CN110776571B

CN110776571B - Metallothionein fusion protein construction, rapid preparation of immobilized carrier and application of metallothionein fusion protein construction and immobilized carrier in heavy metal ion removal

Info

Publication number: CN110776571B
Application number: CN201911064439.XA
Authority: CN
Inventors: 肖庆; 胡娟; 王国增; 韩静; 蒋畅
Original assignee: Fujian Academy Of Traditional Chinese Medicine (fujian Qingcaoyao Development Service Center)
Current assignee: Fujian Academy Of Traditional Chinese Medicine (fujian Qingcaoyao Development Service Center)
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2021-08-10
Anticipated expiration: 2039-11-04
Also published as: CN110776571A

Abstract

The invention provides metallothionein fusion protein construction, rapid preparation of an immobilized carrier and application of the immobilized carrier in heavy metal ion removal. Fusing metallothionein, carbohydrate binding domain and coding gene of super-folding green fluorescent protein by adopting genetic engineering method to obtain fusion genemt‑cbm‑ sfGFP. And constructing an expression vector by the fusion gene, and transforming the expression vector into an escherichia coli expression host for expression. Fixing the protein obtained by expression on an insoluble carbohydrate carrier, and further applying the water-insoluble carbohydrate particles combined with the metallothionein to the removal of heavy metal ions in the traditional Chinese medicine honeysuckle water decoction. The result shows that the lead ions can be quickly and effectively removed, and the main active ingredients in the honeysuckle water decoction are not influenced. The method has the advantages of low cost, rapidness, convenience and visualization, and can be used for rapidly and effectively removing heavy metals in traditional Chinese medicine decoction, liquid food and wastewater.

Description

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

Technical Field

The invention relates to the technical field of genetic engineering, in particular to metallothionein fusion protein construction, rapid preparation of an immobilized carrier and application of the immobilized carrier in heavy metal ion removal.

Background

At present, about 12000 medicinal plants are used in China, and are generally applied to disease prevention, treatment, health care and health preservation. However, the problem of exceeding of heavy metals in traditional Chinese medicine products cannot be ignored. Heavy metal ions are firmly combined with-SH and-S-S bonds on zymoprotein in a human body, so that the protein is denatured, histiocytes are damaged in structure and function, and the heavy metal ions pose serious threats to the health and public health of the human body. 2015 edition of pharmacopoeia of people's republic of China sets heavy metal limit values for 11 kinds of plant medicines including radix astragali, seaweed and radix salviae miltiorrhizae, 10 kinds of animal medicines including oyster, donkey-hide gelatin and bee, and 6 kinds of mineral medicines including talcum powder and borneol.

The treatment of the heavy metal pollution of the traditional Chinese medicinal materials is mainly started from two aspects of soil remediation and traditional Chinese medicinal material processing. Soil remediation includes approaches such as soil-bearing methods, physical remediation, chemical remediation, phytoremediation, and microbial remediation. Compared with physical and chemical methods, the microbial remediation technology can reduce secondary pollution, and the self-propagation of microbes in soil can ensure long-term treatment to achieve the expected effect. However, the treatment cycle of soil pollution is very long and cannot be carried out in a large range in the same time period, otherwise the sustainable development of economy is seriously affected. Therefore, the polluted Chinese herbal medicines can be treated while the soil is repaired, so that the harm of heavy metals to human bodies is reduced. The Chinese herbal medicine is decoction, which is the earliest and most widely applied formulation in clinical application and is prepared by decocting the medicinal materials in water for a certain time, removing residues and taking juice. In the process of decoction, heavy metals enriched in Chinese herbal medicines are dissociated into water decoction due to denaturation of proteins at high temperature. The method for reducing the harm of heavy metals to human bodies and avoiding the waste of polluted traditional Chinese medicine resources has very important research significance.

