Design And Functional Analysis Of HPV16 E2 CTL Epitopes

Cervical cancer is one of the most common tumors of female. It is usually caused by human papillomavirus (HPV), especially by type16. The therapic vaccines aim at E6 and E7 have some curative effect. But recent research has found that: The immune react to E6 and E7 protein of HPV16 have no relation to the regression of pathological changes. This maybe ascribe to the proteins'later expressing in the infected cells. Conversely, E2 protein, in charge of regulating DNA replication and genome expression, express in the infected cells stably and abundantly in the early stage of disease. Cytotoxic T lymphocyte (CTL) plays a very important role in anti-tumor and anti-virus immune reaction. Stimulated by antigen and cytokine, the precursor cell proliferated and differentiated to specificity CTL. So, if we could induce effective CTL which specificity aimed at HPV16 E2 protein, we would found another key to cure HPV16 associated disease. But, the whole HPV16 E2 protein contains not only the CTL epitopes but also the B cell and Th cell epitopes. Those epitopes maybe inhibit each other and lead to the reduction of antigencity.The phenomenons of immune deviation and immune subvertion also suggested that the vaccine constructed by CTL epitopes maybe more excellent than the whole protein. The stability of "MHCâ… -peptide"compound is essentially to the activation of CTL. In our study, the methods to find out the CTL epitopes of E2 are based on this theory. Our study composes of four parts: Part 1 Design of HPV16 E2 CTL epitopes and peptides synthesis HPV16 E2 CTL epitopes were determined by quantization motif method, three peptides with the hightest total binding coefficient were selected as the candidate peptides. All the peptides were synthesized by GL Biochem Ltd (Shanghai), using the method of Fmoc solid-phase synthesis on ABI-431A peptidesynthesizer. The collection peak and chromatographic analysis confirmed the correction of synthesis. Part 2 Construction,identification and expression in Caski cells of pIRES2-HPV16 E2-EGFP The sequence encoding for HPV16 E2 without stop code was amplified from pBR322/HPV16 by PCR technique. Both the PCR product and the vector pIRES2-EGFP were digested by BamHI,EcoRI and relinked. The recombinant plasmid was identified by restriction endonuclease enzyme analysis and DNA sequence analysis.Both detection verificate the correction of construction. Caski cells growing in six-well plates were transfected with pIRES2-HPV16 E2-EGFP by means of GeneJammer transfection reagent. EGFP were observed though fluorescent microscope 48h later. After that, G418 were added regularly for bolting use. Fourteen days later, the Caski cells which expressed EGFP increased to 50%. Part 3 Construction and function analysis of epitope-specifical CTL HLA-A*0201+ peripheral blood were segregated by Ficoll delamination reagent to gain monouclear cells (peripheral blood mononuclear cell, PBMC). All the monouclear cells were stimulated by rhIL-2 and differernt candidate peptides every 7 days. Three days after the third stimulation, suspension cell were collected for standard 51Cr-release assay. The result showed that all the peptides we selected as candidate have the ability to active epitope-specifical CTL. Based on them, a new peptides vaccine maybe constructed.