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Specific human-derived scFv antibody for resisting hemocyanin and application of specific human-derived scFv antibody

A hemocyanin, specific technology, applied in anti-animal/human immunoglobulins, applications, biochemical equipment and methods, etc., can solve problems such as research on KLH protein-specific antibodies that have not yet been found

Active Publication Date: 2022-04-01
普健生物(武汉)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, research on specific antibodies against KLH protein has not yet been found in the prior art

Method used

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  • Specific human-derived scFv antibody for resisting hemocyanin and application of specific human-derived scFv antibody
  • Specific human-derived scFv antibody for resisting hemocyanin and application of specific human-derived scFv antibody
  • Specific human-derived scFv antibody for resisting hemocyanin and application of specific human-derived scFv antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: In vitro rich panning of specific human scFv antibodies against hemocyanin and screening of monoclonal antibodies

[0021] 1. Construction of single-chain scFv phage library

[0022] (1) Extract the total RNA of blood PBMCs cells and reverse transcribe it into cDNA; use cDNA as a template, PCR amplify the antibody variable region sequence, including the heavy chain variable region VH and the light chain variable region VL; 1% agar After sugar gel electrophoresis, the target fragment with a size of 300-500bp is recovered.

[0023] (2) The VH fragment and the VL fragment were connected with a connecting peptide linker (GGGGSGGGGSGGGGSGGGGAS) by the overlap extension PCR method; the target fragment with a size of 700-800 bp was recovered after 1% agarose gel electrophoresis.

[0024] (3) Digest the recovered target fragment and vector pCANTAB5E (Amersham biosciences) with SfiI and NotI, and connect overnight at 16°C under the action of T4 ligase (NEB); transfer...

Embodiment 2

[0089]Example 2: Construction of a full-length IgG antibody specifically recognized by KLH

[0090] 1. Gene synthesis

[0091] The heavy chain and light chain sequences of the two scFv antibodies obtained in Example 1 were gene synthesized, and the recombinant clones were connected to the pcDNA3 vector (purchased from Thermo Company), 1(+); the obtained recombinant plasmids were transferred into the TOP10 clone strain , pick positive clones for sequencing, the specific transfer method is:

[0092] Take 50 μL of Escherichia coli competent cells, add appropriate amount of plasmid (the volume should not exceed 4 μL) and ice-bath for 30 minutes, heat shock at 42°C for 90 seconds, put them back on ice immediately, and put them in ice-bath for 2 minutes; add 400 μL LB medium, shake slowly at 37°C Shake and shake for 45-60 minutes; take 50-100 μL and spread it on LB solid medium containing ampicillin (100 μg / mL), and culture it upside down at 37°C overnight to obtain the desired str...

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Abstract

The invention discloses an anti-hemocyanin specific human scFv antibody and application thereof, and belongs to the technical field of screening and synthesis of anti-hemocyanin monoclonal antibodies. Through panning detection, the invention provides a specific human-derived scFv antibody, also provides a nucleotide sequence for coding the antibody, a corresponding recombinant expression vector and a host cell, and also provides sequences of two anti-KLH detection antibodies (full-length IgG antibodies) obtained through screening. The specific human scFv antibody or a corresponding nucleotide sequence, a recombinant expression vector, a host cell and a full-length IgG antibody can be applied to internal reference labeling of hemocyanin and preparation of a detection kit.

Description

technical field [0001] The invention belongs to the technical field of monoclonal antibody screening and synthesis, in particular to the screening technology of specific monoclonal antibody recognizing hemocyanin (KLH). Background technique [0002] Hemocyanin (Keyhole limpet hemocyanin, hereinafter referred to as KLH) is a free blue respiratory pigment found in the hemolymph of some mollusks and arthropods (spiders and beetles). Hemocyanin contains two copper ions directly connected to the polypeptide chain. Similar to iron-containing hemoglobin, it is easy to bind and dissociate oxygen. It is the only known copper protein that can reversibly bind oxygen. Hemocyanin is turquoise when oxidized and white when reduced, and has the function of carrying oxygen, antiviral activity, antibacterial activity, and coagulation function. [0003] Because of its large mass and high complexity, hemocyanin can elicit a stronger immune response compared with other carrier proteins. It is w...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13G01N33/68G01N33/577
CPCY02A50/30
Inventor 段静秦伏波鲁亮万定一张永霞代腾飞
Owner 普健生物(武汉)科技有限公司
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