CN108220318A - The construction method of the whole-cell biological sensor of lead ion check with high sensitivity - Google Patents
The construction method of the whole-cell biological sensor of lead ion check with high sensitivity Download PDFInfo
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- CN108220318A CN108220318A CN201810032688.XA CN201810032688A CN108220318A CN 108220318 A CN108220318 A CN 108220318A CN 201810032688 A CN201810032688 A CN 201810032688A CN 108220318 A CN108220318 A CN 108220318A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Abstract
The present invention relates to the construction methods of the whole-cell biological sensor of lead ion check with high sensitivity；The pbrR genes in the natural plasmid pMOL30 in copper bacterium Ralstonia metallidurans CH34, the promoter P with self feed back function of plasmid pGN68 are coveted using the resistance to metal of Natural strainsluxRIWith modulin LuxR and green fluorescence protein gene GFP, a variety of structures that quantitative whole-cell biological sensor is carried out to lead ion are realized in engineering colon bacillus DH5 α.Lead ion in biosensor cell detection to water sample, detection limit is respectively less than 1 μM, the background detection value of wherein biosensor cell V is minimum, and sensitivity highest, minimum detection limit can reach 0.001 μM, fluorescent assay signal can be amplified by illustrating the self feed back regulation system of gene Cascaded amplification expression system, improve detection sensitivity, effect is best.
Description
Technical field
The invention belongs to field of environmental biotechnology, are related to a variety of lifes for being used for detecting plumbum ion concentration in aqueous samples
The structure of object sensor and use；The more particularly to construction method of the whole-cell biological sensor of lead ion check with high sensitivity.
Background technology
Lead is a kind of heavy metal element for being prevalent in nature and having severe toxicity, mostly with the form stable of compound
In the presence of.But due to lead be widely used in human lives production every field, such as mining, metal smelt, gasoline,
Construction material, fire coal, storage battery car tail gas etc. have caused lead contamination to some extent in many areas, to natural environment and
Human body health care belt endangers.Lead mainly by the modes such as alimentary canal and respiratory tract enter into the human body, and in human body
Various enzymes and amino acid etc. be combined with each other, interfere the biochemistry and physiological activity of human body.Children, old man and the low people couple of immunity
Lead ratio is more sensitive, and lead poisoning mainly easily causes anemia, nervous disorder and injury of kidney etc..
The detection method of lead is mostly occurred using spectrophotometry, atomic absorption spectrography (AAS) (AAS) and hydride at present former
Sub- fluorescent spectrometry (HG-AFS) etc..Microbiological sensor is currently used for a kind of new method in environmental monitoring, and this method is
The reporter gene easily detected such as fluorescin etc. is transferred to the promoter downstream of pollutant metabolic gene, makes it in pollution sources
It is expressed under induction and is detected and then heavy metal in environment and other pollutants are monitored and carried out above by instrument
The assay method of lead content is compared, this kind of method have the characteristics that it is sensitive, direct, easy, quick, and by microbial cell and
The whole-cell biological sensor of sensor coupling cell inductive signal can be converted into can light signal, and be easy to replicate,
There is very big potential to reduce cost.In MerR families, PbrR albumen has lead ion the binding ability of specificity, and this knot
Conjunction changes the conformation for leading to protein/DNA compounds, makes DNA that the state of exploitation be presented, so as to make under pbr promoters
The expression access of trip is opened, therefore can detect lead ion content by the expression of downstream gene.
Full cell microorganism sensor (Whole-cell biosensor) is a kind of convenient, quick, inexpensive environment
Heavy metal analysis tool, it is transformed living microorganism using technique for gene engineering, can be to heavy metal contaminants
Carry out quantitative analysis.But its specificity is not good enough, not high sensitivity is always to limit its widely applied bottleneck problem.2000
The cellular metal probe of professor's Jouanneaut exploitation of Nantes, France university, reaches the minimum detection limit of associated metal
Nmol is horizontal.Pb (II) sensor detection minimum that Zhao Jing seminars of Nanjing University in 2014 are built based on regulatory factor is reachable
To 0.001 μ Μ, various metals ion selectivity testing result shows that it has preferable selective enumeration method ability to Pb (II).
