沙门菌毒力基因spvB的原核表达及其多克隆抗体的制备

目的:构建沙门菌毒力基因spvB的原核表达载体,诱导表达纯化SpvB蛋白并以其为抗原免疫小鼠,制备多克隆抗体。方法:利用生物信息学软件对SpvB进行分析,选取抗原性较高、易表达的氨基酸序列作为克隆序列,以携带spvB基因的鼠伤寒沙门菌为模板,PCR扩增目的片段后与原核表达载体pET28a(+)连接;将质粒pET28a-SpvB转化大肠埃希菌BL21(DE3)后诱导表达并纯化。目的蛋白免疫小鼠,制备抗SpvB多克隆抗体,Western blot检测抗体特异性。结果:成功构建spvB原核表达载体,经IPTG诱导结果显示,重组蛋白表达且主要存在于包涵体中,将纯化后的蛋白免疫小鼠Western blot检测血清中抗体与SpvB特异性结合。结论:获得具有免疫原性的SpvB蛋白及其多克隆抗体,为进一步研究该基因的功能奠定基础。

Prokaryotic Expression of Gene spvBand Preparation of its Polyclonal Antibody

Objective:To construct the prokaryotic plasmid expressing spvB gene and prepare its antibody.Methods:The amino acid sequences of high antigenicity and easily expression were selected as a pseudo clone sequence according to the bioinformatics analysis. The prokaryotic expression vector pET32a (+)-SpvB was constructed by using spvB fragment fromPCR product and induced to express recombinant protein with IPTG. Then the purified recombinant protein was used to immunize healthy mice for preparing polyclonal antibody against SpvB. The specificity of the antibody was assayed by western blotting.Results:The prokaryotic expression plasmid pET28a-SpvB was successfully constructed, and the SpvB protein was purified by affinity chromatography. The polyclonal antibody against SpvB was obtained and the antibody could specificity conjunct with SpvB protein.Conclusion:The SpvB protein and anti-spvB antibody was obtained, which provide a tool for further investigation of the function of spv B .