Preparation Of Monoclonal Antibody Against Newcastle Disease Virus And Preliminary Development Of Animmunosensor For Determing NDV

Newcastle disease(ND),known as Asian chicken blast,which is one of the most important poultry diseases in the world,is listed as a class of infectious disease by OIE.It will be caused conjunctivitis for human and be significance for controlling ND.By now,there are lots of ways to detect it.With the variation of Newcastle disease,these methods has been not accord with the demand,which makes people begin to turn to the new detection technology.The fiber grating biosensing technology is the focus of optical biochemical trace detection now,we have successfully apply the fiber Biagg grating sensor to detecte PCV2 and NT pro-BNP.In this paper,with the fundamental of great angle tilted fiber bragg grating,we choiced SPA to make NDV monoclonal antibody immobilizing on the surface of Ex-TFG,established a biochemical sensor based on the large angle tilt fiber grating,which was used detecting Newcastle disease virus,this will provide a new method for clinical detection of Newcastle disease.The main contents of this thesis include the following:1.NDV-F gene multiplying extracted RNA,amplified NDV-F gene though RT-PCR and cloned into pET32a(+)vector by gene cloning to select recombinant plasmid,then transformed into E.coli BL21(DE3)and purified of NDV-F by His-tag nickel column,inducing and purifying recombinant protein of F gene.2.Monoclonal antibody was prepared by immunizing mice with antigen of Newcastle disease virus and F gene recombinant protein though the PEG1500 cell fusion and the biological characteristics were identification;3.With the surface modifications of hydroxylating,silanizing,preparating of SPA biosensitive membrane and fixed antibody,we initiallily constructed the biochemical immunosensor of Ex-TFG to detect NDV,and carried on the evaluation.Result:(1)The SDS-PAGE showed a band in 78 kDa after inducibled expression of reconmbinant,accord with expectation,and was main inclusion bodies.The optimum expression condition was tested: 16?,0.1mM IPTG,20 h,Western-blot confirmed the protein stripe was about 78 kDa.(2)The ELISA showed the virus group received 12 hybridoma cell lines secreting Anti-NDV monoclonal antibody,and 3 hybridoma cells of F gene recombinant protein group.SDS-PAGE indicated obtaining high purity monoclonal antibody,and the titer was 1:64000,IgG subtype type.Western-Blot showed that the virus immune preparation of monoclonal antibodies against NDV F protein;and recombinant protein group can react with the whole viruses,this indicated that the prepared monoclonal antibody can recognize the F protein,showed good reavtivity.(3)The change of resonant wavelength indicated that SPA and antibody can stable combine on the surface of Ex-TFG.The detection of Newcastle disease virus concentration was 0.05ng/mL-24ng/mL,and0-1ng/mL range fitting curve turned out that 342 sensitivity,the linearity was R2=0.982,the highest detection limit was 24ng/m L,the lowest detection limit was 0.2ng/m L.The repeated trials showed that that the sensor was stabilize favourable;The specific experiment sensor can specific identification Newcastle disease virus,but no response to AIV.Conclusion:This paper established a great angle tilt fiber grating biochemical sensor for the detection of Newcastle disease virus,related studies have shown that newcastle disease virus immune tilted fiber grating sensor has the sensitive,reproducible and specific,real-time dynamic monitoring,and provides a new method for the clinical detection of Newcastle disease.