Invention content
It is an object of the invention to be directed to the problems such as existing chemical detection method is complicated for operation, expensive, especially carry
Go out the construction method of a variety of detection lead ions of whole-cell biological sensor.
Specific technical solution is as follows：
The construction method of the whole-cell biological sensor of lead ion check with high sensitivity；Covet copper using the resistance to metal of Natural strains
The pbrR genes in natural plasmid pMOL30 in bacterium Ralstonia metallidurans CH34, plasmid pGN68's carries
The promoter P of self feed back functionluxRIWith modulin LuxR and green fluorescence protein gene GFP, in engineering colon bacillus
A variety of structures that quantitative whole-cell biological sensor is carried out to lead ion are realized in DH5 α.
Utilize two-way specificity promoter Ppbr, adjust gene pbrR and green fluorescence protein gene GFP and form successively
The green fluorescence Gene expression and regulation system of lead (II) ion Induction Control, using bacillus coli DH 5 alpha as host strain, constructs energy
Detect the biosensor cell I of lead ion.
Green fluorescence protein gene GFP is placed in two-way specificity promoter PpbrDownstream, improve fluorescence protein gene
Expression intensity, while it is placed in two-way specificity promoter P by gene pbrR is adjustedpbrThe expression of regulatory protein PbrR is improved in upstream
Amount, thus constructs the biosensor cell II that can detect lead ion.
Reduce the background expression of biosensor cell；Second is introduced in the upstream of green fluorescence protein gene GFP
Two-way specificity promoter Ppbr, obtain the biosensor cell III containing double-promoter, the bio-sensing made from the cell
Device cell reduces the autofluorescent background expression of cell in the case where lead ion is not present, so as to improve the sensitive of biosensor
Degree.
Introducing adjusting gene luxR and plasmid pGN68 is collectively constituted in biosensor cell III puts containing gene cascade
The biosensor cell IV of the self feed back regulation system of big expression system.
It is described as follows：
Covet the natural plasmid in copper bacterium Ralstonia metallidurans CH34 using the resistance to metal of Natural strains
PbrR genes in pMOL30, the promoter P with self feed back function of plasmid pGN68luxRIWith modulin LuxR and green
Color fluorescence protein gene GFP, realized in engineering colon bacillus DH5 α it is a variety of have it is highly sensitive, low cost can to lead from
Son carries out the structure of quantitative whole-cell biological sensor, and the whole-cell biological sensor fluorescence intensity for passing through structure
Signal represents the plumbum ion concentration in aqueous samples.
Utilize two-way specificity promoter Ppbr, adjust gene pbrR and green fluorescence protein gene GFP and form successively
The green fluorescence Gene expression and regulation system of lead (II) ion Induction Control, using bacillus coli DH 5 alpha as host strain, we build
The biosensor cell I (attached drawing 1) of lead ion can be detected.It is widely applied that the sensitivity of biosensor limits its
Bottleneck problem, that is, reduce the background expression of biosensor cell and improve the expression quantity of fluorescence in the same circumstances.
The biosensor cell I (attached drawing 1) designed from us there are three types of situation it is found that can improve detection sensitivity.
1) expression quantity of the fluorescence under same experimental conditions is improved.Green fluorescence protein gene GFP is placed in two-way special
Property promoter PpbrDownstream, improve the expression intensity of fluorescence protein gene, while two-way specificity is placed in by gene pbrR is adjusted
Promoter PpbrUpstream improves the expression quantity of regulatory protein PbrR, thus constructs the biosensor cell that can detect lead ion
II (attached drawing 2) improves the detection sensitivity of biosensor cell by improving gene expression amount.
2) the background expression of biosensor cell is reduced.Second is introduced in the upstream of green fluorescence protein gene GFP
A two-way specificity promoter Ppbr, the biosensor cell III (attached drawing 3) containing double-promoter is obtained, is made with the cell
Biosensor cell can be reduced in the case where lead ion is not present cell autofluorescent background expression, so as to improve biology
The sensitivity of sensor.
