soluble recombinant protein. The indirect ELISA method was used to detect the specific antibodies in serum. The sensitivity and specificity of this ELISA method were 80.0%, 97.2% and 88.7%, respectively. The serum levels of Schistosoma japonicum, Paragonimus westermani and cysticercus cellulosae False positive rates were 6.7%, 6.7% and 7.7% respectively. The comparison of nucleotide sequence homology, Clonorchis sinensis GRA2a antigen is so far no functional study of Clonorchis sinensis specific antigen. Conclusion The gene family of GRA2a antigen of Clonorchis sinensis was found. Recombinant plasmids were successfully constructed and recombinant proteins were expressed and purified. The antigen has high immunological diagnostic value.