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

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pLVX-KLF4,并通过PCR、双酶切和测序方法对其进行鉴定.重组质粒与pSPAX2、pMD2.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 mRNA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.EdU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 mRNA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2.05×108 TU/mL.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.EdU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. Methods KLF4 gene was cloned using the measure of polymerase chain reaction (PCR). Then the recombinant transfer vector pLVX-KLF4 (pLVX-KLF4-mCMV-ZsGreen-PGK-Puro) was constructed. The pLVX-KLF4 was confirmed through PCR, restriction enzyme digestion and sequencing. The correct recombinant transfer vector together with its two helper virus vectors (psPAX2 and pMD2.G) were cotransfected into the 293T cells by Lipofectamine? 3000. The supernatant of 293T 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 expression of KLF4 mRNA in RAW264.7 cells was measured by real-time PCR. Results The restriction enzyme digestion, PCR and sequencing confirmed that the transfer lentiviral vector pLVX-KLF4 was constructed successfully. KLF4 mRNA was over-expressed in Lenti-KLF4 transfected RAW264.7 cells than that of wild type RAW264.7 cells (P<0.05). In transfected RAW264.7 cells, KLF4 mRNA was over-expressed (P<0.05). The recombinant lentivirus of KLF4(Lenti-KLF4)titer was 2.05×108 TU/mL 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 EdU showed that the up-regulated expression of KLF4 gene stimulated the proliferation of RAW264.7 cells. Conclusion The recombinant lentiviral vector, which can effectively express KLF4 mRNA, has been successfully constructed. The up-regulated KLF4 gene may increase the proliferation of RAW264.7 cells.