Expression Of N Protein Of Canine Distemper Virus And Application Of Diagnostic Techniques

Canine distemper(CD) is a contagious, incurable, often fatal, multi-systemic viraldisease that affects the respiratory, gastrointestinal, and central nervous systems ofvarious carnivorous animals such as Canidae and Mustelidae. Canine distemper iscaused by the canine distemper virus (CDV), a kind of morbillivirus, of theparamyxovirus family.Canine distemper was treated animal since it was discovered. It occursworldwide even in countries with high animal health level including the USA, theEU, Australia, New Zealand and Japan. It was first described in a wildlife farm in HeLongjiang province, China in 1968 and is now widespread in more than 20 provincesand autonomous regions such as Jilin, Liaoning, Jiangsu, Sichuan, Shandong,Guangxi provinces etc.Canine distemper once was the leading cause of death in unvaccinated puppiesand impacted on the development of animal trade, wildlife protection and Economicanimal farming. With the varying CDV and widowing host spectrum, CD now tendsto present with more complex symptom and more influence on animal health.Especially it can affect endangered species such as panda and Primates. More andmore attention is being paid to CD.Moreover most recent research showed that CDV could infect the humanprecursor osteoclast and propagate in it. It implies that CDV nature host maybe haveincluded human being, and men possibly be the second human virus disease derivedfrom canine.As mentioned above, CD was put on the list of notifiable disease and muchresearch on CD in diagnostic and prevention field has been done in China. Thisproject, funded by AQSIQ, was finished in the key laboratory of Economic AnimalDisease investigation, targeted collecting information, following up the advance,developing testing methods, analysis epidemic trend of CD, and laid the foundationfor further investigation on prevention measures and integraled standard system. Thisstudy includes the followings:1. Isolation and Identification of Canine Distemper Virus from FoxA virus strain, named CDV-FOX-TA, was isolated from the blood of fox, thisvirus can grow well and produce stabilization cytopathic effects in MDCK cells. Itwas demonstrated to be canine distemper viruses by a series of systematic identification such as electron microscope observe morphology physicochemically,animals infection test, RT-PCR and real time RT-PCR.2. Cloning and Sequence Analysis of the N Gene of Canine Distemper VirusTai'an Isolate in a FoxOne pair of primers was designed according to the N genes of canine distempervirus strains reported in Genebank. The N protein gene of CDV-FOX-TA wasamplified with the primers by RT-PCR. The PCR product was cloned into pMD 18-T.The positive recombinant was identified, sequenced and analyzed. As a result, thelarge open reading frame of the N gene of CDV-FOX-TA included 1572bp, whichencoded 523 amino acids. The nucleotide homology of the N genes between CDV-FOX-TA and CDV Ondertepoort strain was 96.0%, and it was 95.9% between CDV-FOX-TA and CDV Convac strain. However, the nucleotide homologies of the Ngenes of CDV-FOX-TA and the CDV wild strains, was from 98.4% to 98.9%. A ninepeptidessequence in the N protein of CDV-FOX-TA was Tyr-Pro-Ala-Leu-Gly-Leu-His-Glu-Phe, which was T lymphocyte defined antigen and senstized target cells. Onthe basis of phylogenetic analysis, it implied that CDV-FOX-TA and the CDV wildstrains had the same ancestor.3. Eukaryotic Expression and Identification of Nucleocapsifl Protein N Geneof Canine Distemper VirusA pair of primers, based on the published sequence of N gene of CDV, wasdesigned, synthesized and can amplify CDV N gene specifically. The N gene's cDNAfragment of about 1.6kb obtained by RT-PCR from FOX TA strain of CDV wascloned into pIREShyg Vector, and then the constructed Eukaryotic Expression Vectornamed as pIRES-N was transfected into CHO-K1 cells with liposome. Positive cellswere selected with hygromycine. The N gene protein expressed in the CHO cells wasidentified by IFA. RT-PCR also showed that N gene was expressed in CHO cells attranscription stage. The success construction of CHO/CDV-N cell strain providedevidence for developing CDV serum detection methods and gene vaccine.4. Prokaryotic Expression and Identification of Nucleocapsid Gene ofCanine Distemper Virus CDV-FOX-TA strain from FoxThe high conservative sequence of Canine Distemper Virus (CDV) N gene wasamplified with RT-PCR, cloned into pMD18-T vector, and then constructed intoupstream site of His Tag coding sequence in prokaryotic expression vector pET24b.The N fusion protein was highly expressed when the recombinant plasmid was transformed E coli Rosetta 2(DE3) strain and induced with IPTG. SDS-PAGE andWestern blot showed the 15kD expressed products reacted with standard positiveserum against CDV. An indirect ELISA demonstrated recombinant expressed proteinhad good antigencity and can distinguish effectively anti-CDV positive serum andnegative serum. The results showed expressed CDV N protein had similarity ofantigencity with natural N protein, and can be used as diagnosis antigen. It providedevidence for establishment of an indirect ELISA method for CDV detection.5 Establishment and Application of Recombinant N Protein Based ELISAfor Detection of Antibody against CDVThe recombinant plasmid pET-N was transformed into E. coli Rosetta 2 (DE3)host cell and the expression products-recombinant nucleocapsid protein of CDV wasobtained under the optimized condition of host cell cultivation and IPTG induction.Subsequently, the expression product was purified by the means of his-bind resinprotein purification procedure. Following SDS-PAGE and Western-blot were used todetect the purification effect and the specificity of purified recombinant nucleocapsidprotein of CDV. On this basis, the purified N protein was used to be coated on thewell of 96-well plate, each following step was optimized, such as coatingconcentration of recombinant nucleocapsid protein, sample diluent, chromogen (TMB)and stop solution and its concentration. As a result, confirmed the determinantstandard and an indirect ELISA were constructed to detect antibody against CDV.About 270 serum samples were detected by this method and Synbiotics ELISA kit,respectively. The agreement ratio between the two methods is 92.4%6. Establishment and Preliminary Application of a TaqMan-based Real-timeRT-PCR method for Detection of Canine Distemper VirusTo establish a TaqMan-based real-time RT-PCR method for detection of caninedistemper virus, the pair of palmers and the TaqMan-based probe was designedaccording to the M protein genes of canine distemper virus strains. The reactiveconditions were optimized to improve the sensitivity and specificity of the method.The specificity and sensitivity of the method were high. Canine distemper virus in thethirty-eight samples had been tested by the TaqMan-based real-time RT-PCR, thecommon RT-PCR and electron microscopy. As a result, the sensitivity of theTaqMan-based real-time RT-PCR was higher than the common RT-PCR and electronmicroscopy. It implied that the method may be used in clinical diagnosis, epizooticstudy and laboratory research.