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Monoclonal antibody of filamentous phage pVIII protein and application thereof

A filamentous bacteriophage and monoclonal antibody technology, applied in the field of biomedicine, can solve the problem of no monoclonal antibody, and achieve the effects of excellent immunogenicity, single biological activity, high specificity and high titer

Active Publication Date: 2021-07-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports of monoclonal antibodies that can be used to recognize filamentous phages in China

Method used

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  • Monoclonal antibody of filamentous phage pVIII protein and application thereof
  • Monoclonal antibody of filamentous phage pVIII protein and application thereof
  • Monoclonal antibody of filamentous phage pVIII protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Synthesis and Identification of Immunogen

[0053] 1. Preparation of immunogen

[0054] (1) The antigenic epitopes for the pVIII protein are all located on the outer surface of the phage capsid (the polypeptide sequences are all at the N-terminus, and the sequence is shown in SEQ ID NO.5: AEGDDPAKAAFDSLQASAT) to design and synthesize the outer membrane of the filamentous phage pVIII capsid protein The polypeptide part Cys-Ahx-AEGDDPAKAAFDSLQASAT, the polypeptide is named TC21;

[0055] (2) Coupling the above synthesized polypeptide with KLH and BSA carrier proteins respectively to obtain the immunogen.

[0056] 2. Immunogen identification

[0057] The polypeptide synthesized in step 1 was identified by mass spectrometry and high performance liquid chromatography, and the identification results were as follows: figure 1 (mass spectrometry) and figure 2 (High Performance Liquid Chromatography), the results showed that the polypeptide was synthesized success...

Embodiment 2

[0059] Example 2 Preparation of Monoclonal Antibody Against Filamentous Phage pVIII Protein

[0060] (1) Immunization of animals

[0061] Animals: Balb / c mice, screening conditions: female, body weight 18-20g, age 6-8 weeks.

[0062] The conjugates of the polypeptide TC21 prepared in Example 1 and the carrier protein (polypeptide group) and the M13KO7 helper phage (phage group) were used as immunogens to immunize animals, and five groups were set up, and 10 BALB / c mice were immunized in each group. Among them, the immunization of the M13KO7 helper phage group was divided into three groups with different dose concentrations (the first group, the second group, and the third group); The immunization was divided into two groups (BSA-CT21 as the fourth group, and KLH-CT21 as the fifth group). The mice were fed for one week before immunization, blood was collected from the orbital venous plexus, centrifuged, and the serum was taken as a negative control.

[0063] The immunization...

Embodiment 3

[0135] Example 3 Subtype Identification and Sequencing of Monoclonal Antibodies

[0136] The subtype identification of the monoclonal antibodies M5G8 and P8E4 prepared in Example 2 was carried out, and the specific method was as follows:

[0137] The subtype classification of the antibody was identified using the Pierce Rapid ELISAMouse mAb Isotyping Kit (product of Sigma, catalog number 19285). The subtype of the monoclonal antibody was detected, and the results of the immunoglobulin subtype of the monoclonal antibody are shown in Table 2.

[0138] Table 2 Antibody subtype identification

[0139]

[0140] The monoclonal antibodies M5G8 and P8E4 were sequenced, wherein the heavy chain variable region sequence of the monoclonal antibody M5G8 was shown in SEQ ID NO.1, and the light chain variable region sequence was shown in SEQ ID NO.3; the monoclonal The heavy chain variable region sequence of antibody P8E4 is shown in SEQ ID NO.2, and the light chain variable region sequen...

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Abstract

The invention relates to the technical field of biomedicine, in particular to a filamentous phage pVIII protein monoclonal antibody and an application thereof. A heavy chain variable region of the filamentous bacteriophage pVIII protein monoclonal antibody provided by the invention has an amino acid sequence as shown in SEQ ID NO.1, and a light chain variable region of the filamentous bacteriophage pVIII protein monoclonal antibody has an amino acid sequence as shown in SEQ ID NO.3; or, the heavy chain variable region has the amino acid sequence as shown in SEQ ID NO.2, and the light chain variable region has the amino acid sequence as shown in SEQ ID NO.4. The monoclonal antibody provided by the invention has the advantages of single biological activity, high titer and high specificity, can efficiently and specifically bind and recognize the filamentous bacteriophage, and has important application value in filamentous bacteriophage recognition and bacteriophage display technologies.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to filamentous phage pVIII protein monoclonal antibody and application thereof. Background technique [0002] Phage display technology is a gene expression screening technology established by George Smith in 1985. This technology inserts the DNA sequence of a foreign protein or polypeptide into the appropriate position of the structural gene of the phage coat protein, so that the foreign gene can be displayed along with the expression of the coat protein. At the same time, foreign proteins are displayed on the surface of the phage following the reassembly of the phage. Phage display technology can combine recombinant protein screening and gene screening, and has been used as a powerful tool in various fields such as antibody and enzyme ligand screening, small molecule receptor screening, and genetic engineering antibody screening. This technology won the 2018 Nobel Prize in Che...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C07K14/01C07K14/765C07K14/795C12N15/11C12N5/20G01N33/569C12R1/91
CPCC07K16/081C07K14/005C07K14/765C07K14/795G01N33/56983C07K2317/56C07K2317/35C07K2317/94C12N2795/14122
Inventor 王战辉于雪芝温凯沈建忠杨慧娟袁婷郭柳川张素霞史为民
Owner CHINA AGRI UNIV
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