HBD-3基因的乳腺特异性表达载体的构建及真核表达

Construction of HBD-3 gene mammary-specific expression vector and eukaryotic expression

摘要:

为了制备高效表达人b-防御素-3基因的转基因奶牛提供可靠的核移植供体细胞, 本试验采用RT-PCR从人胎盘组织获得人b-防御素3 cDNA, 通过双酶切法将目的基因片段hBD插入到质粒pBCP之间, 最后再通过双酶切法将目的片段BCD插入到真核表达载体pEGFP-C1中, 最终构建乳腺特异性表达载体pEBCD。脂质体介导法转染奶牛胎儿成纤维细胞G418, 抗性筛选3~4 周后, 经PCR、RT-PCR 扩增检测和报告基因EGFP表达检测, 得到稳定整合外源基因的转基因供体细胞; 同时检测稳定转染的奶牛乳腺上皮细胞系的上清液, 检测到重组人b-防御素-3蛋白可以在奶牛乳腺上皮细胞组织特异性表达。

关键词:人b-防御素3, 奶牛b-酪蛋白, EGFP报告基因, 供体细胞, 核移植

Abstract:

To establish human β-defensin-3 gene transgenic cell lines as competent donor cells for the production of transgenic animals using somatic cell nuclear transfer (SCNT). Firstly, we obtained human β-defensin-3 by RT-PCR from human placenta, and subsequently inserted the fragment hBD into the corresponding site of the plasmid pBCP. Then we moved the combined fragment BCD (including 5￠ and 3￠ regulating region of β-casein and hBD) into the corresponding site of the plasmid pEGFP-C1. Finally we successfully constructed mammary-specific expression vector pEBCD. We transected pEBCD into Holstein Fetal fibroblast cells by Lipofectamine TM-2000 and selected in medium with G418 for three to four weeks. We identified G418 resistant transfectants by PCR, RT-PCR and EGFP detection. Our results indicated that human β-defensin-3 gene stably was integrated into the open region of the chromatin in G418 resistant fibroblast cells. Meanwhile we identified the expression of human β-defensin-3 in the supernant of stable transfected mammary epithelial cells by Western blotting. This study may provide competent transgenic donor cells for the production of transgenic animals by SCNT and improve the efficiency of transgenic cloning.

Key words:human β-defensin-3, Holstein β-casein, EGFP reporter gene, donor cells, nuclear transfer