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BCR-ABL fusion protein mutant, encoding gene and expression carrier thereof, and construction method and application of the expression carrier

A fusion protein and expression vector technology, applied in application, gene therapy, genetic engineering, etc., to increase sensitivity, overcome potential complications, and promote apoptosis

Inactive Publication Date: 2014-12-17
CHONGQING MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods cannot directly inhibit the combination of RIN1 and BCR-ABL, and RIN1 has different physiological functions in different cells. For example, in breast cells, RIN1 is a tumor suppressor gene, so knocking it out may induce the occurrence of breast cancer

Method used

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  • BCR-ABL fusion protein mutant, encoding gene and expression carrier thereof, and construction method and application of the expression carrier
  • BCR-ABL fusion protein mutant, encoding gene and expression carrier thereof, and construction method and application of the expression carrier
  • BCR-ABL fusion protein mutant, encoding gene and expression carrier thereof, and construction method and application of the expression carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 Computer simulation technology to construct mutant structure and analysis of binding ability

[0049] According to the ABL SH3 structure in the protein structure database PDB (ID: 3EG2, chainsA, ABL60-121 positions, 62 amino acids in length), mutate glutamine (Q) at position 114 to asparagine (N) and change it back to Wild-type ABL SH3 structure, mutating threonine at position 79 of the wild-type ABL SH3 structure to tyrosine to obtain mutant M3 (T79Y), whose structure is as follows figure 1 Shown, its amino acid sequence is shown in SEQ ID NO:2.

[0050] The key amino acids in the combination of the proline-rich domain of RIN1 and the ABL SH3 domain were analyzed by PepSite2 software. These key amino acids include: Pro, Asn, Phe, Trp, Tyr, and these amino acids have a common structural feature that contains a benzene ring ( Pro, Phe, Trp, Tyr) or phenyl-like rings (Asn).

[0051] Use PyMOL1.6 and VMD1.8.7 to complete point mutation and image visualizatio...

Embodiment 2

[0052] Embodiment 2 Obtaining of Mutant Gene Fragment

[0053] (1) Primer design and synthesis

[0054] According to the SH3 fragment in the ABL gene sequence (sequence number NC_000009.12) in NCBI GenBank, the PCR primers of M0, M1, M2, M3, and M4 were designed by Primer5.0 software. After Blast comparison, the primers were provided by Shanghai Yingjun (Invitrogen) Ltd Synthetics. Among them, the upstream 5' end of the ABL SH3 wild-type (M0) amplification primer (also the outer primer for overlap extension PCR) contains a Sal Ⅰ restriction site, and the downstream primer contains a Xho Ⅰ restriction site and a stop codon. Boxes are stop codons, bold italics are restriction sites, and lowercase letters are mutation sites. The primer sequences and mutation points are listed in Table 1.

[0055] Table 1 ABL SH3 wild type and mutant PCR amplification primer sequences

[0056]

[0057] (2) ABL SH3 wild type (M0) PCR amplification

[0058] The pMig210 plasmid (including BCR...

Embodiment 3

[0069] Embodiment 3 recombinant adenovirus vector construction

[0070] (1) The target gene is cloned into the shuttle vector

[0071] The overlap-extension PCR purification products of M0, M1, M2, M3, and M4 obtained in Example 2, that is, the full-length ABL SH3 mutants, were respectively cloned into the shuttle vector pAdTrack-CMV vector.

[0072] ① The PCR purified product and pAdTrack-CMV vector in Example 2 were double-digested with endonucleases Sal Ⅰ and Xho Ⅰ, respectively, at 37°C overnight;

[0073] ②Purify and recover the digested products, ligate with T4DNA ligase, and ligate overnight at 16°C;

[0074] ③Determine the concentration of the connection product, take 100ng and convert it to CaCl 2 The competent DH5α prepared by the method was cultured on LB plates containing kanamycin at 37°C for 12h;

[0075] ④ Pick a single clone, extract the plasmid after enrichment, and store the strain at -80°C;

[0076] ⑤Use ABLSH3 wild-type (M0) primers for PCR amplificatio...

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Abstract

The invention provides an encoding gene of a BCR-ABL fusion protein mutant. A nucleotide sequence of the encoding gene is represented as the SEQ ID No.1. The invention also provides an expression carrier including the nucleotide sequence and a construction method thereof, and also provides the BCR-ABL fusion protein mutant. The BCR-ABL fusion protein mutant is designed on the basis of BCR-ABL / c-ABL SH3 structural domain locus mutation. Threonine at the 79th position of the ABL SH3 structure is mutated into tyrosine, wherein an amino acid sequence of the tyrosine is represented as the SEQ ID No.2. A constructed BCR-ABL fusion protein mutant is competitively combined with R1N1 for directly blocking and reducing combination of intracellular R1N1 and BCR-ABL. With combination of IM, sensitivity of cells on the IM is further increased, apoptosis of tumor cells are promoted and meanwhile potential complications caused by direct knockout of an R1N1 gene are overcome.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a BCR-ABL fusion protein mutant and its encoding gene, an expression vector and a construction method thereof, and also relates to the use of the BCR-ABL fusion protein mutant, its encoding gene, and the expression vector in the treatment of chronic myeloid cells Applications in leukemia. Background technique [0002] BCR-ABL fusion protein has abnormally activated tyrosine kinase activity, which is a typical marker of chronic myelogenous leukemia (CML). More than 90% of CML patients express BCR-ABL fusion gene, while normal cells do not express it BCR-ABL fusion protein, and ABL is located in the nucleus, and RIN1 is mainly located in the cytoplasm. The structure of SH3 located in the ABL segment is directly related to the activation of BCR-ABL protein kinase activity, which can negatively regulate the activity of BCR-ABL. The mutation of its key site or the change of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/63C12N15/66C12N9/12A61K48/00A61K38/45A61P35/02
Inventor 冯文莉刘鑫文良雪黄峥兰李会
Owner CHONGQING MEDICAL UNIVERSITY
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