携带大鼠Foxp3基因慢病毒载体的构建及慢病毒包装的研究

发布时间：2018-08-24 17:22

【摘要】：叉头样转录因子p3(Foxp3)是叉头样转录因子(Forkhead transcription factor)P亚家族的新成员,2001年由Brunkow等首次报道。研究表明,该基因特异性表达于CD4~+CD25~+调节性T细胞(CD4~+CD25~+ regulatory T cells, Tregs),对介导CD4~+CD25~+ Tregs的发育、外周表达以及功能维持起重要作用,被认为是CD4~+CD25~+ Tregs特异性标志物之一。Foxp3具有数个保守区域,包括C端的叉头螺旋(forkhead/winged helix,FKH),C2H2锌指结构,亮氨酸拉链,N端的富含脯氨酸的区域。因为这些特殊的保守区域,使其不能活化相应基因的转录,因而能发挥抑制转录的作用,同是在与靶基因启动子和某些蛋白质结合上亦起到重要作用。近些年,以病毒介导的基因转移在遗传、肿瘤、免疫等方面成为最主要的研究方法之一,人们通过构建Foxp3重组反转录病毒、Foxp3重组腺病毒等方法对该基因的作用机制以及与其相关的临床应用等展开了广泛且深入的研究。实验发现,通过反转录病毒转染Foxp3基因能促使初始T细胞向CD4~+CD25~+ Treg细胞分化;过继转移表达Foxp3的CD4~+CD25-T细胞能防止小鼠自身免疫性胃炎和炎症性肠病;急性和慢性移植排斥反应患者肠黏膜Foxp3~+的天然调节性T细胞数量明显低于健康人群以及炎症感染患者。随着研究的不断深入,该基因在自身免疫性疾病、免疫耐受等方面的作用越来越受到关注,近期的研究还表明,Foxp3与活化T细胞核因子(Nuclear Factor of Activated T cells, NFAT)合作编排一种对免疫耐受性至关重要的遗传程序,这将为研究免疫耐受的基础机制开启一扇新的大门。目的:构建携带Foxp3基因的慢病毒载体,为体外获得CD4~+CD25~+ Tregs,进一步研究转Foxp3基因在治疗自身免疫性疾病、抗移植排斥和防止肿瘤免疫逃逸中的作用奠定基础。方法:1.根据GeneBank上的Foxp3的序列,由Genescript公司化学合成目的基因Foxp3,并在基因的两端插入酶切位点MluI和EcoRI,并将合成好的目的基因克隆于pUC57载体上。 2.Foxp3基因与慢病毒表达载体PWPXL-MOD经MluI和EcoRI双酶切,回收和纯化。T4连接酶连接后转化感受态DH5α,经氨苄青霉素筛选,挑取单克隆菌落,作酶切鉴定及DNA序列测定,抽提阳性菌落PWPXL-Foxp3质粒以包装病毒。 3.磷酸钙共沉淀法将重组慢病毒载体PWPXL-Foxp3和包装质粒共转染人胚肾细胞系来源的293T细胞,收集转染72h的病毒上清液,浓缩后逐孔稀释感染293T细胞,利用流式细胞术测定病毒滴度。结果: 1.重组慢病毒载体PWPXL-Foxp3序列经MluI和EcoRI酶切鉴定,目的基因片段与理论计算片段一致。测序结果与GenBank中的基因序列比对显示为序列一致。 2.通过感染293T细胞,测得的重组慢病毒滴度为3.3×108IU/ml。结论: 1.成功构建了携带大鼠Foxp3基因的重组慢病毒载体PWPXL-Foxp3。 2.重组慢病毒载体PWPXL-Foxp3和包装质粒混合物成功包装成高滴度的慢病毒颗粒。为进一步研究转基因在治疗自身免疫性疾病、抗移植排斥和防止肿瘤免疫逃逸中的作用提供了理论和实验基础。
[Abstract]:Foxp3, a new member of the Forkhead transcription factor P subfamily, was first reported by Brunkow et al. in 2001. Studies have shown that the gene is specifically expressed in CD4~+CD25~+ regulatory T cells (Tregs), which mediates the development of CD4~+CD25~+ Tregs. Peripheral expression of the gene is as follows: Foxp3 has several conserved domains, including forkhead/winged helix (FKH), C2H2 zinc finger structure, leucine zipper, and N-terminal proline-rich domains. Because of these special conserved domains, Foxp3 does not activate the corresponding genes. Transcription, therefore, inhibits transcription and plays an important role in binding to target gene promoters and certain proteins.
In recent years, virus-mediated gene transfer has become one of the most important research methods in genetics, tumor, immunity and so on. Through the construction of recombinant retrovirus Foxp3 and recombinant adenovirus Foxp3, extensive and in-depth studies have been carried out on the mechanism of the gene and its related clinical applications. Foxp3 gene transfection by retrovirus could promote the differentiation of initial T cells into CD4~+CD25~+Treg cells; adoptive transfer of CD4~+CD25-T cells expressing Foxp3 could prevent autoimmune gastritis and inflammatory bowel disease in mice; the number of natural regulatory T cells in intestinal mucosa of patients with acute and chronic transplant rejection was significantly lower than that of healthy people. With the deepening of research, the role of Foxp3 in autoimmune diseases and immune tolerance has attracted more and more attention. Recent studies have also shown that Foxp3 cooperates with activated T cell nuclear factor (NFAT) to compile a genetic program essential to immune tolerance. This will open a new door for studying the basic mechanism of immune tolerance.
AIM: To construct lentiviral vector carrying Foxp3 gene and to obtain CD4~+CD25~+Tregs in vitro, and to further study the role of Foxp3 gene transfection in the treatment of autoimmune diseases, Anti-transplant rejection and prevention of tumor immune escape.
Methods: 1. According to the sequence of Foxp3 on GeneBank, the target gene Foxp3 was synthesized chemically by Genescript Company. MluI and EcoRI were inserted at the two ends of the gene, and the synthesized target gene was cloned into pUC57 vector.
2. Foxp3 gene and lentiviral expression vector PWPXL-MOD were digested by MluI and EcoRI, recovered and purified. T4 ligase was linked and transformed into DH5a. Monoclonal colonies were screened by ampicillin and identified by enzyme digestion and DNA sequencing. The positive colonies PWPXL-Foxp3 plasmid was extracted to package the virus.
3. The recombinant lentiviral vector PWPXL-Foxp 3 and packaged plasmid were co-transfected into 293 T cells derived from human embryonic kidney cell line by calcium phosphate co-precipitation method. The supernatant of the virus transfected for 72 hours was collected and diluted to infect 293 T cells one by one. The virus titer was determined by flow cytometry.
Results: 1. The recombinant lentiviral vector PWPXL-Foxp3 was identified by MluI and EcoRI digestion, and the target gene fragment was identical with the theoretical fragment. The sequencing results were consistent with the gene sequence alignment in GenBank.
2. by infecting 293T cells, the recombinant lentivirus titer was 3.3 * 108IU/ml..
Conclusion: 1. the recombinant lentiviral vector PWPXL-Foxp3. carrying rat Foxp3 gene was successfully constructed.
2. The mixture of recombinant lentiviral vector PWPXL-Foxp3 and packaged plasmid was successfully packaged into high titer lentiviral particles, which provided theoretical and experimental basis for further study of the role of transgene in the treatment of autoimmune diseases, Anti-transplant rejection and prevention of tumor immune escape.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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