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Nanobody, encoding gene thereof, expression vector, host cell and application of nanobody

A technology of nano-antibody and coding gene, which is applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, application, etc. It can solve the problems of high production cost, large molecular weight, difficulty in rapid and uniform dispersion, etc. question

Active Publication Date: 2021-04-09
沈阳荣欣生物制药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Siglec-15 monoclonal antibody reported earlier is a complete antibody molecule. Due to its large molecular weight, it is difficult to quickly and evenly disperse in the internal tumor tissue and tumor stroma, and its structure is complex, suitable for cell expression, and the production cost is high.

Method used

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  • Nanobody, encoding gene thereof, expression vector, host cell and application of nanobody
  • Nanobody, encoding gene thereof, expression vector, host cell and application of nanobody
  • Nanobody, encoding gene thereof, expression vector, host cell and application of nanobody

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Embodiment 1

[0033] This embodiment provides an anti-Siglec-15 nanobody, which is a nanobody directed against a specific extracellular region of Siglec-15, and its amino acid sequence is as SEQ ID NO.8, SEQ ID NO.13, SEQ ID NO.18, As shown in SEQ ID NO.23, the nucleotide sequences encoding them are respectively shown in SEQ ID NO.7, SEQ ID NO.12, SEQ ID NO.17, and SEQ ID NO.22. Specifically, the nucleotide sequences encoding them are composed of 3 framework regions and 3 CDRs, wherein the nucleotide sequences of the 3 framework regions are respectively as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 As shown, the nucleotide sequences of the three CDRs are respectively as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11; SEQ ID NO. NO.14, shown in SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21; the nucleotide sequences of the three framework regions are shown in SEQ ID NO. 24 (corresponding to SEQ ID NO.1), SEQ ID NO.25 (correspon...

Embodiment 2

[0081] This embodiment provides a kind of screening method against Siglec-15 nanobody, it comprises:

[0082] Using the human phage nano-antibody library and using Siglec-15 as the antigen, three rounds of screening were carried out from the antibody library, and each round of screening was carried out according to the steps of adsorption-elution-amplification.

[0083] (1) Amplification of the VH nanobody library: Thaw the frozen phage antibody library on ice, pipette 400 μL into preheated 200 mL of 2×TY, and add 1% Glu and 1% Amp. Cultivate at 37°C, 250 rpm until OD600=0.4. Take 50 mL of bacterial solution and add 2×10 11 KM13 helper phages were incubated in a water bath at 37°C for 30 min. Centrifuge at 3000 g for 10 min to discard the supernatant, resuspend the pellet with 100 mL 2×TY, add 100 μg / mL Amp and 50 μg / mL Kan, and incubate overnight at 30°C and 250 rpm. The next day, the overnight bacterial solution was centrifuged at 3300 g for 30 min, and the precipitate w...

Embodiment 3

[0088] This embodiment provides a method for yeast expression and purification of an anti-Siglec-15 Nanobody, which includes the following steps:

[0089] S1. Three positive clones were sequenced and analyzed, cloned into pPICzαA yeast expression plasmid, and transformed into Pichia pastoris GS115 for soluble expression. The nucleotide and amino acid sequences are respectively SEQ ID: NO.7 and SEQ ID: NO.8; SEQ ID NO.12 and SEQ ID: NO.13; SEQ ID: NO.17 and SEQ ID: NO.18; NO .22 and SEQ ID: NO.23.

[0090] S2. Extract the PIT2-Siglec-15-VH recombinant plasmid and the pPICZα A plasmid respectively, use PIT2-Siglec-15-VH as a template PCR, carry out the amplification of the target fragment VH, use restriction endonucleases to carry out double enzyme digestion, gel After recovering the target fragment and the enzyme-digested vector, use T4 DNA ligase to connect the target gene fragment and the vector. Ethanol precipitated DNA; linearized electrotransformation of 5-20 µg of DNA i...

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Abstract

The invention is applicable to the technical field of biological genes, and provides a nanobody, an encoding gene thereof, an expression vector, a host cell and application of the nanobody. The nanobody is a nanobody aiming at a Siglec-15 extracellular specific region, and comprises a nanobody as shown in at least one of amino acid sequences SEQ ID NO.8, SEQ ID NO.13, SEQ ID NO.18 and SEQ ID NO.23. According to the invention, the nanobody that can be specifically bound with the extracellular region of Siglec-15 is obtained for Siglec-15, and the nanobody has huge potential in research and development of Siglec-15 therapeutic antibodies, preferably anti-tumor antibody therapy. The nanobody has strong and fast tissue penetrating capacity, and can enter compact solid tumor tissue to play a role; and the molecular structure of the nanobody is relatively simple, fermentation production can be performed through an escherichia coli or yeast expression system, and the production cost is greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of biological genes, and in particular relates to a nanobody and its coding gene, expression vector, host cell and application. Background technique [0002] Cancer is the main cause of death from diseases worldwide. According to the statistics of the International Union Against Cancer, about 9.6 million people die of cancer worldwide every year, far exceeding the sum of deaths from diseases such as AIDS, tuberculosis, and malaria. Relevant experts predict that by 2030, the number of people dying of cancer worldwide will reach 13 million every year. Immunotherapy has become the fourth major cancer treatment method after conventional treatments such as surgery, radiotherapy, and chemotherapy because of its small side effects, high antitumor activity, and wide indications. PD1 / PDL1 immunotherapy is currently a high-profile anti-cancer immunotherapy. PDL1 molecules are highly expressed on the surface of tumor ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00C12N15/70C12N15/81G01N33/574G01N33/68C12R1/84C12R1/19
CPCC07K16/2803C07K16/005C12N15/815C12N15/70G01N33/574G01N33/57484G01N33/6854A61P35/00A61K2039/505C07K2317/569C07K2317/565C07K2317/567G01N2333/70503C07K2317/94C07K2317/14
Inventor 王天怡姜海涛
Owner 沈阳荣欣生物制药科技有限公司
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