Influenza virus is globally pathogenic important. It possesses a lipid-bounded segmented genome which encodes at least one biochemically-distinct protein. Its subtype A can be classified according to antigenic differences. NS1 protein is defined as nonstructural protein in the virus. It is known as a multifunctional virulence factor. It only can be detected in the infected cell. In this study, the NS1A gene was successfully cloned into the BamH1/SacI cleaved-pET-32c(+) vector and subsequently electro-transformed to the E.coli BL21(DE3) expressing host. The recombinant NS1A gene has shown identical counterpart with the synthetic NS1A gene and the 3D protein structure was predicted through bioinformatics method. Better protein expression was found at 37Â°C under 5mM lactose induction in E.coli. Protein expressivity in soluble and insoluble fraction was not greatly detected in E.coli. 20% ammonium sulfate saturation was sufficient to concentrate and partially purify the target NS1A protein. The ammonium sulfate precipitated NS1A recombinant protein has characterized a significant immno-response to the polyclonal antibody in the Western blot. A 37kDa-weighed NS1 protein was detected to react with the H1N1 NS polyclonal antibody.
Key Words: Influenza A virus; H1N1 subtype; NS1 protein; Cloning; Overexpression; Purification
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Influenza virus is highly pathogenic and it is usually considered as the causative agent of zoonotic respiratory disease. Its transmission is observed upon either interspecies (Webster et al., 1992) or intra-species. Reassortment may occur in this virus (reviewed by Hampson and Mackenzie, 2006; Gibbs et al., 2009). Its infection is usually associated with cellular alteration, apoptosis and host mortality (Schultz-Cherry et al., 2001).
The influenza viruses, which can be classified into types A, B and C, are included into the family of Orthomyxoviridae (Pringle, 1996; Bouvier and Palese, 2008). The influenza A has shown identical pathogenic potential with influenza B and it extensively assesses pandemic or epidemic threat (reviewed by Pushko, 2009). Recently, HIN1 strain has established its cyclic alternation and reassortment in human. Presumably, the influenza infection is caused by direct and intimate interaction between human and swine. Evidence has shown that the H1N1 subtype remains circulating in the world since its first detected outbreak in 1918. Subsequently, its resurgence was documented in 1950 and 1977 (reviewed by Cox, 1998; Nicholson et al., 2003).
Influenza A virions can appear as spherical shape with 80 to 120nm in diameter (Donatelli et al., 2003; reviewed by Pushko, 2009) or 300nm in length for filamentous form (Suri, 2007). Within the lipid-bound virion, there are eight negative sense single stranded ribonucleic acids (RNA) which are distinguished in length to encode eleven proteins. Influenza A virus nonstructural protein, NS1A protein, which is encoded by segment 8, consists of 230-237 amino acids. The NS1A protein is only can be detected during infection. It is multifunctional, involving significantly in the protein-RNA (Qiu and Krug, 1994) and protein-protein interaction (Xia, Monzingo et al., 2009). Its two functional domains, which are dsRNA-binding domain (RBD) and effector domain (ED), are essential for intracellular and extracellular interaction. This protein is unique and plays a role either as the inhibitor or activator through the association of other internal activator factors or viral proteins in the virus life cycle. In addition, it involves not only in the antiviral response but also in the post transcriptional activity in its host (Lin et al. 2007). Its involvement in cellular signaling pathway also considered important.
To gain more insight of its role in viral life cycle, the cloning and expression of NS1 protein as well as the crystallographic study were carried out purposely for further characteristic-identification and functional-analysis. Regarding the recent proteomic studies, the pET expression vectors are widely used for NS1 gene cloning works. Typically, the NS1 fusion protein was detected at 26kDa but larger molecular weight (Birch-Machin et al. 1997) was also reported. In addition, Ma et al. (2009) has detected the NS1 protein expressing in both soluble and insoluble fraction.
Previously, the NS1 purity was obtained through Ni-NTA purification (Wang et al., 2008), in addition, it was documented that the NS1A proteins were purified by chitin affinity chromatography (Ma et al. 2009) or glutathione S-transferase affinity column (Birch-Machin et al. 1997). Ward et al. (1994) has tried the induction by copper sulfate (CuSO4) on NS1 protein, designing to enhance the expressed NS1 gene to release the toxin in the infected cell. Concluded from the result, the toxicity could affect the cell growth but it was considered vital in yielding nuclear localization signal.
Material and methods
Construction of recombinant plasmid pET32c-NS1A
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E. coli BL21(DE3)-pET-32c and DH5Î±-TOPO-NS1 were purified using the QIAprep Spin Miniprep Kit. The isolated NS1A gene was ligated into BamHI/SacI-cleaved pET-32c using YEA T4 DNA Ligase from yT&A Cloning Vector Kit. Ligation was carried out at 22Â°C for 20 min then followed by 65Â°C for 10 min. The plasmid was electro-transformed into E.coli.
