Aptamers specifically bound with sea anemone cytolysin GT-4 and application thereof
A technology of cytolysin and GT-4, applied in the direction of biochemical equipment and methods, material inspection products, biological testing, etc., to achieve low-cost results
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Embodiment 1
[0025] Example 1: Preparation for filtering library
[0026] Buffer A (1 × PBS pH 7.4, 1MM MGCL 2 , 0.05% Tween20)
[0027] Buffer B (1 × PBS pH 7.4, 1MM MGCL 2 , 0.05% Tween 20, 2Mg / ml BSA)
[0028] 1. Transfer the adapter library (Satheter Biological Engineering Co., Ltd., Item No. 1004728-0000) to 15 ml of centrifuge tube, add 10 ml of buffer B, vortex on the vortex mixer, and centrifuge at 3000 g of 10 min.
[0029] 2. Carefully abandon the supernatant, only about 100 μL of the supernatant (when discarding the supernatant) is retained in the test tube, ensuring that the microspheres remain in the test tube). 3 ml buffer B was added and then mixed on the vortex mixer.
[0030] 3.95 ° C for 5 min. Naturally, it is cooled to room temperature (more than 30 minutes), which is an object to "annealed" to its lowest energy consumption, form a stable three-level structure.
[0031] 4. Add 7ml buffer A. Then vortex on the vortex mixer. Centrifuge at 3000 g of 10 min, carefully discard...
Embodiment 2
[0032] Example 2: Screening of GT-4 adapter
[0033] The screening process mainly includes four steps, namely negative screening, coupling, specific screening, and amplification, the specific process is as follows:
[0034]1. Negative selection
[0035] 1) mixing M-280 streptavidin Dynabeads on a vortex meter (Invitrogen Corporation, Cat. No. 11205D), to draw 250μL 1.5mL tube. On the magnetic rack, after standing 1min, with a precision pipette supernatant was discarded. Add 500μL buffer A, the beads were washed rotation, the supernatant was discarded was repeated twice.
[0036] 2) The beads were resuspended in 50μL of buffer A, then added in its entirety to all of the foregoing aptamer libraries, rotation for 1 hour at room temperature. Magnetic separator removes all magnetic particles and bound aptamer library unadsorbed transferred to a 15mL centrifuge tube. Washing was repeated transfer until all unadsorbed transfer all libraries. Discard all fit beads and strips library adsor...
Embodiment 3
[0053] Example 3: Preparation and Evaluation of GT-4 aptamer sensor
[0054] Biofilm interferometry is a label-free technique, can provide real-time, high-throughput biomolecular interaction information. As a new label-free technology, biofilm interference technology has occupied an important position in the study of the interaction between biological molecules. Since the thickness and quality of the SSA (hyperlink streptavidin) chip surface biofilm denser, more can be assembled on the number of adapter molecules spatial structure, whereby the rear GT-4 interaction may occur due to signal more large changes, so we choose SSA chip having a significant response values, for the preparation of GT-4 aptamer sensor.
[0055] The aptamers 5 'end is modified with biotin, streptavidin and biotin by avidin interaction aptamer immobilized in the sensor surface hyperlink streptavidin (SSA). Fixed before the first of the variable-aptamer complex treatment (95 deg.] C water bath for 10 minutes,...
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