| In the recently poultry industry, ND as a kind of highly contagious diseases caused by NDV, still widely exist in all over the world. Vaccination is a main prevention and control measures. With the development of modern molecular biology, genetic engineering vaccines emerge. The live carrier vaccine of restructuring NDV for its stability, high drop growth characteristics, wide range of immune, immunity means diversity etc, becomes the most development potential of the vaccine. This study by the V4heat resistant strains breeding, adapt to BHK TS09-21cells-C strains as the object, using reverse genetics technology, save a recombinant NDV. To detect its biological characteristics, compared with parental strain, a recombinant strain retains the parental low virulent, high growth characteristics. The first NDV TS09-C heat resistant strains reverse genetic operating system is established. On the basis of established system, we save a recombinant heat-resistant NDV that can express stably green fluorescent protein. It is a solid foundation for other the immunogenicity of virus gene insertion, multivalent vaccine development. At the same time it creates platforms for the researches on the mechanism of the heat.1. NDV TS09-C strains of reverse genetics for the establishment of the operating systemBefore this experiment, we have successfully built full length cDNA of TS09-C strain, called pBR-TS09-C. On the basis of it, we use the full length plasmid as the template and make the amplification of NP, P and L fragments were cloned in pVAX-NP, pVAX-P and pVAX-L.At last, successfully build three auxiliary plasmids. Transfection cells incubated expressing T7RNA polymerase pox virus, then will be transfected with full length plasmid pBR-TS09-C and auxiliary plasmids pVAX-L, pVAX-NP, pVAX-P, using calcium phosphate transfection method. Finally, harvest culture supernatant and vaccination BHK-21cells or age of9days SPF chicken embryo allantoic cavity.In SPF chicken embryo to develop3-5days, recombinant virus allantoic fluid is received. Taking Real-time PCR, HA, IF A, electron microscope, sds-page tests identificated recombinant virus, the results show that the recombinant virus is rescued successfully, and reverse genetic operating system of NDV heat resistant strains is set up.2. The research of biological characteristics of the recombinant NDVFirst restructuring NDV is detected for virulent about the following three aspects:the ICPI, IVPI, MDT. Get results for ICPI is0, IVPI is0, MDT value more than150h. Results can be concluded from this, recombinant strains retained the characteristics of the parent weak poison and low virulent strains. Second, proliferation condition and titer in cells and chicken embryos, HA about the recombinant NDV were determined, drops of the cell proliferation degree is107TCID5o/mL, drops of chicken embryos proliferation degree is107.5EID50/mL, similar to the parent strains. Through the growth curve determination of restructuring NDV, whether at the level of cells, or chicken embryo levels on the growth curve of the overall trend is same as the parent strain. It is retained the parent strains of high degree of growth characteristics.Heat resistance test of restructuring NDV is done, comparing with parental strains, Lasota strains. According to the results, Lasota strains loss of HA after56℃water bath in9min, the parent strains can heat up to75min, recombinant strain56℃water bath function150min remain HA. On this basis, the recombinant strains and parental strains of heat-resisting infectious for testing, results showed that the recombinant strains heat still infectious after120min, than the parent strains heat60min longer. To sum up, recombinant strains not only keep the characteristics of the parent strains heat resistant, but also improve the heat resistant. This feature for as vaccine carrier is a very favorable conditions.The vaccine can make the bondage of break of cold chain, and used in practice widely.3. Construction of expressing green fluorescent protein of recombinant TS09-C vaccine strainOn the basis of NDV TS09-C heat resistant strains reverse genetic operating system, considering the experiences of domestic and foreign in the modification of NDV, we select NP gene upstream region, P and M gene spacer as sides for inserting foreign genes, and try to take advantage of the first heat resistant strains of NDV reverse genetic platform to express exogenous genes; And choose convenient observation and detection of GFP gene as to insert foreign genes.The plasmids pBR-GFP-TS09-C/NP, pBR-GFP-TS09-C/M has been built successfully and without any mutated base. The two plasmids respectively with the three auxiliary plasmids are con-transfection. Harvest virus after infected, were inoculated BHK-21and SPF chicken embryo cell proliferation. By Real-time PCR, HA test, cell fluorescence microscope observation, the results shows that rGFP-TS09-C/M is saved successfully, the GFP inserted in the upstream of the NP scheme is failed. Speculated that failure reasons is that may be the expression of GFP of pBR-GFP-TS09-C/NP has affected the assembly of synthetic recombinant virus particles themselves.Namely, the conclusion is P and M gene spacer is a more suitable site for the insertion of exogenous gene.To sum up, this paper constructed the first NDV TS09-C heat resistant strains reverse genetic operating system, a retained the parent strains were weak poison and low virulent strains, high drop of the growth characteristics restructuring strain. The plant not only keep the characteristics of the parent strains to heat, and heat resistant infectious time increased to120min. Used to establish reverse genetic operation platform, in the interval of P and M gene area insert GFP gene, successful got recombinant Newcastle disease virus to express GFP. Lay a solid foundation for subsequently inserting other immunogenicity of virus gene polyvalent and multivalent vaccine development. At the same time, it also created a platform for the researches on the mechanism of the heat. |
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