Study in molecular biology of Anisakis antigen

利用重组DNA技术对单纯异尖线虫幼虫抗原多肽基因进行了研究。将从来自单纯形线虫幼虫的富含poly（A）的mRNA合成的cDNA连接到噬菌体载体λ gtll DNA中并在体外包装。在大肠杆菌中增殖λ gtll，并构建λ gtll表达文库。通过免疫筛选文库选择编码42 kDa抗原多肽的cDNA克隆，并通过表位选择方法鉴定。分离了含有42 kDa蛋白质的cDNA的克隆。编码该42 kDa抗原性多肽的基因通过使用cDNA作为探针的DNA和RNA印迹分析来表征。该基因被转录为约1400个核苷酸的mRNA，并翻译为42 kDa的多肽。细菌合成的抗原性β-半乳糖苷酶融合蛋白与其它寄生虫感染血清无交叉反应。利用重组DNA技术获得的抗原性β-半乳糖苷酶-cDNA融合蛋白（FP）的酶联免疫吸附试验（ELISA）为异尖孢菌素提供了有用的诊断工具。异尖线虫感染的人血清与用抗β-半乳糖苷酶单克隆抗体固定在微孔板上的FP反应强烈。然而，FP不与其他蠕虫病患者的血清反应。与体细胞粗抗原相比，FP法ELISA检测异尖线虫IgG抗体具有较高的敏感性和特异性。

The gene encoding an antigenic polypeptide of Anisakis simplex larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich MRNA from A simplex larvae was ligated into phage vector lambda gtll DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gtll expression library was constructed. A cDNA clone encoding a 42 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing CDNA for a 42 kDa protein was isolated. The gene encoding this 42 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the CDNA as a probe. The gene was transcribed to MRNA with approximately 1400 nucleotides and translated to 42 kDa polypeptide. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera. An enzyme-linked immunosorbent assay (ELISA) using antigenic beta-galactosidase-cDNA fusion protein (FP) obtained by the recombinant DNA technique provided a useful diagnostic tool for anisakiasis. Anisakis-infected humans sera reacted strongly with FP that was immobilized with anti- beta-galactosidase monoclonal antibody on microplates. However, the FP did not react with sera from patients with other helminthiasis. In detection of anti-Anisakis IgG antibody, ELISA using FP was highly sensitive and specific compared to that using crude somatic antigen.