A peg-modified bifunctional antitumor recombinant protein conjugate based on folate receptor and macropinocytosis
A recombinant protein, folic acid receptor technology, applied in the field of biomedicine, to achieve good application prospects and prolong the half-life effect
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Embodiment 1
[0059] Example 1. Expression and purification of recombinant protein HSA-LDP in Pichia pastoris
[0060] The expression engineered strain of the recombinant protein HSA-LDP was inoculated (sent to the General Microbiology Center of the China Microorganism Culture Collection Management Committee for preservation, with the deposit number CGMCC No. 17450) in BMGY growth medium (10% yeast extract, 20% peptone, 1.34 % YNB, 4 × 10-5% biotin, 100 mM pH 6.0 potassium phosphate buffer, 10% glycerol), 220 rpm, 30 ° C for 36 h, stand at room temperature or centrifuge to collect bacteria, and transfer them to BMMY induction medium (10% yeast extract, 20% peptone, 1.34% YNB, 4×10-5% biotin, 100mM pH 6.0 potassium phosphate buffer, 10% methanol), 250rpm, 28°C for 73h induction (add 100% every 24h % methanol to a final concentration of 1%). The recombinant protein was secreted and expressed in the supernatant of the medium, and the supernatant was collected by centrifugation, filtered throu...
Embodiment 2
[0061] Example 2. Preparation and purification of recombinant protein conjugate F-HSA-LDP
[0062] 1. Preparation of recombinant protein conjugate F-HSA-LDP
[0063] figure 1 -A is the construction flow chart of recombinant protein conjugate F-HSA-LDP and enhanced F-HSA-LDP-AE. Folic acid-PEG propionaldehyde (molecular weight 10KDa, purity >95%) was purchased from Shanghai Pengshuo Biotechnology Co., Ltd., sodium cyanoborohydride (CH 3 BrNa) was purchased from Sigma Company. The purified HSA-LDP protein was prepared into 5mg / ml with pH6.0, 10mmol / L sodium acetate solution, and 0.0126g CH was weighed. 3 The final concentration of BrNa in 10ml of the above-mentioned HSA-LDP solution was 20mmol / L, and 0.038g of folic acid-PEG was weighed in a molar ratio of 1:5 (HSA-LDP: folic acid-PEG) and added to the above-mentioned solution. min stirring reaction for 24h.
[0064] 2. Purification of recombinant protein conjugate F-HSA-LDP
[0065] The reaction mixture was first dialyzed...
Embodiment 3
[0066] Example 3. Analysis of the affinity of recombinant protein conjugate F-HSA-LDP with FR protein and tumor cells 1. ELISA detection of the affinity activity of recombinant protein conjugate F-HSA-LDP on FR protein and pancreatic cancer cells
[0067] ELISA folate receptor detection kit was purchased from Wuhan Huamei Biological Company. Pancreatic cancer cells BxPc-3, MIAPaCa-2, and AsPc-1 at 1 × 10 4 Cells / well density was seeded in 96-well plate, cultured at 37°C for 24h, rinsed twice with PBS, added 50μl / well of 0.05% glutaraldehyde pre-cooled at 4°C, and placed at 4°C to fix the cells for 20min. Rinse 3 times with PBS, shake dry the residual liquid and use it for later use. The 96-well plate pre-coated with folate receptors and the fixed 96-well plate were blocked with 200 μl / well of 5% skim milk solution at room temperature for 2 h; moistened with PBST buffer (PBS containing 0.05% Tween-20). Wash 3 times; Dilute the recombinant protein conjugate with PBS according ...
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