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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。
OBJECTIVE: To clone and prokaryotic express the cathepsin-Z gene of A. cantonensis and evaluate the potential application of the fusion protein in immunodiagnosis. Methods Bioinformatics analysis tools were used to analyze the physicochemical properties, structure and function of cathepsin-Z in C. elegans. The target gene was cloned into prokaryotic expression vector pET30a (+) and identified by PCR and double enzyme digestion. IPTG induced expression The expressed product was identified by SDS-PAGE and the fusion protein was purified by His-Nickel purification column. The sensitivity and specificity of the fusion protein as a diagnostic antigen were determined by ELISA. Results The protein was more stable in physiochemical properties and contained secretory signal peptide. The protein contained three amino acid residues, Cys84, His232 and Asn253, which formed the catalytic center of cysteine protease. The recombinant plasmid was successfully constructed and the target gene was highly expressed in E. coli BL21. The purified fusion protein was obtained by affinity chromatography. The fusion protein could be recognized by the serum of BALB / c mice and the serum of mice infected with A. cantonensis. The sensitivity and specificity of the fusion protein as a coating antigen for detecting the serum of mice were all 100% The specificity was 100% and 97.4%, respectively, for the detection of sera from other parasite patients and normal human sera, which were more specific than crude antigens. Conclusion The angiostrongyliasis cathepsin-Z is homologous to cathepsin-Z in many species. It is a cysteine protease, which contains signal peptide and may be an important secretion and excretion antigen component of parasites. There are potential applications in the diagnosis of the disease.