The Preparation Of The Virus Structural Protein Gene Of Swine Fever And Mouth Disease

Swine fever and Foot and Mouth Disease are viral diseases which can be spread quickly among livestocks,In order to reduce the prevalence of the diseases,developing some genetic engineering subunit vaccines which could resist swine fever and foot-and-month disease virus is particularly necessary.Previous reports found that swine fever virus structural protein E0,E2 and the virus structural protein VP1 protein could induce the pig and artiodactyl produce neutralizing antibody against foot-and-mouth disease virus in the body,Therefore,we packaged lentivirus with recombinant vector which contained the swine fever virus structural protein E0,E2 and foot-and-mouth disease virus structural protein(VP1 protein)gene,we detected the lentivirus transfection efficiency in goat mammary epithelial cells and in order to realize the swine fever virus structural protein E0,E2 and foot-and-mouth disease virus structural protein(VP1 protein)stable expression of the exogenous gene in the host cell and exocrine.Eventually,we collected the exocrine protein of transfected cells,and western blot results showed that exogenous target protein exist.This experiment research content is divided into the following parts.1.Cloning of,CSFV-E0,CSFV-E2,AFMDV-VP1,OFMDV-VP1 gene CDSwith His-Flag tag in the N-terminal.We constructed lentivirus expression vector which could expres s these proteins,we also replaced the promoter with mammary gland specific BLG pr omoters.Enentrully,we constructed lentivirus expression vector PCDH-BP11-CSFV-E2,PCDH-BP5-CSFV-E2,PCDH-BP5-CSFV-E0 PCDH-BP11-CSFV-E0 PCDH-BP5-AFMDVVP1,PCDH-BP11-AFMDV-VP1,PCDH-BP5-OFMDV-VP1,PCDH-BP11-0FMDV-VP1.2 Using lentivirus packaging plasmids PMD2 G PSPAX and lentivirus vector plasmi ds PCDH-BP11-CSFV-E2,PCDH-BP5-CSFV-E2,PCDH-BP5-CSFV-E0 PCDH-BP11-CS FV-E0 PCDH-BP5-AFMDV-VP1,PCDH-BP11-AFMDV-VP1,PCDH-BP5-OFMDV-VP1,PCDH-BP11-0FMDV-VP1 transfected 293-Tcells,and we obtained Lenti-virus with o ur target genes.3 180 goat fertilized egg were obtained using superovulation technology,then we i njected virus into a fertilized egg Oocyte zona pellucida gap,149 living embryos wer e obtained and we transplanted 50 recipients,35 lamps were born.Finally,via PCR,we o btained 4 goats of PCDH-BP5/11-AFMDV-VP1(gm),6 goats of PCDH–BP5/11-OFMDV-VP1(gm),3 goats of PCDH-BP5/11-CSFV E0 genetically modified(gm),3 goats of PC DH-BP5/11-CSFV E2 genetically modified(gm).