Application of FGF-2 nanobody as protein and/or polypeptide protecting agent
A technology of FGF-2 and protein protective agent, applied in the direction of peptide/protein components, animal/human proteins, antibodies, etc., can solve the problems of loss of activity, easy polymerization, and affecting stability, so as to enhance the protective effect and reduce adverse effects Affect and improve the effect of stability
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Embodiment 1
[0040] Embodiment 1 Nanobody Nb4 and FGF-2 affinity determination
[0041] Enzyme-linked immunosorbent assay was used to detect the affinity between Nb4 and FGF-2 (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., product number 10014-HNAE). The specific experimental steps were to add 100 μL, 4 μg / mL FGF-2 to each well of the ELISA plate, and 4 °C Pack overnight. The plate was washed 3 times with PBS-T, each time for 5 min, and different concentrations of Nb4 (starting at 2 μg / mL, 10 gradients at a ratio of 1:4) were added, and incubated at room temperature for 2 h. The plate was washed 3 times with PBS-T, 5 min each time, 100 μL Anti-Flag-HRP antibody (purchased from SIGMA, Cat. No. A8592) was added to each well, incubated at room temperature for 1 h, and the plate was washed 3 times with PBS-T, 5 min each time. The absorbance values were measured at 450nm and 630nm respectively, and the results were analyzed by origin8.0.
[0042] The affinity curve between Nb...
Embodiment 2
[0043] Example 2 Effect of Nanobody Nb4 on FGF-2 Stability
[0044] Since the stability of FGF-2 protein is greatly affected by pH and temperature, this example mainly studies the effect of FGF-2 Nanobody Nb4 on the stability of FGF-2 under different pH and temperature conditions. The experiments were divided into 3 groups:
[0045] Table 1. The mixture of FGF-2 and Nb4 treated under different conditions
[0046]
[0047] According to the grouping in Table 1., an experimental group and a control group are established under each group, and the FGF-2 and Nb4 mixture (final concentration is 200 μg / mL) and the control group sample final concentration of the experimental group are 200 μg / mL. -2 Carry out experimental studies with different pH, different temperature and different treatment time respectively. After the treatment, the protein level of FGF-2 was detected by western blot.
[0048] The specific experimental steps are as follows:
[0049] (1) Add 5× protein loading...
Embodiment 3
[0051] Example 3 Effects of Nanobodies on the Stability of FGF-2 under Different Temperature Conditions
[0052] The concentration of FGF-2 after treatment is detected by ELISA method. The specific experimental steps are based on the anti-human bFGF nano Antibody double-sandwich ELISA method.
[0053] First, dilute FGF-2 into a series of concentrations (400ng / mL, 300ng / mL, 200ng / mL, 100ng / mL, 80ng / mL, 40ng / mL, 20ng / mL, 10ng / mL), and draw the ELISA method to measure FGF-2 Concentration standard curve, the results are as follows image 3 As shown, there is a linear relationship between the concentration of FGF-2 and the absorbance value in the concentration range of 10ng / mL to 400ng / mL.
[0054] The specific steps of the described double-sandwich ELISA method to detect the concentration of FGF-2 are as follows:
[0055] (1) Coating plate: Nb6 protein was prepared into a solution with a final concentration of 5 μg / mL with the coating solution, and 100 μL was added to each well...
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