Enterorrhagia colibacillus 0157:H7 Shiga toxin 2A1 subunit active segment Stx2a1 recombination protein, expression method and application
A technology of Escherichia coli and O157, applied in the field of genetic engineering, to achieve high-efficiency immune response and immune prevention protection, good specificity, and good immunogenicity
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Embodiment 1
[0058] Example 1: Enterohaemorrhagic Escherichia coli O157:H7 Stx2A 1 subunit active fragment Stx2a 1 clone
[0059] 1. Strains and reagents
[0060] Escherichia coli 99A021 was provided by the Jiangsu Provincial Center for Disease Control,
[0061] Plasmid pET-22b and Escherichia coli BL21, DH5α are preserved by the applicant's laboratory,
[0062] ExTaq DNA polymerase, restriction endonucleases Xho I and Nde I, T 4 Both ligase and DNAmarker are products of Dalian TaKaRa Company;
[0063] The plasmid extraction kit is a product of OMEGA;
[0064] The gel recovery kit is a product of BBI;
[0065] Bacterial Genomic DNA Extraction Kit is a product of Tiangen Company.
[0066] 2. According to EHEC O157:H7 Stx2A 1 The full-length gene sequence of the subunit is shown in SEQ ID NIO:3.
[0067] Application of DNAstar software analysis: see Figure 2 for description.
[0068] 3. Design and synthesis of primers (underlined enzyme cleavage sites)
[0069] According to the an...
Embodiment 2
[0101] Example 2: EHEC O157:H7 Stx2A 1 subunit active fragment Stx2a 1 Expression and purification in E. coli
[0102] 1. Induced expression of the target gene:
[0103] The correct active fragment Stx2a identified by Nde I, Xho I double digestion and sequencing 1 The recombinant strains and pET-22b vector / BL21 bacterial liquid were inoculated into the LB liquid culture liquid containing ampicillin, and the expression was induced according to the conventional method, (37°C shaker culture to A 600 0.6-0.8, adding SDS-PAGE loading buffer to each sample for freezing, adding IPTG to a final concentration of 0.1mmol), shaking at 37°C for 1 hour, 3 hours, and 5 hours, sampling and adding SDS-PAGE loading buffer Freeze and store in boiling water for 5 minutes and perform SDS-PAGE gel electrophoresis analysis according to conventional methods (see accompanying drawing 5).
[0104] The results showed that compared with pET-22b empty vector, recombinant Stx2a 1 The protein has a di...
Embodiment 3
[0114] Embodiment 3: Enzyme-linked immunosorbent assay (ELISA) method detects the production of the antibody of EHEC O157:H7 toxoid immunized mice:
[0115] 1. Preparation of EHEC O157:H7 toxoid:
[0116] The natural EHEC O157:H7 toxin protein purified in our laboratory was inactivated by formalin inactivation by adding formaldehyde at a final concentration of 1%, and inactivated at 37°C for 5 days.
[0117] 2. Immunization program:
[0118] Immunize five Balb / c mice aged 5-6 weeks, 100μg / mouse, 100μl inactivated natural toxin, add the same amount of immunogen Freund's adjuvant for the first immunization, and then add incomplete Freund's adjuvant to inject the mouse abdomen and Inguinal subcutaneous immunization. Two weeks after the first immunization, the second immunization was carried out, and then once a week, and the blood supernatant of the mice was collected on the seventh day after the fourth immunization to detect the antibodies produced.
[0119] 3. Using EHEC O15...
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