Goose-array type localized random mutation method and application thereof in monoclonal antibody molecular evolution technology

A technology of random mutation and molecular evolution, applied in the biological field, can solve the problems of long time, huge investment and high risk

Inactive Publication Date: 2011-05-11
DANYANG ZHENGYUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Type ① is already behind, the success rate of Type ② is too low, Type ③ is too complicated, requires huge investment, and takes a long time, high cost and high risk in the preliminary work
It is worth pointing out that these cases are based on the existing commercialized therapeutic monoclonal antibody as the prototype molecule, and the mutation scheme of molecular evolution adopts site-directed mutation (21), random mutation (11-15), shuffling (16 , 20), CDR-grafting (17) and other methods, the effective mutation efficiency is low, the technical difficulty is high, and the workload is large, and the obtained products need to be cross-licensed before they can be commercially produced and sold

Method used

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  • Goose-array type localized random mutation method and application thereof in monoclonal antibody molecular evolution technology
  • Goose-array type localized random mutation method and application thereof in monoclonal antibody molecular evolution technology
  • Goose-array type localized random mutation method and application thereof in monoclonal antibody molecular evolution technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Example 1 Construction of a fully human ultra-large-scale antibody library

[0131] The construction of a fully human ultra-large-scale antibody library was carried out according to the methods in references 1-10. The specific operation is as follows:

[0132] 1. Blood Sample Collection and cDNA Synthesis

[0133] Collect 1ml of peripheral blood from 3000 people, mix them, and separate mononuclear cells with lymphocyte separation medium (produced by Tianjin Institute of Blood, Academy of Medical Sciences). Total mRNA was extracted from isolated human peripheral blood lymphocytes using a kit from GIBCO. The mRNA purification kit from GIBCO was used to purify, and the mRNA obtained above was used as a template to reverse transcribe the first strand of cDNA. The above steps were carried out according to the instructions provided by the manufacturer.

[0134] 2. PCR amplification

[0135] 1. Primer Design

[0136] In order to construct a fully human Fab antibody libra...

Embodiment 2

[0160] The fully human anti-human TNFa monoclonal antibody gene of embodiment 2 people's B cell origin obtains

[0161] In addition to the above-mentioned pathways, there are other sources of fully human monoclonal antibody genes for some antigens, such as human leukocytes that secrete anti-human TNFα monoclonal antibodies.

[0162] 1. Blood samples and their initial screening

[0163] Take 5 milliliters of peripheral blood from patients with active rheumatoid arthritis, separate leukocytes with lymphocyte separation medium, culture them, and identify positive clones according to the results of ELISA.

[0164] In order to obtain human B cells that secrete anti-human TNFα, a 96-well plate was routinely coated with recombinant human TNF (Shanghai Xinbainuo Bioengineering Co., Ltd.), and each well was coated with 250ng of the protein overnight. Then, it was blocked with 5% skimmed milk powder at room temperature for 2 hours, and the milk powder was prepared with pH7.2 PBS. Afte...

Embodiment 3

[0190] Example 3, panning and screening anti-human TNFα monoclonal antibody from a fully human antibody library

[0191] Based on the antibody library established in Examples 1 and 2, the operation steps of panning are as follows. During panning and other processes, the Fab form of Humira was used as a positive control.

[0192] 1. Add 1 ml of recovered antibody library strains to 14 ml of fresh LB medium, and culture in a 50 ml Erlenmeyer flask at 37°C for 16 hours.

[0193] 2. Centrifuge at high speed at 12000rpm for 10 minutes, transfer the supernatant to a sterile 50ml centrifuge tube, and save it for later use. Its titer should be above 2X10E11.

[0194] 3. Using purified recombinant human TNFa (provided by Shanghai Xinbainuo Company) as an antigen, the 25 ml cell culture flask was coated by a conventional method. Add no less than 3X10E10 phage particles to the coated cell bottle and incubate at 37°C for 1 hour.

[0195] 4. Pour off the liquid in the bottle, and wash ...

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Abstract

The invention discloses a goose-array type localized random mutation method and an application thereof in the monoclonal antibody molecular evolution technology. The goose-array type localized random mutation method provided by the invention comprises the following steps: designing a goose-array type primer of a target sequence of random mutation polymerase chain reaction (PCR) amplification, synthesizing the primer, mixing, and performing overlapping PCR to obtain the amplified sequence with random mutation in a designated area. The goose-array type localized random mutation method provided by the invention can be used in the monoclonal antibody molecular evolution. The invention further combines the key amino acid (KA) scanning, the goose-array type localized random mutation method and the phage display technology to provide a monoclonal antibody molecular evolution method. The mutation method provided by the invention is used in molecular evolution and has high speed, high success rate and wide application range.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a random mutation method and its application in the molecular evolution technology of monoclonal antibodies. Background technique [0002] (1) Therapeutic monoclonal antibody [0003] Since the first therapeutic monoclonal antibody drug was born in 1986, the industrialization of therapeutic monoclonal antibodies has achieved great success, which can be clarified from the following data. [0004] The number of products is increasing dramatically. In the U.S., nearly 30 therapeutic monoclonal antibodies have been approved for marketing, and those that have entered clinical research are currently 123 monoclonal antibodies that have entered clinical research, of which 23 have entered phase III clinical trials or new drug application; Therapeutic monoclonal antibody drugs in clinical research are still increasing rapidly. There are more than 700 products under development, of which mor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12N15/13
CPCC07K16/00C12N15/1031C12N15/1034
Inventor 刘庆法吴希美
Owner DANYANG ZHENGYUAN BIOTECH
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