人FasL分子的克隆、表达及多抗制备

Cloning and expression and polyclonal antibody preparation of human FasL

目的获得人 Fas L蛋白 ,并对其功能进行初步研究。方法用 DNA重组法构建了 Fas L c DNA和谷胱甘肽转硫酶融合原核表达质粒 p GEX- KG- h FL。将重组质粒转入大肠杆菌 JM10 9,经 0 .2 mm ol/ L IPTG在 37°C条件下诱导 3h,SDS- PAGE检测。用 8m ol/ L尿素溶解的包涵体作为免疫原免疫家兔制备多抗。结果融合蛋白表达量约占细菌总蛋白的2 0 %。多抗效价达 1∶ 10 2 40 0 ,此多抗可以与重组蛋白发生很好的抗原抗体反应。结论 h FL 在大肠杆菌中得到高效表达 ,为深入研究 Fas L

ObjectiveThe study is aimed at obtaining FasL protein and doing some research about its function. Me thods We constructed E.coli expressed vector pGEX KG hFL, and FasL cDNA fused to the 3’end of the gene encoding the GST protein. The fusion protein was expressed in E.coli JM109 at 37 °C after 0 2 mmol/L IPTG induction for 3 hours. Inclusion bodies dissolved in the solution of 8 mol/L urea were used as immunogen to prepare the polyclonal antibody. ResultsThe fusion protein of high expression has a molecular weight of 46 kd by SDS PAGE. The yield of the FasL fused protein was 20 percent in total proteins. We observed that the polyclonal antibody can recognize fusion protein and GST by immunoblotting. Conclusion The successful expression of FasL fusion protein and preparation of polyclonal antibody will provide material for further studies of FasL. [