牛催乳素cDNA在大肠杆菌中的克隆与表达

CLONING AND EXPRESSION OF cDNA FOR BOVINE PROLACTIN IN ESCHERICHIA COLI

从牛垂体中分离出总的mRNA,经逆转录酶及大肠杆菌DNA聚合酶合成双链cDNA,以pBR322作为克隆载体并用加G.C尾的方法进行重组,把重组质粒导入大肠杆菌中,建立了牛垂体mRNA的cDNA文库。用标记的人工合成的牛催乳素基因片段作为探针进行杂交,并从库中筛选到几个阳性克隆,经酶谱分析和DNA序列分析证明克隆中有一个含有全长的牛催乳素cDNA序列。将所获得的克隆进行'剪切',加上启动子,然后导入大肠杆菌JM 103中并在IPTG诱导下表达。用SDS-PAGE检测,证明有表达产物存在,再通过酶标测定证明该表达产物具有与天然牛催乳素相似的免疫学活性。

The cDNA library prepared from bovine pituitary was constructed, One of the several clones encoding full length bovine prolactin (bPrl) was selected, Synthetic oligodeoxynucleotide was used as hybridization probe to select the positive clone, The insertion of the positive clone has been sequenced. Its sequence was the same as the published sequence of bovine prolactin. Bovine prolactin cDNA was modified and linked to the tac promotor and expressed in E.coli JM103. The method of SDS-PAGE was used to detect the expression product and the result of ELISA showed the product has the immune activity of prolactin.