Objective By prokaryotic expression and purifying the human bocavirus recombinant protein VP2, to establish the indirect enzyme-linked immunoassay for detection of virus. Methods We amplified the human bocavirus recombinant protein VP2 gene fragments from WHL-1 template by PCR , and cloned into the expression vector pET28a, then conversed into the BL21 (DE3) and expressed the fusion protein detected by Western Blot detection , the obtained the antibody and detected the human bocavirus in serum in Guanghzhou area in healthy people. Results The Recombinant prokaryotic expression identified correct by double enzyme, and it could occur specific reaction with the virus positive serum. The best optimal antigen coating concentration were serum multiples and blocking BSA was 2 mg/mL , 1 ∶ 200 and 1%. The best working dilution of enzyme-labeled secondary antibody was 1 ∶ 4 000. The best working hours was 1h. This detection method had good specificity and reproducibility. The cut-off of the indirect ELISA method was 0.1 and the sensitivity and specificity of the developed ELISA method were 92% and 98% respectively. The coincidence rate of determination results by the developed kit and control kit was 97%. Conclusion The competitive ELISA established by prokaryotic expressing and purifying the human bocavirus protein VP2 protein , provides a basis in detecting the human bocavirus serum antibody.%目的：原核表达并纯化人博卡病毒结构蛋白 VP2，并建立间接酶联免疫法，用于病毒感染的检测。方法：本研究采用PCR法从菌种模板WHL-1扩增出人博卡病毒结构蛋白VP2基因片段，克隆至表达载体pET28a中，转化到菌BL21（DE3），经过诱导产生融合蛋白，使用Western blot 检测，并以该蛋白为包被抗原建立检测血清人博卡病毒的间接酶联免疫法。结果：重组原核表达质粒经双酶切鉴定构建正确，并可与人博卡病毒 IgG阳性血清发生特异性反应，建立的间接酶联免疫法最佳抗原包被浓度为2 mg／mL ，血清的最佳稀释倍数为1∶200，封闭以1％BSA封闭效果最佳，酶标二抗的最佳工作稀释度为1∶4000，工作时间最佳为1 h，该检测方法有较好的特异性和重复性。建立的间接 ELISA 方法 Cut-off 值为0．1，灵敏度为92％，特异性为98％，与对照试剂检测结果的符合率为97％。结论：原核表达并纯化了人博卡病毒结构蛋白VP2蛋白建立的间接酶联免疫法，为人博卡病毒的血清抗体检测方法提供了依据。
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