CN113234651A - Construction and application of mercury ion microorganism whole-cell biosensor taking violacein as output signal - Google Patents

Construction and application of mercury ion microorganism whole-cell biosensor taking violacein as output signal Download PDF

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CN113234651A
CN113234651A CN202110345979.6A CN202110345979A CN113234651A CN 113234651 A CN113234651 A CN 113234651A CN 202110345979 A CN202110345979 A CN 202110345979A CN 113234651 A CN113234651 A CN 113234651A
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惠长野
郭妍
杨学琴
林怡然
黄红英
李丽梅
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SHENZHEN PREVENTION AND TREATMENT CENTER FOR OCCUPATIONAL DISEASES
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Abstract

The invention discloses construction and application of a mercury ion microorganism whole-cell biosensor taking violacein as an output signal. The invention provides a recombinant bacterium, which is obtained by introducing a recombinant vector A into a recipient bacterium; the recombinant vector A contains a DNA fragment A; and the DNA fragment A contains a mer bidirectional promoter, one side of the mer bidirectional promoter is a MerR protein coding gene, and the other side of the mer bidirectional promoter is a violacein synthetic gene module. The recombinant strain can be used as a mercury ion microorganism whole-cell biosensor taking violacein as an output signal. On one hand, qualitative indication of target heavy metal exposure can be realized through change of cell color, and on the other hand, intracellular violacein can be extracted through an organic solvent, and target heavy metal quantitative analysis is carried out through colorimetry.

Description

Construction and application of mercury ion microorganism whole-cell biosensor taking violacein as output signal
Technical Field
The invention relates to the technical field of biology, in particular to construction and application of a mercury ion microorganism whole-cell biosensor taking violacein as an output signal.
Background
Mercury is an environmental pollutant that is present in the atmosphere for a long time and has global mobility. Human awareness of mercury toxicity has been around for centuries, and in the last 50 th century, events were generated that had a significant impact on human health due to environmental exposure to mercury, i.e., the present water guarantee event. Mercury has several different chemical forms including elemental mercury, organic mercury, and inorganic mercury. Wherein Hg (II) can be directly taken in by biological cells and metabolized to generate the most toxic organic form of methylmercury. The whole-cell biosensor directly simulates environmental microorganisms to detect target metals in a bioavailable form, so that the whole-cell biosensor has an irreplaceable position in the aspect of environmental pollution monitoring.
Reasonable and accurate monitoring of environmental heavy metal pollutants is an important prerequisite for developing pollution prevention and control work. At present, the detection of the environmental heavy metal poison is mainly a physical and chemical analysis method, such as inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS) and the like, and the method has the advantages of high detection sensitivity and high specificity, but has certain defects, such as expensive equipment, high requirements on professional operation technology, long detection period and the like. Most importantly, the traditional physical and chemical analysis method mainly aims at measuring the total amount of environmental heavy metal elements, and the detection result is difficult to truly reflect the level of toxic heavy metal, especially the bioavailability of the toxic heavy metal.
It is a major challenge to understand and grasp the relationship between heavy metal elements and living bodies in nature in various chemical states or chemical forms, so that the heavy metal elements can be applied to human production and life more sustainably. The advent of biosensing technology based on biological organisms has recently become a focus of research by virtue of its ability to directly reflect the toxicity and impact of contaminants on biological organisms. The microbial cells have the characteristics of rapid propagation, easy storage, high stability and the like, the development of the biosensor taking the microbial cells as a carrier can greatly simplify the detection flow of target molecules, and the detection result directly reflects the level and bioavailability of toxic pollutants.
Violacein is an alcohol-soluble pigment produced by environmental bacteria, particularly Chromobacterium violaceum. Violacein attracts the attention of scholars from a discovery by virtue of its attractive purple color and has now become a powerful tool for studying bacterial quorum sensing. Further studies have shown that violacein has a broad spectrum of antibacterial and antitumor activity, and its demand as a lead compound for pharmaceuticals is increasing. With the elucidation of the pathway for violacein synthesis, heterologous synthesis thereof has become possible.
As the physical and chemical properties of the electron shells, the structures, the valence states and the like of most heavy metal ions are very close, the difficulty of obtaining the high-selectivity metal ion probe through a chemical synthesis method is high. Microorganisms living in severely polluted extreme environments evolve a set of simple and efficient inducible manipulation subsystems to maintain the dynamic balance of toxic heavy metals in thalli. The 'switch' of the induction type operon has delicate functions between target metal ions and biomacromolecules such as regulatory protein (metal binding protein) and DNA sequence (promoter region), and has high efficiency and specificity beyond the range of molecular design which can be carried out by chemists after the natural evolution of hundreds of millions of years. This class of biomacromolecules is an important breakthrough in the development of biosensors that specifically recognize metal ions.
