Aptamer sequence of hepatitis B virus (HBV) core antigen, and applications thereof
A technology of nucleic acid aptamer and hepatitis B virus, which can be applied in antiviral agents, pharmaceutical formulations, microbial measurement/testing, etc., and can solve the problems of nucleic acid aptamers, reports or publications of hepatitis B virus.
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Embodiment 1
[0025] Example 1: HBc gene cloning
[0026] Using known molecular cloning technology, based on the recombinant plasmid pBS_HBV3.6II containing double-copy HBV genome sequence, primers are designed according to the HBc gene sequence:
[0027] HBc_L: 5’-GCCCATATGGACATTGACCCGTA-3’
[0028] HBc_R: 5’-GCCCTCGAGTCAAACAACAGTAGTTT-3’
[0029] Configure PCR system (total volume 50ul): H 2 O 38ul, 10×PCR buffer 5ul, 25mM MgCl 2 3ul, dNTP (10mM) lul, primer mixture 1.5ul (H 2 O:100uM HBc_L:100uM HBc_R=4:0.5:0.5), 1ul DNA template, 0.5ul PrimeStar enzyme. Amplify on a PCR machine under the following conditions: after heating and pre-denaturing at 94°C for 2min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 20s, 30 cycles, extension at 72°C for 8min, and incubation at 4°C for <1h.
[0030] After the reaction, 5ul of the product was electrophoresed in a 1% agarose gel at 100V at a constant pressure. After identifying and amplified DNA fragments that corresponded to t...
Embodiment 2
[0032] Example 2: HBc expression and purification
[0033] A single colony of E. coli containing the recombinant expression plasmid pET28a-HBc was inoculated into 5 mL of LB medium containing Kan antibiotics, and cultured overnight at 37°C. Transfer the overnight culture to 20OmL fresh medium according to the volume ratio of 1:100, and cultivate for 2~4h. 600 When the value reached 0.6, IPTG was added (final concentration is 0.1 mM) and induced and cultured at 30°C for 8 hours. The cells were collected by centrifugation at 4000 rpm for 30 min.
[0034] Resuspend the bacteria with 5mL lysis buffer (50mM Tris-HCl (pH8.5~9.0), 2mM EDTA, 100mM NaCl, 0.5% Triton X-100, 1mg / ml lysozyme), and divide them into 3 2ml tubes, -80 degrees for 30 minutes. 100w ultrasonic breaking in ice bath (2s~5s, 60~80 times). Take 1ml of supernatant and 500ul Ni-NTA Agarose beads and mix them, add to the purification column, use 2ml PBS+Mg(1mM MgCl 2 ) After washing twice, resuspend the mixture of protei...
Embodiment 3
[0036] Example 3: Nucleic acid aptamer screening (one round)
[0037] Sequence of the first round oligo DNA library:
[0038] 5’-acgctcggatgccactacagNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNctcatggacgtgctggtgac-3’
[0039] Primers used for enrichment:
[0040] Aptamer_L: 5’-FAM-acgctcggatgccactacag-3’
[0041] Aptamer_R: 5’-biotin-gtcaccagcacgtccatgag-3’
[0042] Determine the OD of the oligo DNA library 260 Afterwards, it was centrifuged to dry. Dissolve the library with 300ul PBS+Mg, take 200pmol (2.5nmol in the first round) at 95℃ for 8min, quickly place on ice, and then quickly centrifuge.
[0043] Reverse sieve: Aspirate 200pmol of the library into the reverse sieve target equivalent to 40pmol of the positive sieve target, and make up to 800ul with PBS+Mg. Shake at 37°C for 30 min. Suction the Milipore ultrafiltration tube and quickly centrifuge (<1000rpm), and take the filtrate.
[0044] Positive sieve: Take 40pmol positive sieve target, centrifuge at 800rpm, aspirate the supern...
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