| تعداد نشریات | 282 |
| تعداد نشریات سازمان | 80 |
| تعداد شماره‌ها | 3,272 |
| تعداد مقالات | 36,149 |
| تعداد مشاهده مقاله | 55,713,506 |
| تعداد دریافت فایل اصل مقاله | 94,492,257 |
Cloning, Expression and Functionality Evaluation of Recombinant Monoclonal Antibody Against VP1 Capsid Protein of FMD Virus | ||
| Archives of Razi Institute | ||
| مقاله 17، دوره 81، شماره 1، فروردین و اردیبهشت 2026، صفحه 147-156 اصل مقاله (1.31 M) | ||
| نوع مقاله: Original Articles | ||
| شناسه دیجیتال (DOI): 10.66224/ARI.81.1.3491 | ||
| نویسندگان | ||
| Mozhgan Helalinasab1؛ Mohammad Mehdi Ranjbar* 2؛ Hadi Pourtaghi3؛ Mohammad Kazem Shahkarami2 | ||
| 1Department of Pathobiology, SR.C., Islamic Azad University, Tehran, Iran. | ||
| 2Department of Human Viral Vaccines, Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran. | ||
| 3Department of Microbiology, Ka.C., Islamic Azad University, Karaj, Iran. | ||
| چکیده | ||
| Introduction: Foot-and-mouth disease (FMD) is a highly contagious viral disease caused by the FMD virus (FMDV), which belongs to the Aphthovirus genus in the Picornaviridae family. FMDV is a highly variable RNA virus, and there is limited cross-protection between serotypes and strains. The disease can have devastating economic, social, and environmental impacts. The FMDV capsid is composed of VP1-VP4 structural proteins, with VP1 being the most abundant protein consisting of 213 amino acids. The G-H loop (residues 141-160) of the VP1 capsid protein is highly variable and serves as the main antigenic region. It contains a conserved triplet of amino acids, Arg-Gly-Asp (RGD), which induces the production of protective antibodies against various FMDV types. Monoclonal antibodies (mAbs) play a crucial role in detecting and serotyping FMDV in pathological samples, as well as evaluating protection post-vaccination against FMD. Materials & Methods: This study explores the expression and function of an engineered recombinant single-chain variable fragment (scFv-mAb) in Escherichia coli BL21 (DE3) Rosetta strain as a cost-effective prokaryotic system with high yield. The scFv-mAb gene was inserted into the pET28a (+) expression plasmid. E. coli cells were transformed with the plasmid, induced with 0.5 mM IPTG, and incubated at 37 ˚C for 12 hours. The protein was purified using a Ni2+-NTA resin column and analyzed by 12% SDS-PAGE for quality assessment. The efficiency and functionality of the scFv-mAb were confirmed using an indirect sandwich (capture) enzyme-linked immunosorbent assay (ELISA). Results: The purified scFv-mAb concentration was determined to be approximately 2.00 mg/mL using the Bradford assay under optimal conditions. Each well was coated with 400 ng of scFv-mAb based on checkerboard results and the mean of negative serum at a 1:10 dilution. The indirect sandwich ELISA assay yielded an optical density (OD) signal range of 0.3 to 1.5 at a 450 nm wavelength in different positive control treatments. Conclusion: The ELISA results showed that the scFv-mAb fragment successfully detected serotype O of FMDV. Further research could confirm the potential of this recombinant antibody for broader commercial applications in the future. | ||
| کلیدواژه‌ها | ||
| Foot-and-mouth disease (FMD)؛ G- H loop؛ VP1؛ Recombinant scFv-mAb؛ Indirect sandwich enzyme-linked immunosorbent assay (ELISA) | ||
| عنوان مقاله [English] | ||
| کلونینگ، بیان و ارزیابی عملکرد آنتی بادی مونوکلونال نوترکیب علیه پروتئین کپسید VP1 ویروس FMD | ||
| چکیده [English] | ||
| بیماری تب برفکی (FMD) یکی از مسری ترین بیماری های ویروسی با اثرات بالقوه مخرب اقتصادی، اجتماعی و زیستی است که توسط ویروسی از جنس آفتوویروس، خانواده Picornaviridae ایجاد می شود. ویروس FMD (FMDV) یک ویروس RNA بسیار متغییر است و بین سروتیپ‌ها و حتی ساب تایپ های هر سروتایپ، حفاظت متقاطع کمی وجود داشته یا اصلاً وجود ندارد. آنتی بادی مونوکلونال (Mab) نقش مهمی در شناسایی و تعیین سروتیپ FMDV در نمونه های پاتولوژیک و همچنین ارزیابی محافظت در برابر FMD پس از واکسیناسیون را دارد. این مطالعه بیان و عملکرد یک قطعه متغیر تک زنجیره ای نوترکیب مهندسی شده (ScFv-Mab) در سویه روزتا اشریشیا کلی (E.coli) BL21 (DE3) را بررسی می کند. تولید Mab نوترکیب در برابر FMDV در سیستم