Immunoenhancement Of Seven Immunopotentiators And Their Compound To Porcine Circovirus Type 2 Genetic Engineering Vaccine

There is a close relationship between the outbreak of swine infectious diseases and Porcine circovirus type(Porcine circovirus type 2,PCV2).The most common disease is called postweaning multisystemic wasting syndrome(PMWS).Up to now,many scholars have devoted themselves to the research for the development of PCV2 effective vaccine,which can be used to effectively control and eliminate the PCV2 virus.PCV2 Cap protein is the preferred target antigen for PCV2 genetic engineering vaccine.According to the research,immunopotentiator is a kind of immune enhancers and immune stimulating agent,and the main function is to enhance the body's anti-tumor,anti infection,correction immunodeficiency ability;immunopotentiator not only can activate a variety of immune cells in vivo,enhance the specific and nonspecific immune function,but also play the role of adjuvant and antigen immunogenicity,thus accelerating the immune response.The aim of this research is to screen the most effective immune enhancer of PCV2-Cap protein vaccine,including astragalus polysaccharid?ginsenoside?levamisole?squalane?Poly(I:C)?PoIFN-? and PoGM-CSF.1.Immunoenhancement of Astragalus polysaccharide?ginsenoside?levamisole,squalane and Poly(I:C)to PCV2 gene engineering vaccineTo identificate the fuction of immunopotentiators on PCV2-Cap protein vaccine,we prepared five kinds of immunopotentiator+PCV2 subunit vaccine.The volume ratio of PCV2-Cap:Gel 01 vaccine djuvant:immunopotentiator is 75:20:5.Each group of animals was immunized by intraperitioneal injection.After 21 days,we did the challenge test.By indirect ELISA method for the determination of immune serum PCV2 specific IgG antibody at 21 days,the results showed that Poly(I:C)group and squalane group could induce higher levels of PCV2 specific IgG antibody,significantly enhanced the immunogenicity of PCV2 vaccine.We tested the concentration of IL-4?IL-6?IFN-? secreted by spleen lymphocyte after 21 days of vaccination with CUSABIO kit.The result showed that the squalane group could significantily promote the IL-6 and IFN-? secretion.But Poly(I:C)only had a slight enhancement.21 days after challenge of PCV2 virus,we detected PCV2 viremia in different organs of PCV2 viral load by fluorescence quantitative PCR,the results showed that the squalane group was more effective to reduce the level of PCV2 viremia and PCV2 virus loads in spleen.This study proves when squalane and PCV2-Cap admixed with each other,squalane can significantly enhance the protective immunity of PCV2-Cap protein subunit vaccine.2.Immunoenhancement of six kings of compound immunopotentiators on PCV2 gene engineering vaccineIn order to identificate the function of compound immunopotentiators on PCV2-Cap protein vaccine,we selected two or four immunopotentiators from these five immunopotentiators to form six kinds of compound immunopotentiators,which admixed with PCV2 subunit vaccine and Gel 01 vaccine djuvant.The volume ratio of PCV2-Cap:Gel 01 vaccine djuvant:immunopotentiator was 75:20:5.Each group of animals was immunized by intraperitioneal injection.After 21 days,we did the challenge test.By indirect ELISA method for the determination of immune serum PCV2 specific IgG antibody at 21 days.We tested the serum IL-4,IL-6,IFN-? cytokine concentration after 21 days of vaccination with ELISA kit.21 days after challenge of PCV2 virus,we detected PCV2 viremia in serum of PCV2 viral load by fluorescence quantitative PCR,the results showed that three groups were more effective to reduce the level of PCV2 viremia.The formulations are Astragalus polysaccharide+Poly(I:C)+levamisole?Astragalus polysaccharide+Poly(I:C)+levamisole + squalane and Astragalus polysaccharide +Poly(I:C).And the most effective formulation is Astragalus polysaccharide+ Poly(I:C).3.Expression and purification of PoIFN-y and PoGM-CSFThe target gene was amplified by PCR.Five step was displayed as follows:Double edzyme digestion,ligation empty vector,transformation,identification,preparation of PoIFN-?-pCold ?-Fh8 and PoGM-CSF-pCold ?-Fh8 recombinant bacteria.Further more,the recombinant bacteria was induced protein expression,optimization of IPTG concentration and induction time was changed to improve the expression efficiency;PoIFN-y express as Supernatant,but GM-CSF express most as pellets,finally,we use his-tagged GE protein purification column to purify the protein.Then,the concentration of PoIFN-? was 1.6mg/ml,and PoGM-CSF was 2.6 mg/ml.In this study,we lay the foundation for further study on the effect of two cytokines of PoIFN-y and PoGM-CSF on the immune enhancement of PCV2-Cap vaccine.4.Immunoenhancement of PoIFN-? and PoGM-CSF on PCV2 genetic engineering vaccineTo identify the immunity Enhancement of PoIFN-? and PoGM-CSF on PCV2-Cap protein vaccine,we prepared two kinds recombinant protein+PCV2 subunit vaccine.The immune protective effect of two kinds immunopotentiators were identified by immunity and challenge test in mice.The immune serum of PCV2 special antibody at 21 days after vaccine was determined by indirect ELISA method.The results showed that the PoIFN-y increased higher serum antibody level which compared with PCV2-Cap alone immunity,and the difference was statistically significant(p<0.05);PoGM-CSF was not observed as such enhancement effect(p>0.05).The effect of PoIFN-y on the level of serum PCV2 specific antibody was obvious which compared with PCV2-Cap vaccine alone,SYBR quantitative PCR was used to detect rats gene m RNA expression level of spleen lymphocyte cytokines IL-4?IL-6 and IFN-? 21 days after immunization.The results showed that the PoIFN-y as immune strengthening agent,could significantly improve the expression level of IL-4 and IFN-?.21 days after challenge of PCV2b,PCV2 viral load was detected by fluorescence quantitative PCR in viremia and different organs.The results showed that comparied with PCV2-cap group,PoIFN-y protein group could significantly alleviate the PCV2 viremia virusemia,and had obvious inhibitory in PCV2 virus copy in some organ.In this study,we can conclude that PoIFN-y can significantly enhance the immune protective effect of PCV2-Cap protein subunit vaccine when mixed with PCV2-Cap protein,and the PoGM-CSF factor has no obvious immune enhancement effect.