A kind of human single-chain antibody and its application

A single-source antibody technology, applied in human single-chain antibody and its application field, can solve the problems of narrow action range and instability, and achieve the effects of small molecular weight, protection of tissue damage, and promotion of angiogenesis

Active Publication Date: 2020-03-24
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of this, the purpose of the present invention is to address the lack of specificity and tissue targeting of the above-mentioned small molecule inhibitors that inhibit the activity of PHD2. Narrow problems, to provide a low toxicity, high efficiency, specific, stable anti-PHD2 human single-chain antibody, which can be used as a proline hydroxylase inhibitor to achieve its phenotypic function of effectively knocking out PHD2 in cells, thereby Inhibit the hydroxylation of HIF-1α by PHD2, up-regulate the level of HIF-1, promote angiogenesis, and at the same time achieve the purpose of protecting tissue damage and promoting the repair of liver and other tissue damage

Method used

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  • A kind of human single-chain antibody and its application
  • A kind of human single-chain antibody and its application
  • A kind of human single-chain antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: Preparation of recombinant human PHD2 protein

[0049]1. Primers required for PCR

[0050] The PHD2 primers for PCR amplification were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the sequence is as follows:

[0051] Forward primer HumanPHD2FP is 5'-CCGGAATTCATGCTGGCGCTCGAGTACATC-3'

[0052] The reverse primer HumanPHD2RP is 5'-CCCAAGCTTCTATACTTTAGCTCGTGCTCT-3'

[0053] 2. Experimental method

[0054] (1) Total RNA was extracted from MCF-7 cells, and cDNA was obtained by reverse transcription

[0055] MCF-7 cells were cultured in a 6-well plate until the confluence reached 90-100%, the cells were collected, washed twice with pre-cooled PBS, and 0.5ml Trizol extraction solution was added to extract the total RNA of the cells. Using the extracted total RNA as the starting material, according to the instructions of the RT-PCR kit, add various components required for the reverse transcription reaction to carry out the RT ...

Embodiment 2

[0072] Example 2: Preparation of specific anti-PHD2 human single-chain antibody

[0073] The following "specific anti-human PHD2 human single-chain antibody" is referred to as "INP"

[0074] 1. Experimental materials

[0075] Escherichia coli (Escherichia coli) JM109, HB2151 and XL1-Blue were purchased from Beijing Dingguo Biotechnology Co., Ltd.; pUC119 plasmid was purchased from Dalian Bao Biological Engineering Co., Ltd.; HRP / Anti-M13 monoclonal antibody was purchased from Pharmacia.

[0076] 2. Test method

[0077] The acquisition of the phage human single-chain antibody library is carried out with specific reference to the methods of the literature Sblattero D et al. Nat Biotechnol, 2000, 18:74-80 and Sblattero D et al. , the final storage capacity is 1×10 11 phage human single chain antibody library.

[0078] The screening of the antibody library adopts the solid-phase antigen immunoadsorption screening method. The human PHD2 protein is diluted to 100 μg / ml with 0.05...

Embodiment 3

[0087] Example 3: Characterization of the activity of the anti-PHD2 human single-chain antibody INP

[0088] 1. Experimental materials

[0089] Reagents and materials refer to Example 1 and Example 2.

[0090] 2. Experimental method

[0091]1. ELISA analysis of the binding activity of purified anti-PHD2 single-chain antibody INP to recombinant human PHD2.

[0092] 2. Determination of INP affinity of anti-PHD2 single-chain antibody by non-competitive ELISA.

[0093] 3. Competition ELISA assay was used to detect the binding specificity of anti-PHD2 single-chain antibody to PHD2.

[0094] 3. Results and Analysis

[0095] 1. ELISA detection of binding activity of INP and purified PHD2 protein

[0096] In order to identify the binding activity between INP and recombinant human PHD2 protein, the purified recombinant human PHD2 protein and BSA were respectively coated at a concentration of 10 μg / ml. Anti-PHD2 mouse monoclonal antibody was used as a positive control, and INP and ...

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Abstract

The invention belongs to the field of antibody medicine, discloses human single chain antibody namely PHD2-resistant human single chain antibody INP and HD2-resistant human intracellular single chainantibody ER-INP and discloses polynucleotide for encoding the human single chain antibody, a carrier containing the polynucleotide and application. The human single chain antibody is screened by utilizing a phage antibody library technology and prepared by utilizing a genetic engineering method. The human single chain antibody INP and ER-INP disclosed by the invention is a low-toxicity, high-efficiency and specific active molecule for inhibiting PHD2 hydroxylation activity, and toxic and side effects of heterologous antibody which is applied to a human body are avoided. Meanwhile, the human single chain antibody is micromolecular single chain antibody and has the advantages of small molecular weight, low immunogenicity and the like; in addition, endoplasmic reticulum location signals are introduced , so that the human intracellular single chain antibody ER-INP can efficiently and stably inhibit PHD2 hydroxylation activity in cells, can effectively improve HIF protein level, promotes angiogenesis damage repair of tissues like liver and the like and has a protecting effect on tissue damage.

Description

technical field [0001] The invention belongs to the field of antibody drugs, and in particular relates to a human single-chain antibody and its application. Background technique [0002] Oxygen homeostasis plays an important role in the physiology and development of the body. As the oxygen concentration in the environment changes, the body will respond to hypoxia. Under hypoxic conditions, hypoxia-inducible factors (HIFs), as the most important factors of hypoxic stress, can drive the transcription of a series of genes and promote the adaptive response to hypoxia. HIF-1 was first discovered in 1992 by Semenza GL et al. when they performed gel migration analysis on nuclear extracts of hypoxia-treated Hep3B and HepG2 cell lines, and experiments showed that HIF-1 mediated hypoxia-induced Transcription of the erythropoietin (EPO) gene. HIF-1 is the most sensitive factor to acute hypoxic stress, and is also the main transcription factor of the body's hypoxic response. Current r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C07K19/00C12N15/13C12N15/62A61K39/395A61P1/16A61P9/00
CPCA61K2039/505C07K16/40C07K2317/24C07K2317/622C07K2317/76C07K2317/92C07K2319/04C07K2319/40
Inventor 李桂英赵良中郜瑞娟赵佳亮刘子钰琚常青
Owner JILIN UNIV
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