抗人肺癌单克隆抗体及其识别抗原的纯化
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摘要
[目的]：用Protein A亲和层析柱从杂交瘤细胞株P35的培养上清中纯化出抗人肺癌单克隆抗体N-35,并以该抗体为配体制作免疫亲和层析柱获取其特异性识别的肺癌相关抗原N35蛋白。
[方法]：有限稀释法对杂交瘤细胞P35进行克隆化培养,同时将处于对数生长期的云南个旧肺腺癌细胞GLC-82接种于96孔培养板,当肺癌细胞生长铺满孔底时对其固定、封闭；采集GLC-82免疫后的小鼠血,制备阳性血清,96孔肺癌细胞抗原板间接ELISA法对杂交瘤细胞株进行三次阳性克隆的筛选,对稳定分泌单克隆抗体的杂交瘤细胞株扩大培养、冻存,检测上清中抗体效价及亚型,免疫组化法鉴定单克隆抗体的组织特异性：收集足量培养上清用Protein A Sepharose CL-4B亲和层析柱纯化mcAb N-35,鉴定纯化抗体纯度和免疫活性；用纯化后的的单抗与CNBr活化的SepharoseTM4B交联,制备免疫亲和层析柱；收集GLC-82细胞裂解液过柱,获取肺癌相关抗原N35蛋白,SDS-PAGE和Western-blot鉴定纯化后的抗原。
[结果]：筛选出30余株稳定分泌单克隆抗体N-35的杂交瘤细胞株并分别冻存,其中一株上清抗体效价为1：102,亚类为IgG2a,能和肺癌细胞特异性反应；自行纯化出高纯度鼠抗人肺癌单克隆抗体N-35；并成功获取了其特异识别的肺癌相关抗原N35蛋白。
[结论]：经三次克隆化培养及筛选后杂交瘤细胞P35亚克隆分泌抗体的阳性率达到100%：应用Protein A Sepharose CL-4B层析柱纯化的mcAb N-35效价高、纯度好；纯化后的抗体N-35与溴化氰活化的SepharoseTM4B有较高的交联率,自行制作的免疫亲和层析柱能有效工作；纯化所获肺癌相关N35蛋白可能为新的肿瘤相关抗原。
[Objective] The aim of this study to purify the anti-human lung cancer monoclonal antibody N-35from the cell culture supernatant of hybridoma p35, which were used by Protein A Sepharose CL-4B chromatographic column, and employ mcAb N-35as the ligand for the immunoaffinity chromatography. to catch the human lung cancer associated antigen N35protein which be recognized.
[Method] Limited dilution and cloning culture of hybridoma cell P35, at the same time inoculated Yunnan Gejiu lung adenocarcinoma cancer cell GLC-82with logarithmic phase in to96wells plate, after GLC-82bespreading, we mounted and blocked these tumor cells; Prepared the positive mouse immunized serum anti-human lung cancer cells, using the cancer cell GLC-82plate to screen of hybridoma supernatants with ELISA. got the hybridoma cell line that secreted mcAb stably, cultured abundantly and cryopreserved. the potency and subtype of the mcAb was detected, identified the tissue specificity of monoclonal antibody by immunohistochemistry; After the supernatant was sufficiently collected. Protein A Sepharose CL-4B chromatographic column can be used to purify mcAb N-35. identified the purity and activity of N-35; The purified mcAb was coupled with CNBr activated SepharoseTM4B as the ligand for the immunoaffinity chromatography (IAC) to catch the lung cancer associated antigen from the lysate of lung cancer cells GLC-82. The purified antigen of the mcAb was identified by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and Western-blot.
[Result] Screened more than30hybridoma cell lines that secreted monoclonal antibody N-35steadily, cryopreserved them respectively, the potency of mcAb was1:102, subtype was IgG2a, combination to lung cancer was specific; Purified the mouse anti-human lung cancer mcAb N-35, and extracted lung cancer associated antigen N35protein recognized by the antibody successfully.
[Conclusion]100%of P35subclone can secreted mcAb N-35steadily through thrice cloning culture and sieve; The purified mcAb N-35was high potency and high purity by Protein A Sepharose CL-4B chromatographic column; The binding rate of mcAb N-35to CNBr activated SepharoseTM4B was fine. the immune chromatography column we prepared worked effectively. The purified antigen N35could be a new tumor-associated antigen (TAA).
[方法]：有限稀释法对杂交瘤细胞P35进行克隆化培养,同时将处于对数生长期的云南个旧肺腺癌细胞GLC-82接种于96孔培养板,当肺癌细胞生长铺满孔底时对其固定、封闭；采集GLC-82免疫后的小鼠血,制备阳性血清,96孔肺癌细胞抗原板间接ELISA法对杂交瘤细胞株进行三次阳性克隆的筛选,对稳定分泌单克隆抗体的杂交瘤细胞株扩大培养、冻存,检测上清中抗体效价及亚型,免疫组化法鉴定单克隆抗体的组织特异性：收集足量培养上清用Protein A Sepharose CL-4B亲和层析柱纯化mcAb N-35,鉴定纯化抗体纯度和免疫活性；用纯化后的的单抗与CNBr活化的SepharoseTM4B交联,制备免疫亲和层析柱；收集GLC-82细胞裂解液过柱,获取肺癌相关抗原N35蛋白,SDS-PAGE和Western-blot鉴定纯化后的抗原。
[结果]：筛选出30余株稳定分泌单克隆抗体N-35的杂交瘤细胞株并分别冻存,其中一株上清抗体效价为1：102,亚类为IgG2a,能和肺癌细胞特异性反应；自行纯化出高纯度鼠抗人肺癌单克隆抗体N-35；并成功获取了其特异识别的肺癌相关抗原N35蛋白。
[结论]：经三次克隆化培养及筛选后杂交瘤细胞P35亚克隆分泌抗体的阳性率达到100%：应用Protein A Sepharose CL-4B层析柱纯化的mcAb N-35效价高、纯度好；纯化后的抗体N-35与溴化氰活化的SepharoseTM4B有较高的交联率,自行制作的免疫亲和层析柱能有效工作；纯化所获肺癌相关N35蛋白可能为新的肿瘤相关抗原。
[Objective] The aim of this study to purify the anti-human lung cancer monoclonal antibody N-35from the cell culture supernatant of hybridoma p35, which were used by Protein A Sepharose CL-4B chromatographic column, and employ mcAb N-35as the ligand for the immunoaffinity chromatography. to catch the human lung cancer associated antigen N35protein which be recognized.
