CN105200065B - A kind of metallothionein gene and application - Google Patents
A kind of metallothionein gene and application Download PDFInfo
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- CN105200065B CN105200065B CN201510762680.5A CN201510762680A CN105200065B CN 105200065 B CN105200065 B CN 105200065B CN 201510762680 A CN201510762680 A CN 201510762680A CN 105200065 B CN105200065 B CN 105200065B
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Abstract
The present invention provides a kind of new metallothionein genes and its application.The present invention is expanded from tetrahymena Tetrahymena elliotti by round pcr and obtains a kind of new gene, it is mutated terminator codon and obtains mutated gene Te-MTT2 later, mutated gene Te-MTT2 is connected in carrier pSUMO, obtain the recombinant plasmid pSUMO-Te-MTT2 containing the gene, after converting e. coli bl21, it is induced through IPTG, recombination engineering bacteria BL21/pSUMO-Te-MTT2 gives expression to soluble protein SUMO-Te-MTT2.Recombination engineering bacteria BL21/pSUMO-Te-MTT2 has the ability of very strong heavy metal stress tolerance and enriched in metals ion under IPTG induction, and recombination engineering bacteria BL21/pSUMO-Te-MTT2 has wide practical use in terms of administering Heavy Metal Pollution in Water Environment.
Description
Technical field
The present invention relates to a kind of genes, the metallothionein specifically cloned from tetrahymena Tetrahymena elliotti
White gene, the recombination engineering containing the gene have important application prospect in water body environment in terms of Heavy Metal Pollution Control.
Background technique
Metal is increasingly becoming global problem to seriously threatening for environment, and heavy metal runs up to certain limit in water body
Degree will generate serious harm to water body-water plant-aquatic animal system, and may pass through food chain directly or indirectly shadow
Ring the own health for arriving the mankind.How hot spot that heavy metal to the pollution of water body have become research is scientificlly and effectively solved.Mesh
It is preceding for administer the chemical method of heavy metal pollution of water body, physical-chemical process will all generate pollution transportation, easily cause secondary pollution, and
The harmful heavy metal pollution of large watershed, low concentration is difficult to handle.
And bioanalysis has effect is good, small investment and operating costs are low, are easily managed and operate, do not generate secondary pollution etc.
Advantage is increasingly subject to the concern of people.In recent years, microorganism and technique for gene engineering are gradually available for Heavy Metal Pollution in Water Environment
It administers, and plays increasingly important role in terms of the improvement of heavy metal pollution in water body environment.How recombination work is improved
Journey bacterium absorbs the ability of heavy metal ion, is the major issue administering Heavy Metal Pollution in Water Environment and being faced.
Tetrahymena MTs is located at the 7th family, tetrahymena metallothionein gene in metallothionein superfamily categorizing system
In there is no introne, therefore external environment can be quickly responded and coerce and accelerate protein expression.Tetrahymena is a kind of unicellular
Eukaryon model organism, cell-free wall coerce sensitivity for external source environmental contaminants, have quick cellular response mechanism, therefore
It is considered as that toxicology and ecological toxicology study one of good model organism.
Currently, having had both at home and abroad by plant, animal metallothionein is transferred in Escherichia coli for administering water environment weight
The report of metallic pollution recombinates the engineering bacteria absorbable 0.467 × 10 of magnificent river crab metallothionein-4Cd in mol/g water body2+；
Recombinate the engineering bacteria absorbable 1.25 × 10 of cucumber diamond-like coating-4Cd in mol/g water body2+.But the present invention is by tetrahymena
A kind of engineering bacteria BL21/pSUMO-Te- for metallothionein gene building cloned in Tetrahymena elliotti
MTT2 absorbable 3.89 × 10-4Cd in mol/g water body2+, compared with reporting before, the ability of enriching heavy metal ion is stronger.
Summary of the invention
The purpose of the present invention is to provide a kind of new metallothionein genes, provide one kind and contain metallothionein gene
The genetic engineering bacterium of absorbable heavy metal ion and their applications in enrichment heavy metal in water.
A kind of metallothionein gene provided by the invention, nucleotides sequence are classified as SEQ ID NO:1.
A kind of metallothionein mutated gene (Te-MTT2), nucleotides sequence are classified as SEQ ID NO:2.
