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Single domain antibody and detection kit for human epidermal growth factor receptor 2 and application of single domain antibody

A technology of epidermal growth factor and single-domain antibody, which is applied in the direction of antibodies, anti-tumor drugs, measuring devices, etc., and can solve the problems of lack of antibodies, limited detection of tumor treatment, etc.

Active Publication Date: 2020-10-27
圣至润合(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently a lack of antibodies with strong affinity for Her2 protein, which greatly limits the detection of serum Her2 protein levels and the treatment of tumors caused by Her2 protein.

Method used

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  • Single domain antibody and detection kit for human epidermal growth factor receptor 2 and application of single domain antibody
  • Single domain antibody and detection kit for human epidermal growth factor receptor 2 and application of single domain antibody
  • Single domain antibody and detection kit for human epidermal growth factor receptor 2 and application of single domain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Screening process for Her2-specific single domain antibodies:

[0046] (1) Cultivate breast cancer cell line BT474 with T25 cell culture flasks, treat the cells to grow to the logarithmic growth phase, and use PBST (0.1% T 20 PBS) for 2 washes, add 2% BSA-PBST, place at 37°C for 1 hour, suck out the liquid, add 2ml phage (2×10 12 PFU non-immunized alpaca single-domain antibody phage display gene library, 2% BSA-PBST), at room temperature for 1 hour.

[0047] (2) Discard the supernatant, wash 10 times with PBST, and then wash 10 times with PBS.

[0048] (3) Use TEA (triethylamine (7.18M)) solution to elute the phage bound to BT474 cells, infect Escherichia coli TG1 in the logarithmic growth phase, take 2 μl, dilute 100 times, coat 2YT-Amp plates, and calculate The recovery efficiency was screened, and the eluted phages were amplified and purified for the next round of screening. Cell selection was performed for 2 rounds.

[0049] (4) Her2-ECD protein (Cat: 10004-H08H...

Embodiment 2

[0053] Use phage enzyme-linked immunosorbent assay (ELISA) to screen Her2-ECD-specific single positive clones:

[0054] (1) From the bacterial culture dish containing phage after two rounds of screening in step (6) of the above-mentioned embodiment 1, select a single colony and inoculate it in a 96-well deep-well bacterial culture plate, cultivate it for 3 hours, and add helper phage M13 , cultivated overnight, centrifuged at 4000 rpm for 20 minutes.

[0055] (2) Transfer the supernatant to a 96-well ELISA plate pre-coated with Her2-ECD protein and blocked with 2% MPBST, and place it at room temperature or 37 degrees for 1 hour. Unbound phages were washed away with 0.05% PBST.

[0056] (3) Add horseradish peroxidase-labeled anti-phage secondary antibody diluted 1:3000 times with 1% MPBST, and place at room temperature or 37 degrees for 1 hour. Unbound phages were washed away with 0.05% PBST.

[0057] (4) TMB color development, read the absorbance value (OD) at a wavelength ...

Embodiment 3

[0062] Construction method of specific nanobody expression plasmid

[0063] The specific Nanobody gene obtained in PCR amplification embodiment 2, and obtain the PCR product with restriction endonuclease BbsI and BamHI site, process PCR product and carrier (pSJF2 respectively) with restriction endonuclease BbsI and BamHI Vector, kim Is.Biosic Biochem.2002,66(5):1148-51), was connected and recombined by T4 ligase to obtain the plasmid pHxA5 highly expressed in Escherichia coli, and the humanized expression plasmid HpHx5A, and carried out Gene sequence determination to determine the correctness of its sequence. Primers used for PCR amplification: upstream primer: GAAGACACCAGGCCCAGGTRMAGCTGGWGGAGTCT (SEQ ID No. 12); downstream primer gaagatctccggatccTGAGGAGACGGTGACCTGGGT (SEQ ID No. 13). PCR conditions: 94°C, 3 minutes; 94°C, 30 seconds, 72°C, 40 seconds, 55°C, 30 seconds, a total of 30 cycles, then 72°C, 7 minutes, 4°C storage.

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Abstract

The invention provides a single domain antibody and detection kit for a human epidermal growth factor receptor 2 and application of the single domain antibody, and belongs to the technical field of antibodies. The single domain antibody of the human epidermal growth factor receptor 2 comprises the following solutions: A, the single domain antibody is a single domain antibody with amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 shown by SEQ ID No.5-7; B, the single domain antibody is a polypeptide with at least 80% similarity to the amino acid sequence of the single domain antibody in the solution A. The invention further provides a humanized single domain antibody HpHx5A. Based on high affinity of the single domain antibody and the humanized single-domain antibody HpHx5A with Her2-ECD, a material foundation is laid for subsequent application of the single domain antibody to detection of the level of the human epidermal growth factor receptor 2, tumor classification, early judgment and prognosis evaluation of breast carcinoma recurrence and metastasis or preparation of drugs for preventing and / or treating tumors caused by the human epidermal growth factor receptor 2.

Description

technical field [0001] The invention belongs to the technical field of antibodies, and in particular relates to a single-domain antibody of human epidermal growth factor receptor 2, a detection kit and an application thereof. Background technique [0002] Studies have found that the oncogene CerbB2 encodes a transmembrane glycoprotein, and its encoding gene is located on human chromosome 17q21. The transmembrane glycoprotein has a molecular weight of 185KD (P185 for short), called epidermal growth factor receptor 2 (Her2), and belongs to one of the members of the epidermal growth factor receptor (EGFR) family, and its intracellular segment has a receptor tyrosine kinase ( receptor tyrosinekinase (RTK) activity, its extracellular segment is the ligand-binding domain, after the ligand binds to the Her2 receptor, it will autophosphorylate the Her2 receptor and activate its tyrosine kinase activity, ultimately promoting cell proliferation and overexpression of the Her2 gene. Ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/32C12N15/70G01N33/574A61K39/395A61P35/00
CPCA61P35/00C07K16/32C07K2317/14C07K2317/22C07K2317/24C07K2317/565C07K2317/569C07K2317/76C07K2317/92C12N15/70G01N33/574G01N33/57415G01N33/5748
Inventor 徐绍梅李胜华汪劲松许莎莎
Owner 圣至润合(北京)生物科技有限公司
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