پروکاریوتی مقرون به صرفه، ساده و با عملکرد بالا است که می تواند پیچیدگی های تولید را کاهش دهد. ژن ScFv-Mab طراحی شده در پلاسمید بیان pET28a (+) سفارش داده شد. پروتئین بیان شده با استفاده از ستون رزین Ni2+-NTA خالص‌سازی شد و کیفیت آن با 12% SDS-PAGE ارزیابی شد. در نهایت، کارایی و عملکرد ScFv-Mab با روش الایزای غیرمستقیم ساندویچ (گرفتن) تایید شد. سویه روزتا E. coli BL21 (DE3) ترنسفورشده، با 0.5 میلی مولار IPTG القا شد و به مدت 12 ساعت در دمای 37 درجه سانتی گراد انکوبه شد. پروتئین قابل توجهی در هر دو بخش محلول و نامحلول با خلوص بیش از 90٪ بیان شد. غلظت ScFv-Mab خالص شده در شرایط بهینه تقریباً 2 میلی گرم بر میلی لیتر با روش برادفورد محاسبه شد. با آنالیز میانگین سرم منفی و چکربوردها در رقت 1:10، 400 نانوگرم ScFv-Mab در هر چاهک پوشش داده شد. در نهایت چگالی نوری (OD) 0.3 به عنوان برش در روش Sandwich ELISA غیر مستقیم انتخاب شد. نتایج ELISA نشان داد که قطعه ScFv-Mab با موفقیت سروتیپ O - FMDV را با سیگنال‌های در محدوده بالاتر یا مساوی 0.3 و کمتر یا مساوی 1.5 در طول موج 450 نانومتر در تیمارهای مختلف کنترل مثبت شناسایی کرد. | ||
| کلیدواژه‌ها [English] | ||
| FMDV, لوپ G-H, VP1, ScFv-Mab نوترکیب, الایزای ساندویچ غیرمستقیم | ||
|
سایر فایل های مرتبط با مقاله
|
||
| مراجع | ||
|
World Organization for Animal Health (OIE). Chapter 3.1. 8. Foot and mouth disease (infection with foot and mouth disease virus). Manual of diagnostic tests and vaccines for terrestrial animals 2018. 2019 [Updated 2026 Jan 27]. Available from: [Link]
Cavalera S, Russo A, Foglia EA, Grazioli S, Colitti B, Rosati S, et al. Design of multiplexing lateral flow immunoassay for detection and typing of foot-and-mouth disease virus using pan-reactive and serotype-specific monoclonal antibodies: Evidence of a new hook effect. Talanta. 2022; 240:123155. [DOI:10.1016/j.talanta.2021.123155] [PMID]
Ochoa WF, Kalko SG, Mateu MG, Gomes P, Andreu D, Domingo E, et al. A multiply substituted G-H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: A role for water molecules. J Gen Virol. 2000; 81(Pt 6):1495-505. [DOI:10.1099/0022-1317-81-6-1495] [PMID]
Marrero R, Limardo RR, Carrillo E, König GA, Turjanski AG. A computational study of the interaction of the foot and mouth disease virus VP1 with monoclonal antibodies. J Immunol Methods. 2015; 425:51-7. [DOI:10.1016/j.jim.2015.06.008] [PMID]
Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. ScFv antibody: Principles and clinical application. Clin Dev Immunol. 2012; 2012:980250. [DOI:10.1155/2012/980250][PMID]
Gasteiger E, Hoogland C, Gattiker A, Duvaud SE, Wilkins MR, Appel RD, et al. Protein identification and analysis tools on the ExPASy server. In: Walker JM, editor. The Proteomics Protocols Handbook. Springer Protocols Handbooks. New Jersey: Humana Press. [DOI:10.1385/1-59259-890-0:571]
Sambrook J. Plasmid and their usefulness in molecular cloning. Mol Cloning Lab Man. 2001; 1:1-31. [Link]
No Author. Plasmid DNA Extraction Mini Kit, based on kit instructions [Internet]. 2026 [8 April 2026]. Available from: [Link]
Chan CE, Chan AH, Lim AP, Hanson BJ. Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays. J Immunol Methods. 2011; 373(1-2):137-146. [DOI:10.1016/j.jim.2011.08.005][PMID]
Graph Pad. Graph Pad online server regression tools [Internet]. 2026 [Updated 8 April 2026]. Available from: [Link]
Brocchi E, Crosatti ML, Grazioli S, Bugnetti M. Preliminary evaluation of panels of monoclonal antibodies developed against four vaccine strains of FMDVes of serotype A for antigenic profiling and vaccine matching of field isolates. Abstract book of the Open Session of the EuFMD Standing Technical Committee, Erice, Italy, 14-17 October, 2008; Appendix 5, pp. 85. [Link]
Grazioli S, Ferris NP, Dho G, Pezzoni G, Morris AS, Mioulet V, et al. Development and validation of a simplified serotyping ELISA based on monoclonal antibodies for the diagnosis of foot-and-mouth disease virus serotypes O, A, C and Asia 1. Transbound Emerg Dis. 2020; 67(6):3005-15. [DOI:10.1111/tbed.13677] [PMID]
Ferris NP, Grazioli S, Hutchings GH, Brocchi E. Validation of a recombinant integrin αvβ6/monoclonal antibody based antigen ELISA for the diagnosis of foot-and-mouth disease. J Virol Methods. 2011; 175(2):253-60. [DOI:10.1016/j.jviromet.2011.05.026] [PMID]
