天津医药

天津医药 ›› 2019, Vol. 47 ›› Issue (7): 673-677.doi: 10.11958/20190650

• 细胞与分子生物学 •    下一篇

大鼠 paralemmin-3基因重组慢病毒载体构建及其在肺泡巨噬细胞中的表达

唐俭，陈旭昕，樊重阳，韩志海   

  1. 1南方医科大学第二临床医学院（邮编 510515）；2中国人民解放军总医院第六医学中心呼吸与危重症医学科
  • 收稿日期:2019-03-11 修回日期:2019-04-23 出版日期:2019-07-15 发布日期:2019-08-01
  • 通讯作者: 韩志海 E-mail:zhihaihandoctor@163.com

Construction of recombinant lentiviral vectors with rat paralemmin-3 gene and its expression in alveolar macrophages

TANG Jian,CHEN Xu-xin,FAN Chong-yang,HAN Zhi-hai   

  1. 1 The Second School of Clinical Medicine, Southern Medical University, Guangzhou 510515, China; 2 Department of Pulmonary and Critical Care Medicine, the Sixth Medical Center, PLA General Hospital
  • Received:2019-03-11 Revised:2019-04-23 Published:2019-07-15 Online:2019-08-01

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摘要： 目的 构建大鼠 paralemmin-3（PALM3）基因重组慢病毒载体，探讨转染大鼠肺泡巨噬细胞系 NR8383细胞的最佳感染复数（MOI）值及目的基因 PALM3的表达情况。方法 采用聚合酶链反应技术（PCR）扩增鼠 PALM3基因片段，并将其克隆到 pCV186-Cherry载体上；经 PCR及 DNA测序鉴定后，将重组质粒 pCV186-Cherry-PALM3、辅助包装质粒 pHelper 1.0与 pHelper 2.0共转染至 293T细胞进行重组慢病毒 lenti-CV186-Cherry-PALM3的包装，并用荧光标记法行滴度的测定。将不同 MOI值（0、1、10、20、50）的 lenti-CV186-Cherry-PALM3转染 NR8383细胞，依据转染效率得到最佳 MOI 值，以此 MOI 值感染 NR8383细胞，采用 Western blot 法检测目的基因的表达情况。结果 通过 PCR扩增获得了大小约 2 246 bp的目的基因片段，并成功连接到 pCV186-Cherry重组质粒上。PCR及 DNA测序结果证实重组质粒 pCV186-cherry-PALM3 携带正确的鼠 PALM3 基因并成功包装重组慢病毒颗粒。重组慢病毒 lentiCV186-Cherry-PALM3 的滴度测定为 3×108 TU/mL，感染 NR8383 的最佳 MOI 为 20，慢病毒 lenti-CV186-CherryPALM3 感染后 NR8383 细胞中 PALM3 的蛋白表达水平显著上调（P＜0.05）。结论 本实验成功构建了含大鼠PALM3基因的重组慢病毒载体 lenti-CV186-Cherry-PALM3，并能在 NR8383细胞中高效表达，为进一步研究 PALM3对肺泡巨噬细胞极化的影响奠定了基础。

关键词: 巨噬细胞, 肺泡, paralemmin-3, 重组慢病毒载体, 急性肺损伤, 急性呼吸窘迫综合征

Abstract: Objective To construct a recombinant lentiviral vector containing rat paralemmin-3 (PALM3) gene and detect its expression in alveolar macrophages (NR8383 cells). Methods Rat PALM3 gene fragments were amplified and obtained by polymerase chain reaction (PCR), and were cloned to pCV186-Cherry. After identification of pCV186-CherryPALM3 with PCR and DNA sequencing, the recombinant vector, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T packaging cells to generate recombinant lentiviral vector lenti-CV186-Cherry-PALM3. The titer of lenti-CV186-Cherry-PALM3 was determined by fluorescence labeling method. NR8383 cells were transfected with different concentrations of lenti-CV186-Cherry-PALM3 to obtain the optimal MOI according to transfection efficiency. The protein expression of PALM3 was detected by Western blot assay following transfection with lenti-CV186-Cherry-PALM3 under the optimal MOI. Results Rat PALM3 gene fragments were obtained by PCR and ligated into the pCV186 plasmid vector.The PCR and DNA sequencing results demonstrated that the recombinant plasmid pCV186-Cherry-PALM3 was successfully constructed. The recombinant lentiviral vector was successfully packaged, and its titer was 3.0×108 TU/mL. The optimal MOI value for the NR8383 cells was 20. The result of Western blot assay showed that PALM3 protein expression was significantly enhanced after transfection with lenti-CV186-Cherry-PALM3 (P＜0.05). Conclusion The recombinant lentiviral vector lenti-CV186-Cherry-PALM3 has been constructed, and efficiently expressed in alveolar macrophages,which offers a basis for further research of the effect of PALM3 on macrophage polarization.

Key words: macrophages, alveolar, paralemmin-3, recombinant lentiviral vectors, acute lung injury, acute respiratory distress syndrome