The metal binding protein is a key transcription regulating factor of the operon, and can be divided into at least 10 families according to the sequence homology of the transcription factor, the binding of each family member and a target metal ion has high selectivity, and the mutation of individual amino acid can change the specificity of the binding. The existence of target metal ions is the key for the transcription initiation of the regulatory factors, a nucleic acid fluorescent probe is designed by taking a DNA sequence of a promoter region as a blue book, biosensors for specifically identifying Hg (II), Pb (II), Cd (II), Cu (I), Ag (I), Au (I) and the like are developed at present, target metal ions in nanomolar orders can be detected, and the selectivity between the target ions and the ions with the closest background is more than 200 times, and some ions even more than 1000 times.
At present, no report related to a mercury ion microorganism whole cell biosensor taking violacein as an output signal exists.
Disclosure of Invention
The invention claims construction and application of a mercury ion microorganism whole cell biosensor taking violacein as an output signal.
In a first aspect, the invention claims a recombinant bacterium.
The recombinant bacterium claimed by the invention is obtained by introducing the recombinant vector A into a recipient bacterium.
The recombinant vector A contains a DNA fragment A.
And the DNA fragment A contains a mer bidirectional promoter, one side of the mer bidirectional promoter is a MerR protein coding gene, and the other side of the mer bidirectional promoter is a violacein synthetic gene module.
The violacein synthetic gene modules are assembled in polycistronic form and encode a VioA protein, a VioB protein, a VioC protein, a VioD protein and a VioE protein.
The nucleotide sequence of the mer bidirectional promoter is 442-512 th site of SEQ ID No. 1.
The amino acid sequence of the MerR protein is shown as SEQ ID No. 2.
The amino acid sequence of the VioA protein is shown in SEQ ID No. 3.
The amino acid sequence of the VioB protein is shown in SEQ ID No. 4.
The amino acid sequence of the VioC protein is shown in SEQ ID No. 5.
The amino acid sequence of the VioD protein is shown in SEQ ID No. 6.
The amino acid sequence of the VioE protein is shown as SEQ ID No. 7.
The nucleotide sequence of the encoding gene of the MerR protein is the reverse complementary sequence of the 7 th to 441 th positions of SEQ ID No. 1.
The nucleotide sequence of the VioA protein coding gene is the 555-friendly 1811 site of SEQ ID No. 1.
The nucleotide sequence of the VioB protein coding gene is 1829-4825 th site of SEQ ID No. 1.
The nucleotide sequence of the VioC protein coding gene is 4843-6165 site of SEQ ID No. 1.
The nucleotide sequence of the coding gene of the VioD protein is 6183-7334 of SEQ ID No. 1.
The nucleotide sequence of the coding gene of the VioE protein is 7352-7924 of SEQ ID No. 1;
further, the nucleotide sequence of the violacein synthetic gene module is 555-7924 of SEQ ID No. 1.
Further, the nucleotide sequence of the DNA fragment A is SEQ ID No. 1.
The recombinant vector A is obtained by replacing a small fragment between the enzyme cutting sites BglII and SacI of the pET-21a (+) plasmid with the DNA fragment A.
In the present invention, the recipient bacterium is Escherichia coli.
Further, the Escherichia coli is Escherichia coli TOP 10.
In a second aspect, the invention claims the application of the recombinant bacterium in the first aspect as or in the preparation of a mercury ion microorganism whole cell biosensor taking violacein as an output signal.
In a third aspect, the invention claims any of the following products or applications:
p1, recombinant vector a as described in the previous first aspect;
p2, DNA fragment a as described in the previous first aspect;
use of the recombinant vector A of P3 or P1 or the DNA fragment A of P2 in the preparation of the recombinant bacteria of the first aspect;
p4, kit I, containing the recombinant bacterium described in the first aspect above and a water-insoluble alcoholic solution;
p5, kit II, containing the recombinant bacterium described in the first aspect above, a water-insoluble alcoholic solution and mercury ions as standard;
use of a kit II of a kit I or P5 of a DNA fragment a or P4 of a recombinant vector a or P2 of P6, P1 or of a recombinant bacterium of the first aspect in the detection of mercury ions.
