Construction of Eukaryotic Expression Vector pIRES2-ZsGreen1-opLA and Its Expression in Porcine(Sus scrofa) Fibroblasts

• Published in: Nongye shengwu jishu xuebao Vol. 20; no. 8
• Main Authors: Li Yanling, Zhejiang University，Hangzhou(China)，College of Animal Sciences, Wang Ying, Zhejiang University，Hangzhou(China)，College of Animal Sciences, Dang Wenqing, Zhejiang University，Hangzhou(China)，College of Animal Sciences
• Format: Journal Article
• Language: Chinese
• Published: 01.08.2012
• Subjects: ALBUMINAS; ALBUMINE; ALBUMINS; http://www.fao.org/aos/agrovoc#c_247; http://www.fao.org/aos/agrovoc#c_27831; http://www.fao.org/aos/agrovoc#c_8164; TRANSFECCION; TRANSFECTION; VECTEUR DE MALADIE; VECTORES; VECTORS
• ISSN: 1674-7968

α-乳清蛋白(α-lactalbumin，α-LA)是哺乳动物乳汁中一种重要的蛋白质，富含机体必需氨基酸和支链氨基酸，其极佳的氨基酸比例、易吸收性以及功能特异性，使得α-LA在婴幼儿个体正常生长中具有重要的意义。本实验旨在构建人α-乳清蛋白真核表达载体pIRES2-ZsGreen1-opLA，将其转染至猪(Sus scrofa)的成纤维细胞，获得稳定表达人α-LA的细胞。根据猪基因密码子偏爱性优化并合成人α-乳清蛋白基因mRNA序列opLA，并将其定向克隆入pIRES2-ZsGreen1真核表达载体，双酶切及测序方法鉴定重组载体；脂质体介导的方法将载体转染入培养的猪成纤维细胞，荧光显微镜下观察转染效果，G418抗性筛选重组细胞；RT-PCR方法检测目的基因的表达。结果表明，通过酶切以及测序鉴定得到的重组载体pIRES2-ZsGreen1-opLA构建成功；荧光显微镜下观察，转染了pIRES2-ZsGreen1-opLA和pIRES2-ZsGreen1空载体的细胞均发出绿色荧光，且荧光多集中于细胞核；筛选重组细胞的最小G418浓度为400 ng/μL；RT-PCR法检测到与目的基因大小一致的片段，而未转染组和转染空载体组均未检测到。本实验成功构建真核表达载体pIRES2-ZsGreen1-opLA，并获得稳定表达目的基因的猪成纤维细胞，该细胞可作为进一步研究转基因克隆猪的供体细胞。 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