Metallothionein (MT) is a metal binding protein with low molecular weight (6-7 kD), no aromatic amino acid, and unique biological characteristics of metal and cysteine (20-30%) which are widely existed in organisms. The metallothionein has a heavy metal ion chelating function due to a large number of sulfydryl groups. By utilizing the characteristic, the metallothionein not only can be used as a biological index for measuring the heavy metal pollution condition, but also can be used for recovering and removing heavy metals and the like. At present, heavy metal pollution is mainly treated by microorganisms containing metallothionein coding genes, which are transformed by genetic engineering. However, when applying whole-cell organisms to complex environments such as sewage, the whole-cell organisms are not only affected by many factors such as pH, temperature, oxygen content, etc., but also are difficult to separate from the environment. For example, Gupta et al wrap Escherichia coli, which overexpresses a metal binding protein and a metallothionein, with calcium alginate biospheres to treat copper and cadmium contamination in wastewater (Dipite Gupta, Suresh Satpati, Anshuman Dixit, Rajiv Ranjan. Applied Microbiology and Biotechnology, 2019, 103: 5411-. In addition, the application field of the metallothionein for treating heavy metal pollution at present mainly focuses on soil, seawater, wastewater, crops and food, but the application field of the metallothionein for treating heavy metal pollution does not relate to traditional Chinese medicines, particularly traditional Chinese medicine preparations. In addition, because metallothionein is rich in cysteine and has important regulation and control effect on metal ions in host cells, the metallothionein has very low expression level in host cells such as escherichia coli, bacillus, yeast and the like, or exists in the form of inactive inclusion bodies.

In view of the above problems, the present invention provides a fusion gene obtained by fusing a gene encoding a Carbohydrate-binding module (CBM) and a gene encoding a superfolder green fluorescent protein (sfGFP)mt-cbm-sfGFP. The fusion gene component is further expressed by a vector and transferred into a heterologous host for expression. The expressed recombinant fusion protein MT-CBM-sfGFP contains three functional units, metallothionein, carbohydrate binding domain and super-folding green fluorescent protein. Carbohydrates in fusion proteinsThe compound binding domain is capable of specifically binding to the corresponding water-insoluble carbohydrate, and thus the fusion protein can be specifically and rapidly purified and immobilized in one step. The green fluorescent protein in the fusion protein has the characteristic of fluorescence under sunlight, so that whether the fusion protein is expressed or not and whether the fusion protein is combined with water-insoluble carbohydrate such as microcrystalline cellulose, chitin and the like can be directly determined. In addition, the water-insoluble carbohydrate such as microcrystalline cellulose has small particles and large specific surface area, can be more combined with target protein to improve the combination amount of heavy metals, and can be separated from the liquid to be detected by a simple centrifugation or filtration method. In addition, microcrystalline cellulose, chitin and other water insoluble carbohydrates are byproducts of agricultural processing, and have the characteristics of low price, easy acquisition and recyclability. The invention takes microcrystalline cellulose combined with fusion protein as an example, and the microcrystalline cellulose combined with the fusion protein can be used in honeysuckle flower water decoction to effectively remove Pb ions without influencing main functional components of the honeysuckle flower water decoction. Therefore, the method of the invention is an effective means for solving resource waste caused by heavy metal pollution of the traditional Chinese medicinal materials and realizing biological recovery of heavy metals. In addition, the invention can also be applied to the rapid and effective removal of heavy metals in other liquids such as liquid food, wastewater and the like.

Disclosure of Invention

The invention aims to invent a visualized metallothionein fusion protein capable of being rapidly fixed on a carrier and apply the metallothionein fusion protein to heavy metal ion removal. The object of the invention can be achieved by the following measures:

the invention relates to a metallothionein coding genemtCarbohydrate binding domain encoding genecbmAnd coding gene of super-folding green fluorescent proteinsfGFPFusing together, transforming into Escherichia coli, and expressing to obtain metallothionein fusion protein MT-CBM-sfGFP or sfGFP-CBM-MT.

The amino acid sequence of the metallothionein fusion protein MT-CBM-sfGFP is shown as SEQ ID NO. 1; the amino acid sequence of the metallothionein fusion protein sfGFP-CBM-MT is shown as SEQ ID NO. 2; the base sequence of the gene for coding the MT-CBM-sfGFP fusion protein is shown as SEQ ID NO. 3; the base sequence of the gene for coding the sfGFP-CBM-MT fusion protein is shown as SEQ ID NO. 4.

A strain for expressing metallothionein fusion protein, which comprisesmt-cbm-sfGFPGenes orsfGFP-cbm-mtA gene.

