Establishment Of Highly Specific Immunoassay Systems For Staphylococcal Enterotoxin C₂

Staphylococcus aureus is a major human pathogen that produces a wide array of toxins. It contributes to its ability to colonize and cause disease in mammalian hosts. The enterotoxins that secreted by Staphylococcus aureus are a worldwide problem of health. The food poisoning events due to the Staphylococcus enterotoxins occupied 33% of the whole bacterium food poisoning events in the USA. The percentage is larger in Canada, is about 45%. And in China, this kind of events is also happened frequently. Nearly all strains of Staphylococcus aureus secrete a group of enzymes and cytotoxins, which includes hemolysins, nucleases, proteases, lipases, hyaluronidase, collagenase and enterotoxin. The main function of these proteins may be to convert local host tissues into nutrients required for bacterial growth. The TSST-1 and SEs are also known as pyrogenic toxin superantigens.Superantigen is a kind of protein, which can interact with many T cells in a nonspecific manner, and are the most potent activators of T lymphocytes. They require only recognition of specific TCR V β for interaction and cross-link the T-cell receptor and antigen-presenting cells of MHC class II, causing activation. As a superantigen, SEs can have profound effects on the immune system, both acute and long-term. Acute effects include food poisoning and toxic shock syndrome. Long-term effects include autoimmune diseases and immunodeficiency such as atopic allergy, Kawasaki's disease, and periodontal disease. Further, superantigens can cause deregulation of immune responses, resulting in autoimmune disease of immunodeficiency. However, if the burst of T-cell activation that occurs with SEs could be harnessed and exploited, then superantigens can have good effects for thehost, such as enhancement of desirable immune responses.The family of SEs presently includes SEA, SEB, SEC].3, SED, SEE that have been found long before and SEM, SEN, SEO, SEU that are found lately. SEs can produce superantigen reliant cell toxicity to the tumor cells accordingly arose the systematic anti-tumor reaction. At present, the research in SEs fastens on SEA and SEB but less refer to the SEC1-3. The amino acid sequence of SECi, SEC2 and SEC3 are very similar with each other. In the 239 amino acids, the similarity of SEC] and SEC2 are 97.4% and only 7 amino acids in the N-terminal are different. Also, the similarity of SEC 1 and SEC3 are 94.4%. The relationship of protein sequence of the three subtype of SEC determines the relationship of their specific antigenic determinant. Especially, their highly conservative C-terminal determines the bioactivity of the SEC and antigenic determinant of immune cross-reaction between three subtypes. The anti-tumor effect of SEC2 has been applied to clinic. The effective component of an anti-tumor preparation named HAS is SEC2.Therefore, establishing Staphylococcus enterotoxins in food samples is dependent upon rapid, reliable, and sensitive immunological assay system is eagerly required. This system is also useful to be a quality control for the content of the SEs in the curative.Aim at these problems previous content involved primarily, we designed schemes in following study. First, to solve the abounding stock resource in future establishment of examine method of SEC2, we constructed a strain of Escherichia coli., modified by recombinant DNA technology to manufacture SEC2. Second, we hyodermic the BALB/c mice with emulsion of purified SEC2 in Freund adjuvant. The fusion was performed the day after the last immunization. We fused the immune spleen cells with myeloma cells and got the hybridoma that can secrete the specific antibodies steadily. Meanwhile, we also got the polyclonal antibodies by hyodermicing the rabbits with emulsion of another type of purified SEC2 protein that contain 6 histidine tag in the C-terminal. We can get both monoclonal antibodies and polyclonal antibodies without cross-reaction. Then we established a sensitive and rapid immunoassay system using these two antibodies for SEC2primarily. Meanwhile, we also established an electrochemistry examinning method to SEC2 by the monoclonal antibody and immunosensor.1. Cloning and sequencing of the segment of SEC2A pair of primer was designed according to the DNA sequence of