3) the self feed back regulation system of gene Cascaded amplification expression system is introduced.By generating a large amount of tune in a short time
The detection sensitivity of biosensor cell for saving albumen PbrR to improve.In the base of III lead ion of biosensor cell induction
It is opened because green fluorescence protein gene GFP is substituted for the adjusting gene luxR and plasmid pGN68 self feed backs contained in expression system
Mover PluxRI(its nucleotide sequence is as shown in SEQ ID No.4), adjust gene luxR can be in the presence of lead ion
Promote the expression of regulatory protein luxR, while cause self feed back promoter PluxRIStart downstream green fluorescence protein gene GFP's
Great expression in a short time.Adjusting gene luxR and plasmid pGN68 is introduced in biosensor cell III to collectively constitute
The biosensor cell IV (attached drawing 4) of self feed back regulation system containing gene Cascaded amplification expression system.When identical
Interior biosensor cell IV can generate more green fluorescent proteins than biosensor cell I, detect higher
Fluorescence signal, while double-promoter system can reduce the autofluorescent background expression of cell, so as to more sensitively detect lead ion
Presence.
The present invention relates to a kind of whole-cell biological sensors with gene Cascaded amplification expression system of lead ion sensitivity
And construction method；It will artificial fully synthetic bidirectional promoter PpbrAnd lead ion binding proteins specific PbrR such as SEQ ID No.1
The nucleotide sequence of shown nucleotide sequence and the SEQ ID No.4 of self feed back modulin LuxR contained by plasmid pGN68 passes through
Digestion mode connects, and screening positive clone obtains target recombinant vector pET28a-pbr-lux；Plasmid pGN68 contains self feed back and opens
Mover PluxRI, green fluorescence protein gene nucleotide sequence, self feed back modulin LuxR nucleotide sequences, according to digestion mode
It is sequentially connected；By the use of E. coli DH5 α as host, recombinant vector and plasmid pGN68 cotransformations obtain pair
The whole-cell biological sensor of lead ion sensibility.
Description of the drawings
Fig. 1 detects I schematic diagram of biosensor cell of lead contamination.
Fig. 2 detects II schematic diagram of biosensor cell of lead contamination.
Fig. 3 detects III schematic diagram of biosensor cell of lead contamination.
Fig. 4 detects IV schematic diagram of biosensor cell of lead contamination.
Fig. 5-A are the collection of illustrative plates for building I plasmid pET28a-Ppbr of biosensor cell.
Fig. 5-B are the collection of illustrative plates for building II plasmid pET28a-pbrR of biosensor cell.
Fig. 5-C are the collection of illustrative plates for building III plasmid pET28a-Ppbr-pbrR of biosensor cell.
Fig. 5-D are the collection of illustrative plates for building IV plasmid pET28a-luxR of biosensor cell.
Fig. 6-A are plumbum ion concentration-fluorescence response figures of biosensor cell I.
Fig. 6-B are plumbum ion concentration-fluorescence response figures of biosensor cell II.
Fig. 6-C are plumbum ion concentration-fluorescence response figures of biosensor cell III.
Fig. 6-D are plumbum ion concentration-fluorescence response figures of biosensor cell IV.
Specific embodiment
PET28a plasmids used in the present invention are as the structure main carriers of lead ion biosensor and pGN68 plasmid conducts
The expression vector of fluorescence protein gene in self feed back system.
Original chassis cell is bought for E.coli DH5 α from Beijing Quanshijin Biotechnology Co., Ltd.
Below by specific embodiment, the present invention will be further described.
1. the structure of biosensor：
Using PCR (PCR), in artificial fully synthetic two-way specificity promoter Ppbr(its nucleotides sequence
Arrange as shown in SEQ ID No.1) and adjusting gene pbrR (its nucleotide sequence is as shown in SEQ ID No.2) segment both ends add
Enter Hind III, I restriction enzyme sites of BamH, in green fluorescence protein gene GFP (its nucleotide sequence is as shown in SEQ ID No.3) two
End adds in BamH I, III restriction enzyme sites of Hind.By using FastDigest restriction endonucleases to two-way specificity promoter PpbrAnd tune
Section gene pbrR and green fluorescence protein gene GFP above-mentioned twos segment carry out digestion respectively, are then connected with T4DNA ligases
It connects, and converts to competent cell E.coli DH5 α, bacterium colony PCR screening positive clones obtain recombinant plasmid pET28a-Ppbr
(see Fig. 5-A) obtains the green fluorescence Gene expression and regulation system with lead (II) ion Induction Control after sequence verification
Biosensor cell I (see Fig. 1).
