Prediction And Immune Responsibility Identification Of HLA Restrictive Epitopes Derived From HPV58 E6

Objective:Human papillomavirus (HPV) is an etiology that can mainly reduce cervical cancer. Its early genes E6 and E7 stemmed from early region take part in replicating virus by itself, transcription and forming tumor and so on, so it has been ideal target of treating cervical cancer. In our study, we can select human leukocyte antigen (HLA) restrictive cytotoxic lymphocyte (CTL) epitopes derived from HPV58 E6 and E7 gene while synthesizing and identifying their immune responsibility, which can provide candidate epitopes to prepare for therapeutic vaccines which is infecting by HPV58.Methods:1. The prediction of epitope antigen and the identification of the humoral immune response:Through a series of webs and software of looking up predicting epitope antigen around the world, we could predict and select the epitope antigen of HPV58 E6 and E7 gene, synthesis and purify them in other biotechnology companies. And then we were divide C57 female mice into 11 groups:the five synthetic peptides about E6 called P61, P62, P63, P64, P65 and the three of E7 called P71, P72, P73 separately to immune C57 female mice in experimental group, negative peptide called PO in negative group, incomplete freund’s adjuvant alone called P1 in control group, normal phosphate buffer solution (PBS) called P2 as control in blank group. Multi-point injection at the caudal vein in 1,2,3 weeks, prepared for the blood and cellular immunology experiments. All synthesis peptides, adjuvant and PBS supernatant-coated enzyme-linked plate respectively. Conjugated with the various experimental groups of mice serum. The serum of immune mice as the first antibody, the goat anti-mouse IgG as HRP antibody conjugate, TMB (Tetramethylbenzidine) for the substrate. Determination of the absorbance OD at 460nm with microplate.2. Enzyme-linked Immunospot assay (Elispot assay):Extracting lymphocyte of immune mice, the anti-IFN-y antibody coated in the enzyme-linked plate could capture the IFN-y that was secreted by lymphocyte of immune mice and presented to us in spot. We could read the plate to count the spot by company to indirect to count the induced specific T lymphocyte.3.LDH Release Assay: Extracting lymphocyte of immune mice and using it as the effector cell, the sighed C33A/LV-HPV58E6E7 cell as target cell. The effector cell would attack the target cell to make it damaged and release lactic dehydrogenase (LDH), it would calculate the killability of effector cell to target cell to judge the activity of CTL.Results:1. The prediction of epitope antigen and the identification of the humoral immune response:It would be showed from all relative websites and software around the world that the below five nine-peptides of E6 gene and three of E7 gene could be the epitope antigen of HPV58 E6 and E7 (separately called P61, P62, P63, P64, P65, P71, P72 and P73):P61:HPV58 E611-19: TLHDLCQAL; P62:HPV58 E695-103:CLNEILIRC; P63:HPV58 E618-26: ALETSVHEI; P64:HPV58 E665-73:VCLRLLSKI; P65:HPV58 E699-107: ILIRCIICQ; P71:HPV58E769-77:CINSTTTDV; P72:HPV58 E714-22: DLHPEPTDL; P73:HPV58 E772-80:STTTDVRTL. And the five synthetic peptides about E6 called P62, P63, P64, P65 and the three of E7 called P71, P?2, P73 of mice all were positive at the first week after finishing immunization, and the antibody titers of serum were all 1:1600; however, epitope P61 group and P0, P1, P2 group were negative relatively. All experimental groups were all positive at the third week, and there was significant difference with the negative control group P0-P2 (P<0.05), the antibody titers of serum in P62-P65 group of E6 gene and P71-P73 of E7 gene were all 1:3200, P61 of E6 gene was 1:1600.2(P<0.05). ELISPOT assay:Extracting lymphocyte of immune mice for spot assay. It is showed that epitope P62, P63, P64 group of E6 gene and P73 group of E7 gene were positive at the first week after finishing immunization, it would be showed the spot in the plate, the spot was separately counted to 3.2* 107 lymphocytes, 3.05* 107 lymphocytes、1.26* 107 lymphocytes,2.66* 107 lymphocytes. However, others didn’t. There was significant difference with the negative control group (P<0.05).3.LDH release assay:Only three Eitopes of P62, P63 and P64 groups about E6 and E7 gene which were separately P62:HPV58 E695-103: CLNEILIRC; P63:HPV58 E618-26:ALETSVHEI and P64:HPV58 E665-73: VCLRLLSKI after three immunization all could induce specific CTL of C33A /LV-HPV58E6E7 cell.Conclusion:HPV58 E695-103、HPV58 E618-26 and HPV58 E665-73 had the antigenicity to induce immune mice to produce humoral and cellular immune response. It would be candidate target peptide of therapeutic vaccine infected by HPV58.