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Antibodies capable of binding to aav1-13

An antibody, H-CDR3 technology, applied in the field of immunology, can solve the problems of low recovery efficiency and harsh conditions, and achieve the effects of high affinity, good specificity, convenient separation and identification

Active Publication Date: 2021-03-09
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the purification of AAV uses iodixanol density gradient centrifugation. If the purification method of ion exchange chromatography including reverse ion exchange chromatography (including cation and anion chromatography) is used, although the purity is high, the recovery efficiency is low, and Harsh conditions

Method used

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  • Antibodies capable of binding to aav1-13
  • Antibodies capable of binding to aav1-13
  • Antibodies capable of binding to aav1-13

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Preparation of Antibody

[0053] Based on the VP1 of AAV8, compare AAV1-13, remove most of the non-homologous sequences to obtain the VPx sequence, construct it on the pET-21 vector, express and purify the representative, and use it as an antigen to obtain the mouse monoclonal antibody .

[0054] 1. Antigen overexpression prokaryotic vector construction

[0055] (1) Digest the plasmid pET-21(a)+-P24 with Nde1 and Xho I. 50μL enzyme digestion reaction system contains pET-21(a)+-P24 10ug, 10×Cutsmart (NEB) 5μL, Nde1 1μL Xho I 1μL, make up to 50μL with water, digest at 37°C for 4h, and perform 1% agarose gel electrophoresis , Cut off large fragments with a blade under ultraviolet light and recover and purify;

[0056] (2) Synthesize the VPx sequence, add Nde1 and Xho I restriction sites on the upstream and downstream, respectively, construct it into the puc19 vector, and name it puc19-VPx;

[0057] (3) Carry out double digestion of plasmid puc19-VPx with Nde1...

Embodiment 2

[0093] Example 2 Detection of Antibodies

[0094] The antibody prepared in Example 1 is VP-29 antibody after screening. After sequencing, the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO: 15 and 16, respectively. Show.

[0095] 1. Detection of antibodies by Western Blot

[0096] Sample Preparation

[0097] Take 15 μL of AAV1-13 purified virus, add loading buffer at a ratio of 1:4 (loading buffer: cell fluid), mix, cook at 95°C for 5 minutes, place on a dry ice box, and then 12000 g at 4°C , Centrifuge for 5min.

[0098] SDS-PAGE gel preparation: use 10% separating gel and 5% stacking gel.

[0099] SDS-PAGE gel electrophoresis

[0100] a. Fix the gel plate to the inner tank of the electrophoresis device, put the fixed gel plate into the outer tank, and add an appropriate amount of 1×SDS electrophoresis buffer to the outer tank. And add electrophoresis buffer to the inner tank to just submerge the gel sa...

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Abstract

The invention relates to the field of immunology, in particular to an antibody capable of binding to AV1-13. The antibody can recognize VP1, VP2 and VP3 proteins of all serotypes of AV1-13, is high inaffinity and good in specificity; and the antibody can be more conveniently used for separation and identification of adeno-associated viruses due to the fact that the VP proteins are located on thesurfaces of the adeno-associated viruses.

Description

technical field [0001] The present invention relates to the field of immunology, in particular to an antibody capable of binding to AAV1-13. Background technique [0002] Adeno-associated virus (AAV) belongs to the parvoviridae family (parvoviridae). It is a kind of icosahedral microvirus that cannot replicate autonomously and has no envelope. It has a diameter of about 20-26nm and a single-stranded linear DNA structure. The modified rAAV virus tools have been widely used in gene expression, gene manipulation and gene therapy at the animal level. There are currently 13 serotypes of AAV (AAV1-13), and different serotypes have different affinities to tissues or organs. Among them, AAV2, AAV3, and AAV9 are derived from humans and are the most thoroughly studied and most widely used so far. Adeno-associated virus vector. AAV is a simple non-pathogenic single-stranded DNA virus that requires a helper virus to participate in its life cycle. The helper virus is usually adenovirus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08C12N15/13C12N7/00B01D15/38G01N33/569G01N33/577
CPCB01D15/3804C07K16/081C07K2317/56C07K2317/565C12N7/00C12N2750/14151G01N33/56983G01N33/577G01N2333/01
Inventor 杨兴林韦厚良贾国栋杨佳丽夏清梅
Owner OBIO TECH SHANGHAI CORP LTD
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