肠道病毒71型血清学检测方法的建立及应用  

Establishment and application of serological detection method for Enterovirus Type 71

作　　者：马洪滨[1] 侯俊[1] 李鲁平[2] 杨静[1] 郭静霞[1] 刘爱霞[1] 徐军[1] 聂为民[1] 李伯安[1] 毛远丽[1] 

机构地区：[1]解放军302医院,北京100039 [2]沈阳沈阳市第六人民医院,110006

出　　处：《解放军医学杂志》2010年第3期239-240,共2页Medical Journal of Chinese People's Liberation Army

基　　金：军队“十一五”科技攻关项目(06G143)

摘　　要：目的在原核表达系统中对肠道病毒71型(EV71)基因进行克隆、表达、纯化,建立血清IgM抗体的检测方法,用于EV71感染的早期诊断。方法检索EV71 VP1基因序列,设计合成引物,并在引物两端插入酶切位点,采用RT-PCR分离EV71 VP1全基因序列。PCR产物回收、纯化,插入T载体进行序列同源性测定。再插入表达质粒载体pRSET,构建pRSET-EV71重组质粒。利用纯化后的表达产物,采用酶联免疫吸附试验(ELISA)检测EV71IgM抗体。共收集31例临床诊断为手足口病患儿及36例健康儿童的血清标本进行检测。结果PCR产物片段大小约890bp,与预期VP1全基因序列长度一致,并且克隆入T载体后序列测定与GenBank公布序列的同源性达100%;重组蛋白大小约为890bp,与预期蛋白片段一致。ELISA检测结果显示,在手足口病患儿中IgM抗体阳性7例,健康儿童中则未发现阳性,差异有统计学意义。结论成功建立了针对EV71IgM抗体的检测方法。Objective To clone, express and purify the genes of Enterovirus 71 （EV71） in a prokaryotic expression system, and establish a method to detect the IgM antibody in the serum for the early diagnosis of EV71 infection. Methods The gene sequence of EV71 VP1 was retrieved, and the primer was designed and synthesized. The restriction sites were inserted at both ends of the primer. The whole gene sequence of EV71 VP1 was separated by RT-PCR. The PCR products were then extracted, purified, inserted into the vector T after being purified, and then inserted into the expression vector of pRSET after being sequenced, and finally expressed in E. coli. The purified expression products were used to establish the enzyme-linked immunosorbent assay （ELISA） for detecting the IgM antibody to EV71. The sera of 31 children clinically diagnosed as hand-foot-mouth disease （HFMD） and 36 healthy children were then examined. Results The length of the PCR products was around 890bp, and it was consistent with the expected length of the whole gene sequence of VP1. Meanwhile, the homology was 100% between the sequencing result after the PCR products being cloned into T vector and the published sequence. The molecular weight of recombinant protein was about 890bp which was consistent with the expected weight of the protein segment of VP1. ELISA results indicated that 7 samples in the HFMD group showed a positive reaction, while no sample showed positive reaction in the healthy children group, and the differece was significant. Conclusion The method of detecting IgM antibody to EV71 has been established successfully.

关 键 词：肠道病毒71型 基因 VP1 酶联免疫吸附测定 

分 类 号：R446.5[医药卫生—诊断学]