牛αS1酪蛋白5′调控序列驱动报告基因LacZ在奶山羊乳腺上皮细胞中的表达

5'Flanking Sequence of Bovine αS1 Casein Gene Drives Expression of LacZ Report in Goat Mammary Epithelial Cells

αS1酪蛋白是牛乳中含量最高的酪蛋白.本研究克隆了牛αS1酪蛋白5′调控序列约1.2kb的片段,应用基因重组技术将其插入lacZ基因上游,构建真核表达载体gαs1p-psv.胶原酶消化法对奶山羊乳腺上皮细胞进行了分离培养,并用免疫荧光法鉴定了乳腺上皮细胞.将重组载体转染奶山羊乳腺上皮细胞,在细胞培养液中,转染后24h可以检测到lacZ基因的表达,之后表达水平有逐渐升高的趋势,72h开始降低,144h降到最低;在细胞破碎液中,转染后24h表达水平最高,之后表达水平有逐渐降低的趋势,144h降到最低.转染细胞的第1代表达水平最高,随细胞传代表达水平降低,传到第3代时基本检测不到表达产物.对转染后的细胞进行了β-半乳糖苷酶原位细胞染色.实验证明,得到的牛αS1酪蛋白5′调控序列能指导外源基因在奶山羊乳腺上皮细胞中表达.

αS1 casein is the main fraction in the casein of milk. The 5′flanking regulatory regions of 1.2 kb of bovine αS1 casein gene were amplified by PCR and constructed the eukaryotic expression vector gaslp-psv, which was inserted the 5′flanking sequence of bovine αS1 casein gene into the upstream of lacZ gene by the recombinant DNA technology. The goat mammary epithelial ceils could be isolated by collagenase digestion and culture. Furthermore, it was identified by immunofluorescent staining method which detected the expression of keratin 18.The test results showed that the expression of lacZ gene can be detected at 24 hours in the cell culture medium after transfection and expression had a gradually upward trend which declined at 72 hours, and reached the minimum at 144 hours. In the cell-free system, the expression of lacZ gene can be detected at 24 hours after transfection and expression had a gradually downward trend which reach the minimum at 144 hours. On the basis of the expression of transfected cells passage, the first generation reached the highest level, the expression reduced after cells passage, to the third generation the expression could not be detected. Situ staining in the ceils, in which the β-galactosidase could catalyze the disassociation of X-gal, was used to detected the expression of β-galactosidase in the ceils. The cloned 5′flanking sequence of bovine αS1 casein gene has the effect of regulating gene expression specifically.