CONSTRUCTION AND EXPRESSION OF PROKARYOTIC EXPRESSION VECTOR OF TIAASSOCIATED ANTIGENS BY SEREX SCREENING
Keywords:
TIA, SEREX, genetic recombination, prokaryotic expression vector, GST tagged proteinAbstract
Objective: To construct the prokaryotic expressing vectors of cDNA insert fragments for TIA, and recombine GST-fusion proteins.Methods: A phage cDNA library of human aortic endothelial cell line was screened using sera of 19 patients with TIA, the cDNA insert of clones incorporated in pBluescript was cleaved by EcoRI/BamHI/SmalI and XhoI, and the insert fragments were isolated by 1% agarose gel electrophoresis. The expression plasmids of glutathione-S-transferase (GST)-fused proteins were constructed by recombining the cDNA sequence into pGEX-4T-1, 2, 3. The inserted DNA fragments were ligated in frame to pGEX- 4T vectors by using Ligation or In-Fusion protocol. The reaction mixtures were used to transform ECOS? competent E. coli BL-21 and appropriate recombinants were confirmed by DNA sequencing as well as protein expressions through SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The antigenic proteins succeeded in GST-tagged proteins recombinant could be candidate antigens. Results: 21 recombinant pGEX-4T expression vectors containing cDNA sequences of target genes were obtained from 70 SEREX antigens, and the expression of the GST-fusion proteins were shown on SDS-polyacrylamide gels stained with Coomassie staining, and thus, which could be as candidate antigens for further study. Conclusion: Recombination pGEX-4T expression plasmids could be constructed and expressed successfully by Ligation and In-Fusion methods, which may provide basis for the extraction and purification of these antigenic proteins aiming to identify serum antibodies in further study.References
[1] U Sahin, O Türeci, H Schmitt, B Cochlovius, T Johannes, R Schmits, et al. Human neoplasms elicitmultiple specific immune responses in the autologous host. Proc Natl Acad Sci USA, 1995, 92(25): 11810-11813.
[2] Wen-Tao Yue, Zi-Tong Wang, Yue Wang, Lina Zhang. Construction of a T7 human lung cancer cDNA library. Chin J Lung Cancer, 2008, 11(5): 686- 691.
[3] Sang-Yull Lee, Dooil Jeoung. The reverse proteomics for identification of tumor antigens. J Microbiol Biotechnol, 2007, 17(6): 879-890.
[4] Geng Li, Amanda Miles, Aija Line, Robert C Rees. Identification of tumor antigens by serological analysis of cDNA expression cloning. Cancer Immunol Immunother, 2004, 53(3): 139-143.
[5] James A Koziol, Jian-Ying Zhang, Carlos A Casiano, Xuan-Xian Peng, Fu-Dong Shi, Anne C Feng, et al. Recursive partitioning as an approach to selection of immune markers for tumor diagnosis. Clin Cancer Res, 2003, 9(14): 5120-5126.
[6] Sang-Yull Lee, Yuichi Obata, Masahiro Yoshida, Elisabeth Stockert, Barbara Williamson, Achim A Jungbluth, et al. Immunomic analysis of human sarcoma. Proc Natl Acad Sci USA, 2003, 100(5): 2651-2656.
[7] Michael Pfreundschuh. Exploitation of the B cell repertoire for the identification of human tumor antigens. Cancer Chemother Pharmacol, 2000, 46 (Supp l): S3-S7.
[8] Ken-Ichiro Goto, Takao Sugiyama, Ryutaro Matsumura, Xiao-Meng Zhang, Risa Kimura, Akiko Taira, et al. Identification of cerebral infarction- specific antibody markers from autoantibodies detected in patients with systemic lupus erythematosus. J Mol Biomark Diagnos, 2015, 6:2.
[9] Mayumi Muto, Masahiro Mori, Takaki Hiwasa, Masaki Takiguchi, Yasuo Iwadate, Akiyuki Uzawa, et al. Novel serum autoantibodies against talin1 in multiple sclerosis: Possible pathogen tic roles of the antibodies. J Neuroimmunol, 2015, 284:30-36.
