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CH2 structure domain mutant of human IgG antibody Fc segment as well as preparation method and application thereof

A technology of mutants and structural domains, which is applied in the field of human IgG antibody Fc fragment CH2 domain mutants and preparations, can solve the problems of weak anti-aggregation ability and poor stability of Fc fragments, and achieve good stability, good anti-aggregation ability, The effect of reducing the risk of clinical use

Active Publication Date: 2018-10-19
武汉班科生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the defects of poor stability and weak anti-aggregation ability of the Fc fragment in the prior art, and provide a mutant of the CH2 domain of the Fc fragment of a human IgG antibody, its preparation method and application. Compared with wild-type Fc, it is more stable The anti-aggregation ability and anti-aggregation ability are both improved, which is of great significance in the research and development, production and clinical application of miniaturized antibodies

Method used

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  • CH2 structure domain mutant of human IgG antibody Fc segment as well as preparation method and application thereof
  • CH2 structure domain mutant of human IgG antibody Fc segment as well as preparation method and application thereof
  • CH2 structure domain mutant of human IgG antibody Fc segment as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of the C-terminal phage display library of CH2

[0041] According to the CH2 gene sequence of IgG (GenBank: AAC82527.1), analyze its amino acid sequence, design the forward and reverse primers of the CH2 gene to amplify the target fragment (the horizontal line marks the Sfi I restriction site):

[0042] Forward primer (5`end to 3`end):

[0043] GT G GCC CAG GCGGCC GCACCTGAA CTC CTGGGGGGA CCG TCA GTCTTC CTCTTC-

[0044] Reverse primer (5`end to 3`end):

[0045]

[0046] The amplified fragment was digested with SfiI and ligated into the vector pComb3xSS to construct a phage display library.

Embodiment 2

[0047] Embodiment 2: candidate clone screening

[0048] 1. Panning experiment

[0049] 1) Take 5 tubes of TG1 bacteria, 2ml each. Shake to OD in about 4 hours 600 = 0.6.

[0050] 2) The phage library was first heated at 80°C for 10 minutes, and then left at room temperature for 20 minutes.

[0051] 3) Closed Panning orifice plate

[0052] a. Aspirate the coated protein and wash the plate once with PBS.

[0053] b. Add phage to BSA wells, 10 wells in 5 wells 12The final concentration of milk was 2%, 100 μl per well. Add 3% milk to anti-CH2 wells, 200μl per well.

[0054] c. Block for 1 hour at 37°C.

[0055] 4) combine

[0056] BSA well phages were transferred to anti-CH2 wells, 2h, 37°C.

[0057] 5) Elution

[0058] The phage liquid was discarded, and washed 10 times with PBST (5 times for each round, 4 rounds of screening in total).

[0059] 6) Infection

[0060] In the ultra-clean bench, add 100 μl of TG1 bacterial solution to each well of the Panning orifice pl...

Embodiment 3

[0083] Example 3: CH2 and KIKK molecular conformation and existing form (monomer, dimer, etc.)

[0084] AKTA analysis of the existing forms of CH2 and KIKK: Concentrate the purified CH2 and KIKK proteins to 1 mg / ml, use PBS (pH7.4) as the elution buffer, pass through Column Superdex 75Increase 10 / 300GL, and the flow rate is 1ml / min, Then detect its existence form, such as figure 1 , compared with the standard curve, it can be found that the molecular weight of the two proteins is about 14kDa, and the results show that they exist in the form of monomers.

[0085] CD detection of protein molecular conformation: Dilute CH2 and KIKK proteins to 0.3 mg / ml, PBS (pH7.4) as a control, and detect their circular dichroism under different wavelength conditions at the near ultraviolet end (wavelength λ=190nm-260nm). Then analyze the protein secondary structure. like figure 2 As shown, there is an obvious trough at λ=218nm, indicating that the secondary structure of the above protein i...

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Abstract

The invention discloses a CH2 structure domain mutant of a human IgG antibody Fc segment as well as a preparation method and application thereof. A CH2 segment of an IgG antibody is taken as an object, the carboxyl-terminal amino acid of the CH2 segment is modified to obtain a new CH2 framework. The CH2 structure domain mutant has the advantages that the CH2 framework has higher stability than that of wild type CH2, so that more stable C-type single-domain antibodies are developed, and production, purification and storage costs of monoclonal antibodies or Fc fusion protein can be reduced; theanti-aggregating capability is high, and the clinical application risk caused by protein aggregation can be reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant of the CH2 domain of the Fc segment of a human IgG antibody, a preparation method and an application thereof. Background technique [0002] With its strong specificity and high sensitivity, antibodies are widely used in various fields, especially in life science research and clinical treatment such as ELISA, Western blot, immunofluorescence analysis, rapid diagnosis of diseases, and as biological agents to treat various diseases. Therefore, since entering the 21st century, people have begun to pay more and more attention to the research and development and clinical application of therapeutic antibody drugs, because it has less toxic and side effects on the human body, has natural and highly specific curative effects, and has created a huge social benefits and economic benefits. At the same time, the miniaturized antibody developed based on the CH2 fragment has good tissue p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K19/00
CPCC07K16/18C07K2317/524C07K2319/30
Inventor 龚睿
Owner 武汉班科生物技术有限公司
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