本文選題：CD83 + sCD83��； 參考：《蘇州大學(xué)》2009年碩士論文

【摘要】： CD83是表達(dá)于成熟樹(shù)突狀細(xì)胞(DC)和活化的B、T淋巴細(xì)胞的重要功能分子,對(duì)T細(xì)胞在胸腺中的發(fā)育起著重要的作用。未成熟DC表面雖然缺少CD83的表達(dá),但在其細(xì)胞內(nèi)可檢測(cè)到CD83蛋白。膜型CD83(membraneCD83,mCD83)蛋白可以水解形成可溶性CD83(solubleCD83,sCD83),存在于正常人血清中。sCD83能夠影響DC的成熟,以及DC介導(dǎo)的T細(xì)胞增殖,具有免疫抑制活性。sCD83能夠顯著抑制實(shí)驗(yàn)性自身免疫病病變的發(fā)生,而且還能有效地抑制移植排斥反應(yīng)。有研究認(rèn)為與CD83相互作用的分子存在于單核和CD8+T細(xì)胞亞群。最近,一些研究發(fā)現(xiàn)了一些特殊的CD83+細(xì)胞,單核細(xì)胞在IFN-α作用下誘導(dǎo)成CD83+CD14+細(xì)胞群,該細(xì)胞群不具有典型DC的表型,卻具有相似的吞噬功能,而單核細(xì)胞在GM-CSF作用下誘導(dǎo)成CD14lowCD83+ DCSIGN-細(xì)胞群,因此CD83的生物學(xué)特性及其信號(hào)傳導(dǎo)途徑尚待進(jìn)一步研究。 也已表明許多免疫分子的特異性抗體,可模擬或阻斷該分子的信號(hào)。該類抗體可為深入研究免疫分子的生物學(xué)效應(yīng)及分子機(jī)制提供重要的手段。同時(shí)通過(guò)特異抗體增強(qiáng)或減弱協(xié)同信號(hào)轉(zhuǎn)導(dǎo),對(duì)免疫功能低下或異常升高的疾病具有干預(yù)作用。因此,CD83分子轉(zhuǎn)基因細(xì)胞的構(gòu)建及功能性單克隆抗體的研制在基礎(chǔ)研究及臨床應(yīng)用中具有重要價(jià)值。本論文共分為兩個(gè)部分。 1.人CD83轉(zhuǎn)基因細(xì)胞株的構(gòu)建 用TRIZOI從成熟的DC中提取總mRNA,逆轉(zhuǎn)錄成cDNA,以特異性引物進(jìn)行擴(kuò)增,然后進(jìn)行雙酶切裝入pIRES2-EGFP載體,進(jìn)行測(cè)序,測(cè)序正確后用脂質(zhì)體轉(zhuǎn)染L929細(xì)胞,篩選穩(wěn)定表達(dá)株。pIRES2-EGFP載體中既有G418抗性標(biāo)記基因,又有綠色熒光蛋白GFP報(bào)告基因,對(duì)于轉(zhuǎn)基因細(xì)胞具有雙重的篩選作用,大大提高了所篩細(xì)胞的陽(yáng)性率。通過(guò)流式細(xì)胞儀反復(fù)篩選,獲得一株穩(wěn)定表達(dá)CD83的轉(zhuǎn)基因細(xì)胞株,通過(guò)RT-PCR能夠檢測(cè)到目的基因的表達(dá)。 2.一株鼠抗人CD83單克隆抗體的研制及其特性鑒定 以高表達(dá)膜型CD83分子的轉(zhuǎn)基因細(xì)胞株L929/CD83為免疫原,采用B淋巴細(xì)胞融合技術(shù),將免疫小鼠脾臟細(xì)胞與小鼠骨髓瘤細(xì)胞SP2/0進(jìn)行融合。以人CD83基因轉(zhuǎn)染小鼠成纖維細(xì)胞株L929/CD83及293/CD83為陽(yáng)性篩選細(xì)胞,經(jīng)反復(fù)篩選及克隆化培養(yǎng),獲得一株穩(wěn)定分泌特異性鼠抗人CD83單抗的雜交瘤細(xì)胞株,命名為9D8�？焖俣ㄐ栽嚰埛ǚ治�,9D8重鏈為IgG1,輕鏈為κ鏈。經(jīng)體外連續(xù)傳代培養(yǎng),液氮凍存,復(fù)蘇后生長(zhǎng)良好,抗體分泌性能穩(wěn)定。染色體數(shù)目分析顯示,雜交瘤細(xì)胞株的染色體在100條以上。采用本室建立的腹水誘生法制備單抗,腹水形成陽(yáng)性率約為80%以上,腹水的收獲量平均為5 mL/只。經(jīng)Protein G親和層析柱分離純化,腹水中抗體蛋白的得率為1.5~2.0 mg/mL。純化抗體用于間接免疫熒光檢測(cè)的用量為0.2~1μg/1x106細(xì)胞。經(jīng)Western blot分析,9D8能與CD83分子特異性結(jié)合,表明9D8是CD83的特異性單抗。該單抗能識(shí)別人B淋巴瘤細(xì)胞株Daudi,骨髓瘤細(xì)胞株RPMI8226成熟的DC及活化的T淋巴細(xì)胞。 總之,CD83編碼區(qū)全長(zhǎng)基因的成功克隆、重組載體的構(gòu)建和穩(wěn)定表達(dá)人CD83蛋白細(xì)胞株的建立為對(duì)該基因的進(jìn)一步研究奠定了良好的基礎(chǔ)。鑒于CD83在疾病診斷、免疫排斥和自身免疫性疾病中起著重要的作用,CD83轉(zhuǎn)基因細(xì)胞的成功構(gòu)建及鼠抗人CD83功能性單克隆抗體9D8成功研制為以后的研究工作打下了堅(jiān)實(shí)的基礎(chǔ)。
[Abstract]:CD83 is an important functional molecule expressed in mature dendritic cells (DC) and activated B, T lymphocytes. It plays an important role in the development of T cells in the thymus. Although the immature DC surface lacks the expression of CD83, the CD83 protein can be detected in its cells. The membrane type CD83 (membraneCD83, mCD83) protein can be hydrolyzed to form a soluble CD83. BleCD83, sCD83), the existence of.SCD83 in normal human serum can affect the maturation of DC and the proliferation of DC mediated T cells. The immunosuppressive activity.SCD83 can significantly inhibit the occurrence of experimental autoimmune disease and also effectively inhibit the graft rejection. The molecular interaction of CD83 is found to exist in mononuclear and C D8+T cell subsets. Recently, some special CD83+ cells have been found. Monocytes are induced into CD83+CD14+ cell groups under the action of IFN- alpha. The cell group does not have a typical DC phenotype, but has a similar phagocytic function, while monocyte is induced by GM-CSF to form a CD14lowCD83+ DCSIGN- cell group, so the biological special of CD83 is specific. Sex and its signal transduction pathways are still to be further studied.