Further, the detection of the mercury ions is the qualitative and/or quantitative detection of the mercury ions on the liquid sample. Wherein, the kit I can be used for qualitative detection; the kit II can be used for quantitative detection.
Wherein the liquid sample satisfies the following conditions: and (3) placing the recombinant bacteria in the liquid sample for 5-12h, wherein the growth and the propagation of the recombinant bacteria can be normally carried out. For example, 9 volumes of water sample to be tested is mixed with 1 volume of 10 times concentrated LB liquid culture medium to obtain a sample.
In an embodiment of the present invention, the liquid sample is a culture medium for culturing the recombinant bacteria.
In a fourth aspect, the invention claims any of the following methods:
the method comprises the following steps: a method for detecting whether a liquid sample contains mercury ions is the following method A, method B or method C:
the method A comprises the following steps: the thallus observation method comprises the following steps: placing the recombinant bacterium in the first aspect into the liquid sample to be detected, performing shake culture at 37 ℃ and 250rpm for 5-12h, collecting thalli, observing color change of the thalli, and if the color of violacein is presented, determining that the liquid sample to be detected contains or is candidate to contain mercury ions; otherwise, the liquid sample to be detected does not contain or is candidate to contain mercury ions.
The method B comprises the following steps: the bacterial liquid observation method comprises the following steps: placing the recombinant bacterium in the first aspect into the liquid sample to be tested, shake-culturing at 37 ℃ and 250rpm for 5-12h, collecting the thallus, resuspending with an SDS aqueous solution (such as a 2% SDS aqueous solution,% represents g/100ml), adding a water-insoluble alcohol solution, vortex and shake for 5min, standing for layering, observing the color change of an upper organic phase, and if the chromogenic reaction of violacein is observed, determining that the liquid sample to be tested contains or is candidate to contain mercury ions; otherwise, the liquid sample to be detected does not contain or is candidate to contain mercury ions.
The method C comprises the following steps: an A490 value assay comprising the steps of: placing the recombinant bacteria in the first aspect into the liquid sample to be tested, shake-culturing at 37 ℃ and 250rpm for 5-12h, collecting the thallus, resuspending with an SDS aqueous solution (such as a 2% SDS aqueous solution,% represents g/100ml), adding a water-insoluble alcohol solution, vortex-shaking for 5min, centrifuging (such as 3500rpm for 5min), taking the supernatant, and determining the A490 value, which is referred to as the A490 value of the liquid sample group to be tested; if the A490 value of the liquid sample group to be tested is significantly larger than the A490 value of the control group, the liquid sample to be tested contains or is candidate to contain mercury ions; otherwise, the liquid sample to be detected does not contain or is candidate to contain mercury ions.
Wherein the A490 value of the control group is determined by replacing the liquid sample to be tested with a liquid sample not containing mercury ions, compared with the A490 value of the liquid sample group to be tested. The liquid sample that does not contain mercury ions preferably differs from the liquid sample to be tested only in that it does not contain mercury ions.
Further, the method is suitable for the case where the liquid sample to be tested contains more than 0.39 μ M (e.g., more than 0.78 μ M, and further e.g., 0.78-200 μ M) of mercury ions.
When the liquid sample to be tested is colorless or can be distinguished from the self color (can be distinguished by naked eyes) after being developed by superimposing violacein, the method preferably adopts a naked eye observation method (namely the method A or the method B) to carry out result judgment. When the liquid sample to be tested is difficult to distinguish from the self color (can not be distinguished by naked eyes) after being superposed with the violacein color development, the method-one preferably adopts an A490 value measurement method (namely the method C) to carry out result judgment.
The second method comprises the following steps: a method for detecting the content of mercury ions in a liquid sample comprises the following steps:
(A1) placing the recombinant bacteria in the first aspect into a series of mercury ion liquid samples with known concentration, shaking and culturing at 37 ℃ and 250rpm for 5-12h, collecting thalli, re-suspending with SDS aqueous solution (such as 2% SDS aqueous solution,% represents g/100ml), adding water-insoluble alcohol solution, vortex and shaking for 5min, centrifuging (such as 3500rpm and 5min), taking supernatant, determining A490 value, and then drawing a standard curve according to the mercury ion concentration and the A490 value;
(A2) and (D) replacing the series of mercury ion liquid samples with known concentrations in the step (A1) with the liquid sample to be detected, repeating the step (A1) to obtain the A490 value of the liquid sample to be detected, and substituting the A490 value into the standard curve to obtain the mercury ion content in the liquid sample to be detected.
