抗ATP5B单克隆抗体制备及其性质研究

发布时间：2018-06-21 10:48

本文选题：ATP5B + 基因克隆表达　；参考：《福建农林大学》2011年硕士论文

【摘要】：黄曲霉毒素B1是诱发肝脏癌变的主要因素之一。ATP合成酶β亚基(ATP5B)除参与机体各种能量代谢外,近些年国内外研究机构纷纷报道ATP5B还参与了肿瘤组织的信号调控。本实验室前期通过蛋白质组学及荧光定量PCR研究分析了ATP5B蛋白与黄曲霉毒素B1诱发的肝癌演化存在相关性(ATP5B发生上调)。本研究制备了分泌抗ATP5B单克隆抗体的杂交瘤细胞株5C4、6G11,为后期研究ATP5B参与黄曲霉毒素B_1诱发肝癌的信号调控机制奠定基础。本实验把已构建的质粒载体pET-28a(+)-atp5b和pET-32a(+)-atp5b分别转入原核表达系统E. coli BL21(DE3),经IPTG诱导表达及Ni~(2+)-NTA亲和层析得到了纯化的ATP5B-Trx、ATP5B蛋白,为制备抗ATP5B单克隆抗体提供了足量的免疫原及ELISA检测所用的包被抗原。以重组蛋白ATP5B-Trx为抗原免疫BALB/c小鼠。经四次免疫后,采用方阵法建立了间接非竞争ELISA检测体系,抗原ATP5B的最佳工作浓度为1:50,即为4.35μg/mL,IgG-HRP的最佳工作浓度为1:20000。之后以此检测系统测定了小鼠血清效价(1A、2A小鼠效价达1:12800,1B、2B小鼠效价只到1:1600和1:6400,3A、3B小鼠效价还低于1:800),同时采用间接竞争ELISA法评估了6只小鼠血清特异性,最终确定取BALB/c小鼠1A的脾脏细胞与骨髓瘤细胞SP2/0进行融合(50% PEG1450)。通过对杂交瘤细胞的筛选及数次亚克隆,最终获得了2株分泌抗ATP5B单克隆抗体的杂交瘤细胞株5C4、6G11,其染色体条数约为98-102,均大于亲本细胞。对杂交瘤细胞5C4、6G11进行传代、定期冻存复苏,检测其分泌抗体效果,结果证明杂交瘤细胞株性质稳定,其细胞培养上清液效价分别为1:800、1:1600。采用小鼠腹内诱生法制备单克隆抗体,经辛酸-硫酸铵法、DEAE-纤维素离子交换柱层析纯化后,此腹水抗体5C4、6G11效价分别为1:5.12×10~4和1:2.05×10~5,抗体浓度分别为1.14μg/mL和1.72μg/mL。经SDS-PAGE电泳鉴定抗体纯度,电泳图谱显示了抗体的重链和轻链条带,抗体分子量为150 kDa,抗体纯度均大于90 %。抗体亚类鉴定均为IgG1,亲和力常数分别为1.7×10~8 L/moL、2.5×10~8 L/moL,并且通过Western-blot证实了此抗体的高度特异性。
[Abstract]:Aflatoxin B1 (aflatoxin B1) is one of the main factors inducing liver carcinogenesis. ATP synthase 尾 subunit ATP-5B is involved in all kinds of energy metabolism. In recent years, it has been reported that ATP-5B is also involved in the signal regulation of tumor tissue. The relationship between ATP-5B protein and aflatoxin B1 (aflatoxin B1) induced liver cancer evolution was analyzed by proteomics and fluorescence quantitative PCR. In this study, hybridoma cell line 5C4O6G11, secreting monoclonal antibody against ATP-5B, was prepared, which laid a foundation for the study of ATP-5B involved in the signal regulation mechanism of aflatoxin B1-induced hepatocellular carcinoma (HCC). The constructed plasmids pET-28a (pET-28b) and pET-32a (pET-32a) were transferred into the prokaryotic expression system (E. coli BL21DE3b), respectively. The purified AT5B-TrxAT5B protein was obtained by IPTG induced expression and NIT-NTA affinity chromatography. Sufficient amount of immunogen and coated antigen for Elisa detection were provided for the preparation of monoclonal antibody against ATP 5B. BALB / c mice were immunized with recombinant ATP 5 B-Trx as antigen. After four times of immunization, the indirect non-competitive Elisa system was established by square matrix method. The best working concentration of the antigen ATP-5B was 1: 50, that is, 4.35 渭 g / mL IgG-HRP was 1: 20 000. Then, the titer of serum of 1: 12800 and 1B2 B mice was determined by this system. The titers of mice were only 1: 1600 and 1: 6400, 3A0. 3B mice were less than 1: 800, and the specificity of 6 mice was evaluated by indirect competitive Elisa. The spleen cells of BALB / c mouse 1A and myeloma cell SP2 / 0 were selected and fused with 50% PEG1450. Through screening and subcloning of hybridoma cells, two hybridoma cell lines 5C4P6G11 secreting monoclonal antibody against ATP-5B were obtained, the chromosome number of which was about 98-102, which was larger than that of parent cells. The hybridoma cell line 5C4O6G11 was subcultured, frozen and resuscitated periodically. The results showed that the hybridoma cell line was stable in nature, and the supernatant titers of the cell culture supernatant were 1: 800, 1: 1600, respectively. The monoclonal antibody was prepared by the method of mouse abdominal induction. The titers of the antibody 5C4O6G11 were 1: 5.12 脳 10 ~ (4) and 1: 2.05 脳 10 ~ (5) respectively, and the antibody concentration was 1.14 渭 g / mL and 1.72 渭 g / mL, respectively, after purification by DEAE- cellulose ion exchange chromatography with octanoic acid-ammonium sulfate method, the titers of the antibody were 1: 5.12 脳 10 ~ (-4) and 1: 2.05 脳 10 ~ (-1), respectively. The purity of the antibody was identified by SDS-PAGE electrophoresis. The heavy chain and light chain band of the antibody were shown by SDS-PAGE. The molecular weight of antibody was 150 kDa.The purity of antibody was more than 90. The antibody subclasses were identified as IgG1, and the affinity constants were 1.7 脳 10 ~ (8) L / mol ~ (-1) and 2.5 脳 10 ~ (8) L / mol 路L ~ (-1), respectively. The specificity of the antibody was confirmed by Western-blot.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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