突变型Cdh1基因真核表达质粒的构建及鉴定

Construction and identification of a mutated-Cdh1 eukaryotic expressing vector

背景:细胞周期末期促进复合物调节亚基Cdh1的活性受到磷酸化调节,磷酸化的Cdh1不能与细胞周期末期促进复合物结合,从而抑制细胞周期末期促进复合物的活性。目的:构建突变型Cdh1基因真核表达质粒及鉴定。方法:采用RT-PCR方法,从大鼠海马组织扩增出Cdh1基因编码序列。通过限制性内切酶EcoRⅠ和XbaⅠ双酶切PCR回收产物和pBluescript质粒将Cdh1基因克隆到pBluescript质粒上。根据定点突变技术原理,以含Cdh1编码序列的pBluescript-Cdh1质粒为模版,针对Cdh1第40、151、163位丝氨酸(S)和第121位苏氨酸(T)设计4对突变引物,将4个氨基酸位点全部突变为丙氨酸(A)。最后通过测序鉴定。结果与结论:经电泳鉴定PCR扩增产物大小约为1500bp,包括Cdh1基因完整的编码序列、编码序列两端引入的酶切位点以及KOZAK序列,与预期相符。重组质粒pBluescript-Cdh1经EcoRⅠ和XbaⅠ双酶切鉴定与预期结果符合。DNA测序比对发现Cdh1(BC162059.1)编码序列第930位碱基A在重组质粒pBluescript-Cdh1上突变为G,但氨基酸无变化,为同义突变,其他DNA序列无突变。经测序鉴定pBluescript-Cdh1-4A40、121、151、163突变质粒第40、121、151、163位氨基酸全部突变为丙氨酸。提示实验成功构建磷酸化位点突变型Cdh1基因真核表达质粒。

BACKGROUND： The activity of anaphase promoting complex-Cdh1 is regulated by phosphorylation. Phosphorylated Cdh1 cannot be combined with anaphase promoting complex, thereby inhibiting the activity of the anaphase promoting complex. OBJECTIVE： To construct and identify a mutated-Cdh1 eukaryotic expressing vector. METHODS： The entire coding sequence of the Cdh1 gene was amplified from rat hippocampal mRNA by reverse transcription-PCR. Then the PCR product of Cdh1 was cloned into pBluescript plasmid by double digestion with restriction endonucleases EcoR1 and Xba1, and ligation. Based on site-directed mutagenesis, the pBluescript-Cdh1 plasmid containing Cdh1 coding sequence was used as a template. The 40, 151, 163 serine （S） and 121 threonine （T） in Cdh1 gene was mutated to alanine （A） by multiple PCR with four pairs of mutated primers. The mutated Cdh1 vector was identified by DNA sequencing. RESULTS AND CONCLUSION： The PCR product was about 1 500 bp by electrophoresis, including the entire coding sequence of Cdh1, restriction sites at both ends of Cdh1 and KOZAK sequence. The recombinant pBluescript-Cdh1 plasmid was identified by digestion with restriction endonuclease EcoR1 and Xba1, which was consistent with the expected results. DNA sequencing showed that A at the 930th base of Cdh1 （BC162059.1） coding sequence was mutated to G in the recombinant plasmid of pBluescript-Cdh1. But the sequence of amino acids was not affected. The 40, 121, 151, 163 amino acids in pBluescript-Cdh1-4A40, 121, 151, 163 mutant plasmids were all mutated to alanine. The mutated Cdh1 gene expressing plasmid at phosphorylation site was successfully constructed, which provides a good foundation for further studies of Cdh1 gene function.