Polypeptide preventing phase bean lectin polymerization and inhibiting coagulation toxicity, gene and use thereof
A common bean lectin, a technology for inhibiting blood coagulation, applied in the field of medical bioengineering, can solve problems such as economic loss and endanger human health, and achieve the effects of reducing economic loss, strong comprehensiveness, and reducing the risk of human poisoning
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Embodiment 1
[0031] Synthesis of polypeptides that prevent phase bean agglutinin from polymerizing and inhibit coagulation toxicity (hereinafter referred to as: anticoagulant polypeptide or anticoagulant polypeptide SFIVS)
[0032] 1) Theoretical basis for designing anticoagulant polypeptides based on exogenous bean lectin PHA-E:
[0033] Such as figure 1 Shown: There are 7 structures among the top 10 structures with the highest binding intensity between the anticoagulant polypeptide SFIVS and the exogenous phase bean lectin PHA-E monomer, and the anticoagulant polypeptide is located on the dimer binding surface of phase bean lectin. The above results show that the anticoagulant polypeptide SFIVS has a strong competitive relationship with another phase bean agglutinin monomer structure, which can reduce the probability of phase bean agglutinin forming a dimer complex, thereby reducing the coagulation toxicity of phase bean agglutinin.
[0034] 2) Anticoagulant peptide SFIVS peptide design...
Embodiment 2
[0052] Determination method of coagulation toxicity of phase bean agglutinin
[0053] 1) Perform cell quantification on the red blood cell suspension for detection, the specific steps are as follows:
[0054] (A) Fresh rabbit blood was collected and placed in 5% to 10% sodium citrate anticoagulant, mixed with 4 times the volume of Alsever's solution, and then stored in a refrigerator at 4 degrees Celsius for later use.
[0055] (B) Slowly mix the fresh rabbit blood obtained in step (A) with normal saline at a ratio of 5:8, centrifuge at 1000 rpm for 5 minutes, and collect the precipitate.
[0056] (C) Repeat the above steps to wash the erythrocytes until the color of the supernatant is almost transparent, and prepare a 20% erythrocyte suspension according to the volume of erythrocytes.
[0057] (D) Count the erythrocytes after the erythrocyte suspension obtained in step (C) is diluted with normal saline, and adjust the concentration of erythrocytes to 5×10 7 pcs / ml-5×10 8 p...
Embodiment 3
[0069] Anticoagulant polypeptide activity detection, the specific steps are as follows:
[0070] (A) The powder of the anticoagulant polypeptide obtained above is made into a solution with a final concentration of 0.05 mg / mL with PBS buffer, and ultrasonically aided in dissolution;
[0071] (B) After doubling the phase-bean lectin standard solution according to the step (a) above, add 25 μL of 0.05 mg / mL polypeptide solution to each well, continue to shake for 1 min after the addition, and place at room temperature for 30 min until it is fully combined;
[0072] (C)) Repeat step (d), observe the agglutination reaction with naked eyes and judge the agglutination degree compared with the control group.
[0073] In the above method, the agglutination reaction between the standard bean agglutinin, the polypeptide treatment group (3 repetitions), and the blank control group with only the polypeptide and rabbit blood cells was detected, observed and recorded by naked eyes.
[0074]...
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