Halostachys caspica metallothionein gene as well as recombinant protein and application thereof
A technology of metallothionein and salt ear wood, applied in the direction of metallothionein, application, genetic engineering, etc., can solve the problems of complex production process, low MT output, high safety, etc., achieve obvious dose-effect relationship, free oxygen scavenging Basic, wide application field effect
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Embodiment 1
[0036] Embodiment one: the cloning of metallothionein gene
[0037] 1. Extraction of total RNA from Saltwood
[0038] Before the operation, the laboratory bench was irradiated with ultraviolet light for 30 minutes, weighed 0.1 g of the assimilated branches of Saltwood pretreated with 300 mM NaCl, put it into an RNase-free mortar and ground it with liquid nitrogen, and transferred the powder into an Eppendorf tube containing 1 mL of Trizol reagent. Close the lid tightly and shake vigorously for 15 seconds, place at room temperature at 15-30°C for 3-5 minutes, centrifuge at 12,000 r / min at 4°C for 20 minutes, carefully transfer the supernatant into a clean Eppendorf tube, add 0.2 mL of chloroform to extract protein, shake for 15 seconds, and place at room temperature Centrifuge at 4°C and 12000rpm for 10 minutes for 3 to 5 minutes. The solution is divided into upper, middle and lower layers, and the colorless upper layer is the RNA-containing layer. Transfer this layer into a c...
Embodiment 2
[0059] Example 2: Prokaryotic expression of recombinant plasmid pET32a-HcMT
[0060] 5 μL of the extracted plasmid pET32a-HcMT was transformed into competent cells E.coli BL21, and positive clones were obtained by ampicillin screening. Cultivate the positive clone containing the recombinant plasmid overnight at 37°C, then inoculate at 1% and incubate at 37°C for 2-3 hours. 600 When the absorbance value reached 0.6, IPTG was added to a final concentration of 0.5M, and induced at 37°C for 6h. Take 1.5mL of the culture solution, centrifuge at 12000rpm for 3min, collect the precipitate, resuspend the precipitate with 50μL PBS of pH 7.4, add an equal volume of SDS buffer for shaking, put it in a boiling water bath for 5min, centrifuge at 12000rpm for 10min, and detect it by SDS-PAGE electrophoresis See the attached image 3 . M is a standard protein molecular weight marker; Lane 1 is the recombinant transformant containing plasmid pET32a-HcMT without induction; Lane 2 is the exp...
Embodiment 3
[0064] Example 3: High-efficiency expression and purification of recombinant Saltwood metallothionein
[0065] Cultivate the positive clone containing the recombinant Saltwood metallothionein gene carrier at 37°C overnight, then inoculate at 1% ratio, and cultivate at 37°C for 3-4 hours, when the A 600 When the absorbance value reaches 0.6, add IPTG to a final concentration of 0.5-1 M, induce at 37°C for 6 hours, centrifuge at 12,000 rpm for 3 minutes, and collect the precipitate.
[0066] The collected precipitate was dissolved in 50mM NaH 2 PO 4, 300mM NaCl, 10mM imidazole, pH8.0 buffer solution, ultrasonic sterilization at 4°C, ultrasonic ultrasonic sterilization conditions: 400W, every 5sec interval 5sec, a total of 50 times; at 4°C, 12000rpm, 20min, collected after centrifugation clear, and filtered through a 0.45 μm filter membrane for later use.
[0067] Affinity chromatography: the supernatant obtained after sonication was passed through a pretreated Ni-NTA agarose ...
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