牛生长激素基因工程菌的构建及其诱导表达

Construction of Recombinant Bacteria and Expression of Bovine Somatotrophin Gene

为获得牛生长激素(BST)蛋白,构建了工程菌并进行了诱导表达.先设计2对引物,从pUCm-T-BST克隆中扩增出BST成熟蛋白编码序列,双酶切后分别插入表达载体(pET30 a和pET29 a),转化E.coliDH5α并进行阳性克隆鉴定;再将重组质粒分别转化到E.coliBL21(DE3)和RosettaTM(DE3)pLysS中,构建BST原核工程菌;最后进行诱导表达.结果表明,重组表达载体pET30 a-BST和pET29 a-BST中均插入了正确的目的基因;经IPTG诱导后,以BL21(DE3)为宿主的工程菌无目的蛋白表达,而pET30 a-BST/RosettaTM和pET29 a-BST/RosettaTM分别可见分子量约29.5,26.5 kD的融合蛋白表达,约占菌体蛋白的26%,35%.说明稀有密码子限制BST基因在E.coli中的表达,选择适宜的宿主菌可克服此障碍.

To obtain bovine growth hormone protein （BST）, the recombinant bacteria were constructed and induced. Firstly, the DNAs encoding the mature protein of BST were amplified from pUCm-T-BST clones, and inserted into pET30a and pET29a, respectively. After identified, both vectors were transformed into E. coli BL21 （DE3） and RosettaTM （DE3） pLysS. Then the recombinant bacteria were construtted and induced, and desired proteins were examined. The results indicated that the recombinant vectors carried the correct target gene. Only the pET30a-BST/RosettaTM and pET29a-BST/RosettaTM could produce the desired fusion protein with a molecular weight of 29.5, 26.5 kD, accounting for 26% and 35% of the total bacterial protein, respectively. This suggest that some rare codons could restrain the gene expression of BST in E. coli and this problem can be overcome by the utilizing of suirable hosts.