Yaghoobizadeh F, Ardakani MR, Ranjbar MM, Galehdari H, Khosravi M. Expression, purification, and study on the efficiency of a new potent recombinant scFv antibody against the SARS-CoV-2 spike RBD in E. coli BL21. Protein Expr Purif. 2023; 203:106210. [DOI:10.1016/j.pep.2022.106210][PMID]
Dong H, Liu P, Bai M, Wang K, Feng R, Zhu D, et al. Structural and molecular basis for foot-and-mouth disease virus neutralization by two potent protective antibodies. Protein Cell. 2022; 13(6):446-53. [DOI:10.1007/s13238-021-00828-9][PMID]
Baumgarten T, Ytterberg AJ, Zubarev RA, de Gier JW. Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress. Appl Environ Microbiol. 2018; 84(12):e00270-18. [DOI:10.1128/AEM.00270-18][PMID]
Chitray M, Opperman PA, Rotherham L, Fehrsen J, Van Wyngaardt W, Frischmuth J, et al. Diagnostic and epitope mapping potential of single-chain antibody fragments against foot-and-mouth disease virus serotypes A, SAT1, and SAT3. Front Vet Sci. 2020; 7:475. [DOI:10.3389/fvets.2020.00475][PMID]
Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli expression system in producing antibody recombinant fragments. Int J Mol Sci. 2020; 21(17):6324. [DOI:10.3390/ijms21176324][PMID]
Ferris NP, Nordengrahn A, Hutchings GH, Reid SM, King DP, Ebert K, et al. Development and laboratory validation of a lateral flow device for the detection of foot-and-mouth disease virus in clinical samples. J Virol Methods. 2009; 155(1):10-7. [DOI:10.1016/j.jviromet.2008.09.009] [PMID]
Tami C, Taboga O, Berinstein A, Núñez JI, Palma EL, Domingo E, et al. Evidence of the coevolution of antigenicity and host cell tropism of foot-and-mouth disease virus in vivo. J Virol. 2003; 77(2):1219-26. [DOI:10.1128/JVI.77.2.1219-1226.2003][PMID]
Verdaguer N, Sevilla N, Valero ML, Stuart D, Brocchi E, Andreu D, et al. A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: Implications for intratypic antigenic variation.J Virol. 1998; 72(1):739-48. [DOI:10.1128/JVI.72.1.739-748.1998][PMID]
Wang G, Liu Y, Feng H, Chen Y, Yang S, Wei Q, et al. Immunogenicity evaluation of MS2 phage-mediated chimeric nanoparticle displaying an immunodominant B cell epitope of foot-and-mouth disease virus. PeerJ. 2018; 6:e4823. [DOI:10.7717/peerj.4823][PMID]
JacksonT, King AM, Stuart DI, Fry E. Structure and receptor binding. Virus Res. 2003; 91(1):33-46. [DOI:10.1016/S0168-1702(02)00258-7] [PMID]
Brocchi E, Gamba D, Poumarat F, Martel JL, De Simone F. Improvements in the diagnosis of contagious bovine pleuropneumonia through the use of monoclonal antibodies. Rev Sci Tech. 1993; 12(2):559-70. [DOI:10.20506/rst.12.2.702.] [PMID]
Ferris NP, Dawson M. Routine application of enzyme-linked immunosorbent assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular diseases. Vet Microbiol. 1988; 16(3):201-9. [DOI:10.1016/0378-1135(88)90024-7] [PMID]
Roeder PL, Le Blanc Smith PM. Detection and typing of foot-and-mouth disease virus by enzyme-linked immunosorbent assay: A sensitive, rapid and reliable technique for primary diagnosis. Res Vet Sci. 1987; 43(2):225-32. [DOI:10.1016/S0034-5288(18)30778-1] [PMID]
Golchin M. Recombinant antibodies against the K99 colonisation factor of E. coli [PhD dissertation]. University of Glasgow; 2004. [Link]
Zhang W, Lu J, Zhang S, Liu L, Pang X, Lv J. Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: Expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study. Microb Cell Fact. 2018; 17(1):50.[DOI:10.1186/s12934-018-0894-y][PMID]
Sharma GK, Mahajan S, Matura R, Subramaniam S, Mohapatra JK, Pattnaik B. Production and characterization of single-chain antibody (scFv) against 3ABC non-structural protein in Escherichia coli for sero-diagnosis of Foot and Mouth Disease virus. Biologicals. 2014; 42(6):339-45. [DOI:10.1016/j.biologicals.2014.08.005] [PMID]
ShengFeng C, Ping L, Tao S, Xin W, GuoFeng W. Construction, expression, purification, refold and activity assay of a specific scFv fragment against foot and mouth disease virus. Vet Res Commun. 2003; 27(3):243-56. [DOI:10.1023/A:1023300825438] [PMID] | ||
|
آمار تعداد مشاهده مقاله: 996 تعداد دریافت فایل اصل مقاله: 854 |
||
