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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 enterovirus 71 (EV71) gene in prokaryotic expression system and establish a serum IgM antibody detection method for the early diagnosis of EV71 infection. Methods The sequence of EV71 VP1 gene was retrieved and the primers were designed. The EV71 VP1 gene sequence was isolated by RT-PCR. PCR products were recovered, purified and inserted into T vector for sequence homology determination. Then inserted into the expression plasmid vector pRSET to construct pRSET-EV71 recombinant plasmid. The EV71 IgG antibody was detected by enzyme-linked immunosorbent assay (ELISA) using the purified expression product. A total of 31 cases of clinical diagnosis of hand-foot-mouth disease and 36 healthy children serum samples were detected. Results The fragment of PCR product was about 890bp in length, which was consistent with the expected length of VP1 gene. After cloned into T vector, its homology with GenBank was 100%. The size of the recombinant protein was about 890bp, which was consistent with the expected protein fragment. ELISA test results showed that in children with hand-foot-mouth disease IgM antibody positive in 7 cases, healthy children did not find positive, the difference was statistically significant. Conclusion The detection of EV71 IgG antibodies was successfully established.