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Fab fragment of fully-human recombinant anti-CD40L monoclonal antibody and preparation method thereof

A fully human-derived, fragment-based technology, applied in the field of genetic engineering, can solve problems such as unclear immunological effects, achieve good application value, mature preparation technology, and low preparation cost

Active Publication Date: 2017-07-21
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there are natural soluble CD40L in the body, which may be produced by the cleavage of matrix metalloproteinases, but its immunological role in the body is still unclear

Method used

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  • Fab fragment of fully-human recombinant anti-CD40L monoclonal antibody and preparation method thereof
  • Fab fragment of fully-human recombinant anti-CD40L monoclonal antibody and preparation method thereof
  • Fab fragment of fully-human recombinant anti-CD40L monoclonal antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The fully human recombinant CD40L monoclonal antibody Fab fragment provided by this application includes heavy chain Fd and light chain;

[0049] The protein sequence of the heavy chain Fd is shown in SEQ ID NO.1, and the protein sequence of the light chain is shown in SEQ ID NO.2.

[0050] Normally, a Fab fragment consists of a heavy chain Fd and a light chain, which are connected by an interchain disulfide bond to form a heterodimer (eg figure 1 shown). In the rough endoplasmic reticulum of B cells, the variable region is three-dimensionally folded, and intrachain disulfide bonds are formed, so that the light chain and the heavy chain variable region interact with each other to form the correct three-dimensional conformation.

[0051] In order to better express the Fab fragment, during the actual preparation process, the inventors optimized the coding gene sequences of the heavy chain Fd and the light chain. When expressing the protein, a signal peptide PelB was adde...

Embodiment 2

[0053] In the present application, the method for preparing the Fab fragment of the fully human recombinant CD40L monoclonal antibody specifically includes the following steps:

[0054] (1) According to the protein sequence of heavy chain Fd and light chain, reverse transcription into coding gene (such as SEQ ID NO.3, SEQ ID NO.4 respectively);

[0055] Since pETDuet-1 with two independent promoters needs to be selected as the expression vector for co-expression of two genes, the coding gene was further optimized. The principles of gene optimization are as follows:

[0056] An independent leader peptide sequence from the periplasm of Gram-negative bacteria is added to the N-terminus of the light chain and the heavy chain Fd, specifically:

[0057] The amino acid sequence of OmpA was added before the light chain: KKTAIAIAVALAGFATVAQA, after codon optimization according to the E. coli prokaryotic expression system, the full-length light chain DNA was synthesized by the solid-pha...

Embodiment 3

[0096] Using the ELISA method, the CD40L monoclonal antibody antigen-binding fragment Fab prepared in Example 2 was tested for activity analysis, and the related experimental process is briefly introduced as follows.

[0097] The antigen CD40L (5ug / ml) was coated on a polystyrene micro-cell culture plate, and placed in a refrigerator at 4°C for 16 hours;

[0098] After washing, add 200ul blocking solution (5%BSA) and block at room temperature for 2 hours;

[0099] Dilute the purified CD40L monoclonal antibody Fab with diluent (800 ng / mL, 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.5625 ng / mL, 0.7813 ng / mL), 100 μL per well, and at the same time as the dilution control, add horseradish peroxidase-labeled anti-light chain antibody goat anti-rabbit IgG (1: 5000 dilution), incubate at room temperature for 2 hours, add 200 μL o-phenylenediamine solution as a substrate and incubate at room temperature for 30 minutes to develop color, ...

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Abstract

The invention specifically relates to a Fab fragment of a fully-human recombinant anti-CD40L monoclonal antibody and a preparation method thereof, belonging to the field of genetic engineering technology. The Fab fragment comprises a heavy chain Fd and a light chain. During expression and preparation of the Fab fragment, a signal peptide PelB is added to the N terminal of the heavy chain Fd, and the C terminal of the heavy chain Fd is connected with a protease TEV restriction enzyme site and a histidine fusion tag; a signal peptide OmpA is added to the N terminal of the light chain; then a recombinant expression vector is constructed after connection with an Escherichia coli expression vector pETDuet-1 with double promoters; and the Fab fragment is further expressed and prepared. According to the invention, the Fab fragment is optimized and a host cell, the expression vector, expression conditions and purification conditions are reconstructed and optimized, so the Fab fragment is rapidly prepared, has the technical advantages of high purity, low cost, stable effect and the like and has good application value in prevention and control of related diseases.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a patent application for a fully human recombinant CD40L monoclonal antibody Fab fragment and a preparation method thereof. Background technique [0002] CD40 is a type I transmembrane glycoprotein, distributed on the surface of various cells such as B cells, monocytes, dendritic cells, follicular dendritic cells, macrophages, endothelial cells and thymic epithelial cells. It is also highly expressed on the cell surface. CD40L is a type II transmembrane glycoprotein originally identified in activated CD4 + On the surface of T cells, on monocytes, NK cells, mast cells and some CD8 + There are also different levels of expression on the surface of T cells. resting CD4 + T cells do not express CD40L and its expression is transiently upregulated after its activation. In addition, there are natural soluble CD40L in the body, which may be produced by matri...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/70C07K1/22C07K1/18C07K1/16
CPCC07K16/2875C07K2317/51C07K2317/515C07K2317/55C12N15/70
Inventor 张守涛郭亚楠田庆南
Owner ZHENGZHOU UNIV
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