A preparation method of a gene engineering metallothionein fusion protein comprises the following steps:

(1) metallothionein encoding genemtCarbohydrate binding domain encoding genecbmAnd coding gene of super-folding green fluorescent proteinsfGFPFusing together to obtain a fused genemt-cbm-sfGFPOrsfGFP-cbm-mtConnecting the gene sequence to pET22b (+) expression vector to construct recombinant plasmid pET22b-mt-cbm-sfGFPOr pET22b-sfGFP-cbm-mtTransformed into a strainEscherichia coliBL 21-mt-cbm-sfGFPStrain or BL21sfGFP-cbm-mtA strain;

(2) and (2) respectively inoculating the strains prepared in the step (1) into 5mL of LB culture solution containing ampicillin with the final concentration of 100 mug/mL, and culturing overnight at 37 ℃. Respectively inoculating the cultured bacterial liquids into 200 mL LB liquid culture medium containing ampicillin with the final concentration of 100 mu g/mL according to 1% v/v, and carrying out shake culture at 37 ℃ for about 2-3 h and OD₆₀₀Adding inducer IPTG with final concentration of 0.5 mmol/L after reaching 0.5, and shake culturing at 25 deg.C and 180 rpm for 16 h; centrifuging at 12000 rpm for 5 min, and collecting thallus;

(3) suspending the centrifuged bacteria with 50mM Tris-HCl buffer solution with pH7.6, performing ultrasonic disruption, centrifuging at 12000 rpm for 5 min, and collecting the supernatant as the fusion protein.

A method for immobilizing metallothionein fusion protein comprises adsorbing metallothionein fusion protein and microcrystalline cellulose to obtain cellulose with metallothionein fusion protein.

Further, the prepared metallothionein fusion protein is applied to removal of heavy metal ions in traditional Chinese medicine decoction, liquid food and wastewater.

The invention has the beneficial effects that: the invention carries out fusion expression on the metallothionein gene, the carbohydrate binding domain gene and the super-folding green fluorescent protein gene, can promote the soluble expression of the metallothionein and increase the yield, and can directly judge whether the metallothionein is expressed or not and whether the fusion protein is fixed on a carrier or not through the super-folding green fluorescent protein. In addition, the purification and fixation of the metallothionein are carried out in one step through a carbohydrate binding structural domain in the fusion protein, so that the production cost is reduced. Finally, the immobilized metallothionein can quickly chelate the heavy metal in the liquid sample, and the metallothionein chelated with the heavy metal can be recovered by a centrifugation or filtration method, so that the aim of removing the heavy metal in the sample is fulfilled.

Description of the drawings:

FIG. 1 is a schematic representation of a metallothionein fusion protein; wherein A: MT-CBM-sfGFP; b: sfGFP-CBM-MT.

FIG. 2 metallothionein fusion protein expression; wherein 1: not inducing; 2: IPTG induced expression.

FIG. 3 immobilization of metallothionein fusion proteins; wherein 1: microcrystalline cellulose; 2: microcrystalline cellulose incorporating metallothionein fusion proteins.

FIG. 4 shows the comparison effect before and after removing lead ions in the water decoction of honeysuckle.

FIG. 5 the effect of microcrystalline Cellulose and MT-CBM-sfGFP @ Cellulose on chlorogenic acid in honeysuckle; wherein 1: a chlorogenic acid standard substance; 2: decocting flos Lonicerae in water; 3: honeysuckle water decoction and microcrystalline cellulose; 4: honeysuckle decoction + MT-CBM-sfGFP @ Cellulose.

Detailed Description

Test materials and reagents

1. Bacterial strain and carrier: escherichia coli expression vector pET22b (+) and strainEscherichia coliBL21 (DE 3) is available from Novagen.

2. Enzymes and other biochemical reagents: restriction enzymes, T4 DNA ligase, DNA polymerase, and dNTPs are available from TaKaRa, Japan.

3. The genome extraction kit was purchased from Tiangen, Beijing, and the purification and plasmid extraction kit was purchased from OMEGA, USA.

4. Peptone (Tryptone) and Yeast Extract (Yeast Extract) are products of OXOID, UK, and the rest reagents are domestic analytical pure.

5. Culture medium:

(1) LB liquid medium (g/l): 10.0 parts of peptone, 5.0 parts of yeast powder, 10.0 parts of NaCl and 7.0 parts of pHs.

