摘要
目的构建RBD+ODC及其突变体融合表达质粒,并进行验证。方法选取Ras下游效应分子Raf-1中能够与K-Ras蛋白相互作用的RBD+CRD氨基酸片段作为结合结构域,以ODC蛋白分子全长作为功能结构域。以正常人胰腺组织的c DNA为模板扩增相应的目的基因片段,应用分子克隆的方法通过在引物中引入GS片段使结合结构域和功能结构域相连并通过双轮PCR法在ODC功能位点引入突变,所获得的核苷酸片段均连入pc DNA3.1真核表达载体中。结果与结论经测序和Western Bolt验证,成功构建了RBD+ODC及突变体RBD+ODC的重组质粒,2种质粒均能够在人HEK293T细胞中正常表达。
Objective To construct RBD ＋ CRD and mutant RBD ＋ CRD expressing vectors. Methods The RBD ＋ CRD was chosen as the binding domain,and full length ODC as function domain,and the two domain were connected by molecular cloning. The mutant“RBD ＋ ODC”was constructed by two step PCR. All the nucleotide fragments were connected into pcDNA3. 1 vector. Results and Conclusion RBD ＋ CRD and mutant RBD ＋ CRD were constructed successfully by validation of sequencing and Western blot.
出处
《肿瘤基础与临床》
2016年第4期277-280,共4页
journal of basic and clinical oncology
基金
国家自然科学基金青年项目(编号:81401936)
