Mutatant protein of human stem cell growth factor as well as its preparing method and medicine composition
A mutant protein and growth factor technology, which is applied in the field of recombinant human stem cell growth factor mutant protein, can solve the problems of insufficient expression and protein renaturation
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Embodiment 1
[0043] Example 1 Gene Synthesis
[0044] Firstly, a mutant human stem cell growth factor coding sequence (as shown in SEQ ID NO: 4) having the coding sequence of Escherichia coli OmpA signal peptide and codon bias design is provided. In this sequence, the 124th base is changed from C to G, so that the 42nd (corresponding to the 23rd after removing the signal peptide sequence) amino acid is changed from arginine to glycine; the 386th base is changed from A to For C, the 387th base is changed from C to T, so that the 129th (corresponding to the 110th after removing the signal peptide sequence) amino acid of its encoding is changed from tyrosine to serine; the 919th base is changed from C changed to G, and the 921st base was changed from G to T, so that the encoded 307th (corresponding to the 288th after removing the signal peptide sequence) amino acid was changed from proline to alanine. This gene structure is referred to as SCF / ompA. When synthesizing the gene, an EcoRI cutti...
Embodiment 2
[0060] Example 2. Construction of expression vectors
[0061] In this embodiment, the pUC18 vector is used as the expression vector, and the expression vector is purchased from (Promega Company, figure 1 The map of this vector is shown in ). The completely correct DNA of pSCF / ompA was digested with EcoRI and HindIII, separated on 1% agarose electrophoresis, and a fragment of about 1 kb in length was recovered with the gel extraction kit of PROMEGA Company. Then, the SCF / ompA gene fragment was connected to the prokaryotic expression vector pUC18 vector using the connection kit of MBI Company to form the pUC18-SCF / ompA expression vector. The detailed operation process is as follows:
[0062] 1) Digest the expression plasmid pUC18 with EcoRI and HindIII. Add each of the following to a microcentrifuge tube:
[0064] 10× restriction enzyme buffer 3 μl
[0065] EcoRI and HindIII 15U
[0066] 1 mg / ml acetylated BSA 3 µl
[0067] Nuclease-free w...
Embodiment 3
[0083] Example 3. Inducing target gene expression
[0084] Take 100 microliters of the correct cloned bacterial solution and inoculate them into 5 milliliters of LB liquid medium containing ampicillin (100 micrograms / ml), culture at 37°C with shaking at 260 rpm until the OD value reaches 0.4-1.0, add isopropylthio Galactoside (IPTG) to a final concentration of 1 mmol / L. Cultivate for 3 hours, transfer 1 ml from each culture to a centrifuge tube, centrifuge, and remove the supernatant; lyse the bacteria for SDS protein electrophoresis, stain SDS polyacrylamide with Coomassie brilliant blue, and detect the expression of the target protein . The results show that the target product expressed by the strain accounts for 68% of the soluble protein in the thalline, which is 15% of the expression obtained by the method of directly expressing the natural gene that has been published abroad (Chen W, Di X, Li J, Song F, Chen S. cDNA cloning of human stem cell factor and its high level ...
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