大鼠miR-133a-3p慢病毒载体的构建及稳转株建立  

Construction of Rat miR-133a-3p Lentivirus Vector and Establishmentof Stable Strain

作　　者：张志蕊 陈耀平[2] ZHANG Zhirui;CHEN Yaoping(Ningxia Medical University,Yinchuan 750004,China;General Hospital of Ningxia MedicalUniversity,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学,银川750004 [2]宁夏医科大学总医院,银川750004

出　　处：《宁夏医科大学学报》2022年第6期567-572,579,共7页Journal of Ningxia Medical University

基　　金：国家自然科学基金项目(81760267)。

摘　　要：目的构建大鼠micro RNA-133a-3P(miR-133a-3p)过表达及干扰慢病毒载体,筛选建立稳定表达miR-133a-3p的阴茎海绵体平滑肌细胞(CCSMC)稳转细胞株。方法原代及传代培养CCSMCs,并进行细胞免疫荧光染色鉴定。PCR技术扩增相应目的基因片段并进行酶切,回收后与目的基因连接,产物转化细菌感受态细胞,对阳性克隆测序行对比分析后,使用三质粒包装系统将含有目的基因的重组质粒和辅助包装质粒共转染至293T细胞进行慢病毒包装及纯化,并用荧光法测定病毒滴度。设置miR-133a-3p过表达组、miR-133a-3p干扰组、阴性对照组和空白对照组,在感染复数为10的条件下感染CCSMCs。倒置荧光显微镜下观察各组绿色荧光蛋白的表达情况,浓度为2μg·mL^(-1)嘌呤霉素筛选出稳定表达miR-133a-3p的CCSMCs。qRT-PCR检测慢病毒转染后CCSMCs内miR-133a-3p的表达。结果原代培养的大鼠CCSMCsα-SMA阳性细胞率达90%以上。经测序分析证实重组慢病毒构建正确;过表达及干扰慢病毒滴度分别为3×10^(8)和1×10^(9) TU·mL^(-1);qRT-PCR结果显示,与阴性对照组相比,miR-133a-3p过表达组miR-133a-3p的相对表达量增高,miR-133a-3p干扰组miR-133a-3p的相对表达量降低(P均<0.01)。结论本实验成功构建了miR-133a-3p过表达及干扰慢病毒表达载体,对大鼠CCSMCs进行转染和筛选后,可快速、高效、低成本获得过表达及干扰miR-133a-3p的稳转株。Objective To construct rat micro RNA-133a-3p(miR-133a-3p)overexpressed and interfering lentiviral vector.Stable cell lines of corpus cavernosum muscle cell(CCSMC)expressing miR-133a-3p were screened and established.Methods CCSMCs were cultured in primary and subculture and identified by immunofluorescence staining.PCR amplification corresponding purpose gene and enzyme digestion,recycled connection with the purpose gene,the product into bacterial cells,the positive cloning sequencing line after comparison and analysis,using three plasmid packaging systems will contain the purpose gene recombinant plasmid and auxiliary packaging plasmid transfection to 293T cells lentivirus packaging and purification of fluorescence determination of virus drops degree.miR-133a-3p overexpressed group,miR-133a-3p interfering group,negative control group,and control group were set up.CCSMCs were infected when the number of infections was 10.The expression of green fluorescent protein in each group was observed under an inverted fluorescence microscope,and the CCSMCs with stable expression of miR-133a-3p were screened out at a concentration of 2μg·mL^(-1) puromycin.qRT-PCR was used to detect the expression of miR-133a-3p in CCSMCs after lentivirus transfection.Results The rate of CCSMCsα-SMA positive cells in primary culture was more than 90%.Sequencing analysis confirmed that the recombinant lentivirus was constructed correctly.The titers of overexpression and interfering lentivirus were 3×10^(8) and 1×10^(9) TU·mL^(-1),respectively.The results of qRT-PCR showed that the relative expression of miR-133a-3p in the over-expression group was significantly higher than that in the control group,while the relative expression of miR-133a-3p in the interfering group was significantly lower than that in the control group(P all<0.01).Conclusion The expression vector of miR-133a-3p overexpression and interference lentivirus was successfully constructed.After transfection and screening of rat CCSMCs,the stable strain of miR-133a-3p overexpr

关 键 词：micro RNA-133a-3p CCSMCs 慢病毒感染 

分 类 号：R373[医药卫生—病原生物学]