Cloning Of BVDV Complete Gene And The Study Of Its DNA Vaccine

Bovine viral diarrhea-mucosal disease virus (BVDV) is a member of the pestivirus genus,family Flaviviridea, mainly causes bovine viral diarrhea-mucosal disease. Cattle infected by BVDVshows diarrhea, acute and chronic mucosal disease, persistent infection and immunotolerance,immunosupression, pregnant cow abortion, dead fetus and abnormal fetus, etc. BVDV can infectcattle, calf and some of other animals. In the west, the virus causes a variety of clinical diseasesthat adversely affect essentially every stage of production, including decreased milk production,reduced reproductive performance, growth retardation, increased occurrence of other diseases andso. In recent years, control is achieved by implementing systematic control of infection focused onstrict survey and eradicating persistently infected carrier cattle timely. BVDV caused infection inmore than 20 provinces of China. Because the models for assessing impact of BVDV infection onthe cattle industry and proper surveys have not been established, BVDV infection has beenunderestimated for a long time.In the present study, the complete genome of BVDV Oregon C24V strain from IVDC wasamplified by RT-PCR and 5' RACE, then were cloned ,sequenced and analyzed. The full lengthgenome consists of 12305 nucleotides, 5'UTR is 381 nucleotides, and 3'UTR is 182 nucleotides,the open reading frame (OFR) 11742bp containing the start codon (ATG) and the terminal codon(TAA), which encoding 3914 amino acids. Nucleotide of this strain show a high homology withVEDEVAC strain, Ossloss strain and standard Oregon C24V strain that are 99.50％, 92% ,79.41％respectively. The amino acid homology of Oregon C24V strain from IVDC compared withVEDEVAC strain, Ossloss strain and standard Oregon C24V strain is 99.11％, 92.93％, 87.22％respectively. Nucleotide and amino acid phylogenetic analysis for 7 BVDV strains shows thatOregon C24V strain from IVDC is more similar to VEDEVAC strain than other strains. All datasshow that this strain might not be a standard Oregon C24V strain.5′and 3′part cycle of the gene were constructed individually with 9 overlapped fragments byuse of uniqe restriction enzyme in the complete viral genome. The T7 promotor was flanked withupstream 5' end. Restriction enzyme AsuⅡwas introduced as genetic symbol of infectious clone at2920 site of genome by silence mutation. SacⅡwas flanked with 3' end as a tailor signal for run offtranscription in vitro. The fragment consists of entire genome was constructed by long overlappedPCR ,then the low-copy-number vector pOK-12 was ligated with full-length cDNA to constructfull-length infectious BVDV cDNA vector.The structural proteins P14, E2 and non-structural protein NS3 were amplified by PCR. Afteridentification, they were subcloned into an eukaryotic vector pcDNA3.1(+)which containing the CMV promoter and the BGH polyA. DNA vaccines contain three genes were contructedrespectively. In this research, the three DNA vaccines were transfected into monolayer bovineturbinant cell monolayer and the expressed proteins were confirmed by indirectimmunofluorescence assay. The result shows that three eukaryotic expression plasmids can beexpressed in vitro, which provids the solid basis for the research on animal inoculation.In order to evaluate the immune effect of these DNA vaccines, 6-8-week-old female Kun Mingmice were randomly divided into 6 groups and intramuscular inoculated with 100μg of abovevaccines and vector pcDNA3.1(+) respectively, pcDNA3.1(+) is used as control. Every two weeks,the mice were inoculated in the same way for three times. Blood samples were collected beforevaccination to detect the antibody, and the dynamic changes of CD4~+ and CD8~+T cells inperipheral blood lymphocytes were analyzed. The resulted show: (1) The groups immunizedwith recombinant plasmids ofpcDNA3.1 (+)-P14, pcDNA3.1(+)-E2 and pcDNA3.1 (+)-NS3produced positive antibody at different level. (2) The groups of pcDNA3.1(+)-P14,pcDNA3.1 (+)-E2,pcDNA3.1 (+)-NS3 produced higher IFN-γthan groups of pcDNA3.1 (+). (3) Thepercentage of CD4~+ T cell in peripheral blood of pcDNA3.1(+)-P14, pcDNA3.1(+)-E2,pcDNA3.1 (+)-NS3 increased constantly after inoculation by flow cytometry and pcDNA3.1(+)-E2had the same effect on CD8~+T cells. But pcDNA3.1 (+)-NS3 groups display the trend of decrease.The result shows that DNA vaccines can induce humoral and cellular immune responses in mice tosome extent.In conclusion, we have cloned the entire genome of BVDV OregonC24V strain from IVDCand finished the construction of infectious BVDV cDNA clone by genetic recombination and longoverlapped PCR technique. Three DNA vaccines containing P14,E2 and NS3 gene of BVDV wereconstructed and transiently expressed in bovine turbinant cell and animals were inoculated withthem. On the whole, this study provides a scientific evidence for development of BVDV newgeneration vaccine, and provides a foundation of technich and materials for prevention and controlthe disease caused BVDV.