Cloning And Expression Of Influenza A Virus Nucleoprotein

ObjectiveThe genomic nucleic acid of Influenza A virus is a negative sense single-stranded RNA consisted of eight segments. Its genome is assembled with the nucleoprotein and polymerase proteins into viral ribonucleoprotein (vRNP) complexes, which affects the functions of virus such as replication, transcription, packaging and trafficking across the nuclear membrane. The nucleoprotein (NP) is the product coded by the fifth gene segment, and interacts with other macromolecules including RNA, some viral proteins and cellular polypeptides, and involves in whole replication process, e.g. virus invading host cells, and also is the key of the virus causing infection and disease. On the other hand, NP is the major target for Cytotoxic T lymphocytes response. CTLs can recognize NP antigenic peptides combined with major histocompatibility complex (MHC) class I molecules on the surface of infected cells, that benefits to clear the virus from body. Because of highly conservation the NP gene and its products (NP) could cause cross immune response between different strains and even types, i.e. their cellular immune response would not only protect against the same subtypes of influenza virus A strains but also against divergent subtypes. The current influenza subunit vaccines primarily induce humoral immune response against the surface antigens. While neutralizing antibodies are specific to viral strains and the surface antigens are variable, so the defect of current vaccines is significant. The influenza NP might be a potential perfect vaccine candidate due toinducing CTL response which can provide more broad and persistent protection.In view of above this study is to construct a recombinant expression plasmid pGEX-4T-l-NP encoding influenza NP from the virus, SIVA/Swine/Henan/703/ 2001(H3N2), and observe its expression in E. coli BL21, for further research about immune response against NP. Also it is essential to study the virus functions related with NP and subunit vaccine with NP epitope.MethodsTotal RNA was extracted from the virus, A/Swine/Henan/703/2001(H3N2) and the gene fragment encoding NP including a EcoRI site was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from the virus RNA .Then the resulting fragment was cloned into pGEM-T Easy vector and the recombinant plasmid was transformed into JM109.The positive clone was identified by blue-white screening and colony PCR .The sequence was confirmed by DNA sequencing and compared with the influenza NP sequence published in the GenBank. Both recombinant plasmid pGEM-T Easy-NP and prokaryotic plasmid pGEX-4T-l was digested with EcoRI. After purified by gel extraction, the target gene fragment was subcloned into pGEX-4T-l vector correctly. The recombinant plasmid pGEX-4T-l-NP was transformed into E.coli BL21 and the positive clone was identified by colony PCR. The sequence was confirmed by DNA sequencing. The expression of influenza NP was induced with IPTG, and analyzed by 12% SDS-PAGE and Western Blot. Results1. The gene fragment encoding influenza NP was amplified by RT-PCR. The recombinant plasmid pGEM-T Easy-NP and pGEX-4T-l-NP was constructed successfully.2. The results of DNA sequence show that the influenza NP from A/Swine/Henan/703/2001(H3N2) is 1494bp long and encodes 498 amino acid residues. The homology of the NP of A/Swine/Henan/703/2001(H3N2) compared with influenza NP gene published in GenBank in nucleotide acid or amino acidwas 99%. The result of DNA sequence of the influenza virus NP confirmed that NP gene is highly conserved as previously reported.3. After BL21 containing the expression plasmid pGEX-4T-l —NP was induced with IPTG, a 82Kda protein band which should be GST-NP fusion protein as predicted was observed on SDS-PAGE gel.4. The antigenicity of the expressed protein was measured by Western Blot analyses. Result indicated that the fusion protein could be recognized by antiserum against H3N2 influenza viruses.ConclusionsIn this experiment, Cloning and expression of influenza A virus nucleoprotein gene from the A/Swine/Henan/703/2001(H3N2) virus was successful.This study will provide the foundation to analyze the functions of NP related to the replication and infection of virus or immune response to NP. Subunit vaccine containing NP component also can be developed. It might be helpful to understand the pathogenesis of influenza virus and control the disease.