Isolation And Identification Of 2009 H1N1 Influenza Virus And Cloning And Expression Of Its Neuraminidase Gene

ObjectiveThe outbreak of Influenza A H1Nl started in Mexico on March18, 2009. Influenza A H1Nl virus was found to transmit and spread among humans, resulting in outbreaks internationally. According to the report of World Health Organization, the virus caused more than 16,713 deaths worldwide until March 7, 2009. The influenza A H1Nl virus contains a genome composing eight segments of single-stranded, which encodes 10 proteins associated with viral structure and function. The Neuraminidase (NA) is the main Protein of viral surface spikes and a virulence-associated glycoprotein. The antibody against NA can inhibit viral replication and release from the infected cells. The NA active site in both influenza A and B viruses is highly conserved, and its inhibitors develop rapidly for anti-influenza therapy at present.2009 H1N1 influenza virus was isolated from a fecal sample. The whole genome sequences were analyzed. The prokaryotic expression vector (PET-102/D-TOPO-NA) was constructed for the expression of NA protein by genetic engineering technology. The study provided a basis for the further study of NA gene and protein of the 2009 H1N1 influenza virus.Methods The virus was isolated from a fecal sample of the patient with 2009 H1N1 influenza by MDCK cell culture. The specific primers were designed according to the reported nucleotide sequences of 2009 H1N1 influenza virus for generation of the cDNA using RT-PCR. The PCR products were inserted into PMD-19T vector as transformed into DH5αE.coli by heat shock. The recombinant plasmid DNAs were extracted and sequenced. The nucleotide sequences were then submitted to GenBank.For amplification of NA ORF sequence, the upstream primer was designed by adding CACC on its initial sequence for effective connection of the expression vector of PET-102/D-TOPO, while the downstream primer was designed by deleting stop codon sequence for containing 6×HIS tag protein in expressed NA protein. The amplified sequence of the NA gene was cloned into the prokaryotic expression vector PET-102/D-TOPO. The vector was transformed into TOP10 host strain and identified by DNA sequencing. The recombinant plasmid PET-102/D-TOPO-NA was transformed into host strain BL21 (DE3). IPTG induced expression of the NA protein. The expressed products were electrophoresed in SDS-PAGE gel and it's antigenicity was identified by Western Blotting.ResultsPartⅠ1. 2009 H1N1 influenza virus was successfully isolated from the fecal sample using MDCK cell culture method. Slightly CPE was observed within 18～24 hrs after viral inoculation. 70%～100% CPE was observed after 72hrs of the inoculation. The isolate was identified as 2009 A H1N1 virus by RT-PCR and nucleotide sequencing,2. The nucleotide sequences of viral gene were submitted to the GenBank, named as A/Guangdong/SB1/2009(H1N1). The accession numbers are CY064788～CY064795. The homology of the nucleotide sequences of our isolate is more than 98% when compared with the reported nucleotide sequences of 2009 H1N1 influenza virus. HA cleavage sites contained IPSIQSR↓GL amino acid motif, which is a indication of mild pathogenic. Seven mutations were found in the amino acid sequences when compared with that of the fist reported 2009 H1N1 isolate. However, no antigen drift or shift was found in the nucleotide sequences. Three mutations were found on M2 gene indicating the isolate is resistant to M2 protein inhibitors. No"ESEV"sequence was found in NS1, which could bind to the PDZ structure. PB1-F2 protein was found fractured in 11, 57 and 87 amino acids respectively, also indicating that the isolate is a mild pathogenic virus. The amino acid in position 627of PB2 protein is K(Lys), indicating that the isolate is not easy to replicate in mammals.PartⅡ1. RT-PCR product around 1400bp was consistent with the expected size of NA gene.2. The expression vector PET-102/D-TOPO-NA was constructed and confirmed by DNA sequencing.3. NA protein was successfully expressed in E.coli BL21 (DE3). The result of SDS-PAGE showed NA protein was about 64KDa, consisting with the expected size of the protein molecule.4. Antigen activity of the expressed NA protein was confirmed by Western Blotting.ConclusionPart IThe variation of antigen and pathopoiesis in the first 2009 A H1N1 virus which isolated from feces isn't significantly.PartⅡThe isolate is susceptible to NA inhibitors. The optimal IPTG concentration to induce prokaryotic expression of NA is 0.1Mm. The protein needs to be purificated before being further used.