Affinity maturation binding protein bound with CXCR4 and application

A technology of binding protein and affinity, applied in the field of binding protein, can solve problems such as toxic side effects and cell damage, and achieve the effect of promoting application and reducing production cost.

Active Publication Date: 2021-03-19
JINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although these treatments can inhibit or eliminate tumor cells, they

Method used

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  • Affinity maturation binding protein bound with CXCR4 and application
  • Affinity maturation binding protein bound with CXCR4 and application
  • Affinity maturation binding protein bound with CXCR4 and application

Examples

Experimental program
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Example Embodiment

[0064]Example 1 Construction of phage affinity maturation binding protein library

[0065] (1) Using the wild-type binding protein aCX82 as a template (the amino acid sequence encoding the aCX82 binding protein is shown in SEQ ID NO: 26, and the nucleotide sequence encoding the aCX82 binding protein is shown in SEQ ID NO: 34), according to GeneMorph IIRandom Mutagenesis The instruction manual of Kit carries out error-prone PCR, amplifies the target gene by the method of error-prone PCR, and primers are Error-F2 (nucleotide sequence as shown in SEQ ID NO:37) and Error-R2 (nucleotide sequence as shown in SEQ ID NO:37) NO:38).

[0066] (2) After the PCR reaction, use agarose gel electrophoresis to identify error-prone PCR products.

[0067] (3) Perform gel recovery of error-prone PCR products according to the instructions of the EZNA Gel Extraction Kit.

[0068] (4) The pComb3XSS phage display plasmid and the error-prone PCR product were subjected to a double enzyme digestion r...

Example Embodiment

[0074] Example 2 preparation of helper phage

[0075] (1) Line the TG1 Escherichia coli clone strain in three zones on a petri dish with TYE solid medium, and place the marked petri dish upside down in a 37°C incubator for about 14 hours.

[0076] (2) Pick a single colony of TG1 on the petri dish and inoculate it into 5 mL of 2×TY liquid medium, and culture overnight at 37°C and 250 rpm.

[0077] (3) Transfer the bacterial solution obtained in step (2) to another 5mL 2×TY liquid medium at a ratio of 1:100 by volume, and culture it at 37°C and 250rpm until the bacterial solution OD 600nm About 0.5.

[0078] (4) The KM13 helper phage was serially diluted with PBS (10 12 pfu / mL~10 4 pfu / mL).

[0079] (5) Take 200 μL of the bacterial solution cultured in step (3), add 10 μL of the diluted KM13 helper phage, and place the mixture in a 37° C. water bath for 30 minutes to obtain mixture A.

[0080] (6) Mix low-melting point agarose and 2×TY liquid medium, heat until completely m...

Example Embodiment

[0085] Example 3 Amplification of Phage Affinity Maturation Binding Protein Library

[0086] (1) Thaw the bacterial library containing affinity-matured binding protein particles on ice, add 100 μL to 50 ml 2×TY medium (containing 100 μg / mL ampicillin+ and 1% w / v glucose), and shake at 37 ° C and 220 rpm for 2 h , until OD 600nm = 0.5.

[0087] (2) Then add 5×10 11 The pfu KM13 helper phage was placed in a constant temperature water bath at 37°C for 30 minutes. Centrifuge in a small high-speed centrifuge at 3200g for 10min. The pellet was gently resuspended in 2×TY liquid medium (containing 100 μg / mL Amp+ and 50 μg / mL Kana+), and cultured with shaking at 25° C. and 220 rpm for 20 h.

[0088] (3) Centrifuge in a small high-speed centrifuge, centrifuge at 3200g for 20min, retain the supernatant solution, absorb 40ml of the supernatant solution, add 10ml of 20% w / v PEG-NaCl solution, mix gently, and place in ice bath for 2h.

[0089] (4) Centrifuge in a small high-speed centr...

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Abstract

The invention discloses affinity maturation binding protein bound with CXCR4 and application. According to the invention, a human CXCR4 affinity maturation binding protein library is screened to obtain the affinity maturation binding protein bound with the CXCR4. The binding protein is aCX20E5 affinity mature binding protein, or binding protein formed by combining at least one of aCX16C1 affinitymaturation binding protein, aCX17D5 affinity maturation binding protein, aCX20B2 affinity maturation binding protein and aCX13C6 affinity maturation binding protein with the aCX20E5 affinity maturation binding protein. The invention also discloses application of the affinity maturation binding protein bound with the CXCR4 in preparation of autoimmune disease and anti-tumor drugs. The binding protein provided by the invention is humanized, has no immunogenicity in the human body, and can be better applied to the development and clinical application of the autoimmune disease and anti-cancer drugs.

Description

technical field [0001] The invention belongs to the field of binding proteins, in particular to an affinity maturation binding protein combined with CXCR4 and its application. Background technique [0002] Malignant tumors have always been a difficult problem in medicine, and even the "number one killer" of human health. Therefore, the treatment of tumors has always been a hot and difficult research topic. The traditional treatment of tumors is mainly through the combination of radiotherapy, chemotherapy and surgery. Although these treatment methods can inhibit or eliminate tumor cells, they also cause damage and toxic side effects to normal cells in the body. In recent years, targeted therapy for tumors has become a research hotspot. This method of treatment is to bind specific proteins to tumor cells, which can kill tumor cells and minimize the damage of normal cells in the body. [0003] Chemokine receptor 4 (CXCR4) is a seven-time transmembrane glycoprotein consisting ...

Claims

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Application Information

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IPC IPC(8): C07K16/24C12N15/13C12N15/70A61K39/395A61P35/00A61P37/02
CPCC07K16/24C12N15/70A61P35/00A61P37/02C07K2317/56C07K2317/92A61K2039/505
Inventor 魏星周晓欣
Owner JINAN UNIVERSITY
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