天津医药

天津医药 ›› 2018, Vol. 46 ›› Issue (1): 1-6.doi: 10.11958/20170967

• 细胞与分子生物学 •    下一篇

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

杨红艳 1，李鑫 2，向国安 3，王如刚 1，蔡卫红 1，罗虎 1，梁婧 1，姬文婕 4   

  1. 1 北京市疾病预防控制中心、北京市预防医学研究中心（邮编 100013）；2 中国人民武装警察部队总医院急诊科，3 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
  • 收稿日期:2017-09-15 修回日期:2017-11-01 出版日期:2018-01-15 发布日期:2018-01-16
  • 通讯作者: 李鑫 E-mail:lixin9207@sina.com
  • 作者简介:杨红艳（1969），女，副主任医师，硕士，主要从事职业健康监护、职业病诊断研究

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

YANG Hong-yan1, LI Xin2, XIANG Guo-an3, WANG Ru-gang1, CAI Wei-hong1, LUO Hu1, LIANG Jing1, JI Wen-jie4   

  1. 1 Beijing Center for Diseases Prevention and Control, Beijing Research Center for Preventive Medicine, Beijing 100013, China; 2 Emergency Center, 3 Department of Respiratory Medicine, General Hospital of Chinese People Armed Police Forces; 4 Department of Respiratory Medicine, the Affiliated Hospital of Logistics University of Chinese People Armed Police Forces
  • Received:2017-09-15 Revised:2017-11-01 Published:2018-01-15 Online:2018-01-16
  • Contact: Xin LI E-mail:lixin9207@sina.com

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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。

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

Abstract: 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 overexpressed 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.

Key words: Krüppel-like transcription factors, lentivirus infections, genetic vectors, cell cycle, cell proliferation , Krüppel-like factor 4