细粒棘球绦虫转酮醇酶克隆表达及免疫性分析  

Cloning,expression and immunity analysis of transketolase of Echinococcus granulosus

作　　者：曹得萍[1,2] 张耀刚 李超群 刘佳[1] 姜博璠 CAO De-ping;ZHANG Yao-gang;LI Chao-qun;LIU jia;JIANG Bo-fan(Department of Pathogenic Biology, Medical College, Qinghai University, Xining 810001, China;Affiliated Hospital of Qinghai University, Qinghai Province Key Laboratory for Echinococcosis, China)

机构地区：[1]青海大学医学院病原生物学教研室,西宁810001 [2]青海大学附属医院、青海省包虫病研究重点实验室

出　　处：《中国血吸虫病防治杂志》2018年第2期155-160,共6页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金(81360255);青海省科技厅重大专项(2016-SF-A5)

摘　　要：目的通过原核表达获得细粒棘球绦虫转酮醇酶(TK)表达产物,分析其作为棘球蚴病诊断抗原分子的价值。方法 PCR扩增TK基因,克隆至原核载体p MD19-Eg TK,随后亚克隆至表达载体p ET-28a,构建目的基因TK原核表达质粒p ET-28a(+)-Eg TK,转入大肠埃希菌BL21,分离纯化蛋白,经SDS-PAGE、Western blotting鉴定后,收集细粒棘球蚴病(CE组)、多房棘球蚴病(AE组)患者及健康人(健康组)血清,以酶联免疫吸附试验(ELISA)检测重组蛋白作为诊断抗原的价值。结果成功构建p ET-28a(+)-Eg TK质粒,经SDS-PAGE和Western blotting分析,Eg TK蛋白在70 k Da处出现条带,与理论值一致。ELISA显示,CE组、AE组和健康组血清反应吸光度A450值间差异有统计学意义(F=44.47,P<0.01),CE组(1.46±0.41)、AE组A450值(1.28±0.29)高于健康组(0.66±0.23)(P<0.05),但CE组和AE组间A450值差异无统计学意义(P>0.05)。结论重组TK分子可较好地区分棘球蚴病患者和非棘球蚴病患者,但无法区分细粒棘球蚴病和多房棘球蚴病患者。Objective To obtain the prokaryotic expression of transketolase genes and analyze its value as a diagnostic antigen for echinococcosis.Methods TK gene was amplified by PCR and cloned into prokaryotic vector p MD19-Eg TK,and then subcloned into the expression vector p ET-28 a. The target gene TK prokaryotic expression plasmid p ET-28 a was constructed and transferred into BL21. The purified protein was identified by SDS-PAGE and Western blotting. The blood samples of patients with cystic echinococcosis（CE group）,alveolar echinococcosis（AE group）and healthy people（healthy group）were collected and detected by ELISA with the recombinant Eg TK protein as a diagnostic antigen.Results The recombinant plasmid p ET-28 a（＋）-Eg TK was constructed successfully,and there was a band around 70 k Da by using Western blotting. ELISA showed that the difference among the 3 groups of sera reaction A450 was significantly different（F = 44.47,P 〈0. 01）,and the A450 values of the CE group（1.46±0.41）and AE group（1.28±0.29）were higher than that of the healthy group（0.66 ± 0.23）,but there was no significant difference between the former two.Conclusion The recombinant Eg TK protein is better to distinguish the echinococcosis group and healthy group,but it can＇t do a differential diagnosis between CE and AE cases.

关 键 词：细粒棘球绦虫 棘球蚴病 转酮醇酶 重组质粒 诊断抗原 

分 类 号：R383.33[医药卫生—医学寄生虫学]