本文選題：ATP5B + 基因克隆表達��； 參考：《福建農(nóng)林大學》2011年碩士論文

【摘要】：黃曲霉毒素B1是誘發(fā)肝臟癌變的主要因素之一。ATP合成酶β亞基(ATP5B)除參與機體各種能量代謝外,近些年國內(nèi)外研究機構(gòu)紛紛報道ATP5B還參與了腫瘤組織的信號調(diào)控。本實驗室前期通過蛋白質(zhì)組學及熒光定量PCR研究分析了ATP5B蛋白與黃曲霉毒素B1誘發(fā)的肝癌演化存在相關(guān)性(ATP5B發(fā)生上調(diào))。本研究制備了分泌抗ATP5B單克隆抗體的雜交瘤細胞株5C4、6G11,為后期研究ATP5B參與黃曲霉毒素B_1誘發(fā)肝癌的信號調(diào)控機制奠定基礎(chǔ)。 本實驗把已構(gòu)建的質(zhì)粒載體pET-28a(+)-atp5b和pET-32a(+)-atp5b分別轉(zhuǎn)入原核表達系統(tǒng)E. coli BL21(DE3),經(jīng)IPTG誘導表達及Ni~(2+)-NTA親和層析得到了純化的ATP5B-Trx、ATP5B蛋白,為制備抗ATP5B單克隆抗體提供了足量的免疫原及ELISA檢測所用的包被抗原。 以重組蛋白ATP5B-Trx為抗原免疫BALB/c小鼠。經(jīng)四次免疫后,采用方陣法建立了間接非競爭ELISA檢測體系,抗原ATP5B的最佳工作濃度為1:50,即為4.35μg/mL,IgG-HRP的最佳工作濃度為1:20000。之后以此檢測系統(tǒng)測定了小鼠血清效價(1A、2A小鼠效價達1:12800,1B、2B小鼠效價只到1:1600和1:6400,3A、3B小鼠效價還低于1:800),同時采用間接競爭ELISA法評估了6只小鼠血清特異性,最終確定取BALB/c小鼠1A的脾臟細胞與骨髓瘤細胞SP2/0進行融合(50% PEG1450)。通過對雜交瘤細胞的篩選及數(shù)次亞克隆,最終獲得了2株分泌抗ATP5B單克隆抗體的雜交瘤細胞株5C4、6G11,其染色體條數(shù)約為98-102,均大于親本細胞。 對雜交瘤細胞5C4、6G11進行傳代、定期凍存復蘇,檢測其分泌抗體效果,結(jié)果證明雜交瘤細胞株性質(zhì)穩(wěn)定,其細胞培養(yǎng)上清液效價分別為1:800、1:1600。采用小鼠腹內(nèi)誘生法制備單克隆抗體,經(jīng)辛酸-硫酸銨法、DEAE-纖維素離子交換柱層析純化后,此腹水抗體5C4、6G11效價分別為1:5.12×10~4和1:2.05×10~5,抗體濃度分別為1.14μg/mL和1.72μg/mL。經(jīng)SDS-PAGE電泳鑒定抗體純度,電泳圖譜顯示了抗體的重鏈和輕鏈條帶,抗體分子量為150 kDa,抗體純度均大于90 %�？贵w亞類鑒定均為IgG1,親和力常數(shù)分別為1.7×10~8 L/moL、2.5×10~8 L/moL,并且通過Western-blot證實了此抗體的高度特異性。
[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.
【學位授予單位】：福建農(nóng)林大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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