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:: Volume 21, Issue 4 (Bimonthly 2017) ::
Feyz 2017, 21(4): 359-366 Back to browse issues page
Gene cloning and evaluation of the Acinetobacter baumannii nlpD gene expression in human dermal fibroblast cells using RT-PCR
Rasoul Hashemzehi , Abbas Doosti , Mohammad Kargar , Mojtaba Jaafarinia
Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, I. R. Iran. , abbasdoosti@yahoo.com
Abstract:   (6554 Views)
Background:  Acinetobacter baumannii is one of the highly antibiotic-resistant bacteria in the world. This bacterium is a cause of endemic and epidemic nosocomial infections and despite many efforts, there is still no effective vaccine agains it. NlpD is one of the important antigenic agents that stimulate the immune system. So, the aim of this study was to examine gene cloning and expression of the nlpD gene of A. baumannii in human dermal fibroblast (HDF) cells.
Materials and Methods: In this experimental study, the nlpD gene was amplified from A. baumannii genome using polymerase chain reaction (PCR). Then, the nlpD gene was cloned and sub-cloned in pTZ57R/T and pIRES2-EGFP vectors, respectively. Confirmation of gene cloning was performed by PCR, restriction endonuclease and sequencing methods. The final pIRES2-EGFP-nlpD recombinant vector was transformed into HDF cells using electroporation and the expression of target gene was evaluated by RT-PCR.
Results: In this study, the 831 bp nlpD gene of A. baumannii was amplified successfully. Also, the results of the study showed that the recombinant pIRES2-EGFP-nlpD final construct was produced. Observation of the 831 bp band on agarose gel in transformed cells compared to control cells confirmed the nlpD gene expression in HDF cells.
Conclusion: The final construct that generates in this study can express the nlpD gene of A. baumannii in eukaryotic cells. Successful expression of the target gene can be used as a new recombinant vaccine in animal model. The pIRES2-EGFP-nlpD recombinant vector has also the potential as a gene vaccine for future research.
Keywords: Acinetobacter baumannii, nlpD, Gene expression, RT-PCR
Full-Text [PDF 317 kb]   (2471 Downloads)    
Type of Study: Research | Subject: General
Received: 2017/03/27 | Revised: 2017/10/7 | Accepted: 2017/07/4 | Published: 2017/10/7
References
1. Pourhajibagher M, Hashemi FB, Pourakbari B, Aziemzadeh M, Bahador A. Antimicrobial resistance of Acinetobacter baumannii to imipenem in Iran: a systematic review and meta-analysis. Open Microbiol J 2016; 10: 32-42.
2. Howard A, O'Donoghue M, Feeney A, Sleator RD. Acinetobacter baumannii: an emerging opportunistic pathogen. Virulence 2012; 3(3): 243-50.
3. Kroger C, Kary SC, Schauer K, Cameron AD. Genetic regulation of virulence and antibiotic resistance in Acinetobacter baumannii. Genes (Basel) 2016; 8(1): pii: E12.
4. Ni Z, Chen Y, Ong E, He Y. Antibiotic resistance determinant-focused Acinetobacter baumannii vaccine designed using reverse vaccinology. Int J Mol Sci 2017; 18(2): pii: E458.
5. Ahmad TA, Tawfik DM, Sheweita SA, Haroun M, El-Sayed LH. Development of immunization trials against Acinetobacter baumannii. Trials Vaccinol 2016; 5: 53-60.
6. Hua X, Liu L, Fang Y, Shi Q, Li X, Chen Q, et al. Colistin resistance in Acinetobacter baumannii MDR-ZJ06 revealed by a multiomics approach. Front Cell Infect Microbiol 2017; 7: 45.
7. Singh R, Capalash N, Sharma P. Reverse vaccinology: developing vaccine against MDR Acinetobacter baumannii. J Vaccines Vaccin 2016; 7(3): 1-3.
8. Eslami E, Doosti A. Cloning and expression study of the hcpD gene of Helicobacter pylori. J Ardabil Uni Med Sci 2017; 17(1): 499-510. [in Persian]
9. Moriel DG, Beatson SA, Wurpel DJ, Lipman J, Nimmo GR, Paterson DL, et al. Identification of novel vaccine candidates against multidrug-resistant Acinetobacter baumannii. PLoS One 2013; 8(10): e77631.
10. Tidhar A, Flashner Y, Cohen S, Levi Y, Zauberman A, Gur D, et al. The NlpD lipoprotein is a novel Yersinia pestis virulence factor essential for the development of plague. PLoS One 2009; 4(9): e7023.
11. Doosti A, Ghasemi-Dehkordi P, Kargar M, Sharifi A. Generation of divalent DNA vaccine based on p39 and shiga-like toxin 2 (stx2) genes. Genetika 2015; 47(2): 499-507.
12. Ghorbani M, Doosti A. Isolation and cloning of Helicobacter Pylori ureE gene into pIRES2-DSRed expression vector to generate a gene vaccine. J Birjand Uni Med Sci 2016; 23(4): 286-97. [in Persian]
13. Snitkin ES, Zelazny AM, Montero CI, Stock F, Mijares L, Murray PR, et al. Genome-wide recombination drives diversification of epidemic strains of Acinetobacter baumannii. Proc Natl Acad Sci USA 2011; 108(33): 13758-63.
14. Taiwo AA, Falilat AJ, Ezemuel YS. Computational design of peptide vaccine against Acinetobacter baumannii infection using comparative genomic approach. Comput Biol Bioinform 2014; 2(1): 13-18.
15. Jorritsma SH, Gowans EJ, Grubor-Bauk B, Wijesundara DK. Delivery methods to increase cellular uptake and immunogenicity of DNA vaccines. Vaccine 2016; 34(46): 5488-94.
16. Luo G, Lin L, Ibrahim AS, Baquir B, Pantapalangkoor P, Bonomo RA, et al. Active and passive immunization protects against lethal, extreme drug resistant-Acinetobacter baumannii infection. PLoS One 2012; 7(1): e29446.
17. Lin L, Tan B, Pantapalangkoor P, Ho T, Hujer AM, Taracila MA, et al. Acinetobacter baumannii rOmpA vaccine dose alters immune polarization and immunodominant epitopes. Vaccine 2013; 31(2): 313-8.
18. Singh R, Garg N, Shukla G, Capalash N, Sharma P. Immunoprotective efficacy of Acinetobacter baumannii outer membrane protein, FilF, predicted In silico as a potential vaccine candidate. Front Microbiol 2016; 7: 158.
19. Huang W, Wang S, Yao Y, Xia Y, Yang X, Long Q, et al. OmpW is a potential target for eliciting protective immunity against Acinetobacter baumannii infections. Vaccine 2015; 33(36): 4479-85.
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Hashemzehi R, Doosti A, Kargar M, Jaafarinia M. Gene cloning and evaluation of the Acinetobacter baumannii nlpD gene expression in human dermal fibroblast cells using RT-PCR. Feyz 2017; 21 (4) :359-366
URL: http://feyz.kaums.ac.ir/article-1-3308-en.html


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Volume 21, Issue 4 (Bimonthly 2017) Back to browse issues page
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