大鼠EZH2基因过表达质粒以及催化亚基缺失质粒构建及鉴定

Construction and identification for the EZH2 gene overexpression plasmid and EZH2 catalytic subunit SET domain deletion plasmid in rats

目的 构建特异性的大鼠EZH2过表达质粒以及EZH2催化亚基SET domain缺失质粒，并在293T细胞中评估其表达水平。 方法 提取大鼠心脏组织RNA，逆转录为cDNA，重叠PCR扩增催化亚基SET domain缺失的EZH2的cDNA，以其为模板进行PCR扩增，将PCR产物和载体双酶切，切胶回收目的片段，经T4连接酶连接，连接产物转入感受态细胞，涂板挑取单克隆摇菌测序，提取质粒，采用脂质体转染法转染293T细胞。 结果 Western Blot 法和RT-PCR法检测结果表明，质粒在293T细胞中实现EZH2基因的过表达。 结论 构建的2种质粒为进一步研究EZH2基因与心肌肥厚的关系及EZH2催化亚基的作用奠定了基础。

Objective To constructe the rat-specific EZH2 overexpression plasmid and the EZH2 catalytic subunit SET domain deletion plasmid, as well as assess their levels of expression in 293T cells. Methods The RNA extracted from rat cardiac tissue was reverse-transcribed into cDNA, the cDNA of catalytic subunit SET domain deleted EZH2 was amplified by overlap PCR, then the PCR amplification was achieved using the EZH2 gene as template. Products through PCR amplification and vector were cut with double enzyme, and then ligated the target fragments, purified by gel extraction, using T4 ligase. The ligated products were transferred into competent cells. After plate coating, monoclonal picking, shaking cultivation and sequencing procedures, the plasmids were extracted and transfected into 293T cells using liposome-mediated method. Results Western Blot and RT-PCR assays showed recombinant plasmids DNA can successfully overexpress EZH2 gene in 293T cells. Conclusions Two plasmids were successfully constructed. This study laid the foundation for the further study on the relationship between EZH2 and myocardial hypertrophy, and the function of EZH2 catalytic subunit.