Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Veterinary Infectious Diseases > Virus disease

Study on Construction and Experimental Immunity of DNA Vaccine Against Genotype Ⅶ Newcastle Disease Virus

Author YangZuo
Tutor LiuYuFen; LiuHuaiRan
School Harbin Normal University
Course Biochemistry and Molecular Biology
Keywords Newcastle disease virus Genotype Ⅶ HN gene DNA vaccine
CLC S855.3
Type Master's thesis
Year 2012
Downloads 74
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Newcastle disease(ND)is an acute and highly contiguous avian infectious diseasebrought by Newcastle disease virus(NDV).Since the first outbreak in1926inIndonesia’s Java Island and the United Kingdom’s new town, Newcastle disease hasbrought great harm to poultry industry, and is a violent contagion classified by OIE.In recent years, with the changes of Newcastle disease epidemiology, the genotypeⅦ Newcastle disease virus that can cause immune chickens" atypical Newcastledisease" and goose and duck die has appeared. Although the mortality rate of theAtypical Newcastle disease is not high, it can lead to the decrease of the poultryproduction performance, so its control is particularly important. Currently, theapplication of ND vaccines is mainly about geneⅠ,Ⅱ,Ⅲ, which controls theprevalence of the Newcastle disease to a large extent, but its effect on the gene type VIINewcastle disease virus is not good, therefore, developing a new vaccine to deal withthe genotype Ⅶ NDV is imperative. The DNA vaccine is the third generation ofvaccines following the traditional vaccine and gene engineering subunit vaccine, canalso stimulate the humoral immunity and cellular immunity, and has relatively longimmunity duration. With such an advantage incomparable by many other geneticvaccines, it can be used as a vaccine candidate to control the spread of the Newcastledisease effectively.This research selects the death goose NDV strain Go/CH/HLJ/LL01/08(gene Ⅶstrain, LL01strain) isolated from Heilongjiang province Lalin area, and afteroptimization of the F, HN gene in eukaryotic expression system, they were cloned intoeukaryotic expression vector pCDNA3.1R screening identified containing F, HN generecombinant plasmids, named for pCDNAR-F and pCDNAR-HN respectively. Theplasmid was transfected into293T cells, and by indirect immunofluorescence,Western-blot detection, the target protein can be expressed.In order to detect recombinant Newcastle disease virus F, HN DNA vaccineimmune effect, an experiment has been conducted on the SPF chicken. First prepare thefollowing immunosuppressive doses: pCDNAR-F200μg,100μg, pCDNAR-HN200μg,100μg, pCDNAR-F+pCDNAR-HN50μg,100μg to immune the SPF chicken, with an interval of14days, a total of3immunocompetent, and on the23th day afterimmunization, use LL01strain virus in105EID50,106EID50to challenge through muscleinjection. The results show that, only from pCDNAR-F plasmid and saline control SPFchicken all died, and the recombinant plasmid pCDNAR-HN50μg,100μg,200μgimmunization against SPF chicken got100%protection. On the basis of this, againconstructed the unoptimized F gene recombinant plasmid pCDNAR-WF, SPF immunechicken and attack, validated and plasmid pCDNAR-F, plasmid pCDNAR-WF onchicken no immunogenicity.The recombinant plasmid pCDNAR-HN minimum immune dose for threeimmunizations, with every time30μg, give the virulent strain100%protection afterchallenging. Based on the above immune procedure, recombinant antibody durationreached260days, HI antibody effect value can reach24-25.70days after immunity, thedistribution of plasmids can be detected in the brain, lung, heart, liver, spleen, kidney,stomach, intestine, muscle (in addition to the thymus).Research results show that, the construction of recombinant HN gene DNA vaccinecan stimulate the chicken body to produce efficient humoral immune response and resistlethal genotype Ⅶ NDV virulent, and the duration is longer, so it is expected to be thevaccine candidates of the Newcastle disease virus genotype Ⅶ.

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