Single-domain heavy chain antibody L5-78 for Listeria monocytogenes

A single-domain heavy chain antibody, Listeria monocytogenes technology, applied in bacteria, recombinant DNA technology, anti-bacterial immunoglobulin and other directions, can solve the problems of high production cost and complex preparation process

Inactive Publication Date: 2014-01-08
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with single-domain heavy chain antibodies, these antibodies have disadvantages such as relatively high production costs and complicated preparation processes

Method used

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  • Single-domain heavy chain antibody L5-78 for Listeria monocytogenes
  • Single-domain heavy chain antibody L5-78 for Listeria monocytogenes
  • Single-domain heavy chain antibody L5-78 for Listeria monocytogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Panning and identification of anti-Listeria monocytogenes single domain heavy chain antibody

[0029] The solid phase panning method was used to pan the single domain heavy chain antibody against Listeria monocytogenes from the camel-derived natural antibody phage display library. The culture of Listeria monocytogenes was carried out according to the national standard GB4789.30-2010, and the cultured Listeria monocytogenes was diluted to 2×10 with sterile phosphate buffer solution (PBS, pH7.4) 9 CFU / mL, add it to the wells of the ELISA plate, 100 μL / well, and coat overnight at 4°C, coat 3 wells in the first round of panning, coat 1 well in the subsequent panning, aspirate the coating solution, and wash the plate with PBS 3 times. During several rounds of panning, 3% BSA or 3% OVA was used as a blocking agent alternately, blocked at 37°C for 2 h, washed with PBS 3 times, and added 100 μL of the phage antibody library (about 2×10 11 PFU), incubate at 37°C for 1.5h,...

Embodiment 2

[0037] Example 2 Expression of single domain heavy chain antibody against Listeria monocytogenes in E. coli

[0038] Obtaining of DNA fragments encoding anti-Listeria monocytogenes: 1. Using restriction endonuclease Not I, single-enzyme digestion of phagemid pHEN-L5-78 ( image 3 ), and use agarose gel electrophoresis to recover, then use restriction enzyme NcoI to incomplete digestion of the recovered fragments, and recover the target fragments by agarose gel electrophoresis. 2. According to the sequence information provided by the present invention, chemically synthesized by a biotechnology service company; 3. Design specific primers from alpaca ( Lama pacos ) Amplify from the source cDNA library.

[0039] The obtained L5-78 double-enzyme digested gene fragment was cloned into the expression vector PET-25b, and after sequencing verification, it was named PET25-L5-78( Figure 4 ).

[0040] The recombinant plasmid PET25-L5-78 was transformed into E. coli BL21, and a single colony w...

Embodiment 3

[0043] Example 3 ELISA detection of Listeria monocytogenes

[0044] Based on the principle of indirect enzyme-linked immunosorbent assay, a detection method for Listeria monocytogenes was established. The processing of the test sample was carried out in accordance with the national standard GB 4789.30-2010 for sample collection and enrichment culture. 1 mL of the enrichment solution was put into a sterilized small test tube and heated in boiling water for 15 minutes. The remaining enrichment solution is stored at 4°C for positive confirmation. Refer to Application Example 2 for the preparation of recombinant anti-Listeria monocytogenes single domain heavy chain antibody. Take negative control OD 450 2.1 times the value is regarded as the positive judgment value. The color reagent is 3,3',5,5'-tetramethylbenzidine (TMB), which can be purchased through the biological reagent company.

[0045] In the ELISA plate, coat the rabbit anti-Listeria polyclonal antibody according to 100 μL...

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Abstract

The invention relates to a single-domain heavy chain antibody and polypeptide for Listeria monocytogenes. The antibody has the protein or polypeptide with the amino acid sequence disclosed as SEQ ID NO:1, and can be used in the fields of immune detection, antigen enrichment purification and the like. The amino acid sequence provided by the invention can be used as a precursor, can be modified by using a random or site-directed mutagenesis technique to obtain a mutant with better properties (affinity, specificity, stability and the like), and is used for developing proteins or polypeptides which are further used in medicine, industry and agriculture.

Description

Technical field [0001] The present invention relates to single-domain heavy-chain antibody technology (also known as nanobody technology) and genetic engineering antibody technology, especially single-domain heavy-chain antibodies or polypeptides against Listeria monocytogenes. technical background [0002] Single-domain antibodies refer to genetically engineered antibodies composed of common antibody variable regions (VH or VL). Single-domain heavy-chain antibody (also known as nanobody, VHH antibody, variable domain of heavy chain of heavy-chain antibody) refers to genetic engineering consisting only of the variable region of heavy-chain antibody (heavy-chain antibody) Antibodies, among them, heavy-chain antibody (heavy-chain antibody) is an antibody that naturally lacks light chains in animals such as camels and sharks. Single-domain heavy chain antibodies have the characteristics of small molecular weight and good permeability, and have been widely used in basic research, me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/70C12N1/21G01N33/569
Inventor 涂追陈奇陶勇熊勇华许杨
Owner NANCHANG UNIV
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