(2) LB solid Medium (g/l): 5.0 parts of yeast powder, 10.0 parts of peptone, 10.0 parts of NaCl, 15.0 parts of agar and 7.0 parts of pHs.

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Example 1 design of metallothionein fusion Gene

One key step in the expression of metallothionein fusion proteins is the order and junction regions between individual monomers of the fusion protein. The metallothionein fusion protein comprises 3 functional units, namely 1 metallothionein; 2. a carbohydrate-binding domain; 3. superfolder green fluorescent protein. Therefore, there are 6 combinations in the permutation and combination. Since the carbohydrate-binding domain functions as an adsorption carrier, the metallothionein and the superfolder green fluorescent protein are located at both ends of the carbohydrate-binding domain (shown in FIGS. 1, A and B), respectively, and thus can function as both functional units, respectively.

The present invention selects metallothionein gene derived from common marmoset as a representative (GenBank accession number: BN 178946), cellulose binding domain gene derived from trichoderma as a representative (GenBank accession number: AY 234176), and sfGFP coding gene is obtained from plasmid pG1AK-sfGFP (Addgene, accession number: 71739). Optimizing the metallothionein gene sequence and the cellulose binding structure domain gene sequence according to the codon preference of escherichia coli, and finally obtaining the fusion protein with the gene sequence as follows:

1. mt-cbm-sfGFPthe gene sequence is as follows:

ATGCCGGACCCGTGCTGCAAGGATAAATGCGAGTGCAAGGAAGGTGGCTGCAAGGCGGGTTGCAAATGCACCGCGTGCTGCTGCAGCCCGTGCGACAAATGCACCAGCGGCTGCAAGTGCACCAACAAAGATGAATGCAGCAAGACCTGCAGCAAACCGTGCAGCTGCTGCCCGGGTAGCGGTGGCGCGGGTGGCAGCGCGCCGGGTTGCCGTGTGGACTATGCGGTTACCAACCAATGGCCGGGTGGCTTCGGTGCGAACGTGACCATCACCAACCTGGGTGACCCGGTTAGCAGCTGGAAGCTGGATTGGACCTATACCGCGGGCCAGCGTATTCAGCAACTGTGGAACGGTACCGCGAGCACCAACGGTGGCCAAGTGAGCGTTACCAGCCTGCCGTGGAACGGTAGCATTCCGACCGGTGGCACCGCGAGCTTCGGCTTTAACGGTAGCTGGGCGGGTAGCAACCCGACCCCGGCGAGCTTCAGCCTGAACGGTACCACCTGCACCGGTGGTGGCGGTAGCCCGACCGGCGGTCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTACCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATGA

2. sfGFP-cbm-mtthe gene sequence is as follows:

ATGCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTACCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAAGGTAGCGGTGGCGCGGGTGGCAGCGCGCCGGGTTGCCGTGTGGACTATGCGGTTACCAACCAATGGCCGGGTGGCTTCGGTGCGAACGTGACCATCACCAACCTGGGTGACCCGGTTAGCAGCTGGAAGCTGGATTGGACCTATACCGCGGGCCAGCGTATTCAGCAACTGTGGAACGGTACCGCGAGCACCAACGGTGGCCAAGTGAGCGTTACCAGCCTGCCGTGGAACGGTAGCATTCCGACCGGTGGCACCGCGAGCTTCGGCTTTAACGGTAGCTGGGCGGGTAGCAACCCGACCCCGGCGAGCTTCAGCCTGAACGGTACCACCTGCACCGGTGGTGGCGGTAGCCCGACCGGCGGTCCGGACCCGTGCTGCAAGGATAAATGCGAGTGCAAGGAAGGTGGCTGCAAGGCGGGTTGCAAATGCACCGCGTGCTGCTGCAGCCCGTGCGACAAATGCACCAGCGGCTGCAAGTGCACCAACAAAGATGAATGCAGCAAGACCTGCAGCAAACCGTGCAGCTGCTGCCCGTGA

the amino acid sequences corresponding to the two fusion genes are as follows:

1. protein sequence of MT-CBM-sfGFP

MPDPCCKDKCECKEGGCKAGCKCTACCCSPCDKCTSGCKCTNKDECSKTCSKPCSCCPGSGGAGGSAPGCRVDYAVTNQWPGGFGANVTITNLGDPVSSWKLDWTYTAGQRIQQLWNGTASTNGGQVSVTSLPWNGSIPTGGTASFGFNGSWAGSNPTPASFSLNGTTCTGGGGSPTGGRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

