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Staphylococcal enterotoxin gene engineering reshaped antibody and its preparation method and use

A staphylococcal enterotoxin genetic engineering technology, which is applied in the field of staphylococcal enterotoxin genetic engineering remodeling antibody and preparation, can solve the problems of disappearance of secretion ability, stability defect, specific binding ability to be improved, etc., and achieve strong affinity and high-efficiency expression specific effect

Inactive Publication Date: 2013-07-31
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2009, Kalinina et al. established a prokaryotic expression system for anti-SEC1 gene remodeling antibody, but due to the lack of protein modification function of the expression system, its protein activity was blocked. The antibody was different from SEA, SEB, SED, SEE, SEG and SEI. degree of cross-reactivity, and the specific binding ability needs to be improved; in 2010, Pawan et al. prepared an anti-SEB single-chain antibody with strong specificity and high affinity through the pCANTAB5E vector system, but the study found that the secretion ability of the expression system gradually disappeared over time, and the stability Defect highlights

Method used

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  • Staphylococcal enterotoxin gene engineering reshaped antibody and its preparation method and use
  • Staphylococcal enterotoxin gene engineering reshaped antibody and its preparation method and use
  • Staphylococcal enterotoxin gene engineering reshaped antibody and its preparation method and use

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Using SEK-GST fusion protein as the immunization antigen, immunize 6-week-old Balb / c mice 4 times, 2 weeks / time: the initial immunization adopts the method of adding the same amount of Freund's complete adjuvant to subcutaneously inject multiple points on the back of the neck, and the dose of immunization antigen is 100 μg / rat; the second immunization adopts the method of adding the same amount of Freund’s incomplete adjuvant into multiple subcutaneous injections on the back of the neck, and the dose of immune antigen is 100 μg / rat; the third and final immunization does not add adjuvant, and the tail vein is used to immunize The dose of antigen was 50 μg / monkey. The Balb / c mice that had been immunized were sacrificed, the spleen was aseptically removed, and the immune spleen cell suspension was prepared for future use. In vitro fusion of immune spleen cells and myeloma SP2 / 0 cells by the PEG method, the steps are as follows: draw and mix 1×10 8 immune spleen cells and ...

Embodiment 2

[0093] (1) Cellular indirect immunofluorescence (IFA): BHK-21 cells transfected with p2C2HILO and pcDNA3.1(+) for 45 days were collected separately, and the concentration was 1×10 6 cell / mL suspension, dropped on a sterile coverslip, at 37°C, 5% CO 2 Cultivate for 24h. Discard the culture medium, wash with PBS twice, drop a few drops of methanol on the slide, and let it stand at -20°C for 10 minutes. Wash once with PBS, add a few drops of pre-cooled acetone solution, and let stand at -20°C for 1 min. Wash once with PBS, add 10 μL / mL of natural staphylococcal enterotoxin SEA and recombinant staphylococcal enterotoxin SEA-His, SEG-His, SEK-His, SEO-His, SEQ-His, SEU-His respectively, 4°C Let stand for 45min. Wash once with PBS, add 5% FCS blocking solution dropwise, and let stand at 4°C for 30min. Wash once with PBS, add dropwise 1:100 dilution of anti-SEs chicken polyclonal antibody, and let stand at 4°C for 45min. Wash once with PBS, add dropwise 1:100 dilution of mouse a...

Embodiment 3

[0097] Collect a large number of stable expression secretory cells obtained in Example 1, wash the cells twice with pre-cooled PBS, add RIPA lysate, ice-bath for 30 minutes, centrifuge at 1000r / min for 5 minutes, centrifuge to get the supernatant, and freeze for later use. The lysed supernatant was diluted 1:10 with carbonate coating solution (pH 9.6), coated at 100 μL / well, and left overnight at 4°C. After taking it out, wash it twice with PBS, 1 min each time. Block with 5% fetal bovine serum, 150 μL / well, incubate at 37°C for 30 min and wash as before. Add natural enterotoxin SEA and recombinant antigens SEA-his, SEK-his, SEG-his, SEO-his, SEQ-his, SEU-his respectively from 10 μg / mL to 2× doubling dilution, 100 μL / well, each group Repeat for 3 wells, incubate at 37°C for 30 min and wash as before. Add 1:100 diluted chicken polyclonal antibody, 100 μL / well, incubate at 37°C for 30 min and wash as before. Add enzyme-labeled secondary antibody Anti-chicken-HRP, 100 μL / well,...

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Abstract

The invention discloses a staphylococcal enterotoxin gene engineering reshaped antibody and its preparation method and use. The staphylococcal enterotoxin gene engineering reshaped antibody has an amino acid sequence shown in the formula of SEQ ID No.1 in the sequence table. Through construction of light and heavy chain eukaryotic co-expression vectors of the staphylococcal enterotoxin monoclonal antibody, a high-efficiency expression and stable-secretion mammalian cell line and the gene engineering reshaped antibody having high singularity and strong affinity are obtained. The staphylococcal enterotoxin gene engineering reshaped antibody can be used in staphylococcal enterotoxin detection, cell indirect immunofluorescence detection and flow cytometry detection.

Description

technical field [0001] The invention relates to a recombinant antibody, in particular to a staphylococcal enterotoxin genetically engineered recombinant antibody and its preparation method and use. Background technique [0002] Staphylococcus aureus (Staphyloccocus aureus, SA) is an important pathogenic bacterium of zoonosis and one of the most common bacterial food poisoning pathogens at home and abroad. The bacterium is widely distributed in nature, including air, water, dust, and human and animal excreta, especially food is highly susceptible to contamination, and the clinical manifestations of food poisoning caused by it are mainly nausea, vomiting and abdominal pain, and a small number of patients have Diarrhea, headache, dizziness, fever, dehydration and other symptoms. So far, many countries in the world have reported outbreaks of Staphylococcus aureus, which not only seriously endanger human health, but also cause huge direct and indirect economic losses. The stron...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/85G01N33/68G01N33/577
Inventor 黄金海刘鹏翀
Owner TIANJIN UNIV
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