Anti-helicobacter pylori recombinant antibody, preparation method and application

A Helicobacter pylori and recombinant antibody technology, which is applied in the field of antibody engineering and biomedicine, can solve the problems of low stability and cumbersome Fab antibody preparation process, and achieve the effect of small molecular weight, easy operation and strong penetrability

Active Publication Date: 2021-10-08
甘肃中科博瑞生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a recombinant antibody against Helicobacter pylori, its preparation method and application, so as to solve the defects of cumbersome preparation process and low stability of the existing Fab antibody

Method used

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  • Anti-helicobacter pylori recombinant antibody, preparation method and application
  • Anti-helicobacter pylori recombinant antibody, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Culture against Helicobacter pylori

[0031] A. Helicobacter pylori medium: Weigh 39g of Columbia agar and add it to 1L double distilled water, sterilize at 121°C for 20min, cool to 50°C, add incubating defiberized sheep blood and Helicobacter pylori selective supplement respectively The final concentrations were 7% and 4%.

[0032] B. Take the cryopreserved Helicobacter pylori and immediately put it in a 37°C water bath for 30s, take 100μL and spread it on the Columbia selective medium, and put it in CO 2 Incubator, 37°C (O 2 5%, CO 2 10%, N 2 85%) activated for 24h. After two activations, they were transferred to the newly configured Columbia selective medium and cultured for 12 hours.

Embodiment 2

[0034] Screening Anti-Helicobacter Pylori Single Chain Antibody Using Phage Display Antibody Library Technology

[0035] Our laboratory has constructed a natural fully human scFv antibody library in the early stage, with a library capacity of 2.5×10 8 , good diversity. Using Helicobacter pylori as antigen, the natural fully human scFv antibody library was enriched by phage display by immunomagnetic bead method, clones were randomly selected from the enriched scFv antibody library, and monoclonal phage amplification and expression were performed. The expressed scFv was detected by phage ELISA with anti-M13-HRP monoclonal antibody. The concrete experiment of this embodiment is as follows:

[0036] (1) Add 1.5 mL of 5% MPBS to the EP tube to block overnight, then discard the MPBS and wash with PBS for 3 times.

[0037] (2) Take 100μL concentration to be 1.72×10 13 The CFU phage single-chain antibody library and 200 μL of 5% MPBS were added to the sealed EP tube, mixed evenly,...

Embodiment 3

[0052] ELISA detection of screening results

[0053] (1) The Helicobacter pylori cultured in step B (Example 2) was scraped into a 1.5mL EP tube containing 1mL PBS buffer, mixed evenly, and washed 3 times repeatedly.

[0054] (2) Add 100 μL of 0.25% glutaraldehyde to each well, incubate at 37° C. for 2 hours, and fix the cells.

[0055] (3) Wash each well with 200 μL of PBS to remove unfixed helical rods and excess glutaraldehyde, and wash repeatedly 5 times.

[0056] (4) Add 300 μL of 2% skimmed milk to each well to block the plate, and incubate at 37° C. for 2 hours; wash off excess skimmed milk with 200 μL of PBS, and wash 5 times repeatedly.

[0057] (5) Add 200 μL skimmed milk to each well, and add 10 μL KM13-rescued phage with scFv monoclonal to each well, shake and mix well, and float and incubate at 37° C. for 1 hour.

[0058] (6) Each well was repeatedly washed 5 times with 200 μL of 0.1% Tween20 PBS to remove non-specifically bound phages.

[0059] (7) Add 200 μL ...

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Abstract

The invention discloses an anti-helicobacter pylori recombinant antibody, a preparation method and application, belongs to the technical field of biological medicine, and particularly relates to the technical field of antibody engineering.The antibody is rFab and comprises VH and VL, the VH and the VL are formed by fusing one end of a fully human CH1-CL fragment, the amino acid sequence of the VH is shown as SEQ ID NO: 1, the amino acid sequence of the VL is shown as SEQ ID NO: 2, the amino acid sequence of the CH1-CL is shown as SEQ ID NO: 3, the rFab is formed by connecting a light chain and a heavy chain in the Fab antibody through a full human CH1-CL fragment by virtue of a gene recombination technology, and meanwhile, the defects of tedious preparation process and low stability of the Fab antibody are overcome.

Description

technical field [0001] An anti-helicobacter pylori recombinant antibody, preparation method and application, the invention belongs to the technical field of biomedicine, and specifically relates to the technical field of antibody engineering. Background technique [0002] Helicobacter pylori, referred to as Hp, is a Gram-negative microaerophilic bacterium that lives in various areas of the stomach and duodenum. It is associated with chronic gastritis, peptic ulcer, gastric cancer and gastric It is associated with gastric diseases such as mucosa-associated tissue lymphoma (MALT), which is classified as a class I carcinogen by the World Health Organization. The infection rate of Helicobacter pylori in my country is as high as 58.07%, which belongs to the region with a high infection rate in the world. Although the "triple" drug therapy developed based on the combination of antibiotics, proton pump inhibitors and bismuth is currently an effective method for eradicating Hp infe...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13C12N15/81C12N1/19C07K1/22G01N33/569A61K39/40A61P31/04C12R1/84
CPCC07K16/1203C12N15/815G01N33/56911A61P31/04C07K2317/55C07K2317/33C07K2317/76G01N2469/10
Inventor 雷天柱张生银马建忠
Owner 甘肃中科博瑞生物工程有限公司
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