Fused protein restructuring target lethal leukemia cells and preparation method and use thereof
A technology of leukemia cells and fusion proteins, which is used in drug combinations, peptide/protein components, recombinant DNA technology, etc., to achieve the effect of broad application prospects
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0030] Example 1: Preparation method of plasmid pGEX-4T-1-PE38KDEL
[0031] 1. Amplification of PEA gene
[0032] Chromosomal DNA of Pseudomonas aeruginosa PA103 was extracted by conventional methods. A pair of primers located on both sides of the PEA structural gene were designed with reference to the sequence published by Gray et al.
[0033] 5' primer: 5'-GAT CAG CCT CAT CCT TCA C-3',
[0034] 3' Primer: 5'-GCT CGC GGC AGT TAC TT-3'.
[0035] Reaction system: 5 μl DNA template (0.5 μg), 5 μl 10× PCR amplification buffer, 2 μl (100 ng) each of 5’ primer and 3’ primer, 1 μl of 10 mmol / L dNTP mixture, 5 μl of 50% glycerol, 2.5 μl of DMSO, sterilized Bacteria water 27μl, TaqDNA polymerase 0.5μl (2.5u), total volume 50μl. Reaction conditions: denaturation at 94°C for 4 win, followed by cycling, denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 3 min, a total of 30 cycles. Final extension at 72°C for 30min.
[0036] 2. PE40 gene fragme...
Embodiment 2
[0052] Example 2: Expression and activity identification of recombinant protein IL3-PE38KDEL
[0053] 1. Materials and methods
[0054] 1.1 Reagents
[0055] Various restriction enzymes, TaqDNA polymerase, PrimeSTARTM HS DNA Polymerase, DL2000Marker, 500bpMarker, protein Marker, T4 ligase, plasmid mini-suction kit, and gel recovery reagents were purchased from Takara Company, and the primers were synthesized by Shanghai Yingjun company completed.
[0056] IPTG, Coomassie brilliant blue R250 were purchased from Takara Company.
[0057] Nickel ion affinity chromatography column (Ni2+-NTA) was purchased from Novagen Company.
[0058] RPMI1640 was purchased from GIBCO Company.
[0059] Calf serum was purchased from Hangzhou Sijiqing Biological Materials Co., Ltd.
[0060] MTT is a product of Amresco.
[0061] 1.2 Construction of fusion gene PQE30-IL3-Linker-PE38KDEL
[0062] 1.2.1 Construction of recombinant plasmid PQE30-Linker
[0063] In order to ensure that the protein...
Embodiment 3
[0094] Example 3: Expression and activity identification of recombinant protein IL3-Linker-PE38KDEL
[0095] 1. Materials and methods
[0096] 1.1 Reagents
[0097] Various restriction enzymes, TaqDNA polymerase, PrimeSTAR TM HS DNA Polymerase enzyme, DL2000Marker, 500bpMarker, protein marker, T4 ligase, plasmid mini-pump kit, and gel recovery reagent were purchased from Takara Company.
[0098] The synthesis of primers was completed by Shanghai Yingjun Company.
[0099] IPTG, Coomassie brilliant blue R250 were purchased from Takara Company.
[0100] Nickel ion affinity chromatography column (Ni 2+ -NTA) was purchased from Novagen.
[0101] RPMI1640 was purchased from GIBCO Company.
[0102] Calf serum was purchased from Hangzhou Sijiqing Biological Materials Co., Ltd.
[0103] MTT is a product of Amresco.
[0104] 1.2 Synthetic primers:
[0105] Primer1 (upstream) 5'-TCGC GCATGC GCTCCCATGACCCAG-3' (underlined is the sph I restriction site),
[0106] Primer2 (do...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More