A kind of ssDNA nucleic acid aptamer and application thereof
A nucleic acid aptamer, peptide nucleic acid technology, applied in the field of genetic resource detection, can solve the problems of lack of effective prevention and detection methods for neuronecrosis virus, and achieve the effects of easy synthesis and preservation, high affinity and high specificity
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[0024] The ssDNA nucleic acid aptamer sequence of the present invention comprises any one or more of the DNA fragments shown in SEQ ID NO: 1-3.
[0025] The ssDNA nucleic acid aptamer of the present invention is mainly screened by the following methods:
[0026] (1) Synthesize a random single-stranded DNA library and primers shown in the following sequences:
[0027] The sequence of the random library Library50 is shown in SEQ ID NO: 4; the sequences of the primers are shown in SEQ ID NO: 5-7.
[0028] (2) Cell-SELEX screening:
[0029] i. Culture GB cells in L15 medium containing 10% fetal bovine serum to 95% of the bottom of the culture bottle, add grouper neuronecrosis virus (GNNV) and infect for 48 hours, wash the cells with PBS buffer 3-5 times and prepare for use for filtering.
[0030] ii. Dissolve 10nmol random library in screening buffer, bathe in constant temperature water at 95°C for 5min, then quickly insert into ice, and bathe in ice for 10min.
[0031] iii. A...
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[0039] Experimental example The specific binding of aptamers to RGNNV-infected GB cells was obtained by flow cytometry detection and screening
[0040] The RGNNV-infected GB cells were blown down with a gun, centrifuged at 300 g to remove the supernatant, and washed 3 times with 1 mL of PBS. Dissolve 300 nM fluorescein isothiocyanate (FITC)-labeled aptamer (SEQ ID NO: 1-3) in 1 mL of binding buffer, and then incubate with the above-treated cells on ice for 30 min, 300 g Centrifuge to remove the supernatant, wash with 1mL PBS for 3 times, and finally mix the cells in 400ul PBS for flow cytometry detection. The aptamer combined with normal GB cells was used as a control. This result confirms that the aptamer has high specificity and affinity to RGNNV-infected GB cells ( figure 1 ).
[0041] Experimental example Fluorescence microscopy detection of the specific binding of carboxytetramethylrhodamine (TAMRA)-labeled nucleic acid aptamers to RGNNV-infected GB cells in this examp...
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