Molecular Cloning And Analysis Of The Fusion Glycoprotein Of NDV Sddy-02 Isolate And The Applied Studies On E.coli CpG-DNA In ND Vaccines

In order to find the molecular biological Characteristics of virulent NDV strain and to find the preventive and remedial technology of new vaccine, one NDV strain (Chicken/Shandong/Dongying/2002, NDV Sddy-02) was isolated and identified. Its biological Characteristics were studied and the important fragment of its F gene was cloned and analyzed. On the basis of these studies, a compound proplis inactivated vaccine against ND was developed with this isolate and the C30 strain. At the same time the different doses of E.coli CpG-DNA were added into this compound proplis inactivated vaccine and C30 live vaccine to compare the results caused by E.coli CpG-DNA in these two kinds of vaccines. It is the aim that lay the foundations of producing a more efficient and more safe vaccine. PartⅠ:Isolation and identification of NDV Sddy-02 strain and studies on its biological Characteristics One strain was isolated and identified from a certain chicken house in Shandong province, with its biological characteristics studied. Cultured by SPF chicken embryo, its HA is 27-8, HI is 28, ICPI is 1.87, IVPI is 2.62, MDT is 53.4, the dehaemagglutination time is fast and the haemagglutinin heat-stability is poor. By all these characteristics the isolate was confirmed to be virulent NDV strain.PartⅡ:Molecular Cloning and Analysis of the Fusion Glycoprotein of NDV Sddy-02 Isolate A pair of primers was designed specially and synthetized. The fragment of F gene covering cleavage site region was amplified by RT-PCR, cloned and compared. Basing on published sequences of representative NDV strains, a phylogenetic tree was constructed to analyse their variation relation. Comparisons of F gene (ShD-1.99, Qd-1, Sdbz-s99, Clone30 and F48E9) showed that the homologies of nucletide sequence were97.5%, 86.5%, 98.4%, 84.1% and 86% respectively. Their homologies in deduced amino acid sequence were 99.2%, 92.6%, 100%, 89.3% and 91.7% separately. Because the amino acid sequence of the cleavage site is made up of 112R-R-Q-K-R-F117, which match to that of all virulent NDV strains, the isolate was proved to be an virulent strain. The constructed phylogenetic tree showed that the Sddy-02, ShD-1.99 and Sdbz-s99 belonged to NDV genetype Ⅶ. From the molecular results it is further demonstrated that the ND caused by genetype Ⅶ also prevail in Shandong province.Part Ⅲ:Applied Studies on E.coli CpG-DNA in ND Vaccines The compound proplis inactivated vaccine against ND was developed with NDV Sddy-02 strain and the C30 strain. Meanwhile the E.coli CpG-DNA extracted by CTAB method was used as a new adjuvant with dose of 10μg and 30μg added into this compound proplis inactivated vaccine and C30 live vaccine. Then 14-day-age chickens were inoculated by the two kinds of vaccines including different CpG-DNA doses respectively. At different time HI was performed to find whether the E.coli CpG-DNA has enhanced the immunostimulatory results to these two kinds of vaccines. The results indicated that the dose of 10μg had an enhanced effect to the compound proplis inactivated vaccine, while the dose of 30μg had no more effect to it. Both 10μg dose and 30μg dose were no aidant to C30 live vaccine. The detailed reasons are to be further studied in the future. It is the first study that the E.coli CpG-DNA is able to enhance the immune response in compound proplis inactivated vaccine against ND. This research laid foundations of developing new compound adjuvants in ND vaccines.