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| Expression, Purification and Identification of Thymidine Kinase 1 Recombinant Protein |
| WANG Yun-long1,2, ZHAO Er-xia1, LI Yu-lin2,3 |
1. School of Life Science, Zhengzhou University, Zhengzhou 450001, China;
2. Henan Biotechnology Research Center, Zhengzhou 450001, China;
3. Zhengzhou Biopharmaceutical Key Laboratory, Zhengzhou 450001, China |
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Abstract In order to obtain the TK1 recombinant protein with immunogenicity. The target gene was amplified by PCR, double digested with XhoI/EcoRI restriction endonuclease, linked to the expression vector pET32a, and then transferred into competent cells to prepare BL21-pET32a-TK1 recombinant strain. The TK1 protein was expressed by IPTG induced BL21-pET32a-TK1 recombinant bacteria and optimized from IPTG concentration, culture temperature and time. The expression level of recombinant protein TK1 was tested by SDS-PAGE, and determined the optimal induction conditions. The expressed product was purified by nickel ion affinity chromatography, used SDS-PAGE to detect the purity of the target protein, and the antigenicity of the target protein was detected by Western blot. Used the obtained TK1 recombinant protein to immunize the mice, took the spleen cells of the best immune effect mice to fuse with SP2/0. Screened monoclonal cell lines that secreting specific antibodies stably. And then detected the subtype and specificity of antibody. The experimental results showed that the condition of 37℃,when the concentration of IPTG was 0.2mmol/L and induced for 6h meanwhile, the recombinant protein expression quantity of TK1 was highest. Under the condition of nickel ion affinity chromatography gradient elution, in 80mmol/L imidazole, the content of recombinant protein TK1 was the highest purity. It was 87.3%. After concentrating, the protein concentration was 5.96mg/ml. With indirect ELISA detection,10 strains of cell that can stable secretion specificity TK1 antibody by hybridoma technology were obtained. this suggests that TK1 recombinant protein have immunogenicity. It can provide the material base for the clinical research of related tumors.
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Received: 23 February 2017
Published: 25 September 2017
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[1] Munch-Petersen B, Cloos L, Jensen H K, et al. Human thymidine kinase 1. Regulation in normal and malignant cells. Adv Enzyme Regul, 1995, 35(1):69-89.
[2] Kumar J K, Aronsson A C, Pilko G, et al. A clinical evaluation of the TK 210 ELISA in sera from breast cancer patients demonstrates high sensitivity and specificity in all stages of disease. Tumour Biol, 2016, 37(9):11937-11945.
[3] Gasparri F, Wang N N, Skog S, et al. Thymidine kinase 1 expression defines an activated G1 state of the cell cycle as revealed with site-specific antibodies and ArrayScanTM assays. European Journal of Cell Biology, 2009, 88(1):779-785.
[4] Ke P Y, Yang C C, Tsai I C, et al. Degradation of human thymidine kinase is dependent on serine-13 phosphorylation:involvement of the SCF-mediated pathway. Biochem J, 2003, 370(1):265-273.
[5] 周际, 李劲, 斯文·斯库格,等. 一种多表位TK1抗体的制备及其在评估肿瘤患者治疗效果中的应用:中国,CN102516390A. 2012-06-27. Zhou J, Li J, Skog S, et al. Preparation of A Multi Epitope TK1 Antibody and Its Application in Evaluating Therapeutic Effect of Tumor Patients:China, CN102516390A. 2012-06-27.
[6] He E, Xu X H, Guan H, et al. Thymidine kinase 1 is a potential marker for prognosis and monitoring the response to treatment of patients with breast,lung,and esophageal cancer and non-Hodgkin's lymphoma. Nucleosides Nucleotides Nucleic Acids, 2010, 29(1):4-6.
[7] Rausch S, Hennenlotter J, Teepe K, et al. Muscle-invasive bladder cancer is characterized by overexpression of thymidine kinase 1. Urol Oncol, 2015, 33(10):426-426.
[8] Chen G, He C, Li L, et al. Nuclear TK1 expression is an independent prognostic factor for survival in pre-malignant and malignant lesions of the cervix. BMC Cancer, 2013, 13(1):249-249.
