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Apoptotic protein fusion type anti-HER-2 single-chain antibody as well as preparation method and application thereof

A HER-2, single-chain antibody technology, applied in the direction of immunoglobulin, peptide/protein components, anti-enzyme immunoglobulin, etc., can solve the problems of poor stability of single-chain antibodies, cell death, high immunogenic side effects, etc. Achieve the effect of avoiding toxic side effects, high immunogenicity and low immunogenicity

Active Publication Date: 2020-11-17
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have successfully prepared anti-HER-2 single-chain antibodies for tumor targeting. However, single-chain antibodies have poor stability, easy aggregation, and short half-life.
[0004] Single-chain antibody scFv is also used as a drug delivery platform to deliver effector molecules to kill target cells, but the killing domains of many existing immunotoxins are mainly derived from plant or bacterial toxins, which have too large molecular weight, low tissue permeability, and high Immunogenicity and strong side effects limit its clinical application
In order to solve these problems, it has been reported that a series of endogenous apoptosis-related molecules can replace exogenous toxins. After a large number of literature studies, it was found that cytochrome c (Cytc) and DNA fragmentation factor 40 (DFF40) are involved in apoptosis. In the presence of dATP, cytochrome C released from Cytc to the cytoplasm can bind to apoptosis-related factor 1 (Apaf-1), making it form a multimer, and prompting caspase-9 to combine with it to form a The apoptotic body, caspase-9 is activated, and the activated caspase-9 can activate other caspases such as caspase-3, etc., thereby inducing cell apoptosis; DFF40 in apoptotic cells, after being activated by caspase-3, this The protein cleaves internucleosomal DNA, resulting in the characteristic DNA ladder, and since DFF40 is a downstream component in the apoptotic cascade, its expression and activation will lead to irreversible DNA damage leading to definitive cell death

Method used

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  • Apoptotic protein fusion type anti-HER-2 single-chain antibody as well as preparation method and application thereof
  • Apoptotic protein fusion type anti-HER-2 single-chain antibody as well as preparation method and application thereof
  • Apoptotic protein fusion type anti-HER-2 single-chain antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of pET-32a(+)-scFv recombinant plasmid

[0037] 1. Obtaining of scFv homologous recombination gene fragment of anti-HER-2 single chain antibody

[0038] 1.1 Heavy chain variable region VH, light chain variable region VL and flexible peptide (G 4 S) 3 sequence acquisition

[0039] Through NCBI and Uniprot database search and literature search, compare the trastuzumab single-chain antibody sequence and linker sequence, and determine the amino acid sequence of the heavy chain variable region VH shown in SEQ ID NO.1, such as SEQ ID NO.2 The amino acid sequence of the light chain variable region VL shown and the flexible peptide (G) shown in SEQ ID NO.3 4 S) 3 amino acid sequence;

[0040] Heavy chain variable region VH sequence (SEQ ID NO.1): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS;

[0041] Light chain variable region VL sequence (SEQ ID NO.2): DIQMTQSPSSLSASVGDRV...

Embodiment 2

[0054] Example 2: Construction of pET-32a(+)-Cytc-scFv, pET-32a(+)-3Cytc-scFv and pET-32a(+)-DFF40-scFv recombinant plasmids

[0055] 1. Experimental materials

[0056] 1. Primers required for PCR

[0057] Search and determine the cytochrome C (Cytc) amino acid sequence as shown in SEQ ID NO.6 and the cDNA sequence of coding described cytochrome C as shown in SEQ ID NO.7 by NCBI and Uniprot database search; Search by NCBI and Uniprot database Determine the amino acid sequence of DNA Fragmentation Factor 40 (DFF40) as shown in SEQ ID NO.8 and the cDNA sequence of encoding said DFF40 as shown in SEQ ID NO.9; According to the cDNA sequence of Cytc and DFF40 and pET-32a ( +) the sequence on the vector, design and synthesize PCR primers Cytc-F and Cytc-R for amplifying the gene sequence of the above-mentioned Cytc for homologous recombination with the pET-32a (+) vector; and amplify DFF40 for use with The PCR primers DFF40-F and DFF40-R of the gene sequence of vector homologous r...

Embodiment 3

[0081] Example 3: Induced expression and identification of scFv, Cytc-scFv, 3Cytc-scFv and DFF40-scFv proteins

[0082] 1. Experimental materials

[0083] Escherichia coli expression strain competent cells BL21 (DE3) were purchased from Beijing Tiangen Biochemical Technology; Isopropyl-L-thio-β-D-galactopyranoside (IPTG) was purchased from China Aladdin; NI-NTA Resin was purchased from From Beijing Quanshijin Biotechnology;

[0084] 2. Experimental method

[0085] 1. Induced expression of scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv proteins

[0086] The positive plasmid pET28a-scFv obtained in the above-mentioned Example 1 and the positive plasmid pET-32a(+)-Cytc-scFv, pET-32a(+)-3Cytc-scFv and pET-32a(+)- DFF40-scFv was transformed into Escherichia coli expression strain competent cells BL21(DE3), induced by IPTG and explored corresponding conditions to induce scFv, Cytc–scFv, 3Cytc–scFv and DFF40-scFv protein expression, and passed NI-NTA Resin Proteins purified by affini...

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Abstract

The invention discloses an apoptotic protein fusion type anti-HER-2 single-chain antibody as well as a preparation method and application thereof. The apoptotic protein fusion type anti-HER-2 single-chain antibody is formed by coupling an anti-HER2 single-chain antibody with apoptotic protein in different series modes, and the apoptotic protein is a cytochrome C or DNA fragmentation factor 40, canbe specifically coupled with the anti-HER-2 single-chain antibody to form a DFF40-scFv fusion type single-chain antibody and an nCytc (series)-scFv fusion type single-chain antibody. The apoptotic protein fusion type anti-HER-2 single-chain antibody is inserted into the upstream of scFv through a cDNA sequence of the apoptotic protein and cloned into an expression vector to construct recombinantplasmids, the recombinant plasmids are transferred into cells to induce expression of the apoptotic protein fusion type anti-HER-2 single-chain antibody, the preparation method is easy to construct and express, the constructed apoptotic protein fusion type anti-HER-2 single-chain antibody can specifically target HER-2 high-expression malignant tumors and mediate cancer cell apoptosis, and can be used for preparing drugs for targeted therapy of HER-2 high-expression cancers.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to an apoptosis protein fusion type anti-HER-2 single-chain antibody and its preparation method and application. Background technique [0002] Breast cancer is the most common malignant tumor in women all over the world, and the global incidence of breast cancer has been on the rise since the late 1970s. On March 23, 2018, the latest data released by the National Cancer Center showed that new cases of female breast cancer accounted for 16.51% of female malignant tumors nationwide, ranking first among female malignant tumors. HER-2 (HumanEpidermal Growth Factor receptor 2, also known as neu, ErBb-2, HER-2) is a member of the epidermal growth factor receptor superfamily and is one of the most frequently altered oncogenes in human tumors. Its gene expression Both levels and gene copy number are significantly increased in several human tumor cells, especially in breast, ovarian a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K47/68A61K38/17A61P35/00
CPCA61K38/1703A61K47/6811A61K47/6849A61K47/6871A61P35/00C07K14/4702C07K16/2863C07K16/32C07K16/40C07K2317/56C07K2317/622C07K2319/21C07K2319/33C12N15/70
Inventor 郭军陆丹丹郭艺辰郑子慧陈佳慧王婷婷王敏
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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