山羊β-casein位点打靶载体在乳腺上皮细胞中的表达研究  

Expression of Goat β-casein Gene Targeting Vector in Mammary Gland Cell

作　　者：俞慧清 李志国 刘红茹 吴国祥 成国祥 

机构地区：[1]上海转基因研究中心,上海201203

出　　处：《生物工程学报》2004年第1期21-24,共4页Chinese Journal of Biotechnology

基　　金：国家"863"高技术项目 (No .AA2 0 665 2 )~~

摘　　要：以本地山羊基因组DNA为模板 ,通过长链PCR扩增出山羊 β casein上游包括启动子 ,外显子 1及部分外显子 2的 6 1kb的调控序列及下游 3 3kb的序列 ,将来自质粒pCDNA3的neo基因以及来自质粒pNEOZTK2 的tk基因 ,经克隆重组后构建了本地山羊乳腺特异性定点打靶载体 ,并在其中克隆人乳铁蛋白mini基因 ,采用脂质体法转染小鼠乳腺上皮癌化细胞系C12 7,以进行打靶载体的表达功能检测 ,双夹心ELISA测得诱导液中乳铁蛋白表达量为 0 2 μg mL ,Western blot显示重组蛋白分子量比标准品略小 ,约为 76kD ,结果说明本载体能够指导外源基因在动物乳腺细胞内正确表达。The study of mammary gland bioreactor is in the ascendant. In order to generate transgenic goats of well-controlled expression of exogenic genes, we constructed a human lactoferrin (hLF) gene targeting vector containing promoter, exon 1, intron1 and some of exon 2 (about 6.1kb fragment) and exon 6～9 (about 3.3kb fragment) of the goat beta-casein gene as well as hLF minigene, neo gene inserted into them and tk gene ligated to the 3′end of the construct. The 9.4kb goat genomic sequences as homologous arms were initially amplified by PCR with local goat tissue DNA. The expression vector was named pBC-tk-neo-hlf. Then the recombinant plasmid pBC-tk-neo-hlf containing hLF minigene was transfected into mice mammary tumor cell line C127 by liposome, cell clones were selected with G418. After proliferating, the transfected cells were induced with insulin, luteotropic hormone and hydrocortisone. The result of Western-blotting analysis showed that the transfected cells can secrete hLF protein, and the recombinant protein expressed in cultured cell supernatant has the similar molecular weight as the native protein. The expression level detected by ELISA was 0.2μg/mL. This result indicated that the targeting vector could efficiently direct the expression of hLF in mammary cells,and it confirmed the validity of the constructed vector. At the same time, C127 cell line proved to be useful for evaluating the regulation of a foreign gene expression in mammary gland specific expression vector.

关 键 词：山羊 打靶载体 人乳铁蛋白 C127细胞 表达 β-casein位点 

分 类 号：Q789[生物学—分子生物学]