[9] Du Y Y, Zhang Q J, Sun G P. Expression and clinical significance of cytokeratin-19 and thymidine kinase-1 in advanced gastrointestinal cancer. Chinese Medical Journal, 2016, 129(18):2168-2172.
[10] 朱艳哲, 马泰, 张从军,等. 血清胸苷激酶1在肿瘤患者中的表达及临床意义. 安徽医科大学学报, 2015, 50(7):1012-1015. Zhu Y Z, Ma T, Zhang C J, et al. The expression and clinical significance of serum thymidine kinase 1 in cancer patients. Acta Universitatis Medicinalis Anhui, 2015, 50(7):1012-1015.
[11] 刘秀菊,周际,李远,等. 一种新肿瘤生长相关生物标志物胸苷激酶1临床应用进展. 中国药理学与毒理学杂志, 2011, 25(1):123-126. Liu X J, Zhou J, Li Y,et al. Progress in clinical application of a new tumor growth related biomarker thymidine kinase 1. Chin J Pharmacol Toxicol, 2011, 25(1):123-126.
[12] O'Neill K L, Zhang F, Li H, et al. Thymidine kinase 1——a prognostic and diagnostic indicator in ALL and AML patients. Leukemia, 2007, 21(3):560-563.
[13] Hallek M, Wanders L, Strohmeyer S, et al. Thymidine kinase:a tumor marker with prognostic value for non-Hodgkin's lymphoma and a broad range of potential clinical applications. Ann Hematol, 1992, 65(1):1-5.
[14] He Q, Zou L, Li H, et al. Concentration of thymidine kinase 1 in serum (S-TK1) is a more sensitive proliferation marker in human solid tumors than its activity. Oncology Reports, 2005, 14(4):1013-1019.
[15] Wang Y, Jiang X, Dong S, et al. Serum TK1 is a more reliable marker than CEA and AFP for cancer screening in a study of 56,286 people. Cancer Biomark, 2016, 16(4):529-536.
[16] 庞丽君,王珊珊,吴云,等.胸苷激酶1原核表达载体的构建表达及鉴定. 医药论坛杂志, 2015, 36(8):6-8. Pang L J, Wang S S, Wu Y, et al. Construction, expression and identification of Thymidine kinase 1 prokaryotic expression vector. J Medical Forum, 2015, 36(8):6-8.
[17] 陈爱春,彭伟,汪生鹏. 亲和标签在重组蛋白表达与纯化中的应用.中国生物工程杂志, 2012, 32(12):93-103. Chen A C, Peng W, Wang S P. Application of affinity tags in the expression and purification of recombinant proteins. China Biotechnology, 2012, 32(12):93-103.
[18] 任广威. 组氨酸标签蛋白纯化介质的合成及其应用研究. 杭州:浙江工商大学, 食品科学与生物技术学院, 2011. Ren G W. Sythesis and Application of His-tagged Protein Purification Medium. Hangzhou:Zhejiang Gongshang University, College of Food Science and Biotechnology, 2011.
[19] Birringer M S, Perozzo R, Kut E, et al. High-level expression and purification of human thymidine kinase 1:quaternary structure,stability,and kinetics. Protein Expression and Purification, 2006, 47(1):506-515.
[20] 王云龙, 张春艳, 邓黎黎, 等. EB病毒融合蛋白Zta-P54在大肠杆菌中的表达、纯化及鉴定. 生物技术通报, 2014, 7(1):173-178. Wang Y L, Zhang C Y, Deng L L, et al. Expression,purification and identification of Zta-P54 fusion protein in escherichia coli of epstein-barr virus. Biotechnology Bulletin, 2014, 7(1):173-178.
[21] 葛新杰. NT-proBNP单克隆抗体的研制及ELISA定量检测方法的建立. 郑州:郑州大学, 生命科学学院, 2015. Ge X J. Development of Monoclonal Antibody NT-proBNP and Establishment of ELISA Quantitative Detection Method. Zhengzhou:Zhengzhou University, School of Life Science, 2015.
[22] 赵群莉. 血清胸苷激酶单克隆抗体酶促化学发光试剂盒的研制. 天津:天津医科大学, 免疫学, 2013. Zhao Q L. The Development of the Kit of the Enzyme Chemiluminescence Detection for Serum Thymidine Kinase with Monoclonal Antibody. Tianjin:Tianjin Medical University, Immunology, 2013. |
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