| 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。 |
| 关键词: piggyBac转座子 A-FABP基因 表达载体 牛成纤维细胞 |
| DOI： |
| 分类号: |
| 基金项目: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) |
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| Construction and identification of expression vector of A-FABP mediated by piggyBac transposon |
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YANG Ning, ZAN Linsen, CHENG Gong
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NorthwestA&FUniversity
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| Abstract: |
| 【Objective】The special expression vector pPB-AFABP of A-FABP gene,containing piggyBac (PB) as transposon component,green fluorescence protein (EGFP) as reporter gene,Neomycin (NEO) as resistance gene,and α-actin protein as promoter,was constructed and the transposition activity was verified.【Method】The transgene vector pPB-AFABP of A-FABP gene sequence was connected with PB transposon backbone sequence,NEO-EGFP sequence,and α-actin promoter sequence.These sequences were synthesized 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 fibroblasts 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. |
| Key words: piggyBac transposon A-FABP gene expression vector bovine fibroblasts |
