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Contactin 6 protein mutant as well as coding gene, expression vector and application thereof

A protein mutant, encoding gene technology, applied in the field of biomedicine, can solve the problems of inconvenient screening and identification of monoclonal antibodies, lack of monoclonal antibodies, etc., achieve good application value, stable expression, and promote the effect of axon regeneration

Active Publication Date: 2022-05-13
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently a lack of mutants of the contactin 6 domain, which cannot be used to directly identify the domain that mediates the homology interaction of contactin 6 and study the function of the domain; Mutants of cloned antibodies are inconvenient for the screening and identification of monoclonal antibodies

Method used

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  • Contactin 6 protein mutant as well as coding gene, expression vector and application thereof
  • Contactin 6 protein mutant as well as coding gene, expression vector and application thereof
  • Contactin 6 protein mutant as well as coding gene, expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Construction of recombinant expression vector

[0044] 1. Construction of pcDNA3.1-contactin 6ΔIgG2-Myc vector

[0045] 1. PCR primer design and target fragment amplification and recovery

[0046] Primers F2a, R2a, F2b, R2b were designed by Primer5.0 software, F3a, R3a, F3b, R3b and F2-3a, R2-3a, F2-3b, R2-3b primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. Principle: Carry out linearization at the cloning site of the vector, and introduce a 15-25 bp homologous sequence that is completely consistent with both ends of the vector cloning site at the 5' end of the insert fragment PCR primer (such as figure 1 shown).

[0047] contactin 6-F2a (shown in SEQ ID NO.5);

[0048] contactin 6-R2a (shown in SEQ ID NO.6);

[0049] contactin 6-F2b (shown in SEQ ID NO.7);

[0050] contactin 6-R2b (shown in SEQ ID NO.8);

[0051] contactin 6-F3a (shown in SEQ ID NO.9);

[0052] contactin 6-R3a (shown in SEQ ID NO.10);

[0053] contactin 6-F3b (...

Embodiment 2

[0141] Example 2: Application to identify structural domains that mediate contactin 6 homology interactions

[0142] 1. Co-immunoprecipitation

[0143] 1. Co-transfect the following plasmids in 293T cells (10cm culture dish):

[0144] (1) 19.2 μg empty plasmid (Vector); 9.6 μg Vector+9.6 μg contactin 6-HA; 9.6 μg Vector+9.6 μg contactin 6-Myc plasmid; 9.6 μg contactin 6-HA+9.6 μg contactin 6-Myc plasmid;

[0145] (2) 19.2 μg empty plasmid (Vector); 9.6 μg Vector+9.6 μg contactin 6-HA; 9.6 μg Vector+9.6 μg contactin 6-ΔIgG2-Myc recombinant plasmid; 9.6 μg contactin 6-HA+9.6 μg contactin 6-ΔIgG2- Myc recombinant plasmid;

[0146] (3) 19.2 μg empty plasmid (Vector); 9.6 μg Vector+9.6 μg contactin 6-HA; 9.6 μg Vector+9.6 μg contactin 6-ΔIgG3-Myc recombinant plasmid; 9.6 μg contactin 6-HA+9.6 μg contactin 6-ΔIgG3- Myc recombinant plasmid;

[0147] (4) 19.2 μg empty plasmid (Vector); 9.6 μg Vector+9.6 μg contactin 6-HA; 9.6 μg Vector+9.6 μg contactin 6-ΔIgG2-3-Myc recombinant pl...

Embodiment 3

[0178] Example 3: Western blot identification of monoclonal antibodies that can recognize contactin 6 protein IgG2 and IgG3 domains

[0179] 1. 293T cells were transiently transfected with pcDNA3.1-contactin 6-Myc, pcDNA3.1-contactin 6ΔIgG2-Myc, pcDNA3.1-contactin 6ΔIgG3-Myc and pcDNA3.1-contactin 6ΔIgG2-3-Myc plasmids. After 48 hours, add RIPA lysate for protease inhibitors, after lysing, centrifuge to get the supernatant.

[0180] 2. Protein samples were quantified by BCA method, loaded on SDS-PAGE gel electrophoresis, and analyzed by western blot. The primary antibody was incubated with two monoclonal antibodies (numbers 20H9 and 23F8) prepared by immunizing mice with the contactin 6 protein and hybridoma technology, and the secondary antibody was incubated with HRP anti-mouse IgG. Such as Figure 6 As shown, the monoclonal antibody 20H9 can recognize not only the full-length contactin 6 protein, but also contactin 6ΔIgG2, contactin 6ΔIgG3, and contactin 6ΔIgG2-3 mutants,...

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Abstract

The invention discloses a contactin 6 protein mutant as well as a coding gene, an expression vector and application thereof, the contactin 6 mutant disclosed by the invention is used for identifying a structural domain mediating contactin 6 homology interaction and a function of the structural domain, and is also used for screening and identifying a contactin 6 monoclonal antibody capable of identifying an IgG2 structural domain, an IgG3 structural domain and an IgG2-3 structural domain of the contactin 6. The method has the advantages of high expression efficiency, stable expression, high transfection efficiency and the like, and has good application value. The method can be used for screening and accurately blocking contactin 6 homologous interaction and promoting spinal cord injury posterior process regeneration and functional repair of specific monoclonal antibodies or small molecule drugs.

Description

technical field [0001] The invention relates to a contactin 6 protein mutant and its coding gene, expression vector and application, belonging to the technical field of biomedicine. Background technique [0002] Spinal cord injuries caused by various diseases and traumas (car accidents, violence, falls, etc.) have damaged the upper and lower sensory motor pathways of the spinal cord to varying degrees, resulting in serious sensory and motor nerve functions below the spinal cord injury level. Handicap, or even complete loss of function paraplegia. After spinal cord injury, it is difficult for damaged axons to pass through the scar tissue at the injury site to re-establish synaptic connections with target neurons below the injury level, so the sensorimotor neural circuit cannot be rebuilt. At present, in the basic and clinical research on the treatment of spinal cord injury, important progress has been made in gene regulation to improve the injury environment, improve the int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C07K16/18A61K39/395A61P25/00
CPCC07K14/47C07K16/18A61P25/00A61K2039/505Y02A50/30
Inventor 刘耀波孙玉慧孙晨贾欣仪李永振
Owner SUZHOU UNIV
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