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Barzegar H, Sharifi H, Langroudi L, Azadmanesh K, Arashkia A. Human papillomavirus genotype 16 pseudovirus production and purification in HEK-293FT cells. vacres 2017; 4 (3 and 4) :46-50
URL: http://vacres.pasteur.ac.ir/article-1-111-fa.html

Introduction: Human papillomavirus (HPV) is the main causative agent of cervical cancer worldwide leading to a big health problem, especially in the developing countries. Among 14 common high-risk genotypes, HPV16 accounts for more than 50% of all cervical cancers. The current prophylactic vaccines against HPV infection are based on L1 protein. Due to some drawbacks in the current vaccines such as their restrictions to genotype and relatively high costs, second-generation HPV vaccines, based on L2 protein, are under development. The evaluation of the neutralization efficiency of the antibodies against the virus needs pseudoviruses consisting of L1 and L2 capsid proteins harboring a reporter gene. The aim of this study was assembling HPV16 L1 and L2 proteins into a pseudovirion that contains a reporter plasmid. Methods: HPV16 L1 and L2 coding plasmid along with pEGFP-N1 were amplified in E. coli host and were extracted before transfection with DNA-calcium phosphate deposition method into the HEK 293FT cells. The expression of HPV16 pseudoviruses was confirmed by fluorescence microscopy and flow cytometry. Pseudoviruses were partially purified with gel chromatography and then were visualized by atomic force microscopy. Results: Simultaneous transfection of HPV16 L1 and L2 coding plasmid along with pEGFP-N1 into the HEK 293FT cells resulted in self-assembly of 45-55 nm pseudoviruses, harboring the reporter gene coding plasmid. Conclusion: In this project, HPV16 pseudoviruses, harboring a reporter gene were produced and partially purified, and their infecting capability was assessed. The pseudovirions could be used for neutralization assay and also as vectors for gene therapy.