2. Protein sequence of sfGFP-CBM-MT

MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSGGAGGSAPGCRVDYAVTNQWPGGFGANVTITNLGDPVSSWKLDWTYTAGQRIQQLWNGTASTNGGQVSVTSLPWNGSIPTGGTASFGFNGSWAGSNPTPASFSLNGTTCTGGGGSPTGGPDPCCKDKCECKEGGCKAGCKCTACCCSPCDKCTSGCKCTNKDECSKTCSKPCSCCP

Example 2 expression of metallothionein fusion Gene and immobilization of fusion protein

The coding gene of the fusion protein constructed according to the design thought is sent to Nanjing Kingsrei Biotech CoAnd (4) synthesizing. To introduce at 5 'and 3' ends of gene respectivelySacI andXhothe primer pair of the enzyme cutting site I (see table 1) and the plasmid obtained by gene synthesis as a template are subjected to PCR amplification. The PCR reaction parameters are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 sec, annealing at 60 ℃ for 30 sec, extension at 72 ℃ for 1.5 min, 30 cycles, and heat preservation at 72 ℃ for 5 min. The expression vector pET22b (+) was subjected to double digestion (SacI+ XhoI) Simultaneously fusing metallothionein with the gene of the proteinmt-cbm-sfGFPAndsfGFP- cbm-mtcarrying out double enzyme digestion (SacI+ XhoI) Gene of the excised metallothionein fusion proteinmt-cbm-sfGFPAndsfGFP-cbm-mtthe fragment was ligated with an expression vector pET28a (+) to obtain a gene containing a fusion proteinmt-cbm-sfGFPAndsfGFP-cbm-mtthe recombinant plasmid pET22b of (1)-mt-cbm-sfGFPAnd pET22b-sfGFP-cbm-mtAnd transforming Escherichia coli BL21 (DE 3) to obtain the recombinant Escherichia coli strain BL21mt-cbm-sfGFPAnd BL21sfGFP-cbm-mt。

TABLE 1 primer sequences used in this study

Taking a plasmid containing the recombinant plasmid pET22b-mt-cbm-sfGFPAnd pET22b-sfGFP-cbm-mtThe BL21 strains (A) were inoculated in 5mL of LB medium (containing ampicillin at a final concentration of 100. mu.g/mL) and cultured overnight at 37 ℃. Then respectively inoculating the cultured bacterial liquids into 200 mL LB liquid culture medium (added with ampicillin with the final concentration of 100 mug/mL) according to 1% (v/v), and carrying out shake culture at 37 ℃ for about 2-3 h (OD)₆₀₀Reaching 0.5), adding inducer IPTG with the final concentration of 0.5 mmol/L, and culturing for 16 h at 25 ℃ and 180 rpm with shaking. Coli not induced was used as a control. Centrifugation was carried out at 12000 rpm for 5 min to collect the cells. As shown in FIG. 2, the cells were white without the fusion protein expression in the non-induced cells, and the cells were green in sunlight with the sfGFP present in the cells after the induction due to the fusion protein expression.

The cells obtained by centrifugation were suspended in Tris-HCl buffer (50mM, pH7.6), sonicated, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. Microcrystalline cellulose was added to the obtained supernatant in an amount of 0.5 g per ml, and left at 4 ℃ for 12 hours, during which time it was taken out at 1 hour intervals and gently shaken for 1 minute to promote the binding of the fusion protein to microcrystalline cellulose. As shown in FIG. 3, microcrystalline cellulose bound to the fusion protein appeared green (FIG. 3-2), while the control was white (FIG. 3-1). The Cellulose to which the metallothionein fusion protein was bound was named MT-CBM-sfGFP @ Cellulose.

