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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,为研究纤维蛋白原Bβ链的
OBJECTIVE: To provide a simple and convenient method for constructing variant clones by replacing the KGD structure of carboxyl end of fibrinogen Bβ chain, which may be related to thrombosis, with EGS structure by gene recombination technique. Methods: In the solution of pMLP plasmid containing normal Bβ cDNA (p668 for short), mutagenic primers were added to screen for primers and dNTPs. Under the action of T4 polymerase and T4 ligase, the mutant Bβ chain whose bases are complementary to the original normal Bβ chain except for the variant bases was synthesized, and after two times of expression and screening in different strains of E. coli, Get variant clones. Results: The new cloning site of BstBI was introduced into the mutant clones. The success of the construction of the mutant clones can be verified by electrophoresis. In the present study, electrophoresis and DNA sequence analysis confirmed that the recombinant clones contained the mutant EGS structure. Conclusion: This method is a molecular biology technique for constructing variant clones, which can be used to construct any clone. In this study, we successfully obtained the variant of KGD to convert C-terminal KGD of fibrinogen to EGS. In order to study the Bβ chain of fibrinogen