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Enhanced CH2 structure domain mutant combined with newborn Fc receptor as well as preparation method and application thereof

A receptor binding and structural domain technology, applied in the biological field, can solve the problems of weak CH2 stability and achieve the effect of pH-dependent binding enhancement, good stability, and good anti-aggregation ability

Active Publication Date: 2018-06-01
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

[0005] CH2 is less stable and needs to be optimized

Method used

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  • Enhanced CH2 structure domain mutant combined with newborn Fc receptor as well as preparation method and application thereof
  • Enhanced CH2 structure domain mutant combined with newborn Fc receptor as well as preparation method and application thereof
  • Enhanced CH2 structure domain mutant combined with newborn Fc receptor as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction of yeast display library

[0027] Using the base sequence of the m01s backbone as a template (its gene sequence is Seq ID No.2 and its amino acid sequence is Seq ID No.1), through the combination of site-directed mutation and random mutation, according to the existing literature (Chao G, et al ., NatProtoc., 2006), constructing a yeast display library.

Embodiment 2

[0028] Embodiment 2: flow cytometry sorting

[0029] With the soluble expressed recombinant human FcRn protein as the target, the yeast cells that can bind to FcRn are sorted out by flow cytometry, and the next round of flow sorting is carried out after expanding culture, and the final four rounds of screening Finally, the polyclonal yeast was analyzed by flow cytometry, the enriched clones were further identified, and a mutant was identified, which was named m01sHLEm1.

[0030] The amino acid sequence of m01sHLEm1 is Seq ID No.3, and the gene sequence is Seq ID No.4.

Embodiment 3

[0031] Example 3: Prokaryotic expression and purification of m01sHLEm1 skeleton protein

[0032] The enriched clones were amplified by PCR to obtain the target fragment, that is, the base sequence of m01sHLEm1, and then the target fragment and the pCom empty load were digested by the restriction endonuclease sfi I, and the digested target fragment and the empty load were recovered. , under the action of T4 ligase, ligate the target fragment with empty load overnight at 16°C.

[0033] After the clone to be expressed was constructed, the recombinant plasmid was transferred to Escherichia coli HB2151 competent cells, and SB medium was used for protein expression.

[0034] The prokaryotic expressed m01sHLEm1 protein was purified by nickel column, the eluate of different imidazoles was detected by SDS-PAGE, and the m01sHLEm1 protein eluate was concentrated by ultrafiltration and exchanged according to the results of Coomassie brilliant blue staining.

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Abstract

The invention discloses an enhanced CH2 structure domain mutant combined with a newborn Fc receptor as well as a preparation method and application thereof. According to the enhanced CH2 structure domain mutant combined with the newborn Fc receptor as well as the preparation method and application thereof disclosed by the invention, amino acid neighbouring to a helical area which interacts with FcRn in a CH2 structure domain of a constant area of a human antibody IgG is mutated to obtain the enhanced CH2 structure domain mutant. Relative to a wild type CH2 structure domain, the mutant disclosed by the invention has higher stability and anti-gathering ability, and has the advantages that the production, purification and storage costs of a monoclonal antibody or Fc fusion protein can be reduced; the CH2 structure domain mutant is combined with pH dependency of FcRn so that enhancement is achieved, and a good basis is laid for searching a novel single-domain antibody drug using the enhanced CH2 structure domain mutant as a framework.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a CH2 domain mutant with enhanced binding to newborn Fc receptors and its preparation method and application. Background technique [0002] With its strong specificity and high sensitivity, monoclonal 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. Since the beginning of the 21st century, people have also begun to pay more and more attention to the development and clinical application of therapeutic monoclonal antibody drugs, mainly because it has the following advantages: less toxic and side effects on the human body; natural effector function and high specificity to the target. Therefore, more and more monoclonal antibodies are approved for clinical use. [0003] With the deepening of an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13
CPCC07K16/283C07K2317/21C07K2317/524C07K2317/94
Inventor 龚睿
Owner 武汉班科生物技术有限公司
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