Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 20 5 2021 09 28 525 536 EN Somayeh Shokri Infectious and Tropical Disease Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran AND Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Shahab Mahmoudvand Infectious and Tropical Disease Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran AND Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Manoochehr Makvandi Infectious and Tropical Disease Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran AND Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Reza Taherkhani Department of Virology, School of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran Mohammad Rashno Department of Immunology, Medicine Faculty, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Farid Azizi Jalilian Department of Virology, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran Kambiz Ahmadi Angali Department of Biostatistics, School of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Mohammad Ali Foroughi Department of Pathobiology, Section Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad Iran 2021 01 10 2021 05 22 More than 99% of cervical cancers are associated with human papillomaviruses (HPVs) worldwide. Current HPV vaccines are safe, highly immunogenic, with effective immunity against specific HPV types. However, DNA vaccines are a new appealing platform which can be considered for designing the HPV vaccines. This study aimed to construct a recombinant eukaryotic expression plasmid containing L1 of HPV-18, tissue plasminogen activators (tPA), and pan HLA DR-binding epitope (PADRE) genes into the pVAX1 vector. The L1, tPA, and PADRE genes were amplified in a thermocycler. The polymerase chain reaction (PCR) products were cloned and insertion of the genes was confirmed using colony PCR, restriction enzymes analysis, and sequencing methods. Indirect immunofluorescence, RT-PCR, and western blot assays were applied to identify the target gene in HEK-293 cells. Total IgG and its isotypes in immunized mice were measured by enzyme-linked immunosorbent assay technique. Western blot analysis showed a protein band of about 67.5 kDa in supernatant and cell lysate of transfected cells. The results of mice immunization with different constructs (group 1: the pVAX-L1, group 2: pVAX-tPA-PADRE-L1, group 3: pVAX1, and group 4: PBS as controls) indicated that the pVAX1-tPA-PADRE-L1 construct induced a significantly higher level of total IgG than pVAX1-L1 (p=0.003). In conclusion, pVAX1-tPA-PADRE-L1 recombinant plasmid is a highly immunogenic construct and suggests as a promising candidate for vaccine development against HPV type 18 in low-middle-income countries. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/3092 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/3092/1730