Sequence and application of Escherichia coli outer membrane protein tolc nucleic acid aptamer
A nucleic acid aptamer and Escherichia coli technology, which is applied in the fields of molecular biology and medical microorganisms, can solve the problems of reporting or publishing aptamers without bacterial outer membrane proteins, and achieves easy acquisition, strong specificity and simple preparation method. Effect
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Embodiment 1
[0049] Example 1: Outer Membrane Protein TolC Gene Amplification
[0050] Molecular cloning technology was used to design specific primers (upstream primer Pa and downstream primer Pb) according to the genome sequence of the outer membrane protein TolC of E. coli, and the full-length cDNA was used as a template for PCR amplification. PCR reaction conditions: 94°C, pre-denaturation for 5min, then denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, 30 cycles, and finally extension at 72°C for 10min. The PCR product was subjected to 1wt% agarose gel electrophoresis, and the target fragment was recovered by cutting the gel.
Embodiment 2
[0051] Embodiment 2: Construction of recombinant expression plasmid
[0052] (1) The target gene was double digested with Bam HI and Xho I. The digestion system was as follows: 25 μl plasmid, 2 μl Bam HI, 2 μl Xho I, 8 μl 10×Buffer, 43 μl ddH 2 O, the mixture was reacted overnight at 37°C, and the digested product was purified with a quick gel extraction kit.
[0053] (2) The PET-30a plasmid was digested with Bam H I and Xho I, and the digestion system was as follows: 5 μl vector (pET-30a) plasmid, 2 μl BamH I, 2 μl Xho I, 8 μl 10×Buffer, 63 μl ddH 2 O, the mixture was reacted overnight at 37°C, and the digested product was purified with a quick gel extraction kit.
[0054] (3) T4 ligase was used to connect the gene of Escherichia coli outer membrane protein TolC and the PET-30a vector to obtain a recombinant plasmid. The ligation system is as follows: 1 ul vector (pET30a), 3 ul PCR recovery product, 4 ul ligase (Takara, DNA ligation Kit Ver2.0), mix well, and react at roo...
Embodiment 3
[0056] Example 3: Expression of TolC protein in Escherichia coli
[0057] Pick a single clone from the transformed plate and put it into 1.5ml LB liquid medium, culture at 37°C and 200rpm. The cultured bacteria liquid was transferred to 500 mL LB liquid medium and mixed, 37° C., 200 rpm, cultivated to OD=0.6-0.8, and induced by IPTG (0.5 mM) for 4 hours. Centrifuge at 6000rpm for 5min with a 400m large centrifuge, and take the supernatant. The precipitate was blown away with 20-30ml of 10mM Tris-HCl (pH8.0) solution, and ultrasonically crushed (500W, 30 times, each time for 10s, with an interval of 15s). Take 100 μl of the sonicated bacterial suspension, centrifuge at 12,000 rpm for 10 min to obtain TolC supernatant and TolC precipitate respectively, take 50 μl of TolC supernatant to another EP tube, blow the TolC precipitate with 50 μl of 10 mM Tris-HCl (pH8.0) solution, add 50 μl of 2× Loadingbuffer, cook at 100°C for 5min, electrophoresis. Electropherogram as figure 2...
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