[Method] Limited dilution and cloning culture of hybridoma cell P35, at the same time inoculated Yunnan Gejiu lung adenocarcinoma cancer cell GLC-82with logarithmic phase in to96wells plate, after GLC-82bespreading, we mounted and blocked these tumor cells; Prepared the positive mouse immunized serum anti-human lung cancer cells, using the cancer cell GLC-82plate to screen of hybridoma supernatants with ELISA. got the hybridoma cell line that secreted mcAb stably, cultured abundantly and cryopreserved. the potency and subtype of the mcAb was detected, identified the tissue specificity of monoclonal antibody by immunohistochemistry; After the supernatant was sufficiently collected. Protein A Sepharose CL-4B chromatographic column can be used to purify mcAb N-35. identified the purity and activity of N-35; The purified mcAb was coupled with CNBr activated SepharoseTM4B as the ligand for the immunoaffinity chromatography (IAC) to catch the lung cancer associated antigen from the lysate of lung cancer cells GLC-82. The purified antigen of the mcAb was identified by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and Western-blot.
[Result] Screened more than30hybridoma cell lines that secreted monoclonal antibody N-35steadily, cryopreserved them respectively, the potency of mcAb was1:102, subtype was IgG2a, combination to lung cancer was specific; Purified the mouse anti-human lung cancer mcAb N-35, and extracted lung cancer associated antigen N35protein recognized by the antibody successfully.
[Conclusion]100%of P35subclone can secreted mcAb N-35steadily through thrice cloning culture and sieve; The purified mcAb N-35was high potency and high purity by Protein A Sepharose CL-4B chromatographic column; The binding rate of mcAb N-35to CNBr activated SepharoseTM4B was fine. the immune chromatography column we prepared worked effectively. The purified antigen N35could be a new tumor-associated antigen (TAA).
引文
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[2]Ahmedin Jemal, Freddie Bray, Melissa M, et al. Global cancer statistics. CA CANCER J CLIN,2011,61:69-90
[3]支修益.我国肺癌流行病学现状分析.中国处方药,2009,2(83)：56-57
[4]Irem Dogan, Ahmet Cumaoglu, Aysel Aricioglu, et al. Inhibition of ErbB2 by Herceptin reduces viability and survival, induces apoptosis and oxidative stress in Calu-3 cell line. Mol Cell Biochem,2011,347:41-51
[5]孙思凡,张部昌,靳彦文.治疗性单克隆抗体研究进展.生物技术通讯,2009,20(2)258-262
[6]吴炜霖,仲人前.人源化抗体的演进发展及应用现状.现代免疫学,2009,29(4)：337-340
[7]马玲娟,严俊,张晓莉.单克隆抗体偶联物治疗肿瘤的研究与进展,国际检验医学杂志.2010,31(10)：1142-1144
[8]BarryH.G, RichardK.J.B, GregoryP.K. Optimal anatomic coverage for CT in staging lung cancer:Lessons from PET-CT correlation. Lung cancer,2010,10,021
[9]Rafael Molina, Jose Maria Auge, Jose Miguel Escudero, et al. Mucins CA 125, CA 19.9, CA 15.3 and TAG-72.3 as Tumor Markers in Patients with Lung Cancer:Comparison with CYFRA 21-1, CEA, SCC and NSE. Tumor Biol.2008,29:371-380
[10]李娟,李延团,李晓明等.分子靶向抗肿瘤药物进展.生物技术通讯,2009,20(3)：411-416
[11]ANNELIES H, JOS H. B, JAN H.M. Trastuzumab. The Oncologist,2011,16:800-810
[12]殷宁，杜明华.非小细胞肺癌靶向性治疗的进展.医学综述,2009,15(22)：3415-3417
[13]GIORGIO SCAGLIOTTI, RAMASWAMY GOVINDAN. Targeting Angiogenesis with Multitargeted Tyrosine Kinase Inhibitors in the Treatment of Non-Small Cell Lung Cancer. The Oncologist,2010,15:436-446
[14]Robert Pirker, MartinFilipits. Monoclonal antibodies against EGFR in non-small cell lung cancer. Oncology,2010,23 (11):54-58
[15]Melarkode S, Ramakrishnan, Anand Eswaraiah, et al. Nimotuzumab, a promising therapeutic monoclonal for treatment of tumors of epithelial origin. Landes Bioscience,2011,21 (1):41-48
[16]方慧云,陈敬国,王秦秦.肺癌相关蛋白N35亚细胞结构定位的研究.山东医药,2008,48(38)：59-60
[17]王秦秦,姜平,李继梅.肺癌细胞相关蛋白分子N35的实验研究.细胞与分子免疫学杂志,2000,16(2)：152-155
[18]工秦秦,马丽菊,李海红,等.突变型人蛋白磷酸化酶2Aa催化亚基的基因序列.细胞与分子免疫学杂志,2004,20(2)：152-156
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