RESUMO
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.
Assuntos
Anticorpos Antivirais/sangue , Proteínas do Capsídeo/imunologia , Proteínas Oncogênicas Virais/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vírion/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Epitopos/imunologia , Humanos , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus , Pichia/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismoRESUMO
In this study, bovine enteric caliciviruses (BECs) were detected in 49.4% of a total of 253 stool specimens for diarrheic calves collected from 42 industrial dairy farms from March 2010 to February 2012. Genogroup III norovirus (NoVsGIII) were more prevalent (39.5%) than neboviruses (NBs) (15%), and coinfections were observed in 5.1% of the samples tested. Sequence analysis of the partial polymerase gene from 13 NoVsGIII samples indicated the circulation of both genotype 1 and genotype 2 strains. Among the six NB strains sequenced, five were related to the Bo/Nebraska/80/US strain, while one was related to the Bo/Newbury1/76/UK strain.