Using PCR (PCR), Hind III, Bgl are added at artificial fully synthetic adjusting gene pbrR both ends
II restriction enzyme site, in two-way specificity promoter PpbrBoth ends add in Hind III, I restriction enzyme sites of BamH and green fluorescent protein
Gene GFP both ends add in BamH I, III restriction enzyme sites of Hind (its nucleotide sequence is same as above), by using FastDigest inscribes
Enzyme is to adjusting gene pbrR, two-way specificity promoter PpbrAnd green fluorescence protein gene GFP above threes segment respectively into
Row digestion is connected by T4 ligases, and is converted to competent cell E.coli DH5 α, and bacterium colony PCR screening positive clones obtain
To recombinant plasmid pET28a-pbrR (see Fig. 5-B), obtained after sequence verification by green fluorescence protein gene GFP and adjusting
Gene pbrR is respectively placed in two-way specificity promoter PpbrUpstream and downstream, it is high by the expression intensity for improving fluorescence protein gene
Divalent inorganic lead ion biosensor cell II in sensitive detection aqueous solution (see Fig. 2).
Using PCR (PCR), in artificial fully synthetic two-way specificity promoter Ppbr(its nucleotides sequence
Arrange as shown in SEQ ID No.1) and adjusting gene pbrR (its nucleotide sequence is as shown in SEQ ID No.2) segment both ends add
Enter Hind III, I restriction enzyme sites of BamH, in two-way specificity promoter PpbrBoth ends add in Hind III, I restriction enzyme sites of BamH and
Green fluorescence protein gene GFP both ends add in BamH I, III restriction enzyme sites of Hind, by using FastDigest restriction endonucleases to double
To specificity promoter Ppbr, adjust gene pbrR, two-way specificity promoter PpbrAnd green fluorescence protein gene GFP (its
Nucleotide sequence is same as above) digestion is carried out, it is connected by T4 ligases, and convert to competent cell E.coli DH5 α, bacterium colony
PCR screening positive clones obtain recombinant plasmid pET28a-Ppbr-pbrR (see Fig. 5-C), are obtained after sequence verification in green
The upstream of fluorescence protein gene GFP introduces second two-way specificity promoter Ppbr, obtain lead (II) ion Induction Control
The green fluorescence Gene expression and regulation system biological sensor cell III that dual two-way specificity promoter starts (see Fig. 3).
Using PCR (PCR), in artificial fully synthetic two-way specificity promoter Ppbr(its nucleotides sequence
Arrange as shown in SEQ ID No.1) and adjusting gene pbrR (its nucleotide sequence is as shown in SEQ ID No.2) segment both ends add
Enter Hind III, I restriction enzyme sites of BamH, in two-way specificity promoter PpbrBoth ends add in Hind III, I restriction enzyme sites of BamH and
It adjusts gene luxR (its nucleotide sequence is as shown in SEQ ID No.4) both ends and adds in BamH I, III restriction enzyme sites of Hind.Pass through
Using FastDigest restriction endonucleases to two-way specificity promoter Ppbr, adjust gene pbrR, two-way specificity promoter PpbrWith
And adjust gene luxR and carry out digestion, it is connected by T4 ligases, and convert to competent cell E.coli DH5 α, bacterium colony PCR
Screening positive clone receives recombinant plasmid pET28a-luxR after sequence verification (see Fig. 5-D).
By PCR, in PluxRIGene order both ends add in I restriction enzyme site of Sal I and EcoR, adjust
The gene order both ends of gene luxR add in I restriction enzyme site of EcoR I and BamH.Artificial fully synthetic green fluorescence protein gene
GFP, and add in I restriction enzyme site of BamH I and Not at sequence both ends.By using FastDigest restriction endonucleases to above three
Segment carries out digestion respectively, is then connected and is converted to competent cell E.coli DH5 α, bacterium colony PCR with T4DNA ligases and sieved
Positive colony is selected, sequence verification obtains plasmid pGN68.Recombinant plasmid pET28a has base with what plasmid pGN68 cotransformations obtained
Because of the biosensor cell IV of the self feed back regulation system of Cascaded amplification expression system (see Fig. 4).
