摘要
目的原核表达并纯化人细小病毒B19(Human parvovirus B19,HPVB19)结构蛋白VP1,并初步建立IgM ELISA检测方法,用于B19病毒感染的检测。方法采用PCR法从B19 IgM阳性血清中扩增VP1基因片段,亚克隆至表达载体pGEX-2T中,构建重组表达质粒pGEX-2T-VP1,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经疏水纯化后进行Western blot分析。以纯化的重组蛋白包被酶标板,建立IgM间接ELISA检测方法,确定Cut-off值,并进行初步应用。结果重组原核表达质粒经双酶切鉴定构建正确;表达的重组融合蛋白相对分子质量为61 000,主要以包涵体形式存在,纯度为96%,可与B19 IgG阳性血清发生特异性反应;建立的间接ELISA方法 Cut-off值为0.102,灵敏度为91.7%,特异性为98.6%,与对照试剂检测结果的符合率为97.1%;用建立的方法检测4 500份健康献血者及39份类风湿性关节炎患者血清的B19 IgM抗体,阳性率分别为1.04%和10.3%。结论原核表达并纯化了HPVB19 VP1蛋白,并初步建立了IgM间接ELISA检测方法,为B19病毒感染的早期诊断提供了参考。
Objective To express the structural protein VP1 of human parvovirus B19（HPVB19） in prokaryotic cells,purify the expressed product,and preliminarily develop an ELISA method for IgM to monitor HPVB19 infection.Methods VP1 gene fragment was amplified by PCR and subcloned into prokaryotic expression vector pGEX-2T.The constructed recombinant plasmid pGEX-2T-VP1 was transformed to E.coli BL21（DE3） and induced with IPTG.The expressed product was purified by hydrophobic chromatography and analyzed by Western blot.An indirect ELISA method for IgM was developed by coating the microtiter plate with the purified recombinant protein,then determined for cut-off value and applied preliminarily.Results Restriction analysis proved that recombinant plasmid pGEX-2T-VP1 was constructed correctly.The expressed recombinant fusion protein,with a relative molecular mass of 61 000,mainly existed in a form of inclusion body,which reached a purity of 96% and showed specific reaction with B19 IgG positive serum.The cut-off value,sensitivity and specificity of the developed ELISA method were 0.102,91.7% and 98.6% respectively.The coincidence rate of determination results by the developed kit and control kit was 97.1%.A total of 4 500 serum samples from healthy donors and 39 from patients with rheumatoid arthritis were determined by the developed ELISA method,in which the positive rates of B19 IgM were 1.04% and 10.3% respectively.Conclusion The VP1 protein of HPVB19 was expressed in prokaryotic cells and purified,and an indirect ELISA method for IgM was preliminarily developed,which provided a reference for early diagnosis of HPVB19 infection
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第3期345-348,356,共5页
Chinese Journal of Biologicals
