Establishment And Application Of RT-PCR For Bvdv And Gene Cloning Expression Of GP48 In Prokaryotic

Bovine viral diarrhoea - mucosal disease （BVD-MD）, is composed of bovine viral diarrhea virus by a fever, mucosal erosion, ulcer, diarrhea, leukopenia, cough and a pregnant cow abortion or output malformation fetus as the main features of infectious diseases.Because it can cause the cow reproductive system disorders and can form a persistent infection, so the cattle industry has an important influence on.In this experiment, the proliferation of MDBK cells of BVDVOregon C₂₄V cell, in 80% lesions, cell harvesting viral culture liquid.On the standard strain Oregon C₂₄V TCID₅₀ were measured, the measured TCID₅₀ 105.52.On the basis of GenBank published in Oregon C₂₄V （AF091605）, NADL （M3118）, ZM95 （AF526381）, Singer （DQ088995）, 13 strains of BVDV gene sequence alignment, using Primer 5 software to design a pair of primers for amplification, in standard strain Oregon C₂₄V got big small for 330 BP fragments, and the expected tentative agreement.On CSFV, IBRV, BTV, PRV, PRRSV, MDBK cells and RT-PCR amplification, are not amplified fragment, it shows that this method has high specificity.The BVDV TCID₅₀ diluted were detected, 106 times diluted can still be detected by the virus, shows that this method has higher sensitivity.The collected clinical 70 calves with diarrhea stool samples tested, 17 were positive, the positive rate was 24.28%.It shows that this method can be used for BVDV laboratory testing.The above results indicate that the method has high practical value, can be BVDV laboratory rapid diagnosis and provide an effective method for.The test on the basis of GenBank published in BVDV gene sequence alignment, using Primer 5 software to design a pair of gP48 gene amplification primers, application of RT-PCR amplification technology on BVDVC₂₄V strains were amplified and cloned and identified by enzyme digestion, pMD18-T-gP48 vector was constructed.The cloning vector sequence, sequence comparison and published consensus sequences, show that pMD18-T-gP48 vector was constructed successfully.Test the gP48 gene was inserted into pET32a （+） plasmid, construction of prokaryotic expression vector.The constructed prokaryotic expression vector of enzyme digestion.Detection and analysis of SDS-PAGE, about 46kDa at the protein band.The expression of protein after purification was analyzed by Western-Blotting, about 46kDa at specific immunoblot band.The results indicate that gP48 gene protein in a prokaryotic expression vector for expression, and can be identified by the BVDV positive serum.Demonstrated success to construct PET32a （+） prokaryotic expression vector, to further establish and detection of BVDV ELISA methods to lay the foundation.