| The injection of vaccine plays an important role in prevention and treatment of swine viral diseases. Moreover, with the advancement of diagnostic techniques, we find that mixed infection is a common phenomenon in pig diseases, so the new vaccine needs for "Immunizing one time, preventing many diseases". It has been proven that using the live Pseudorabies virus as a vector and inserted heterologous gene is a new and effective method in the research of multivalent vaccine for pig diseases. Pseudorabies virus and porcine parvovirus are the main pathogens of reproductive disorders, and cause huge economic losses in pig industry. We know porcine parvovirus can enhance the postweaning multisystemic wasting syndrome clinical symptoms caused by the the porcine circovirus 2, and it is interesting that porcine bocavirus is found in pigs with PMWS in the first time. It is also reported porcine bocavirus has some relationship with piglets respiratory infections, diarrhea and other clinical symptoms, but still there is a gap in development of porcine bocavirus vaccine. So the vaccine that can prevent pseudorabies virus, porcine parvovirus and porcine bocavirus at the same time has great value in research and daily use. In this experiment we use attenuated pseudorabies virus as vector to develop this kind of virus. By the way, now there is only PCR method to diagnose porcine bocavirus, here we also develop the monoclonal antibody against the porcine bocavirus structural protein VP2 to lay a foundation for the follow-developed porcine bocavirus ELISA diagnostic kits. Here is the main content of experiment.1. The monoclonal antibody against porcine bocavirus VP2 protein:First we clone the VP2 gene from the positive materials of porcine bocavirus, and then insert it into the prokaryotic expression plasmid pET-30a. After identification, the recombinant plasmid is transformed into BL21 cells and induced by IPTG.We find there is an expression strip around 48 kDa, the purified protein is injected to the Balb/c mouse. After the titer reach the requirement of fusion, spleen cells are fused with SP2/0 cells. By the experiment of indirect ELISA and Western-blot, we finally succeed in developing 4 strains of monoclonal antibody.2. Construction of recombinant virusWe use pseudorabies virus TK’/glacZ+ as the live virus vector and pIECMV as the middle transfer vector. The genes including PBoV-VP2, PPV-VP2, as well as CMV promoter, PloyA terminator are inserted into the pIECMV transfer vector. IBRS-2 cells are co-transfected with genomic DNA of PRV TK’/gE’/lacZ+ which is digested by restriction enzyme EcoR I and recombinant plasmid. By the experiment of PCR, plaque-formation assay and western-blot, we find that the expression of PBoV-VP2 strip is around 62kDa, and PPV-VP2 is around 64 kDa, finally by detecting the growth curve of virus, we make sure than the insert of VP2 genes do not affect the growth of pseudorabies virus.3. The immune assessment of recombinant virusThe mice were divided into three groups and injected 105.0TCID50 pseudorabies virus TK-/gE-/lacZ+, recombinant virus and MEM medium respectively. The second immunization was at four weeks after the first time. The sera were collected at 0,14,28, 42 and 56 days after immunization for immunoassays. We found that the group of recombinant virus induced the antibody against the porcine parvovirus and porcine bocavirus, at the same time we found that the group of pseudorabies virus TK-/gE-/lacZ+ and recombinant virus had similar ability in PRV neutralization. |
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