Gene cloning method, expressing method and application of psychrophilic yeast metallothionein

A technology of metallothionein and cloning method, applied in the field of gene cloning of psychrophilic yeast metallothionein, can solve the problem of no polar yeast, and achieve the effect of strong binding ability of heavy metals and broad prospects

Inactive Publication Date: 2017-03-22
HARBIN INST OF TECH AT WEIHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, heavy metal pollution in Antarctica has attracted more and more attention. The research on the resistance of Antarctic organisms to heavy metals and the metallothionein induced by them is in its infancy, and most of the research focuses on shellfish and fish. Reports on related research on polar yeast

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  • Gene cloning method, expressing method and application of psychrophilic yeast metallothionein
  • Gene cloning method, expressing method and application of psychrophilic yeast metallothionein
  • Gene cloning method, expressing method and application of psychrophilic yeast metallothionein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Cloning of a metallothionein gene of psychrophilic yeast

[0037] (1) Extraction of psychrotrophic yeast genome RNA:

[0038] ①Inoculate the activated RhodotorμLa mucilaginosa AN5 strain into a 250mL Erlenmeyer flask containing 100mL YEPD liquid medium, while adding 8mM CuSO 4 , Induction. Incubate at 10°C at 120 rpm for 3 days. Centrifuge at 8000 rpm to obtain bacterial cells.

[0039] ② Wash the bacteria collected by centrifugation with 5 times the volume of distilled water, shake the pellet and mix well, then centrifuge at 4°C and 8000rpm for 8min, discard the supernatant; repeat once.

[0040] ③Add anhydrous alcohol to the mortar to fully burn and eliminate the RNase that may exist in the mortar.

[0041] ④Put the cells obtained by centrifugation into a mortar pre-cooled with liquid nitrogen and grind them thoroughly.

[0042] ⑤Add 50-100mg of the grinded bacteria into a 1.5mL RNase-free centrifuge tube with 1mL Buffer Rlysis-F, and mix well.

[0043] ⑥ Add 200μL o...

Embodiment 2

[0065] Example 2: Expression and purification of a psychrophilic yeast metallothionein

[0066] (1) Induced expression of metallothionein gene

[0067] ① Pick a single colony containing an empty expression vector (positive control plasmid) and a recombinant expression vector, respectively, and inoculate them in a 250mL Erlenmeyer flask containing 50mL LB liquid medium containing kanamycin, and cultivate overnight at 37°C;

[0068] ②Take 5mL of the overnight bacterial solution in a 500mL Erlenmeyer flask containing 250mL LB liquid medium (containing kanamycin), and shake culture to OD at 200r / min. 600 The value is about 0.6;

[0069] ③Add 100mM IPTG to the final concentration of 0.1mM in the induction flask, culture in a shaker at 37℃ to induce expression for 2h;

[0070] ④ Take the well-growing recombinant strain and the empty expression vector bacteria solution at 4℃, 12000r / min, and centrifuge for 15min. The centrifuged cells were added to silica and 6 mL of pre-cooled 50 mM phosphat...

Embodiment 3

[0073] Example 3: Test and analysis of the resistance of the recombinant protein to metal ions

[0074] The Escherichia coli BL21 transferred into the recombinant plasmid was inoculated into LB liquid medium containing kanamycin and cultivated, OD 680 When it is 0.42, add Cu with a final concentration of 1mM 2+ , Continue to culture for 2 days, and detect the OD of the bacteria 680 , The results are as attached Figure 5 As shown, cK: control, that is, bacteria without metallothionein gene transfer; mt-BL21: bacteria with metallothionein gene transfer. The growth rate of Escherichia coli transfected with metallothionein gene in the medium containing copper ions was significantly higher than that of the control, and it had higher resistance to heavy copper ions.

[0075] The novel metallothionein cloned in the present invention contains 16 cysteines, and the abundant sulfhydryl groups can chelate a variety of heavy metal ions. It is stronger than the heavy metal binding capacity of ...

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Abstract

The invention provides a gene cloning method, expressing method and application of psychrophilic yeast metallothionein. Metallothionein genes are extracted, a primer F1 and a primer R1 are designed, subjected to PCR amplification and then connected with an expression carrier, and a recombinant expression carrier is constructed; the recombinant expression carrier is converted into an expression strain, and a recombination expression transformant is constructed; the expressing and purifying method of recombinant protein is built. The nucleotide sequence and the coded amino acid sequence of the metallothionein genes are shown in the sequence table SEQ ID NO:1 and the sequence table SEQ ID NO:2. The novel metallothionein obtained through cloning comprises 16 pieces of cysteine, multiple metal and heavy metal ions can be chelated through rich sulfydryl, and the novel metallothionein has broad prospects in the medicine field, the food health caring field, the cosmetic additive field, the genetic engineering reagent field, the chemical engineering field, the environmental protection field, the animal field, the agriculture field and the like.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a gene cloning, expression method and application of psychrophilic yeast metallothionein. Background technique [0002] Heavy metal pollution has become a worldwide problem. Japan’s Minamata disease and Toai-in disease are caused by the severe excess of mercury and cadmium in the environment. Heavy metal elements have strong biological toxicity, even in a very low concentration range, they may still cause harm to the natural ecosystem, and heavy metal pollution is concealed, long-term and irreversible. Heavy metals can be absorbed by organisms and are concentrated at the end of the food chain to extremely high concentrations, which can cause various malformations, poisoning, and even cancer. Therefore, it is imperative to clarify the impact of heavy metals on organisms and reveal the adaptation mechanism of organisms to heavy metals, so as to effectively prevent and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/825C12N15/31C12N15/70C12N15/66C12N1/21C12P21/02
CPCC07K14/825C12N15/66C12N15/70C12P21/02
Inventor 阚光锋王晓飞史翠娟解秋菊任丹丹文华魏轲
Owner HARBIN INST OF TECH AT WEIHAI
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