转B2C/拟蜘蛛拖丝蛋白基因绵羊成纤维细胞株的筛选  

Screening of sheep fibroblast cell line transferred with B2C gene and spider dragline silk protein gene

作　　者：安铁洙[1] 李昊[1] 王春生[1] 王子竹[1] 宋红卫[1] 郑仁玖 朴善花[1] 

机构地区：[1]东北林业大学生命科学学院,哈尔滨150040 [2]长白山科学研究院,吉林安图133613

出　　处：《黑龙江畜牧兽医》2016年第4期23-26,29,283,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(30771538;31272441)

摘　　要：为了建立绵羊被毛特异表达蜘蛛拖丝蛋白基因的方法,研究利用前期工作获得的绵羊高硫角蛋白结合蛋白基因B2C启动子和人工合成的拟蜘蛛拖丝蛋白基因二聚体(2S),构建真核表达载体pc DNA3.1-B2C-2S,进行线性化后,采用脂质体法转染进绵羊成纤维细胞并经G418筛选获得转基因阳性细胞,再进行PCR检测和冷冻复苏后进行生物学特性检测。结果表明:成功构建真核表达载体pc DNA3.1-B2C-2S,将其整合入转基因阳性细胞的基因组中;转基因阳性细胞经冷冻复苏后,其细胞形态仍保持成纤维细胞的梭形形态,且在续培养中呈现正常细胞相近的"S"型生长曲线。说明试验成功获得转pc DNA3.1-B2C-2S绵羊成纤维细胞株。To establish a specific expression method of spider dragline silk protein gene in the hair coat of sheep,an eukaryotic expression vector pc DNA3. 1- B2C- 2S was constructed using the B2 C promoter of high sulfur keratin- associated protein gene from sheep that obtained by the previous work and a synthetic dimer of spider drag silk protein gene（ 2S）. The linearized pc DNA3. 1- B2C- 2S was transfected into sheep fibroblasts by liposome method,and then the transgenic- positive cells were obtained by G418 screening. The transgenic- positive cells were used for PCR detection and the detection of biological characteristics after freezing and thawing,respectively. The results the eukaryotic expression vector pc DNA3. 1- B2C- 2S was successfully established,and then it was integrated into the genome of the transgenic- positive cells.The morphology of transgenic- positive cells still remained spindle morphology of fibroblasts after freezing and thawing,and presented＂S＂type growth curve that was similar with normal cells in the continuous culture. The results indicate that pc DNA3. 1- B2C- 2S- transferred sheep fibroblast cell line is successfully obtained.

关 键 词：蜘蛛拖丝蛋白基因 B2C启动子 绵羊成纤维细胞 转染 细胞株 

分 类 号：S826.1[农业科学—畜牧学]