|
|
|
| Preparation of MAGE-A3 polyclonal antibody and its application in detection of human gastric cancer cell |
| CAI Yiqi1, CHENG Kai1, CHEN Xudong1, CHEN Xiaodong1, CEN Danwei2, ZHANG Chanqiong2, SONG Yiling2, MAO Shanshan2, YE Xiaoxian2, ZHANG Lifang2, ZHU Guanbao1. |
1.Department of Gastroenterological Surgery, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015; 2.Instiute of Molecular Virology and Immunology, Department of Medical Microbiology and Immunology, Wenzhou Medical University, Wenzhou, 325035
|
|
| Cite this article: |
|
CAI Yiqi,CHENG Kai,CHEN Xudong, et al. Preparation of MAGE-A3 polyclonal antibody and its application in detection of human gastric cancer cell[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2017, 47(5): 318-324.
|
|
|
Abstract Objective: To prepare Human MAGE-A3 (MAGE-A3) protein by prokaryotic expression system. At the same time, a polyclonal antibody was prepared to against it, after preliminary identification and application. Methods: The MAGE-A3 full-length nucleic acid sequences were synthetized and cloned into pET21a (+) plasmid to construct pET21a (+)/MAGE-A3 restructuring plasmids. After sequencing, the recombinant plasmid was transformed into E.coli BL21 (DE3). The MAGE-A3 protein was expressed through Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducing. Then the recombinant protein of MAGE-A3 was purified by Ni-NTA affinity chromatography and confirmed by SDS-PAGE and Western blot analysis, the rabbits were immunized with the purified protein MAGE-A3 in order to prepare the polyclonal antibody. The titers of specific antibodies were detected by ELISA. The specificity activity was further detected by Western blot and immunofluorescence technique analysis. Its preliminary application was detected in human gastric cancer cells by immunohistochemical techniques. Results: The recombinant plasmids pET21a(+)/MAGE-A3 (full-length) were successfully constructed. The MAGE-A3 recombinant protein could be expressed and purified in the prokaryotic expression system. SDS-PAGE showed the size of the protein about 48 kDa was consistent with the expected size of the protein and identified by Western blot. The polyclonal antibodies were produced in the rabbits immunized with the MAGE-A3 recombinant protein. The Antigen-antibody reactions reached a peak in the eighth week. Western blot analysis indicated that the polyclonal antibody could specifically recognize the MAGE-A3 recombinant protein. The immunofluorescence showed that fluorescent clumps appeared in intracytoplasm of MAGE-A3 positive cells A-375 (melanoma cell line) and SGC-7901 cells (gastric cancer cell line). It confirmed that polyclonal antibody could specifically recognizes natural MAGE-A3 protein. ELISA analysis indicated the titer of the polyclonal IgG was up to 1:40 000 in the eighth week; Immunohistochemistry display appeared in the cytoplasm A-375 and SGC-7901 tumor tissue lumpy brown granular precipitate, while a negative in BGC-823 tumor tissues, suggesting that the preparation of the MAGE-A3 rabbit polyclonal antibody was capable identifing MAGE-A3 protein specifically in tumor tissue. Conclusion: MAGE-A3 recombinant protein is expressed by the prokaryotic expression system and its polyclonal antibody is prepared successfully. And the polyclonal antibody can specifically recognize MAGE-A3 protein in human gastric cancer cells.
|
|
Received: 22 June 2016
|
| |
|
|
|
[1] BOEL P, WILDMANN C, SENSI M L, et al. BAGE: a new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes[J]. Immunity, 1995, 2(2): 167-175.
[2] BRICHARD V G, GODECHAL Q. MAGE-A3-specific anticancer immunotherapy in the clinical practice[J]. Oncoim-munology, 2013, 2(10): e25995.
[3] 汪国华, 徐磊, 王慧明, 等. 肿瘤特异性MAGE-A3基因重组质粒的构建[J]. 肿瘤学杂志, 2008, 14(11): 899-901.
[4] ROGNER U C, WILKE K, STECK E, et al. The melanoma antigen gene (MAGE) family is clustered in the chromosomal band Xq28[J]. Genomics, 1995, 29(3): 725-731.
[5] WANG Y, WU X J, ZHAO A L, et al. Cancer/testis antigen expression and autologous humoral immunity to NY-ESO-1 in gastric cancer[J]. Cancer Immun, 2004, 4: 11.
[6] SADANAGA N, NAGASHIMA H, TAHARA K, et al. The heterogeneous expression of MAGE-3 protein: difference between primary lesions and metastatic lymph nodes in gastric carcinoma[J]. Oncol Rep, 1999, 6(5): 975-977.
[7] SCHULTZ E S, LETHE B, CAMBIASO C L, et al. A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD+ cytolytic T lymphocytes[J]. Cancer Res, 2000,60(22): 6272-6275.
[8] 金劲激, 丁玉杰, 王冰冰, 等. MAGE-A家族抗原共同B细胞表位预测分析[J]. 温州医科大学学报, 2013, 43(11): 707-710.
[9] SHEN X, JIN J, DING Y, et al. Novel immunodominant epitopes derived from MAGE-A3 and its significance in serological diagnosis of gastric cancer[J]. J Cancer Res Clin Oncol, 2013, 139(9): 1529-1538.
[10] SCHNURR M, CHEN Q, SHIN A, et al. Tumor antigen processing and presentation depend critically on dendritic cell type and the mode of antigen delivery[J]. Blood, 2005, 105(6): 2465-2472.
[11] MOSTAFA A A, MORRIS D G. Immunotherapy for lung cancer: has it finally arrived[J]. Front Oncol, 2014, 4: 288.
[12] MANICI S, STURNIOLO T, IMRO M A, et al. Melanoma cells present a MAGE-3 epitope to CD4(+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11[J]. J Exp Med, 1999, 189(5): 871-876.
[13] 叶菁, 陈广生, 黄亚渝, 等. 人MAGE-1基因的克隆、原核表达及其抗体的制备[J]. 免疫学杂志, 2004, 20(4): 259-262.
[14] 王冰冰, 涂建欣, 吕艳, 等. HPV16型E7重组蛋白的表达及其多克隆抗体的制备[J]. 细胞与分子免疫学杂志, 2014, 30(2): 167-170, 175
|
|
|
|
