Construction Of Expression Vector Of Newcastle Disease Virus F Gene And Expression In Lactobacilli

Newcastle disease(ND),caused by Newcastle disease virus(NDV),was a kind of highly contagious disease with worldwide distribution.NDV could infect many kinds of poultry such as chicken,turkeys,ducks,Pigeons,Parrots and so on.Because of its great economic loss to the poultry industry, the World Organization for Animal Health(OIE) determined ND as classâ… of animal infectious disease.Vaccination was still one of the main measures to control ND.But the application of traditional vaccines existed the disadvantages which inherit such as higher cost,inconvenience to operate,short protection period,the danger that virulence might return stronger and so on.Along with development of molecular biology,main exterior protein F is not only the base of NDV serotype typing,but also the main protective antigen.In this study,Lactobacilli was selected as receptor strain to express F gene,which combined physiological function with specific immunity together.The aim was to lay foundation for developping the gene engineering Lactobacilli oral vaccine of Newcastle disease virus.In this study,the total RNA of NDV as template,F gene was cloned by the reverse transcription-polymerase chain reaction(RT-PCR).The length of target gene was 1662bp.The products of RT-PCR were ligated with Vector pMD-18-T,and the ligation product was used to transform E.coli JM 109.By the analyses of restriction endonuclease,PCR and sequencing,it showed that the inserted gene had been a positive reading frame.Then, F gene of NDV was cloned into expressing vector with Erythromycin and ThyA double labellings,which can shuttle and express in E.coli and Lactobacilli.After optimizing the condition of electroporation,the recombinant plasmid pW425et-Fwas transformed into the competence of Lactobacilli.And the positive clone was detected by plasmid extraction,enzyme digestion and PCR identification.F gene was confirmed to express in the by SDS-PAGE and Western blotting.The result showed that the expression vector of Newcastle disease virus F gene was constructed and F gene expressed in Lactobacilli.The study was expected to lay the foundation for further studies on the gene engineering L.acidlophilus oral vaccine in their prevention of NDV.