SEC2. EcoR I site was introduced at the 5' terminus of the up terminus whereas a stop codon and Xho I site was added to the 5' terminus of the down terminus. The genome of Staphylococcus aureus was extracted and was amplified by PCR. The aim gene segment was regained and inserted into the clonal vector pGEM-T. After gene sequencing, the correct segment was cut from the clonal vector by EcoR I and Xho I digestion.2. Construction and characterization of recombinant Escherichia coli. strain expressing SEC22.1 Construction of recombinant pGEX-4T-SEC2 expression plasmidThe correct segment was inserted into linear pGEX-4T-l vector and the joint product was transformed to competent E. coli. BL21 (DE3) cells.2.2 Induced expression of recombinant strain and solubility analysisE. coli. BL21(DE3) transformed with pGEX-4T-SEC2 was grown at 37°C and induced by 0.5mM IPTG. The collected bacteria were sonicated in the presence of 0.05% Tritox-100. After centrifugation, the supernatant and sediment sample were checked by SDS-PAGE to analysis attribution of soluble fusion protein. GST-SEC2 with molecular weight 30,000 was highly expressed in E.coli BL21(DE3) with 0.5mM IPTG inducing, which occupied 30% of germ protein in soluble form.2.3 Purification of fusion proteinThe GST-tag in the fusion protein allowed it to be purified by Glutathione Sepharose 4B. The GST-SEC2 protein was eluted down by the reduced-glutathione. The purified fusion protein showed a single band on 12% SDS-PAGE with the yield of 0.1 gram per liter culture was obtained after quantified by BCA protein determination method.2.4 Cleavage of GST-SEC2 with thrombinThere is a thrombin digestion site between the sequence of GST and SEC2 thatthe SEC2 was cleavaged from purified GST-SEC2 with thrombin incubation at 20°C overnight. The expected peptides were collected through Glutathione Sepharose 4B. The purity of SEC2 is above 90%.2.5 Western blotting analysis of fusion proteinWestern blotting result displayed that SEC2 recombinant protein can be recognized by rabbit anti-SEC2 antibodies. The rabbit anti-SEC2 polyclonal antibodies were obtained by immunizing the New Zealand white rabbit with SEC2-His protein (part 2). It approved SEC2 gene had been inserted into expression vector correctly.2.6 SEC2 bioactivity assayWe used MTT method to examine the bioactivity of this recombinant protein. The test was applied to observing the activation and the lethal effects on tumor cells of mice lymphocyte, which had been stimulated by SEC2. In this test, three kinds of tumor cells was used as the operated cells and the results indicated that SEC2 had strong ability to stimulate mice lymphocyte to proliferate, which was a dose-dependent effect. 3. Production and bioactivity analysis of monoclonal and polyclonal antibodies toStaphylococcal enterotoxin C2 3.1 Preparation of McAb to SEC2Ten-week-old BALB/c female mice were hyperimmunized by hyodermic injection of an emulsion of purified SEC2 in complete Freund adjuvant. 10 days later, a booster injection of SEC2 with incomplete Freund adjuvant was given. Fusion was performed the day after the last immunization. The myeloma cells were fused with the immune spleen cell through adding PEG4000 into the cell pellet. The HAT medium was used to screen the correct clones. An indirect ELISA system was used to detect antibody in the hybridoma growth medium. Positive clones were sub clone three times by the limiting dilution procedure. Intraperitoneal injection with the myeloma was performed and the ascites was got after 7-10days. 3.2 Nature and reactivity of the antibodyWestern blotting was performed to examine the specificity of the McAb. The SEC2 protein was electrophoresed in a 12% SDS-PAGE. Then the protein from thegel was transferred by electroblotting onto activated nitrocellulose. The McAb was added to the test system and incubated for 3h at 37°C. The results indicated that two lines of hybridoma could secrete the specific McAb steadily.3.3 Purification of the McAbThe protein A can bind the Fc region of IgG that it was used to purify the McAb from the ascites. The ascites was applied on the protein A affinity column. After washing the column volume with the binding buffer, the McAb was eluted by the elution buffer. The result of SDS-PAGE indicated that pure McAb was obtained.3.4 Preparation of the polyclonal antibodies to SEC2Three