Construction And Expression Of ND Virulent Strain F Protein Schizolysis Sites Gene Cascade Connection And Identification Of Low Virulent And Virulent Strain Of NDV

Newcastle disease(ND) is a acute, high contact infection disease , which is caused by Newcastle disease virus (NDV). Its typical symptoms are dyspnea, alo laxata, disorder of neurologic function, bleeding of mucous membrane and ectoptygma, and the sick avian often appear septico. ND spreads all over the world and is a great hazard to the development of poultry industry. But recent years, the epidemic NDV appears new characteristics, such as the appearance of atypical form in many areas and occasionally transoid virulent strain. All of these situation make the prevention and cure of ND more difficult.The study has showed that F protein was essential to the cell infection of NDV. F protein can not only facilitate the confluence of virus peplos and host cell envelope, but also the confluence between the host cells. Thus F protein is important to the virulence of NDV. Through the molecular biology study , the virulence of NDV is determined by the conjunction capability of the disulfide between F1,F2 created by the protease split of F0 protein. The split exposes the F1 hydrophilia N-term, causing the confluence of the virus and the host cells. The schizolysis site of F protein locates in amino acids 112-117 where the sequence and schizolysis capability is key point to the virus toxicity. The sequence of low virulent strain is G-R/K-Q-G-R-L, and velogenic strain is R-R-Q-K/R-R-F.Based on the degeneracy of codons and preference of Bacterium coli coding codons,the nucleotide sequence of F protein schizolysis site is optimized. To the current prevailing NDV , the nucleotide sequence of F protein schizolysis site is GRRQRRF and GRRQKRF.And to the new mutated ND velogenic strain, the sequence is RRRQKRF,GRRKKRF. All these sequences were merged by the linker GGGGS, then 2, 4 copies were reconstructed. The prokaryotic expression plasmids pET-28a(+)/2Fe including 2 copies and pET-28a(+)/4Fe including 4 copies were constructed. The study showed 2Fe protein and 4Fe protein could express efficiently. Through the identification of Western-blotting, both of 2Fe protein and 4Fe protein could react with F48E9(multipartial serum of ND velogenic strain), but not V4(multipartial serum of ND low virulent strain).The multi-clone antibody was collected by the rabbit immunized with purified 2Fe protein and 4Fe protein, respectively. Based on 2Fe and 4Fe multi-clone antibody and optimization of correlated conditions, the ELISA identification method of ND velogenic strain and low virulent strain was set up, and got ideal effect. The immunological identification method of ND velogenic strain and low virulent strain was established. Also, the method settled foundation for ELISA identification kit of ND velogenic strain and low virulent strain.