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Ramie metallothionein gene and recombinant protein and application thereof

A technology of metallothionein and ramie, applied in the field of bioengineering, can solve the problems of limited heavy metal ion binding capacity, large molecular weight, and low cysteine ​​content, and achieve strong binding force, small molecular weight, and high cysteine ​​content Effect

Active Publication Date: 2018-02-16
INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The metallothionein obtained in the above patents has the defects of relatively large molecular weight and low cysteine ​​content, so its ability to bind heavy metal ions is limited

Method used

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  • Ramie metallothionein gene and recombinant protein and application thereof
  • Ramie metallothionein gene and recombinant protein and application thereof
  • Ramie metallothionein gene and recombinant protein and application thereof

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Experimental program
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Effect test

Embodiment 1

[0039] The ramie metallothionein gene is the nucleotide sequence shown in SEQ ID NO.1 of the sequence table; the ramie metallothionein is the amino acid sequence shown in the sequence table SEQ ID NO.2.

[0040] 1. Synthesize the nucleotide sequence shown in SEQ ID NO.1 in the sequence table

[0041] (1) Extraction of total RNA from ramie root samples

[0042]The total RNA of ramie root samples was extracted according to the RNA extraction kit of Promega Company The instructions for Total RNAKit II (50) were carried out with minor modifications. The plastic utensils used were treated with DEPC (diethylpyrocarbonate) water; the glass utensils were baked at 180°C for 6h; all other reagents were also prepared with DEPC treated water, and sterilized at 121°C for 20min

[0043] ①Take about 100 mg of ramie root samples, wash them with DEPC treated water, put them into a liquid nitrogen pre-cooled mortar, and immediately add liquid nitrogen to grind them into powder;

[0044] ② I...

Embodiment 2

[0070] Tolerance analysis of a ramie metallothionein recombinant transformation engineered strain to cadmium ions

[0071] The escherichia coli engineering strain BL21 that will be transformed into the ramie metallothionein recombinant plasmid is inoculated in LB liquid medium (containing kanamycin 50mg / L) by 1:50 ratio and cultivates; When the absorbance value OD 600At about 0.2, add IPTG with a final concentration of 1mM for induction for 30 minutes, and then add cadmium chloride solutions with a final concentration of 50mg / L, 100mg / L, and 200mg / L, respectively, and culture with shaking at 200rpm at 37°C. Hourly detection of bacterial solution OD 600 value. The control group was the Escherichia coli control strain without the ramie metallothionein gene. Experimental results such as Figure 4 As shown, A is the Escherichia coli control strain (pET-30a / BL21) that has not been transferred to the ramie metallothionein gene, and B is a recombinant strain (pET-30a-bnmtl / BL21) t...

Embodiment 3

[0073] Enrichment effect of a ramie metallothionein recombinant transformation engineering bacteria on cadmium ions

[0074] The Escherichia coli engineering strain BL21 transformed into the ramie metallothionein recombinant plasmid was inoculated into LB liquid medium (containing kanamycin 50mg / L) in a ratio of 1:50 for cultivation; when OD 600 When the value is 0.2, add IPTG with a final concentration of 1mM for induction for 30 minutes, then add a cadmium chloride solution with a final concentration of 100mg / L, and collect the bacteria after shaking at 37°C and 200rpm for 6 hours; take 0.1g of the bacteria and add Put it into a ceramic crucible, heat it in a muffle furnace at 500°C for 8 hours, and then add 5ml of nitric acid / chloric acid mixture (HNO 3 :HClO 4 =3:4); reheating (100° C.) until no carbon residue is visible, add 10ml concentration of 8.3% saline solution to dissolve the residue, and use flame atomic absorption spectrometry to measure the cadmium ion content ...

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Abstract

The invention discloses a ramie metallothionein gene and recombinant protein and an application thereof. The ramie metallothionein gene is a nucleotide sequence shown as the sequence table SEQ ID NO.1, and the ramie metallothionein is an amino acid sequence shown as the sequence table SEQ ID NO.2. A new metallothionein gene is obtained from ramie which is a herbaceous plant through in-vivo cloning, a recombinant expression engineering strain is constructed, and the recombinant metallothionein is obtained through expression and purification. The recombinant protein contains 12 cysteines (Cys),is abundant in mercapto groups that can chelate with cadmium and other heavy metal ions, and has wide application prospects in the fields of preparation of a heavy metal chelating antidote, chemical or medicine or heavy metal polluted cultivated land and pollution treatment of agricultural non-point sources.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a ramie metallothionein gene, its recombinant protein and its application. Background technique [0002] With the rapid development of economy and industry, the accumulation and continuous diffusion of heavy metals in the ecological environment have led to the increasingly serious problem of heavy metal pollution worldwide. At present, the total area of ​​cultivated land polluted by heavy metals such as cadmium, arsenic, chromium, and lead in my country reaches 20 million hm 2 , the cultivated land area polluted by cadmium is close to 13,300 hm 2 , Bringing a severe test to my country's ecological economic construction and sustainable development. Metallothionein (MT), which is rich in cysteine ​​residues (10-30%), can chelate cadmium, copper, lead, zinc and other ions through the aggregation of sulfhydryl clusters, and is a natural ligand for heavy metal ion...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/825C12N15/70B09C1/08
CPCB09C1/08C07K14/825C12N15/70
Inventor 高钢朱爱国喻春明陈平陈坤梅陈继康熊和平
Owner INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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