| PART 1Production of The Human Papillomavirus Type 18 L1 Protein by Insect Cell Baculovirus Expression System and Investigation The Bio-function and Immune Response of Purified VLP in VitroObjective:To produce and purified human papillomavirus type 18(HPV 18) L1 protein and investigate it’s effectiveness in vitro.Methods: Using Bac to Bac expression system , generated a recombinant baculovirus expressing plasmid ,which contains HPV18L1 gene sequence .The HPV18 L1 gene was amplified by polymerase chain reaction(PCR) and cloned into plasmid pFastHTb .Then , the recombinant plasmid pFastHTb -L1 was used to transform DH10Bac cells ,constructing recombinant Baculovirus ,next the recombinant virus was used to infect Sf-9 insect cells. The interested protein was identified to self-assemble into VLPs by transmission electron microscope and purified by Ni-NTA His?Bind? Resins purification system, The bio-function and immune response of purified VLPs were analyzed by the mouse erythrocyte haemagglutination assay and the IFN-γELISPOT.Results:After incubating at 27℃for 72 hours, the infected cells were collected and total cellular proteins were extracted SDS-PAGE assay revealed a roughly 63 KDa expressed protein and be confirmed by Western blotting that the expressed protein was HPV18 L1. The proteins could form VLP by self-assemble in vitro and primarily locate at nucelus which were identified by transmission electron microscope; it can induce murine erythrocyte hemagglutination, and had an effective immune activity in vitro. The level of IFN-áin the specific HPV18 positive group was significantly higher than that of the mixedhigh-risk HPV infected group and non-HPV infection one in vitro. Conclusions:HPV18 later protein L1 could be efficiently expressed with Bac to Bac expression system, and the expressed L1 protein has good immunoreactivity and bio-activity in vitro. The Ni-NTA purification system could purify the protein with 6×His tag efficiently and conveniently. Furthermore, the ELISPOT results support the hypothesis that the cell-mediated immunity generated by HPV18 L1-VLP may cross-type among high risk HPV types. PART 2Explore the malignancy and mechanism of Human papilloma- virus 16/18 (HPV16/18) E5BACKGROUND & OBJECTIVE: The human papillomavirus type 16/18 (HPV16/18) early gene (E5) could stimulate cell proliferation and transformation in different ways, and complement or enhancement the function of E6 and E7. It is closely sociated with the carcinogenes of cervical cancer. This study was to investigate the effects of HPV16/18 E5 on the HPV positive/negative cells or cervical cancer cells/normal cells and explore the pass membrane direction of E5 porotein (N extreme or C extreme).METHODS: The sense sequence of HPV16/18 E5 was cloned into eukaryotic expression vector pEGFP-N1/C1 to prepare recombinant plasmid, which was stable transfected into SiHa,HeLa,C33A and 293 cells; The mRNA and protein levels of E5 and some related genes were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR); the E5 position was observed by fluorescent microscope and confocal microscopy; Cell proliferation after stable transfection was evaluated by MTT assay; The tumorigenesis of SiHa/16E5 cells was assessed by soft agar clone form test in vitro; The cell cycle change and the level of KI67 after stable transfection was tested by FACS; The invasion potential of cells after stable transfection were explored with Transwell; To study the possible mechanism of HPV16 E5 we used IP; The IHC was used to prove the different expression level of the interesting genes with the development of the cervical disease.RESULTS: After stable transfection of HPV16/18 E5 sense RNA, the mRNA and protein levels of E5 in all the cells were obviously increased, but that of P21 ,BUB1 and MAD2 were decreased; All the pEGFP C1-E5 plasmids sited at cell membrane;The proliferation activity of those cells were significantly higher than that of MOCKs (stable transfection with empty plasmids)and no-transfected cells (P<0.05); The clone numbers of SiHa/16E5 cells, were significantly more than SiHa / pEGFP-C1 and SiHa cells [ (33.4±1.6)vs (15.1±3.1), (16.3±2.5),P <0.05]in vitro; The results of FACS inferred that the percents of S period were higher (P<0.05)after transfection and the level of KI67 was more(P<0.05) than the MOCK and no-transfect one; The Transwell outcome confirmed a significant strengthen invasion ability after transfection(P<0.05); The interaction between HPV16E5 and BUB1 was verificated by IP; In the tissue level, the expression of BUB1 and MAD2 were decreased with the development of the cervical diseases.CONCLUSION: Construction both of the pEGFP-N1 and C1 plasmids can effect ly test the direction of the membrane protein pass membrane site; HPV16/18 E5 may influence the cell cycle through impair the expression or assembling of spindle checkpoint proteins, meanwhile, decreased the expression of P21, then enhance the proliferation of cells and the ability of tumorigenesis; HPV16/18 E5 could intensify the invasion ability of cells after stable transfection, and play an important role in the early stage of cervical cancer. PART 3Production the poly peptide vaccine to HPV16 positive cervical cancerBACKGROUND & OBJECTIVE: Construction the animal model of HPV 16 positive tumor with E5,E6 and E7 stable expression by AAV-TC-1(E5);production the poly peptide vaccine directly to HPV16 positive vaccine based on the HPV16 E5,E6 and E7.METHODS: Constructed AAV-TC-1(E5)cell line which stable expressed HPV 16 E5,E6,E7 with adenovirus-associated virus expression systerm ,then injected into C57BL/6 mouse to form the animal model about HPV 16 positive tumor; depending on the bioinformatics condition of our lab,according to the sequence of HPV16 E5,E6,E7,analyzed and screened the peptides which identified the MHC1 agretope ,then prepared and purified in vitro. Used the three peptides plus CpG sequence as vaccine treated the mouses from both prevation and therapy aspects, we maily divided the test animals into five groups,such as three peptides groups , two peptides groups,one peptides groups,CpG group and control groups. Observed the tumor growth in test groups and controls; obtained the mouse blood behind the eyeball and tested the expression change of related immune factors by ELISA; abstracted the splenic lymphocyte co-culture with the AAV-TC-1(E5)cell ,then analyized the lethal effect of splenic lymphocyte by FACS; desquamated the tumor , then assaied the expression of some related immue factors on RNA and protein levels; paraffin imbedding the tumor tissues, tested the level of CD4 and CD8 with IHC.RESULTS: Constructed HPV 16 positive tumor animal model with HPV 16 E5, E6, E7;the mouse in tripeptide groups all lived without tumor;the type 1 cytokine such as : IL-2, IFN-γet al were promoted greatly in tripeptide group than those in the one peptides groups,CpG group,control groups(p<0.01) and two peptides groups(p<0.05) tested by ELISA; the lethal effects of splenic lymphocyte were evidently higher in the treatment groups than the controls proved by FACS;the level of those immune factors in the tumor microenvironment significant higher in the test group than in the controls evidenced by Realtime-RT PCR, Western Blot and IHC.CONCLUSION: The tripeptide vaccine of HPV 16 positive cervical cancer based on HPV16 E5,E6,E7 could efficiently prevent and treat the HPV16 positive tumors, furthermore , it can be used in clinical with excellent prospects. |
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