EGFR-targeted TRAIL fusion protein as well as preparation method and application thereof
A fusion protein and targeting technology, applied in the field of tumor treatment, can solve the problems of loss of protein biological activity, low renaturation yield, cumbersome inclusion body renaturation process, etc., and achieves strong targeting, simple purification process, and production cost. low cost effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0043] Preparation of embodiment 1 fusion protein Z-TRAIL
[0044] 1) Molecular design of Z-TRAIL
[0045] The TRAIL of the present invention is the truncated human TRAIL extracellular segment protein sequence (114-281 amino acids, see Table 1). Affimer Z that recognizes EGFR EGFR Consists of 58 amino acids (Table 1). Since the region where TRAIL binds to its receptor is close to the C-terminus, in order to avoid the impact of steric hindrance on its activity, the present invention designs Z EGFR Linked to the N-terminus of TRAIL, and a flexible linker (G4S) was added between the two fragments 3 ( figure 1 shown). The fusion protein is called Z-TRAIL for short.
[0046] 2) Construction of Z-TRAIL recombinant expression vector
[0047] Using nucleic acid analysis software, the amino acid sequence of Z-TRAIL was reverse-translated into nucleic acid sequence, and codon optimization was performed to obtain the coding gene of Z-TRAIL, which was submitted to Qingdao Qingke Bi...
Embodiment 2
[0056] Example 2 Targeting and killing effect of Z-TRAIL on tumor cells in vitro
[0057] 1) Targeting analysis of Z-TRAIL on EGFR positive tumor cells
[0058] It has been reported that human skin squamous cell carcinoma A431 highly expresses EGFR. A431 cells were incubated with FITC-labeled EGFR antibody, and detected by flow cytometry, it was confirmed that A431 was a high expression cell of EGFR ( Figure 4 A), the positive expression rate is greater than 95%. Therefore, the present invention selects this cell as the object of Z-TRAIL targeting research in vivo and in vitro.
[0059] TRAIL and Z-TRAIL proteins were labeled with 6-carboxyfluorescein (6-FAM), and 200nM of the two proteins were added to A431 cells, incubated at 37°C for 30 minutes, washed with PBS, and then detected by flow cytometry. Such as Figure 4 As shown in B, the fluorescence intensity of the Z-TRAIL incubation group was significantly higher than that of the TRAIL group, indicating that Z-TRAIL ca...
Embodiment 3
[0065] Example 3 Targeting and killing effect of Z-TRAIL on tumor in vivo
[0066] 1) Z-TRAIL in vivo tumor targeting analysis
[0067] A431 tumor cells (1×10 6 / only) subcutaneously in the back of BALB / c nude nude mice, until the tumor volume grows to 100-200mm 3 , to obtain a mouse xenograft tumor model. The TRAIL and Z-TRAIL proteins were labeled with the near-infrared fluorescent dye Sulfo-Cy7NHS ester (purchased from Xi'an Ruixi Biology) respectively, and the two protein solutions prepared in Example 1 were added to 1M NaHCO 3 The pH of the solution was adjusted to 8.0, and Sulfo-Cy7 NHS ester dye was added according to the molar ratio of protein and dye at 1:10, and reacted at room temperature for 2 hours, and then dialyzed with PBS to remove unreacted dye. Inject 100 μg of the two labeled proteins into the tumor-bearing mice through the tail vein, and use The Spectrum small animal in vivo imaging system (Perkin Elmer) scanned the mouse fluorescence signal (excitati...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More