Construction of metallothionein fusion protein, rapid preparation of immobilized carrier and application of metallothionein fusion protein in heavy metal ion removal

A metallothionein and fusion protein technology, which is applied in the removal of heavy metal ions, the construction of metallothionein fusion proteins, and the rapid preparation of immobilized carriers, can solve the problems of difficult separation, small specific surface area, and large biological beads. Achieve the effect of increasing yield, reducing production cost, and promoting soluble expression

Active Publication Date: 2020-02-11
福建省中医药科学院
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  • Abstract
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Problems solved by technology

However, when whole-cell organisms are applied in complex environments such as sewage, not only the whole-cell organisms will be affected by many factors such as pH, temperature, and oxygen content, but it is also difficult to separate them from the environment.
For example, Gupta et al. wrapped Escherichia coli that can overexpress metal-binding protein and metallothionein with calcium alginate biobeads to treat copper and cadmium pollution in sewage (Dipinte Gupta, Suresh Satpati, Anshuman Dixit, Rajiv Ranjan. Applied Microbiology andBiotechnology, 2019, 103: 5411-5420), although the above method can separate the biological beads adsorbing heavy metals from sewage, but the biological beads have large particles and relatively small specific surface area
In addition, the current application fields of metallothionein to control heavy metal pollution are mainly concentrated in soil, seawater, wastewater, crops, and food, while Chinese herbal medicines, especially Chinese herbal medicine preparations, have not been involved.

Method used

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  • Construction of metallothionein fusion protein, rapid preparation of immobilized carrier and application of metallothionein fusion protein in heavy metal ion removal
  • Construction of metallothionein fusion protein, rapid preparation of immobilized carrier and application of metallothionein fusion protein in heavy metal ion removal
  • Construction of metallothionein fusion protein, rapid preparation of immobilized carrier and application of metallothionein fusion protein in heavy metal ion removal

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Design of metallothionein fusion gene

[0032] A key step in the expression of the metallothionein fusion protein lies in the order of the individual monomers of the fusion protein and the linking regions between them. The metallothionein fusion protein of the present invention includes three functional units: 1. metallothionein; 2. carbohydrate binding domain; 3. superfolded green fluorescent protein. Therefore, there are a total of 6 combinations according to the way of permutation and combination. Since the carbohydrate-binding domain acts as an adsorption carrier, the carbohydrate-binding domain is placed in the middle, and metallothionein and superfolded green fluorescent protein can be located at its two ends ( figure 1 , shown in A and B), which can play the roles of these two functional units respectively.

[0033] The present invention selects the metallothionein gene derived from common marmoset as a representative (GenBank accession number: BN178...

Embodiment 2

[0043] Example 2 Expression of metallothionein fusion gene and immobilization of fusion protein

[0044] The gene encoding the fusion protein constructed according to the design ideas above was sent to Nanjing GenScript Biotechnology Co., Ltd. for synthesis. Introduced at the 5' and 3' ends of the gene, respectively Sac I and xho The primer pair for I restriction site (see Table 1), and the plasmid obtained by gene synthesis was used as a template for PCR amplification. The PCR reaction parameters were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 1.5 min, 30 cycles, and incubation at 72°C for 5 min. The expression vector pET22b (+) was double digested ( Sac I+ xho I), while the metallothionein fusion protein gene mt-cbm-sfGFP and sfGFP-cbm-mt double enzyme digestion ( Sac I+ xho I), Chopped metallothionein fusion protein gene mt-cbm-sfGFP and sfGFP- cbm-mt The fragment was connecte...

Embodiment 3

[0049] Example 3 Application of fusion protein in removing heavy metals in honeysuckle water decoction

[0050] 3.1 Preparation of honeysuckle water decoction

[0051] Accurately weigh 25 g of honeysuckle, add 250 ml of double-distilled water to soak for one hour, boil and reflux for 1 hour, cool to room temperature, and filter with gauze to obtain a decoction. The decoction was centrifuged at 10000 rpm / min for 10 min to obtain the supernatant. Take the supernatant, add an equal volume of water or 0.5 g / ml MT-CBM-sfGFP@Cellulose suspension, shake for half an hour and centrifuge to get 1ml of the supernatant, add 2w / v% ammonium dihydrogen phosphate and 0.4 w / v % magnesium nitrate each 0.25ml, the absorption value was determined by the graphite furnace method.

[0052] Determination of Pb in Water Decoction of Honeysuckle by Graphite Furnace Method 2+

[0053] Take the supernatant and add an equal volume of water or 0.5 g / ml MT-CBM-sfGFP@Cellulose suspension, shake for half ...

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Abstract

The invention provides construction of metallothionein fusion protein, rapid preparation of an immobilized carrier and application of the metallothionein fusion protein in heavy metal ion removal. A genetic engineering method is used for fusing encoding genes of metallothionein, carbohydrate binding domain and superfolded green fluorescent protein to obtain a fusion gene mt-cbm-sfGFP. An expression vector is constructed from the fusion gene and transformed into an escherichia coli expression host to be expressed. The expressed protein is immobilized onto an insoluble carbohydrate carrier, andwater-insoluble carbohydrate particles bound with the metallothionein are further applied to remove heavy metal ions in traditional Chinese medicine flos lonicerae japonicae water decoction. The results show that lead ions can be removed quickly and effectively without affecting main active ingredients in the flos lonicerae japonicae water decoction. The method has the advantages of low cost, quickness and convenience and visualization, and can be used for the rapid and effective removal of heavy metals in traditional Chinese medicine water decoction, liquid food and waste water.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the construction of a metallothionein fusion protein, the rapid preparation of an immobilized carrier and its application in the removal of heavy metal ions. Background technique [0002] At present, there are about 12,000 kinds of medicinal plants in China, which are widely used in disease prevention, treatment and health care. However, the problem of excessive heavy metals in traditional Chinese medicinal materials cannot be ignored. Heavy metal ions bind firmly to the -SH and -S-S bonds on enzyme proteins in the human body, thereby denaturing proteins, causing structural and functional damage to tissue cells, and posing a serious threat to human health and public health. The 2015 edition of the Pharmacopoeia of the People's Republic of China includes 11 kinds of herbal medicines including astragalus, seaweed, and salvia miltiorrhiza, 10 kinds of animal medicines i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C07K17/12B01J20/24B01J20/30C02F1/28C02F101/20
CPCB01J20/24C02F1/286C02F1/288C02F2101/20C07K14/43595C07K14/825C07K17/12C07K2319/00C12N15/70
Inventor 肖庆胡娟王国增韩静蒋畅
Owner 福建省中医药科学院
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