pEGFP-N1-BRCC3重组质粒的构建、鉴定及其表达  

The construction, identification and expression of pEGFP-N1-BRCC3 recombinant plasmid

作　　者：程芯育 钟海凤 徐绍业 张聪慧 韦睿妮 邵晓云[4] CHEN Xin -yu;ZHONG Hai-feng;XU Shao-ye;ZHANG Cong-hui;WEI Rui-ni;SHAO Xiao-yun(Graduate School of Guilin Medical University,Guilin 541004,Guangxi,China)

机构地区：[1]桂林医学院研究生学院,广西桂林541004 [2]桂林医学院生物技术学院,广西桂林541004 [3]桂林医学院科学实验中心,广西桂林541004 [4]桂林医学院人体解剖学教研室、广西脑与认知神经科学重点实验室,广西桂林541004

出　　处：《广东医学》2019年第19期2705-2709,共5页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:31660269);广西自然科学基金青年基金项目(编号:2016GXNSFBA380098);广西脑与认知神经科学重点实验室自由探索课题(编号:GKLBCN-20170107)

摘　　要：目的构建pEGFP-N1-BRCC3真核表达重组质粒,鉴定并检测其在HEK293细胞中的表达。方法根据去泛素化酶BRCC3(人的同源物称为BRCC36)基因cDNA克隆基因序列和表达载体增强绿色荧光蛋白真核表达载体(pEGFP-N1)质粒上的多克隆位点设计引物,利用RT-PCR从小鼠脑组织中提取BRCC3全长基因作为目的DNA片段,选择HindⅢ/SalⅠ内切酶将目的片段与pEGFP-N1真核表达载体进行双酶切,然后用T4连接酶连接,导入大肠杆菌经过转化、抗性筛选、质粒提取等过程获得融合的重组质粒,并再次双酶切电泳后初步筛选出可能正确的重组质粒,进而寄送公司进行DNA测序分析,通过DNAMAN软件比对,鉴定出目的基因BRCC3成功插入pEGFP-N1的重组质粒,以脂质体介导法转染HEK293细胞,通过Western blot检测BRCC3蛋白的表达。结果 DNA测序结果显示,pEGFP-N1-BRCC3重组质粒中目的DNA序列及方向完全正确,开放阅读框正确无误,表明重组pEGFP-N1-BRCC3质粒构建成功,将其转染HEK293细胞后,Western blot检测BRCC3蛋白表达明显增加。结论成功构建了pEGFP-N1-BRCC3真核表达重组质粒,并能在HEK293真核细胞中有效表达,为深入研究BRCC3基因在神经生物学领域的功能提供了实验基础。Objective To construct and verify the eukaryotic expression plasmid pEGFP-N1-BRCC3, and to detect its expression in HEK293 cells. Methods Based on the cDNA gene sequence of the deubiquitin enzyme BRCC3(human homologue BRCC36) and the multiple clone sites of the expression vector pEGFP-N1 plasmid, two specific pairs primers were designed and synthesized. The full-length gene of BRCC3 was extracted from mouse brain and amplified by RT-PCR, which was as target DNA fragment. This target fragment and pEGFP-N1 vector were all digested with the restriction enzymes HindⅢ/SalⅠ, then ligated with T4 ligase under the right conditions. The BRCC3 fragment was inserted into the downstream of pEGFP-N1 vector promoter. The recombinant plasmid was identified by restriction enzyme digestion and gene sequencing. And it was transfected to HEK293 cells with lipofectamine, the expression of BRCC3 protein was detected by Western blot. Results The results of sequence analysis of the inserted part of pEGFP-N1-BRCC3 plasmid showed that the direction, sequence and open reading frame of BRCC3 were correct, which indicated that the recombinant pEGFP-N1-BRCC3 plasmid was successfully constructed. After transfection of HEK293 cells, the expression of BRCC3 protein was significantly increased compared with the control group. Conclusion The eukaryotic expression plasmid of pEGFP-N1-BRCC3 is successfully constructed. It can be expressed effectively in HEK293 eukaryotic cells, which provides an experimental basis for further study of the function of BRCC3 gene in the field of neurobiology.

关 键 词：BRCC3基因 绿色荧光蛋白 基因重组 真核表达质粒 

分 类 号：Q78[生物学—分子生物学] R34[医药卫生—基础医学]