Clone Identification and determination
The colony PCR was performed using the fresh colony as the template then amplified by T7 promoter (100Î¼M) and T7 terminator (100Î¼M). PCR reaction involved 95Â°C for 5mins, and followed by 25 PCR cycles of 30s at 95Â°C, 30s 55Â°C and 1min at 72Â°C, additionally elongated at 72Â°C for 7min. The presence of insertion in the selected clones was verified by BamHI and SacI restriction enzyme double digestion. respectively, then further sequenced using the universal primer, T7 terminator. Few bioinformatics tools were applied to validate identify and characterize the target protein.
Expression and purification of NS1A recombinant protein
The cells were induced with 1mMIPTG or 5mM lactose when the bacterial growth reached an OD600nm of 1.5. The cells were harvested by centrifugation at 5000 rpm for 30min after 4-hour-induction. Enzymatic-ultrasonication was used for cell disruption. Centrifugation at 12,000 rpm at 4Â°C for 30 minutes was applied to separate the homogenates components into cytoplasmic fraction (supernatant) and inclusion bodies fraction (cell pellet). The cell pellet was resuspended by using denaturing lysis buffer containing urea (100mM NaH2PO4, 10mM Tris-HCl, 8M urea, pH7.4) and incubated at 4Â°C for overnight.
Ammonium sulfate precipitation has been applied to partially purify the NS1A recombinant protein. 0.113g/ml of ammonium sulfate (20%) was added slowly into the sample. The sample was kept stirring for at least 1 hour. The harvested precipitate was resuspended with 1X sterile PBS solution.
SDS-PAGE and Western blot analysis
Purified NS1A fusion protein was analyzed in SDS-PAGE. The electrophoresed proteins were transferred to a nitrocellulose and subsequently blocked with 1% BSA. The membrane was probed with polyclonal antibody to influenza A H1N1 NS at 1:1250 in PBS, and the antibody-antigen complex was detected with secondary antibodies, goat anti-rabbit IgG (H+L) HRP conjugate in 1:5000 dilution. 3-times-washing with PBS-Tween 20 was applied on the membrane after each process of incubation. After that, the membrane was subjected to colour development.
NS1A Gene Cloning and sequencing analysis
There were three clones, clone 97, clone 100 and clone 104, were assumed as positive clones carrying gene of interest. The correct orientation of pET-32c (+) vector and NS1A gene were identified by BamHI and SacI restriction enzyme double digestion, which respectively gave 5901bp DNA fragment to the pET-32c (+) vector and 698bp to the NS1A gene (Fig 1).
Figure 1: Clone identification by BamHI and SacI restriction enzyme. Lane 1: GeneRulerâ„¢ 1kb DNA Ladder; lane 2-4: pET32c-NS1 plasmid isolated from clone 97, clone 100 and clone 104.
Significant identical part was found between NS1A recombinant protein and Influenza virus A/California/04/2009 H1N1 segment 8 nuclear export protein (NEP) and nonstructural protein 1 (NS1) genes (complete cds; gi: 227809838; gb: FJ966086.1). 3D protein structure was predicted using the comparative modeling (Fig 2).
Figure 2: Predicted structure of NS1A recombinant protein.
Expression Analysis of NS1A Recombinant Protein
The SDS-PAGE analysis result has established that the expression products contained a band of fusion protein at approximately 45 kDa (Fig 3).
Figure 4. Batch II induction: SDS-PAGE profile of expressed NS1A fusion protein extracted from E. coli clones after 1mM IPTG induction at 37Â°C.
Lane 1: MW marker (SDS-Page Molecular Weight Standards, Low Range, Bio-Rad Catalog No.: 161-0304); lane 2, 6 and 10: cytoplasmic fraction of uninduced clone 97, clone 100 and clone 104; lane 4, 8 and 12: inclusion bodies fraction from uninduced clone 97, clone 100 and clone 104; lane 3, 7 and 11: cytoplasmic fraction of induced clone 97, clone 100 and clone 104; lane 5, 9 and 13: inclusion bodies fraction of induced clone 97, clone 100 and clone 104.
Figure 4. The SDS-PAGE pattern of the expression product. and 20% ammonium precipitation on inclusion bodies fraction of NS1A recombinant protein expressed in E.coli after 5mM lactose induction at 37Â°C.
Lane 1: MW marker (SDS-Page Molecular Weight Standards, Low Range), lane 2: crude lysate before buffer exchange, lane 3: crude lysate after buffer exchanged, lane 4: 20% ammonium precipitated supernatant, lane 5: 20% ammonium precipitated pellet.
