Prediction And Identification Of B-Cell Epitopes Derived From HPV18E7

Objective:Human papillomavirus (HPV) is a type of DNA virus which infects squamous cell of epidermis and mucous membranes. High-risk HPV18 is correlate with condyloma acuminatum(CA), cervical intraepithelial neoplasia(CIN) and other epithelial tumors. As a distinctive tumor antigen, HPV18E7 could be an ideal target for antitumor immunotherapy. In this study, computer-assisted algorithms were used to screen the B-cell epitopes derived from E7 antigen of human papillomavirus (HPV) type 18. The aim of this study is to provide candidate epitopes for the preparation of peptide-based therapeutic vaccine which could be expected to prevent the infection of HPV18. Methods:①Computer-assisted algorithms. The B-cell epitopes candidates was predicted basing on analysis of the hydrophilic regions, flexible regions, antigen index and surface probability of the HPV18 E7 antigen.②The predicted epitope was synthesized with standard Fmoc strategy and purified by reverse-phase high performance liquid chromatogram-phy(RP-HPLC). Mass spectrometry was used to identify its nature.③The immunogenicity evaluation of Peptide epitope. the three synthetic peptide called P1, P2, P3 separately to immune Balb/c mice in experimental group, normal saline as control in Negative group. Multi-point injection at the armpits, groin and abdominal in 0, 2, 4 weeks, After the initial immunization, Acquisition and Preparation of the experimental group and negative control mouse serum in 1, 3, 5 weeks.④Collection of HPV18 virus. Collect one case of fresh cervical cancer specimens (Detection of HPV18 positive with PCR), Crushed the specimens, ultrasonic rupture of membrane, and centrifuge to collect HPV virus.⑤Identify with Indirect ELISA. Synthetic peptide, and cervical carcinoma supernatant-coated enzyme-linked plate respectively. Conjugated with the various experimental groups of mice serum, and the serum which without immune for negative control. The sheep anti- mouse IgG as HRP antibody conjugate, TMB (tetramethylbenzidine) for the substrate. Determination of the absorbance A at 492nm with microplate reader. Results:①General analysis, the B-cell epitopes of the HPV18E7 may be located at No. 15-22, 29-44, 51-59. That is, E715-22 ( LEPQNEIP ), E729-44 ( EQLSDSE EENDEIDGV),E751-59(ARRAEPQRH). Named P1, P2 and P3.②The synthesized peptide was beyond 90% in purity and the measured value of molecular weight conformed to its theoretical value.③In the immunogenicity evaluation of Peptide epitopes. Antibody titers of mice were negative after immunization at the 1st week. epitope peptide P2, P3 group were positive at the third week, and there was significant difference with the negative control group(P<0.05), and P1 group was negative(P>0.05); At the fifth week, all the groups showed positive results, and there was significant difference with the negative control group(P<0.05).④Cervical carcinoma supernatant-coated plate enzyme- linked. The antibody induced by P1 and P2 could react with the supernatant of human cervical cancer tissue(P<0.05),but the antibody induced by P1 could not have a positive react. Conclusion :①The combination of our four computer-assisted algorithms can improve the prediction efficiency and avoid synthesizing peptide aimlessly about HPV18E7 antigen.②HPV18E714-21 and HPV18E726-37 had antigenicity, and may be used for the development of peptide vaccine against HPV infection.