关键词: 基因克隆, 融合基因, 双片段连接法, 周期蛋白E, 周期蛋白依赖性激酶2

Abstract: In order to reduce random mutation that was accompanied by DNA amplification in vitro, PCR was only used to attach a short linker sequence to the 5′ end of CDK2 (cyclin dependent kinase 2). Because of the interference of double BamHⅠ sites, cyclin E and CDK2 sequences could not be inserted into vector in an orderly way. Therefore, two gene fragments were linked together into vector, so as to construct the fusion gene. Employing similar strategy, CDK2 mutant sequence was used to displace the corresponding wild type sequence of the fusion gene in order to rebuild its mutant. After transformation of competent Escherichia coli with both double fragments ligation product, several colonies were screened out and some target clones were identified successfully. Double fragments ligation broke the rigorous demand of restriction sites of step by step ligation, and expanded the application of enzyme cleave-ligation in DNA assembly. On these grounds, it was convenient to clone fusion gene and its mutant by replacement of DNA elements with the assistance of the identified recombinant plasmid.

Key words: gene clone, fusion gene, double fragments ligation, cyclin E, CDK2