口蹄疫病毒VP1基因的原核表达及免疫原性检测

将口蹄疫病毒VP1基因克隆至原核表达载体 pBAD/TOPO中 ,经鉴定后得到了重组质粒 pBAD VP1。将此重组质粒转化到受体菌TOP10中 ,分别以不同浓度的诱导剂L 阿拉伯醛糖进行诱导 ,并在不同诱导时间进行采样 ,经处理后做SDS PAGE和蛋白质印迹分析。结果 ,以终浓度为 0 .0 2 g/L的L 阿拉伯醛糖进行诱导 ,4h后表达可达到高峰 ,表达产物大小约为 4 0ku。软件扫描结果显示 ,VP1融合蛋白的表达量占细菌总蛋白的 2 6 .3% ,能与抗FMDV抗体发生特异性反应 ,融合蛋白以包涵体和可溶形式存在。抽提融合蛋白的包涵体 ,经过洗涤后制成油乳剂疫苗 ,经皮下注射免疫豚鼠 ,用乳鼠中和试验测定豚鼠血清中和指数 ,并用口蹄疫病毒对豚鼠进行攻毒。结果表明 ,用此融合蛋白的包涵体免疫豚鼠能诱导产生中和抗体 ,并对病毒的攻击提供免疫保护。

Prokaryotic expression of VP1 gene of foot-and-mouth disease virus and detection of expression product immunogenicity

The recombinant expression vector pBAD-VP1 was constructed by cloning VP1 gene of foot-and-mouth disease virus into the prokaryotic expression plasmid pBAD/TOPO. The recombinant vector was transformed into the E.coli TOP10 strain. Samples were collected at different induction time after (induction) with L-arabinose .The specificity of the expressed fusion protein was identified with SDS-PAGE and Western-blotting. The results showed that the optimal amount of the expressed fusion protein is (26.3%) of total bacterial protein after being induced with L-arabinose at 0.02 g/L concentration for 4 hours. The (fusion) protein is about 40 ku in size and can effect specific reaction with the antibody. The (fusion) protein inclusion body was extracted to prepare the oil-emulsion vaccine, which was used to immunize cavians subcutaneously. The sera were collected from the cavians and tested for the presence of neutralizing antibody, and the cavians were challenged with FMDV. The results showed that the fusion (protein) can yield antibodies with high neutralizing activity as well as provide protection against challenge with virulent virus.