Construction Of TGEV、PEDV And ProV Virus-Like Particles With Hepatitis B Core Antigen As Skeleton And Its Immunogenicity Evaluation

Among many of the infectious diseases of swine,porcine viral diarrhea which caused by transmissible gastroenteritis virus(TGEV),porcine epidemic diarrhea virus(PEDV)and porcine rotavirus(PRoV)is the "first killer" of suckling piglets.They are often mixed infection,and can lead to 100% mortality of piglets which are under 7days of age.Vaccine immunization is the most economical and effective way to prevent and control procine viral diarrhea,but the epidemic trend of this disease has deviated from the vaccine strains.To come over the challenges of genetic diversity and high variability of the disease,as well as the inadequacy of traditional vaccines,a strategy for vaccine construction which is short in time and induces systemic immunity is urgently needed.hepatitis B virus core protein antigen(HBcAg)can self-assemble into virus-like particles(VLPs)in vitro.It also can display foreign antigens on its surface by using a high degree of order,so it can induce a strong specific cellular and humoral immune response.HBcAg-VLPs not only improves the weak immunogenicity of subunit vaccine,but also has the characteristics of high efficiency,safety,economy and good biocompatibility of this type of vaccine preparation,so it is a very ideal exogenous antigen delivery carrier and vaccine synthesis platform.In this study,the tandem hepatitis B virus core capsid protein was used as the skeleton to carry the conservative linear neutralizing epitopes of TGEV,PEDV and PRoV respectively,so as to construct the triple chimeric VLPs,aiming at obtaining a one-dose three-prevention porcine viral diarrhea particles vaccine which can quickly respond to the change of epidemic trend.The specific construction process is: Three recombinant plasmids,p ET-28b-HBT/AD(+),p ET-28b-HBP/270(+)and p ET-28b-HBR/VP4(+),were constructed and transformed into Rosetta(DE3)receptor cells,respectively,induced at 37°C for 8h,using final concentration of 0.2m M IPTG.After centrifuge,the inclusion body proteins were dissolved in urea-containing buffer solution after ultrasonic crushing.The three proteins were purified by Ni-NTA and then regenerated by urea concentration gradient of 4M-2M-1M-0M.Western blot analysis showed that all three chimeric proteins could react with corresponding antiviral sera.Under transmission electron microscopy(TEM),all virus-like particles were formed with a diameter of about 20 nm.These results indicate that three kinds of virus-like particles of porcine viral diarrhea have been successfully constructed.To verify the immunogenicity of the prepared triple VLPs,mice were immunized with doses of 25μg and 50μg respectively,and the cellular immunity and humoral immunity levels of the immunized mice were monitored.The serum levels of cytokines IFN-γ and IL-4 were detected by indirect ELISA.The results showed that the levels of cytokines in VLPs group increased significantly at 28 days after immunization,and the levels of IFN-γ and IL-4 in high-dose group were higher than those in commercial vaccine group at 35 days after immunization.These results indicated that the triple VLPs preparation could induce good cellular immunity in mice.In order to detect the specific Ig G content in serum of immunized mice,TGEV,PEDV and PRoV viruses were used as coated antigens to perform indirect ELISA tests.The results showed that the specific antibody level in serum of mice showed an increasing trend over time within 35 days after immunization,and the immune dose was positively correlated with the antibody titer.After immunization,the antibody level of the commercial vaccine group decreased slightly,and the specific antibody level of the VLPs group continued to increase.At the 35 th day,the specific antibody level of the high-dose group reached the peak and was higher than that of the commercial vaccine group,indicating that the triple VLPs had good immunogenicity.In conclusion,the triple chimeric hepatitis B core VLPs designed and prepared in this study can induce good cellular and humoral immunity in mice,and has good immunogenicity.This study provides technical reserve for the development of vaccine for novel procine viral diarrhea.