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  

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

作　　者：唐俭 陈旭昕 樊重阳 韩志海 TANG Jian;CHEN Xu-xin;FAN Chong-yang;HAN Zhi-hai(The Second School of Clinical Medicine,Southern Medical University,Guangzhou 510515,China;Department of Pulmonary and Critical Care Medicine,the Sixth Medical Center,PLA General Hospital)

机构地区：[1]南方医科大学第二临床医学院,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

出　　处：《天津医药》2019年第7期673-677,共5页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81300050);北京市自然科学基金项目(7182163);海军总医院创新培养基金项目(CXPY201417);军队后勤科研计划重点项目(BHJ16J011)

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(M OI)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pHelper1.0与pHelper2.0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Westernblot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2246bp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lenti-CV186-Cherry-PALM3的滴度测定为3×10^8TU/mL,感染NR8383的最佳MOI为20,慢病毒lenti-CV186-Cherry-PALM3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。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-Cherry- PALM3 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 3.0×10^8 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.

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 

分 类 号：R383.1[医药卫生—医学寄生虫学]