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| Construction of Eukaryotic Expression Vector pIRES2-ZsGreen1-opLA and Its Expression in Porcine(Sus scrofa) Fibroblasts |
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Abstract α-lactalbumin(α-LA) is an important protein in mammalian milk, and contains body essential amino acids and branched-chain amino acids. For its excellent amino acids ratio, easy absorption and functional specificity, α-LA has a great significance for the infant individuals' growth. In this study we aimed to construct human α-LA eukaryotic expression vector pIRES2-ZsGreen1-opLA, and transfected the human α-LA gene into pig(Sus scrofa) fibroblasts to get cells of stable expression of human α-LA. We optimized and synthesized human α-LA mRNA sequence according to the codon preference of pig α-LA gene, and cloned the sequence into the eukaryotic expression vector pIRES2-ZsGreen1, and then proved the correction of the recombinant vector by restriction enzyme digestion and sequencing. The plasmid was transfected into pig fibroblasts cultured by liposome-mediated method, and the cells were divided into three groups: Negative control of transfection group, positive control of empty plasmid transfection group and recombinant plasmid transfection group. The effect of transfection was detected by florescence observation, and the stably transfected cells were selected and cultured by G418, and the target gene expression was detected by RT-PCR. The results of restriction enzyme digestion and sequencing showed that the recombinant plasmid pIRES2-ZsGreen1-opLA was constructed successfully. Observed by fluorescence microscopy, cells of the empty plasmid pIRES2-ZsGreen1 transfection group and the recombinant plasmid pIRES2-ZsGreen1-opLA transfection group both emitted green fluorescence, and the fluorescence was concentrated in the nucleus. The minimum concentration of G418 for screening the recombinant cells was 400 ng/μL. There was an objective gene fragment of the recombinant plasmid transfection cells after RT-PCR, on the contrary, the other two groups cells were not detected such fragments. The eukaryotic expression vector pIRES2-ZsGreen1-opLA has been successfully constructed, and the human α-LA gene can be steadily expressed in pig fibroblasts. These fibroblasts act as the donor cells for the further study of transgenic cloned pigs
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Received: 27 March 2012
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ZHU Xiao-Feng, XU Hou-Qiang, CHEN Wei, CHEN Ying-Lian, NI Kai, WU Xiao-Min, NI Meng-Meng, LU Xian-Jun. Cloning, Expression and Bioinformatics Analysis of IGF-1 and IGF-2 Genes in Congjiangxiang Pigs (Sus scrofa)[J]. 农业生物技术学报, 2019, 27(8): 1382-1391. |
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