陈思思,周恒,王继春,谢菁,周艳丽,卢帅,卢力
CHEN Sisi,ZHOU Heng,WANG Jichun,XIE Jing,ZHOU Yanli,LU Shuai,LU Li

• 1:武汉大学人民医院心内科
• 2:武汉大学心血管病研究所
• 3:心血管病湖北省重点实验室

摘要(Abstract)：

目的:构建大鼠β-抑制蛋白2(β-arrestin2)基因过表达慢病毒载体并鉴定。方法:取Pubmed数据库中大鼠β-arrestin2的c DNA序列合成引物,采用聚合酶链式反应(PCR)扩增获取大鼠β-arrestin2基因;采用琼脂糖凝胶电泳鉴定,回收PCR产物并对其进行纯化;取限制性内切酶Bam HI/Age I酶切载体质粒和纯化后的PCR产物,将二者实施连接后转化感受态细胞,挑取菌落进行PCR鉴定,并对PCR鉴定阳性的克隆进行测序和比对抽提质粒;将β-arrestin2重组质粒与慢病毒辅助包装质粒共转染至293T细胞,获取病毒粗提液并进行扩增、纯化,稀释法测定病毒滴度、荧光显微镜下观察慢病毒感染效率。结果:PCR扩增所得产物大小为1 274 bp,与Pubmed数据库中该基因片段大小一致;阳性转化子的PCR产物大小为728 bp,对比Pubmed数据库中大鼠Arrb2基因序列,上下游引物基因序列大小与转化子PCR产物结果一致;慢病毒包装完成后病毒滴度为5×1011TU/L,病毒感染效率较高。结论:成功构建了携带大鼠β-arrestin2基因的慢病毒载体,目的基因在转染细胞内能稳定高表达。
Objective: To construct β-arrestin2 gene in rats overexpressing recombinant lentivirus vector and identify it. Methods: The target gene β-arrestin2 in rats was amplified by PCR and identified by agarose gel electrophoresis according to the cDNA sequence of rat β-arrestin2 in Pubmed database. The gels were recovered and PCR products were purified. The purified PCR products and digested plasmid vector were connected by using restriction endonuclease BamHI and AgeI and added to competent cells. The cultured colonies were identified by PCR and sequenced. β-arrestin2 recombinant plasmid and lentivirus packaging plasmid were co-transfected into 293 T cells,and the obtained lentivirus was amplified and purified. Virus titer was detected by dilution assay,and the transfection efficiency was observed under the fluorescence microscope. Results: According to the results of agarose gel electrophoresis,the length of PCR product was 1 274 bp. Then,the target gene product was validated by PCR and agarose gel electrophoresis. The results showed that the length of PCR product of the effective transformant was 728 bp. Through analyzing the Arrb2 gene in the Pubmed database,it was found that the length of transformant PCR product was in accordance with the gene size between the primers. Moreover,the virus titer was 5 × 1011 TU/L and the transfection efficiency was high under fluorescence microscope. Conclusion: The lentivirus vector carrying rat β-arrestin2 gene is constructed successfully,and the target gene is highly expressed in the transfected cells.

关键词(KeyWords)： 质粒;慢病毒属;β-抑制蛋白2基因;基因过表达;聚合酶链式反应
plasmids;lentivirus;β-arrestin2 gene;gene overexpression;polymerase chain reaction(PCR)

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金青年科学基金项目(81600226)

作者(Author): 陈思思,周恒,王继春,谢菁,周艳丽,卢帅,卢力
CHEN Sisi,ZHOU Heng,WANG Jichun,XIE Jing,ZHOU Yanli,LU Shuai,LU Li

DOI: 10.19367/j.cnki.2096-8388.2020.09.007

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