VLP Production from Recombinant L1/L2 HPV-16 Protein Expressed in Pichia Pastoris

Abstract

Background: Human papillomavirus 16 is considered a causative agent of genital cancers. Since there is no decisive treatment, the only approach is vaccination of high-risk group.

Objective: This study aimed to produce a chimeric L1/L2 protein in Pichia Pastoris system.

Method: To develop VLPs of chimeric L1/L2 protein HPV-16, first, a cross-neutralizing epitope of HPV-16 L2 gene was inserted into L1 HPV-16 gene. Then the chimeric L1/L2 HPV-16 was inserted in pPICZA plasmid and expressed in Pichia pastoris (P. pastoris). The final purification of VLPs was carried out by ultra-centrifugation (130000 g) using 10-40% sucrose density gradient for 4 h at 4 °C. The SDS-PAGE and western blot assay was carried out for L1-HPV-16 and L2-HPV- 16 proteins separately. Amount of 55ng of the purified VLPs was coated to the wells of ELISA for detection of L1 HPV-16 antibody and L2-HPV-16 antibody by ELISA test separately using commercial L1-HPV-16 and L2-HPV-16 antibodies. The sera of 16 patients positive for HPV-16 and 85 sera negative for HPV infections were tested for detection of HPV-16 antibody by ELISA test and the results were compared with commercial test kit.

Results: The formation and purified VLPs were observed by TEM and AFM. The result of purified VLPs by SDS-PAGE showed a band of 60 KD and confirmed by western blot assay. The results of ELISA for detection of L1-HPV-16 antibody and L2 -HPV-16 antibody showed positive reaction which displayed similar sensitivity with commercial test kit.

Conclusion: The present study will pave the way for producing recombinant pan-HPV vaccine.

Keywords: Chimeric protein; Pichia pastoris; Virus-Like Particle (VLP); crossneutralizing epitope; genital cancer; human papillomavirus..