Volume 25, Issue 5 (Iranian South Medical Journal 2023)                   Iran South Med J 2023, 25(5): 408-421 | Back to browse issues page

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Rahimpour M, Naeimi S, Rahimpour A, Farshadpour F, Taherkhani R. Design and Cloning of the Optimized L1 Gene from Human Papilloma virus 18 into the Expression Vector PcDNA3 and Evaluating its Expression in a Eukaryotic System. Iran South Med J 2023; 25 (5) :408-421
URL: http://ismj.bpums.ac.ir/article-1-1657-en.html
1- Department of Genetics, Colleague of Science, Kazerun Branch, Islamic Azad University, Kazerun, Iran
2- Department of Tissue Engineering, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3- The Persian Gulf Tropical Medicine Research Center, The Persian Gulf Biomedical Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran
4- The Persian Gulf Tropical Medicine Research Center, The Persian Gulf Biomedical Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran , taherkhanireza2005@yahoo.com
Abstract:   (174 Views)
Background: Vaccines have played a special role in controlling and reducing mortality from infectious diseases. In this regard, DNA vaccines were developed to ease the production and reduce the risks of traditional vaccines. Human papillomavirus (HPV) has been introduced as the causing agent of cervical cancer. The capsid protein (L1) of HPV has been used to produce subunit and DNA vaccines. The aim of this experimental research is to design and construct the L1 expression system of HPV 18 and to investigate its expression in eukaryotic cells.
Method and Materials: In this experimental study, the L1 gene of HPV 18 was subcloned in the expression vector pcDNA 3.1 Hygro after optimization and synthesis. Cloning was confirmed through colony PCR test and enzyme digestion reaction. The expression vector was transfected into HEK293 cells using the Turbofect reagent. After 72 hours, total RNA was extracted from transfected cells and control cells and cDNA was synthesized. Gene expression was examined at the mRNA level in cells by performing PCR on cDNA.
Results: The results showed that following the optimization of the L1 gene sequence, the CAI and Fop indices increased to an ideal level. The cloning of the optimized HPV 18-L1 gene in the pcDNA3 expression vector was successfully confirmed by colony PCR test and enzyme digestion reaction, and the results indicate the production of recombinant plasmid pCDNA3.1-L1. Finally, the evaluation of the L1 gene at the mRNA expression level showed the successful expression of the L1 gene in the eukaryotic system.
Conclusion: The results of this research show the effectiveness of the constructed expression vector in the effective expression of the L1 gene in vitro. This expression vector can be used as a DNA vaccine in future studies.
Full-Text [PDF 610 kb]   (56 Downloads)    
Type of Study: Original | Subject: Microbiology and Immunology
Received: 2022/07/20 | Accepted: 2023/01/10 | Published: 2023/02/19

