Human tropomyosin receptor kinase A mutant and application

A technology of tropomyosin and receptor kinase, which is applied in the application field of tropomyosin receptor kinase A mutant and its mutant in tumor treatment, and can solve the problem of high requirements for exogenous fragments, limited application, and TrkA transformation Site limitations and other issues

Inactive Publication Date: 2019-04-16
周越
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commonly used method of adding adjuvants can be used to improve immunogenicity, but it is difficult to effectively induce the production of specific antibodies; or TrkA can be modified to fuse some exogenous T cell epitopes to improve immunity. Originality, although this method can produce antibodies, it has high requirements for foreign fragments, and the modification site of TrkA is limited, so the application is limited

Method used

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  • Human tropomyosin receptor kinase A mutant and application
  • Human tropomyosin receptor kinase A mutant and application
  • Human tropomyosin receptor kinase A mutant and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] pBR322-mNTRK1 plasmid construction

[0025] (1) According to the TrkA wild-type amino acid sequence ( figure 1 ), through software analysis, the active region in the sequence is screened out. One or more amino acids in this region are mutated, and the amino acid sequence of the mutated tropomyosin receptor kinase A mutant is SEQ ID NO:1.

[0026] (2) Design of NTRK1 mutant gene (mNTRK1): According to the amino acid sequence of tropomyosin receptor kinase A mutant, the wild-type gene (NTRK1) encoding tropomyosin receptor kinase A was site-directed mutation, the gene The nucleotide sequence of is SEQ ID NO:2.

[0027] (3) The designed NTRK1 mutant gene was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the designed gene was on the plasmid HF392 provided by the company. Primers were designed according to the gene sequence, and the rationality was evaluated with the computer software OLIGO. Primer sequence I: upstream primer P-F (5'-TCTG...

Embodiment 2

[0030] Screening of strains with high expression of tropomyosin receptor kinase A mutant

[0031] (1) Transform the pBR322-mNTRK1 plasmid into E.coli DH5α competent cells, inoculate them in LB medium containing ampicillin, shake overnight at 37°C, and re-inoculate the activated bacteria at a ratio of 1% to new culture cells the next day In the solution, continue to culture and shake at 37°C. When the absorbance A value (formerly known as optical density OD value) (600nm) reaches 0.5-2, add IPTG to a final concentration of 1 mmol / L, continue to culture for 5 hours, and reserve a sample for identification. , to screen high-expression cells.

[0032] (2) Detection of tropomyosin receptor kinase A mutant: high-expression cells were collected by centrifugation, disrupted by ultrasonication, and the precipitate was collected by centrifugation. Wash with 50mmol / L Tris-HCl (pH8.0), 1mmol / L EDTA solution containing 0.5% triton X-100 for 2-3 times, and centrifuge at 10000r / min to colle...

Embodiment 3

[0034] Expression and purification of tropomyosin receptor kinase A mutant

[0035] The obtained tropomyosin receptor kinase A mutant culture solution was centrifuged to obtain bacterial pellet, ultrasonically broken, centrifuged to obtain supernatant, and the supernatant was purified. First pass the supernatant through the Q Sepharose Fast Flow under the monitoring of the AKTA protein purification instrument,

[0036] The main protein was eluted using pH 8.0, 20 mM Tris-HCl buffer containing 0.2 M NaCl. Secondly, the eluate containing the main protein was purified by Ni-NTA, and the impurity protein and the main protein were eluted with 50 mM imidazole, pH 8.0 of 500 mM imidazole, and 20 mM Tris-HCl buffer, respectively. The obtained protein purification solution was determined by indirect ELISA method and Masie brilliant blue method, and the detection purity reached 98%.

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Abstract

The invention discloses a human tropomyosin receptor kinase A mutant, and relates to the field of genetically-engineered drugs. The human tropomyosin receptor kinase A mutant is characterized in thatthe mutant is obtained through amino acid mutation of one or more loci of the prototype of human tropomyosin receptor kinase A, and the amino acid mutation loci are located in the ligand binding areaof the tropomyosin receptor kinase A and include LEU486, GLY488, HIS489, ILE490, PHE521, LYS544, GLU560, LEU564, VAL573, PHE589, GLY667, ASP668, PHE669 and GLY670. The human tropomyosin receptor kinase A mutant has the advantages that generation of corresponding antibodies generated in vitro and in vivo can be induced, the corresponding antibodies inhibit multiple positive cancer cells of tropomyosin receptor kinase A, and the human tropomyosin receptor kinase A mutant is safe and free of cytotoxicity.

Description

technical field [0001] The invention belongs to the field of genetic engineering drugs, and in particular relates to a mutant of tropomyosin receptor kinase A and the application of the mutant in tumor treatment. Background technique [0002] Tropomyosin receptor kinase A (TrkA), also known as neurotrophic factor tyrosine kinase receptor type 1 (NTRK1), is a 140kDa spanning protein encoded by the NTRK1 gene. Membrane glycoproteins. TrkA is a specific high-affinity receptor for nerve growth factor (NGF), mediates most of the biological functions of NGF, and is its functional receptor. Binding of NGF to TrkA receptor can lead to receptor dimerization and activation of intrinsic kinase activity, thereby causing the activation of various signaling pathways, including Ras / MAPK, PI3K / Akt and PLCγ pathways, mediating various pathologies such as nerves, blood vessels and tumors Physiological function. [0003] Studies have shown that NGF-TrkA is related to tumorigenesis, prolifer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12A61K38/45A61P35/00
CPCA61K38/00A61P35/00C12N9/12C12Y207/10001
Inventor 周越
Owner 周越
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