Further, the second method is suitable for the case that the liquid sample to be tested contains 0.78 μ M to 12.5 μ M of mercury ions.
In the two methods, the content of the recombinant bacteria in the liquid sample to be detected or the mercury ion liquid samples with known concentrations is OD600＝0.6。
In the above aspects, the mercury ions are divalent mercury ions (hg (ii)).
In a particular embodiment of the invention, the divalent mercury ions (hg (ii)) are in particular mercury chloride (HgCl)2) Is present in the form of (1).
In the above aspects, the water-insoluble alcohol solution is n-butanol.
The invention builds a series of artificial mer operons by a synthetic biology means, and assembles a microorganism whole-cell sensor which specifically responds to the expression of a pigment reporter gene of a target heavy metal. The exposure of the target heavy metal can be used for opening the expression of the violacein synthetic gene, and the qualitative and quantitative detection of the heavy metal can be respectively realized by the characteristic that the gene expression level and the target metal concentration are in positive correlation. By replacing the heavy metal resistance gene downstream of the promoter with a synthetic gene module of a natural pigment product, namely violacein, the exposure of target heavy metal induces the synthesis of alcohol-soluble violacein, on one hand, the change of cell color can realize the qualitative indication of the exposure of target heavy metal, and on the other hand, the intracellular violacein is extracted by an organic solvent, and can be quantitatively analyzed by colorimetry.
Drawings
FIG. 1 is a molecular mechanical diagram of a mercury ion microbial whole-cell biosensor using violacein as an output signal according to the present invention.
FIG. 2 shows the identification of recombinant bacterium TOP10/pPmer-vio, specifically the identification result of SphI and XbaI double digestion of the objective plasmid extracted therefrom.
FIG. 3 shows the response results of recombinant bacterium TOP10/pPmer-vio to different concentrations of mercury ions. A is dose response; b is a linear response in the range of 0.78-12.5 μ M; c is the color change of the organic phase corresponding to each point in B.
FIG. 4 shows the response results of the recombinant strain TOP10/pPmer-vio to different metal ions.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 construction and application of Mercury ion microorganism whole-cell biosensor using violacein as output signal
Firstly, construction of recombinant plasmid pPmer-vio
In the first step, a violacein synthetic gene module (at position 555-7924 of SEQ ID No. 1) is synthesized in a whole gene by using pET-21a (+) as a vector, and is inserted into pET-21a (+) by using NdeI/SacI, and the recombinant plasmid which is verified to be correct by sequencing is named as pET-vio.
The structure of pET-vio is described as: a recombinant plasmid in which a small segment between NdeI/SacI of the pET-21a (+) vector is replaced with a DNA fragment shown in the 555-7924 position of SEQ ID No. 1.
In the second step, the merR-mer promoter gene module (positions 7-512 of SEQ ID No. 1) was synthesized in its entirety, inserted into pET-vio using BglII/XbaI, and the recombinant plasmid, which was confirmed to be correct by sequencing, was named pPmer-vio.
The structure of pPmer-vio is described as: the small fragment between BglII/SacI of pET-21a (+) vector was replaced with a recombinant plasmid of the DNA fragment shown in SEQ ID No. 1.
The 7 th to 512 th positions of SEQ ID No.1 are MerR-mer bidirectional promoter sequences and are bivalent mercury ions Hg (II) sensing elements. The 442-512 th site of SEQ ID No.1 is a mer bidirectional promoter, and the 7-441 th site of SEQ ID No.1 is a reverse complementary sequence of the merR gene, and encodes the MerR protein shown in SEQ ID No. 2.
Position 555-7924 of SEQ ID No.1 is vioA-vioB-vioC-vioD-vioE, which is a violacein synthetic gene cluster, assembled in polycistronic form and encodes five proteins. The 555-nd 1811 site of SEQ ID No.1 is the vioA gene and codes the VioA protein shown in SEQ ID No. 3. The 1829-4825 site of SEQ ID No.1 is the vioB gene, which encodes the VioB protein shown in SEQ ID No. 4. The 4843-6165 locus of SEQ ID No.1 is the vioC gene, which encodes the VioC protein shown in SEQ ID No. 5. The 6183-7334 site of SEQ ID No.1 is a vioD gene which encodes the vioD protein shown in SEQ ID No. 6. The 7352-7924 position of SEQ ID No.1 is the vioE gene, encoding the vioE protein shown in SEQ ID No. 7.
