Research Of Isatis Root Polysaccharide On Immune Effect Of HSV-2 DNA Vaccine

HSV-2 currently has no effective method of prevention and treatment.Although the drug can control the symptoms,the drug cannot function when the virus is in a latent state,so the development of related vaccines became the focus of research.The DNA vaccine is highly specific and can be continuously expressed in the body to stimulate long-lasting cellular and humoral immunity,thereby effectively preventing HSV-2 infection.However,the immunogenicity of DNA vaccines is weak,and suitable vaccine adjuvants are needed to enhance the protection of the body.Isatis root polysaccharide(IRPS)is a good immune adjuvant,which is related to many factors.In recent years,studies have found that IRPS can promote DC cell maturation,activate macrophages and release related cytokines(such as TNF-?),regulate T cell immune types,and promote immunoglobulin production.The extraction methods of IRPS mainly include decoction alcohol precipitation method,ultrasonic method,microwave method,etc.Compared with the traditional decoction alcohol precipitation method,ultrasonic method and microwave method in the extraction of polysaccharides to reduce the extraction temperature,shorten the time,improve the yield and ensure the advantages of product structure and stability of physical and chemical properties,but whether microwave method and ultrasonic method will affect the structure or activity of IRPS have not been reported,and need to be further explored.Afterwards,in order to explore the effect of IRPS on the immune effect of HSV-2 DNA vaccine,this study co-immunized BALB/c mice with the IRPS and the DNA vaccine pgD in our laboratory,including:1)The different extraction methods of IRPS are divided into Water(water decoction and alcohol precipitation method),US-1(ultrasonic wave 75 W,0? ice bath,30min),US-2(ultrasonic 100W,0? ice bath,30min),US-3(ultrasonic 125W,0? ice bath,30min),MW-1(microwave 350W,6min),MW-2(microwave 450W,6min),MW-3(microwave 600W,6min),compare the yield,and then in vitro study the effects of different extraction methods of polysaccharides on the proliferation of mouse T lymphocytes and the effects of cytokines secreted by lymphocytes.2)Divide 40 BALB/c mice into PBS control group,pgD vaccine group,pgD+IRPS adjuvant group,the pKan+IRPS empty vector group and IRPS non-vaccinated group were injected into the hind leg muscle of mice,and immunized again after 2 weeks after the initial immunization.Two weeks after the last immunization,blood was collected from the eyeballs of the mice,serum was separated,the IgG levels and IgGl,IgG2a subtype antibody titers were determined by ELISA;the spleen cells of mice were prepared to be determined by MTS T cell proliferation ability;The content of cytokines IFN-? and IL-10 was determined by ELISA.The in vitro results showed that the polysaccharide yield of all US groups and MW groups was higher than that of the Water group,the Splenic lymphocyte proliferation capacity of all US groups was higher than that of the Water group(P<0.05),and the concentration of IFN-? in the US-2 group was higher than that of the Water group(P<0.05).The IL-10 concentration of US-2 and US-3 group was higher than that of Water group(P<0.05).The in vivo results showed that the IgG level of the pgD+IRPS vaccine adjuvant group in the mouse serum was significantly higher than that of the other 4 groups(P<0.01),the titers of IgGl and IgG2a was significantly higher than the pgD vaccine group(P<0.01),and the ratio of IgG2a/IgGl was also higher than that of pgD group(P<0.05);under the stimulation of HSV-2,the splenic T cell proliferation ability of pgD+IRPS vaccine adjuvant group was stronger than that of pgD vaccine group(P<0.05);After ELISA,the IFN-? content of the pgD+IRPS vaccine adjuvant group was significantly higher than that of the pgD vaccine group(P<0.01),and the content of IL-10 was also significantly higher than that of the pgD vaccine group(P<0.01).In summary,the ultrasonic method and microwave method can improve the yield of Isatis root polysaccharides,and under certain conditions can improve the immune activity of polysaccharides.IRPS as a vaccine adjuvant can significantly enhance the IgG level of HSV-2 DNA vaccine pgD,improve the proliferation of T cells,and mainly use cellular immunity to mediate Th1 and Th2 type mixed immunity.This shows that the IRPS is a good DNA vaccine adjuvant and has broad application prospects.