Heptapeptide capable of axially and specifically bonding with surface of tooth enamel and use thereof
A specific, enamel technology, applied in the field of heptapeptide sequences, to achieve the effect of promoting remineralization
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Embodiment 1
[0020] Example 1: Screening of recombinant phage that specifically binds axially to the surface of tooth enamel
[0021] 1. The first round of screening and elution:
[0022] 1) The premolars were extracted for orthodontic reasons, and the soft tissue attached to the tooth surface was removed. The premolars were ultrasonically cleaned in distilled water for 30 minutes, and sterilized with ethylene oxide before use.
[0023] 2) Put the sterilized premolars into the blocking buffer (0.1M NaHCO 3 [pH 8.6], 5 mg / ml BSA, 0.02% NaN 3 ), blocked overnight at 4°C.
[0024] 3) On the next day, the premolars that had been blocked overnight were taken out, put into sterilized water, and blocked for 1 hour at 4°C.
[0025] 4) The tooth was suspended in one well of a 24-well plate, and the tooth was quickly rinsed 6 times with 500 uL of TBST (TBS+0.1% [v / v] Tween-20) buffer solution. Note that after rinsing and absorbing the TBST buffer solution each time, add the TBST buffer solution ...
Embodiment 2
[0053] Example 2: Isolation and preparation of monoclonal phage
[0054] 1) According to Example 1, in the third and fourth rounds of screening, the phages had been bound to tooth enamel to a maximum extent. Therefore, about 15 single clones were picked respectively from the phage obtained in the third and fourth rounds of screening.
[0055] 2) The ER2738 overnight culture was diluted 1:100 and inoculated in LB medium, and divided into 5 mL centrifuge tubes.
[0056] 3) Use a sterilized toothpick or pipette tip to pick up one blue phage plaque at a time, and transfer it to the above-mentioned 5mL centrifuge tube. Note that it should be picked from a plate with a total of less than 100 plaques to ensure that each picked plaque contains only one DNA sequence.
[0057] 4) 37°C constant temperature shaker, 230rpm, culture for 4-5 hours.
[0058] 5) Centrifuge the culture at 10,000 rpm for 10 minutes at 4°C.
[0059] 6) Transfer the upper 80% of the supernatant to a new centri...
Embodiment 3
[0064] Example 3: Extraction of phage single-stranded genomic DNA and acquisition of heptapeptide amino acid sequence
[0065] 1) Take 800 uL of the amplified monoclonal phage solution obtained in Example 2, add 300 uL of PEG / NaCl solution, mix by inverting, and place at room temperature for 30 minutes.
[0066] 2) Centrifuge at 10,000 rpm for 10 minutes, discard the supernatant, centrifuge briefly, and carefully aspirate the remaining supernatant.
[0067] 3) The precipitate obtained in the previous step was resuspended in 100uL of iodide buffer, and then 250uL of absolute ethanol was added, and incubated at room temperature for 10 minutes.
[0068] 4) Centrifuge at 10,000 rpm for 10 minutes, discard the supernatant. Wash the pellet with 70% ethanol.
[0069] 5) Centrifuge at 10,000 rpm for 10 minutes, discard the supernatant, and dry.
[0070] 6) The pellet was resuspended in 30 uL TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA), which is the single-stranded genomic DNA of the mo...
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