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ALK nano antibody developed based on phage display technology and application thereof

A nano-antibody and antibody technology, applied in the field of biomedicine, can solve the problems of restricting the development of ALK antibody, long research and development cycle, and high production cost, and achieve the effect of optimizing prokaryotic expression system, high sensitivity and specificity, and reducing development and production costs

Active Publication Date: 2021-05-14
SHENZHEN HUADA GENE INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at "the development of ALK antibody in the prior art is concentrated on the traditional monoclonal antibody derived from rabbits, the traditional monoclonal antibody screening method is time-consuming and laborious, the traditional antibody cannot be expressed in the prokaryotic system, the research and development cycle is long, the production cost is high, and the batch-to-batch difference is large. , seriously restricting the development of ALK antibody in my country, and completely unable to meet the needs of diagnosis and treatment of NSCLC patients in my country.” This problem provides an ALK nanobody developed based on phage display technology and its application

Method used

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  • ALK nano antibody developed based on phage display technology and application thereof
  • ALK nano antibody developed based on phage display technology and application thereof
  • ALK nano antibody developed based on phage display technology and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1. Amplification and rescue of camelid natural nanobody phage display library

[0085] 1. Construction of phage recombinant plasmid library

[0086] Collect camel peripheral blood cells (PBMC), extract total RNA, use Nest-PCR technology to clone the V region of camel heavy chain antibody, insert it into phage plasmid pMECS, and construct a phage recombinant plasmid library.

[0087] The specific operation is as follows:

[0088] 1. Separation of blood lymphocyte samples The collected camel blood samples are separated into lymphocytes. The separation method is as follows:

[0089] (1) Add 7ml of lymphocyte separation medium Ficoll (GE, Cat. No. 17-1440-02) into a 15ml centrifuge tube;

[0090] (2) Add an equal volume of PBS (Sanko, Cat. No. B548117-0500) (1×) or normal saline to fresh whole blood that has been added with anticoagulant (EDTA), and mix thoroughly;

[0091] (3) Take the 15ml centrifuge tube added with lymphocyte separation medium, carefully and s...

Embodiment 2

[0116] Example 2, panning ALK fusion protein nanobodies with phage display technology

[0117] (1) Affinity ALK nanobody phage library panning

[0118] Take 500ng of ALK antigen (product of Shanghai Anyan Company, AY-78081P-1mg) to coat the ELISA plate, and incubate overnight at 4°C. The next day, add the natural nanobody phage display library rescued in Example 1, incubate at room temperature for 2 hours; wash the wells 10 times with PBST, add 100 μl triethylamine, incubate at room temperature for 15 minutes, and the collected phages are ALK obtained by affinity panning Nanobody phage library: Take 10 μl of infected E. coli TG1 cells to coat the plate for determining the number of clones after screening, and the remaining screened phages are used for amplification.

[0119] (2) Amplification and rescue of phage after screening

[0120] The amplification and rescue method is the same as in Example 1. The obtained PBS suspension is the amplified phage after the first round of...

Embodiment 3

[0129] Example 3, Induced Expression and Purification of ALK Nanobody

[0130] Since pMECS has an amber terminator (TAG) between the HA tag and the M13 GIII gene, ordinary expression systems cannot effectively recognize the terminator, thereby effectively expressing the Nanobody protein. The invention optimizes the prokaryotic expression system, and uses the Escherichia coli prokaryotic expression system ALK nanobody F10 to induce expression and purify.

[0131] 1. Expression of ALK Nanobody

[0132] The recombinant Escherichia coli expressing Nanobody monoclonal F10 obtained in Example 2 was inoculated into 15 ml of TB culture medium (INVITROGEN, product number 22711022), and cultured at 37°C overnight to OD 6000.6-0.8, transfer the inoculated 15ml TB culture medium to 400ml TB culture medium, add Ampicillin resistance and incubate at 37°C for 2h, then add IPTG with a final concentration of 1mM to induce expression at 25°C for 5h. Centrifuge the bacterial liquid, collect th...

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Abstract

The invention discloses an ALK nano antibody developed based on a phage display technology and an application of the ALK nano antibody. The nano antibody provided by the invention comprises a complementarity determining region consisting of a CDR1, a CDR2 and a CDR3, the amino acid sequence of the CDR1 is the 21st-28th site of SEQ ID No.1; the amino acid sequence of the CDR2 is from the 46th-52nd site of SEQ ID No.1; and the amino acid sequence of the CDR3 is the 91th-107th sites of SEQ ID No.1. A camel nano antibody natural library is constructed by utilizing camel PBMC cells, and high-affinity ALK nano antibody phage clones are screened from the library by utilizing a phage display technology and are converted into an escherichia coli expression system for mass expression, so that the development and production cost of the ALK antibody is effectively reduced, the immunohistochemical verification of a clinical sample shows that the ALK nano antibody disclosed by the invention has higher sensitivity and specificity.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an ALK nanobody developed based on phage display technology and an application thereof. Background technique [0002] In the past decade, lung cancer has become one of the common malignant tumors that seriously endanger human health, and its morbidity and mortality have been increasing. According to the 2018 Health Statistical Yearbook, there are more than 700,000 new cases of lung cancer in my country every year, and the death rate ranks first in the death rate of malignant tumors in my country. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for about 85%, and the 5-year survival rate is only 15%. According to different histological types, NSCLC can be divided into adenocarcinoma (≥40%), squamous cell carcinoma (30%) and relatively rare large cell carcinoma (LCC) (10%). With the increasingly different individualized treatment options for lung a...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N15/13G01N33/574
CPCC07K16/40G01N33/57484G01N33/57423C07K2317/569C07K2317/22C07K2317/92C07K2317/565C07K2317/567
Inventor 任丙昭欧阳冰洁于颖佳范广益王媚娘李新洋杨乃波刘心
Owner SHENZHEN HUADA GENE INST
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