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A nanobody gn1 specifically binding to gpc3 protein and its preparation method and application

A nanobody, GN1 technology, applied in the field of nanobody GN1 and its preparation, can solve the problems of low effective concentration in the tumor area, insufficient stability, large molecular mass, etc. good thermal performance

Active Publication Date: 2022-06-17
GUANGXI UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional monoclonal antibodies (150kD) have too large a molecular weight and poor tumor tissue penetration, resulting in a low effective concentration in the tumor area, insufficient therapeutic effect, and high immunogenicity
It is difficult for the engineered antibody to achieve the original affinity, which hinders the sensitivity of antibody detection
In addition, many factors such as the long development cycle of fully humanized traditional antibodies, high production costs, and insufficient stability limit their clinical application.

Method used

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  • A nanobody gn1 specifically binding to gpc3 protein and its preparation method and application
  • A nanobody gn1 specifically binding to gpc3 protein and its preparation method and application
  • A nanobody gn1 specifically binding to gpc3 protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Preparation of Nanobody GN1

[0066] The preparation process of Nanobody GN1 includes the following steps:

[0067] (1) Immune alpacas:

[0068] 1 mg of GPC3 protein expressed by eukaryotic cells HEK293 cells was emulsified with Freund's complete adjuvant, a total of 2 mL, and a healthy adult alpaca was vaccinated for the first time by subcutaneous multi-point injection; on the 15th day, 0.5 mg of GPC3 protein Emulsified with Freund's complete adjuvant, a total of 2 mL, subcutaneously injected at multiple points for the second immunization; after that, 0.5 mg of GPC3 protein was emulsified with Freund's incomplete adjuvant every 7 days to obtain a total of 2 mL of emulsified injection for immunization. One immunization. A total of 6 immunizations were performed, and blood was collected on the 7th day after each immunization to detect the titer. After the serum titer was detected, 100 mL of peripheral blood was collected. The inventors found that the prote...

Embodiment 2

[0092] Example 2: Nanobody GN1 thermal stability experiment:

[0093] (1) Coating GPC3 protein, 1 μg / mL GPC3 protein, 100 μL per well was added to the ELISA plate, and coated overnight at 4°C.

[0094] (2) After the plate was washed three times with PBST, 300 μL of 5% skim milk was added to each well, and the plate was blocked at 37° C. for 1 hour.

[0095] (3) The nanobody GN1 of the present invention and the commercial GPC3 monoclonal antibody (GPC3-mAb, Invitrogen Company) treated at 4°C, 37°C, 60°C, 70°C, 80°C, and 90°C for 2 hours were added to each well. . 100 μL per well, after incubation at room temperature for 1 hour, the plate was washed 3 times with PBST.

[0096] (4) GN1 group, because the GN1 antibody has HA label, HRP enzyme-labeled HA-mAb (SANTA CRUZ company) was added to each well, incubated at room temperature for 40 minutes, washed three times with PBST, added TMB for color development for 10 minutes, and used 2M After terminating the reaction with sulfuri...

Embodiment 3

[0098] Example 3: Establishment of a sandwich ELISA method for the detection of serum GPC3 protein by GN1-luciferase fusion protein based on GN1 nanobody:

[0099] (1) Amplify GN1 gene. Using the plasmid obtained in step (4) of Example 1 as a template, the GN1 nucleotide sequence was amplified by PCR; the PCR product was digested with Nco I and Sfi I, and the PCR product purification kit was used to purify and recover the digested product.

[0100] (2) Construction of fusion gene GN1-Luc. A synthetic fluorescent reporter gene, Nano-luciferase (Luc for short) (the amino acid sequence of fluorescent protein reporter gene luciferase is shown in SEQ ID NO. 14): subcloned into the NotI and SalI sites of the vector pET22b Between, Nco I and Sfi I double enzyme cut the carrier pET22b containing the fluorescent reporter gene nano-luciferase, and cut the gel to recover the pET22b vector backbone sequence containing the luciferase gene; step (2) The recovered vector backbone sequence a...

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Abstract

The present invention relates to the field of biotechnology, in particular to a nanobody GN1 specifically binding to GPC3 protein; the nanobody GN1 is composed of a variable region of a heavy chain antibody; the variable region of the heavy chain antibody includes an epitope complementary region and framework region; said framework region is selected from the group consisting of FR1, FR2, FR3 and FR4 and homologous sequences thereof, said epitope complementary region is selected from the group consisting of CDR1, CDR2 and CDR3 and homologous sequences thereof; The amino acid sequence of CDR1 to CDR3 has the sequence shown in SEQ ID NO.1-3; the amino acid sequence of FR1-4 has the sequence shown in SEQ ID NO.4-7. The amino acid sequence of the antibody is SEQ ID NO.8, and the nucleotide sequence encoding the amino acid is SEQ ID NO.9. The nanobody GN1 of the present invention can specifically bind to liver cancer cells that highly express GPC3 protein, and inhibit the proliferation of liver cancer cells. The amino acid sequence of Nanobody GN1 or the nucleotide sequence of Nanobody GN1 or the recombinant plasmid containing its nucleotide sequence or the recombinant cell containing its nucleotide sequence recombinant plasmid can be applied to the research and development of the preparation of diagnostic reagents and the treatment of liver cancer. drug.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nanobody GN1 that specifically binds to GPC3 protein and a preparation method and application thereof. Background technique [0002] Glypican-3 (GPC3) is a member of the Glypican family and is attached to the cell surface through glycosylphosphatidylinositol (GPI) anchoring. GPC3 is abnormally highly expressed in hepatoma cells, but limited in normal tissues. The high expression of GPC3 is positively correlated with the poor prognosis of hepatocellular carcinoma. In addition, secreted GPC3 protein can be detected in the blood of liver cancer patients. Therefore, GPC3 has become a new target for the diagnosis and treatment of liver cancer. [0003] At present, the successful application of monoclonal antibodies in cancer detection and targeted therapy has led to a revolution in tumor therapy. However, the traditional monoclonal antibody (150kD) has too large molecular mass and po...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/70C12N1/21G01N33/574A61K39/395A61P35/00
CPCC07K16/303C07K16/005C12N15/70G01N33/57438A61P35/00C07K2317/565C07K2317/55C07K2317/569A61K2039/505
Inventor 段斯亮于声桂雄于亚婷
Owner GUANGXI UNIVERSITY OF TECHNOLOGY
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