小鼠膜型抗衰老蛋白Klotho基因特异片段的克隆、原核表达及多克隆抗体制备

目的克隆小鼠膜型抗衰老蛋白Klotho基因特异片段，制备小鼠Klotho多克隆抗体。方法以小鼠基因组为模板进行PCR，克隆了小鼠膜型抗衰老蛋白Klotho基因外显子Ⅳ部分序列，经BamH I和Nhe I双酶切后定向克隆到质粒pET-GST中，构建原核表达质粒pET-GST-Klotho，转化大肠埃希菌BL21（DE3），用IPTG诱导表达。以重组GST-Klotho融合蛋白免疫家兔，制备Klotho多克隆抗体。结果表达产物经SDS-PAGE检测表明，在大肠埃希菌中成功表达了GST-Klotho融合蛋白，GST-Klotho融合蛋白表达量占菌体总蛋白的15％左右；另外通过ELISA法测得抗血清抗体效价约为1：10000，Western印迹分析验证了抗体特异性。结论GST-Klotho融合蛋白的表达和Klotho多克隆抗体的制备为进一步研究Klotho蛋白在小鼠体内的表达模式以及相关抗衰老药物的研制奠定了基础。

Establishment of Prokaryotic Construct Expressimg Mouse Membrane Anti-aging Protein Klotho Gene Specific Fragment and Preparation of Polyclonal Antibody against Klotho

Objective To Clone mouse membrane anti-aging protein Klotho gene specific fragment, and prepare polyclonal antibody against mouse Klotho. Methods By use of mouse genome DNA as a template, PCR was conducted with the primers designed according to mouse Klotho gene sequence to clone exon 4 fragment of Klotho gene. The PCR products were digested by restriction endonuclease BamH I and Nhe I, and inserted into prokaryotic expression plasmid pET-GST. The GST-Klotho fusion protein was then expressed in E. coli BL21（DE3） by IPTG induction. Rabbits were immunized with GST-Klotho recombinant fusion protein to prepare polyclonal antibody against mouse Klotho. Results GST-Klotho fusion protein was produced to account for about 15% of total bacterial protein. ELISA experiment showed that the titer of the polyclonal antibody against mouse Klotho was up to 1 ： 10000. And the immunological specificity of the antibody was confirmed by Western blot analysis. Conclusion The produced GST-Klotho fusion protein expression and prepared polyclonal antibody against mouse Klotho may be used for further studying mouse Klotho gene expression pattern and developing anti-aging medicine.