Construction Of The Eukaryotic Expression Vector For NP Gene Of Influenza A Virus And Its Immune Effects

ObjectiveInfluenza is the acute respiratory infection which is caused by influenzavirus A, B and C types respectively. Among the viruses, influenza A virus(IAV) is always appear by means of popularity, especially pandemic influenza.In nature influenza A virus could not only take place antigenic drift, but alsohappen antigenic shift which could be attributed to gene reassortment. Theimmunity against influenza is constantly in force, or less and even invalid toprevent IAV invasion because the antigen variate easily. It is essential todevelop a more broadly protective vaccine for preventing influenza.The genome of influenza A virus is single-stranded RNA, which iscomposed of eight segments. The nucleoprotein (NP) is the product coded bythe fifth segment. NP participates in multiple stages of virus replication cycle,affects multiple functions of virus, such as transcription, packaging andtrafficking across the nuclear membrane. In other words, NP also is the majorepitope for cytotoxic T lymphocytes (CTL) response. CTL could recognize NP antigenic peptides combined with major histocompatibility complexⅠ(MHC-Ⅰ)molecules on the cells surface of infected by IAV, that benefits to clear thevirus. Thus it could cause cross-protection between different strains and eventypes of IAV.In this study, we wanted to construct an eukaryotic expression vector,pcDNA3.1 (+)/NP, which could express NP in eukaryotic cells. We alsowanted to explore the immune effects, especially the cross-protection againstIAV challenge when the mice were vaccinated with pcDNA3.1 (+)/NP DNA,and observe the union protective effects aider the mice were immunized withpcDNA3.1 (+)/NP and pcDNA3.1 (+)/HA DNA vaccines.Methods1. The construction of pcDNA3.1(+)/NP: IAV RNA was extracted frominfluenza virus which had been purified from allantoic fluid of chickenembryo inoculated with an influenza virus strain, A/PR/8/34 (H1N1). NP genewas amplified by RT-PCR and inserted into the eukaryotic expression vectorpcDNA3.1(+) for constructing the recombinant plasmid pcDNA3.1(+)/NP.The pcDNA3.1 (+)/NP was identified by endonuclease digestion, PCR for NPgene and sequencing analyses.2. The expression of pcDNA3.1(+)/NP in eukaryotic cells: ThepcDNA3.1 (+)/NP plasmid DNA was transfected into HEK293 cells by usingLipofectamine^TM 2000 Transfection Reagent at first. Immunofluorescenceassay was used to detect the transient expressing of NP gene. Then the DNA ofpcDNA3.1(+)/NP was injected into the experimental mice by intramuscular.The muscular tissues were taken out and prepared histological slides atdifferent time points. The immunohistochemistry was performed to observethe expression of NP genes in muscle cells.3. The study on the immune effects of pcDNA3.1(+)/NP as DNAvaccines: The mice were immunized with the pcDNA3.1(+)/NP plasmid DNA twice through intramuscular injection of hind-limbs with two weeks interval.After that, the mice were challenged with 10×LD50 IAVs, including H1N1and H3N2. The observing indicators included lymphocyte proliferation assay,protection rates, lung index and lung index inhibitory rates. The immunizationresults of pcDNA3.1(+)/NP plasmid DNA separately and together withpcDNA3.1 (+)/HA plasmid DNA also were studied.Results1. The whole sequence of NP gene, about 1500 bp, was gotten throughRT-PCR from IAV A/PR/8/34 (H1N1). The fragment of NP gene was insertedinto peDNA3.1(+) by ligation, and pcDNA3.1(+)/NP, the eukaryoticexpression vector of NP gene, was confirmed by endonuclease digestion, PCR,and sequencing identification.2. Fluorescence microscopy revealed strong expression of NP genes inHEK293 cells transfected with pcDNA3.1(+)/NP plasmid DNA transiently.NP protein was also detected in the muscle tissues post-intramuscular injectionwith pcDNA3.1 (+)/NP at different intervals.3. Lymphocyte proliferation assay showed that the antigen specific forIAV could lead an active proliferative response after inoculation withpcDNA3.1(+)/NP DNA in experimental animals. The protection rate was 50％after the mice were immunized with pcDNA3.1 (+)/NP plasmid DNA andchallenged with A/PR/8/34, and it was 37.5％after challenged withA/ShenZhen/283/02. Lung index and lung index inhibitory rate withpcDNA3.1(+)/NP and pcDNA3.1(+)/HA were also better than that ofpcDNA3.1 (+)/NP alone.Conclusions1. The eukaryotic expressing vectors containing NP gene of IAV wasconstructed successfully, and it was named as pcDNA3.1 (+)/NP. 2. The recombinant plasmids pcDNA3.1 (+)/NP could express NP proteinin mammalian cell, HEK 293 cell line. It was observed that NP gene could beexpressed in the muscle tissues which were the locations for the plasmid DNAinoculation.3. The result of lymphocyte proliferation assay showed the recombinantplasmids pcDNA3.1 (+)/NP could stimulate the cellular immune responseagainst IAV antigen. The cross-protections against influenza A virus wereproved in the mice immunized with pcDNA3.1 (+)/NP and pcDNA3.1 (+)/NPwith pcDNA3.1 (+)/HA.