Nano antibody and preparation method thereof

A nanobody and antibody technology, applied in the direction of antibodies, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as the advent of antibody drugs, and achieve the effects of high stability, high expression, and low immunogenicity

Active Publication Date: 2019-06-18
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, for ion channel targets, there is no mature antibody drug available so far

Method used

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  • Nano antibody and preparation method thereof
  • Nano antibody and preparation method thereof
  • Nano antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Embodiment 1 TRPV3 antigen preparation and animal immunization

[0109] (1) Antigen synthesis. Through chemical synthesis, two extramembrane regions of human TRPV3 protein were synthesized, namely CSYYRPREEEAIPHP (SEQ ID NO: 19) and SYGSFSDAVLELFKLTIC (SEQ ID NO: 20), as antigens for animal immunization.

[0110] (2) Alpacas are immune. Take 800ug of the polypeptide and mix it with an equal volume of Freund's adjuvant, and inject it into the hind leg muscle of the alpaca. Before immunization, 10 mL of blood was collected from the alpaca's marginal ear vein. Immunization was performed every two weeks, and a total of 4 immunizations were carried out. At the same time, the peripheral blood of the alpaca was collected, placed on ice, and transported back to the laboratory.

[0111] (3) ELISA detection of TRPV3 antibody production. After the 4 times of immunization, the ELISA method was used to detect whether the serum contained the enrichment of antibodies against TRPV3...

Embodiment 2

[0112] Phage display library construction of embodiment 2TRPV3 nanobody

[0113] (1) PBMC separation. Alpaca blood samples were taken before and after immunization, and the lymphocytes in the peripheral blood of the alpaca were separated and purified by Percoll density gradient centrifugation. After the cells were washed two to three times with PBS, RNA later was added for RNA extraction.

[0114] (2) TRPV3 nanobody library construction.

[0115] Total RNA extraction. Take the lymphocytes separated in (1), add 1ml Trizol, let stand at room temperature for 10min, add 0.2ml chloroform, shake vigorously, let stand at room temperature, wait for the solution to separate layers (about 10min), centrifuge at 12,000rpm, and collect the upper aqueous phase , add an equal volume of isopropanol, mix well, and let stand at room temperature for 15 minutes. After the nucleic acid is precipitated, centrifuge at a high speed to remove the supernatant. Add 1 ml of 75% ethanol (prepared with D...

Embodiment 3

[0127] Example 3 Obtaining High Affinity TRPV3 Nanobodies Using Phage Display Technology

[0128](1) Affinity TRPV3 nanobody phage library panning. Take 100ng of antigen to coat the ELISA plate and incubate overnight at 4°C. The next day, add the TRPV3 nanobody phage library obtained above, at room temperature, and incubate for 2 hours; wash the wells 10 times with PBST, add 100 μl triethylamine, room temperature, and incubate for 30 minutes. The collected phages are the TRPV3 nanobody phage library obtained by affinity panning ; Take 10 μl of infected TG cells to coat the plate for determining the number of clones after screening, and the remaining screened phage library is used for further amplification and rescue.

[0129] (2) Amplification and rescue of phage library after screening. The amplification and rescue method is the same as in Example 2 (4). The obtained phage library after the first round of screening is stored at 4°C and used for the second round of screening...

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Abstract

The invention provides a nano antibody. As for the nano antibody, (1) an amino acid sequence shown in SEQ ID NO:1 is achieved; or (2) compared with the (1), the at least 70% identity, the at least 75%identity, the at least 80% identity, the at least 85% identity, the at least 90% identity, the at least 95% identity, and the at least 99% identity are achieved. According to the embodiments, the nano antibody is an antibody specifically targeting an ion channel TRPV3, the native conformation TRPV3 can be combined, and the nano antibody has the high water solubility, high tolerance, high stability, high antigen bondability, low immunogenicity, high tissue penetrating power, and high expressivity.

Description

technical field [0001] The present invention relates to the field of biotechnology, specifically, the present invention relates to nanobodies and their preparation methods and applications, more specifically, the present invention relates to nanobodies, isolated nucleic acids, nucleic acid constructs, expression vectors, host cells, phages, drug combinations The use of substances, nanobodies in the preparation of medicines and methods for preparing nanobodies. Background technique [0002] Mammalian transient receptor potential vanilloid receptor 3 (transient receptor potential vanilloid, TRPV3), as a member of the transient receptor potential (TRP) family, is a type of ion channel that exists on the cell membrane or intracellular organelle membrane protein. Studies have found that TRPV3 plays an important role in mediating skin sensation, affecting the proliferation and differentiation of epidermal keratinocytes, hair growth, participating in inflammatory responses, and ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P17/00A61P1/02
Inventor 王媚娘刘楚新杨乃波曹丽霞丁权黄谧马莹莹
Owner SHENZHEN HUADA GENE INST
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