Construction And Immunogenicity Evaluation Of Bivalent Chimeric Virus-like Particles In Brucellosis

Brucellosis is a zoonotic disease caused by Brucella,which can infect a variety of animals including cattle,pigs,sheep,goats,horses,dogs and other animals,causing abortion and stillbirth of infected mothers,and can also infect humans by eating unprocessed infected animal meat or eating unpasteurized milk and other dairy products,causing symptoms such as human orchitis and reproductive system damage,and is considered by the World Health Organization to be one of the seven neglected zoonotic diseases.It is one of the key zoonotic infectious diseases in China at present.Therefore,vaccination through vaccination is essential to control and eventually eliminate the disease.At present,the traditional vaccines commonly used for brucellosis are mainly live attenuated vaccines such as Rev-1,S19 and S2,but the above-mentioned weak live vaccines have the disadvantages of strong side reactions,large species limitations,interference with serological diagnosis after vaccination and possible virulence.Therefore,in view of the limitations of traditional vaccines,the inability to cause effective cellular immune response and the relatively limited protective effect,a green,safe,efficient and multi-directional immunity new vaccine for brucellosis was developed,which plays an important role in the prevention and control of brucellosis.L7/L12 protein is widely present in various species of Brucella,and is highly conserved among various species,and is one of the dominant antigenic proteins of Brucella.The BCSP31 gene can be expressed in different species of Brucella(except Brucella epididymis sheep),which can stimulate the body’s protective immune response to Brucella infection,and the above two proteins are ideal candidate antigens for the development of new Brucella vaccines.IL-2 can promote the proliferation and differentiation of T cells,promote the activity of natural killer cells and B lymphocytes,participate in regulating the balance and stability of immune response,and enhance cellular immunity and humoral immunity.Clinically,IL-2 can also be used as an immune booster to enhance vaccine protection and as a candidate immune booster factor to enhance the protective efficacy of novel vaccines.Therefore,the main research contents of this study are as follows:1.Construction of bivalent chimeric virus-like particles BCSP31-L7/L12-IL-2c VLPs for brucellosisBased on the ND Virus like particles(ND VLPs)vector platform,this study used the insect baculovirus expression system and GPI anchoring strategy to construct a green,Safe bivalent chimeric virus-like particles novel vaccine candidate BCSP31-L7/L12-IL-2c VLPs.The results of PCR and immunofluorescence showed that recombinant baculoviruses expressing IL-2 and L7/L12 proteins were successfully constructed,the western blot results showed that the components of the c VLPs were correctly expressed,and the particles with fibroid structure and size of about 100 nm were visible by transmission electron microscopy,indicating that the bivalent chimeric virus-like particles BCSP31-L7/L12-IL-2 c VLPs of brucellosis were successfully constructed.2.Study on the immunogenicity of bivalent chimeric virus-like particles BCSP31-L7/L12-IL-2 c VLPs in brucellosisIn this study,BCSP31-L7/L12-IL-2 c VLPs were used as the experimental group,and soluble protein control group and PBS group were set up to immunize BALB/c mice.In order to evaluate the immune response of mice,on the one hand,serum antibodies were prepared by mouse tail vein blood collection,and ELISA was used to detect the humoral immune response of mice,on the other hand,lymphocytes were isolated by the spleen of each group of mice,and the ratio of CD3+CD4+ T cells to CD3+CD8+ T cells was analyzed by flow cytometry to evaluate the immune response of mouse cells.The results showed that the BCSP31-L7/L12-IL-2 c VLPs group exhibited a faster and higher level of antibody response and an increased ratio of CD3+CD4+ T cells to CD3+CD8+ T cells compared with the same dose soluble proteome.In summary,BCSP31-L7/L12-IL-2 c VLPs exhibit better humoral and cellular immunity and better immunogenicity compared with the same dose of soluble proteome,and can be used as a new vaccine candidate to make up for the shortcomings of traditional vaccines and provide a new strategy for the prevention and treatment of brucellosis.