Apoptotic protein fusion type anti-her-2 single chain antibody and its preparation method and application

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, easy aggregation, and insignificant effect of single-chain antibodies

Active Publication Date: 2021-08-13
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Abstract
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  • 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 and its preparation method and application
  • Apoptotic protein fusion type anti-her-2 single chain antibody and its preparation method and application
  • Apoptotic protein fusion type anti-her-2 single chain antibody and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of PET-32A (+) - SCFV recombinant plasmid

[0037] First, anti-HER-2 single-chain antibody SCFV homologous recombinant gene fragment

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

[0039] The NCBI and UNIPROT database find and document retrieval of the traston single antibody sequence and the Linker sequence, 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 shown in the light chain variable region VL and the flexible peptide shown in SEQ ID NO.3 (G 4 S) 3 Amino acid sequence;

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

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

Embodiment 2

[0054] Example 2: Construction of PET-32A (+) - CYTC-SCFV, PET-32A (+) - 3CYTC-SCFV and PET-32A (+) - DFF40-SCFV recombinant plasmid

[0055] First, experimental materials

[0056] 1. Primers required by PCR

[0057] The NCBI and UNIPROT database lookups that determine the cytochrome C (CYTC) amino acid sequence shown in SEQ ID NO.6 and cDNA sequences encoding the cytochrome C, such as SEQ ID NO., Is found by NCBI and UNIPROT database. It is determined that the DNA fragmentation factor 40 (DF40) amino acid sequence as shown in SEQ ID NO. The cDNA sequence encoding the DFF40 as shown in SEQ ID NO is; according to CDNA sequences of CYTC and DFF40 and PET-32A ( +) The sequence on the vector is designed and synthesized to amplify the PCR primer CYTC-F and CYTC-R for gene sequences of the above CYTC homologous recombination of the PET-32A (+) vector; and amplify the DFF40 for use in The PCR primer DFF40-F and DFF40-R of the gene sequence of the vector homologous recombination, the spec...

Embodiment 3

[0081] Example 3: Induced Expression and Identification of SCFV, CYTC-SCFV, 3CYTC-SCFV and DFF40-SCFV proteins

[0082] First, experimental materials

[0083] Escherichia coli expression strain sensitive cells BL21 (DE3) purchased from Beijing Tiangen biochemical technology; isopropyl-L-thio-β-D-pyran galactoside (IPTG) purchased from China Aladdin; Ni-NTA Resin Self-style gold biotechnology from Beijing;

[0084] Second, the experimental method

[0085] 1, SCFV, CYTC-SCFV, 3CYTC-SCFV and DFF40-SCFV protein induced expression

[0086] The yang-type PETs 32a (+)-CYTC-SCFV, PET-32A (+)-Cytc-Scfv, PET-32A (+)-3Cytc-ScFv and PET-32A (+) obtained in the first embodiment obtained. DFF40-SCFV is converted to Escherichia coli expression strain sensing state cells BL21 (DE3), induced by IPTG, induces SCFV, CYTC-SCFV, 3CYTC-SCFV, CYTC-SCFV, 3CYTC-SCFV and DFF40-SCFV protein expression, and passed Ni-NTA Resin. The two-dimensional structure of the purified protein, SCFV, CYTC-SCFV, 3CYTC-SCF...

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Abstract

The invention discloses an apoptosis protein fusion type anti-HER-2 single chain antibody and its preparation method and application. The apoptotic protein is cytochrome C or DNA fragmentation factor 40, which can be conjugated with anti-HER-2 single-chain antibody to form DFF40-scFv fusion single-chain antibody, nCytc (tandem)-scFv fusion single-chain antibody. The apoptotic protein fusion anti-HER-2 single chain antibody is inserted into the upstream of the scFv by the cDNA sequence of the apoptotic protein and cloned into the expression vector to construct a recombinant plasmid, and the recombinant plasmid is transferred into cells to induce the expression of the apoptotic protein fusion anti-HER-2 single chain antibody, the preparation method is easy to construct and express, and the constructed apoptotic protein fusion anti-HER-2 single-chain antibody can specifically target malignant tumors with high expression of HER-2 and mediate apoptosis of cancer cells, and can be used to prepare targeted Drugs to treat cancers with high expression of HER‑2.

Description

Technical field [0001] The present invention belongs to the field of biomedicine, and is specifically related to apoptotic protein fusion anti-HER-2 single-chain antibody and its preparation methods and applications. Background technique [0002] Breast cancer is the most common malignant tumor of women in the world, and the incidence of breast cancer in the 1970s has been an upward trend. On March 23, 2018, the latest data released by the National Cancer Center showed that there was 16.51% of women's malignant tumors in the country, ranging from the first place in women's 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, which is one of the most frequent cancer genes in human tumors, and its gene expression The number of levels and gene copies has significantly increased in several human tumor cells, especially in breast cancer, ovarian cancer and gastric cancer. Studies have s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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