山羊α-乳清蛋白乳腺特异性表达载体的构建及核供体细胞的转染

Construction of mammary specific gland expression vector and transfection of nuclear donor cells

为了构建α-乳清蛋白乳腺特异性表达载体及提供山羊乳腺生物反应器α-乳清蛋白核移植供体细胞,提取乳腺组织中的总RNA,采用RT-PCR技术获得α-乳清蛋白基因cDNA,测序酶切后连接乳腺特异性表达载体pBCⅠ-Neo,并对重组载体进行酶切测序鉴定;取重组正确的载体酶切线性化后利用脂质体介导转染至山羊胎儿成纤维细胞,通过G418筛选培养转染了α-乳清蛋白乳腺特异性表达载体的山羊胎儿成纤维细胞。结果显示:经过RT-PCR特异性扩增克隆出α-乳清蛋白基因;克隆的基因碱基组成序列经测序与预期完全一致,α-乳清蛋白基因片段正向插入乳腺特异性表达载体;重组载体在山羊胎儿成纤维细胞中稳定转染,转入α-乳清蛋白乳腺特异性表达载体的山羊胎儿成纤维细胞生长迅速,仍保持原有的细胞生长形态。成功构建了α-乳清蛋白乳腺特异性表达载体,获得稳定转染的山羊胎儿成纤维细胞可用于核移植。

The aim of this study was to construct a mammary gland-specific expression vector with a-lactalbumin gene and provide donor cells for mammary gland bioreactor. Total RNA was extracted from goat breast tissue and cDNA of a-lactalbumin gene was obtained by using RT-PCR technology. After the sequence of a-lactalbumin gene was identified by enzyme digestion, it was ligated into mammary gland-specific vector of pBC I -Neo. The recombinant vector sequence was identified by the enzyme digestion,and then transfected into goat fetal fibroblast by lipsome-mediated transfection. The transfected goat fetal fibroblast cells with mammary gland specific expression vector was screened by G418. As a result, the fraction of a-lactalbumin gene was properly placed into a mammary gland-specific expression vector, and recombint vector was steadily transfected into goat fetal fibro- blast cells. Remaining the original morphology, the goat fetal fibroblast cells with transfected mammary gland-specific expression vector growed rapidly. In conclusion, mammary gland- specific expression vector was successfully constructed and steadily transfected goat fetal fibroblast cells for nuclear transfer were obtained.