Expression And Purification Of Nonstructural Protein (nsi) Of Influenza A/h1n1 Virus In E. Coli, Application In Differentiating Infected From Vaccinated Mice And Evaluation Of Its Immunological

Influenza viruses belong to the family Orthomyxoviridae. Influenza viruses are spherically shaped enveloped particles with an up to eight-fold segmented, single-stranded RNA genome of negative polarity. Influenza viruses are classified into types A, B or C. Influenza A viruses are further subdivided into sixteen H (H1-H16) and nine N (N1-N9) subtypes. Influenza A virus has caused four great pandemics in the last century. Highly pathogenic avian influenza (HPAI) emerged in Hong Kong in 1997 and has spread in humans ever since 2003. By now HPAI has infected 423 people, 258 of them dead, with mortality rate up to 61.0%. On April 26, 2009, Influenza A/H1N1 viruse outbreak took place in Mexico. Within a few days, 33 countries have officially reported 5728 cases of influenza A(H1N1) infection. WHO expressed serious concern about the situation, as it had the potential to become flu pandemic due to the novelty of the influenza strain and its transmission from human to human.Circulating influenza viruses undergo rapid mutation and easy recombination owing to the low fidelity of the viral RNA polymerase and its multi-fold segmented genome. The migration of wild birds and its connection with domestic birds both accelerate this process. As the reservoir of influenza virus, livestock like swine and dog also play an important role in recombination of the viruses. So, global surveillance system is needed for the control and prevention of influenza pandemic. In fact, influenza is also the first viral diseases that have global monitoring system. Vaccination has been widely practiced to reduce the incidence of clinical disease and in some cases has been used as part of an eradication strategy. Vaccination has many potential benefits, but it also has many potential drawbacks to use. The main negative aspect to vaccination is the effect that it has on serologic surveillance. The killed vaccines that are available today result in measurable antibody titers to all the structural proteins, including the matrix and nucleoproteins. Therefore, vaccinated birds cannot be distinguished from naturally infected birds using routine approach. It is vital important not only to differentiate naturally infected and vaccinated animals(DIVA), but also to identify vaccinated animals that become infected. An alternative DIVA strategy that has recently been described looks at a differential immune response to the influenza nonstructural protein 1 (NS1).Vaccination is the cornerstone of prevention. By now there is a new way for us to design an effective vaccine against influenza infection that is to find the viral conservative protein which also play key role in vial replication and pathogenesis. Animals immunized with the vaccine of these kind may get infected as well, but which can mitigate animal condition, reduce mortality rate. This kind of vaccination is more important in high pathogenic virus infection, like HPAI. Extensive investigation has been made on influenza M2 protein. NS1 protein is also conservative protein and is closed linked with the pathogenesis of influenza virus. But until now, little work has been done about this. It seems there is a long way to go before we know whether it will actually work and what immunoprotective efficiency it has.The main results of this research are as following:1. Expression and purification of influenza virus nonstructural protein (NS1) in E. coli and preparation of its muticlonal antibody.The NS1 gene of influenza virus A/FortMonmouth /1/47 was amplified by RT-PCR and cloned into pET28a to construct a recombinant expression plasmid. The constructed vector which had been identified by PCR, enzyme digestion and nucleotide sequences analysis was transformed into BL21. After inducing with IPTG, the recombinant protein was purified with Ni~+ affinity chromatography. SDS-PAGE and Western blot analysis showed that the recombinant protein was about 26 kDa. We got high-titer serum after immunizing New Zealand rabbits with the protein. The ELISA titer of the antibody was over 1×10~5. Western blot assay show that the antibody was bound to NS1 protein with high specificity.2. Application of the recombinant protein NS1 in differentiating infected from vaccinated mice with NS1-ELISA assay. The purified recombinant protein NS1 was used as an antigen to detect specific antibodies in the sera from virus infected and vaccinated mice. Between 15 to 30 days after viral infection, this method can differentiate infected mice from vaccinated mice for the former has a much higher NS1 antibody titer. The sensitivity and specificity of this assay were 94.4% and 88.9%, respectively.3. Evaluation of immunoprotective efficiency of the NS1 protein Mice were immunized with purified NS1 protein twice, the positive rate of serum was 84.6% by day 30. The survival rate of NS1 immunized mice was only 37.5% after15 days postinfection, which is much lower than the mice immunized with inactivated whole virus vaccine (81.3%). The median survival time of NS1 immunized mice was 5 day which is the same as the mice of infection control group. Pulmonary index and pathologic changes in lung tissues of NS1 immunized mice have little improvement compared with the mice of infection control. All the data strongly suggest that the NS1 protein is not a good candidate antigen for protection mice from influenza infection.In conclusion, we obtained the recombinant NS1 protein and it could be expressed with high performance in E.coli. We utilized the NS1 protein in differentiating infected mice from vaccinated mice which may be helpful in the global influenza surveillance monitor system. We also found that the NS1 protein can not protect mice from lethal infection by FM1. Eventually we generated a multiclonal antibody recognizing NS1 which may be used to study the biological activity of NS1.