| Rabbit hemorrhagic disease(RHD)is a highly contagious and fetal disease in adult rabbits caused by rabbit hemorrhagic disease virus(RHDV).And the disease have already caused great economic loss for rabbit industry and associated productions.RHDV is a member of Caliciviridae family,a positive-sense single-stranded RNA virus containing a genomic RNA(gRNA)with 7.4 kb and an additional RNA species with 2.2 kb designated subgenomic RNA(sgRNA).VP60 was a major structural protein of virus capsid and a special antigen of RHDV in stimulating immune response.And because of these,it is a research hotspot in detection of RHDV and development of RHD vaccine.There are no effective drugs for curing RHD in clinic yet.Disease control in rabbitries relies mainly on vaccination.In order to better control and prevent RHD,we studied the RHDV from two parts:the detection method of RHDV and the genetic engineering vaccine.A new detection method,SYBR Green RT-PCR of RHDV,was developed and applied to detect the course of RHDV infection in main target organs.Then we primary studied the protective mechanism of genetic engineering vaccine of RHD through the evaluations of humoral immunity and cell-mediated immunity.1.Establishing and Application of SYBR Green RT-PCR Method for Detection of Rabbit Haemorrhagic Disease Virus(RHDV)The primers used in RT-PCR were designed according to the VP60 gene sequence of RHDV.The established SYBR Green RT-PCR of RHDV was developed by optimization of parameters.The results showed that the method had good repeatability and sensitivity.The minimum detection limit of the method was 14.6 copies/?L.Additionally,the method was applied to detect the RDHV in serum,liver and spleen samples at different time(6h,12h,24h,30h,36h).The results displayed that the contents of RHDV in serum,liver and spleen all showed a time-dependent effect,increasing with the time.The copies in serum at different time were 105,106,107 and 108,respectively.The copies in liver at different time were 105,108,108 and 109,respectively.And the copies in spleen were 105,108,108 and 108,respectively.From the results at 36h,we could know that the content of RHDV in liver was higher than others,and this indicated that liver was the target tissue of RHDV.2.Research on the Protection Mechanism of Genetic Engineering Vaccine of RHDVThe aim of this part was to study the protection mechanism of genetic engineering vaccine of RHD through evaluating the immunity in humoral and cellular immunity.In experiment group,the genetic engineering vaccine was injected to rabbits.In positive group,inactivated vaccine was injected to rabbits.And in control group,PBS solution instead of the vaccine was injected to rabbits.The blood samples of rabbits were collected at 0,3,7,14,21 and 28 days post immunization.The antibodies(IgG and IgM)and cytokines(IL-2,IL-4 and IFN-?)in serum were test by using ELISA kits.Meanwhile,hemagglutination inhibition test was performed to evaluate the level of total antibody.To evaluate the protective effect of the genetic engineering vaccine,the challenge test was carried out at 21st day in all groups.The results displayed that the IgG and IgM antibodies in genetic engineering vaccine group and inactivated vaccine group got a significant(P<0.05)increase compared with those of the control group at 7,14 and21 days post immunization.The IL-2,IL-4 and IFN-y in genetic engineering vaccine group were also significantly(P<0.05)increased compared with those of the control group 3,7 and 14 days post immunization.There was a significant difference(P<0.05)in IL-4 and IFN-? between inactivated vaccine group and control group,but not in IL-2(P>0.05).The haemagglutination titres in genetic engineering vaccine group and inactivated vaccine group also significantly(P<0.05)higher than that of the control group at 7,14 and 21 days post immunization.Additionally,through comparing the levels of antibodies and cytokines in the genetic engineering vaccine group and inactivated vaccine group,we found that the levels of IgG,IgM,IL-4 and IFN-?induced by genetic engineering vaccine were all higher than that induced by inactivated vaccine,and the levels of IL-2 in two groups had a significant difference(P<0.05)at 3,7,14,21 and 28 days post immunization.After infection of RHDV at 21st day,the two vaccine groups had 100%survival rate,and the control group had a 0%survival rate.In conclusion,genetic engineering vaccine could induce effective humoral immunity and cellular immunity and effectively prevent RHD. |
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