2. lead ion quantitative detecting method：
(1) it is cultivated under the conditions of 220rpm, 37 DEG C with the fluid nutrient medium LB of addition kanamycins (50 milligrams per liter) above-mentioned
Five kinds of biosensor cell pellet overnights (12-14 hours).
(2) and then the above-mentioned seed bacterium solutions of 1mL will be taken, and will measure its absorbance under 600nm, be diluted to LB culture mediums
OD600=1, and 1% is seeded in LB culture mediums of the 100mL without resistance, 220rpm, 2.5 hours of 37 DEG C of shaking flask cultures, directly
To OD600About 0.6, that is, reach the growth logarithmic phase of biosensor cell.
(3) the lead ion standard solution of various concentration is added in into conical flask, measures fluorescence intensity (RFU, the survey of each solution
Fixed excitation wavelength is 480nm, launch wavelength 520nm).
The result shows that the plumbum ion concentration and fluorescence intensity block diagram (see Fig. 6-A) measured by biosensor cell I are aobvious
Show that the lead ion standard solution of various concentration obtains different fluorescence intensities, and when plumbum ion concentration is less than 100 μM with lead
Ion concentration increases, and fluorescence intensity also increases；Plumbum ion concentration and fluorescence intensity measured by biosensor cell II
Fluorescence intensity is maximum when block diagram is shown in 50 μM (see Fig. 6-B)；Plumbum ion concentration measured by biosensor cell III with
Fluorescence intensity block diagram (see Fig. 6-C) display its background value compared with biosensor I, II is smaller, is in plumbum ion concentration
There are higher fluorescence response, sensitivity enhancing at 0.001 μM；Plumbum ion concentration measured by biosensor cell IV with it is glimmering
Luminous intensity block diagram (see Fig. 6-D) display measured fluorescent value enhancing, chassis compared with traditional biosensor I (see Fig. 1)
Background value is smaller, high sensitivity, and minimum detection limit can reach 0.001 μM.
To sum up result shows that above-mentioned four kinds of biosensor cells can detect the lead ion in water sample, and detection limit is equal
Less than 1 μM, the background detection value of wherein biosensor cell V is minimum, and sensitivity highest, and minimum detection limit can reach
0.001 μM, fluorescent assay signal can be amplified by illustrating the self feed back regulation system of gene Cascaded amplification expression system, improve detection
Sensitivity, effect are best.Illustrate that above-mentioned four kinds of biosensor cells can be used in lead (II) ion in detection aqueous solution simultaneously,
And the degree of lead ion pollution can be quantitatively determined in a certain range.
SEQ ID NO.01
TTCTGGGTTGCGCGTCGCAACGGAAGCGCAGCCACATCCCTTCGAGCCACATTCGCTCATGGCAACCCC
TTGTGTGTATTCATCTCGCGTTGCCGATTTAACACCCTCTAGTTACTATAGAGTCAAGACATCTCCCATCCGACGCC
SEQ ID NO.02
ATGAATATCCAGATCGGCGAGCTTGCCAAGCGCACCGCATGCCCGGTGGTGACCATTCGCTTCTACGAA
CAAGAAGGGCTGTTGCCGCCGCCGGGCCGCAGCCGGGGGAATTTTCGCCTGTATGGCGAGGAGCACGTGGAGCGCTT
GCAGTTCATTCGTCACTGCCGGTCTCTGGATATGCCGTTGAGCGACGTACGGACCTTATTGAGTTACCGGAAGCGGC
CCGACCAGGATTGCGGTGAAGTCAATATGCTCTTGGATGAGCACATCCGTCAGGTCGAATCTCGGATCGGAGCTTTG
CTCGAACTGAAGCACCATTTGGTGGAACTGCGCGAAGCCTGTTCTGGTGCCAGGCCCGCCCAATCGTGCGGGATTCT
GCAGGGACTGTCGGACTGCGTGTGTGATACGCGGGGGACCACCGCCCATCCAAGCGACTAG
SEQ ID NO.03
AGTAAAGGAGAAGAACTTTTCACTGGAGTTGTGACAATTCTTGTTGAATTAGATGGTGATGTTAATGGT
CACAAATTTTCTGTTAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGTACTAC
