Fusion protein and nucleic acid containing peptide carrier and epidermal growth factor and its uses
A technology of epidermal growth factor and fusion protein, applied in the field of modified epidermal growth factor, can solve the problems of difficult to penetrate mucous membrane, difficult to penetrate skin, restricting deep tissue and systemic drug use, etc.
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Embodiment 1
[0146] Construction and Identification of Expression Vector pPTD-hEGF
[0147] The synthetic EGF gene (SEQ ID NO: 1) was connected to the pBS-T carrier (purchased from Beijing Tianwei Times Company), and the target gene was excised from the T carrier with Kpn I+EcoR I, recovered and purified; I+EcoR I double-enzyme cut expression vector pPTD (the nucleotide sequence (SEQ ID NO: 3) of the synthetic PTD was connected to the carrier pRSET (purchased from Invitrogen, San Diego, CA) and constructed), and the purified vector was reclaimed Fragment; Take the appropriate ratio of the recovered target gene fragment and the target carrier fragment, and at T 4 Under the action of DNA ligase, connect overnight at 16°C, take appropriate ligation solution to transform competent cell TOP10 (purchased from Beijing Tianwei Times Company), and select a few from the clone plaques grown on the screening medium for culture and identification.
[0148] The plasmids of the recombinant colonies were...
Embodiment 2
[0150] Expression of PTD-hEGF fusion protein
[0151] Extract the correct expression vector constructed above to transform Escherichia coli BL21(DE3), pick multiple clones for induced expression, figure 2 is the expression status of one of the typical clones, and it can be seen from the figure that the plasmid has been effectively expressed. According to biological software analysis, the expression amount accounts for 40% of the total bacterial protein.
Embodiment 3
[0153] Determination of expression form of recombinant protein
[0154] After the cells induced to express were collected by centrifugation, they were washed with PBS buffer, and then the washed cells were dissolved in an appropriate lysate, the cells were disrupted by ultrasonic waves, and the supernatant and precipitate were separated by centrifugation. The results of SDS-PAGE analysis showed that , the target protein content in the supernatant is very small, and most of them exist as insoluble inclusion bodies. Take equal amounts of lysate precipitates and dissolve them in 2, 4, 6, and 8M urea respectively. Electrophoresis analysis shows that the solubility of the inclusion body is the largest in 8M urea ( image 3 ).
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