Repeated Expression Of Fusion Glycoprotein Cleavage-Activation Site Region Of NDV Virulent Strain And The Study On The Application Of Its Expressed Product

In this paper, the gene of fusion glycoprotein cleavage-activation site region of NDV virulent strain was repeated expressed in E.coli prokaryotic expression system. The expressed product was used to detect the antisera of virulent and avirulent strain of NDV by indirect ELISA. In addition, the antipeptide antibody of the fusion glycoprotein was used to detect the ivrulent and avirulent strain of NDV by indirect ELISA. This study indcludes:1. In this study, the synthesized oligonucleotide (coding two same 14aa of fusion glycoprotein cleavage-activation site region of NDV virulent strain and two hydrophobic amino acids in between) was cloned into fusion-expression vector pET-32a(+) to construct the recombinant vector pET-F. The pET-F was then transformed into E.coli BL21 and the objective fusion protein was highly expressed by IPTG inducing. The expressed product of positive clone could be proved to be about 23.6kD by SDS-PAGE. The result of sequencing corresponded with what designed.2. Recombinant fusion expression vector was transformed into E.coli strain BL21. The positive clone was selected to culture. The optimized expression conditions of the fusion protein was:30℃, 0.1 mM IPTG, incubating 2~3hrs after induction. For the molecular weight of the fusion protein is small and the temperature of culture is low, the expressed protein is soluble in E.coli. The result of SDS-PAGE showed that the protein was in supemate. The protein was purified by metal chelated chromatography. The purified protein will be utilized for further study. In addition, the expressed product which was reclaimed by SDS-PAGE was used to inoculate tame rabbit to raise antibodies. The result of Western-blot showed that the liter of the antibodies was rather high.3. In the study, the purified protein was used to detect the anti-serum of virulent and avirulent strain of NDV by indirect ELISA. The aim is to detect their reactivity. In the same time, the results of the substrate of OPD and TMB were contrasted. The result showed that the reaction ladder of antisera of virulent and avirulent strain was obvious, and the OD data of antisera of avirulent strain were higher than the antisera of virulent strain; The OD datausing OPD as substrate were higher than using TMB as substrate. In addition, the antipeptide antibody of FCASR of NDV virulent strain was used to detect the virulent and avirulent strain of NDV by indirect ELISA. The reaction results (OD data) were: IV strain >F48E10 strain > F48E10 strain (treated with trypsin)> IV strain (treated with trypsin).