重组人淋巴细胞激活基因3慢病毒载体的构建及其在小鼠成纤维细胞中的稳定表达  

Recombinant human LAG3 lentiviral vector and its stable expression in mouse fibroblast cells

作　　者：李欣悦 金月[1] 蔡秀 LI Xinyue;JIN Yue;CAI Xiu(Northern Jiangsu People's Hospital,Yangzhou,Jiangsu 225001,China;School of Medicine,Jiangsu University,Zhenjiang,Jiangsu 21201,China;The First people's Hospital of Yancheng,Yancheng,Jiangsu 224000,China)

机构地区：[1]江苏省苏北人民医院,江苏扬州225001 [2]江苏大学医学院,江苏镇江212013 [3]盐城市第一人民医院,江苏盐城224000

出　　处：《中国热带医学》2025年第3期328-332,共5页China Tropical Medicine

基　　金：江苏省卓越博士后计划项目(No.2022ZB895)。

摘　　要：目的 构建人淋巴细胞激活基因3(lymphocyte activation gene 3, LAG3)慢病毒重组表达载体,并转染小鼠成纤维细胞3T3以获得LAG3优势表达的单克隆细胞株。方法 提取人外周血单核细胞总RNA并逆转录为cDNA,以高保真DNA聚合酶PCR扩增LAG3胞外区和跨膜区基因序列。PCR产物采用限制性内切酶Eco RⅠ和NotⅠ双酶切后,与慢病毒载体pTSB-copGFP连接构建重组表达载体pTSB-LAG3-copGFP,并转化大肠埃希菌DH5α。PCR法筛选出阳性克隆菌,提取质粒DNA测序验证。pTSB-LAG3-copGFP重组载体与包装质粒psPAX2、pMD2.0G共同转染人胚肾293T细胞以包装、分泌病毒,收集病毒感染3T3细胞,采用嘌呤霉素筛选的同时有限稀释法获取LAG3稳定表达的3T3单克隆细胞。实时荧光定量PCR、免疫荧光法和流式细胞术分别检测细胞中LAG3 mRNA的转录及LAG3蛋白的表达。结果 重组pTSB-LAG3-copGFP慢病毒载体测序结果显示,扩增LAG3基因序列与GenBank中LAG3标准序列(序列号NM_002286.6)比对仅在跨膜区1 697 bp碱基位存在一处His氨基酸密码子同义突变。pTSB-LAG3-copGFP包装病毒感染3T3细胞经嘌呤霉素筛选,共获得20株LAG3+copGFP+-3T3单克隆细胞,均可检测出LAG3 mRNA的转录,其中MC-6单克隆细胞株LAG3转录水平最高。免疫荧光法与流式细胞术检测结果提示LAG3蛋白在MC-6中有效表达并分布于细胞膜和胞质细胞器膜上。结论 成功构建pTSB-LAG3-copGFP慢病毒载体,筛选获得淋巴细胞激活因子3高效表达的LAG3+copGFP+-3T3单克隆细胞株。为后续开展LAG3与乙型肝炎等慢性病毒感染性疾病发展的关系以及LAG3干预治疗等实验研究打下基础。Objective To construct a recombinant lentiviral expression vector for human lymphocyte activation gene 3(LAG3)and generation of monoclonal cell lines that preferentially express LAG3 by transfection of the vector into mouse fibroblast cells 3T3.Methods After extracting total RNA extracted from human peripheral blood mononuclear cells,the RNA is reversely transcribed into cDNA.The LAG3 extracellular and transmembrane region sequences are amplified by PCR using high-fidelity DNA polymerase.The PCR products are double-digested with the restriction endonucleases EcoRⅠand NotⅠ,then ligated with the lentiviral vector pTSB-copGFP to construct the recombinant expression vector pTSB-LAG3-copGFP,which is subsequently transformed into Escherichia coli DH5α.Positive clonal bacteria are selected by PCR,and the plasmids are extracted and sequenced for verification.The recombinant vector pTSB-LAG3-copGFP,along with packaging plasmids psPAX2 and pMD2.0G,are co-transfected into human embryonic kidney 293T cells to assemble and release virus particles,the virus infected 3T3 cells were collected.During the puromycin selection of infected 3T3 cells,the limited dilution method is used to obtain 3T3 monoclonal cells that stably express LAG3.Real-time fluorescent quantitative PCR,immunofluorescence and flow cytometry were utilized to verify the transcription of LAG3 mRNA and the expression of LAG3 protein respectively.Results Sequencing of the recombinant pTSB-LAG3-copGFP lentiviral vector plasmid reveals that the amplified LAG3 sequence contains a synonymous mutation in the His codon at nucleotide position 1697 bp within the LAG3 transmembrane region,which aligns with the standard LAG3 sequence(accession number NM_002286.6)in GenBank.The 3T3 cells infected by pTSB-LAG3-copGFP packaging virus screened with puromycin. A total of 20 LAG3+copGFP+-3T3 monoclonal cell lineswere obtained, all of which exhibited transcription of LAG3 mRNA. The monoclonal cell line MC-6 exhibits the highesttranscriptional level of LAG3. Effective expressi

关 键 词：重组慢病毒载体 淋巴细胞激活基因 免疫抑制性受体 慢性病毒感染 

分 类 号：R373[医药卫生—病原生物学]