Construction Of Eukaryotic Expressing Plasmids For NP Of Influenza A Virus And Their Expression

The influenza A viruses are the causative agents of yearly epidemics of respiratory disease and occasionally more severe pandemics。Influenza A virus is a negative-sense RNA virus. Its genome comprises eight segments of RNA encoding eleven proteins, including haemagglutinin (HA), neuraminidase(NA), matrix protein M1, ion channel M2,non-structural proteins NS1 and NS2, nucleoprotein(NP), pro-apoptotic protein PB1-F2 and RNA-dependent RNA polymerase PA, PB1 and PB2. Influenza virus nucleoprotein (NP), serologically characterized as a type-specific antigen that distinguishes type A, B, and C viruses,constitutes a major internal component of the virion。The influenza A virus nucleoprotein (NP) is a 56-kDa basic RNA-binding protein encoded by segment 5 that plays an essential structural role, encapsidating the segmented viral genome into ribonucleoproteins (RNPs). RNPs are organized in a unique pattern in the virus and for transcription and replication of the viral genome. NP also is one of the major antigens targeted by cytotoxic T lymphocytes (CTLs) which are able to recognize conserve depitopes.Since the viral NP is a multifunctional protein, which plays a central role in influenza virus replication, so, nucleoprotein gene of influenza virus A can be used as prevention and treatment of an effective target.Objective :This experiment by constructing influenza A virus(A/PR/8/34(H1N1)) NP eukaryotic expressing plasmids. Then transfected it into Hela cells and using Flu A Rapid Test Kit study their expression in Hela cells. So the study provides a basis for further study of protein function and the role of the relevant physical and chemical impact on the protein to lay the foundation for influenza A virus.Methods:Based on the published influenza A virus A / PR / 8 / 34 (H1N1) (GenBank / NCB, GI: 8486129) strain of full-length sequence of the nucleoprotein which published in NCBI database to designed two specially primers of the NP by Primer Primier5 software. The NP gene was cloned by PCR from cDNA of influenza A virus and then inserted into the eukaryotic expression vector pcDNA3.1(+).Then identification with restriction enzyme digestion ,PCR assay and sequencing. After identification transfected the recombinant plasmid into Hela cells with lipofectamine 2000 induction. The transient expression of target protein was observed by Flu A Rapid Test Kit。Results :By PCR cloning of the influenza virus A / PR / 8 / 34 (H1N1) NP gene, by digestion connected to construct a new recombinant plasmid pcDNA3. 1 (+) / NP, identification with restriction enzyme digestion ,PCR assay and sequencing show that the successful construction of the pcDNA3. 1 (+) / NP. The NP gene fragment at a length of 1432bp was amplified ,which was consistent with that expected. The sequence of amplified NP gene was identical to that reported in GenBank. Flu A Rapid Test Kit proved the expression of Recombinant plasmid's NP expression at 24h, 48h, 72h both inside and outside cells.Conclusion :The experiment is a success in the construction of eukaryotic expressing plasmids for NP gene, thus providing a basis for further probing into the mechanism of virus infection and physical and chemical factors which can affect the gene.