| The term superantigen was coined by John Kappler in 1989 to denote its ability to stimulate T cells, although this was an immunologist's redefinition of a group of toxins which had been studied for many years prior as staphylococcal enterotoxins (SE) that caused toxic shock and food poisoning and the streptococcal pyrogenic exotoxins (SPEs), agents causing scarlet fever, pyrogenicity, and streptococcal TSS (STSS).Staphylococcal enterotoxins are series of exotoxins secreted by Staphylococcus aureus, they are also typical superantigen (SAg). Dissimilar with the mechanism of conventional antigen, they require only recognition of specific TCR V(3 for interaction and cross-link the T-cell receptor and antigen-presenting cells of MHC class II, without the processing of antigen presenting cells, causing activation and proliferation. Meanwhile, large amounts of cytokines are elevated to toxic levels, but interferon-y (IFN-y), interleukin-2 (IL-2), and tumor necrosis factor a (TNF-a) are generally believed to be the main culprits in toxicity. With this characteristic, SEs and other SAg have a wild prospect of application in anti-tumor field.Staphylococcal enterotoxin A (SEA) was first identified by Merlin Bergdoll from the culture of an isolate from a food poisoning outbreak. Ingestion of microgram amounts of SEA elicited classic staphylococcal food poisoning, characterized by vomiting and diarrhea within 1-2 hr since ingestion and lasting 6-12 hr. At present, 20 serologically distinct staphylococcal SAgs have been described, including TSS toxin-1 (TSST-1), the SEA-SAE, SEG-SEJ, the staphylococcal enterotoxin-like (enterotoxicity unproven) toxins SEl-K-SEl-R and SEl-U, and the recently identified SEl-U2 and SEl-V. Not all SAgs are enterotoxic and a new nomenclature was introduced in 2004 to distinguish those with proven emetic activity (SEs) with those that remain unconfirmed (SEls).Previously researches have shown that the staphylococcal PTSAgs share a number of genetic and biochemical characteristics. The genes of these toxins are carried by plasmids, bacteriophages, or heterologous genetic elements, referred to as enterotoxin gene cluster and pathogenicity islands. The molecular weight for SEs is about 20000-30000Da. There is remarkable sequence variation among members of the staphylococcal SAg family. The most distant members, SEB and SEK, have only 21.8% amino acid identity. Excluding allelic variants, the most similar are SEA and SEE with 81.7%amino acid identity. All the staphylococcal SAgs are potent T cells mitogens but exhibit different preferences for MHC classâ…¡alleles and also produce distinct TCRVβprofiles. This suggests that their diversity has been driven by a need to stimulate as many different T cells as possible while retaining MHC and TCR as target molecules.The SEs have reasonable theoretics of anti-tumor effect. So far, the lethal effect of superantigen is reacted in following two ways:(1) Superantigen-dependent cell mediated cytotoxicity (SDCC) and superantigen antibody dependent cell-mediated cytolysis (SADCC):SEs are not processed but bind to major histocompatibility complex (MHC)classâ…¡molecules outside of the peptide-binding groove and form a trimolecular complex with the TCR which can directly kill the tumor cells expressing MHC-â…¡on their surface (SDCC). And for the tumor cells expressing low level of MHC-â…¡, antibody conjugated with SEs can effectively cause activation of T-cells, and attain the goal of killing tumor cells, consequently (SADCC).(2) High levels of cytokines are released from both macrophages and Tcells as a result of SAg-induced T cell proliferation, including IFN-β, TNF-a, IL-la, IL-1β,IL-2, IL-6, IL-12, etc. These cytokines not only kill the tumor cell, but also increase the expression of MHC-II, but also stimulus T cell.Previous researches about SEs have almost focused on the classical SEA-SAE, there are few reports about staphylococcal enterotoxin-like toxins. SEO pertains to staphylococcal enterotoxin-like toxins. With the molecular weight about 26700Da, the gene for SEO is carried by egc. Among members of the staphylococcal SAg family, SEO shares the highest homology of amino acid identity,43.2%, with SEN, but the lowest,23.7%, with SEM. At present, there has been no report about crystal structure of SEO. We have successfully cloned and expressed the recombinant SEO in the GST-fusion system. The proliferation of murine lymphocytes assay demonstrated the superantigen activity of rSEO. Besides, rSEO was demonstrated the similar bioactivites with SEC which is the possible active constituent of Jinpuye Preparation, suggesting rSEO has the potential medicinal value. However, there are still problems in the expression of rSEO. Comparing with other members of the staphylococcal SAg family, rSEO only got a low