【摘要】： 目的:在本科室成功構(gòu)建的抗人B7-1人-鼠嵌合抗體重組表達(dá)質(zhì)粒pIRES/ch-4E5的基礎(chǔ)上,將其轉(zhuǎn)染CHO細(xì)胞,獲得持續(xù)穩(wěn)定分泌嵌合抗體的真核表達(dá)細(xì)胞株CHO-ch-4E5并制備嵌合抗體。進(jìn)而分析抗體的生物學(xué)功能。 方法:(1)采用小量質(zhì)粒抽提試劑盒提取抗人B7-1人-鼠嵌合抗體的重組表達(dá)質(zhì)粒pIRES/ch-4E5,經(jīng)脂質(zhì)體法轉(zhuǎn)染CHO細(xì)胞,用G418選擇培養(yǎng)基加壓培養(yǎng)約2周,收集培養(yǎng)上清,經(jīng)流式細(xì)胞術(shù)(FCM)進(jìn)行檢測。陽性孔細(xì)胞再經(jīng)亞克隆篩選;(2)收集穩(wěn)定分泌嵌合抗體的CHO細(xì)胞,采用Trizol法抽提總RNA,經(jīng)RT-PCR方法合成cDNA,再加入特異性引物經(jīng)PCR擴(kuò)增嵌合重、輕鏈基因;(3)大規(guī)模無血清培養(yǎng)穩(wěn)定分泌嵌合抗體的CHO細(xì)胞,收集上清,經(jīng)高速離心、超濾濃縮及PBS透析過夜,采用Protein G親和層析柱進(jìn)行分離純化,Lowry法定量,間接免疫熒光及FCM法分析抗體對Raji、Daudi、U937、K562、L929-B7-1及Jurkat等多種細(xì)胞表面膜型B7-1分子的結(jié)合;(4)將Raji和Daudi細(xì)胞與終濃度為10μg/ml的ch-4E5共培養(yǎng),經(jīng)FCM動態(tài)檢測腫瘤細(xì)胞表面Fas及FasL的表達(dá),MTT法分析ch-4E5對Raji和Daudi細(xì)胞體外增殖的抑制作用;(5)針對嵌合抗體的Fc段功能,以人外周血PBMC為效應(yīng)細(xì)胞,以上述腫瘤細(xì)胞為靶細(xì)胞,按效靶比20:1混合細(xì)胞,加入終濃度10μg/ml的ch-4E5共培養(yǎng),MTT法分析嵌合抗體介導(dǎo)的ADCC效應(yīng);(6)在上述體外研究的基礎(chǔ)上,選取4～6周齡,雌性BALB/c裸鼠,隨機(jī)分組,10只/組。按體重10 mg/kg的抗體量分別將抗體(ch-4E5、親本4E5)與Raji細(xì)胞(5×10~6/只)混合于37℃、5%CO2培養(yǎng)箱中孵育30 min,按下列組別接種于裸鼠左前肢腋下。①組:Raji;②組:Raji+Hu-IgG1;③組:Raji+ch-4E5;④組:Raji+4E5。逐日觀察并記錄荷瘤裸鼠的生存狀況、成瘤時間及成瘤率。 結(jié)果:(1)成功獲得了持續(xù)穩(wěn)定分泌抗人B7-1人-鼠嵌合抗體的真核表達(dá)細(xì)胞株CHO-ch-4E5,PCR鑒定結(jié)果證實(shí)目的基因正確;(2)經(jīng)Lowry法定量,從CHO-ch-4E5培養(yǎng)上清中獲取嵌合抗體的量約30mg/L;(3) FCM分析表明,ch-4E5能夠特異性結(jié)合多種細(xì)胞表面膜型B7-1分子,與Raji、Daudi、U937、K562、L929-B7-1及Jurkat細(xì)胞的陽性結(jié)合率分別為98.6%、96.4%、2.2%、29.6%、98.8%及10.1%,與親本鼠源性抗體的結(jié)合率相當(dāng);(4)ch-4E5與腫瘤細(xì)胞共培養(yǎng)后,能夠明顯抑制Raji及Daudi細(xì)胞的體外增殖,抑制率分別為34.60%及32.64%,進(jìn)一步的分析表明腫瘤細(xì)胞Fas及FasL的表達(dá)上調(diào);(5) ch-4E5 Fc段介導(dǎo)PBMC對Raji及Daudi的殺傷率分別為55.61%及54.42%;(6)體內(nèi)荷瘤實(shí)驗(yàn)的結(jié)果表明,Raji細(xì)胞經(jīng)ch-4E5及4E5抗體處理的實(shí)驗(yàn)組均未見腫瘤形成,小鼠成活率為100%。而接種Raji及Raji+Hu-IgG1組一周后,裸鼠開始出現(xiàn)消瘦、活動減弱、食量減少、脊柱隆起等生長狀態(tài)不佳的表現(xiàn)。至30天時,出現(xiàn)肉眼可見的腫瘤,90天時,成瘤率達(dá)80%,瘤體體積為(1404.70±47.23)mm~3,腫瘤生長速度為(15.53±0.58)mm~3/d。 結(jié)論:本組自行研制的抗人B7-1人-鼠嵌合抗體具有生物學(xué)功能,對B7-1分子相關(guān)腫瘤的臨床治療具有潛在意義。
[Abstract]:Objective: to transfect the recombinant expression plasmid pIRES/ch-4E5 against human B7-1 human mouse chimeric antibody into CHO cells. The eukaryotic expression cell line CHO-ch-4E5 was obtained and the chimeric antibody was prepared. Then the biological function of antibody was analyzed. Methods: (1) Recombinant expression plasmid pIRES/ch-4E5, against human B7-1 human-mouse chimeric antibody was extracted by a small amount of plasmid extraction kit and transfected into CHO cells by liposome method. The supernatants were collected and cultured in G418 selected medium for 2 weeks. (FCM) was detected by flow cytometry. (2) CHO cells secreting stable chimeric antibodies were collected, and total RNA, was extracted by Trizol method. CDNA, was synthesized by RT-PCR method and then amplified by PCR with specific primers, and the chimeric weight and light chain genes were amplified by PCR. (3) Large-scale serum-free CHO cells secreting stable chimeric antibodies were collected and supernatant was collected and purified by high speed centrifugation, ultrafiltration concentration and PBS dialysis overnight. Protein G affinity chromatography was used to isolate and purify the cells. Lowry assay was used. Indirect immunofluorescence and FCM assay were used to analyze the binding of antibodies to Raji,Daudi,U937,K562,L929-B7-1 and Jurkat. (4) Raji and Daudi cells were co-cultured with ch-4E5 with final concentration of 10 渭 g/ml. The expression of Fas and FasL on tumor cells was dynamically detected by FCM. The inhibitory effect of ch-4E5 on the proliferation of Raji and Daudi cells in vitro was analyzed by MTT assay. (5) aiming at the Fc segment function of chimeric antibody, the PBMC of human peripheral blood was used as effector cells, the tumor cells were used as target cells, and the cells were mixed with ch-4E5 at 10 渭 g/ml final concentration according to the effect target ratio of 20:1, and the final concentration of ch-4E5 was 10 渭 g/ml. The ADCC effect mediated by chimeric antibody was analyzed by MTT. (6) on the basis of the above in vitro study, 10 female BALB/c nude mice were randomly divided into 10 groups at the age of 4 and 6 weeks. The antibodies (ch-4E5, parent 4E5) and Raji cells (5 脳 10 ~ (6) / mouse) were mixed at 37 鈩，

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