Design, Construction, And Application Of A Novel Prokaryotic Fusion Expression Vector Based On Enterohaemorrhagic Escherichia Coli O157:H7 Secreted Protein A

It is an important and key element to make use of expression vectors to prepare recombinant proteins in life science research and production of biological products and pharmaceutical products. Production of large amounts of proteins is a requisition for subsequent research and further production. Construction of effective expression vector is required for expression of target genes and also has an important impact on genetic expression level and biological activity of protein. Although a number of commercialized expression vectors are available for expression, production and research of recombinant proteins, the lack of cost-effective and stable recombinant protein expression system which also has independent intellectual property rights is a major bottleneck in research and production of genetically engineered drugs (or vaccines) and correlated biotechnology.Escherichia coli secreted protein A(EspA) is secreted via the Enterohaemorrhagic (EHEC) Escherichia coli TTSS system. A large body of data showed that EspA is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected cell surface and is required for the translocation of effectors into infected epithelial cells. EspA as membrane protein has confirmed to be of nice immuno-reactivity and immunogenicity, becoming a vaccine antigen candidate. During the study of EHEC O157:H7 subunit vaccine, we found that EspA is expressed in high level in E.coli. In our previous study, we failed to obtain Stx2A1 protein in many prokaryotic expression vector: pET-22b, pQE-30, pET-28a(+). We overcame the extreme difficulty in stx2A1's prokaryotic expression by construction of a recombinant plasmid pET-28a::EspA-Stx2A1 which directs the synthesis of Stx2A1 N-terminally fused to E. coli secreted protein A (EspA). Therefore, we speculated that EspA might be used to increase the expression of foreign proteins. In view of these, my study aimed at design, construction, and application of a novel prokaryotic fusion expression vector based on enterohaemorrhagic Escherichia coli O157:H7 secreted protein A.Methods1. Design and construction of vector pEspA based on EspA. The Linker which is composed of a flexible part (YAPQDP), multiple cloning sites and a cleavage sequence for enterokinase is designed at the C-terminus of EspA. The espA gene encoding the EspA was amplified by PCR. Overlapping PCR was applied to develop the espA-Linker fusion gene using two pairs of specific primers. The PCR product was subcloned into pMD-18T vector. The sequence of the plasmid pMD-18T-espA-Linker was confirmed by DNA sequencing (Shanghai Bioengineering). Finally, this insert was transferred from pMD-18T vector into pET-28a(+) prokarrotic expression vector via NcoⅠand XhoⅠcleavage sites to create a pEspA vector. The expression of recombinant proteins was induced by an addition of 1mM (final concentration) IPTG at 37℃. Whole cell lysate of pEspA/BL21 was analyzed on SDS-PAGE, and then western blotting was carried out to identify the specific reactivity of recombinant protein with the McAb anti-EspA.2. Expression of target proteins using pEspA vector. Six different proteins were used to test pEspA vector. stx2A1,IL-24,GFP,stx2B,S1,intiminC300 were cloned into pEspA , respectively, and the expression of fusion protein were induced at 16,25,37℃with IPTG at concentration of 0.1,0.2,0.5 or 1mM after transforming into E.coli BL21. Western blotting was carried out to identify the specific reactivity of recombinant protein with the McAb anti-EspA. The expression quantities and style of fusion protein was analyzed by SDS-PAGE.3. Removal of EspA after site-specific proteolysis from fusion proteins. EspA-IL-24 and EspA-GFP were purified by affinity chromatography. To determine if EspA could be cleaved off, we treated EspA-IL-24 and EspA-GFP with EK. In brief, the protein solutions were adjusted to pH 7.0-8.0, followed by cleaved by EK for 8h. Following digestion, the samples were centrifuged for 10 min and the supernatants were examined using SDS-PAGE.4. Study on biological activities of EspA-Stx2A1 fusion protein expressed using pEspA vector. 4.1 Purification and renaturation of the fusion proteins. After inclusion bodies were washed, denatured, and resolved, the solubilized inclusion bodies were purified by affinity chromatography. 