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:: Volume 23, Issue 2 (Iranian South Medical Journal 2020) ::
Iran South Med J 2020, 23(2): 143-152 Back to browse issues page
Comparative Evaluation of Expression Vectors (pET32a and pET25b) in Expression of Polyepitopic Sequence Synthesized from Leishmania infantum Antigens
Marzeah Taherzadeh1 , MoradAli Fouladvand 2, Bahram Kazemi3
1- Department of microbiology, School of Agriculture and Basic Sciences, Shiraz Branch, Islamic Azad University, Shiraz, Iran
2- The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran , mfooladvand39@yahoo.com
3- Cellular and Molecular Biology Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract:   (137 Views)
Background: Molecular methods are nowadays used to diagnose diseases, produce vaccines, drugs and recombinant proteins. Therefore, one of the important steps in these procedures is selecting the appropriate vector for cloning and expression of the target genes. Because the expression of synthetic polygenes or so-called chimeric antigens with high molecular weight requires special conditions, the aim of this study was to evaluate the importance of vector type for expression of such antigens.
Materials and Methods: The 1250bp sequence consisting of epitopes from eight important antigens of Leishmania infantum parasite was designed and synthesized by Biomatic Company (Cambridge, Canada). The sequence was cloned separately in two expression vectors pET25b and pET32a and then transformed into E.coli BL21 (DE3) and expressed under similar appropriate conditions. Bacterial lysates were analyzed by SDS PAGE and evaluated by Western blotting.
Results: of SDS- PAGE and Western blot analysis showed that intact recombinant protein production by pET25b vector was not successful. However, the recombinant protein resulted from the expression of the aforementioned poly-epitope in pET32a vector was successfully produced and confirmed.  
Conclusion: According to the successful expression confirmation of the poly-epitopic sequence within the pET32a vector and furthermore the failure to obtain protein in the pET25b vector showed that in the case of some specific mosaic sequences expression, protein isolation will be difficult because of low solubility. So, the expression vector choose should be made more carefully.
 
Keywords: Poly-epitopic structure, Leishmania infantum, Expression level, pET25b vector, pET32a vector
Full-Text [PDF 576 kb]   (45 Downloads)    
Type of Study: Original | Subject: Parasitology
Received: 2019/08/11 | Accepted: 2020/05/15 | Published: 2020/07/1
References
1. Mesa-Pereira B, Rea MC, Cotter PD, et al. Heterologous Expression Of Biopreservative Bacteriocins With A View To Low Cost Production. Front Microbiol 2018; 9: 1654.
2. Rosano GL, Ceccarelli EA. Recombinant Protein Expression In Escherichia Coli: Advances And Challenges. Front Microbiol 2014; 5: 172.
3. Faria AR, De Castro Veloso L, Coura-Vital W, et al. Novel Recombinant Multiepitope Proteins For The Diagnosis Of Asymptomatic Leishmania Infantum-Infected Dogs. Plos Negl Trop Dis 2015; 9(1): e3429.
4. Oliveira GG, Magalhães FB, Teixeira MC, et al. Characterization Of Novel Leishmania Infantum Recombinant Proteins Encoded By Genes From Five Families With Distinct Capacities For Serodiagnosis Of Canine And Human Visceral Leishmaniasis. Am J Trop Med Hyg 2011; 85(6): 1025-34.
5. Alibakhshi A, Bandehpour M, Kazemi B. Cloning, Expression And Purification Of A Polytopic Antigen Comprising Of Surface Antigens Of Toxoplasma Gondii. Iran J Microbiol 2017; 9(4): 251-6.
6. Liu ZQ, Yang PC. Construction Of Pet-32 Α (+) Vector For Protein Expression And Purification. N Am J Med Sci 2012; 4(12): 651-5.
7. Rietsch A, Belin D, Martin N, et al. An In Vivo Pathway For Disulfide Bond Isomerization In Escherichia Coli. Proc Natl Acad Sci U S A 1996; 93(23): 13048-53.
8. Mierendorf RC, Morris BB, Hammer B, et al. Expression And Purification Of Recombinant Proteins Using The pET System. Totowa NJ: Humana Press, The Nucleic Acid Protocols Handbook, 2000, 947-77.
9. Geum L, Huber R, Leung N, et al. Construction Of Recombinant Expression Vectors To Study The Effect Of Thioredoxin On Heterologous Protein Solubility. J Microbiol Immunol Infect 2015; 19: 1-5.
10. Chen X, Zaro JL, Shen WC. Fusion Protein Linkers: Property, Design And Functionality. Adv Drug Deliv Rev 2013; 65(10): 1357-69.
11. Sadeghi M, Doosti A. Cloning And Study Of Expression Of Helicobacter Pylori FlaAgene In Eukaryotic System. Iran South Med J 2017; 20(3): 245-56. (Persian)
12. Lizier M, Sarra PG, Cauda R, et al. Comparison Of Expression Vectors In Lactobacillus Reuteri Strains. FEMS Microbiol Lett 2010; 308(1): 8-15.
13. Mahmood N, Xie J. An Endogenous 'NonSpecific' Protein Detected By A His-Tag Antibody Is Human Transcription Regulator YY1. Data Brief 2015; 2: 52-5.
14. Western Blot Troubleshooting: Unusual or Unexpected Bands. Western Blot Troubleshooting: Unusual or Unexpected Bands BioRad. (Accessed June 27, 2020, at https://www.bio-rad-antibodies.com/westernblot-unusual-unexpected-bands-western-blotting.html)
15. Tobias AM, Toska D, Lange K, et al. Expression, Purification, And Inhibition Profile Of Dihydrofolate Reductase From The Filarial Nematode Wuchereria Bancrofti. PloS One 2018; 13(5): e0197173.
16. Wang J, LI Y, Guo F, et al. Expression And Activity Identification Of A Human Nasopharyngeal Carcinoma I50 Anti-Idiotype Antibody. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2011; 36(3): 185-91.
17. Lu Q, Li X, Zhao J, et al. Nanobody-Horseradish Peroxidase And-EGFP Fusions As Reagents To Detect Porcine Parvovirus In The Immunoassays. J Nanobiotechnol 2020; 18(1): 7.
18. Lee HB, Piao DC, Lee JY, et al. Artificially Designed Recombinant Protein Composed Of Multiple Epitopes Of Foot-And-Mouth Disease Virus As A Vaccine Candidate. Microb Cell Fact 2017; 16(1): 33.
19. Wang X, Xie G, Liao J, et al. Design And Evaluation Of A Multi-Epitope Assembly Peptide (MEAP) Against Herpes Simplex Virus Type 2 Infection In BALB/c Mice. Virol J 2011; 8: 232.
20. Boarino A, Scalone A, Gradoni L, et al. Development Of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes Of Leishmania Infantum For Serodiagnosis Of Visceral Leishmaniasis. Clin Diagn Lab Immunol 2005; 12(5): 647-53.
21. Jaramillo Ortiz JM, Montenegro VN, De La Fournière SA, et al. Development Of An Indirect ELISA Based On A Recombinant Chimeric Protein For The Detection Of Antibodies Against Bovine Babesiosis. Vet Sci 2018; 5(1): 13.
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Taherzadeh M, Fouladvand M, Kazemi B. Comparative Evaluation of Expression Vectors (pET32a and pET25b) in Expression of Polyepitopic Sequence Synthesized from Leishmania infantum Antigens. Iran South Med J. 2020; 23 (2) :143-152
URL: http://ismj.bpums.ac.ir/article-1-1295-en.html


Volume 23, Issue 2 (Iranian South Medical Journal 2020) Back to browse issues page
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