Cloning And Expression Of Hemagglutinin-neuraminidase Gene Of Newcastle Disease Virus In Lactobacillus

Newcastle disease virus(NDV) is classified as a member of the newly defined genus Avulavirus in the family of Paramyxoviridae.It is a causative agent of Newcastle disease(ND),which is widely distributed and has causes large economic loss.Hemagglutinin-neuramidinase(HN) is a structural glycoprotein which constructes the apophysis of peplos surface.This structural glycoprotein has two activities which are hemagglutinin and neuramidinase.It is a protective antigen of NDV and has favorable immunogenicity.It could discriminate acceptor when NDV infested organism.The experiment confirmed that the mechanism of chicken counteracted NDV in nonage mainly depended on the local immunity contribution of respiratory tract and digestive tract.NDV has a special rodent for mucous membrane and infects through respiratory tract and digestive tract easily as the paramyxovirus.The method of immunizing live vaccine by nose,eyes,water and air could make organism to produce S-IgA and set up mucosa immunity.S-IgA has main effects on local immunity of respiratory tract and digestive tract.Local immunity is the best measure after NDV premonition. Therefore,the study of mucosa immunity has significant meaning.In this research,we clone the Hemagglutinin-neuramillidase genes from NDV(F48E9 starin) by RT-PCR. Then the protein was expressed in the E.coli and Lactobacillus.It found important base for the research of NDV recombination gene engineering Lactobacillus vaccine and mucosa immunity by Lactobacillus mediate. The following reseaerhes were explored:1.Cloning and sequence analysis of Hemagglutinin-neurmainidase geneAccording to published HN gene sequence of NDV starin F48E9,primers with restriction sites were designed and synthesized.1 716 bp fragments were amplified from Newcastle disease virus RNA by RT-PCR and cloned into the pMD18-T vector,then it was inverted to recipient germ JM109.The method of cloning technique,restriction enzyme correspond restriction endonuclease,PCR,sequencing and so on were used to identify the recombinant masculine cloning plasmid pMD18-T-HN.The nucleotide sequence could be analyzed by DNASTAR software,the consequence manifest that this gene has 99.4%homology with the HN gene order in the Genbangk' record.2.Construction of Recombinant Plasmid pW425et-HN of Newcastle disease virusThe purified HN gene was subcloned into the vector pW425et.Thus,the recombinant pW425et-HN was constructed.Analyzed by SDS-PAGE and western blotting,HN-GST fusion proteins presented a 66 KD and with immunogenieity.3.Expression of Recombinant Plasmid pW425et-HN in LactobacillusThe recombinant plasmid pW425et-HN of Newcastle disease virus was electroporated into L.acidlophilus,after optimizating condition of electroporation.The positive clone was selected by SDS-PAGE and Western blotting.The result certified that the HN gene could be expressed in L.acidlophilus and had the reactionogenicity with the polyclonal antibody of Newcastle disease virus.The experiment result showed,NDV-HN gene expressed in Lactobacillus successfully,it made foundation for advanced development of Lactobacillus vaccine against NDV,conduction channel and action mechanism of mucosa immunity by Lactobacillus mediate.