本文選題：CD80 + 嵌合抗體��； 參考：《蘇州大學(xué)》2008年碩士論文

【摘要】： 本研究在自行成功研制的鼠抗人CD80阻斷型單克隆抗體(4E5)的基礎(chǔ)上,采用嵌合抗體構(gòu)建方法,在真核細(xì)胞CHO中實(shí)現(xiàn)穩(wěn)定表達(dá),并對(duì)嵌合抗體生物學(xué)功能進(jìn)行初步研究。 第一部分:CD80鼠-人嵌合抗體的構(gòu)建及表達(dá) 從4E5雜交瘤細(xì)胞中抽取總RNA,常規(guī)逆轉(zhuǎn)錄,應(yīng)用簡(jiǎn)并引物進(jìn)行PCR擴(kuò)增。根據(jù)重、輕鏈基因序列設(shè)計(jì)引物,應(yīng)用SMART-PCR方法擴(kuò)增含信號(hào)肽序列的V_H和V_L基因。同時(shí)用特異性引物從pIRES/hu5C11質(zhì)粒中擴(kuò)增人IgG1γ鏈的Fc、CH1及κ鏈的Cκ基因。再利用TP-PCR方法分別將Fc、CH1和含信號(hào)肽序列的V_H序列進(jìn)行拼接得到嵌合重鏈,Cκ與含信號(hào)肽序列的V_L序列進(jìn)行拼接獲得嵌合輕鏈。構(gòu)建共表達(dá)嵌合重、輕鏈的重組質(zhì)粒pIRES/ch4E5。pIRES/ch4E5重組質(zhì)粒用脂質(zhì)體法轉(zhuǎn)染293T細(xì)胞,經(jīng)FCM檢測(cè)培養(yǎng)上清中嵌合抗體的瞬時(shí)表達(dá)后,再轉(zhuǎn)染CHO細(xì)胞,經(jīng)G418加壓篩選,獲取持續(xù)穩(wěn)定分泌嵌合抗體的CHO-ch4E5細(xì)胞。研究結(jié)果表明:成功構(gòu)建了含嵌合重、輕鏈基因的真核表達(dá)載體pIRES/ch4E5,并分別在293T及CHO細(xì)胞中得到瞬時(shí)表達(dá)及穩(wěn)定表達(dá)。 第二部分:CD80鼠-人嵌合抗體生物學(xué)功能的初步研究 大量收集CHO-ch4E5細(xì)胞無(wú)血清培養(yǎng)上清,經(jīng)protein G親和層析法純化及Lowry法定量,SDS-PAGE鑒定嵌合抗體的純度及分子量,FCM分析嵌合抗體對(duì)多種腫瘤細(xì)胞Daudi、SH2-1及U937等細(xì)胞膜型CD80分子的識(shí)別。選擇天然高表達(dá)CD80分子的人B淋巴瘤細(xì)胞株Daudi(陽(yáng)性表達(dá)率＞90%)與ch4E5(終濃度為10μg/ml)共培養(yǎng),MTT和FCM分析ch4E5對(duì)Daudi細(xì)胞的生長(zhǎng)與存活的影響。采用競(jìng)爭(zhēng)抑制法分析ch4E5與母本抗體的競(jìng)爭(zhēng)抑制作用及MTT對(duì)MLR的影響作用。ELISA檢測(cè)ch4E5與PBLs共培養(yǎng)后IL-2、INF-γ及IL-10的水平。結(jié)果表明:CHO-ch4E5細(xì)胞培養(yǎng)上清中嵌合抗體的得率為4～5.8 mg/L。ch4E5能夠與4E5相互競(jìng)爭(zhēng)抑制抗原抗體結(jié)合,并有效識(shí)別Daudi細(xì)胞膜型CD80分子(結(jié)合率為95.5%)。ch4E5能有效抑制Daudi細(xì)胞體外增殖(P=0.000067＜0.05),并誘導(dǎo)其凋亡。ch4E5抑制PBLs體外增殖(P=0.000012＜0.05),下調(diào)分泌IL-2(P=0.000156＜0.05)及INF-γ(P=0.000001＜0.05),上調(diào)分泌IL-10(P=0.000035＜0.05)。提示CD80嵌合抗體具有良好的生物學(xué)活性。 本研究獲得的成果:1、成功構(gòu)建了抗人CD80鼠-人嵌合抗體(ch4E5)真核表達(dá)質(zhì)粒。2、獲得了穩(wěn)定分泌嵌合抗體的細(xì)胞株CHO-ch4E5及相應(yīng)的純品抗體。3、CD80嵌合抗體可有效抑制天然高表達(dá)CD80分子的人B淋巴瘤細(xì)胞株Daudi的體外增殖;抑制混合淋巴細(xì)胞反應(yīng)。該抗體在某些腫瘤的免疫治療及移植抗排異中具有潛在的應(yīng)用價(jià)值。
[Abstract]:On the basis of the mouse anti-human CD80 blocking monoclonal antibody 4E5), the stable expression of chimeric antibody in eukaryotic CHO was achieved by using chimeric antibody construction method, and the biological function of chimeric antibody was preliminarily studied. Part one: construction and expression of murine-human chimeric antibody against CD80 Total RNAs were extracted from 4E5 hybridoma cells and were amplified by PCR using degenerate primers. According to the sequence of heavy and light chain genes, primers were designed and amplified by SMART-PCR method. At the same time, specific primers were used to amplify human IgG1 緯 chain Fctch1 and 魏 chain C 魏 genes from pIRES/hu5C11 plasmids. Using TP-PCR method, the chimeric heavy chain C 魏 and the signal peptide sequence VL sequence were spliced to obtain the chimeric light chain. The recombinant plasmid pIRES/ch4E5.pIRES/ch4E5 was constructed and transfected into 293T cells by liposome method. The transient expression of chimeric antibody in the supernatant was detected by