β-catenin基因克隆及pcDNA3.1(-)myc/hisA载体的构建

Cloning of β-catenin gene and the recombination of pcDNA3.1(-) myc/hisA vector

目的:通过RT-PCR技术获得β-catenin基因,构建重组的克隆载体,再进行亚克隆构建重组pcD-NA3.1(-)myc/hisA表达载体,为进一步研究细胞增殖的信号传导提供基础。方法:应用TRIZO试剂盒从胰岛瘤NIT细胞中提取总RNA,经RT-PCR获得β-catenin目的基因,胶回收后的PCR产物与pGEM-T克隆载体相连,酶切鉴定后再亚克隆至pcDNA3.1(-)myc/hisA表达载体,重组的表达载体转化E.coli工程菌JM109,获得大量的含目的基因pcDNA3.1(-)myc/hisA表达载体。结果:经过氨苄青霉素的筛选、重组质粒的单酶切和双酶切鉴定表明,β-catenin成功连入载体pcDNA3.1(-)表达载体中,同时得到了大量含目的基因的克隆子。结论:成功构建pcDNA3.1(-)myc/hisA-βcatenin的重组质粒,为进一步研究β-catenin功能和Wnt细胞信号传导通路奠定了基础。

Objective： To obtain the β-catenin gene through RT-PCR,then inserted it to the vector of pcDNA3.1（-） myc / hisA in terms of subcloning method.The construction of recombinated pcDNA3.1（-） myc / hisA vector would provide the basis for the further study of cell proliferation signal transduction pathway.Methods： The total RNA was extracted from NIT cells by using the TRIZOL kit,RT-PCR was used to synthesize β-catenin gene,the gel extraction production of β-catenin gene was firstly integrated to the pGEM-T vector,and then subcloned to the vector pcDNA3.1（-） myc / hisA after that it was verified by enzyme digestion.Finally,the recombinated pcDNA3.1（-） myc / hisA was used to transformated the E.Coli JM109 for the amplification of recombinated pcDNA3.1（-） myc / hisA expression vector.Results： The elimination of Ampicilin and the restriction enzyme digestion results both indicated that the recombination of the pcDNA3.1（-） had been successful and a considerable amount of recombinated vector had been obtained.Conclusion： With the success of recombinated pcDNA3.1（-） myc / hisA-beta catenin,some fundamental work has been done for the function of β-catenin and Wnt signal transduction pathway.