Nanobody GN2 composed of variable region of heavy chain antibody and preparation method and application of nanobody GN2

A nanobody and heavy chain antibody technology, applied in the biological field, can solve the problems of high production cost, strong immunogenicity, and large molecular weight

Active Publication Date: 2020-06-23
GUANGXI UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to constraints such as high production costs, strong immunogenicity, large molecular weight, and poo...

Method used

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  • Nanobody GN2 composed of variable region of heavy chain antibody and preparation method and application of nanobody GN2
  • Nanobody GN2 composed of variable region of heavy chain antibody and preparation method and application of nanobody GN2
  • Nanobody GN2 composed of variable region of heavy chain antibody and preparation method and application of nanobody GN2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Preparation of Nanobody GN2

[0075] Concrete preparation process comprises the following steps:

[0076] (1) Immune alpaca:

[0077] Take 1 mg of GPC3 protein expressed by eukaryotic HEK293 cells and emulsify with complete Freund's adjuvant, totaling 2 mL, and immunize 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 eukaryotic cells u...

Embodiment 2

[0104] Embodiment 2: nanobody GN2 thermal stability experiment:

[0105] Coat GPC3 protein on the ELISA well plate, 1 μg / mL GPC3 protein, add 100 μL per well to the microtiter plate, and coat overnight at 4°C. After the plate was washed three times with PBST, 300 μL of 5% skimmed milk was added to each well, and blocked for 1 hour at 37°C. Nanobody GN2 of the present invention and commercialized GPC3 monoclonal antibody (GPC3-mAb, Invitrogen) treated at different temperatures (4°C, 37°C, 60°C, 70°C, 80°C, 90°C) for 2 hours were added to each well. Add 100 μL to each well, incubate at room temperature for 1 hour, and wash the plate 3 times with PBST.

[0106] In the GN2 group, because the GN2 antibody has an HA tag, add HRP enzyme-labeled HA-mAb (SANTACRUZ company) to each well, incubate at room temperature for 40 minutes, wash the plate three times with PBST, add TMB for 10 minutes to develop color, and stop the reaction with 2M sulfuric acid. , the microplate reader detects...

Embodiment 3

[0108] Example 3: Establishment of a sandwich ELISA method for detecting serum GPC3 protein with GN2 nanobody-based GN2-luciferase fusion protein:

[0109] (1) Amplify the GN2 gene. Using the plasmid obtained in step (4) of Example 1 as a template, the GN2 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.

[0110] (2) Construction of fusion gene GN2-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 present invention relates to a nanobody GN2 composed of a variable region of a heavy chain antibody; and the variable region of the heavy chain antibody comprises an epitope complementary region selected from a group consisting of CDR1, CDR2 and CDR3 and homologous sequences of the CDR1, CDR2 and CDR3 and a skeleton region selected from a group consisting of FR1, FR2, FR3 and FR4 and homologous sequences of the FR1, FR2, FR3 and FR4. The present invention also relates to a preparation method and an application of the nanobody GN2. The nanobody GN2 can specifically bind to hepatocarcinoma cells that highly express GPC3 protein, inhibits hepatocarcinoma cell proliferation, and is small in molecular weight, stable in structure, good in heat resistance, high in affinity, easy for storage and transportation, weak in immunogenicity to human body, also strong in tumor tissue penetrating power, liable to express and genetically engineered, and particularly suitable for use as a diagnosticreagent or a therapeutic antibody. The method can retain more antigen epitopes, and is high in carrier quality, good in an enzyme digestion effect, high in connection efficiency, low in self-connection efficiency and also low in cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nanobody composed of the variable region of a heavy chain antibody and its preparation method and application. Background technique [0002] Glypican 3 (GPC3) is a glycoprotein attached to the cell surface via glycosylphosphatidylinositol (GPI) anchors, belonging to the heparan sulfate glycoprotein (HSPG) family. GPC3 is related to the occurrence and existence of liver cancer. GPC3 is highly expressed in the tissues and serum of patients with liver cancer, but not in normal liver tissue or serum. Compared with alpha-fetoprotein, GPC3 may have better practical value in detecting early liver cancer. Therefore, GPC3 has become an auxiliary diagnostic marker and therapeutic target for liver cancer. [0003] Monoclonal antibodies have shown broad application prospects in the diagnosis and treatment of diseases. However, due to constraints such as high production costs, s...

Claims

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Application Information

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IPC IPC(8): C07K16/30C07K19/00C12N15/13C12N15/70A61K39/395A61P35/00A61P1/16G01N33/574
CPCC07K16/303C12N15/70A61P35/00A61P1/16G01N33/57438G01N33/57492C07K2317/569C07K2317/565C07K2317/567C07K2317/22C07K2317/94C07K2317/92C07K2317/73C07K2319/60G01N2333/705
Inventor 段斯亮于声桂雄
Owner GUANGXI UNIVERSITY OF TECHNOLOGY
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