FCM, and then transfected into CHO cells. CHO-ch4E5 cells secreting chimeric antibodies were obtained. The results showed that the eukaryotic expression vector pIRESr / ch4E5 containing chimeric weight and light chain gene was successfully constructed, and transient expression and stable expression were obtained in 293T and CHO cells, respectively. Part two: a preliminary study on the biological function of mouse human chimeric antibody against CD80 A large number of supernatants of serum-free culture of CHO-ch4E5 cells were collected and purified by protein G affinity chromatography. The purity of chimeric antibodies and the recognition of cell membrane CD80 molecules such as Daudio SH2-1 and U937 were identified by Lowry quantitative SDS-PAGE. The natural human B-lymphoma cell lines with high expression of CD80 (positive rate > 90) and ch4E5 (final concentration 10 渭 g / ml) were cocultured. The effects of ch4E5 on the growth and survival of Daudi cells were analyzed. The competitive inhibition of ch4E5 and maternal antibody and the effect of MTT on MLR were analyzed by competitive inhibition method. Elisa was used to detect the levels of IL-2INF- 緯 and IL-10 after co-culture of ch4E5 and PBLs. The results showed that the yield of chimeric antibody in the supernatant of Cho ch4E5 cell culture was 45.8 mg/L.ch4E5, which could bind to 4E5. The Daudi cell membrane type CD80 molecule (the binding rate was 95.5%).ch4E5) could effectively inhibit the proliferation of Daudi cells in vitro and induce its apoptosis. Ch4E5 could inhibit the proliferation of PBLs in vitro and down-regulate the secretion of IL-2(P=0.000156 < 0.05), and INF- 緯 -P0. 0001 < 0. 05, and up-regulate the secretion of IL-10(P=0.000035 < 0. 05. The results suggest that CD80 chimeric antibody has good biological activity. The result of this study was: 1, successfully constructed the eukaryotic expression plasmid of anti-human CD80 murine-human chimeric antibody ch4E5). The cell line CHO-ch4E5 and the corresponding purified antibody, .3mCD80 chimeric antibody, which secreted the chimeric antibody stably, could effectively inhibit the natural high surface. Proliferation of human B lymphoma cell line Daudi with CD80 molecule in vitro; Inhibition of mixed lymphocyte reaction. This antibody has potential application value in immunotherapy and transplantation of some tumors.
【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】：2008
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 朱艷,陳永井,邱玉華,鄭峰豐,朱江;TP-PCR法構(gòu)建抗人CD28嵌合抗體雙啟動(dòng)子昆蟲(chóng)桿狀病毒重組轉(zhuǎn)移載體[J];生物工程學(xué)報(bào);2005年05期

2 瞿秋霞;陳永井;葛彥;王勤;陳成;楊明峰;張學(xué)光;;抗人CD40人-鼠嵌合抗體的構(gòu)建及其表達(dá)[J];細(xì)胞與分子免疫學(xué)雜志;2006年02期

3 瞿秋霞;陳成;王勤;葛彥;陳永井;邱玉華;張學(xué)光;;抗人CD40人-鼠嵌合抗體在CHO細(xì)胞中的表達(dá)及其功能研究[J];細(xì)胞與分子免疫學(xué)雜志;2007年06期

4 邱玉華,季玉紅,郭玲,周照華,王月丹,傅晉翔,張學(xué)光;鼠抗人B7-1分子功能性單克隆抗體的制備及生物學(xué)特性[J];中國(guó)免疫學(xué)雜志;2000年11期

，

本文編號(hào)：1820625