發(fā)布時(shí)間：2018-08-24 17:22

【摘要】：叉頭樣轉(zhuǎn)錄因子p3(Foxp3)是叉頭樣轉(zhuǎn)錄因子(Forkhead transcription factor)P亞家族的新成員,2001年由Brunkow等首次報(bào)道。研究表明,該基因特異性表達(dá)于CD4~+CD25~+調(diào)節(jié)性T細(xì)胞(CD4~+CD25~+ regulatory T cells, Tregs),對介導(dǎo)CD4~+CD25~+ Tregs的發(fā)育、外周表達(dá)以及功能維持起重要作用,被認(rèn)為是CD4~+CD25~+ Tregs特異性標(biāo)志物之一。Foxp3具有數(shù)個(gè)保守區(qū)域,包括C端的叉頭螺旋(forkhead/winged helix,FKH),C2H2鋅指結(jié)構(gòu),亮氨酸拉鏈,N端的富含脯氨酸的區(qū)域。因?yàn)檫@些特殊的保守區(qū)域,使其不能活化相應(yīng)基因的轉(zhuǎn)錄,因而能發(fā)揮抑制轉(zhuǎn)錄的作用,同是在與靶基因啟動(dòng)子和某些蛋白質(zhì)結(jié)合上亦起到重要作用。 近些年,以病毒介導(dǎo)的基因轉(zhuǎn)移在遺傳、腫瘤、免疫等方面成為最主要的研究方法之一,人們通過構(gòu)建Foxp3重組反轉(zhuǎn)錄病毒、Foxp3重組腺病毒等方法對該基因的作用機(jī)制以及與其相關(guān)的臨床應(yīng)用等展開了廣泛且深入的研究。實(shí)驗(yàn)發(fā)現(xiàn),通過反轉(zhuǎn)錄病毒轉(zhuǎn)染Foxp3基因能促使初始T細(xì)胞向CD4~+CD25~+ Treg細(xì)胞分化;過繼轉(zhuǎn)移表達(dá)Foxp3的CD4~+CD25-T細(xì)胞能防止小鼠自身免疫性胃炎和炎癥性腸病;急性和慢性移植排斥反應(yīng)患者腸黏膜Foxp3~+的天然調(diào)節(jié)性T細(xì)胞數(shù)量明顯低于健康人群以及炎癥感染患者。隨著研究的不斷深入,該基因在自身免疫性疾病、免疫耐受等方面的作用越來越受到關(guān)注,近期的研究還表明,Foxp3與活化T細(xì)胞核因子(Nuclear Factor of Activated T cells, NFAT)合作編排一種對免疫耐受性至關(guān)重要的遺傳程序,這將為研究免疫耐受的基礎(chǔ)機(jī)制開啟一扇新的大門。 目的:構(gòu)建攜帶Foxp3基因的慢病毒載體,為體外獲得CD4~+CD25~+ Tregs,進(jìn)一步研究轉(zhuǎn)Foxp3基因在治療自身免疫性疾病、抗移植排斥和防止腫瘤免疫逃逸中的作用奠定基礎(chǔ)。 方法:1.根據(jù)GeneBank上的Foxp3的序列,由Genescript公司化學(xué)合成目的基因Foxp3,并在基因的兩端插入酶切位點(diǎn)MluI和EcoRI,并將合成好的目的基因克隆于pUC57載體上。 2.Foxp3基因與慢病毒表達(dá)載體PWPXL-MOD經(jīng)MluI和EcoRI雙酶切,回收和純化。T4連接酶連接后轉(zhuǎn)化感受態(tài)DH5α,經(jīng)氨芐青霉素篩選,挑取單克隆菌落,作酶切鑒定及DNA序列測定,抽提陽性菌落PWPXL-Foxp3質(zhì)粒以包裝病毒。 3.磷酸鈣共沉淀法將重組慢病毒載體PWPXL-Foxp3和包裝質(zhì)粒共轉(zhuǎn)染人胚腎細(xì)胞系來源的293T細(xì)胞,收集轉(zhuǎn)染72h的病毒上清液,濃縮后逐孔稀釋感染293T細(xì)胞,利用流式細(xì)胞術(shù)測定病毒滴度。 結(jié)果: 1.重組慢病毒載體PWPXL-Foxp3序列經(jīng)MluI和EcoRI酶切鑒定,目的基因片段與理論計(jì)算片段一致。測序結(jié)果與GenBank中的基因序列比對顯示為序列一致。 2.通過感染293T細(xì)胞,測得的重組慢病毒滴度為3.3×108IU/ml。 結(jié)論: 1.成功構(gòu)建了攜帶大鼠Foxp3基因的重組慢病毒載體PWPXL-Foxp3。 2.重組慢病毒載體PWPXL-Foxp3和包裝質(zhì)�；旌衔锍晒Πb成高滴度的慢病毒顆粒。為進(jìn)一步研究轉(zhuǎn)基因在治療自身免疫性疾病、抗移植排斥和防止腫瘤免疫逃逸中的作用提供了理論和實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

【共引文獻(xiàn)】

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