copGFP和Puro^(R)双标记基因慢病毒融合表达载体的构建及应用  

Construction and application of lentivirus overexpression vector with two labeling genes fused with CopGFP and Puro^(R)

作　　者：张浩 朱文琦 赵思捷 李永利 侯俊 卞成蓉 鲍春梅 卫桢 孙杰 李波 李伯安 ZHANG Hao;ZHU Wen-qi;ZHAO Si-jie;LI Yong-li;HOU Jun;BIAN Cheng-rong;BAO Chun-mei;WEI Zhen;SUN Jie;LI Bo;LI Bo-an(Center for Clinical Laboratory,the Fifth Medical Center,General Hospital of Chinese PLA,Beijing 100039,China;Infectious Medicine Institute,the Fifth Medical Center,General Hospital of Chinese PLA,Beijing 100039,China)

机构地区：[1]解放军总医院第五医学中心检验科,北京100039 [2]解放军总医院第五医学中心感染病研究所,北京100039

出　　处：《中国应用生理学杂志》2022年第2期108-112,153,共6页Chinese Journal of Applied Physiology

基　　金：军队重大科研项目(AWS14C003)。

摘　　要：目的:构建具有绿色荧光蛋白(copGFP)和嘌呤霉素抗性基因(PuroR)融合表达双筛选标记的慢病毒过表达载体,检测其嘌呤霉素抗性和绿色荧光蛋白表达的特性。方法:从pCDH-CMV-MCS-copGFP载体中扩增copGFP编码区DNA序列,从pLKO.1载体中扩增Puro^(R)编码区DNA序列,运用重组PCR方法,扩增copGFP与Puro^(R)基因融合编码序列并克隆至经BamHⅠ+SalⅠ双酶切的pCDH-CMV-MCS-copGFP载体片段中,构建含copGFP和Puro^(R)融合表达双筛选标记的慢病毒过表达载体;载体的融合标签序列进行测序确证;将该载体用辅助包装质粒PLP1、PLP2、VSVG在293T细胞中包装成慢病毒后感染肝癌细胞MHCC97H,检测感染细胞对嘌呤霉素的抵抗作用以及绿色荧光蛋白的表达情况;为验证该载体表达外源目的基因的有效性,将Sp1编码区DNA序列插入该载体中包装成慢病毒,用对照及表达Sp1的慢病毒感染肝癌细胞MHCC97H,感染细胞经1 mg/ml嘌呤霉素筛选7 d后获得稳定感染细胞株,提取稳定感染细胞的总RNA及总蛋白,分别运用RT-qPCR和Western blot方法检测Sp1在对照及表达Sp1的慢病毒感染的肝癌MHCC97H细胞中的mRNA和蛋白表达水平的差异。结果:成功构建含copGFP和Puro^(R)融合表达双筛选标记的慢病毒过表达载体;该载体与辅助质粒包装出的慢病毒感染肝癌细胞后,感染细胞同时具有嘌呤霉素抗性和表达绿色荧光蛋白特性;将Sp1编码序列插入该载体,包装慢病毒并肝癌细胞,Sp1的mRNA水平对照细胞相比分别升高3.3倍,蛋白水平升高2.2倍(P<0.01)。结论:成功构建含copGFP和Puro^(R)融合表达双筛选标记的慢病毒过表达载体,该载体编码的融合双标记基因具有嘌呤霉素抗性和绿色荧光蛋白表达的特性,可高水平表达长片段的目的基因。Objective:To construct the lentivirus overexpression vector with two label genes fused with CopGFP and Puro^(R) and to detect the emission of green fluorescence as well as resistance to puromycin in liver cancer cells infected with lentivirus packaged with the above vector.Methods:Firstly,two fragments containing copGFP and Puro^(R) coding sequences were amplified from pCDH-CMV-MCS-copGFP and pLKO.1 respectively;secondly,the two amplified regions were fused with each other by recombinant PCR;thirdly,the fusion DNA fragment was cut and inserted into pCDH-CMV-MCS-copGFP vector,which was linearized with the same restriction endonuclease as used to digest fusion DNA fragment:BamHⅠand SalⅠ.The fusion region in the constructed vector was confirmed by DNA sequencing.The checked vector was co-transfected with package assistant plasmids,namely PLP1,PLP2 and VSVG into in 293T cells and the culture supernatant was subjected to centrifuge and infect liver cancer MHCC97H cells,which were then used to detect their resistance to puromycin(infected cells were treated with 1 mg/ml puromycin for 7 days after infection)and to observe green fluorescence emission in microscope.To determine its efficiency in expressing foreign target protein,the Sp1 coding region was inserted into the MCS sites of the vector,and Sp1 mRNA and protein expression levels were compared with the vehicle vector by RT-qPCR and Western blot.Results:The lentivirus overexpression vector with two label genes fused with CopGFP and Puro^(R) was successfully constructed,and the liver cancer cells infected with lentivirus packaged with the vector expressing two labeling genes fused with CopGFP and Puro^(R) showed both emission of green fluorescence and resistance to puromycin simultaneously,while cells containing with the vector inserted with Sp1 coding region improved Sp1 mRNA level with 3.3 fold and protein level with 2.2 fold higher in comparison with cells containing the vehicle vector(P<0.01).Conclusion:The fused label genes consisting of copGFP and Puro^(R)

关 键 词：桡足类绿色荧光蛋白 嘌呤霉素抗性基因 融合表达 慢病毒载体 特异性蛋白1 

分 类 号：Q78[生物学—分子生物学]