When mercury ions appear, coupling transcription of five genes of the vioA-vioB-vioC-vioD-vioE is activated, and 5 enzymes generated by translation catalyze the production of violacein by taking tryptophan as a substrate. The molecular mechanism is shown in FIG. 1.
Second, construction of recombinant bacterium TOP10/pPmer-vio
And (3) transforming the recombinant plasmid pPmer-vio constructed in the first step into Escherichia coli TOP10 to obtain a recombinant bacterium TOP 10/pPmer-vio.
The target plasmid is extracted from the recombinant bacterium TOP10/pPmer-vio and subjected to SphI and XbaI double enzyme digestion, and the result is shown in figure 2. As can be seen, two bands of size approximately 12K and 740bp were obtained, consistent with expectations. The correctness is further confirmed by sequencing verification.
Thirdly, the response of the recombinant bacterium TOP10/pPmer-vio to mercury ions with different concentrations
1. Inoculating the recombinant bacterium TOP10/pPmer-vio constructed in the step two into a liquid LB culture medium containing 50mg/L ampicillin, and carrying out shake culture at 37 ℃ and 250rpm until OD is achieved600nmThe value is 0.6.
2. Mercury chloride (HgCl) was added2) To a final concentration of 0, 0.098, 0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 or 200. mu. mol/L Hg (II), respectively, and cultured at 37 ℃ for 5h with shaking at 250 rpm.
3. The bacterial liquid of each group is collected, 3500rpm is carried out, centrifugation is carried out for 5min, and the supernatant is discarded.
4. The cells were resuspended in 200. mu.L of 2% (2g/100mL) SDS aqueous solution, 400. mu.L n-butanol was added, and vortexed for 5 min.
5. 3500rpm, 5min of centrifugation. On the one hand, after standing for layering, the color change of the upper organic phase was observed. On the other hand, 150. mu.L of the supernatant organic phase was aspirated, placed in a 96-well plate, and the absorbance at 490nm was measured.
The results are shown in table 1 and fig. 3. It can be seen that the A490 values vary linearly (R) in the Hg (II) concentration range from 0.78 to 12.5. mu.M20.9955) with a minimum detection limit of 0.39 μ M.
TABLE 1 Absorbance value at 490nm for each group
Hg(II)μM A490 SD
0 0 0
0.098 0 0.002
0.195 0.003 0.002
0.39 0.028 0.006
0.78 0.072 0.012
1.56 0.108 0.015
3.125 0.241 0.04
6.25 0.601 0.051
12.5 1.122 0.076
25 0.974 0.082
50 0.831 0.11
100 0.74 0.12
200 0.706 0.13
Thirdly, the response of the recombinant bacterium TOP10/pPmer-vio to different metal ions
1. Inoculating the recombinant bacterium TOP10/pPmer-vio constructed in the step two into a liquid LB culture medium containing 50mg/L ampicillin, and carrying out shake culture at 37 ℃ and 250rpm until OD is achieved600nmThe value is 0.6.
2. The final concentration was 8. mu.M of Mn (II), Ni (II), Cu (II), Zn (II), Pb (II), Cd (II) or Hg (II) added to the above culture, and the control was cultured at 37 ℃ and 250rpm for 12 hours with shaking without adding a metal salt.
3. The bacterial liquid of each group is collected, 3500rpm is carried out, centrifugation is carried out for 5min, and the supernatant is discarded.
4. The cells were resuspended in 200. mu.L of 2% (2g/100mL) SDS aqueous solution, 400. mu.L n-butanol was added, and vortexed for 5 min.
5. 3500rpm, 5min centrifugation, standing for layering, and observing the color change of the upper organic phase. On the other hand, 150. mu.L of the supernatant organic phase was aspirated, placed in a 96-well plate, and the absorbance at 490nm was measured.
The results are shown in table 2 and fig. 4. It can be seen that only hg (ii) exposed group had violacein coloration, a490 was significantly higher than the other metal groups.
