牛酪蛋白cDNA基因克隆的改建

RECONSTRUCTION OF BOVINE CASEINB cDNA WITH THE PLANT EXPRESSION VECTOR

牛酪蛋白全长cDNA克隆pB_aS_1C184经Bgl Ⅰ和EcoR Ⅰ双酶切,回收其中的酪蛋白基因编码片段,与经BamH Ⅰ、SmaⅠ双酶切的植物表达载体pBI121.2分两步连接:第一步载体的BamHⅠ末端与酪蛋白基因的BglⅠ粘性末端连接;第二步用T_4DNA多聚酶补平酪蛋白基因的EcoR Ⅰ末端,再与载体SmaⅠ进行平末端连接而环化,转化JM109菌株,从得到的33个转化子中,通过^(32)P标记的酪蛋白cDNA基因为探针,点杂交筛选到一个阳性克隆。经酶切鉴定,确认酪蛋白cDNA基因编码区已正确插入到pBI121.2中。

The plant expression vector pBI121. 2 was digested with BamH I and Sma I , the bovine casein B gene cDNA code fragment was seperated from pBα slC184 plasmid with Bgl Ⅱ and EcoR I, the cloning process includes two steps: the first, the Bgl Ⅱ sticky end of casein B gene was ligased to BamH Ⅲ sticky end of pBI 121. 2; the second, the EcoR I sticky end of casein B gene has been filled up with T4DNA polymerase, then the blund end was ligased to Sma I end of pBI 121. 2 After transformation of JM109, the recombinant has been selected by dot hybridization with 32plabelled casein B gene, the positive cloning plasmid (We name it pAS-2) DNA was digested with Hind Ⅲ and EcoR I, it shows that there is a single copy of casein B gene inserted, and the pAS-2 can't digest with BamH I and SmaI. These results show that the bovine casein B gene coding region has been correctly cloned into the plant expression vector pBI121. 2.