Example 3 application of fusion protein in removing heavy metals in honeysuckle flower water decoction

3.1 preparation of honeysuckle Water decoction

Accurately weighing 25 g of honeysuckle, adding 250 ml of redistilled water, soaking for one hour, boiling, refluxing for 1 hour, cooling to room temperature, and filtering with gauze to obtain decoction. Centrifuging the decoction at 10000rpm/min for 10 min to obtain supernatant. And (3) taking the supernatant, respectively adding water with the same volume or 0.5 g/ml MT-CBM-sfGFP @ Cellulose suspension, shaking for half an hour, centrifuging to take 1 ml of the supernatant, adding 0.25ml of each of 2w/v% ammonium dihydrogen phosphate and 0.4 w/v% magnesium nitrate, and measuring the absorption value by adopting a graphite furnace method.

Graphite furnace method for measuring Pb in honeysuckle water decoction²⁺

And (3) adding equal volume of water or 0.5 g/ml MT-CBM-sfGFP @ Cellulose suspension into the supernatant respectively, shaking for half an hour, centrifuging to obtain 1 ml of the supernatant, adding 0.25ml of each of 2w/v% ammonium dihydrogen phosphate and 0.4 w/v% magnesium nitrate, and measuring the absorption value by adopting a graphite furnace method.

As can be seen from FIG. 4, Pb in the water decoction of flos Lonicerae was free²⁺Relatively much Pb in solution after interaction with MT-CBM-sfGFP @ Cellulose²⁺The significant reduction (3.64 ng/ml → 1.11 ng/ml) indicates that the MT-CBM-sfGFP @ Cellulose can be used for treating Pb in the honeysuckle water decoction²⁺Has strong adsorption effect.

Determining the influence of MT-CBM-sfGFP @ Cellulose on the main active ingredients in the honeysuckle water decoction

The flos Lonicerae is Lonicera Japonica of CaprifoliaceaeLonicera japonica Thunb.The dried buds or the flowers to be bloomed,can be used as raw material of medicine, health product, cosmetic and food, and has antibacterial, antiinflammatory, heat and toxic materials clearing away, and cold wind and heat dissipating effects. Chlorogenic acid is one of the indexes for controlling the quality of honeysuckle in pharmacopoeia, and has broad-spectrum antibacterial activity. Pb in honeysuckle water decoction by MT-CBM-sfGFP @ Cellulose²⁺Has strong adsorption effect, and further examination is needed to determine whether influence is caused on chlorogenic acid.

(1) Chromatographic conditions are as follows: elitet hypersil ODS 2C₁₈Columns (250 mm. times.4.6 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid water (gradient elution); column temperature: 30 ℃; the sample amount is 10 mul; the detection wavelength is 327 nm.

TABLE 2 acetonitrile-0.1% phosphoric acid water ratio and elution time

(2) Preparing a chlorogenic acid reference substance solution and a test solution: accurately weighing 1mg chlorogenic acid reference substance, and dissolving in distilled water to obtain 1mg/ml reference substance solution. Taking the supernatant of the honeysuckle water decoction, respectively adding water, 0.5 g/ml Cellulose and 0.5 g/ml MT-CBM-sfGFP @ Cellulose suspension in the same volume, stirring for 0.5 h, centrifuging to take the supernatant, diluting by 10 times, and filtering to obtain the sample solution.

As can be seen from FIG. 5, chlorogenic acid peaks at 13.07 min, supernatant of water decoction of flos Lonicerae was added with equal volume of water, 0.5 g/ml Cellulose and 0.5 g/ml MT-CBM-sfGFP @ Cellulose suspension, and after stirring for 0.5 h, the supernatant was centrifuged, and after 10-fold dilution, the chlorogenic acid in the solution was filtered, whether the retention time/min (13.093, 13.073, 13.07, respectively) or the peak area/mAU.min (72.4629, 72.1709, 72.1408) was almost the same, which indicates that the use of MT-CBM-sfGFP @ Cellulose to remove lead ions from water decoction of flos Lonicerae did not affect the main active ingredient chlorogenic acid.