It has also been shown that specific antibodies against many immune molecules can mimic or block the molecules' signals. This kind of antibody can provide an important means to further study the biological effects and molecular mechanisms of immune molecules. At the same time, it can interfere with diseases with low immune function or abnormal increase by enhancing or weakening the cooperative signal transduction by specific antibodies. Therefore, the construction of CD83 molecular transgenic cells and the development of functional monoclonal antibodies are of great value in basic research and clinical application. This paper is divided into two parts.
Construction of 1. CD83 transgenic cell lines
Using TRIZOI to extract total mRNA from mature DC, reverse transcriptase cDNA, amplify with specific primers, then carry out double enzyme cut into pIRES2-EGFP vector, sequence and transfect L929 cells with liposome after sequencing, and select both G418 resistance marker gene and GFP reporting basis of green fluorescent protein in stable expression strain.PIRES2-EGFP vector. Because of the double screening effect on the transgenic cells, the positive rate of the screened cells was greatly improved. A transgenic cell line with stable expression of CD83 was obtained through the flow cytometry, and the expression of the target gene could be detected by RT-PCR.
2. preparation and characterization of a mouse anti human CD83 monoclonal antibody
The transgenic cell line L929/CD83 with high expression of membrane CD83 molecule was used as immunogen, and B lymphocyte fusion technique was used to fuse the spleen cells of the mice with the murine myeloma cells SP2/0. The transfected mouse fibroblast cell line of human CD83 gene, L929/CD83 and 293/CD83 as positive screening cells, was screened and cloned by repeated screening and culturing. A hybridoma cell line that secretes the specific anti human CD83 monoclonal antibody, named 9D8. fast qualitative test paper method, 9D8 heavy chain IgG1, light chain kappa chain. After continuous subculture in vitro, liquid nitrogen is frozen, after resuscitation, the antibody secretion performance is stable. Chromosome number analysis shows that the chromosomes of hybridoma cell lines are 100 The positive rate of ascites was more than 80%, and the average harvest of ascites was 5 mL/, and purified by Protein G affinity chromatography column. The yield of antibody protein in ascites was 1.5~2.0 mg/mL. purified antibody used for indirect immunofluorescence detection using 0.2~1 mu g/1x106 cells. RN blot analysis shows that 9D8 can specifically bind to CD83 molecules, indicating that 9D8 is a specific monoclonal antibody to CD83. This monoclonal antibody can identify human B lymphoma cell line Daudi, myeloma cell line RPMI8226 mature DC and activated T lymphocyte.
In conclusion, the successful cloning of the full-length gene in the CD83 coding region, the construction of the recombinant vector and the establishment of a stable expression of the human CD83 protein cell line lay a good foundation for further research on this gene. In view of the important role of CD83 in the diagnosis of disease, immune rejection and autoimmune diseases, the successful construction of CD83 transgenic cells and the successful construction of the transgenic cells, The successful development of mouse anti human CD83 functional monoclonal antibody 9D8 laid a solid foundation for future research.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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