转甜味蛋白Brazzein基因乳腺特异表达载体的山羊体细胞株筛选  

The Screening and Identification of Transgenic Goat Fibroblast Cells with Sweet Protein Brazzein Gene

作　　者：王春生[1] 李咏乐 仇雨歌 朴善花[1] 安铁洙[1] 

机构地区：[1]东北林业大学生命科学学院,黑龙江哈尔滨150040

出　　处：《安徽农业科学》2014年第24期8099-8101,8166,共4页Journal of Anhui Agricultural Sciences

基　　金：中央高校基本科研业务费专项资金项目(DL13EA06-02);国家自然科学基金项目(30771538)

摘　　要：[目的]建立通过山羊乳汁获取甜味蛋白Brazzein的方法。[方法]将前期工作中构建的Brazzein基因山羊乳腺特异表达载体STP-pBC1进行线性化,采用脂质体法转染山羊成纤维细胞,以期获得转基因阳性细胞。[结果]采用脂质体法转染山羊成纤维细胞后,通过最适G418浓度(400μg/ml)筛选获得转基因阳性细胞;转基因阳性细胞形态呈现梭形且核仁清晰,其生长曲线呈现正常的"S";转基因阳性细胞经冷冻复苏后,仍呈现与冷冻前新鲜转基因细胞相似的形态和生长曲线;PCR鉴定结果表明,STP-pBC1已整合入转基因细胞的基因组中。[结论]成功获得乳腺特异的转甜味蛋白基因Brazzein的山羊成纤维细胞株。[Objective] In order to obtain transgenic goat fibroblast cells with sweet protein Brazzein gene.[Method] Goat fibroblast cells were transfected with mammary gland specific expression vector STP-pBC1 constructed in the previous work by cationic liposome method and cultured G418-resistant cell.After PCR identified,transgenic cell line with Tem was established.The G418 positive cells were detected.[Result] Transgenic cell could be obtained by the optimum concentration of G418.Morphology of the transgenic cell and transgenic cells after freezing-thawing was similar with normal mammary epithelial cells,the cell growth curve was ＂S＂ shape,PCR results showed that the vector constructed was integrated into genome.[Conclusion] The transgenic cells with temporin-GFP were filtrated,and lay the foundation for the transgenic goat with expression temporin in mammary.

关 键 词：山羊 乳腺特异表达载体 BRAZZEIN基因 转基因细胞 

分 类 号：S827[农业科学—畜牧学]