细粒棘球绦虫H3蛋白的克隆表达及囊型包虫病检测效果评价  

Cloning,expression and serological evaluation of H3 protein from Echinococcus granulosus

作　　者：朱慧慧[1] 高春花[1] 汪俊云[1] 杨玥涛[1] 石锋[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,科技部国家级热带病国际联合研究中心,世界卫生组织热带病合作中心,上海200025

出　　处：《中国血吸虫病防治杂志》2016年第5期541-544,593,共5页Chinese Journal of Schistosomiasis Control

基　　金：国家重大科技专项(2012ZX10004-220);上海市卫计委科研项目(20134235)

摘　　要：目的克隆、表达细粒棘球绦虫基底膜特异性硫酸乙酰肝素聚糖核心蛋白（H3）,并评价其检测囊型包虫病的效果。方法将从细粒棘球绦虫原头节cDNA文库中免疫筛选的H3基因,克隆入pGEX-4T表达载体,将重组质粒转化大肠杆菌BL21细胞,用异丙基硫代半乳糖苷（IPTG）进行诱导表达,用亲和层析法纯化重组蛋白,用ELISA方法检测囊型包虫病患者血清、其他寄生虫病患者血清及健康人血清,评价其检测效能,并与本课题组制备的细粒棘球蚴包囊液粗抗原（Hydatid cyst fluid,HCF）和重组Ag B8/2进行比较。结果成功构建细粒棘球蚴pGEX-4T-H3重组质粒,并在原核细胞中成功表达。重组H3抗原、纯化HCF和rAgB8/2检测囊型包虫病患者血清的敏感度分别为84.0%（68/81）、90.1%（73/81）、77.8%（63/81）,3者差异无统计学意义（χ2=4.58,P〉0.05）。重组H3抗原与泡型包虫病患者、囊虫病患者及血吸虫病患者血清分别存在63.3%（19/30）、16.7%（5/30）和5.0%（1/20）的交叉反应,与50份健康者血清无交叉反应;H3检测总特异度为80.8%（105/130）,H3、纯化HCF[71.5%（93/130）]和rAgB8/2[82.3%（107/130）]特异度间差异无统计学意义（χ2=5.71,P〉0.05）。结论重组H3抗原在囊型包虫病诊断上具有潜在的应用价值。Objective To clone and express the basement membrane specific heparan sulfate proteoglycan core protein（H3）,and to evaluate its effect in detection of human cystic echinococcosis（CE）. Methods The H3 gene immunoscreened from the cDNA library was cloned into pGEX-4T vector. The recombinant plasmid pGEX-3X-Ag B8/3 was transformed into Escherichia coli BL21 strains and induced by isopropyl-β-D-thiogalactopyranoside（IPTG）. Then the expressed recombinant protein was purified by affinity chromatography and its effect in the detection of CE was evaluated by ELISA. Meanwhile,the effects of H3 and two antigens that the research group prepared before（purified HCF and rAgB8/2）in CE detection were compared.Results The plasmid pGEX-4T-H3 was successfully constructed and H3 was successfully expressed in prokaryotic cells. The sensitivities of the recombinant H3,purified HCF and rAgB8/2 in CE detection were 84.0%（68/81）,90.1%（73/81）and 77.8%（63/81）respectively,and there was no statistical difference among them（ χ~2= 4.58,P 〉0.05）. The cross reactions of recombinant H3 with the sera of the patients with CE,cysticercosis and schistosomiasis were 63.3%（19/30）,16.7%（5/30）and 5.0%（1/20）respectively,and the cross reaction was 0 with the sera of healthy people. The specificities of recombinant H3,purified HCF,and rAgB8/2 were 80.8%（105/130）,71.5%（93/130）and 82.3%（107/130）respectively,and there were no statistical difference among them（χ2= 5.71,P 〉0.05）. Conclusion The recombinant H3 is a potential diagnostic antigen for CE detecting.

关 键 词：细粒棘球绦虫 基底膜特异性硫酸乙酰肝素聚糖核心蛋白(H3) 基因克隆 表达 诊断抗原 酶联免疫吸附试验 敏感度 特异度 交叉反应 

分 类 号：R383.33[医药卫生—医学寄生虫学]