FMDV免疫活性串联片段FA的表达及免疫原性测定

Expression and immunogenicity detecting of immunogenic tandem fragments FA of FMDV

将口蹄疫病毒免疫串联片段FA克隆至原核表达载体pBAD/TOPO中 ,经鉴定后得到重组质粒pBAD_FA ,将此重组质粒转化到受体菌TOP10中 ,用诱导剂阿拉伯醛糖分别以不同的浓度进行诱导 ,并在不同诱导时间进行采样 ,经处理后做SDS_PAGE、Westernblot分析。结果发现以终浓度为 0 0 0 2 %的阿拉伯醛糖进行诱导 ,4h后表达可达到高峰 ,其大小约为 2 3kD ,软件扫描结果显示 ,FB融合蛋白的表达量占细菌总蛋白的 2 9 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。将融合蛋白的可溶性组分用 5 0 %Ni_NTA树脂过柱纯化并抽提融合蛋白的包涵体 ,经过洗涤后分别制成油乳剂疫苗 ,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 ,用此融合蛋白可溶部分的纯化产物和包涵体免疫豚鼠能诱导产生高滴度的中和抗体 ,对病毒的攻击分别提供 10 0 %和 75

The recombinant expression vector pBAD-FA was constructed by cloning immunogenic tandem Fragments FA of foot-and-mouth disease virus into a prokaryotic expression plasmid pBAD/TOP.The recombinant vector was transformed into E.coli TOP10 strain.Samples were collected at different induction time after induction with arabinose.The specificity of the expressed fusion protein was identified with SDS-PAGE and Western Blot. The results showed that the optimal amount of expressed fusion protein is 29.3 % of total bacterial protein after induced with arabinose at 0.002 % concentration for 4 hours.The fusion protein is about 23 kDa in size and could effect specific reaction with antibodies.The soluble fraction of fusion protein was purified with 50 % Ni-NTA resin affinity chromatography, and fusion protein inclusion body was extracted.Both were used to prepare oil-emulsion vaccine separately,which were used to immunize guinea pigs subcutaneously.The sera were collected from the guinea pigs used in suckling mice to test for the presence of neutralizing antibody,and the guinea pigs were challenged with FMDV.The results showed fusion protein could yield antibodies with high neutralizing activity as well as provide 100 % and 75 % protection against challenge with virulent virus.