1. Yousefi Z, Aria H, Ghaedrahmati F, et al. An Update on Human Papilloma Virus Vaccines: History, Types, Protection, and Efficacy. Front Immunol 2022; 12: 805695. [DOI]
2. Magalhaes GM, Vieira EC, Garcia LC, et al. Update on human papilloma virus - part I: epidemiology, pathogenesis, and clinical spectrum. An Bras Dermatol 2021; 96(1): 1-16. [DOI]
3. Araldi RP, Sant'Ana TA, Modolo DG, et al. The human papillomavirus (HPV)-related cancer biology: An overview. Biomed Pharmacother 2018; 106: 1537-56. [DOI]
4. Soheili M, Keyvani H, Soheili M, et al. Human papilloma virus: A review study of pidemiology, carcinogenesis, diagnostic methods, and treatment of all HPV-related cancers. Med J Islam Repub Iran 2021; 35: 65. [DOI]
5. Crosbie EJ, Einstein MH, Franceschi S, et al. Human papillomavirus and cervical cancer. Lancet 2013; 382(9895): 889-99. [DOI]
6. Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021; 71(3): 209-49. [DOI]
7. Balasubramaniam SD, Balakrishnan V, Oon CE, et al. Key Molecular Events in Cervical Cancer Development. Medicina (Kaunas) 2019; 55(7): 384. [DOI]
8. Wilting SM, Steenbergen RDM. Molecular events leading to HPV-induced high grade neoplasia. Papillomavirus Res 2016; 2: 85-8. [DOI]
9. Roden RBS, Stern PL. Opportunities and challenges for human papillomavirus vaccination in cancer. Nat Rev Cancer 2018; 18(4): 240-54. [DOI]
10. Lee J, Arun Kumar S, Jhan YY, et al. Engineering DNA vaccines against infectious diseases. Acta Biomater 2018; 80: 31-47. [DOI]
11. Marc MA, Dominguez-Alvarez E, Gamazo C. Nucleic acid vaccination strategies against infectious diseases. Expert Opin Drug Deliv 2015; 12(12): 1851-65. [DOI]
12. Williams JA. Vector Design for Improved DNA Vaccine Efficacy, Safety and Production. Vaccines (Basel) 2013; 1(3): 225-49. [DOI]
13. Stenler S, Blomberg P, Smith CI. Safety and efficacy of DNA vaccines: plasmids vs. minicircles. Hum Vaccin Immunother 2014; 10(5): 1306-8. [DOI]
14. 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) [Article]
15. Taherkhani R, Farshadpour F, Makvandi M, et al. Cloning of fliC Gene From Salmonella typhimurium in the Expression Vector pVAX1 and Evaluation of its Expression in Eukaryotic Cells. Jundishapur J Microbiol 2014; 7(11): e12351. [DOI]
16. Taherkhani R, Farzaneh MR, Taherkhani S, et al. Molecular Detection of Epstein-Barr virus in Biopsy Samples of Patients Suffering from Bladder Cancer in Bushehr Province, Iran. Iran South Med J 2022; 25(4): 326-39. [Article]
17. Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol 1981; 151(3): 389-409. [DOI]
18. Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes. J Mol Biol 1981; 146(1): 1-21. [DOI]
19. Farshadpour F, Makvandi M, Taherkhani R. Design, Construction and Cloning of Truncated ORF2 and tPAsp-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector. Jundishapur J Microbiol 2015; 8(12): e26035. [DOI]
20. Farshadpour F, Taherkhani R, Makvandi M, et al. Codon-Optimized Expression and Purification of Truncated ORF2 Protein of Hepatitis E Virus in Escherichia coli. Jundishapur J Microbiol 2014; 7(7): e11261. [DOI]
21. Simabuco FM, Tamura RE, Carromeu C, et al. Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice. J Virol Methods 2009; 158(1-2): 93-9. [DOI]
22. Harper DM, DeMars LR. HPV vaccines - A review of the first decade. Gynecol Oncol 2017; 146(1): 196-204. [DOI]
23. Yang B, Yang A, Peng S, et al. Coadministration with DNA encoding papillomavirus capsid proteins enhances the antitumor effects generated by therapeutic HPV DNA vaccination. Cell Biosci 2015; 5: 35. [DOI]
24. Kumar S, Biswas M, Jose T. HPV vaccine: Current status and future directions. Med J Armed Forces India 2015; 71(2): 171-7. [DOI]
25. Lalonde ME, Durocher Y. Therapeutic glycoprotein production in mammalian cells. J Biotechnol 2017; 251: 128-40. [DOI]
26. Zhu J. Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv 2012; 30(5): 1158-70. [DOI]
27. Nettleship JE, Watson PJ, Rahman-Huq N, et al. Transient expression in HEK 293 cells: an alternative to E. coli for the production of secreted and intracellular mammalian proteins. Methods Mol Biol 2015; 1258: 209-22. [DOI]
28. Namvar A, Bolhassani A, Javadi G, et al. In silico/In vivo analysis of high-risk papillomavirus L1 and L2 conserved sequences for development of cross-subtype prophylactic vaccine. Sci Rep 2019; 9(1): 15225. [DOI]

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