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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 the problems of toxic and side effects of anti-PCSK9 protein monoclonal antibodies, and achieve low immunogenicity and high water solubility. , the effect of high antigen binding

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

So far, no obvious side effects have been found with anti-PCSK9 protein monoclonal antibodies, only minor side effects such as local injection reactions, diarrhea and headache have been reported

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

[0099] Example 1 PCSK9 nanobody phage display library construction;

[0100] (1) PCSK9 immune camel

[0101] Mix 1mg of PCSK9 with an equal volume of Freund's adjuvant to 5ml, inject it at 3-5 points under the skin of the camel's neck, and collect blood from the ear vein of the camel before immunization. Immunize once a month, and immunize 4 times in total; take 10ml of peripheral blood of camels for each immunization. Fix the camel's head to one side when collecting blood, shave (pluck) the skin of the animal's blood collection site first, disinfect with 75% alcohol, collect blood after drying, and press the jugular vein groove with your fingers. The site was sterilized and the blood was drawn through the needle, and 10ml of blood was collected in a 15ml EDTA anticoagulant tube, which was shaken continuously and slowly, mixed thoroughly, placed on ice, and transported back to the laboratory.

[0102] (2) Separation of blood lymphocyte samples

[0103] Lymphocytes were sepa...

Embodiment 2

[0141] Example 2 Elutriation of PCSK9 Nanobody Antibody Using Phage Display Technology

[0142] (1) Affinity PCSK9 nanobody phage library panning

[0143] Take 100ng PCSK9 antigen coated ELISA plate, 4 ℃, overnight incubation. The next day, add the rescued PCSK9 nanobody phage, 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 PCSK9 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 phages are used for amplification.

[0144] (2) Amplification and rescue of phage after screening

[0145] The amplification and rescue method is the same as in Example 1 (7). The obtained PBS suspension is the amplified phage after the first round of screening, stored at 4°C, and used for the next round of screening; according to the...

Embodiment 3

[0164] Example 3 Induced Expression and Purification of PCSK9 Nanobody

[0165] (1) Construction of PCSK9 Nanobody Expression Bacteria

[0166] First, culture the PCSK9 nanobody monoclonal transfer medium at 37°C overnight; the next day, use Plasmid Mini Kit I (OMEGA) was used to extract the plasmid, and after agarose gel electrophoresis and concentration determination, the plasmid containing the PCSK9 nanobody sequence was transformed into expression bacteria HB2151, and the plate was spread and incubated overnight at 37°C.

[0167] (2) Induced expression of PCSK9 nanobody

[0168] The next day, pick 5 clones from the plate for clone PCR to verify whether the plasmid is transferred into the expression strain; pick positive clones and culture them at 37°C until OD 600 0.6-0.8, add IPTG to induce expression. Centrifuge the bacterial liquid, collect the bacterial pellet, resuspend the pellet with lysis buffer, disrupt the bacterial cells by ultrasonic, and collect the supern...

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Abstract

The invention provides a nano antibody. The nano antibody comprises the characteristics that (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. The nano antibody is an antibody specifically targeting PCSK9, the native conformation PCSK9 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] The drugs currently on the market for lowering cholesterol mainly include statins, cholesterol absorption inhibitors, probucol, etc. Although statins have excellent performance in the treatment of cardiovascular diseases, with their widespread use, possible disadvantages have gradually been discovered. First, patients with intensive statin therapy still have a high residual risk of cardiovascular events, with a risk of 22.4% within 2 years; second, a large number of patients cannot tolera...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N15/13A61K39/395A61P9/00A61P3/06A61P9/10
Inventor 王媚娘杨乃波曹丽霞黄谧刘楚新张婷婷李新洋李波侯勇
Owner SHENZHEN HUADA GENE INST
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