Construction And Characterization Of A Recombinant Live Vectored Vaccine And A DNA Vaccine Against Classical Swine Fever

Classical swine fever(CSF) is one of the most important diseases affecting swine industry in China. Hog cholera lapinized virus (HCLV) vaccine has been used for a long time and played a key role in the control of epizootic CSF. However, an atypical form of CSF has emerged in China since the 1970s, especially in the recent years. Pseudorabies(PR) and porcine reproductive and respiratory syndrome (PRRS) are also responsible for major economic losses especially in some countries with an industrialized pig production. The above three diseases have been recognized worldwide as the most economically important pathogens of swine production, and cross infections or multi-infection with the pathogens are often seen in pig herds. Therefore, developing efficacious combined vaccines and reasonable immune procedure is extremely significant in control of the diseases.1, Construction and immune efficacy of a DNA-based vaccine against classical swine fever The VP22 protein of bovine herpesvirus-1 (BHV-1) as reported can facilitate proteins to transduce in cells. In this study, we amplified the E2 gene of CSF virus (CSFV) and VP22 gene of BHV-1 by PCR at first, then we inserted the E2 gene into a eukaryotic expression vector pcDNA3.1+ downstream the CMV promoter to acquire the recombinant plasmid named as pcDE2. Another recombinant plasmid pcDEV was constructed based on pcDE2 by inserting the VP22 gene at downstream of the E2 gene to express two proteins in fusion form. We transfected these two recombinant plasmids into 293T cell. The E2 gene in two plasmids could be expressed, which was demonstrated by Western blot. But in cells transfected with pcDEV, the E2 protein was diffuse when detected by indirect immunofluorescent antibody test, indicated that the VP22 protein can transduce protein endocellularly. In further study we evaluated the immune efficacy of the two plasmids in mice and rabbits. In the first experiment, 21 BALB/c mice were divided into three groups (7 mice in each group), and injected in the tibialis anterior of each hind leg with pcDNA3.1, pcDE2, pcDEV, respectively. Each group was injected three times at a two-week interval with 100μg per mouse in each time. Blood was collected at 2, 4, and 6 week post immunization. The detection of antibodies against CSFV were performed using IDEXX antibody detection kit. After the third immunization, two groups injected with pcDE2 and pcDEV developed E2-specific antibody, but the antibody level of the pcDEV group was higher, indicated that VP22 protein might have enhanced the expression or presentation of the E2 protein. In another experiment, we divided 9 rabbits into three groups (3 rabbits in each group), the immunization and the antibody detection procedures were similar to those of the mice model, 500μg plasmids were injected each time per rabbit. After the second immunization, the two groups immunized with pcDE2 and pcDEV exhibited seroconversion, and the percentage of positive rabbits and the antibody level in pcDEV immunized group were higher than those of the pcDE2 group. Seven weeks after prime immunization, all rabbits were challenged intravascularly with the 484^th rabbit attenuated CSFV (collected from the lymph and spleen) diluted by 1:40, and each rabbit was injected 1 ml. The body temperature was monitored, 3 daysbefore challenge temperature was measured at 12-hour interval, 24 hours after challenge temperature was measured at 6-hour interval for 96 hours. The results indicated three rabbits in the group immunized with pcDNA3.1 began to have fever about 24-48 hours after challenge, the febrile episode lasted for 36 hours with typical CSFV infection feature. The temperature, appetite and spirit of the three rabbits in the group immunized with pcDEV were all normal. But in the group immunized with pcDE2, only one rabbit was normal, the other rabbits had a fever for one time at 48 hours after challenge which lasted for 12 hours, and the temperature was 0.5 °C higher than normal. All the results demonstrated that VP22 protein of BHV-1 when fused to E2 protein of CSFV could facilitate the transduction of E2 protein and enhance the immune efficacy.2 > Construction and biological characterization of a recombinant pseudorabies virus expressing multiple foreign genes A recombinant PR virus (PRV) expressing the GP5 of PRRSV had been constructed by our laboratory, animal experiments had demonstrated that pigs immunized with this recombinant virus could be protected against the challenges of PRV and PRRSV. In this experiment, based on the success of the above recombinant PRV we inserted the E2 gene of CSFV into PRV genome, in order to get another recombinant PRV to express E2 and GP5 proteins at the same time. Firstly, we constructed a recombinant plasmid to carry TK gene of PRV which would be used as homologous arms subsequently. Then we constructed three eukaryotic expression cassette: the LacZ gene under the SV40 promoter, the E2 gene and the GP5 gene under the CMV promoter, respectively. We inserted the three cassettes into the TK gene to construct the transfer vector. This vector when transfected with the genome of Bartha-K.61, could let the three cassettes be integrated into the genome by homologous recombination. We sifted the recombinant virus by blue plague, a recombinant virus was identified and purified which was named as rPRV-E2-GP5. The expression of E2 and GP5 in the cells infected by rPRV-E2-GP5 was demonstrated by Western blot and indirect immunofluorescent assay, the antigenicity of the two proteins were similar to their parent viruses. Then we compared the replication efficacy and the CPE of rPRV-E2-GP5 with it's parent PRV in different cell types such as Vero, IBRS2, PK15, CEF, no obvious difference was found. These results indicated that at least 9.4 Kb fragment could be inserted into the genome of PRV to express three foreign proteins without changing their biological characterizationsThe new type DNA vaccine against CSFV and the recombinant PRV expressing multiple foreign genes constructed in this study will help to set down combined immunization and comprehensive prevention strategies to diseases associated with reproduction.