转人乳铁蛋白cDNA山羊胎儿成纤维细胞系的建立  

Establishment of Human Lactoferrin cDNA Transgenic Caprine Fetal Fibroblast Cell Lines

作　　者：林慧[1] 赵明涛[1] 张玉玲[1] 刘风军[1] 王国华[1] 张涌[1] 

机构地区：[1]西北农林科技大学生物工程研究所,农业部家畜生殖内分泌与胚胎工程重点开放实验室,杨凌712100

出　　处：《农业生物技术学报》2008年第3期436-442,共7页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.2004AA213072)资助

摘　　要：采用RT-PCR获得人乳铁蛋白cDNA,通过Long and Acute PCR扩增山羊(Capra hircus)β-酪蛋白5'端6.5kb的调控序列,以pEGFP-C1为骨架构建乳腺特异性表达载体p6.5hLF-EGFP。脂质体介导法转染山羊胎儿成纤维细胞,G418抗性筛选3-4周后,经PCR扩增和报告基因EGFP表达检测,得到稳定整合外源基因的转基因供体细胞系;同时在稳定转染p6.5hLF-EGFP的乳腺上皮细胞系的上清中检测到重组人乳铁蛋白的组织特异性表达。这为制备高效表达人乳铁蛋白的转基因山羊乳腺生物反应器提供可靠的核移植供体细胞。To establish human lactoferrin cDNA transgenic donor cell lines and prepare competent donor cells for the production of transgenie animals by somatic cell nuclear transfer （SCNT） and detect the tissue-specific expression of human lactoferrin eDNA, Human lactoferrin eDNA was obtained by RT-PCR while 6.5 kb caprine（Capra hircus） β-casein regulatory elements were amplified by Long and Acute PCR. Subsequently the two fragments were inserted into the corresponding site of the plasmid pEGFP-C1, resulting in the construction of mammary specific expression vector p6.5hLF-EGFP. Then the eaprine fetal fibroblast cells were transfeeted with p6.5hLF-EGFP by LipofectamineTM-2000 and selected with G418 for 3 to 4 weeks. The G418 resistant transfeetants were identified by PCR and EGFP detection. The results indicated that the transgene was stably integrated into the open region of the ehromatin of G418 resistant fibroblast cells. The expression of human lactoferrin was identified in the supernant of stable transfeeted mammary epithelial cells by Western blot. This study may provide competent transgenic donor cells for the production of transgenie animals by SCNT and improve the efficiency of transgenic cloning.

关 键 词：人乳铁蛋白 山羊β-酪蛋白 EGFP报告基因 供体细胞 核移植 

分 类 号：S188[农业科学—农业基础科学] S185