expression in this system, less than 10%of total protein of induced E.coli BL21 (DE3), therefore the subsequent work is difficult to launch. Thus, our study aimed at enhancing the expression level of SEO by codon optimization and the choice of appropriate expression system, and also to validate the bioacitivities of the optimized SEO.Aim at these problems previous content involved primarily, we designed schemes in following studies:The gene of SEO-his was amplifiered by PCR technology from PG4X-4T-1-SEO, then cloned to the plasmid pET28a.15 rare codons on the gene were optimized by PCR technology, using 10 pairs of primers. These recombinant plasmids then were transformed into E.coli BL21 (DE3), respectively. After IPTG induced, the expression level of those mutants was analyzed by SDS-PAGE. Meanwhile, the fusion protein SEO-his was purified by affinity chromatography, anion exchange chromatography and ultrafiltration and identified by western blotting analysis. MTT method was applied to testing the bioactivity of SEO-his(include the one which is optimized).1. Construction of recombinant pET-28a-SEO-his expression plasmidA pair of primer was designed according to the DNA sequence of SEO. EcoR I site was introduced at the 5'terminus of the up terminus whereas a stop codon, Xho I site and his-tag were added to the 5'terminus of the down terminus. After PCR amplification, the target gene segment and pET-28a plasmid were digested by Xhoâ… / EcoR I, then linked by T4 ligase. The recombinant plasmid was transformed into E.coli DH5a. Gene sequencing showed that we obtained the correct gene.2. Codon optimization on the SEO-his gene by Quick ChangeAccording to the result of SEO sequence analysis:the SEO gene has 15 rare codons, including Arg, Leu, Ile, Gly, Thr.Codon adaption index is 0.68 and average GC content is 26.69%.10 pairs of primers covering 15 rare codons were designed, including:#1:ataâ†'att(21);#2:ataâ†'att(87), cgaâ†'cgc(96), cgaâ†'cgc(102);#3:ggaâ†'ggc (183), ataâ†'att(186);#4:ctaâ†'ctg(249), ggaâ†'ggc(252);#5:gggâ†'ggc(336);#6: agaâ†'cgc(478,480);#7:agaâ†'cgc(514,516);#8:ggaâ†'ggc(609);#9:ataâ†'att(657);#10: ataâ†'att(381), ggaâ†'ggc(384).Using pET-28a-SEO-his as original template. After digested by Dpn I, the PCR products transformed to E.coli DH5a. Using the sequenced mutant plasmid as the second template, we ordinally obtained the 10 mutant plasmids by Quick Change PCR.3. Induced expression of recombinant strain and solubility analysisE.coli. BL21 (DE3) transformed with pET-28a-SEO-his was grown at 37℃and induced by 1mM IPTG,28℃,6hr. After centrifugation, the supernatant and sediment sample were checked by SDS-PAGE to analysis attribution of soluble fusion protein.4. Comparison of the protein expression level before and after codon optimization The 10 mutationally recombinant plasmids then were transformed into Escherichia coli BL21 (DE3), respectively. E.coli. BL21 (DE3) transformed related plasmid was grown at 37℃and induced by 1mM IPTG,28℃,6hr. SDS-PAGE analysis shows that after the optimization of rare codons, the expression level had increased from 7.49%to 19.8%.5. Purification of the fusion proteinCollected E. coli was breaked up by FRENCH pressure, and ultrasonic was applied to decrease viscosity. After filtering by ultrafiltration membrane, the fusion protein SEO-his was purified by affinity chromatography, Anion exchange chromatography (dislodge the 20kD impurities) and ultrafiltration (dislodge the 70kD impurities). We obtained the purifiered SEO-his which is more than 90%(SDS-PAGE).6. Western blotting analysis of fusion proteinWestern blotting result displayed that SEO-his recombinant protein can be recognized by mouse anti-his-tag antibodies. It approved SEO-his gene had been inserted into expression vector correctly.7. SEO bioactivity assayMTT method was used to examine the bioactivity of this recombinant protein. The test was applied to observing the activation of mice lymphocyte, which had been stimulated by SEO. The results indicated that SEO had strong ability to stimulate mice lymphocyte even when the concentration is lOng/mL.Conclusion:This study has successfully cloned pET28a-SEO-his and 10 mutant plasmids. The fusion protein was soluble in this expression system. After the optimization of rare codons, the expression level had increased from 7.49%to 19.8%. Besides, we obtained the purifiered SEO-his which is of more than 90%purity. Western blotting result displayed that SEO-his recombinant protein can be recognized by mouse anti-his-tag antibodies and MTT assay results indicated that SEO-his had strong ability to stimulate mice lymphocyte even when the concentration was lOng/mL. Both SEO-his and rSEO exhibited the similar bioacitvities in mice lymphocyte proliferation assay. |
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