The polypeptide of above-mentioned mutated gene coding, amino acid sequence are SEQ ID NO:3.
A kind of recombinant plasmid pSUMO-Te-MTT2, contains the mutated gene.
A kind of recombination engineering bacteria is the recombinant bacterium for obtaining above-mentioned recombinant plasmid transformed to Escherichia coli.
The construction method of engineering bacteria of the present invention containing metallothionein gene (Te-MTT2), includes the following steps:
(1) clone obtains MTT2 gene from tetrahymena Tetrahymena elliotti, respectively at the 5 ' ends of Te-MTT2
BamH I and Xho I restriction enzyme site are added with 3 ' ends；And it is terminated by two Escherichia coli in PCR method mutant gene sequence
Codon；Te-MTT2 gene after mutation is connected in expression vector pSUMO, recombinant plasmid pSUMO-Te-MTT2 is obtained；
(2) recombinant plasmid pSUMO-Te-MTT2 is transferred to e. coli bl21, goes out destination protein with IPTG inducing expression
SUMO-Te-MTT2, and with the method for Western blot detection albumen expression；
Test experience: the Cd of various concentration is used2+Handle e. coli bl21/pSUMO-Te- after IPTG is induced
MTT2 detects OD600Data, and construct dosage structure-activity relationship, compare for handled under the same terms e. coli bl21/
PSUMO, every group experiment three parallel.
E. coli bl21/pSUMO and e. coli bl21/pSUMO-Te-MTT2 warp are detected with Xray fluorescence spectrometer
Cd is chelated after IPTG induction2+Ability, every group three are parallel.
Experiment shows that the genetic engineering bacterium provided by the present invention containing Te-MTT2 gene has gold in chelating water environment
Belong to the ability of ion, rapid reaction can efficiently apply in processing heavy metal (cadmium, copper, lead or mercury) sewage.The mutation
The polypeptide of gene and its coding can also be applied in heavy metal pollution reparation.
Detailed description of the invention
The identification of Fig. 1 recombinant plasmid pSUMO-Te-MTT2.(Mr is Trans 2K DNA Marker；Swimming lane 1 is recombination
Plasmid pSUMO-Te-MTT2；Swimming lane 2 is the PCR product of Te-MTT2；Swimming lane 3 is the BamH of recombinant plasmid pSUMO-Te-MTT2
The double digestion of I and Xho I is identified)
The building schematic diagram of Fig. 2 recombinant plasmid pSUMO-Te-MTT2.
The detection of Fig. 3 destination protein SUMO-Te-MTT2 expression.(Mr is Premixed Protein Marker；
Swimming lane 1 is the whole bacterial protein that BL21/SUMO-Te-MTT2 is expressed when 0.05mM IPTG is induced；Swimming lane 2 is BL21/SUMO-
The whole bacterial protein of the non-inducing expression of Te-MTT2；Swimming lane 3 is what BL21/SUMO-Te-MTT2 was expressed when 0.05mM IPTG is induced
Soluble protein)
The expression of Fig. 4 Western blot testing goal Protein S UMO-Te-MTT2.(swimming lane 1 is the full bacterium of BL21/SUMO,
Swimming lane 2 is BL21/SUMO supernatant；Swimming lane 3 is the whole bacterial protein that BL21/SUMO-Te-MTT2 is not induced；Swimming lane 4 is BL21/
The supernatant protein that SUMO-Te-MTT2 is not induced；Swimming lane 5 is BL21/SUMO-Te-MTT2 whole bacterial protein under IPTG induction；Swimming
Road 4 is supernatant protein of the BL21/SUMO-Te-MTT2 under IPTG induction)
Fig. 5 Cd2+After handling e. coli bl21/pE-SUMO and BL21/pSUMO-Te-MTT2, OD600—Cd2+Concentration
Relational graph.
Fig. 6 e. coli bl21/pSUMO and BL21/pSUMO-Te-MTT2 is enriched with Cd2+Compare.
