Construction And Vaccination Test Of DNA Vaccine Of NDV Strain Isolated From Guangxi And Study On The Distribution Of DNA Vaccine After Delivery

Newcastle disease is a highly contagious and fatal viral disease that affectsall species of birds, which was listed into the Group A diseases by OfficeInternational des Epizooties. The disease has a worldwide distribution, and is amajor threat to the poultry industries due to the huge economic loss associatedwith it. Newcastle disease is caused by the Newcastle disease virus (NDV), thehotspot of NDV study were biological characteristics, new type vaccines andmolecular epidemiology. The identification of biological characteristics,construction, vaccination test and delivery of DNA vaccine of NDV GX7/02strain were studied.The biological characteristics of NDV strain GX7/02 were identifiedaccording to international standerd and method ruled by OIE. The resultsshowed that ELD₅₀was 10^-9.42/0.1mL, EID₅₀was 10^-9.57/0.1mL, MDT was 54.0h,TCID₅₀was 10^-9.50/0.1mL, ICPI was 1.925, IVPI was 2.43. and the resultsillustrated that GX7/02 virus was belonged to virulent strains. The 16-dayschickens, ducks and geese are attacked by the GX7/02 strain, the incidence were100%, 40%, 100%, and the mortality were 100%, 0, 60%. The results alsoproved that GX7/02 belonged to fast type of agglutination-separation and wasnot stable to heat.Two pairs of special primer were designed to amplify the F and HN genes ofNDV isolated from Guangxi. And the F and HN genes were inserted into thepTracer-CMV2 and controlled by the PCMV promoter individually to constructthe recombined plasmid pTracer-CMV2F and pTracer-CMV2HN. The IL2 geneof Guangxi Sanhuang chicken were amplified by another pair of primer andwere inserted into the plasmids pTracer-CMV2F and pTracer-CMV2HNindividually controlled by PEF1 promoter, pTracer-CMV2FIL2 andpTracer-CMV2HNIL2 were constructed and were transfected into BHK-21 cells by Lipofectamine 2000 Transfection Kit individually. Expressions of F and HNgenes were detected by indirect immuno-fluorescence experiment, and themRNA transmitted from IL2 gene were detected by RT-PCR. DNA vaccinespTracer-CMV2FIL2 and pTracer-CMV2HNIL2 were injected into two-weeksSPF chickens individually or together. The dose of vaccines is 200ug perchicken. The combine immunized chickens were divided into three groupswhich immunized once, twice and three times with pTracer-CMV2FIL2 andpTracer-CMV2HNIL2. Blood serum were taken once a week after immunization,and the antibody were monitored by ELISA method. The chickens werechallenged by DNV GX7/02 after six weeks from immunization. The resultsshowed that: the antibody levels of groups which immunized with DNAvaccines were higher very significantly than that of the negative control(P＜0.01); there is not denotes significant difference in antibody levels betweenthe group immunized with pTracer-CMV2FIL2 and the group immunized withpTracer-CMV2HNIL2(P＞0.05); the antibody levels of groups whichimmunized with pTracer-CMV2FIL2 and pTracer-CMV2HNIL2 together werehigher significantly than the groups immunized with those two vaccinesindividually; and the level was becoming higher along with the rising of theimmunization numbers(P＜0.05). The results of protective immunity showedthat the groups immunized with pTracer-CMV2FIL2 and pTracer-CMV2HNIL2together provided significant protection compared with the groups immunizedwith those two vaccines individually.The distribution, expression and persistence of DNA vaccine has beenexamined following intramuscular administration with 200μgpTracer-CMV2FIL2 in 3-week-old chickens. The recombinant plasmidpTracer-CMV2FIL2 in heart, liver, spleen, lung, bursa, brain, bone marrow,thymus and inoculated muscle was detected by Nested PCR technique. Thereal-time fluorescent quantitative PCR technique was used to quantitate therecombinant plasmid in serum at different time point, and the RT-Nested PCRwas used to detect RNA expression in different time tissues. The results showedthat the recombinant plasmid was observed in serum between 2 to 3 hours post inoculation, highest concentration was detected at 6h, and can't be detected at 7dpost inoculation. The recombinant plasmid was found in the tissues of heart,liver, spleen, lung, bursa, brain, bone marrow, thymus at different time after 4hpost inoculation, and disappeared at different time after 13d. The mRNAtransmitted from pTracer-CMV2FIL2 was observed in tissues except forinoculated muscle by RT-PCR at different time after 1d post inoculation, anddisappeared at different time after 13d. And the results also showed that therecombinant plasmid was detected at 2h and the mRNA was found at 10h in thecell of inoculated muscle, and both of them can also be detected at 150d.Three inactivated Newcastle disease virus (NDV) oil emulsion adjuvantvaccines were prepared respectively using NDV GX1/06 strain isolated fromGuangxi, Lasota strain and F48E9 strain. And three groups of three-weeks oldspecific pathogen free chickens were inoculated by the three vaccinesrespectively, another group is the negative control. NDV titers were detectedevery week after immunization. All chickens were challenged by NDV GX1/06strain in the 5th week after immunization. The result showed that three vaccinescan induce 100% protection from lethal attack by NDV isolated strain GX1/06.