含hBDNF cDNA和HTH_1 cDNA两个转录单位表达质粒的构建及在CHO细胞的表达  

Construction of Expression Plasmid Containing Both hBDNF and HTH 1 cDNA Transcriptional Units and Its Expression in CHO Cells

作　　者：应其龙[1,2] 彭鲁英[1,2] 王德春 张乃娴[1,2] 宋后燕 

机构地区：[1]上海医科大学基础医学院分子遗传学教研室 [2]中山医院骨科

出　　处：《上海医科大学学报》1999年第3期157-160,共4页Journal of Fudan University(Medical Science)

基　　金：国家自然科学基金

摘　　要：在ＣＨＯ细胞同时表达ｈＢＤＮＦ和ＨＴＨ１ｃＤＮＡ两个外源基因。方法构建同时含ｈＢＤＮＦｃＤＮＡ和ＨＴＨ１ｃＤＮＡ两个转录单位的表达质粒并转染ＣＨＯ细胞，筛选出同时表达ｈＢＤＮＦ和ＨＴＨ１的克隆命名为ＣＨＯｈＢＤＮＦＨＴＨ１细胞；用双重免疫组化、ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ及活性测定等方法检测外源ＨＴＨ１、ｈＢＤＮＦ基因在ＣＨＯｈＢＤＮＦＨＴＨ１细胞的表达。结果ｈＢＤＮＦ、ＨＴＨ１同时在ＣＨＯｈＢＤＮＦＨＴＨ１细胞得到表达；ＨＰＬＣ－ＥＣＤ法检测到ＣＨＯ－ｈＢＤＮＦ－ＨＴＨ１细胞培液中Ｌ－ＤＯＰＡ含量为每２４ｈ（２５．３±６．７）ｎｇ／１０６ｃｅｌｓ；ＣＨＯ－ｈＢＤＮＦ－ＨＴＨ１细胞培液可明显促进人胚脊神经根节细胞的存活和生长。结论采用多基因表达技术可以使ｈＢＤＮＦ和ＨＴＨ１ｃＤＮＡ两个外源基因同时在一个细胞中得到表达，而且表达的ｈＢＤＮＦ。Purpose Co express both foreign hBDNF and HTH 1 genes in CHO cells. Methods The multiple genes expression plasmid containing both hBDNF and HTH 1 cDNA transcriptional units was constructed and transfected into CHO cells.The clone which expressed both hBDNF and HTH 1 was screened and named CHOhBDNFHTH 1 cells.The expression of hBDNF and HTH 1 in CHOhBDNFHTH 1 cells were detected by double immunohistochemical staining,western blotting and biological activity assay. Results hBDNF and HTH 1 were co expressed in CHO hBDNFHTH 1 cells;the level of LDOPA in the culture medium of CHO hBDNFHTH 1 cells was detected by HPLCECD and was about (25.3±6.7)ng/10 6 cells every 24h;the culture medium of CHO hBDNFHTH 1 cells significantly enhanced survival and growth of cultured dorsal root ganglia of human fetus. Conclusions Foreign hBDNF and HTH 1 genes can be co expressed in one cell using multiple genes expression techniques and the expression products of hBDNF and HTH 1 have biological activity.

关 键 词：酪氨酸羟化酶 脑源性 神经营养因子 基因表达 

分 类 号：Q753[生物学—分子生物学]