The Study On Molecular Biology Of HPV16 L1 Protein

【BackgrounBackground】Human papillomaviruses (HPVs) are small, non-enveloped DNA viruses, whichcan elicit epithelial hyperplastic lesions of human skin and mucosa. Some high risktypes, including HPV16 and HPV18, have been proved an important initiator ofcervix cancer and are associated with other cancer. HPV virion (55-60 nm in diameter)is contained within an icosahedral capsid, which comprises 360 molecules of L1major capsid proteins and 12 molecules of L2 minor capsid proteins. During the viruslife cycle, L1 proteins seem to enter the nuclei of host cells twice. In the initial stageof HPV infection, immediately after the virions impinge the undifferentiatedproliferating epithelial cells, L1 proteins are transported into the nuclei ofproliferating epithelial cells together with the viral genome. During the late stage ofHPV infection, the newly synthesized L1 proteins in cytoplasm are transported intothe nuclei of terminally differentiated keratinocytes, to package the replicated HPVgenomic DNAs and to assemble into infectious virions, together with L2 proteins. L1proteins form the most lineal epitopes and conformational epitopes of HPV, whichinduce protective immune response to HPV infection. This would suggest that the L1proteins should play a very important role in HPV infection and production, especiallyin inducing protective humoral immunity response for HPV infection.So in this program, the bioactivity of HPV L1 protein was mainly investigated.This achievement would stride the study on the mechanism for HPV16 infection and provide valuable experience for preparation of HPV 16 prophylactic vaccine.【PurposPurpose】(1) In order to discover whether there was some novel NLSs, which weredissimilar from the known NLSs, The NLSs of HPVs L1 proteins were analyzed andpredicted, by bioinformatics technology.(2) In order to study and discuss the feasibility of using NLS HPV16L1 (NLS ofHPV16 L1 protein) as targeting drug carrier in Targeting drug delivery system(TDDS), the nuclear import dynamic process of HPV16 L1 protein was imaged.(3) The different intracellular localization of various truncated HPV 16 L1proteins were expressed by DNA vaccine. The effect of it on the humoral immunerespons was analyzed by gene immunization technique.(4) In order to prepare anti-HPV16L1 IgY(egg yolk immunoglobulin)polyclonal antibody, hens were immunized using HPV16 L1 proteins.(5) In order to provide valuable experience for preparation of HPV 16prophylactic vaccine, the antigenic epitopes were screened by phage display randompeptide library.【Methods】(1) The HPVs L1 amino acid sequences were obtained fromhttp://cubic.bioc.columbia.edu/predictNLS/, http://www.stdgen.lanl.gov/stdgen/virus/,http://www.ncbi.nlm.nih.gov/, and http://ca.expasy.org/. PredictNLS software wasused to analyze and to predict the NLSs of HPVs L1 proteins. According to thecharacteristics, homology of NLSs predicted by PredictNLS, and the general rule ofclassical NLSs (cNLSs), the NLSs of HPVs L1 proteins were classified into differentcategories.(2) The recombinant Ac-EGFP-HPV16L1△NLS, Ac-EGFP-HPV16L1, Ac-EGFP, Ac-EGFP-NLSHPV16L1 baculoviruses were obtained respectively by applyingthe classical bio-technique. After Sf-9 cells had been transfected with the aboverecombinant baculoviruses respectively, intracellular localization of various truncatedHPV16 L1 fusion proteins tagged with EGFP in Sf-9 cells, were imaged byfluorescence microscopy and laser confocal microscopy and immunohistochemistyassay (IHC).(3) Recombinant pcDNA-EGFP-HPV16L1 and pcDNA-EGFP-HPV16L1△NLS plasmids were generated respectively. Then, they were injected into the tibial musle ofBALB/c mice. The titer of the sera anti-EGFP IgG polyclonal antibody wasdetermined by ELISA (Enzyme-linked immunosorbent assay).(4) The hens were immunized with purified HPV16 L1 protein. Then IgY of eggyolk was separated and purified. Then IgY bioactivity was tested by ELISA and IHCand hemagglutination inhibition(HAI).(5) The Protein G Magnetic Beads and purified anti-HPV16 L1 polyclonalantibody were used in screening the phage display random peptide library by affinityselective method. After three rounds screening, 35 clones were selected and analyzedwith the bioinformative softwares (DNAClub and Blast softwares), and identified byimmunity blot test.【ResultResults】(1) There were 107 types HPVs were obtained from above databases. The NLSsof 107 HPVs L1 proteins were classified into 15 categories. There were many novelNLSs, which were dissimilar from the known cNLSs.(2) Green fluorescence congregated in the nuclei of Sf-9 cells transfected withthe recombinant Ac-EGFP-HPV16L1 and Ac-EGFP-NLSHPV16L1 baculovirusesrespectively, while green fluorescence resorted in the cytoplasm of Sf-9 cellstransfected with recombinant Ac-EGFP-HPV16L1△NLS viruses at all times. ButGreen fluorescence spreaded through the nuclei and cytoplasm was seen in Sf-9 cellstransfected with the recombinant Ac-EGFP viruses.(3) After three times immunization, the titer of anti-EGFP IgG polyclonalantibody was higher in pcDNA -EGFP-HPV16L1△NLS plasmids immunized micegroup than that in recombinant pcDNA-EGFP-HPV16L1 immunized mice (P<0.001).(4) Anti-HPV16L1 IgY polyclonal antibodies from the eggs yolk of immunizedhens could react specifically with EGFP-HPV16 L1 proteins expressed in CHO cells,and could inhibite HA mediated by HPV16 L1 especially.(5) Though there was no mimic epitopes homologous with HPV16 L1 in aminoacid sequence could be found from phage clones,but the five peptides"TNLDLYG","IFDNHP","LTFKPQ","GIDS","NHGLLYSPLPT"displayed on 29 of 35 phageclones were identified, which could bind to anti-HPV16L1 polyclonal antibody inimmunity blot test. 【ConclusionConclusions】(1) Predicting and classifying the NLSs of 107 HPVs L1 would offer animportant guidance for investigating the HPV L1 nucleocytoplasmic transportation.(2) The novel pFB-EGFP vector could express EGFP proteins efficiently, whichcould become a useful EGFP tagged fusion proteins expression system for tracing theinteraction and transportation of proteins in vitro and in vivo investigation.(3) The NLS of HPV16 L1 has the full function. It could paly an important rolein the nuclear import of HPV L1.The NLSHPV16L1 could be as a kind of potentialytargeting drug carrier in Targeting drug delivery system(TDDS).(4) Double function EGFP-HPV16L1 and EGFP-HPV16L1△NLS fusionproteins had been proved, which could self-assemble into VLPs (virus-like particles)and emit green fluorescence. It could suggest that it could be used in follow-upinvestegation of HPV infection mimicry。(5) Whether a long gene was fused in the N-terminal or the NLS in the Cterminalwas deleted, which did not affect HPV16 L1 self-assembling into VLP.(6) The humoral immuno-response could be affected by the intracellularlocalization of the proteins expressed by the DNA vaccine. A strong humoralimmuno-response could be induced by the proteins localized in cytoplasm not thatlocalized in nuclei. It might be useful in the design of DNA vaccine.(7) High titer anti-HPV16 L1 IgY polyclonal antibody could be prepared byimmunizing hens with HPV16 L1 protein.(8) The five peptides"TNLDLYG","IFDNHP","LTFKPQ","GIDS", and"NHGLLYSPLPT"might be the mimic conformation epitopes of HPV16 L1 protein.