摘要
目的:构建β-半乳糖苷结合凝集素-9(Gal-9)的真核表达载体,在CHO细胞中表达,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Gal-9基因,插入真核表达载体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pGal-9瞬时转染CHO细胞,通过Western blot和RT-PCR方法检测Gal-9的表达,MTT法检测Gal-9对同种异体淋巴细胞增殖的影响。结果:DNA测序和酶切鉴定证明Gal-9基因正确克隆至pcDNA3.1(+)的多克隆位点;以重组质粒pGal-9瞬时转染CHO细胞,通过Western blot和RT-PCR方法,在分子和蛋白水平证实Gal-9的表达,MTT法检测显示,Gal-9明显抑制同种异体淋巴细胞增殖反应,平均抑制率达85.43%。结论:成功构建pGal-9的真核表达载体,证实其在CHO细胞中可以成功表达,并抑制同种异体淋巴细胞增殖反应。
Objective：To construct the eukaryotic expression vector of Gal-9 and to express it in CHO cells and detect its product.Methods：Gal-9 gene was obtained by PCR and DNA recombination from PBMC.PCR product of Gal-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 pGal-9 was transfected into CHO and then detected by Western blot and RT-PCR.MTT colorimetry was used to detect allogeneic lymphocyte proliferation.Results：DNA sequencing and restriction enzyme digestion verified the correction of recombinant plasmid pGal-9.After plasmid pGal-9 was transfected into CHO,the expressed product of Gal-9 was detected by Western blot and RT-PCR.The result showed that Gal-9 significantly inhibited lymphocyte proliferation response,the level was 85.43%.Conclusion：The eukaryotic expression vector pGal-9 has been successfully constructed and successfully expressed in CHO cell line,and Gal-9 inhibited lymphocyte proliferation response.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2011年第9期775-778,共4页
Chinese Journal of Immunology
基金
湖北省科技厅研究项目(No.2007ABA186)
湖北省教育厅研究项目(No.Q200734002)
武汉市高校科研重点资助项目(No.2006Z02)
关键词
Gal-9
构建
表达
真核表达载体
Gal-9
Construction
Expression
Eukaryotic expression vector
