纤维蛋白原Bβ链变异基因的体外重组

Extrinsic Recombination of Mutagenic Gene in ? Chain of Fibrinogen B

目的:采用基因重组技术将可能与血栓形成有关的纤维蛋白原Bβ链羧基端的KGD结构置换为EGS结构,提供一种简单方便的构建变异克隆的方法。方法:在含有正常BβcDNA的pMLP质粒(简称p668)的溶液中,加入变异引物、筛选引物和dNTP。在T4聚合酶和T4连接酶的作用下,合成除变异碱基外,其他碱基均与原正常Bβ链互补的变异Bβ链,经过两次在不同菌系的大肠杆菌中的表达与筛选,获取变异克隆。结果:由于变异克隆引入了BstBI这个新的酶切位点,可通过电泳证实变异克隆构建的成功与否,本研究经电泳及DNA序列分析证实重组体确系已含变异EGS结构的克隆。结论:本方法为一构建变异克隆的分子生物学技术,可用于任何克隆的构建。本研究成功地获得了纤维蛋白原Bβ链羧基端KGD转换为EGS的变异体。

Objective:To use a gene recombination technology to contribute a mutagenic clone of β chain of fibrinogen B (to substi-tute EGS from KGD). Methods:To add mutagenic primer,selected primer,dNTP,T4 polymorase and ligase to pMLP plas-mid which included normal B β cDNA. It could synthesize a new B β cDNA chain with a few mutagenic bases. The mutagen-ic clone could be obtained through two times of transformation in different E. coli lines. Results: As a BstBI cut site was in-troduced in the mutagenic clone,it was easy to determine which was the new mutagenic clone through electrophoresis. The mutagenic clone had been obtained through the DNA sequence analysis. Conclusion:This is a good method to contribute mu-tagenic clone. It can be used to any clone contribution. When got the B β fibrinogen mutagenic clone successfully, it will be useful for the function research of B β fibrinogen KGD area.