| H5 highly pathogenic avian influenza virus(H5 HPAIV)is highly infectious and pathogenic in poultry,and must be operated in BSL-3 laboratory,so the experimental environment has always been a bottleneck of research.In this study,nineteen strains of H5 HPAIV pseudovirus were packaged and the H5 HPAIV pseudovirus library was constructed;The recombinant virus with high hemagglutination titer was obtained by using the pseudovirus model,which provides a convenient method for influenza virus research,vaccine preparation,vaccine evaluation and drug screening in the BSL-2 experimental environment.In this study,three transfection methods of PEI,calcium phosphate and liposome transfection were used to package influenza pseudovirus.After comparing the transfection efficiency,calcium phosphate transfection was used to package influenza pseudovirus;the HA or NA gene fragments of four H5 HPAIV strains were inserted into pCMV/R vector to obtain three HA and four NA recombinant plasmids,then the HA,NA plasmids were randomly combined,and with the p CMV/RΔ8.9 Plasmids and p HR-CMV-Luc plasmids constitute a four-plasmid system.The four plasmids were transfected into HEK293T cells by calcium phosphate method to obtain twelve pseudoviruses;hemagglutination assay showed that the hemagglutination titer of pseudoviruses between 100～400 HAU/m L,Western blot showed that there were specific bands at 75 k D,54 k D,50 k D and 25 k D,and luciferase reagent test showed that the relative Luciferase activity(RLA)of pseudoviruses between 50～4.5×10~5.The results showed that the matching of HA and NA of different H5 HPAIVs would affect the hemagglutination titer and RLA of pseudoviruses,;nineteen representative viruses from 0 to 9 clade were selected from the candidate H5 HPAIV vaccine strains recommended by WHO,and construct an evolutionary tree using their HA nucleotide sequence,then the corresponding pseudoviruses were packaged by calcium phosphate transfection method.After detection,it was found that the ratio of the hemagglutination titer of nineteen pseudovirus particles to the content of p24 protein was normal,but the hemagglutination titer and relative luciferase activity(RLA)of some pseudoviruses were low;A/Thailand(KAN-1)/2004(TH)strain with high RLA and A/blackbird/Hunan/1/2004(HN)strain with low RLA were selected from nineteen viruses,and the HA and NA plasmids of the two strains were exchanged to pack chimeric pseudoviruses.It was found that the RLA of TH chimeric pseudovirus was still higher than HN chimeric pseudovirus;Comparing the amino acid sequence of HA of TH strain and HN strain,there are seventeen different amino acids in total,and then taking the HA protein of the two strains of virus as the backbone protein to mutate seventeen amino acids,comparing the function of the mutated pseudovirus,42,44 and 53 were identified as the key sites;In order to determine whether the three sites will increase the hemagglutination titer of the recombinant virus,the HN mutant recombinant virus and HN recombinant virus were saved by reverse genetics.After the expansion of cells and chicken embryos,it was found that the hemagglutination titer of HN mutant recombinant virus was higher than that of HN recombinant virus;Mice were immunized with HN mutant and HN recombinant virus inactivated vaccine to obtain immune serum.Pseudovirus neutralization(PN)assays were used to detect the neutralization titer of immune serum.It was found that there was no difference in the neutralization titer of immune sera between HN recombinant vaccine and HN mutant recombinant inactived vaccine.To sum up,this study constructed a H5 HPAIV pseudovirus library,and used the pseudovirus model to improve the hemagglutination titer of the recombinant virus without changing the immunogenicity of the inactivated vaccine of the recombinant virus,providing a convenient method for the subsequent research and development of H5 HPAIV vaccine and drugs. |
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