Construction And Immunogenicity Of Chikungunya Virus And Nipah Virus-like Particles

As a class of preventive or therapeutic biological products,vaccines are becoming more and more important to humans.In terms of classification,vaccines can be divided into traditional inactivated vaccines,live attenuated vaccines and new genetically engineered vaccines.The virus-like particles vaccine studied in this thesis is a kind of genetic engineering vaccine.Because this vaccine does not contain viral nucleic acid,it can stimulate the body to produce strong humoral and cellular immune responses.Therefore,it has high safety and strong immunity.This paper constructed two recombinant baculoviruses and expressed structural proteins of Chikungunya and Nipah viruse,respectively.After infecting Sf9 cells with recombinant baculovirus,we obtained the VLPs of Chikungunya virus and Nipah virus by concentration,ultracentrifugation and de-sugarization.The research work of this paper laid the foundation for the development and production of two viral VLPs vaccines.This paper is divided into four chapters:Chapter 1: Preface.This chapter mainly introduces the development of VLPs vaccines and the different expression systems used in vaccine production.Different expression systems include E.coli expression system,yeast expression system,baculovirus expression system,mammalian cell expression system,and plant bioreactor.As a new vaccine,VLPs vaccine has its unique advantages,but it also has many shortcomings.In this chapter,we specifically describe the advantages and disadvantages of each expression system,and also summarize the development of Chikungunya virus and Nipah virus vaccine.Chapter 2: Construction of Chikungunya virus VLPs.We obtained the correct recombinant baculovirus containing the complete structural protein of Chikungunya virus using baculovirus expression system that is currently widely used and is easy to mass produce.We infected Sf9 insect cells with recombinant baculovirus and determined the expression of Chikungunya structural proteins and the formation of virus-like particles through a series of experiments including SDS-PAGE,Western blotting,immunofluorescence and transmission electron microscopy.The morphology and size of the particles we observed were similar to the wild-type Chikungunya virus.Finally,we infected Sf9 suspension cells with recombinant baculovirus and successfully purified the virus-like particles of Chikungunya virus by sucrose density gradient centrifugation.Chapter 3: Immunogenicity of Chikungunya virus VLPs.We immunized mice with samples and isolated mouse serum and spleen cells to detect changes in specific antibodies and cytokines in mice before and after immunization,and finally to determine the immunogenicity of virus-like particles.Chapter 4: Construction of Nipah virus VLPs.We successfully constructed and obtained two recombinant baculoviruses containing Nipah virus matrix protein M,fusion protein F and glycoprotein G using baculovirus expression system.Similar to the previous study of Chikungunya virus,we also determined the formation of VLPs by detecting proteins expression;Finally,we purified the VLPs in large quantities.After negative staining experiments,we can observe the virus-like particles of Nipah virus.Chapter 5: Outlook.This chapter mainly analyzes the research progress and future development of virus-like particles vaccine.