Specific embodiment
The building of amplification, the mutation and recombinant plasmid pSUMO-Te-MTT2 of 1 metallothionein Te-MTT2 gene of embodiment
And identification
Design primer:
(1) primer of clone gene Te-MTT2 design:
Te-MTT2-F:GGATCCATGGACCCTCAAACTCAAAATA
Te-MTT2-R:CTCGAGTCAGCATTTGCATTCTGAGCA
(2) gene Te-MTT2 is mutated the primer of terminator codon design:
It is mutated the primer of first terminator codon
Tubian-Te-MTT2-F-1:GCAACTGTCAACCTTGTGAAAACTG
Tubian-Te-MTT2-R-1:CAGTTTTCACAAGGTTGACAGTTGC
It is mutated the primer of second terminator codon
Tubian-Te-MTT2-F-2:CTGAAAATTGCCAATGCAACCCTTG
Tubian-Te-MTT2-R-2:CAAGGGTTGCATTGGCAATTTTCAG
Above-mentioned primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Using Te-MTT2-F/Te-MTT2-R, expanded from tetrahymena Tetrahymena elliotti by round pcr
New gene is obtained, nucleotides sequence is classified as SEQ ID NO:1.Using Tubian-Te-MTT2-F-1/Tubian-Te-MTT2-R-1,
And Tubian-Te-MTT2-F-2/Tubian-Te-MTT2-R-2, pass through two large intestine bars in PCR method mutant gene sequence
Bacterium terminator codon (TAA) obtains mutated gene Te-MTT2, and nucleotides sequence is classified as SEQ ID NO:2.Recycling PCR product simultaneously will
It is mixed with intermediate vector pMD-18T according to proper ratio, and 16 DEG C of connections overnight, it is a large amount of that connection product converts bacillus coli DH 5 а
Amplification extracts plasmid pMD-18T-Te-MTT2 and carries out PCR and digestion identification.
BamH I digestion and Xho I digestion are carried out to pMD-18T-Te-MTT2 and expression pSUMO respectively, digestion products return
It receives, with the Te-MTT2 and carrier pSUMO after digestion, mixes in proportion, with the 16 DEG C of connections overnight of T4 ligase, connection product
PSUMO-Te-MTT2 converts bacillus coli DH 5 а, and the LB solid medium tablets of kanamycins, 37 DEG C are incubated overnight, picking list
Bacterium colony mass propgation in LB liquid medium, plasmid purification are by PCR and BamH I and Xho I digestion identification (Fig. 1), Mr
The overall length of Trans2K Plus II DNA Marker, Te-MTT2 are 273bp, and the segment after pSUMO-Te-MTT2 digestion is respectively
5628bp and 273bp.The schematic diagram (Fig. 2) of recombinant plasmid pSUMO-Te-MTT2.
The vivoexpression of 2 metallothionein gene Te-MTT2 of embodiment and identification
Recombinant plasmid pSUMO-Te-MTT2 is transferred to e. coli bl21, picking single colonie is trained in LB liquid medium
It supports to logarithmic growth phase, continues 4h, 1000rpm, 4 DEG C of centrifugation 5min of culture after being induced with IPTG and collect cell, cell is resuspended
In PBS buffer solution, ultrasonic cell disruption instrument ultrasonication thallus collects full bacterium solution and supernatant, expression product is carried out
The albumen of 12%SDS-PAGE analysis, the Bacillus coli cells strain expression of unused IPTG induction compares.Give expression to destination protein
SUMO-Te-MTT2 predicts that its size is 30KD, as a result can primarily determine that SUMO-Te-MTT2 expresses successfully (Fig. 3).
The Western blotting of recombinant protein SUMO-Te-MTT2 is detected.By BL21/pSUMO and BL21/pSUMO-
The full bacterium of Te-MTT2 and supernatant carry out 12%SDS-PAGE respectively；Transferring film；Closing；Primary antibody Anti-His (1:5000) is added to carry out
Antigen-antibody reaction；Two anti-igg (1:15000) are incubated for；LI-COR Ddyssey IR fluorescence scanning imagery.Western
Blotting is the results show that band, control BL21/ occur at 30KD in the full bacterium of BL21/pSUMO-Te-MTT2 and supernatant protein
There is band at 20KD in the full bacterium of pSUMO and supernatant protein, show recombinant protein SUMO-Te-MTT2 in expression in escherichia coli
Success (Fig. 4).