[10] Philippe Krebs, Michael Kurrer, Ugur Sahin, Ozlem Türeci, Burkhard Ludewig. Autoimmunity seen through the SEREX-scope. Autoimmunity Reviews, 2003, 2(6):339-345.
[11] J Chris Bradberry, Susan C Fagan, David R Gray, Yong S K Moon. New perspectives on the pharmacotherapy of ischemic stroke. J Am Pharm Assoc, 2004, 44(2S l1): S46-S56.
[12] Peter M Rothwell, Charles P Warlow. Timing of TIAs preceding stroke: time window for prevention is very short. Neurology, 2005, 64(5): 817-820.
[13] Caren M Wu, Kevin McLaughlin, Diane L Lorenzetti, Michael D Hill, Braden J Manns, William A Ghali. Early Risk of Stroke After Transient Ischemic Attack: A Systematic Review and Meta-analysis. Archives of Internal Medicine, 2008, 167(22):2417-2422.
[14] Dominic Tse, Michael D. Hill, Shelagh B. Coutts. Early Secondary Prevention in Transient Ischemic Attack (TIA) and Minor Stroke. Current Neurology and Neuroscience Reports, 2019, 19(6).
[15] Peter M Rothwell, Matthew F Giles, Arvind Chandratheva, Lars Marquardt, Olivia Geraghty, Jessica N E Redgrave, et al. Effect of urgent treatment of transient ischaemic attack and minor stroke on early recurrent stroke (EXPRESS study): a prospective population-based sequential comparison. Lancet, 2007, 370(9596):1432.
[16] Philippa C Lavallée, Elena Meseguer, Halim Abboud, Lucie Cabrejo, Jean- Marc Olivot, Olivier Simon, et al. A transient ischaemic attack clinic with round-the-clock access (SOS-TIA): feasibility and effects. Lancet Neurology, 2007, 6(11):953-960.
[17] Glen C Jickling, Frank R Sharp. Biomarker Panels in Ischemic Stroke. Stroke, 2015, 46(3):915-920.
[18] Emmanuel Touzé, Olivier Varenne, David Calvet, Jean-Louis Mas. Coronary risk stratification in patients with ischemic stroke or transient ischemic stroke attack. International Journal of Stroke, 2010, 2(3):177-183.
[19] R Donnelly, A M Emslie-Smith, I D Gardner, A D Morris. ABC of arterial and venous disease: Vascular complications of diabetes. BMJ, 2000, 320(7241):1062-1066.
[20] Desheng Hu, Changjun Yin, Shanshan Luo, Andreas J R Habenicht, Sarajo K. Mohanta. Vascular Smooth Muscle Cells Contribute to Atherosclerosis Immunity. Frontiers in immunology,2019,10:1101.
[21] Hideaki Shimada, Masaaki Ito, Akiko Kagaya, Tooru Shiratori, Mari Kuboshima, Masae Suzuki, et al. Elevated serum antibody levels against cyclin L2 in patients with esophageal squamous cell carcinoma. J Cancer Sci Ther, 2015, 7(2): 60-66.
[22] Hideaki Shimada, Satoshi Yajima, Yoko Oshima, Takaki Hiwasa, Masatoshi Tagawa, Kazuyuki Matsushita, Fumio Nomura. Impact of serum biomarkers on esophageal squamous cell carcinoma. Esophagus, 2012, 9: 131-140.
[23] Jaehong Park, Andrea L Throop, Joshua LaBaer. Site-Specific Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes. Current protocols in molecular biology, 2015, 110:3.20.1- 3.20.23.
[24] Chad R Irwin, Andrew Farmer, David O Willer, David H Evans. In-fusion? cloning with vaccinia virus DNA polymerase. Methods Mol Biol, 2012;890:23‐35.
[25] Chao-Guang Chen, Louis J. Fabri, Michael J. Wilson, and Con Panousis. One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification. Nucleic Acids Res, 2014;42(4): e26.
[26] Roxane Lestini, Sergey P Laptenok, Jo?lle Kühn, Mark A Hink, Marie- Claire Schanne-Klein, Ursula Liebl, Hannu Myllykallio. Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication. Nucleic Acids Res, 2013;41(22):10358‐10370.