Cadmium ion targeted metal-binding peptide Z1 and application thereof
A cadmium ion and peptide-binding technology, which is applied in the fields of peptides, introduction of foreign genetic material using carriers, water/sludge/sewage treatment, etc., can solve the problems of unstable expression of exogenous peptides, limited applications, short half-life, etc., and achieve good results The effect of applying the effect
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Embodiment 1
[0063] In this example, the process of screening and obtaining cadmium ion metal-binding peptides is briefly introduced as follows.
[0064] (1) First prepare cadmium ion chelating resin
[0065] First, take 150 µL of nickel ion chelating resin in a 1.5 ml EP tube in a sterile ultra-clean bench, centrifuge at 10,000 rpm for 10 min, discard the supernatant; add 5 ml of 30 μg / mL CdSO 4 solution;
[0066] Put 500 µL of TBS in the chelating resin for washing, centrifuge at 8000 rpm for 5 min, and discard the supernatant;
[0067] Add 1 ml of 0.5mol / L EDTA (pH 8.0) to the chelating resin, centrifuge at 8000 rpm for 5 min, and discard the supernatant;
[0068] Add 0.5 ml of 0.1% TBST to the resin, centrifuge at 12000 rpm for 5 min, and discard the supernatant;
[0069] Add an equal volume of TBST to each EP tube, pipette the suspended resin, and quickly dispense into new EP tubes. Each tube contains 200 µL of resin mixture, and store in a refrigerator at 4°C.
[0070] This is th...
Embodiment 2
[0130] On the basis of the sequence of the cadmium ion metal-binding peptide gene obtained in Example 1, this example mainly introduces the related experiments of integrating the gene into Saccharomyces cerevisiae cells and preparing a cadmium ion adsorbent.
[0131] (1) Construction of plasmid vector
[0132] According to the dodecapeptide metal-binding sequence obtained in step (1), the corresponding base sequence was artificially synthesized, and the gene fragment was combined with the pYD1 plasmid ( Eco R and xho double enzyme digestion) for ligation and transformation into Escherichia coli 116 to construct recombinant plasmids pYD1-Z1, pYD1-Z2, pYD1-Z3, and pYD1-Z4, respectively.
[0133] After positive resistance screening of the transformed product, a small amount of plasmid was extracted and verified by enzyme digestion (results such as figure 1 shown), and then colony PCR verification (results as figure 2 As shown), the bands are about 5kb and 0.4kb respecti...
Embodiment 3
[0147] This example mainly introduces the actual adsorption effect experiment of cadmium ions in wastewater.
[0148] First, draw a cadmium ion standard curve (eg Figure 4 shown), the linear equation Y=0.733X-0.023 was obtained in the range of 1 to 10 μg / mL, where R was 0.99984, indicating that the linear relationship was good.
[0149] Secondly, the correctly identified transgenic Saccharomyces cerevisiae prepared in Example 2 was directly or immobilized for cadmium ion adsorption, and the adsorption efficiency was measured.
[0150] Saccharomyces cerevisiae directly conducts cadmium ion adsorption and the determination of cadmium ion adsorption efficiency, refer to the following steps for operation:
[0151] (1) Inoculate Saccharomyces cerevisiae (or transgenic Saccharomyces cerevisiae) on YNB plates (EBY100 needs to add tryptophan to YNB medium), and culture at 30°C for 48 hours;
[0152] Pick a single colony on the plate, inoculate it in YNB medium, and culture it on a ...
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