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The Acc-apisimin-2 gene cleaved from the pMD/Acc-apisimin-2 using BamHâ… and Xhoâ… was inserted into vector pGEX-4T-2. The recombinant expression vector with the insertion of Acc-apisimin-2 was identified by
PCR and digestion with BamHâ… and Xhoâ… . The pGEX/Acc-apisimin-2 was then transformed into E. coli BL21 (DE3) for expression. The SDS-PAGE analysis results showed that the expression products contained a band of fusion protein of about 31 kDa (Fig 3) which was identical to the predicted molecular weight of the recombinant protein composed of GST (25 kDa) and Acc-apisimin-2 (5.9 kDa), and half of the expressed fusion protein was soluble ( Fig 3). The scanning result of SDS-PAGE gel profile showed that the expressed fusion protein accumulated up to about 22.1% of total protein of bacterial cells.
Western blot analysis with GST-antibodies as the first antibody showed that the expressed fusion protein was recognized by the GST-antibody (Fig 4), which confirmed that this product of expression was the expected GST-Acc-apisimin-2 fusion protein.
Purification of the recombinant protein and its cleavage
Recombinant Acc-apisimin-2 was expressed in E. coli as a fusion protein containing GST for affinity purification. The purified GST-Acc-apisimn-2 fusion protein achieved up to 90% purity in a single step from the soluble portion of the total proteins using affinity chromatography of Glutathione Sepharose 4B. The results are shown in Figure 4 after GST-Acc-apisimin-2 fusion protein has been cleaved by thrombin protease at 37â„ƒ for more than 10 h. The purified GST band could be seen clearly on SDS-PAGE profiles. But Acc-apisimin-2 band could not be shown because its molecular weight is only 5.9 kDa which is difficult to be detected with this method.
Figure 4. The SDS-PAGE pattern of expression product, purified protein and Western blot analysis. A. Lane M: protein marker; 1: expressed soluble GST-Acc-apisimin-2 fusion protein; 2: the purified GST-Acc-apisimin-2; 3: the purified thrombin-cleaved GST-Acc- apisimin-2; 4: proteins from BL21 transformed with pGEX-4T-2 plasmid ; 5: bacterial proteins from BL21 cells ; B. Western blot analysis using anti-GST antibody
Statistics of ESTs revealed that apisimin gene was richly expressed in the Chinese honeybee head. The analysis result was based on our ongoing project, analysis and functional annotation of a ESTs collection from the brain of the Chinese honeybee. Another project conducted by Robinson's team on European honeybee in America was completed and obtained 15311 high-quality ESTs. They have annotated the ESTs on their homology to the well-characterized Drosophilia gene set. This approach led to the identification of honeybee orthologues18. These ESTs has been employed on DNA microarrays to study the messenger RNA changes associated with worker behavior of nurse and labor.21 As an important work, MRJPs and peptides, enzymes in RJ should be analyzed and expressed in E. coli and insect cells, because these components have high nutritional value and biological activities, and potential economic value for the beekeeping industry which has a very important status in China. In recent 10 years, China has about 300 million colonies of honeybee every year, and has produced the most output of honey and RJ and has been the largest beekeeping country in the world.12-13 It has shown that the EST data of Chinese honeybee head will offer valuable information for the research on RJ and behavior of honeybee.
The sequence analysis showed that the Acc-apisimin-2 gene in this paper is consistent with Am-apisimin, but different from Aci-apisimin and the previously reported Acc-apisimin-1 in nucleotide and amino acid level, so it was considered to be a new apisimin gene. This result revealed an important information that the apisimin gene shows polymorphism which has been found in MRJPs families, e.g., MJP3, MRJP2 and MRJP5 of Chinese honeybee22, and will be further studied.
It can be concluded that the 20% ammonium sulfate saturation was sufficient to concentrate and partially purify the target NS1A protein. Another attempt was made to precipitate the NS1A recombinant protein from 5mM lactose-induced crude insoluble extract of IB fraction using 20% ammonium sulfate (see Figure 4.22). Undoubtedly, antigenicity of the crude extract of IB fraction which harvested from pre- and post-buffer exchange, as well as the 20% ammonium sulfate precipitate retained, showing positive signal to the probing polyclonal antibody during immunodetection (see Figure 4.23). Therefore, it can be concluded that ammonium sulfate precipitation at 20% saturation is a suitable step to partially purify NS1A recombinant protein from the solubilized inclusion bodies fraction.
This work was supported by grants from the Natural Science Foundation of Zhejiang province (No.Y305115). We thank Prof Songnian Hu and his research group in Waston Institute of Genome Science, Zhejiang University for the construction and sequencing of the cDNA library of worker heads of Chinese honeybee. We also thank Dr. Songkun Su and Professor Shenglu Cheng for the supply of honeybee colonies, Professor Longjiang Fang for aid of the analysis of EST data.