Development And Immunological Evaluation In Mice Of Hepatitis C Virus DNA Vaccines

Hepatitis C Virus(HCV)is a global pandemic affecting over 170 million individuals worldwide.Currently there is no vaccine for HCV however it is believed that an effective vaccine must be able to induce strong HCV T cell based immunity.In this thesis,we havetaken a multi-step approach to develop four distinct HCV DNA vaccines encoding HCV genotype 2a proteins,Core,NS3,NS4B,and fussion protein of Core,NS3 and NS4B.We show that these constructs are expressed in vitro and are able to induce potent peripheral humoral and cell-mediated immunological responses in BALB/c mice.The main contents were as followed:1.The construction of HCV DNA vaccines encoding Core,NS3,NS4B,and the fussion protein of Core,NS3 and NS4B.The RT-PCR amplified Core,NS3,NS4B,were respectively cloned into pVAX1 simple vector to construct recombinant of plasmid pVAXl-Core,pVAXl-NS3 and pVAX1-NS4B.The amplified Core gene,NS3 gene,and NS4B gene were 573 bp,1893 bp,and 783 bp in length.The eukaryotic plasmid pVAXl-Core?pVAXl-NS3?pVAX1-NS4B and pVAXl-NS3-Core-NS4B were constructed and tranfected into Huh7.5.1 cells.Vitro expression of the recombinant plasmids were confirmed via RT-PCR,which indicated that the eukaryoti-c expression plasmids were correctly constructed and the mRNA of the relative proteins were successfully detected in the transfected Huh7.5.1 cells,demonstrating that the four contr-usted plamids pVAX1-Core?pVAX1-NS3?pVAXl-NS4B and pVAXl-NS3-Core-NS4B could successfully express in vitro,these research offered a foundation on which to further develop HCV DNA vaccines.2.The immunization in BALB/c mice of the four HCV DNA vaccinesBalb/c mice were immuned three times at 3 weeks intervals with 100 ?g pVAXl-Core?pVAXl-NS3?pVAXl-NS4B,pVAXl-NS3-Core-NS4B,the mixture of pVAXl-Core?pVAXl-NS3?pVAXl-NS4B,and empty plasmid by direct intramuscular injection,saline as natural control.The mice were bled from the eyes and serum was prepared at 2th week after each DNA immunization.T lymphocytes type in peripheral blood were detected by flow cytomet-ery.The antibody level and the cytokines as IL-2,IL-4 and IFN-y in the serum were determined by ELISA.The result of serum blocking HCV viral infection was detected by quantitative PCR.The results indicated that the pVAXl-Core,pVAX1-NS3 and pVAXl--NS4B can induce potent peripheral humoral and cell-mediated immunological responses in BALB/cmice,while pVAXl-NS3-Core-NS4B coud induce cell-mediated immunological responses.This study laid a foundation for the research and application of HCV DNA vaccine.