小鼠Semcap2分子的克隆、表达与抗血清制备

Cloning and expression and anti-serum preparation of mouse Semcap 2

目的 获得小鼠Semcap 2蛋白 ,并对其功能进行初步研究。方法 用DNA重组法构建了mSemcap 2cDNA的原核表达质粒pGEX KG mSemcap 2。将重组质粒转入大肠杆菌DH5α,经 0 .3mmol LIPTG在 37℃条件下诱导 3 .5h,行SDS PAGE检测。用 8mol L尿素溶解的包涵体作为免疫原免疫家兔制备多抗。结果 蛋白表达量约占细菌总蛋白的 1 5 %。多抗效价达 1∶1 0 2 4 0 0 ,此多抗可以与重组蛋白及真核转染细胞中的蛋白发生很好的抗原抗体反应。结论 小鼠Semcap 2在大肠杆菌中得到高效表达 ,其抗体的制备为深入研究mSemcap

Objective To obtain mouse Semcap 2 protein and study its function. Methods we constructed E.coli expressed vector pGEX KG mSemcap 2, and mSemcap 2 cDNA fused to the 3'end of the gene encoding the GST.The protein was expressed in E.coli DH5 α at 37 ℃ after 0.3mmol/L IPTG induction for 3.5 h. Inclusion bodies dissolved in the solution of 8 mol/L urea were used as immunogen to prepare the polyclonal antibody.Results Fusion protein of high expression has a molecular weight of 63 000 u by SDS PAGE. The yield of the mSemcap 2 fused protein was 15 percent of total proteins. We observed that the anti serum could recognize recombinant protein and natural protein by immunoblotting. Conclusion Successful expression of mSemcap 2 protein and preparation of polyclonal antibody will provide materials for further study of mSemcap 2.