Iranian South Medical Journal (Feb 2023)
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
Abstract
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.
