鼠伤寒沙门菌pagC启动子的克隆与活性分析

Cloning and Functional Analysis of Promoter pagC from Attenuated Salmonella typhimurium

从鼠伤寒沙门菌中克隆出pagC启动子 (PpagC) ,构建体内激活的表达质粒pZW ,插入庚型肝炎病毒 (HGV)NS3基因 ,构建出表达质粒pZWNS3。以其转化减毒鼠伤寒沙门菌SL72 0 7,观察PpagC 的启动活性。结果表明 :Mg2 +可抑制PpagC 的启动活性 ,Mg2 +浓度小于50mmol L时培养的重组菌经SDS PAGE和Westernblot检测能高水平表达HGVNS3蛋白。Mg2 +浓度升至 50mmol L时 ,NS3蛋白表达量明显降低。收集以 50mmol LMg2 +的培养基扩增的重组菌 ,灌胃接种C57小鼠 ,检测小鼠的血清抗体、T细胞增殖和CTL反应。结果显示PpagC是一个强的宿主体内激活的启动子 ,为构建以伤寒沙门氏菌为载体的高效免疫口服疫苗提供了一个新的途径。

This study describes the cloning and function of promoter pagC(P pagC) from Salmonella typhimurium. The expression plasmid containing the in vivo-inducible promoter pagC and HGV-NS3 gene was introduced into the attenuated Salmonella typhimurium SL7207 to investigate the function of P pagC. The expressed HGV-NS3 protein was detectable by SDS-PAGE and Western blotting in the recombinant bacteria in the presence of low concentration of Mg 2+ (<50mmol/L). When the concentration of Mg 2+ reached to 50mmol/L, the amount of expressed HGV-NS3 was decreased significantly. The recombinant bacteria were multiplied in LB medium containing 50mmol/L of Mg 2+ and used as a DNA vaccine to orally inoculate C57 mice for three times. The results of serum antibodies, T lymphocyte proliferative response and cytotoxic T lymphocyte response of immunized mice showed that the oral vaccine could iuduce strong humoral and cellular immune responses in mice, which indicates that the P pagC is a strong in vivo-inducible promoter and can be used in attenuated Salmonella typhimurium to construct an effective oral vaccine.