3 e. coli bl21s of embodiment/pSUMO and e. coli bl21/pSUMO-Te-MTT2 are to Cd2+Tolerance point
Analysis
E. coli bl21/pSUMO and e. coli bl21/pSUMO-Te-MTT2 are gone to containing 100 μ g/mL respectively
When culture is to OD600=0.7 in the LB culture medium (5mL) of kanamycins, IPTG to final concentration of 0.05mM is added, continues to cultivate
4h induces the expression of SUMO label protein and SUMO-Te-MTT2 albumen respectively.Two kinds of cell strains are gone into multitube LB training respectively
It supports in base (5mL), making its final concentration is OD600=0.1, while respectively into the multitube culture medium for be inoculated with both bacterial strains
The Cd of concentration gradient is added2+, continue to cultivate 5h, detection bacterium increment.
Data processing: the setting three of every group of test sample is parallel, draws bacteria concentration-test sample Cd2+Concentration relationship figure
(Fig. 5).
4 e. coli bl21s of embodiment/pSUMO-Te-MTT2 is enriched with heavy metal in water
E. coli bl21/pSUMO and e. coli bl21/pSUMO-Te-MTT2 are gone to containing 100 μ g/mL respectively
In the LB culture medium (100mL) of kanamycins, 37 DEG C, when 180 turns of cultures are to OD600=0.7, IPTG is added to final concentration of
0.05mM continues to cultivate 4h, induces the expression of SUMO label protein and SUMO-Te-MTT2 albumen respectively.In strain cultured solution all
The Cd of 80ug/ml is added2+, continue to cultivate the culture solution taken out under aseptic condition after 5h in 50mL culture, 13000rpm centrifugation takes
Supernatant is spare；Remaining 50mL culture solution continues culture to 17h, and the same terms take final proof spare, use Xray fluorescence spectrometer
Detection e. coli bl21/pSUMO and e. coli bl21/pSUMO-Te-MTT2 chelates Cd after IPTG is induced2+Ability.
Data processing: given the test agent repeats three times, and thallus chelates Cd2+Ability-given the test agent reaction time relational graph (figure
6)。
Claims (8)
1. a kind of metallothionein gene, nucleotides sequence are classified as SEQIDNO:1.
2. a kind of metallothionein mutated gene Te-MTT2, nucleotides sequence are classified as SEQIDNO:2.
3. the polypeptide of mutated gene coding as claimed in claim 2, amino acid sequence SEQIDNO:3.
4. a kind of recombinant plasmid pSUMO-Te-MTT2, contains mutated gene as claimed in claim 2.
5. a kind of recombination engineering bacteria is that recombinant plasmid pSUMO-Te-MTT2 as claimed in claim 4 is transformed into large intestine bar
The recombination engineering bacteria BL21/pSUMO-Te-MTT2 that bacterium BL21 is obtained.
6. application of the recombination engineering bacteria BL21/pSUMO-Te-MTT2 as claimed in claim 5 in processing cadmium sewage.
7. application of the mutated gene Te-MTT2 as claimed in claim 2 in cadmium pollution reparation.
8. application of the polypeptide as claimed in claim 3 in cadmium pollution reparation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003006480A1 (en) * | 2001-07-13 | 2003-01-23 | University Of Rochester | Tetrahymena metallothionein gene promoter and its use |
| CN104342451A (en) * | 2014-10-15 | 2015-02-11 | 王清路 | Optimization of long-chain metallothionein gene MTT1 and establishment of plant expression vector of long-chain metallothionein gene MTT1 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003006480A1 (en) * | 2001-07-13 | 2003-01-23 | University Of Rochester | Tetrahymena metallothionein gene promoter and its use |
| CN104342451A (en) * | 2014-10-15 | 2015-02-11 | 王清路 | Optimization of long-chain metallothionein gene MTT1 and establishment of plant expression vector of long-chain metallothionein gene MTT1 |
Non-Patent Citations (3)
| Title |
|---|
| Tetrahymena sp. 1.7 CuMT gene for metallothionein, isolate 1.7;Zahid,M.T.等;《GenBank: HE820725.1》;20130717;序列说明 |
| 嗜热四膜虫金属硫蛋白MTT1和MTT2的结构与功能分析;王清路;《中国博士学位论文全文数据库 基础科学辑》;20121215;摘要，第1页第1.1.1节，第4页，第6页第2段，第20-24页第2.2节，第29页，第53-54页 |
| 四膜虫金属硫蛋白 ＭＴＴ２在大肠杆菌中的表达与纯化;郭荣等;《山西大学学报（自然科学版）》;20160515;301-306 |
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