摘要
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 heterologously express VMH protein of Vibrio mimicus and prepare its polyclonal antibody. Methods Vmh gene from V. mimicus was amplified by PCR using the primers designed according to the vmh gene sequence in GenBank. The purified PCR products were digested with nucleic acid restriction enzyme, and then inserted into PET-32a vector to construct recombinant expression plasmid. The positive recombinant plasmid was transformed into Rosetta （DE3） E. coli-competent cells upon induction with isopropyl-β-D-thiogalactopyranoside （IPTG）. The expression form of rVMH protein was analyzed by SDS-PAGE. The hemolytic activity and immune reactivity of rVMH protein were detected by rabbit blood agar diffusion method and Western blot, respectively. A New Zealand white rabbit was vaccinated with the purified rVMH protein. And then antisera were collected and purified by the combination of saturated ammonium sulfate precipitation and a HiTrap Protein GHP column after three immunizations. The purity and titer of the purified antibody were detected by SDS-PAGE, indirect ELISA as well as agar diffusion test, respectively. Results SDS-PAGE analysis showed that a 77.8 kDa inclusion form of rVMH protein band appeared in the original and purified expression products. This protein possossed hemolytic activity and immune reactivity after denaturation and renaturation. The purity of purified polyclonal antibody against rVMH protein reached 95%; the titer of purified antibody was 1∶26843545600 and 1∶32 as detected by indirect ELISA and agar diffusion test, respectively. Conclusion rVMH protein and its polyclonal antibody were successfully prepared, which provided substantial basis for further identification of VMH protein epitope using phage peptide library screening technology.
出处
《中国微生态学杂志》
CAS
CSCD
2016年第7期770-774,共5页
Chinese Journal of Microecology
基金
国家自然科学基金(31272696)
关键词
拟态弧菌
VMH蛋白
原核表达
多克隆抗体
Vibrio mimicus
VMH protein
Prokaryotic expression
Polyclonal antibody