SEQUENCE LISTING

<110> department of Fujian province of traditional Chinese medicine research (development service center of green grass medicine of Fujian province)

<120> metallothionein fusion protein construction, rapid preparation of immobilized carrier and removal of heavy metal ions

In (1)

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Met Pro Asp Pro Cys Cys Lys Asp Lys Cys Glu Cys Lys Glu Gly Gly

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Lys Cys Thr Ser Gly Cys Lys Cys Thr Asn Lys Asp Glu Cys Ser Lys

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Ser Phe Ser Leu Asn Gly Thr Thr Cys Thr Gly Gly Gly Gly Ser Pro

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Thr Leu Thr Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met

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Glu Arg Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala

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Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys

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145 150 155 160

Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

180 185 190

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Val Leu Ser

195 200 205

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

210 215 220

Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Gly Ser

225 230 235 240

Gly Gly Ala Gly Gly Ser Ala Pro Gly Cys Arg Val Asp Tyr Ala Val

245 250 255

Thr Asn Gln Trp Pro Gly Gly Phe Gly Ala Asn Val Thr Ile Thr Asn

260 265 270

Leu Gly Asp Pro Val Ser Ser Trp Lys Leu Asp Trp Thr Tyr Thr Ala

275 280 285

Gly Gln Arg Ile Gln Gln Leu Trp Asn Gly Thr Ala Ser Thr Asn Gly

290 295 300

Gly Gln Val Ser Val Thr Ser Leu Pro Trp Asn Gly Ser Ile Pro Thr

305 310 315 320

Gly Gly Thr Ala Ser Phe Gly Phe Asn Gly Ser Trp Ala Gly Ser Asn

325 330 335

Pro Thr Pro Ala Ser Phe Ser Leu Asn Gly Thr Thr Cys Thr Gly Gly

340 345 350

Gly Gly Ser Pro Thr Gly Gly Pro Asp Pro Cys Cys Lys Asp Lys Cys

355 360 365

Glu Cys Lys Glu Gly Gly Cys Lys Ala Gly Cys Lys Cys Thr Ala Cys

370 375 380

Cys Cys Ser Pro Cys Asp Lys Cys Thr Ser Gly Cys Lys Cys Thr Asn

385 390 395 400

Lys Asp Glu Cys Ser Lys Thr Cys Ser Lys Pro Cys Ser Cys Cys Pro

405 410 415

<210> 3

<211> 1251

<212> DNA

<213> Artificial sequence

<400> 3

atgccggacc cgtgctgcaa ggataaatgc gagtgcaagg aaggtggctg caaggcgggt 60

tgcaaatgca ccgcgtgctg ctgcagcccg tgcgacaaat gcaccagcgg ctgcaagtgc 120

accaacaaag atgaatgcag caagacctgc agcaaaccgt gcagctgctg cccgggtagc 180

ggtggcgcgg gtggcagcgc gccgggttgc cgtgtggact atgcggttac caaccaatgg 240

ccgggtggct tcggtgcgaa cgtgaccatc accaacctgg gtgacccggt tagcagctgg 300

aagctggatt ggacctatac cgcgggccag cgtattcagc aactgtggaa cggtaccgcg 360

agcaccaacg gtggccaagt gagcgttacc agcctgccgt ggaacggtag cattccgacc 420

ggtggcaccg cgagcttcgg ctttaacggt agctgggcgg gtagcaaccc gaccccggcg 480

agcttcagcc tgaacggtac cacctgcacc ggtggtggcg gtagcccgac cggcggtcgt 540

aaaggcgaag agctgttcac tggtgtcgtc cctattctgg tggaactgga tggtgatgtc 600

aacggtcata agttttccgt gcgtggcgag ggtgaaggtg acgcaactaa tggtaaactg 660

acgctgaagt tcatctgtac tactggtaaa ctgccggtac cttggccgac tctggtaacg 720

acgctgactt atggtgttca gtgctttgct cgttatccgg accatatgaa gcagcatgac 780

ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac gcacgatttc ctttaaggat 840

gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag gcgataccct ggtaaaccgc 900

attgagctga aaggcattga ctttaaagaa gacggcaata tcctgggcca taagctggaa 960

tacaatttta acagccacaa tgtttacatc accgccgata aacaaaaaaa tggcattaaa 1020

gcgaatttta aaattcgcca caacgtggag gatggcagcg tgcagctggc tgatcactac 1080

cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc cagacaatca ctatctgagc 1140

acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg atcatatggt tctgctggag 1200

