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Therapeutic monoclonal antibody for resisting escherichia coli infection, hybridoma cell strain generating monoclonal antibody and use of therapeutic monoclonal antibody

A hybridoma cell line, monoclonal antibody technology, applied in antibacterial immunoglobulins, applications, microorganisms, etc., can solve problems such as slow progress in research on bacteriolysis mechanism

Inactive Publication Date: 2014-12-24
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, research on the mechanism of bacteriolysis has been slow and controversial

Method used

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  • Therapeutic monoclonal antibody for resisting escherichia coli infection, hybridoma cell strain generating monoclonal antibody and use of therapeutic monoclonal antibody
  • Therapeutic monoclonal antibody for resisting escherichia coli infection, hybridoma cell strain generating monoclonal antibody and use of therapeutic monoclonal antibody
  • Therapeutic monoclonal antibody for resisting escherichia coli infection, hybridoma cell strain generating monoclonal antibody and use of therapeutic monoclonal antibody

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1: Construction of MraY hydrophilic region fusion protein prokaryotic expression vector

[0045] Using Escherichia coli DH5α genomic DNA as a template, a 96bp fragment a (shown in SEQ ID NO.3) and a 99bp b fragment (shown in SEQ ID NO.4) were amplified by PCR, and the two fragments were amplified by overlapping PCR. The fragments were fused, cut with restriction enzymes (Bam H I and Sal I), ligated with pGEX-6P-1 (purchased from Amersham), and transformed into E. coli BL21 (DE3) (purchased from Merck). And the recombinant plasmid pGEX-6p-M-ab was obtained after digestion and PCR identification.

Embodiment 2

[0046] Expression and purification of embodiment 2 fusion protein

[0047] (1) Induction of recombinant protein

[0048] Pick out the single clone colonies containing the plasmid, inoculate them in 5ml of LB medium containing ampicillin, shake overnight at 220rpm at 37°C. The next day, dilute 1:100 into 500ml LB medium containing the corresponding antibiotics, culture at 37°C until OD 600 About 0.6, add IPTG to make the concentration 1mM, induce for 4 hours at 37°C, and collect the precipitate by centrifugation.

[0049] (2) Deformation of urea to extract inclusion bodies

[0050] 1) Suspend the precipitate with 12mL resuspension solution (20mM Tris, 150mM NaCl) according to the ratio of 4ml / 100ml bacterial solution (the bacterial solution volume is the bacterial solution volume during IPTG induction);

[0051] 2) Ultrasound for 30 minutes, each ultrasonic wave is 5 seconds, the interval is 15 seconds, the amplitude is 30%, and the operation is performed on ice;

[0052] 3...

Embodiment 3

[0064] Preparation of embodiment 3 antiserum

[0065] After mixing and emulsifying the purified GST-M-ab fusion protein with an equal volume of Freund's complete adjuvant (purchased from Sigma Company), subcutaneously immunized 6-8 week-old female BALB / c mice (purchased from China Agricultural Experimental Animal Center, Harbin Veterinary Research Institute, Academy of Sciences), the injection dose was 100 μg / animal; a total of three immunizations, the latter two with an equal volume of Freund’s incomplete adjuvant (purchased from Sigma Company), the immunization dose was also 100 μg / animal. Seven days after the third immunization, blood was collected from the tail vein, and serum was separated as positive serum. Measure titer by indirect ELISA method, when reaching 10 -4 Then ready to blend. Three days before fusion, booster immunization was carried out by intraperitoneal injection of protein without adjuvant, and the dose was also 100 μg per mouse.

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Abstract

The invention discloses a therapeutic monoclonal antibody for resisting escherichia coli infection, a hybridoma cell strain for generating the monoclonal antibody and use of the therapeutic monoclonal antibody. The invention also discloses an epitope polypeptide of MraY protein which is specifically combined with the monoclonal antibody. The monoclonal antibody disclosed by the invention is generated by a hybridoma cell named M-H11 and has specificity for polypeptide which is encoded by a nucleotide sequence shown in SEQ ID No.1. The invention provides a novel monoclonal antibody for resisting escherichia coli, and a hybridoma cell strain generating the monoclonal antibody; the epitope and in vitro antibacterial properties and the in-vivo treatment effect are identified; the invention has a great significance in application of the therapeutic monoclonal antibody for preventing and treating escherichia coli infection and also lays a foundation for treatment of gram-negative bacterial infection and intensive study of bacterial ghost vaccines.

Description

technical field [0001] The present invention relates to a therapeutic monoclonal antibody against Escherichia coli infection, a hybridoma cell line producing the monoclonal antibody, and an epitope polypeptide of MraY protein of Escherichia coli specifically combined with the monoclonal antibody. The invention belongs to the field of biotechnology. Background technique [0002] Gram-negative bacteria phosphate-N-acetylmuramate-thymopentin translocase (MraY, translocase Ⅰ) is encoded by the mraY gene and is one of the key enzymes in bacterial cell wall biosynthesis, catalyzing the first step lipid It is a member of UDP-GlcNAc / MurNAc enzyme family: polyisoprenyl-P GlcNAc / MurNAc 1-P translocase family, which is called GPTase family. MraY is the attack target of some antibacterial drugs such as tunicamycin, cytoplasmin A and lipasein B. Cytoplasmin can lead to the lysis of Gram-negative bacteria such as Pseudomonas aeruginosa by inhibiting the activity of MraY. This shows the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12G01N33/577G01N33/569C12N5/20C07K7/08C12N15/31C12R1/91
Inventor 于申业刘思国
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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