rabbits were hyperimmunized by hyodermic injection of an emulsion of SEC2-His in complete Freund adjuvant. After 4-times immunization, we used double-gel immunodiffusion assay to test the titer of the serum. Collect the rabbit blood from its heart, the polyclonal antibodies were in the serum we collected.3.5 Purification of polyclonal antibodiesThe polyclonal antibodies were purified by two-step method using caprylic acid and amine sulphate from the serum. The caprylic acid was used to eliminate the albumin and the solid amine sulphate was used to deposit the IgG from the serum. Then the deposition was dissolved into the PBS and dialyzed in the PBS for 48h. 4. Establishment of the immunoassay system for SEC24.1 Sandwich ELISA methodThe purified polyclonal antibodies were coated onto a solid-phase support to remove the antigen SEC2 from the test material. The SEC2-specific McAb is used to probe for the captured SEC2. A third anti-mouse antibodies, which is conjugated to HRP enzyme, was use to probe the McAb that had been binded with the captured antigen. Subsequent reaction of the enzyme with the substrate results in a quantitative, colorimetric measurement of the SEC2 present.4.2 Detecte SEC2 protein content using immunosensorThe immunosensor can be used to detect the signal changing before and after the combination of antigen and antibody. In the study, we fix the monoclonal antibody up on the electrode and obtain the protein content signal using the CV andEIS.Altogether, concluding as following:1. A fusion protein GST-SEC2 was successfully expressed and purified from E. coll and proved to possess remarkable bioactivity. It provides a convenient method to produce large amount of SEC2 for the next application. For the characteristic of the express vector, there are 5 amino acid left on the N-terminus of the recombinant protein SEC2 because there are 5 amino acids between the thrombin site and the SEC2. The bioactivity site is located in the C-terminal that the bioactivity of the SEC2 was barely affected owing to the increased amino acids. Furthermore, by testing the immune activity and bioactivity of recombinant SEC2, it showed that we had obtained the active recombinant SEC2 protein. The GST fusion tag not only enhances the solubility of recombinant proteins but also provides a good method of moderate purification. Since the fusion protein was not expressed in inclusion bodies form, a series of tough manipulation for denature and refolding was avoided.2. In this study, a high purity of recombinant enterotoxins SEC2 was used as the immunogen to immunize the BABL/c. The spleen cells from the immunized mouse are fused with the myeloma cell line by adding polyethylene glycol to promoter membrane fusion. The myeloma cells used for hybridiztion lack a purine salvage enzyme, hypoxanthine phosphoribosyl transferase (HGPRT). This absence of HGPRT results in a defect of the alternated pathway to synthesize nucleic acids, so the cells will die in HAT medium. The fused cell survives in HAT medium, although the spleen cell and the myeloma cell would die on their own in HAT medium. These hybrid cells, or hybridomas, are maintained in vitro and will continuously secrete monoclonal antibodies with a defined specificity. At the same time, we also prepared to immunize the New Zealand white rabbits using another type of recombinant SEC2, which have a his tag on the N-terminal to obtain the polyclonal antibodies. These two immunogens were expressed by the different expression system and purified from the different affinity principle because of their different tag that the antibodies from these two immunogens will not take the cross-reactionwith each other in the sandwich ELISA system.3. In this study, we established a sandwich ELISA method on testng the SEC2. SEC2-sepicific polyclonal antibodies were coated onto a solid-phase support to capture the SEC2 in the sample. And the McAb was used to bind with another site of the SEC2. A third antibodie conjugated to HRP enzyme was used to probe for the capture the McAb. Subsequent reaction of the enzyme with the substrate results in a quantitative, colorimetric measurement of the SEC2. For comparing the sandwich ELISA method, we also tried to use MEMS to establish another method for SEC2. This method used immunosensor and monoclonal antibody to detect the protein content.