Gene cloning and evaluation of the Acinetobacter baumannii nlpD gene expression in human dermal fibroblast cells using RT-PCR

• Main Authors: Rasoul Hashemzehi, Abbas Doosti, Mohammad Kargar, Mojtaba Jaafarinia
• Format: Article
• Language: fas
• Published: Kashan University of Medical Sciences and Health Services 2017-08-01
• Series: Fiyz̤
• Subjects: Acinetobacter baumannii; nlpD; Gene expression
• Online Access: http://feyz.kaums.ac.ir/article-1-3308-en.html

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.