The Research Of Optimization And Immunization Of Thermostable Newcastle Disease Vaccine Carrier Expressing GFP

Newcastle Disease(ND) is highly infectious and pathogenic to all kinds of poultry, being pandemic throughout the world several times and has impacted the poultry industry. The causative agent of Newcastle Disease is Newcastle Disease Virus (NDV) which possesses one single negative RNA and six structural proteins including Nucleoprotein(NP),Phosphoprotein(P),Matrixprotein(M),Fusionprotein(F),Haemagglutin in-Neuraminidase(HN), Larger protein(L). At present, vaccine is efficient to prevent poultry from infecting NDV. The attenuated live vaccine provides not only an efficient immunization procedure, but also a little side-effect, which save both time and money. Therefore, these attenuated live vaccines prefer to breeding enterprises, especially those household farms that locate in rural areas.Our previous work had obtained a attenuated thermostable NDV strain TS09-C by serial passaging of NDV V4strain in BHK-21cells, subsequently established the reverse genetic system of TS09-C strain, finally the green fluorescence protein (GFP) was inserted and expressed into the recombinant virus TS09-C. Our previous work facilitated us to search the suitable expressing site of foreign gene in the genome of rTS09-C. By referring to the relevant literatures, we choose three candidate sites which locate in the transcription start sites of structural genes NP, M and L respectively to insert the GFP ORF. We want to try a novel strategy to express the foreign gene. Briefly, the GFP ORF was expressed by the way of fusing GFP into the structural gene. In order to reduce impact of the inserting GFP gene on structural protein expression, the5’ terminal of GFP ORF are added with the first60bp sequence of structural gene. Therefore, GFP gene and structural gene are integrated into only one transcription cassette5UTR-60Head-GFP-2ABbi-SG-3’UTR, rather increasing the number of transcription cassette than transcripting into a poly-cistron mRNA possessing additional1146bp sequence, Theoretically, it reduces the impact of the transcription "polar attenuation effect".We use PCR to amplify the inserted segment60bp-GFP and2AUbi, restriction enzyme to linearize the plasmid pTS09-C, The fragments of the60bp-GFP,2AUbi and linearized vectors were assembled by in-fusion clone kit. The constructed full-length cDNA clones were further confirmed by DNA sequencing, enzyme digesting and PCR. The3full length plasmids and helper plasmids are co-transfected into BHK-21cells. The BHK-21cells transfected with DNA plasmids were lysated by3time of freeze and thawing. The rescued virus was tested by HA, real-time PCR and IFA assays. The results confirmed that all3recombinant virus were rescued and could express GFP successfully. They are named as rTS-GFP/NP、rTS-GFP/M、rTS-GFP/L based on GFP relative site to different structural genes. Now we reported firstly that GFP was fusion expressed by integrating it into the transcriptional unit of NDV genome and self-cleaved by the unique feature of2AUbi.We compare the biological characteristics of3NDV strains expressing GFP including cell multiplication efficiency and GFP expression efficiency. We use0.1MOI virus to infect BHK-21cell. At24,48,72,96h post infection, the fluorescent figures were snapped, and the suspension of media and the lysates of infected cells were collected. By analyzing TCID50of cell culture suspension, depicting the cell growth kinetics of each strain, we find that rTS-GFP/M grow more efficiently than others, which consist with the rTS09-C. By comparing the fluorescent figures, we find that rTS-GFP/M fluorescent intensity is stronger than others. The similar results were obtained by western blot analyzing of lysates from infected cells. By taking into account, we confirm that the transcription start of gene M is a suitable site to express foreign gene. The Western-Blot results to analyze GFP of purify rTS-GFP/M are positive, the Confocal observed in somatic cell nuclear transfer also have GFP, but rTS-GFP/M exists only in cells, the GFP exists in embedded or free forms.To test the protection efficiency of rTS-GFP/M against NDV challenge,1-day-old SPF chickens were immunized with105.0EID50of rTS-GFP/M and V4. At14days post immunization, the serum samples were collected, then the immunized birds were challenged with106.0EID50dose of virulent NDV strain CA02. The results confirmed that the HI antibody of rTS-GFP/M group was a little lower, but protection rate of both rTS-GFP/M and V4groups were100%.The recombinant virus rTS-GFP/M can provide complete protection against virulent NDV challenge. In summary, we compare and analyze the GFP expression level and viral growth rates of these three viruses, rTS-GFP/NP、rTS-GFP/M、rTS-GFP/L. We confirm the transcription start site of gene M(rTS-GFP/M) is optimal site to express foreign genes. The SPF chickens immunized with rTS-GFP/M have completely been protected from virulent NDV challenge, which show the potential usage of being vaccine vector. The recombinant virus rTS09-C can be used to develop the bivalent thermostable vaccine against NDV and other avian diseases. However, the serum antibody titer (HI) of are a little low. It is worth to considering how to evaluate the vaccine performance, other protective mechanisms beside the humoral immunity may be involved in and need further investigation.