我国细粒棘球绦虫新疆分离株AgB8/3亚基克隆表达及其检测囊型包虫病价值分析

摘要

目的 评价重组细粒棘球蚴AgB8/3亚基检测囊型包虫病价值.方法 从新疆源细粒棘球蚴原头节中提取总RNA,通过RT-PCR扩增目的基因片段并克隆入pGEX-3X表达载体,重组质粒转化大肠杆菌BL21细胞,IPTG诱导表达,用亲和层析法纯化重组蛋白,用ELISA方法评价其诊断价值并与本课题组制备的重组AgB8/1和AgB8/2进行比较.结果 酶切鉴定及测序分析表明,成功构建细粒棘球蚴pGEX-3X-AgB8/3重组质粒,并在原核细胞用IPTG成功诱导目的蛋白表达.rAgB8/3检测囊型包虫病患者血清的敏感度为55.9％(66/118)；检测泡型包虫病患者血清阳性率为44.12％(15/34)；59份其它寄生虫病患者血清中除3份囊尾蚴病人血清阳性外,其他均为阴性；50份健康者血清全部为阴性,总特异度为87.4％(125/143).rAgB8/3检测的敏感度与rAgB8/1相当(P＞0.05)而低于rAgB8/2 (P＜0.05)；rAgB8/3检测的特异度与rAgB8/1和rAgB8/2均无统计学差异(P＞0.05).结论 成功构建新疆源细粒棘球蚴pGEX-3X-AgB8/3重组质粒并在原核细胞表达,rAgB8/3检测囊型包虫病结果与rAgB8/1无统计学差异.%In the present study, total RNA was extracted from E. granulosus protoscoleces collected from a sheep hyda-tid cyst from Xinjiang, and AgB8/3 fragments were obtained by RT-PCR using the extrcted RNA and cloned into pGEX-3X vector. The recombinant plasmid pGEX-3X- AgB8/3was transformed into Escherichia coli BL21 strains and induced by isopro-pyl-(3-D- thiogalactopyranoside (IPTG). The expressed recombinant protein was purified by affinity chromatography and was e-valuated for the diagnosis of CE by ELISA compared with rAgB8/l and rAgB8/2. The results of restriction enzymes digestion and sequencing analysis showed that plasmid pGEX~3X-AgB8/3 was successfully constructed and rAgB8/3 was successfully expressed in prokaryotic cell induced by IPTG. Detection on 118 serum samples of patients with cyst echinococcosis showed a sensitivity of 55. 9% (66/118) , while 15 positives out of 34 alveolar echinococcosis, 3 positives out of 59 patients with other parasitic disease and none of 60 healthy persons with a specificity of 87. 4% (125/143). There was no statistical difference in the sensitivity between rAgB8/3 and rAgB8/l(P>0. 05), but sensitivity of rAgB8/2 was higher than that of rAgB8/3(P<0. 05). There was no statistical difference in the specificity among the three recombinant subunits (P>0. 05).