人sCD40L基因的克隆及表达

Cloning and expression of extracellura regin of human CD40L

目的 克隆人ｓＣＤ４０Ｌ基因片段，并在原核细胞中表达。方法用ＲＴ－ＰＣＲ技术，从激活的人外周血淋巴细胞总ＲＮＡ中，扩增ＣＤ４０Ｌ胞外区ｃＤＮＡ，并克隆至载体ｐＧＥＭ－Ｔ。测序验证后，转至载体ＰＱＥ３１中，并在大肠杆菌Ｍ１５中进行表达，最后通过亲和层析柱得到纯化蛋白。结果纯化所得的产物经Ｗｅｓｔｅｒｎ ｂｌｏｔ证实，确为人ｓＣＤ４０Ｌ蛋白。结论 应用基因重组法构建了人ｓＣＤ４０Ｌ基因片段，并在原核细胞内成功地进行了表达，为今后进一步研究ＣＤ４０Ｌ与凋亡、疾病的发病机制及临床治疗奠定了基础。

Aim To clone extracellular region of human CD40L gene and express it in prokaryotic cells. Methods RT-PCR was used to amplify the cDNA of CD40L extracellular region from the total RNA of activated human peripheral blood lymphocytes. The DNA fragment was then cloned into vector pGEM-T. After verification sequencing, it was transferred into vector PQE31 and expressed in E.Coli M15. The expressed product was purified through affinity chromatography column. Results The purified protein was proved to be extracellular region of soluble human CD40L protein by Western blot. Conclusion Extracellular region of human CD40L gene was cloned , and expressed in prokaryotic cells successfully. It is a good basis established for further study on relationship between CD40L and apoptosis,pathogenesis of illness and clinical treatment.