12% SDS-PAGE was used to test its purity. The purified fusion proteins were slowly dripped into 20 volume of refolding buffer (50mM Tris-HCl, 1mM arginine monohydrochloride, 1mM EDTA, 1mM reduced form, and 0.5mM oxidized form of glutathione, pH 8.0) under vigorous stirring at 4℃for 20h. The refolding fusion protein was subsequently concentrated by protein concentration tube. By the use of the McAb anti-EspA and anti-Stx2A1 western blotting was carried out to identify the specific reactivity of fusion protein EspA-Stx2A1.4.2 ELISA analysis. To determine the antigenicity of the fusion protein, Balb/c mice (4~5 weeks) were inoculated with the protein of EspA-Stx2A1 or EspA in the dose of 100μg/mouse/time. This step was repeated for triplicates with interval of 1 week (0, 1st, 2nd week). The control group was injected with 200ul mixture of PBS and Complete Freund's Adjuvant at a ratio of 1:1 at the same schedule. The collected sera were subjected to ELISA-based antibody titer assays in a series of dilution. EspA protein and Stx2 toxin were used with BSA as negative control.4.3 Immunoprotection test of recombination fusion protein EspA-Stx2A1 in vivo. Balb/c mice (4-5weeks) were subcutaneously inoculated with the fusion protein of EspA-Stx2A1 or EspA protein in the dose of 100ug/mouse/time emulsified with an equal amount of Freund's complete adjuvant three times on days 0, 7, and 14. Two weeks after the last administration, the immunized mice were challenged intra-peritoneally with 50ug of Stx2 toxin and the number of dead mice was determined every day. PBS-treated animals were taken as negative controls.4.4 In vitro neutralization assay. The Stx2 toxin was found to be toxic to HeLa cells less than 50pg/ml. Stx2 at a dilution of killing 70% of HeLa cells was pre-incubated with anti-sera collected from mice immunized with EspA-Stx2A1 fusion protein (or EspA protein) for 1h at 37℃in 5% CO2, and then the mixture was added to the cells and incubated 3d at 37℃in 5% CO2. The ability of anti-sera to neutralize Stx2 toxin was determined by MTT method.4.5 Fluorescence actin staining (FAS). Fluorescence actin staining test was carried out to detect the formation of"attaching and effacing"(A/E) lesion and the adherence between O157:H7 and HeLa cells in the presence of anti-EspA-Stx2A1 sera.Results1. Under the induction of 1mM IPTG at 37℃, EspA protein containing Linker was expressed. The His-tagged EspA protein was present as a predominant band at approximately 25KDa. The expression level was about 25% of total cell proteins. Our results showed the insert of Linker sequence did not interfere with the EspA protein's own expression. Immunodetection was achieved by using EspA-specific McAb as primary antibody and HRP-conjugated rabbit anti-mouse immunoglobulins as secondary antibody. A band of approximate 25KDa showed the strong and specific reaction between whole cell lysate and EspA-specific McAb.2. Six fusions were employed to evaluate the suitability of pEspA vector for expression and preparation of target proteins. Optimal expression of the fusion proteins was obtained at a cell density of OD600=0.8 with 1mM IPTG after 8h incubation at 25℃. Immunoblot analysis of fusion proteins demonstrated that six fusion proteins were all recognized by anti-EspA monoclonal antibody. Lowing cultivation temperature exhibited pronounced effects on improving solubility of EspA-GFP, EspA-Stx2B, EspA-intiminC300. Although expression temperature and concentration of inducer were manipulated to increase the solubility of EspA-S1, EspA-IL-24, EspA-Stx2A1 fusion protein, they were shown to be expressed in insoluble form and almost none was found in the supernatant portion.3. Digestion results indicated that EspA can be removed by the digestion of the fusion protein with EK at 23℃for 16 h and most of the cleaved protein remained in the soluble fraction.4. Affinity chromatography has been proved to be efficient and it can result in high purity (90%) of EspA-Stx2A1 fusion protein. The yield of fusion protein was about 120mg/L of culture. After refolded, the yield of fusion protein was about 30mg/L of culture. ELISA results demonstrated that subcutaneous administration of EspA-Stx2A1 fusion protein could elicite immune response against both EspA and Stx2A1 in mice. Most importantly, anti-Stx2A1 antibodies induced by fusion protein displayed high cytotoxicity neutralizing activities in vitro and could protect mice against Stx2 toxin challenge. FAS results suggested that anti-EspA-Stx2A1 sera could not block the adhesion between EHEC O157:H7 and HeLa cell but had a marked effect upon host cell actin rearrangement.Conclusions1. The newly designed prokaryotic expression vector pEspA has been successfully constructed. The pEspA system has the following properties: T7 promoter, Kanamycin resistance gene, a EK cleavage site to remove EspA from fusion protein, multiple clone site to facilitate target genes to insert, 6×his-tag for Ni-NTA affinity chromatography.2. pEspA system significantly enhances the expression yields of target proteins. EspA-fusion expression may be advantageous in solubilizing some target proteins which were difficult to express in soluble form using nonfusion expression expression vectors. Digestion results indicated that EspA can be removed from fusion proteins and most of the cleaved protein remained in the soluble fraction.3. EspA-Stx2A1 fusion protein has been demonstrated to be of nice immuno-reactivity and immunogenicity. Anti-sera from Balb/c mice immunized with the EspA-Stx2A1 fusion protein were found to exhibit strong neutralization activity and protection capability in vitro and in vivo. As a result, it can be of implications for the development of vaccines candidate.4. This study not only makes it possible to further development of expression system which has independent intellectual property rights, but also provides the basis for the development of multiple-subunit vaccines for EHEC O157:H7 infection.