| Newcastle disease (ND) is a paramyxovirus of Newcastle disease virus (NDV) which caused a fatal infectious disease in poultry, and it is one of A-class animal diseases in China. Newcastle disease virus F protein is one of the major structural proteins. The function of F protein is to make the virus envelope and host cell plasma membrane fusion. The most important function of F protein is involved in virus penetration, cell fusion and hemolysis. F protein is antigen protein which is also able to induce antibody, and plays an important role in immune.F gene can express F protein. So, in the way of recombination technology, the development of new vaccines by using F gene as the target gene becomes the hotspot of attention and studying extensively.In this study, gene sequences which was saved in plasmid pMD-NDV-F in our laboratory as reference sequence, were used designed a pairs of specific primers which contained HA-tag sequences for PCR by using DNAstar analysis software. After amplifying HA-F gene, the HA-F gene was cloned into pMD18-T vector and analysised by restriction enzyme. Then the HA-F gene was analysised and identificated by using nucleotide sequence technology in order to prove to be a Newcastle disease virus F gene containing HA-tag correctly. In the end, the recombinant plasmid pMD-NDV-HA-F was named pTYL-HA-F.By using restriction enzyme digestion and T4-ligase technology, pTYL-HA-F contained the HA-F gene was constructed into eukaryotic expression plasmid pLM, pLM-ITRs, pLM (-), pSD, pSD-ITRs, pSD (-) which were saved in our lab respectively and the recombinant transfer plasmids were named pYL-LM, pYL-LM-ITRs, pYL-LM (-), pYL-SD, pYL-SD-ITRs, pYL-SD (-). Then the recombinant transfer vectors were transformed into E.coli DH10bac competent cells and the recombinant Bacmid were named bYL-LM, bYL-LM-ITRs, bYL-LM (-), bYL-SD, bYL-SD-ITRs, bYL-SD (-). Then the extracted recombinant bacmid DNA transfected Sf9 insect cells in order to acquire recombinant baculovirus as P1 virus stock and the P1 virus stock was named vYL-LM, vYL-LM-ITRs, vYL-LM (-), vYL-SD, vYL-SD-ITRs, vYL-SD(-) respectively. After P1 virus stock, P2 and P3 virus stocks were amplified in order to get high titer virus and the titer of stock was determined by a plaque assay determination experiments. Finally the titer of stock must be 108 pfu/mL at least.The next, high titer virus stock was used to infect CHO-K1 in the condition of MOI= 40 and adding 10mmol/ml butyrate and protein could be collected after 72h culture. Then F protein antigenicity was identified by using SDS-PAGE and Western blotting analysis, and was compared by using software gel-pro analyzer 4.5. In the end, best recombinant transfer plasmid can be picked out. It is an impotant foundation of preparating Newcastle disease vaccine.Inclusion, first of all, this study can demonstrate that the six recombinant plasmids all can express F antigen by using western blotting. Secondly, pYL-SD, pYL-SD-ITRs, pYL-SD (-) with the same CBA promoter can express stronger F protein than pYL-LM, pYL-LM-ITRs, pYL-LM (-) with the same promoter CMV. Thirdly, pYL-SD, pYL-LM can express F protein stronger than control plasmid pYL-SD (-), pYL-LM (-) with comparing of two groups pYL-SD and pYL-SD (-), pYL-LM and pYL-LM (-). The inclusion is that adding the regulatory element WPRE can increase the efficiency of F protein. The fourth, about the role of ITRs components, the expression of pYL-LM and pYL-LM-ITRs, pYL-SD and pYL-SD-ITRs can be used to demonstrate. Howerer, according to the literature reported, the expression can be extended up to 90 days with the influence of ITRs components. So chicken immune experiments should be used to prove that。With the limination of condition, this study was not involved in this fact.
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