VLP production from recombinant L1/L2 HPV-16 protein expressed in Pichia Pastoris.

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. Due to the high cost, the current HPV vaccines are not affordable in developing countries. This study aimed to produce a chimeric L1/L2 protein in Pichia Pastoris system. To develop the VLPs retrieved from chimeric L1/L2 protein HPV-16, first, a cross-neutralizing epitope of HPV-16 L2 gene was inserted into L1 HPV-16 gene. The chimeric L1/L2 HPV-16 was inserted in pPICZA plasmid and expressed in Pichia pastoris (P. pastoris). Then, the expression of recombinant protein was tested by SDS-PAGE and western blot analyses. The final purification of VLPs was carried out by ultra-centrifugation (130000 g) using a 10-40% sucrose density gradient for 4 h at 4 °C. The purified VLPs were visualized using atomic-force and electron microscopies. The western blot assay was carried out for L1-HPV-16 and L2-HPV-16 antibodies separately. Amount of 55ng of the purified VLPs were 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. In addition to 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. The purified VLPs was observed by TEM and AFM. Further, the visualizing of formation of VLPs by TEM and AFM had distinctive advantages, which complement each other. The result of purified VLPs by SDS-PAGE showed a band of 60 KD which was also confirmed by western blot assay. The results proved the expression of chimeric L1/L2 HPV-16 protein in P. pastoris. The results of ELISA for both L1-HPV-16 and L2 -HPV-16 showed positive reaction. In addition to the results of ELISA test for detection of HPV-16 antibody in 16 patients positive for HPV-16 showed similar sensitivity with commercial test kit. In conclusion, the present study will pave the way for producing recombinant pan-HPV vaccine.