赖氨酸去甲基化酶2A全长及截短重组质粒构建及其在细胞中的定位

Construction of recombinant plasmid encoding full-length and truncated mutations of KDM2A and subcellular distribution of recombinant proteins

目的:构建赖氨酸去甲基化酶2A(lysine demethylase 2A,KDM2A)全长及截短重组表达质粒,并初步探究其在前列腺癌CWR22Rv1细胞中的定位及机制。方法:根据KDM2A的mRNA编码序列及蛋白结构域特点设计合成KDM2A全长及截短表达序列的引物,以含有KDM2A全长编码序列的质粒作为模板,通过PCR扩增目的片段,再用限制性内切酶NotⅠ和XbaⅠ进行双酶切,连接转化后,挑取单克隆菌落扩增,并提取质粒进行酶切鉴定及测序比对。将序列比对正确的KDM2A全长及截短重组质粒转染到HEK293细胞中,进行Western Blot实验检测KDM2A全长及截短蛋白在HEK293细胞中的表达。将构建好的KDM2A全长及截短重组质粒转染到前列腺癌CWR22Rv1细胞中,进行免疫荧光染色检测外源的KDM2A全长及截短蛋白在细胞中的定位。结果:构建了KDM2A全长及截短重组表达质粒;重组质粒能够在细胞中表达;KDM2A全长蛋白主要分布在细胞核,少量分布在细胞质;C1截短蛋白分布在细胞核;N1、C2截短蛋白分布在细胞质。结论:KDM2A蛋白的核定位序列位于564-888位氨基酸之间,本实验为后续深入研究KDM2A在肿瘤中的作用及分子机制提供了实验基础。

Objective:To construct recombinant expression plasmids expressing full-length and truncated of lysine demethylase 2 A(KDM2 A),and to explore its location in prostate cancer CWR22 Rv1 cells.Methods:Based on the full-length CDS sequence and domain characteristics of KDM2 A,the primers of full-length and truncated KDM2 A were designed and synthesized.The expression vector containing the full-length KDM2 A coding sequence was used as a template by PCR to amplify the target fragments.The endonucleases NotⅠand XbaⅠwere double-digested.After the ligation and transformation,the constructed plasmids were digested,identified and sequenced,which proved that the full-length and truncated plasmid of KDM2 A was successfully constructed.The plasmids were transfected into HEK293 cells,and Western Blot experiments were performed to detect the expression of full-length KDM2 A and truncated proteins in HEK293 cells.The constructed full-length and truncated expression plasmids of KDM2 A were transfected into prostate cancer CWR22 Rv1 cells,and the confocal experiment was performed to detect the localization of the full-length and truncated mutants of KDM2 A.Results:KDM2 A full-length and truncated expression plasmids were constructed,and its protein expression was verified in HEK293.Immunofluorescence experiments showed that the full-length KDM2 A was distributed in the nucleus and cytoplasm.C1 truncated protein was distributed in the nucleus.N1 and C2 truncated protein were distributed in the cytoplasm.Conclusion:In prostate cancer CWR22 Rv1 cells,564-888 aa of KDM2 A may play a vital role in its nuclear localization,which provides experimental basis for investigation of KDM2 A’s function and molecular mechanism in tumor.