ttcgtaaccg cagcgggcat cacgcatggt atggatgaac tgtacaaatg a 1251

<210> 4

<211> 1251

<212> DNA

<213> Artificial sequence

<400> 4

atgcgtaaag gcgaagagct gttcactggt gtcgtcccta ttctggtgga actggatggt 60

gatgtcaacg gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt 120

aaactgacgc tgaagttcat ctgtactact ggtaaactgc cggtaccttg gccgactctg 180

gtaacgacgc tgacttatgg tgttcagtgc tttgctcgtt atccggacca tatgaagcag 240

catgacttct tcaagtccgc catgccggaa ggctatgtgc aggaacgcac gatttccttt 300

aaggatgacg gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta 360

aaccgcattg agctgaaagg cattgacttt aaagaagacg gcaatatcct gggccataag 420

ctggaataca attttaacag ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc 480

attaaagcga attttaaaat tcgccacaac gtggaggatg gcagcgtgca gctggctgat 540

cactaccagc aaaacactcc aatcggtgat ggtcctgttc tgctgccaga caatcactat 600

ctgagcacgc aaagcgttct gtctaaagat ccgaacgaga aacgcgatca tatggttctg 660

ctggagttcg taaccgcagc gggcatcacg catggtatgg atgaactgta caaaggtagc 720

ggtggcgcgg gtggcagcgc gccgggttgc cgtgtggact atgcggttac caaccaatgg 780

ccgggtggct tcggtgcgaa cgtgaccatc accaacctgg gtgacccggt tagcagctgg 840

aagctggatt ggacctatac cgcgggccag cgtattcagc aactgtggaa cggtaccgcg 900

agcaccaacg gtggccaagt gagcgttacc agcctgccgt ggaacggtag cattccgacc 960

ggtggcaccg cgagcttcgg ctttaacggt agctgggcgg gtagcaaccc gaccccggcg 1020

agcttcagcc tgaacggtac cacctgcacc ggtggtggcg gtagcccgac cggcggtccg 1080

gacccgtgct gcaaggataa atgcgagtgc aaggaaggtg gctgcaaggc gggttgcaaa 1140

tgcaccgcgt gctgctgcag cccgtgcgac aaatgcacca gcggctgcaa gtgcaccaac 1200

aaagatgaat gcagcaagac ctgcagcaaa ccgtgcagct gctgcccgtg a 1251

<210> 5

<211> 31

<212> DNA

<213> Artificial sequence

<400> 5

ttcgagctca tgccggaccc gtgctgcaag g 31

<210> 6

<211> 30

<212> DNA

<213> Artificial sequence

<400> 6

gtgctcgagt cacgggcagc agctgcacgg 30

<210> 7

<211> 37

<212> DNA

<213> Artificial sequence

<400> 7

ttcgagctca tgcgtaaagg cgaagagctg ttcactg 37

<210> 8

<211> 34

<212> DNA

<213> Artificial sequence

<400> 8

gtgctcgagt cacgggcagc agctgcacgg tttg 34

Claims

1. A genetically engineered metallothionein fusion protein is characterized in that metallothionein coding gene is usedmtCarbohydrate binding domain encoding genecbmAnd coding gene of super-folding green fluorescent proteinsfGFPFusing together, transforming into Escherichia coli, and expressing to obtain metallothionein fusion protein MT-CBM-sfGFP or sfGFP-CBM-MT;

2. A strain expressing the metallothionein fusion protein of claim 1, wherein the strain comprisesmt-cbm-sfGFPGenes orsfGFP-cbm-mtA gene.

3. A method for preparing the genetically engineered metallothionein fusion protein according to claim 1, comprising the steps of:

(2) respectively inoculating the strains prepared in the step (1) into 5mL of LB culture solution containing ampicillin with the final concentration of 100 mug/mL, and culturing overnight at 37 ℃; respectively inoculating the cultured bacterial liquids into 200 mL LB liquid culture medium containing ampicillin with the final concentration of 100 mug/mL according to 1% v/v, and carrying out shake culture at 37 ℃ for 2-3 h and OD₆₀₀Adding inducer IPTG with final concentration of 0.5 mmol/L after reaching 0.5, and shake culturing at 25 deg.C and 180 rpm for 16 h; centrifuging at 12000 rpm for 5 min, and collecting thallus;

4. The method for immobilizing metallothionein fusion protein according to claim 1, wherein the metallothionein fusion protein is adsorbed and bound to microcrystalline cellulose to obtain the cellulose having metallothionein fusion protein.

5. The use of the metallothionein fusion protein of claim 1 to remove heavy metal ions from traditional Chinese medicine water decoctions, liquid foods, and wastewater.