Serological Cross Reaction Between Influenza A Virus Subtype Characteristics Research And Optimization Of Reverse Genetics System

Because of annually recurring epidemics and the enduring threat of a pandemic, influenza remains a serious public health problem. Recent outbreaks of highly pathogenic avian influenza A virus (H5N1subtype) infections in poultry and humans (through direct contact with infected birds) have raised concerns that a new influenza pandemic might occur in the near future. For the characteristics of numerous subtypes and frequent variations of influenza virus, it brings great difficulty to influenza in the prevention and control. Thus, the most pressing matter of the moment is to reinforce the research in pathogenesis and transmission mechanism, as well as to develop new type vaccines. Then my research about investigating the rule of interrelation of all subtype of A influenza virus and used the strong SCP I promoter to improve the effect of reverse genetic system.Hemagglutinin and neuraminidase are the two surface glycoproteins of influenza A virus, which are further subtyped based on the antigenicity of HA and NA. Currently,16HA and9NA subtypes have been identified among type A viruses.All sequences of16HA and9NA in NCBI genbank and influenza sequence database were downloaded and aligned, then clustalW method was applied to construct phylogenetic tree and analyzed the homology. Sequence analysis shows that homology of HA gene of16subtypes between36.2%～78.5%in amino acids and NA gene of9subtypes between37.9%～66.6%in amino acids.The sequence of cleavage site of the anino acid deduced from the gene was the characteristic sequence of influenza virus with low pathogenicity except for HA of H5and H7.The codons of16HA and9NA were changed to the codon usage of highly expressed mammal and Sf9cell gene. The opti-HA gene of16subtypes and opti-NA gene of9subtypes were PCR amplified from the templates using specific primers. The PCR products were used to construct opti-HA-bac and opti-NA-bac recombinant baculovirus using Bac-to-Bac baculovirus expressing system. Expression of opti-HA and opti-NA were confirmed by SDS-PAGE, Western blot and protein mass spectra. High level and excrete of protein expression was detected in Sf9cells following baculaovirus infection.The modified HA and NA genes were cloned into shuttle-vector pShuttle-CMV respectively, to generate recombinant plasmid pAd-HA(NA). and then the constructed plasmids containing target gene was transformed into E.coli BJ5183. Transfection of the recombinant AdEasy plasmids into293cells were performed to obtain recombinant adenovirus. The mice were immunized with the recombinant adenoviruses. Their immunogenicity were evaluated by testing antibody of immunized mice.An indirect enzyme-linked immunosorbent assay (ELISA) for detecting the rule of interrelation of all subtype of A influenza virus. The assay used recombinant HA/NA antigen expressed in Sf9cells and polyclonal antibody from immunized mice. The results indicate multiplicity between HA of16subtypes and NA of9subtypes, which are a cue for influenza A virus control direction.Recently, the development of reverse genetics technique breaks a new path for influenza virus in research of function analysis and preparation of vaccines. Reverse genetics techniques to rescue influenza viruses have thus far been based on the use of bidirectional RNA Pol Ⅰ-Pol Ⅱ transcription system, which can not meet to the need of human vaccine production because of low yield. Here we report improved the RNA polymerase Ⅱ (pol Ⅱ) promoter of mRNA synthesis have led to a significant higher-yielding vaccine strain production. These improvements pave the way for the reproducible generation of influenza virus vaccine strain and may be applicable to other reverse genetics systems.