过表达溶质载体家族1成员5和敲低慢病毒载体构建及稳定转染RAW264.7细胞株  

Construction of lentiviral vectors for solute carrier family 1 member 5 overexpression and knockdown and stably transfected RAW264.7 cell line

作　　者：郭大鑫 范苏苏 朱振东 侯建红 张旋 Guo Daxin;Fan Susu;Zhu Zhendong;Hou Jianhong;Zhang Xuan(Third People’s Hospital of Yunnan Province/Second Affiliated Hospital of Dali University,Kunming 650011,Yunnan Province,China;School of Pharmaceutical Sciences&Yunnan Key Laboratory of Pharmacology for Natural Products/College of Modern Biomedical Industry,Kunming Medical University,Kunming 650500,Yunnan Province,China)

机构地区：[1]云南省第三人民医院/大理大学第二附属医院,云南省昆明市650011 [2]昆明医科大学药学院暨云南省天然药物药理重点实验室/现代生物医药产业学院,云南省昆明市650500

出　　处：《中国组织工程研究》2025年第7期1414-1421,共8页Chinese Journal of Tissue Engineering Research

基　　金：云南省科技厅科技计划项目(202101BA070001-228),项目负责人:侯建红;昆明医科大学抗炎与免疫调节药物研究科技创新团队(CXTD2022003),项目负责人:张旋。

摘　　要：背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。BACKGROUND:Solute carrier family 1 member 5(SLC1A5)plays a potential role in a variety of diseases,but the exact mechanism of action is unclear.The construction of stable SLC1A5 overexpression and knockdown cell models can provide a powerful experimental tool for in-depth study of the exact role and mechanism of SLC1A5 in diseases and the discovery of potential therapeutic targets.OBJECTIVE:To construct lentiviral vectors for overexpression and knockdown of mouse SLC1A5 and establish stable transfected RAW264.7 cell lines,so as to provide an experimental foundation for further investigation of the role of SLC1A5 in inflammation.METHODS:Primers were designed and synthesized based on the SLC1A5 gene sequence,and the gene segment was amplified using polymerase chain reaction.Subsequently,the target gene segment was directionally inserted into the GV492 vector plasmid,which had been digested with Age I/Nhe I enzymes,to construct recombinant lentiviral plasmids.Positive clones were further selected,and their sequences were confirmed.The pHelper1.0 plasmid vector and pHelper2.0 plasmid vector,along with the target plasmid vector,was co-cultured with 293T cells for transfection,resulting in the production and titration of lentiviral stocks.Furthermore,RAW264.7 cells were cultured in vitro,and the working concentration of puromycin was determined.Lentiviruses were separately co-cultured with RAW264.7 cells,and transfection efficiency was determined by measuring fluorescence intensity.Stable transfected cells were selected using puromycin,and real-time fluorescence quantitative PCR and western blot assay were employed to assess the gene and protein expression levels of SLC1A5 in stably transfected cell lines.RESULTS AND CONCLUSION:(1)Sequencing results indicated a perfect match between the sequencing and target sequences,confirming the successful construction of recombinant lentiviral vectors.(2)The titer for the overexpression SLC1A5 lentivirus was 1×10~9 TU/mL,while the titer for the knockdown SLC1A5 lentivirus was 3×

关 键 词：慢病毒载体 溶质载体家族1成员5 SLC1A5 过表达 敲低 RAW264.7细胞 稳转细胞株 

分 类 号：R459.9[医药卫生—治疗学] R373[医药卫生—临床医学] R684.3