Preparation Of BVDV E0Gene Restructuring L.acidophilus And Detection Of Protective Immunity

Bovine Viral Diarrhea Virus(BVDV) is one of the main mumber of the pestivirusgenus flaviviridea family.Presented with fever, mucosal erosion, ulcer, leucopenia, diarrhea,female abortion or production defects fetus as the main feature.The disease has becomeone of the serious harm of aquaculture infectious diseases in China after recent years ourcountry dairy import dramatically increased, caused for the animal husbandry andaquaculture. At present, dueing to the patent medicine is not manufactured, so theprevention is more important to strengthen.A pair of specific primers was designed on the Oregon C24V strain E0nucleotidesequence of Bovine viral diarrhea virus published in Genbank and amplification of BVDVof HB-DCZ strain E0gene by Polymerase chain reaction(PCR).The PCR product wascloned into pMD18-T vector,cloned products were sequenced and analysis.Results:specificbands about the size of687bp was amplified in vitro on cDNA of HB-DCZ.Sequencedresults showed that the E0gene of HB-DCZ strains BVDV composed of681nu-cleotidesand encoding227amino acids.Compared with nucleotide and deduced amino acidsequence homology of the other strains BVDV has been published was that:CCSYD99%,QHZK1098.4%,VEDEVAC98.2%,Oregon C24V80.9%,Yak74.2%.The geneticdistance of HB-DCZ strain is close to CCSYD,QHZK10,and VEDEVAC strain,fartherfrom Oregon C24V with international standar-ds and the Yak strain.The hydropHilicityplot of E0was predicted by the Kyte-Doolittle based on E0sequence and the flexibilityplot, the antigenic index and the surface probability plot were by the methods ofKarplus-Schulz,Jameson-Wolf and Emini,respectively. The B cell epitopes of E0by thesepredicted results was analyzed which are most likely localized in or adjacent to itsN-teminal No.6-17,23-29,47-53,59-68,70-81,97-109,114-122,127-134,137-143,165-172,186-198,213-220.To study the construction of bovine viral diarrhea virus Lactococcal expression vectorpMG36e-E0,digested of the target gene E0by restriction endonuclease SacI and HindIIIand integrated into shuttle expression vector pMG36e,the ligation products weretransferred into E.coli by KCM to get the recombinant vector.Positive E.colipMG36e-E0/DH5α was extracted and verified by double enzyme digestion and PCR amplification. Analysis of the E0protein expression by SDS-PAGE andWestern-blotting.The results showed that the recombinant plasmid pMG36e-E0wassuccessfully constructed,and the E0expression in E.coli was successfullydetected.Therefore,BVDV E0gene of E.coli recombinant strain was successfullyconstructed and expressed．Built the bovine viral diarrhea virus E0gene recombinant expression plasmidpMG36e-E0into L.acidopHilus use Electricity into law..restriction analysis and PCRidentification after extracted the positive of Lactobacillus acidopHilus pMG36e-E0/LA-5plasmid, analysis of the E0protein expression by SDS-PAGE electropHoresis andWestern-blotting.The results show that the BVDV E0gene lactic acid bacteria recombinantexpression plasmid pMG36e-E0successfully constructed in the reorganization ofL.acidopHilus LA-5and successfully detected.Therefore,BVDV E0gene of L.acidopHilusrecombinant strain was successfully constructed and expressed.Drench the restructure pMG36e-E0/LA-5for6~8weeks of BALB/c mice. Thecontent of intestinal tract was gathered, preimmune,1d,2d,7d For after the first timeimmunization, before the second and1d,2d,7d For after the second time immunization,before the third and1d,2d,7d For after the third time immunization. The mucosa wasgathered before and7d after first, second and third time immunization.Theabove-mentioned sample was detected by indirect ELISA for the level of the sIgA andantibody of blood. Virus test to BVDV immune group and the control group afterimmune.Results showed that the irrigation uniform recombinant L.acidopHilus groupcompared with control group,serum antibody and sIgA antibody levels increasedsignificantly(P<0.05); challenge experiment recombinant lactic acid bacteria bovineviralprotection rate of diarrhea (9/10) was significantly higher than the group of lactic acid(7/10), empty vector recombinant lactic acid bacteria group (6/10) and saline group (3/10),indicating that recombinant Lactobacillus acidopHilus bovine viraldiarrhea play a goodrole in immune protection..The result show that E0gene was expressed in L.acidopHilus expression system andhad good antigenicity.Furthermore BVDV E0gene engineering lactobacillus mayenhanced immunity of mouse, which is certificated by immunoprotection experiment. Thestudy is expected to lay the foundation for further studies on the gene engineeringL.acidlopHilus oral vaccine in their prevention of BVDV.