转KAP6.1-GFP-蜘蛛拖丝蛋白基因核心序列4S绵羊成纤维细胞株的筛选  

Filtration of Transgenic Sheep Skin Fibroblasts with KAP6.1-GFP-polymerized Spider Dragline Silk Protein Gene(4S)

作　　者：王春生[1] 原璐[2] 宁方勇[1,3] 吴治昊[1] 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,哈尔滨150040 [2]沈阳医学院何氏视觉科学学院,沈阳110163 [3]东北农业大学动物科技学院,哈尔滨150030

出　　处：《中国农业科学》2011年第12期2561-2566,共6页Scientia Agricultura Sinica

基　　金：国家自然科学基金资助项目(30771538);国家转基因生物新品种培育科技重大专项(2009ZX08008-004B)

摘　　要：【目的】通过体细胞核移植为获得皮肤特异表达蜘蛛拖丝蛋白的绵羊奠定基础。【方法】将pcDNA3.1和带有角蛋白结合蛋白启动子质粒pGM-T-KAP6.1分别用BglⅡ和Hin dⅢ双酶切后连接,再与蜘蛛拖丝蛋白基因核心序列4S连接,最后通过酶切与pIRES2-EGFP质粒连接后构建真核表达载体pIRES2-EGFP-4S;将此载体线性化后,采用脂质体法转染绵羊皮肤成纤维细胞,通过G418筛选获得转基因阳性细胞。【结果】筛选得到转pIRES2-EGFP-4S的阳性细胞。对阳性细胞经体外培养后的检测显示:(1)细胞形态(长梭形)、细胞生长曲线(呈S形)、群体倍增时间(随培养时间增加逐渐缩短)和细胞接种率(细胞贴壁率及存活率在24 h内逐渐升高,并达到最高值125%)等均具有正常绵羊成纤维细胞的生物学特征;(2)阳性细胞经冷冻复苏后具有与新鲜阳性细胞相似的生物学特征;(3)PCR检测结果显示,pIRES2-EGFP-4S在阳性细胞的基因组中整合。【结论】获得具有在绵羊皮肤特异表达,且便于检测的转蜘蛛拖丝蛋白4S的绵羊成纤维细胞株。【Objective】This study aims to establish transgenic sheep fibroblast cell line and lay a foundation for transgenic sheep with expression spider dragline silk protein gene in hair follicle by nuclear transplantation.【Method】pcDNA3.1 and pGM-T-KAP6.1（hair follicle-specific promoter） were digested by BglⅡand Hind Ⅲ,and linked each other.The recombinant plasmid was linked with spider dragline silk protein gene and then linked with pIRES2-EGFP by a series of molecular methods.The eukaryotic expression vector pIRES2-EGFP-4S was constructed.Sheep fibroblasts were transfected with the plasmid by cationic liposome method and G418 was used to filtrate them.After identified,transgenic cell line with spider dragline silk protein gene was established.【Result】The G418 positive cells were detected in vitro.The results showed that cellular morphology was similar with normal fibroblast,cell growth curve was ＂S＂ shape,population doubling time（PDT） shorten gradually along with culture time increase,plating efficiency was gradually increasing in 24h.These biological analyses showed the transgenic cells were similar with normal sheep fibroblast.Biological characters of the transgenic cells after freezing-thawing were similar with fresh sheep fibroblast.The PCR result showed that the vector constructed was integrated into sheep genome.【Conclusion】The transgenic cells with polymerized spider dragline silk protein gene were filtrated,and thus laid a foundation for the transgenic sheep with expression spider dragline silk protein in hair follicle.

关 键 词：蜘蛛拖丝蛋白基因 角蛋白启动子 真核表达载体构建 绵羊成纤维细胞 转基因 

分 类 号：Q78[生物学—分子生物学]