PEG-modified bifunctional anti-tumor recombinant protein conjugate based on folate receptor and macropinocytosis
A recombinant protein, folic acid receptor technology, applied in the field of biomedicine to achieve the effect of prolonging half-life and good application prospects
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Embodiment 1
[0064] Embodiment 1, expression and purification of recombinant protein HSA-LDP in Pichia pastoris
[0065] Inoculate the engineering strain expressing the recombinant protein HSA-LDP (sent to the General Microorganism Center of China Microbiological Culture Collection Management Committee for preservation, preservation number CGMCC No.17450) in BMGY growth medium (10% yeast extract, 20% peptone, 1.34 %YNB, 4×10-5% biotin, 100mM pH 6.0 potassium phosphate buffer, 10% glycerol), 220rpm, cultured at 30°C for 36h, left standing at room temperature or centrifuged to collect the bacteria, and transferred 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 (add 100 % methanol to a final concentration of 1%). The recombinant protein was secreted and expressed in the supernatant of the culture medium, and the supernatant was collected by centrifugation, filtered through a 0...
Embodiment 2
[0066] Example 2, Preparation and Purification of Recombinant Protein Conjugate F-HSA-LDP
[0067] 1. Preparation of recombinant protein conjugate F-HSA-LDP
[0068] figure 1 -A is a flow chart for the construction 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. Prepare the purified HSA-LDP protein with pH6.0, 10mmol / L sodium acetate solution to make 5mg / ml, weigh 0.0126g CH 3 BrNa was added to 10ml of the above HSA-LDP solution to a final concentration of 20mmol / L, and 0.038g of folic acid-PEG was weighed at a molar ratio of 1:5 (HSA-LDP: folic acid-PEG) and added to the above solution. Min stirring reaction 24h.
[0069] 2. Purification of recombinant protein conjugate F-HSA-LDP
[0070] The reaction mixture was first dialyzed against 20mM Tris-HCl bu...
Embodiment 3
[0071] Example 3. Analysis of affinity activity between recombinant protein conjugate F-HSA-LDP and FR protein and tumor cells 1. ELISA detection of affinity activity of recombinant protein conjugate F-HSA-LDP on FR protein and pancreatic cancer cells
[0072] ELISA folic acid receptor detection kit was purchased from Wuhan Huamei Biological Company. Pancreatic cancer cells BxPc-3, MIAPaCa-2 and AsPc-1 were treated with 1×10 4 Cells / well density were seeded in a 96-well plate, cultured at 37°C for 24 hours, rinsed twice with PBS, added 50 μl / well of pre-cooled 0.05% glutaraldehyde at 4°C, fixed the cells at 4°C for 20 minutes, and fixed the cells Rinse with PBS 3 times, dry the residual liquid and set aside. The 96-well plate pre-coated with folic acid receptors and the 96-well plate after cell fixation were blocked with 5% skim milk solution at 200 μl / well at room temperature for 2 h; moistened with PBST buffer (0.05% Tween-20 in PBS) Wash 3 times; dilute the recombinant pr...
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