本文關(guān)鍵詞：人DC-SIGN蛋白克隆及免疫學(xué)功能初探　出處：《重慶醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： DC-SIGN 轉(zhuǎn)染 COS7細(xì)胞 免疫熒光

【摘要】： DC-SIGN全稱是樹突狀細(xì)胞特異性細(xì)胞間粘附分子-3-結(jié)合非整合素分子(dendritic cell-specific intercellular adhesion molecule-3- grabbing nonintergrin,DC-SIGN),又稱CD209,表達(dá)在未成熟的樹突狀細(xì)胞(immature dendritic cells,iDCs)表面,主要作用是捕獲抗原和參與細(xì)胞間粘附。多種病原體通過自身不同結(jié)構(gòu)成分與DC-SIGN結(jié)合,從而進(jìn)入樹突狀細(xì)胞(dendritic cells,DCs),包括HIV、BCG、HCV、CMV、埃博拉病毒、利什曼原蟲等[1-4]。其中,結(jié)核分枝桿菌(Mycobacterium tuberculosis,Mtb)通過其胞壁成分—帶甘露糖帽的脂阿拉伯甘露聚糖(mannose-capped lipoarabinomannan, ManLAM)與DC-SIGN分子進(jìn)行強(qiáng)有力結(jié)合,介導(dǎo)Mtb黏附、感染DCs,并經(jīng)信號內(nèi)傳和交流,抑制iDCs成熟。Mtb隱藏在DCs中,通過某些機(jī)制,形成非溶酶體酸性隔室作為寄生屏障,從而逃避免疫監(jiān)視和藥物攻擊,潛伏下來,成為日后結(jié)核復(fù)發(fā)的因素[4、5]。 本研究首先預(yù)備以C57BL/6J小鼠為動物模型進(jìn)行小鼠DC-SIGN研究。對小鼠DCs體外培養(yǎng)和流式細(xì)胞儀分析后,我們發(fā)現(xiàn)隨著培養(yǎng)時間的推移,流式細(xì)胞儀所檢測到的小鼠CD209表達(dá)量逐漸減少,在培養(yǎng)的第3天,流式細(xì)胞儀檢測CD209的表達(dá)量極小,可以認(rèn)為是0。在2007年之前的文獻(xiàn)中,國外研究人員發(fā)現(xiàn)小鼠DC-SIGN相關(guān)蛋白組成復(fù)雜,具有多個同系物,在功能和結(jié)構(gòu)上同人差異很大,從而指出小鼠作為模型研究DC-SIGN的價值還須進(jìn)一步深入探討[6-8]。經(jīng)過討論和專家指導(dǎo),課題組將實驗對象改為人的DCs。從人外周血分離DCs,培養(yǎng)后提取總RNA,RT-PCR得到編碼DC-SIGN蛋白的基因片段,總長度1240bp,同表達(dá)綠色熒光的質(zhì)粒pEGFP-C1重組,獲得總長度為5938bp的重組質(zhì)粒(命名為:DS-pEGFP-C1)。Lipofectamine2000脂質(zhì)體轉(zhuǎn)染COS7細(xì)胞,表達(dá)DC-SIGN綠色熒光融合蛋白(命名為:DS-EGFP)。Real-Time PCR定量工具細(xì)胞中mRNA的轉(zhuǎn)錄量為4.52×1011copies/ml。標(biāo)記物綠色熒光蛋白連接在DC-SIGN胞內(nèi)氨基端,在假設(shè)不影響其胞外碳端的抗原結(jié)合功能的情況下,我們最后用免疫熒光法進(jìn)行了DS-EGFP與BCG結(jié)合的初步鑒定。 研究結(jié)果:用RT-PCR擴(kuò)增得到正確的目的基因片段并構(gòu)建成真核表達(dá)載體DS-pEGFP-C1,轉(zhuǎn)染COS7細(xì)胞,熒光定量PCR檢測DS-pEGFP-C1轉(zhuǎn)錄mRNA拷貝量是4.52×1011copies/ml,激光掃描共焦顯微鏡檢測證實DS-EGFP在COS7細(xì)胞表面表達(dá),同時具有攝取BCG的功能。 研究結(jié)論:本研究成功構(gòu)建人DC-SIGN綠色熒光融合蛋白的真核表達(dá)載體DS-pEGFP-C1,表達(dá)了融合蛋白DS-EGFP。標(biāo)記物連接在DC-SIGN蛋白氨基端,不影響DC-SIGN結(jié)合抗原。重組質(zhì)�？捎糜谶M(jìn)一步篩選DC-SIGN基因序列中最佳RNAi干擾片段,為有效的RNAi打下基礎(chǔ)。
[Abstract]:DC-SIGN is a dendritic cell-specific intercellular adhesion molecule-3-binding non-integrin molecule. Dendritic cell-specific intercellular adhesion molecule-3- grabbing. Nonintergrin. DC-SIGN, also called CD209, is expressed on the surface of immature dendritic cells, immature dendritic cells / iDCs. The main function is to capture antigens and participate in intercellular adhesion. Many pathogens bind to DC-SIGN through their own different structural components, thus entering dendritic cells into dendritic cells. DCsN, including HIV BCGV / CMV, Ebola virus, Leishmania protozoa, etc. [1-4] .Mycobacterium tuberculosis. The cell wall component of Mtb was mannose-capped lipoarabinomannan with mannose-capped capped arabinan with mannose cap. ManLAM) strongly binds to DC-SIGN molecules, mediates Mtb adhesion, infects DCS, and inhibits iDCs maturation. MTB is hidden in DCs through signal