BLNK基因慢病毒载体构建及其稳定表达的人乳腺癌MDA-MB-231细胞株的筛选  

Construction of BLNK gene lentivirus vector and screening of stably expressed human breast cancer cell line MDA-MB-231

作　　者：肖斌[1] 庄玉格 侯贤德 刘萍[1] 陈建芸 孙朝晖 李林海 XIAO Bin;ZHUANG Yuge;HOU Xiande;LIU Ping;CHEN Jianyun;SUN Zhaohui;LI Linhai(Department of Laboratory Medicine,Guangzhou General Hospital of PLA,Guangzhou 510010,China)

机构地区：[1]中国人民解放军广州总医院检验科,广州510010 [2]南方医科大学南方医院检验系,广州510515

出　　处：《实用医学杂志》2018年第13期2137-2140,共4页The Journal of Practical Medicine

基　　金：军队后勤科研项目(编号:CWH17C017);广州市科技计划项目(编号:201804010186)

摘　　要：目的构建人BLNK基因慢病毒载体,筛选稳定表达BLNK的人乳腺癌MDA-MB-231细胞株,为探讨BLNK对乳腺癌的调控作用提供细胞株模型。方法采用PCR法从3×Flag-BLNK质粒中扩增BLNK基因片段,连接到带有绿色荧光蛋白(GFP)标签的慢病毒载体表达质粒中,与包膜质粒pMD2.G、包装质粒psPAX2共转染293T包装细胞48 h,获得携带FLAG-BLNK的重组慢病毒,同时以慢病毒空载体FLAG-vector作为阴性对照。以重组慢病毒及阴性对照慢病毒分别感染人乳腺癌MDA-MB-231细胞,1周后在高倍镜100×视野下观察被感染细胞数及细胞状态,在荧光显微镜下通过观察GFP的表达情况,筛选稳定表达BLNK的细胞株。采用反转录实时荧光定量PCR(RT-qPCR)的方法和Western Blotting法检测该细胞系中BLNK基因的表达情况。结果 PCR成功扩增出BLNK基因片段,插入慢病毒表达载体后,利用核酸序列测定证实重组慢病毒质粒构建成功。通过慢病毒包装三质粒表达系统获得表达BLNK的重组慢病毒,感染MDA-MB-231细胞后,在荧光显微镜下能够明显观察到绿色荧光且细胞形态正常。RT-qPCR检测结果显示,感染表达BLNK慢病毒的MDA-MB-231细胞中BLNK表达水平显著高于对照组,差异有统计学意义(P<0.01)。Western blotting检测结果表明,筛选出的GFP阳性的人乳腺癌MDA-MB-231细胞株可过表达BLNK。结论成功构建了含BLNK基因的慢病毒表达载体,并筛选出稳定表达BLNK的人乳腺癌MDA-MB-231细胞株,为进一步明确乳腺癌的发生、发展机制奠定了基础。Objective To construct the lentivirus vector of human BLNK gene,screen the stably expressed human breast cancer cell line MDA-MB-231,and provide a cell model for exploring the regulation of BLNK on breast cancer.Methods The gene fragment of BLNK was cloned from the 3×Flag-BLNK plasmid by PCR and then was inserted into a lentivirus vector with GFP tag.The recombinant lentivirus plasmid,envelope plasmid pMD2.G and packaging plasmid psPAX2 were co-transfected into 293T cells for 48h to obtain the recombi-nant lentivirus carrying FLAG-BLNK,and the lentivirus empty vector(FLAG-vector)was served as a negative con-trol.The MDA-MB-231 cells were infected with the recombinant lentivirus and negative control lentivirus respec-tively.One week later,the quantity and status of the infected cells were observed under the high magnification with the 100×visual field and the expression of green fluorescent protein(GFP)was observed under the fluorescence microscope to screen for a cell line with stable expression of BLNK.The expression of BLNK gene in this cell line was detected by RT-qPCR and Western Blotting.Results The gene fragment of BLNK was successfully amplified by PCR.After inserting the lentivirus expression vector,the recombinant lentivirus plasmid was successfully con-structed,which was confirmed by the nucleic acid sequence determination.The BLNK-expressing recombinant len-tivirus was obtained by lentivirus-packaging three plasmid expression system.After infection,the green fluores-cence and normal cell morphology were observed under the fluorescence microscope.RT-qPCR showed that the BLNK expression in the MDA-MB-231 cells of human breast cancer infected with BLNK lentivirus was significantly higher than that in the control group(P<0.01).Western blotting results showed that the selected GFP-positive MDA-MB-231 cell line of human breast cancer over-expressed BLNK.Conclusion The lentivirus vector contain-ing BLNK gene is successfully constructed and the MDA-MB-231 cell line of human breast cancer stably expressing BL

关 键 词：BLNK 慢病毒载体 乳腺癌 MDA-MB-231 

分 类 号：R737.9[医药卫生—肿瘤]