抗人B7-1人—鼠嵌合抗体的制备及生物学功能研究
发布时间：2018-11-21 13:29
【摘要】： 目的:在本科室成功构建的抗人B7-1人-鼠嵌合抗体重组表达质粒pIRES/ch-4E5的基础上,将其转染CHO细胞,获得持续稳定分泌嵌合抗体的真核表达细胞株CHO-ch-4E5并制备嵌合抗体。进而分析抗体的生物学功能。 方法:(1)采用小量质粒抽提试剂盒提取抗人B7-1人-鼠嵌合抗体的重组表达质粒pIRES/ch-4E5,经脂质体法转染CHO细胞,用G418选择培养基加压培养约2周,收集培养上清,经流式细胞术(FCM)进行检测。阳性孔细胞再经亚克隆筛选;(2)收集稳定分泌嵌合抗体的CHO细胞,采用Trizol法抽提总RNA,经RT-PCR方法合成cDNA,再加入特异性引物经PCR扩增嵌合重、轻链基因;(3)大规模无血清培养稳定分泌嵌合抗体的CHO细胞,收集上清,经高速离心、超滤浓缩及PBS透析过夜,采用Protein G亲和层析柱进行分离纯化,Lowry法定量,间接免疫荧光及FCM法分析抗体对Raji、Daudi、U937、K562、L929-B7-1及Jurkat等多种细胞表面膜型B7-1分子的结合;(4)将Raji和Daudi细胞与终浓度为10μg/ml的ch-4E5共培养,经FCM动态检测肿瘤细胞表面Fas及FasL的表达,MTT法分析ch-4E5对Raji和Daudi细胞体外增殖的抑制作用;(5)针对嵌合抗体的Fc段功能,以人外周血PBMC为效应细胞,以上述肿瘤细胞为靶细胞,按效靶比20:1混合细胞,加入终浓度10μg/ml的ch-4E5共培养,MTT法分析嵌合抗体介导的ADCC效应;(6)在上述体外研究的基础上,选取4～6周龄,雌性BALB/c裸鼠,随机分组,10只/组。按体重10 mg/kg的抗体量分别将抗体(ch-4E5、亲本4E5)与Raji细胞(5×10~6/只)混合于37℃、5%CO2培养箱中孵育30 min,按下列组别接种于裸鼠左前肢腋下。①组:Raji;②组:Raji+Hu-IgG1;③组:Raji+ch-4E5;④组:Raji+4E5。逐日观察并记录荷瘤裸鼠的生存状况、成瘤时间及成瘤率。 结果:(1)成功获得了持续稳定分泌抗人B7-1人-鼠嵌合抗体的真核表达细胞株CHO-ch-4E5,PCR鉴定结果证实目的基因正确;(2)经Lowry法定量,从CHO-ch-4E5培养上清中获取嵌合抗体的量约30mg/L;(3) FCM分析表明,ch-4E5能够特异性结合多种细胞表面膜型B7-1分子,与Raji、Daudi、U937、K562、L929-B7-1及Jurkat细胞的阳性结合率分别为98.6%、96.4%、2.2%、29.6%、98.8%及10.1%,与亲本鼠源性抗体的结合率相当;(4)ch-4E5与肿瘤细胞共培养后,能够明显抑制Raji及Daudi细胞的体外增殖,抑制率分别为34.60%及32.64%,进一步的分析表明肿瘤细胞Fas及FasL的表达上调;(5) ch-4E5 Fc段介导PBMC对Raji及Daudi的杀伤率分别为55.61%及54.42%;(6)体内荷瘤实验的结果表明,Raji细胞经ch-4E5及4E5抗体处理的实验组均未见肿瘤形成,小鼠成活率为100%。而接种Raji及Raji+Hu-IgG1组一周后,裸鼠开始出现消瘦、活动减弱、食量减少、脊柱隆起等生长状态不佳的表现。至30天时,出现肉眼可见的肿瘤,90天时,成瘤率达80%,瘤体体积为(1404.70±47.23)mm~3,肿瘤生长速度为(15.53±0.58)mm~3/d。 结论:本组自行研制的抗人B7-1人-鼠嵌合抗体具有生物学功能,对B7-1分子相关肿瘤的临床治疗具有潜在意义。
[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，
本文编号：2347097
[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，
本文编号：2347097
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