transmission and communication. Through some mechanisms, a non-lysosomal acidic compartment is formed as a parasitic barrier, thus avoiding immune surveillance and drug attacks, lurking down, and becoming a factor for the recurrence of tuberculosis in the future. [4,5]. In this study, C57BL / 6J mice were used as the animal model to study the DC-SIGN of mice. The mouse DCs was cultured in vitro and analyzed by flow cytometry. We found that with the passage of culture time, the expression of CD209 in mice detected by flow cytometry gradually decreased. On the third day of culture, the expression of CD209 detected by flow cytometry was very small. In the literature before 2007, foreign researchers found that mouse DC-SIGN related proteins have complex composition, have multiple homologues, and differ greatly in function and structure. It is pointed out that the value of studying DC-SIGN in mice as a model should be further explored. [After discussion and expert guidance, our group changed the experimental object into human DCs.isolate DCS from human peripheral blood and extract total RNA after culture. The gene fragment encoding DC-SIGN protein was obtained by RT-PCR. The total length of the gene was 1240bp. the plasmid pEGFP-C1 was recombined with the expression of green fluorescence. A total length of 5938 BP recombinant plasmid was obtained and transfected into COS7 cells with liposome: 1. DS-pEGFP-C1. Lipofectamine2000. Expression of DC-SIGN green fluorescent fusion protein. Real-Time. The transcription amount of mRNA in PCR quantitative tool cells was 4.52 脳 10 11 copes / ml. The green fluorescent protein (GFP) was linked to the amino terminal of DC-SIGN. Under the assumption that the antigen-binding function of the extracellular carbon terminal was not affected, we finally identified the binding of DS-EGFP to BCG by immunofluorescence method. Results: the correct target gene fragment was obtained by RT-PCR amplification and the eukaryotic expression vector DS-pEGFP-C1 was constructed and transfected into COS7 cells. The copy amount of DS-pEGFP-C1 transcribed mRNA detected by fluorescence quantitative PCR was 4.52 脳 1011copes / ml. Laser scanning confocal microscopy confirmed that DS-EGFP was expressed on the surface of COS7 cells and had the function of uptake of BCG. Conclusion: in this study, the eukaryotic expression vector DS-pEGFP-C1 of human DC-SIGN green fluorescent fusion protein was successfully constructed. The fusion protein DS-EGFP. was linked to the amino terminal of DC-SIGN protein. The recombinant plasmid can be used to further screen the best RNAi interference fragment in the DC-SIGN gene sequence and lay the foundation for effective RNAi.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392.111

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