| Newcastle disease is a highly contagious disease caused by Newcastle disease virus (NDV) that leads to substantial economic losses in the poultry industry around the world. Genotype Ⅶ NDV is predominant in China, Japan and Korea. The conventional ND vaccine strains and the current circulating NDV strains belong to different genotypes, so it is required to develop new vaccines which are match with current circulating strains. In this study, we constructed the recombinant viruses expressing the glycoproteins F and HN of Genotype VII NDV strain by using Marek’s disease viruses (MDV) CV1988/Rispens vaccine strain as the vector, and the protective efficacy of two recombinant viruses was evaluated. The aim is to provide a new vaccine to prevent and control Genotype VII ND.In order to make the purpose gene efficiently express in recombinant MDV, the promoters of purpose gene were screened firstly. Recombinant plasmids were constructed to express the green fluorescent protein (GFP) which contains either Pec promoter or CMV promoter. Then the two plasmids were transfected to DF1 cells respectively. The activities of promoters were determined by observing fluorescence and western blot test. The result showed that the activity of CMV promoter was higher than Pec promoter.Then F and HN genes of Genotype Ⅶ NDV strain(DT-2014) isolated from Jiangsu area amplified by RT-PCR and cloned into pEASY TM-M1 expression vector which contains CMV promoter. Positive plasmids severally were named pEASY-M1-F and pEASY-M1-HN. And then the expression boxes of F and HN were respectively inserted into the pU/D vector which contained the sorfl/sorf2 sequence of the CVI988 genome and got the transfer plasmids pU/D-F and pU/D-HN. The transfer plasmids and the recombinant MDV express GFP (rMDV-GFP) genome were co-transfected into CEF cells separately. GFP gene was removed from recombinant viruses through limiting dilution method. Results of PCR, western-blot and IFA showed that the F and HN gene were correctly inserted into the genome of Marek’s disease virus and expressed steadily.In this study, we also evaluated the protective efficacy of rMDV-F and rMDV-HN against both Newcastle disease strain DT-2014 and vvMDV RB1B. The experiment of recombinant viruses against with NDV,One hundred,1-day-old SPF chickens were randomly divided into 5 groups:rMDV-F immune group, rMDV-HN immune group, attenuated vaccine immune group, challenge control group and blank control group. The first two groups were inoculated subcutaneously with 5000 PFU of rMDV-F or rMDV-HN. Attenuated vaccine group vaccinated LaSota attenuated vaccine in the 7-day-old. Every group except blank control group was challenged with 105 EID50 DT-2014 NDV strain intraocularly and intranasally in the 21-day-old. Continuous observation for 7 days after challenge, the results showed that the difference between every immune group and challenge control group, whose mortality rate was 100%, was significant extremely.Both of recombinant virus immune groups and attenuated vaccine immune group can provide effective protection against Newcastle disease, the protection ratio of rMDV-F immune group, rMDV-HN immune group and attenuated vaccine immune group was 85%,80%, 90%,respectively. The antibody growth of every group showed that antibody levels continued rising after immunization in two recombinant virus immune groups and antibody level was rapidly rising one week after immunization in the attenuated vaccine immune group. Moreover, the experiment of recombinant viruses against with MDV showed that two recombinant viruses as well as CVI988/Rispens provided effective protection when challenged with vvMDV RB1B strain.In conclusion, the two recombinant virus strains rMDV-F and rMDV-HN were built successfully, they can provide adequate protection against the challenge of Newcastle disease virulent strain and become new vaccines to prevent and control ND. |
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