TGGAAAACTACCTGTTCCCTGGCCAACACTTGTTACTACTTTGACTTATGGTGTTCAATGTTTTTCAAGATACCCAG
ATCACATGAAACGGCACGACTTTTTCAAGAGTGCAATGCCCGAAGGTTATGTACAAGAAAGAACTATTTTTTTCAAA
GATGACGGTAACTACAAGACACGTGCTGAAGTTAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGG
TATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATTA
TGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGTGTTCAACTA
GCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTTCTTTTACCAGACAACCATTACCTGTCCACACA
ATCTGCTCTTTCTAAAGATCCCAACGAAAAGAGAGACCATATGGTGCTTCTTGAGTTTGTAACAGCTGCTGGTATTA
CACACGGTATGGATGAACTATACAAACACCATCACCATCACCATCACTAG
SEQ ID NO.04
ATGCCTTCTCTAGTTGATAATTATCGAAAAATAAATATAGCAAATAATAAATCAAACAACGATTTAACC
AAAAGAGAAAAAGAATGTTTAGCGTGGGCATGCGAAGGAAAAAGCTCTTGGGATATTTCAAAAATATTAGGCTGCAG
TGAGCGTACTGTCACTTTCCATTTAACCAATGTGCAAATGAAACTCAATACAACAAACCGCTGCCAAAGTATTTCTA
AAGCAATTTTAACAGGAGCAATTGATTGCCCATACTTTAAAAATTAA
Claims (5)
1. the construction method of the whole-cell biological sensor of lead ion check with high sensitivity；It is characterized in that utilize the resistance to gold of Natural strains
Belong to the pbrR genes in the natural plasmid pMOL30 in greedy copper bacterium Ralstonia metallidurans CH34, plasmid pGN68
The promoter P with self feed back functionluxRIWith modulin LuxR and green fluorescence protein gene GFP, in engineering large intestine
A variety of structures that quantitative whole-cell biological sensor is carried out to lead ion are realized in bacillus DH5 α.
2. the method as described in claim 1, it is characterized in that utilizing two-way specificity promoter Ppbr, adjust gene pbrR and
The green fluorescence Gene expression and regulation system for the lead (II) ion Induction Control that green fluorescence protein gene GFP is formed successively, with
Bacillus coli DH 5 alpha is host strain, constructs the biosensor cell I that can detect lead ion.
3. method as claimed in claim 2, it is characterized in that green fluorescence protein gene GFP is placed in two-way specificity promoter
PpbrDownstream, improve the expression intensity of fluorescence protein gene, while two-way specificity promoter is placed in by gene pbrR is adjusted
PpbrUpstream improves the expression quantity of regulatory protein pbrR, thus constructs the biosensor cell II that can detect lead ion.
4. method as claimed in claim 2, it is characterized in that reducing the background expression of biosensor cell；In green fluorescence egg
The upstream of white gene GFP introduces second two-way specificity promoter Ppbr, it is thin to obtain the biosensor containing double-promoter
Born of the same parents III, and the biosensor cell made from the cell reduces the autofluorescent background table of cell in the case where lead ion is not present
It reaches, so as to improve the sensitivity of biosensor.
5. method as claimed in claim 4 adjusts gene luxR and matter it is characterized in that being introduced in biosensor cell III
Grain pGN68 collectively constitutes the biosensor cell IV of the self feed back regulation system containing gene Cascaded amplification expression system.
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| CN110007072A (en) * | 2019-05-07 | 2019-07-12 | 北京理工大学 | A kind of construction method and its application method of microbiological sensor |
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| CN110684792A (en) * | 2019-10-14 | 2020-01-14 | 江南大学 | Glycolic acid sensor capable of being eliminated repeatedly and construction method thereof |
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Application publication date: 20180629 |