携带小鼠KLF4基因重组慢病毒的构建及对RAW264.7细胞增殖的影响  

Construction and identification of mouse Krüppel-like factor 4 recombinant lentiviral expression vector and its effect on proliferation of RAW264.7 cells

作　　者：杨红艳[1] 李鑫 向国安 王如刚[1] 蔡卫红[1] 罗虎[1] 梁婧[1] 姬文婕[4] 

机构地区：[1]北京市疾病预防控制中心北京市预防医学研究中心,100013 [2]中国人民武装警察部队总医院急诊科 [3]中国人民武装警察部队总医院呼吸科 [4]中国人民武装警察部队后勤学院附属医院呼吸与重症医学科

出　　处：《天津医药》2018年第1期1-6,共6页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81441101;81570335)

摘　　要：目的构建小鼠Krüppel样因子4(KLF4)慢病毒表达载体,并建立KLF4过表达小鼠腹腔巨噬细胞RAW264.7细胞株。方法采用聚合酶链式反应(PCR)技术扩增目的基因KLF4后,构建重组质粒p LVX-KLF4,并通过PCR、双酶切和测序方法对其进行鉴定。重组质粒与p SPAX2、p MD2.G辅助质粒通过Lipofectamine~?3000共转染293T细胞,包装病毒并测定病毒滴度。将获得的慢病毒感染RAW264.7细胞,实时定量PCR法检测KLF4 m RNA的表达。分选流式细胞仪分选GFP阳性RAW264.7细胞。流式细胞术检测KLF4对RAW264.7细胞周期的影响。Ed U法检测KLF4对RAW264.7细胞增殖的影响。结果经PCR、双酶切鉴定和测序证实,成功构建了包含小鼠KLF4基因的慢病毒穿梭质粒,RT-PCR证实Lenti-KLF4感染的RAW264.7细胞中KLF4 m RNA表达高于未感染的对照组RAW264.7细胞(P<0.05)。初次浓缩后测定小鼠KLF4基因重组慢病毒的滴度为2.05×10~8TU/m L。分选出KLF4过表达的RAW264.7细胞。KLF4过表达的RAW264.7细胞周期变化显示为S期延长,G0/G1期缩短。Ed U检测显示KLF4过表达的RAW264.7细胞增殖活性增高。结论成功构建了KLF4的慢病毒表达载体,并建立KLF4过表达的RAW264.7细胞株,KLF4过表达促进了RAW264.7细胞的增殖。Objective To construct and identify a lentiviral vector carrying mouse Krüppel-like factor 4(KLF4) gene,and establish RAW264.7 cell line of peritoneal macrophages that over-expressed KLF4.MethodsKLF4 gene was clonedusing the measure of polymerase chain reaction(PCR). Then the recombinant transfer vector p LVX-KLF4(p LVX-KLF4-m CMV-Zs Green-PGK-Puro) was constructed. The p LVX-KLF4 was confirmed through PCR, restriction enzyme digestionand sequencing. The correct recombinant transfer vector together with its two helper virus vectors(ps PAX2 and p MD2.G)were cotransfected into the 293 T cells by Lipofectamine~?3000. The supernatant of 293 T was harvested to infect RAW264.7 cells. Flow cytometry(FCM) was used to test the viral titer of the expression level of green fluorescent protein. The expressionof KLF4 m RNA in RAW264.7 cells was measured by real-time PCR.ResultsThe restriction enzyme digestion, PCR andsequencing confirmed that the transfer lentiviral vector p LVX-KLF4 was constructed successfully. KLF4 m RNA was over-expressed in Lenti-KLF4 transfected RAW264.7 cells than that of wild type RAW264.7 cells(P<0.05). In transfectedRAW264.7 cells, KLF4 m RNA was over-expressed(P<0.05). The recombinant lentivirus of KLF4(Lenti-KLF4)titer was2.05×10~8 TU/m L measured by FCM.The flow cytometry results showed that the S phase fraction was prolonged and G0/G1 was arrested in the over-expressed KLF4 of RAW264.7 cells. The Ed U showed that the up-regulated expression of KLF4 gene stimulated the proliferation of RAW264.7 cells.ConclusionThe recombinant lentiviral vector, which can effectivelyexpress KLF4 m RNA, has been successfully constructed. The up-regulated KLF4 gene may increase the proliferation of RAW264.7 cells.

关 键 词：Krüppel样转录因子类 慢病毒感染 遗传载体 细胞周期 细胞增殖 Krüppel样因子4 

分 类 号：R392.12[医药卫生—免疫学]