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Nano-antibody GN1 specifically combined with GPC3 protein and preparation method and application thereof

A nanobody, GN1 technology, applied in the field of nanobody GN1 and its preparation, can solve the problems of insufficient stability, hindering the sensitivity of the antibody, and it is difficult for the antibody to achieve affinity.

Active Publication Date: 2020-03-10
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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  • Nano-antibody GN1 specifically combined with GPC3 protein and preparation method and application thereof
  • Nano-antibody GN1 specifically combined with GPC3 protein and preparation method and application thereof
  • Nano-antibody GN1 specifically combined with GPC3 protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Preparation of Nanobody GN1

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

[0067] (1) Immune alpaca:

[0068] Take 1 mg of GPC3 protein expressed by eukaryotic HEK293 cells and emulsify with complete Freund's adjuvant, totaling 2 mL, and inject a healthy adult alpaca 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 2mL, subcutaneous multi-point injection for the second immunization; then use 0.5mg GPC3 protein emulsified with Freund's incomplete adjuvant to obtain a total of 2mL of immunization emulsified injection every 7 days for next immunization Immunity once. A total of 6 immunizations were performed, and blood was collected on the 7th day after each immunization to detect the titer. After testing the serum titer, 100 mL of peripheral blood was collected. The inventors found that the protein expressed by eukaryoti...

Embodiment 2

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

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

[0094] (2) After washing the plate three times with PBST, add 300 μL of 5% skimmed milk to each well, and block at 37° C. for 1 hour.

[0095] (3) Add Nanobody GN1 of the present invention and commercialized GPC3 monoclonal antibody (GPC3-mAb, Invitrogen) to each well for 2 hours at 4°C, 37°C, 60°C, 70°C, 80°C, and 90°C. . 100 μL per well, after incubating at room temperature for 1 hour, wash the plate 3 times with PBST.

[0096] (4) In the GN1 group, because the GN1 antibody has a HA tag, add HRP enzyme-labeled HA-mAb (SANTA CRUZ company) to each well, incubate at room temperature for 40 minutes, wash the plate three times with PBST, add TMB for 10 minutes, and use 2M After sulfuric acid terminates the reaction, the microplate reader detects the ultraviolet absorbance (OD45...

Embodiment 3

[0098] Example 3: Establishment of a sandwich ELISA method for detecting serum GPC3 protein with GN1 nanobody-based GN1-luciferase fusion protein:

[0099] (1) Amplify the 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. The synthetic fluorescent reporter gene is Nano-luciferase gene (Nano-luciferase, referred to as Luc) (the amino acid sequence of the 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 digest the vector pET22b that contains fluorescent reporter gene nano-luciferase, cut the gel and reclaim the pET22b vector backbone sequence that contains luciferase gene; The vector backbone sequence ...

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Abstract

The invention relates to the technical field of biology, in particular to a nano-antibody GN1 specifically combined with a GPC3 protein. The nano-antibody GN1 is composed of a variable region of a heavy-chain antibody, the variable region of the heavy-chain antibody comprises an antigenic determinant complementary region and a skeleton region, the skeleton region is selected from the group consisting of FR1, FR2, FR3 and FR4 and homologous sequences thereof, the antigenic determinant complementary region is selected from the group consisting of CDR1, CDR2 and CDR3 and homologous sequences thereof, the amino acid sequences of the CDR1 to the CDR3 are shown in the formulas of SEQ ID NO. 1 to SEQ ID NO. 3, the amino acid sequences of the FR1-4 are shown in formulas of SEQ ID NO. 4-7, the amino acid sequence of the antibody is shown in the formula of SEQ ID NO.8, and the nucleotide sequence for encoding the amino acid is shown in the formula of SEQ ID NO.9. The nano-antibody GN1 can be specifically combined with hepatoma carcinoma cells highly expressing the GPC3 protein to inhibit hepatoma carcinoma cell proliferation. The amino acid sequence of the nano-antibody GN1 or the nucleotidesequence of the nano-antibody GN1 or the recombinant plasmid containing the nucleotide sequence of the nano-antibody GN1 or the recombinant cell containing the recombinant plasmid containing the nucleotide sequence of the nano-antibody GN1 can be applied to research and development of diagnostic reagents and drugs for treating liver cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nanobody GN1 specifically binding to GPC3 protein and its preparation method and application. Background technique [0002] Glypican 3 (glypican-3, GPC3) is a member of the glypican (Glypican) family, which is anchored on the cell surface by glycosylphosphatidylinositol (GPI). GPC3 is abnormally highly expressed in liver cancer cells, but its expression is limited in normal tissues. The high expression of GPC3 is positively correlated with the poor prognosis of liver cancer. 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 caused a revolution in tumor treatment. However, the traditional monoclonal antibody (150kD) has too large molecul...

Claims

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Application Information

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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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