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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。
OBJECTIVE: To construct prokaryotic expression vector of Salmonella virulence gene spvB, express purified SpvB protein and immunize mice with it as antigen to prepare polyclonal antibody. Methods: SpvB was analyzed by using bioinformatics software. The amino acid sequence with high antigenicity and high expression was selected as the cloned sequence. Salmonella typhimurium harboring spvB gene was used as a template. The target fragment was amplified by PCR and ligated with prokaryotic expression vector pET28a (+). The plasmid pET28a-SpvB was transformed into Escherichia coli BL21 (DE3) and then induced and purified. The target protein was used to immunize mice to prepare anti-SpvB polyclonal antibodies, and the antibody specificity was detected by Western blot. Results: The prokaryotic expression vector of spvB was successfully constructed. The recombinant protein was expressed mainly in inclusion bodies by IPTG induction. Western blot was used to detect the specific antibody binding to SpvB in the purified protein immunized mice. Conclusion: Acquired immunogenicity of SpvB protein and its polyclonal antibody lay the foundation for further study on the function of this gene.