Cloning The Hemagglutinin-Neuraminidase Gene Of Newcastle Disease Virus And Expressing It's Main Antigen Region In Prokaryotic Cell

AbstractNewcastle disease,caused by Newcastle disease virus,was an acute height contagious disease of chicken and many avians.After infected with velogenic strain of Newcastle disease virus,susceptivity avain often showed septicaemia which clinical feature was dyspneic respiration,alo laxata,nerves disorder,hemorrhage of mucosa and tunica serosa.Poultry industry had enormous economics loss by Newcastle disease currently.Therefore,precaution and control Newcastle disease was an difficult mission at present and a stage in our country.At present,our country prevented and controlled Newcastle disease with vaccine.Hemagglutinin Neuraminidase of Newcastle disease virus could stimulus organism to produce protective antibody.In order to analysis epitope of Hemagglutinin Neuraminidase protein,we had tests:First:the virions of the Newcastle Disease of the La Sota vaccine strain was prolifered in the SPF chick embryos,purified,extracted the whole RNA of the Newcastle Disease Virus by the whole RNA separating kit,and amplify the gene of NDV HN by using the RT-PCR technology.After cloned the product of the PCR to the pGEM-T easy vector,we named the new plasimid pTHN.The results showed:1.The cDNA of NDV HN gene was amplified successfully from the whole RNA of the NDV by RT-PCR.The cDNA was clonged successfully into pGEM-T easy vector by PCR and EcoR I enzyme.2.After checked the recombinant plasmid pTHN by PCR and sequencing,we had the Hemagglutinin Neuraminidase gene and its nucleotide sequence was coincidence with the classical low virulent strain La Sota(Y18898).The vaccine strain doesn't take place mutation after sequence verifying.Second:The main epitop gene of Hemagglutinin-Neuraminidase gene,1239bp,was cloned into prokaryotic expression vector pET-28a and identified with enzyme and PCR.We named new vector pETHN.The recombinant plasmid was transformated into competent cell,BL21(DE3),and the recombinant bacteria was cultured with isopropy-β-D-thiogalactoside.The reaction of the expression product was identified by Western bloting.The results showed:1.The main epitop gene of Hemagglutinin-Neuraminidase gene was clonged successfully into pET-28a,which was identified by PCR and enzyme.And the sequencing result showed clonged gene was not mutation.2.28kD protein,detected by SDS-PAGE,was expressied after IPTG induced.The expression product could reacted specifically with antiserum of Newcastle disease virus.