piggyBac转座子介导的牛A-FABP表达载体的构建与鉴定  

Construction and identification of expression vector of A-FABP mediated by piggyBac transposon

作　　者：杨宁[1] 昝林森[1,2] 成功[1] 付常振[1] 李耀坤[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]国家肉牛改良中心,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2013年第9期8-14,20,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家"十二五"转基因育种重大专项(2011ZX08007-002);国家"十二五"863计划项目(2011AA100307)

摘　　要：【目的】构建以piggyBac(PB)转座子为载体元件,绿色荧光蛋白(EGFP)为报告基因,Neomycin(NEO)为抗性基因,alpha-肌动蛋白(α-actin)为启动子的A-FABP基因特异性表达载体pPB-AFABP,并验证其转座活性。【方法】通过PCR方法合成PB转座子骨架序列、NEO-EGFP、α-actin启动子以及A-FABP基因序列,并将各个序列连接构建成转基因载体pPB-AFABP;用脂质体法将供体质粒pPB-AFABP和辅助质粒pCAG-PBase组成的PB二元系统及质粒pPB-AFABP分别转染牛成纤维细胞,通过G418筛选得到转基因细胞。【结果】经过酶切和测序鉴定,载体pPB-AFABP构建正确;PB二元系统的阳性克隆数明显多于转座子载体pPB-AFABP的克隆数;PCR鉴定结果表明,牛成纤维细胞中存在目的基因A-FABP。【结论】成功构建了转基因表达载体pPB-AFABP,且证实PB转座子在牛成纤维细胞中具有较高的转座活性。[Objective] The special expression vector pPB-AFABP of A-FABP gene,containing piggy- Bac （PB） as transposon component,green fluorescence protein （EGFP） as reporter gene, Neomycin （NEO） as resistance gene,and a-actin protein as promoter,was constructed and the transposition activity was veri- fied. [Method] The transgene vector pPB-AFABP of A-FABP gene sequence was connected with PB trans- poson backbone sequence, NEO-EGFP sequence, and a-actin promoter sequence. These sequences were syn- thesized by PCR amplification. PB binary system consisting of the plasmid pPB-AFABP and helper plasmid pCAG-PBase and vector pPB-AFABP were transfected into bovine fibroblasts by liposomes, respectively. The transgenic cells after screened by G418 was observed. [Result] The final transgene vector pPB AFABP was verified by restrictive enzyme and sequencing. The number of the positive colonies in bovine fi- broblasts transfected with PB binary system was larger than that transfected with the plasmid pPB-AFABP alone. PCR results showed that the A-FABP gene existed in bovine transgenic fibroblast. [Conclusion] A transgene vector pPB-AFABP was successfully constructed, and a high transposition activity of the PB transposon in bovine fibroblasts was verified.

关 键 词：PIGGYBAC转座子 A-FABP基因 表达载体 牛成纤维细胞 

分 类 号：S823.2[农业科学—畜牧学]