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目的实现拟态弧菌热不稳定性溶血素(VMH)的原核表达,制备兔抗VMH蛋白的多克隆抗体。方法根据GenBank上已有的拟态弧菌vmh基因序列,设计并合成引物,通过PCR方法扩增vmh基因。PCR纯化产物酶切后,定向插入到PET-32a表达载体构建重组表达质粒PET-32a-vmh。重组表达质粒转化至E.coli Rosetta感受态细胞,在IPTG诱导下进行VMH蛋白表达。SDS-PAGE分析重组VMH蛋白(rVMH)的表达形式,并分别采用兔血平板扩散法和Western blot检测其溶血活性和免疫反应性。用纯化的rVMH蛋白免疫新西兰大白兔,3次免疫后采集免疫血清,采用饱和硫酸铵分级沉淀结合亲和层析法纯化多克隆抗体,并检测其纯度与效价。结果重组表达质粒PET-32a-vmh诱导表达后,经SDS-PAGE分析发现分子量约为77.8kDa的rVMH蛋白主要以包涵体形式表达,该蛋白经变性复性后具有溶血活性和免疫反应性。兔抗rVMH蛋白的多克隆抗体经纯化后,其纯度达95%,ELISA效价为1∶26843545600,琼扩效价为1∶32。结论成功制备了rVMH蛋白及其多克隆抗体,为进一步采用噬菌体肽库筛选技术鉴定VMH蛋白表位提供了物质基础。
Objective To construct prokaryotic expression vector of VMH and prepare polyclonal anti-VMH protein. Methods Based on the vmh gene sequence of Vibrio mimicus in GenBank, primers were designed and synthesized. The vmh gene was amplified by PCR. The PCR product was digested and inserted into PET-32a expression vector to construct the recombinant expression plasmid PET-32a-vmh. The recombinant plasmid was transformed into E.coli Rosetta competent cells and VMH protein was induced by IPTG. The expression of recombinant VMH protein (rVMH) was analyzed by SDS-PAGE. The hemolytic activity and immunoreactivity of rVMH were detected by rabbit blood plate diffusion assay and Western blot respectively. The purified rVMH protein was used to immunize New Zealand white rabbits. The immune serum was collected after three immunizations and the polyclonal antibody was purified by saturated ammonium sulfate fractionation and affinity chromatography. The purity and the potency of the purified polyclonal antibody were determined. Results After induced by the recombinant expression plasmid PET-32a-vmh, the rVMH protein with a molecular weight of about 77.8 kDa was mainly expressed in inclusion bodies by SDS-PAGE. The protein was hemololytic and immunoreactive after denaturalization. The purity of rabbit polyclonal anti-rVMH protein was 95%, the titer of ELISA was 1:26843545600, and the titer of agar was 1:32. Conclusion The rVMH protein and its polyclonal antibody were successfully prepared, which provided a material basis for the further identification of VMH protein epitope by phage peptide library screening.