Study in molecular biology of Anisakis antigen

用重组DNA技术研究了单纯异尖线虫幼虫抗原多肽基因。从单纯形虫幼虫中合成富含PolyA(A)的mRNA，将其连接到噬菌体载体lambda gtll DNA中，体外包装。在大肠杆菌上扩增噬菌体，构建了lambda gtll表达文库。通过对文库的免疫筛选，获得了编码42 kDa抗原多肽的cDNA克隆，并用表位选择法进行了鉴定。分离到一个含42 kDa蛋白CDNA的克隆。以CDNA为探针，对该42 kDa抗原性多肽的编码基因进行了DNA和RNA印迹分析。该基因转录成约1400个核苷酸的信使核糖核酸，翻译成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他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-c DNA融合蛋白(FP)的酶联免疫吸附试验(EL ISA)为异尖线虫病的诊断提供了一种有用的工具。异尖线虫感染者血清与用抗β-半乳糖苷酶单抗固定在微板上的FP发生强烈反应。然而，FP与其他蠕虫病患者的血清不发生反应。在抗异尖线虫抗体检测中，与粗制体细胞抗原法相比，FP法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.