重组人半乳糖苷结合凝集素-9质粒的构建及表达

摘要

目的 构建人半乳糖苷结合凝集素-9(Galectin-9)的真核表达质粒pGalectin-9,并检测其表达.方法 通过DNA重组技术和PCR方法 从人外周血单个核细胞克隆Galectin-9基因,插入真核表达质粒pcDNA3.1(+)中,通过PCR、酶切及测序鉴定重组载体的正确性;采用脂质体转染技术将重组质粒pGalectin-9瞬时转染中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞,通过Western blot、RT-PCR和间接免疫荧光方法 检测Galectin-9的表达.结果 DNA测序和酶切鉴定证明Galectin-9基因正确克隆至pcDNA3.1(+)的多克隆位点;以重组质粒pGalectin-9瞬时转染CHO细胞,通过Western blot、RT-PCR和间接免疫荧光方法,在分子和细胞水平证实Galectin-9的表达.结论 成功构建pGalectin-9的重组质粒,并证实其在CHO细胞中可以成功表达.%Objective To construct the eukaryotic expression vector of Galectin-9 and to detect its expression. Methods Galectin-9 gene was obtained by PCR and DNA recombination from peripheral blood mononuclear cells(PBMCs). PCR product of Galectin-9 was then inserted into plasmid pcDNA3. 1( + ). The recombinant vector was identified by PCR,restriction enzyme digestion and sequencing. Using LipofectamineTM 2000, the plasmid pGalectin-9 was transfected into Chinese hamster ovary (CHO)cells and then detected by Western blot,RT-PCR and immunofluorescence. Results DNA sequencing and restriction enzyme digestion verified the correction of recombinant plasmid pGalectin-9. After plasmid pGalectin-9 was transfected into CHO cells,the expressed product of Galectin-9 was detected by Western blot,RT-PCR and immunofluorescence. Conclusion The eukaryotic expression vector pGalectin-9 has been successfully constructed and successfully expressed in CHO cells.