广州管圆线虫雌性成虫肌蛋白-1基因的克隆表达及免疫学效应分析

目的 对广州管圆线虫雌性成虫肌蛋白-1(Ac-fmp-1)基因的开放读码框进行克隆、表达及重组蛋白的免疫学效应分析,评价其重组蛋白在免疫学诊断中的应用前景。方法 逆转录聚合酶链反应扩增目的基因,克隆至原核表达载体pET-30a-c(+),重组质粒转化至大肠埃希菌E.coli BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,镍离子亲和层析(Ni-IDA)纯化分离表达产物。免疫印记(Western-blot)和酶联免疫吸附试验(ELISA)分析重组蛋白的免疫性。结果 广州管圆线虫Ac-fmp-1基因的编码区有1 251个碱基,编码417个氨基酸。原核表达载体pET-Ac-fmp-1构建成功,IPTG诱导表达获得了体外高效表达的重组融合蛋白,经亲和层析得到纯化蛋白。融合蛋白可被感染广州管圆线虫的SD大鼠血清及病人血清所识别;作为包被抗原用于ELISA检测大鼠血清其敏感性与特异性均为100%,与粗抗原无差别;检测广州管圆线虫病人血清敏感性为100%,与粗抗原有差别;检测其他寄生虫病人血清与正常病人血清其特异性为100%和98%,与粗抗原相比特异性较高。结论 广州管圆线虫Ac-fmp-1基因在原核表达系统获得高效表达,编码蛋白可能是虫体的重要抗原成分,在免疫学诊断方面有潜在的应用前景。

Cloning,expression and immunological efficacy analysis of female muscle protein-1 from Angiostrongylus cantonensis

The purpose of this study is to clone and express the opening reading frame （ORF） of female muscle protein- 1 （Ac-fmp-1） gene from Angiostrongylus cantonensis, and analyze the immunological efficacy of the recombinant protein for evaluation of its prospects in immunodiagnosis. The ORF of Ac-fmp-1 gene was amplified by RT-PCR. The PCR product was then cloned into the prokaryotic expression vector pET-30a-c（＋） and the recombinant plasmid pET-Ac-fmp-1 was transformed into E. coli BL21（DE3） induced by IPTG. After that, the protein was purified by Ni-IDA affinity chromatography, and then identified by SDS-PAGE. The immunoreactivity of purified protein was analyzed by Western-blotting and ELISA. Results showed that the coding sequence of Ac-fmp-1 gene contained 1 251 base pairs coding 417 amino acids. The recombinant plasmid pET-Ac-fmp-1 was constructed successfully and expressed in E. coli BL21 efficiently by IPTG inducing. Purified protein was obtained by using affinity chromatography and could be recognized by the serum of SD rats and patients infected with A. cantonensis by Western-blotting. ELISA results showed that the sensitivity and specificity were 100M in testing SD rats serum by using fusion protein as coating antigen, and had no difference compared with crude antigen. But in the test for human serum, the sensitivity and specificity were higher than that of crude antigen. In conclusion, the Ac-fmp-1 gene has been efficiently expressed in prokaryotic expression in vitro. The protein is probably an important component of antigen, and might be a promising candidate for immunologic diagnosis of Angiostrongyliasis.