含心脏阿霉素反应蛋白基因的重组慢病毒过表达和RNA干扰载体的构建及鉴定  

Construction and identification of recombinant lentivirus overexpression and RNA interference vector containing cardiac adriamycin reactive protein gene

作　　者：梁春梅 许旭三 余华军 周霞 温霞 李友 KOJIC Snezana 汪亚君 马国达 LIANG Chunmei;XU Xusan;YU Huajun;ZHOU Xia;WEN Xia;LI You;KOJIC Snezana;WANG Yajun;MA Guoda(Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases,Affiliated Hospital,Guangdong Medical University,Zhanjiang 524001,China;Experiment Animal Center,Guangdong Medical University,Zhanjiang 524023,China;Institute of Molecular Genetics and Genetic Engineering,University of Belgrade,Belgrade 11010,Serbia;Clinical Research Center,Affiliated Hospital,Guangdong Medical University,Zhanjiang 524001,China;College of Life Science & Technology,Heilongjiang Bayi Agricultural University,Daqing 163319,China)

机构地区：[1]广东医科大学附属医院广东省衰老相关心脑疾病重点实验室,广东湛江524001 [2]广东医科大学实验动物中心,广东湛江524023 [3]塞尔维亚贝尔格莱德大学分子遗传学及遗传工程研究所,塞尔维亚贝尔格莱德11010 [4]广东医科大学附属医院临床医学研究中心,广东湛江524001 [5]黑龙江八一农垦大学生命科学与技术学院,黑龙江大庆163319

出　　处：《吉林大学学报（医学版）》2019年第4期766-771,I0002,共7页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(81670252,81770034,81571157);黑龙江省教育厅基金资助课题(12521379);广东省科技厅自然科学基金资助课题(2015A030313523);中国-塞尔维亚科技合作委员会第3届例会项目资助课题(3-13);2016年度中共广东省委组织部与广东省人力资源与社会保障厅“扬帆计划”培养高层次人才项目资助课题(4YF17006G)

摘　　要：目的:构建心脏阿霉素反应蛋白(CARP)基因重组慢病毒过表达和RNA干扰载体并进一步包装慢病毒,为研究CARP在阿霉素(ADR)心肌病中的作用及机制奠定基础。方法:将PCR扩增的大鼠CARP基因序列和设计合成的短发夹RNA(shRNA)序列分别插入GV-358和GV-248表达载体,行DNA测序鉴定。采用重组CARP过表达和shRNA表达穿梭载体与Helper 1.0和Helper 2.0辅助包装质粒共转染人胚肾细胞HEK293T,进行病毒包装和扩增,采用终点稀释法测定病毒滴度。采用重组慢病毒感染HEK293T细胞,荧光显微镜观察绿色荧光强度。采用重组慢病毒感染H9C2细胞,分为对照组、Scramble RNA组、CARP过表达组和shRNA CARP组,Western blotting法检测各组细胞中CARP蛋白表达水平。结果:基因测序,CARP过表达及shRNA载体序列与原设计序列完全相符。载体转染HEK293T细胞后,在荧光显微镜下可见绿色荧光蛋白表达。包装的CARP过表达及shRNA病毒感染H9C2细胞后,与对照组比较,CARP过表达组细胞中CARP蛋白表达水平升高5.3倍(P=0.01),shRNA CARP组细胞中CARP蛋白表达水平降低53%(P=0.02)。结论:成功构建了CARP过表达和特异性shRNA慢病毒表达载体并获得高感染效率的过表达和RNA干扰慢病毒,后者在H9C2细胞中能够干预CARP表达。Objective: To construct the recombinant lentiviral vector containing cardiac adriamycin responsive protein (CARP) gene and small-hairpin RNA (shRNA) targeting CARP gene and to pack the lentivirus,and to lay the foundation for further study on the function and mechanism of CARP in adriamycin(ADR)-induced cardiomyopathy.Methods:After the rat CARP gene was amplified by PCR and shRNAs targeting CARP were designed and synthesized,they were inserted into the shuttle plasmids GV-358 or GV-248,respectively.After confirmed by sequencing,the recombinant shuttle vectors containing CARP gene or shRNA and auxiliary packaging plasmid Helper 1.0 and Helper 2.0 were co-transfected into the HEK293T cells for virus packaging and amplification;the viral titer was detected by end-point dilution.The HEK293T cells were infected with the recombinant lentiviruses,and the green fluoresence intensity was observed by fluorescence mircroscope.The H9C2 cells were infected with the recombinant lentivirues and divided into control group,CARP overexpression group and shRNA group;Western blotting method was used to detect the CARP expression levels in the cells in various groups.Results:The DNA sequencing results showed that the sequences of CARP overexpression and shRNA vectors were in accordance with the designed sequences.The expression of green fluorescence protein was seen under fluorescence microscope after transfection of the vectors of the HEK293T cells.After infection of the H9C2 cells,the expression level of CARP protein in CARP overexpression group was 5.3 times higher than that in control group (P=0.01);while it was down-regulated by 53% in CARP shRNA group compared with control group (P=0.02).Conclusion: The lentivirus expression vectors carrying CARP or shRNA targeting CARP are successfully constructed and the lentiviruses obtained could significantly interfere the expression of CARP in the H9C2 cells.

关 键 词：心脏阿霉素反应蛋白 RNA干扰 慢病毒 H9C2细胞 

分 类 号：Q78[生物学—分子生物学]