重组人半乳糖苷结合凝集素-9质粒的构建及表达

Construction and Expression of Human Galectin-9 Recombinant Plasmid

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(Chinesehamster 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pGa-lectin-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 eu karyotic expression vector pGalectin-9 has been successfully constructed and successfully expressed in CHO cells.