Expression Of HPV16 L1 Proteins And Study On Their Immunogenicity

The causal association between Human papillomavirus (HPV) infection and cervical cancer has been demonstrated; the development of a prophylactic vaccine to protect against HPV infection may therefore reduce the incidence of this cancer worldwide. Noninfectious HPV-like particles (VLPs), composed of the L1 major capsid protein, are current candidate vaccines for prevention of HPV infection and cervical neoplasia. Since approximately 50% of cervical cancers are associated with HPV16 infection, the administration of this type of vaccine to young women could reduce the incidence of HPV16 infection. HPV16 VLP vaccine has shown to be 91% protective effect against HPV16 infections in the first phase III study. HPV16 L1 VLPs are usually expressed in yeast, insect cell or prokaryotic systems and need the costly purification procedure. Here, for the first time, we display HPV16 L1 on the surface of yeast EBY100 and lactobacillus DM 8909, both are probiotics. these modified probiotics may be directly delivered as vaccines, without the need of purification of recombinant antigen proteins. There are three parts in this paper: 1.High expression of HPV16 L1 inE.coli BL21(DE3)HPV16 L1 fragment with modified restriction enzyme sites are amplified by PCR, and cloned into T-easy vectors. After being confirmed correct by DNA sequencing, the vectors were digested by Kpn1 and Xho1. Desired fragments were obtained and purified. The gene of interest is cloned into the PET32a expression vector. The resulting construct is transformed into E.coli BL21(DE3). the expression of target protein was mainly in the inclusion body as shown by SDS-PAGE. These antigen proteins would be used to detect the serum antibody titer elicited by PYD1- HPV16 L1/ EBY100 and PSC111 AE- HPV16 L1/ lactobacillus DM 8909 administered orally, nasally or subcutaneously. 2.Construction of expression plasmid PYD1- HPV16 L1 and display of HPV16 L1 on the surface of yeast EBY100HPV16 L1fragment with modified restriction enzyme sites was amplified by PCR, and cloned into T-easy vectors. After being confirmed correct by DNA sequencing, the vectors were digested by Kpnl and Xhol .Desired fragments were obtained and purified. The gene of interest is cloned into the pYD1 vector. The resulting construct is transformed into the EBY100. Induced by galactose, HPV16 L1 was expressed with the tandemly-linked C-terminal half of alpha-agglutinin on the surface of EBY100 as identified by immunofluorescence assay . 3.Construction of expression plasmid PSC111 AE- HPV16 L1 and display of HPV16 L1 on the surface of lactobacillus DM 8909HPV16 L1 fragment with modified restriction enzyme sites were amplified by PCR, and cloned into T-easy vectors. After being confirmed correct by DNA sequencing, the vectors were digested by Sfil and Ascl. Desired fragments were obtained and purified. The gene of interest is cloned into the PSC111 AE expression vector. The resulting construct is electrotransformed into the lactobacillus DM 8909. Induced by lactose, HPV-16 L1 was secreted and anchored on the cell wall was as identified by ELISA and Western Blot, with HPV16 L1 specific monoclonal antibodies.In summary, we have expressed HPV16 L1 on the surface of two probiotics: yeast EBY100 and lactobacillus DM 8909. If proven effective as vaccines in animal models, these live recombinant probiotics may provide a novel route for vaccine delivery.