Construction Of Newcastle Disease Virus Strain La Sota C5Infecitous Clone And Preparation Of Monoclonal Antibody Against Interferon β Of Gallus

Newcastle disease (ND) is one of the most serious avian diseases. ND can cause substantialeconomic losses and remains a major threat to the poultry industry worldwide. The causative agent ofthe disease is Newcastle disease virus (NDV), which is an enveloped virus belonging to the genusAvulavirus within the family Paramyxoviridae. NDV contains a single-stranded, negative-sense,non-segmented genome of approximately15.2kb. The NDV strains La Sota was one of the lentogenicND vaccines in the world. However, due to the age-old and repeated passaging, the50%embryoinfection dose and hemagglutination titer was decreased sharply. We applied the embryo limited dilutionmethod to passage and purify the La Sota strain5times, then obtained a NDV clone strain and namedLa Sota C5. The clone strain showed the highly hemagglutination titer and low pathogenicity thanparental virus. Here, we report the cDNA clone and sequence of the entire NDV La Sota C5genome aswell as the establishment of a system that allows the genetic manipulation of NDV. We show that thegrowth characteristics and pathogenicity of the rescued NDV are identical with parental virus. Thisrecovery system, which is based on a vaccine strain of NDV, not only provides the possibility forexperimental investigation of NDV molecular biology, but also serves as a basis for the development ofimproved marker vector vaccines. Monoclonal antibody against gallus interferon β was alsosuccessfully produced in this study, which can facilitate the research work of NDV and innate immunesystem in chicken.1. Purification and entire genome sequencing of NDV vaccine strain La SotaThe La Sota viruses were passaged in embryos and virus in allantoic fluid with the highesthemagglutination titer were passaged subsequently. After5times of passage, the La Sota C5wasobtained with high hemagglutination titer. We designed12pairs of primers based on the La Sotagenome sequences have been published on GenBank. The fragments amplified from allantoic fluidsamples according to RT-PCR method. Then the fragments were inserted into pGEM-T Easy vectors,and each fragment choosed5clones to sequence. The genome sequence of La Sota C5showed52nucleotides mutation comparing with La Sota strain which was sequenced in2005.2. Establishment of reverse genetic manipulation for La Sota C5The genome was divided into8fragments to amplify the whole genome. The3’and5’ sequences ofgenome were firstly cloned into TVT (0.0) vector to obtain TVT-LT plasmids. Then subsequently clonedthe other fragments into pMD-20T vectors gradually and obtained the transitive plasmids RLBESABwhich containing the main parts of the La Sota C5genome sequence. RLBESAB and TVT-LT weredigested by restriction enzyme BglⅡ, the target fragments were purified and ligated, then the cDNAclone of La Sota C5were constructed successfully. Meanwhile, the sequence of NP, P and L gene of LaSota C5were cloned into eukaryotic expression plasmids and obtained PCI-NP, PCI-P and PCI-L3helper plasmids. Cotransfection of3helper plasmids with the TVT-La Sota C5into BSR-T7/5cellswhich can express T7RNA polymerase. The transfected cells were collected at72h post-transfectionand freeze thawing for3times.The mixtures of the collected liquids were incubated into SPF embryos. The HA titers of the collected allantoic fluid were detected according the OIE standard method. Therecused virus was confirmed by RT-PCR and gene sequencing analysis. The HA titers of the firstpassage of the allantoic fluid is28, after3passages the HA titres was climb to211. All the resultsindicated the La Sota C5reverse genetic system was successfully constructed, which provided anadvanced platform for the future research of NDV and the associated NDV-based vector vaccinesdevelopment.3. Preparation of monoclonal antibody against IFN β from gallusA pair of primers were designed referring the sequence of gallus IFN β published on GenBank(accession no. GU119897). Then the fragment of gallus IFN β was cloned into pCold TF DNA vector,obtained the recombined prokaryotic expression plasmid pCold-ChIFN-β. The identified positiveplasmids were transformed into BL21competent cells. The correct bacteria liquid was subsequentlyincubated with0.5mmoL/L IPTG，15℃for24h. The recombined ChIFN-β was purified usingNi-NTA according the instruction. Immune the8weeks old Balb/c mice each time per3weeks. After4times immunization, collected the spleen of the mice, and the hybridoma cells were produced.4timesof subclone process were performed to get the anti-ChIFN-β cell clone strain. Finally, we obtained2anti-ChIFN-β positive cell clones. The cells were cultured and amplified. According the method ofpreparation of monoclonal antibody, we got the ascites. The ascites supernatant were identified byELISA detection. The antibody titers reached to10-3. Western blot also confirmed the responsespecificity of2produced antibodies. One of the antibodies can detect the minimal concentration of125pg/mL of gallus IFN β. The successfull production of anti-gallus IFN β provided an advanced tool forchicken disease and immune response research in chicken, which also paved the way for vaccineevaluation and the related gallus IFN β ELISA kits development.