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目的在大肠杆菌中表达沙门菌属pad蛋白,纯化后制备兔抗pad抗体。方法利用PCR方法从肠炎沙门菌中扩增出pad基因,构建pET-32a(+)-pad重组表达质粒;将重组表达质粒转化至大肠杆菌BL21(DE3)中,筛选阳性克隆,测序鉴定后,优化IPTG浓度、诱导温度和诱导时间以诱导重组蛋白的表达;以纯化的pad蛋白免疫家兔,制备抗pad多克隆抗体并进行鉴定。结果与结论成功扩增得到pad基因,经双酶切和测序证实重组表达质粒构建成功,经过诱导条件的优化,在大肠杆菌中表达出相对分子质量为41×103的目的蛋白;纯化的重组蛋白免疫家兔后,能够有效地刺激家兔产生特异性抗体,经琼脂糖双向扩散测定抗血清效价达到1:32以上。为进一步研制沙门菌属体外快速检测试剂打下了基础。
Objective To express Salmonella pad protein in Escherichia coli and prepare rabbit anti-pad antibody after purification. Methods The pad gene was amplified from Salmonella enteritidis by polymerase chain reaction (PCR), and the recombinant plasmid pET-32a (+) - pad was constructed. The recombinant plasmid was transformed into E. coli BL21 (DE3) The concentration of IPTG, induction temperature and induction time were optimized to induce the expression of recombinant protein. The purified pad protein was used to immunize rabbits to prepare and identify anti-pad polyclonal antibody. RESULTS AND CONCLUSION: The pad gene was successfully amplified and confirmed by double enzyme digestion and sequencing. The recombinant plasmid was successfully constructed. The recombinant protein was expressed in E.coli with the molecular weight of 41 × 103. The purified recombinant protein Rabbit immunization can effectively stimulate rabbits to produce specific antibodies, antisera titer determined by agarose two-way diffusion of 1:32 or more. Which laid the foundation for the further development of Salmonella in vitro rapid detection reagent.