广州管圆线虫组织蛋白酶Z基因的原核表达及免疫学分析

Prokaryotic expression of cathepsin Z gene from Angiostrongylus cantonensis and the immunological analysis of the fusion protein

目的克隆并原核表达广州管圆线虫组织蛋白酶-Z基因,评价其融合蛋白在免疫诊断中的应用前景。方法利用生物信息学分析工具,分析广州管圆线虫组织蛋白酶-Z的理化性质、结构与功能特征;克隆目的基因至原核表达载体pET30a(+),经PCR、双酶切鉴定后,IPTG诱导表达,表达产物通过SDS-PAGE鉴定,融合蛋白用His-镍蛋白纯化柱纯化;ELISA检测融合蛋白作为诊断抗原的敏感性及特异性。结果该蛋白理化性质较稳定,含有分泌型信号肽;含有构成半胱氨酸蛋白酶催化中心的Cys84、His232和Asn253三个氨基酸残基。成功构建了重组质粒且目的基因在E.coliBL21中获得高效表达,经亲和层析获得了纯化的融合蛋白。融合蛋白可被其免疫的BALB/c小鼠血清及感染广州管圆线虫的小鼠血清识别;作为包被抗原用于ELISA检测小鼠血清其敏感性及特异性均为100%与粗抗原无差别,检测其他寄生虫病人血清及正常人血清其特异性分别为100%和97.4%与粗抗原相比特异性较高。结论广州管圆线虫组织蛋白酶-Z与多个物种组织蛋白酶-Z基因同源,是一种半胱氨酸蛋白酶,含有信号肽,可能是重要的虫体分泌排泄抗原成分,在广州管圆线虫病的免疫诊断方面有潜在的应用前景。

In order to clone and express cathepsin Z gene of Angiostrongylus cantonensis（A.cantonensis） to evaluate its prospects in immunodiagnosis,bioinformatics analysis tools were applied to analyze physicochemical properties,structural,and functional characteristics of gene encoded protein.Target gene was cloned into prokaryotic expression vector pET30a（＋） and the recombinant was identified by PCR and double enzyme digestion respectively.When the recombinant was expressed in E.coli BL21 by IPTG induction,the expressed product was identified by SDS-PAGE and the fusion protein was purified by Ni-IDA affinity chromatography.As fusion protein was used as diagnostic antigen,the sensitivity and specificity of that were tested by ELISA.The protein contained a secretory signal peptide with stable physicochemical property and three key amino acids for cysteine proteinase,which contained Cys84,His232 and Asn253 to form the catalytic sites.The recombined plasmid was successfully constructed and expressed in E.coli BL21 effectively.Purified protein was obtained by affinity chromatography and could react with serum of mouse that infected with A.cantonensis or immunized with the fusion protein.ELISA results showed that the sensitivity and specificity were 100% in testing serum of mouse by using fusion protein as the coating antigen,and has no difference compared with crude antigen.But in the test for human serum,its specificity was higher than that of crude antigen.In conclusion,cathepsin Z of A.cantonensis is homologous with cathepsin Z from other species and contained a secretory signal peptide.It is probably a crucial component of excretory-secretory antigen and might be a promising candidate for immunodiagnosis of Angiostrongyliasis.