bcr-abl fusion protein mutant and its coding gene, expression vector and its construction method and application
A fusion protein and expression vector technology, applied in application, gene therapy, genetic engineering, etc., to increase sensitivity, overcome potential complications, and promote apoptosis
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Embodiment 1
[0048] Example 1 Computer simulation technology to construct mutant structure and analysis of binding ability
[0049] According to the structure of ABL SH3 in the protein structure database PDB (ID: 3EG2, chainsA, ABL60-121, full-length 62 amino acids), the glutamine (Q) at position 114 was mutated to asparagine (N) to change it back The wild-type ABL SH3 structure, mutated the threonine at position 79 of the wild-type ABL SH3 structure to tyrosine to obtain the mutant M3 (T79Y), whose structure is as follows figure 1 As shown, its amino acid sequence is shown in SEQ ID NO:2.
[0050] The key amino acids that bind the proline-rich domain of RIN1 to the ABL SH3 domain were analyzed by PepSite2 software, these key amino acids include: Pro, Asn, Phe, Trp, Tyr, these amino acids have common structural features that contain a benzene ring ( Pro, Phe, Trp, Tyr) or a benzene-like ring (Asn).
[0051] Using PyMOL1.6 and VMD1.8.7 to complete point mutation and image visualization, N...
Embodiment 2
[0052] Example 2 Acquisition of mutant gene fragments
[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, Primer5.0 software designed PCR primers for M0, M1, M2, M3, and M4. After Blast alignment, the primers were prepared by Shanghai Invitrogen Biotechnology Ltd. Synthetic. Among them, the ABL SH3 wild-type (M0) amplification primer (also the outer primer of the overlap extension PCR) contains a Sal I restriction site at the upstream 5' end, and the downstream primer contains an 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 shown in Table 1.
[0055] Table 1ABL SH3 wild-type and mutant PCR amplification primer sequences
[0056]
[0057] (2) ABL SH3 wild type (M0) PCR amplification
[0058] The pMig210 plasmid (containing BCR-ABL ...
Embodiment 3
[0069] Example 3 Construction of recombinant adenovirus vector
[0070] (1) Cloning the target gene 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 mutant, were cloned into the shuttle vector pAdTrack-CMV vector, respectively.
[0072] ① The PCR-purified product and pAdTrack-CMV vector in Example 2 were double digested with endonuclease Sal I and Xho I respectively, overnight at 37°C;
[0073] ②The digestion product was purified and recovered, ligated with T4 DNA ligase, and ligated overnight at 16°C;
[0074] ③ Determine the concentration of the ligation product, and take 100 ng to convert it by CaCl 2 The DH5α competent prepared by the method was cultured on LB plates containing kanamycin at 37°C for 12h;
[0075] ④Pick the single clone, extract the plasmid after enrichment, and store the strain at -80℃ at the same time;
[0076] ⑤ Use ABLSH3 wild-type (M0) ...
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