牛凝乳酶基因的克隆与表达

Cloning and expression of the bovine chymosin gene

为了获得大量高纯度高活性的凝乳酶制剂,采用基因工程方法,从犊牛皱胃黏膜细胞中克隆得到凝乳酶基因,然后将此基因插入原核表达载体pTWlN1中,使之与几丁质结合域(CBD)一内含肽(intein)融合,从而获得原核表达质粒pTWIN1/EchybF2。经转化大肠杆菌BL21(DE3)后,在IPTG诱导下进行凝乳酶的表达。SDS-PAGE电泳分析和酶活性实验结果显示,CBD-intein-EchybF2融合蛋白在BL21(DE3)中获得高效表达,在低温诱导时主要以可溶性蛋白的形式存在,并具有凝乳活性。

Traditionally cheese is made mainly by the use of bovine chymosin, but the source of supply of this enzyme is limited due to decreasing number of calves. In order to obtain large quantities of pure and active enzyme, the gene of bovine chymosin was cloned from the rnucosal cells of the abomasum of calf. The DNA fragment was then inserted into the prokaryotic expression vector pTWIN1, resulting in the expression plasmid pTWIN1/EchybF2,wbich was further transformed into Eo coli BL21 （DE3）. Expression of the enzyme was performed at different temperatures with IPTG induction. SDS- PAGE and activity tests showed that chymosin was expressed in soluble form at a low temperature, and the recombinant protein had milk-clotting activities.