| The SPF chicken whole blood, peripheral blood mononuclear cells (PBMCs) from low-speed centrifugation of heparinized blood, PBMCs by Ficoll gradient centrifugation procedure, splenic lymphocytes by low-speed centrifugation procedure and splenic lymphocytes by Ficoll gradient centrifugation procedure were prepared and stimulated by mitogens and CpG oligodeoxynucleotides (ODN) 19and 21, respectively. The stimulated lymphocytes were cultured at different incubation temperatures and different cell concentrations. The transformation effect of the stimulated lymphocytes was further measured by using 3H-TdR incorporation assay to determine and compare the stimulation index (SI) of the stimulated lymphocytes from different procedures and conditions. And the chicken lymphocyte transformation assay based on 96-well flat-bottomed plastic microplates and suitable for large scale screening of CpG ODNs with strong immnunostimulatory activity was established. Then the library of 38 CpG ODNs designed and synthesized by ourselves were screened by using the above established assay. It was demonstrated that CpG ODNs 19, 21 and 23CpG ODNs really have stimulation activity on chicken lymphocytes. Inactivated vaccine for chicken Newcastle disease combined with the above mentioned CpG ODNs 19, 21 and 23 employed to inoculate 7-days health chickens. Serum was isolated from chicken wing vein at 7D, 17D, 27D and 37D after inoculation respectively and to be valued for titer with HI test. Meanwhile inoculated chicken at 38D was challenged with Beijing strain of Newcastle disease virus (NDV) . It was showed that protection rate of groups immunized by inactivated vaccine combined with CpG ODNs 19, 21and 23, alone inactivated vaccine, and inactivated vaccine with oil adjuvant was 100%, 80%, and 80%, respectively while challenged by NDV virulent Beijing strain.Meantime, 38 CpG ODNs were designed and synthesized with considerations of ligation bases between CpG motifs, number of CpG motif, base at the end of CpG ODN, decoration of phosphodiester backbones to obtain CpG ODNs with the strongest immunostimulatory activityon porcine peripheral blood mononuclear cells. The proliferation of porcine PBMC with the addition of CpG ODNs was determined by 3H incorporation assay and the level of IFN-r from the supernatant of PBMC culture was measured by swIFN-r kit. It was showed that CpG ODNs which have effective immunostimulatory function relate to characterization of their structure, but the amount of cytokines from the supernatant of PBMC culture has not direct proportion of stimulatory index(SI). The CpG ODNs which have strongest immnostimulatory activity have been screened.In order to save the cost of CpG DNA, we tried to construct the CpG DNA recombinant by molecular methods. 6 CpG ODNs were designed and synthesized based on the previously screened specific CpG ODN sequences and 3 fragments after annealing were inserted into T vector respectively by the T-A and directional cloning. Then 3 recombinants were constructed which contain different numbers of CpG motif. PCR primers were designed with the help of computer and synthesized and the conditions and components of PCR were defined by experiment to prepare the great amount of DNA fragments full of CpG ODN motif. The DNA fragments full of CpG motifs were amplified by using PCR and recombinant plasmid as template. The proliferation of porcine PBMC with the addition of amplified DNA fragments full of CpG motifs was determined and SI was determined by 3H incorporation assay of porcine PBMCs to analyze the immunostimulatory effect of DNA fragments on porcine PBMCs. It was showed that DNA fragments with SI>4.5 can really stimulate porcine PBMCs to proliferate more strongly than that of recombinant plasmids with SI<2 as control.
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