TABLE 2 Absorbance value at 490nm for each group
Metal species A490 SD
Mn(II) 0.055 0.021
Ni(II) 0.072 0.031
Cu(II) 0.068 0.023
Zn(II) 0.075 0.028
Pb(II) 0.086 0.025
Cd(II) 0.089 0.036
Hg(II) 0.75 0.062
Control 0.056 0.023
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> Shenzhen market occupational disease prevention and treatment hospital
<120> construction and application of mercury ion microorganism whole-cell biosensor taking violacein as output signal
<130> GNCLN210752
<160> 7
<170> PatentIn version 3.5
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cagactgaac cccagccgct gtgccgattt cacgaatttc acccgaacca cgtccgcctc 300
cccatagcgg cggatgctgc cgtaaggctt gtccggttcc cgcaacaggc ccttgcgctg 360
atagaagcgg attgtctcca cgttgacccc ggccgccttg gcaaaaacgc caatggtcag 420
gttttccaaa ttattttcca tatcgcttga ctccgtacat gagtacggaa gtaaggttac 480
gctatccaat ccaaattcaa aagggccaac gttctagaaa taattttgtt taactttaag 540
aaggagatat acatatgaaa cacagctctg atatctgcat cgttggtgcg ggcatctctg 600
gtctgacctg cgcgagccac ctgctggatt ctccggcgtg ccgtggtctg agcctgcgta 660
tcttcgatat gcagcaggaa gcgggtggcc gtatccgttc taaaatgctg gatggtaaag 720
cgagcattga actgggtgcg ggtcgttaca gcccgcagct gcacccgcac ttccagtccg 780
ctatgcagca ctacagccag aaatccgaag tttacccgtt tacccagctg aaattcaaat 840
ctcacgttca gcagaaactg aaacgcgcga tgaacgaact gtctccgcgt ctgaaagaac 900
acggtaaaga atctttcctg cagttcgttt cccgttacca gggccacgat agcgcggttg 960
gcatgatccg ttctatgggt tacgatgcgc tgttcctgcc ggatatctct gcggaaatgg 1020
cgtatgatat cgttggtaaa cacccggaaa tccagagcgt taccgataac gatgcgaacc 1080
agtggttcgc ggcggaaacc ggtttcgcgg gtctgatcca gggcatcaaa gcgaaagtaa 1140
aagctgctgg tgcacgtttc tctctgggtt accgtctgct gagcgttcgc accgatggcg 1200
atggttacct gctgcagctg gcgggcgacg atggctggaa actggaacac cgtacccgtc 1260
acctgatcct ggcgatcccg ccgtctgcga tggcgggcct gaacgttgat ttcccggaag 1320
cgtggtccgg cgcgcgttac ggttctctgc cgctgttcaa aggtttcctg acctacggtg 1380
aaccgtggtg gctggattat aaactggatg atcaggtgct gatcgttgat aacccgctgc 1440
gtaaaatcta cttcaaaggt gataaatacc tgttcttcta cactgatagc gaaatggcta 1500
actactggcg tggttgcgtt gctgaaggtg aagatggtta cctggaacag atccgtaccc 1560
acctggcgtc tgctctgggc atcgttcgtg aacgtatccc gcagccgctg gcgcacgttc 1620
acaaatactg ggcgcacggc gttgaatttt gccgtgatag cgatattgat cacccgtccg 1680
cgctgagcca ccgtgattct ggtatcatcg cttgctctga tgcttacacc gaacactgcg 1740
gctggatgga aggcggtctg ctgagcgcgc gtgaagcgag ccgtctgctg ctgcagcgta 1800
tcgcggcgta attaaggagg taaaaaaaat gagcatcctg gatttcccgc gtatccattt 1860
ccgtggctgg gcgcgtgtta acgccccgac tgcgaaccgt gacccgcacg gccacattga 1920
tatggcgtct aataccgtcg caatggcggg tgaaccgttc gatctggcgc gccacccgac 1980
cgaatttcac cgtcacctgc gttctctggg cccgcgtttc ggcctggatg gccgtgcaga 2040
cccggaaggc ccgttcagcc tggcggaagg ctataacgca gcgggtaaca accatttttc 2100
ttgggaatct gcgaccgtgt ctcatgtgca gtgggatggc ggcgaagcag atcgtggtga 2160
cggtctggtc ggcgctcgcc tggcgctgtg gggtcactat aacgactacc tccgtaccac 2220
cttcaaccgt gcgcgttggg tggactctga cccgacacgt cgtgatgcgg cccagatcta 2280
cgcaggtcaa ttcactatca gcccggctgg tgcgggccct ggcaccccgt ggctgttcac 2340
tgcggatatc gacgactccc acggcgcgcg ctggacccgt ggtggccaca tcgcagaacg 2400
cggtggccat ttcctggacg aagagttcgg cctggcgcgt ctgttccaat ttagcgtgcc 2460
aaaagatcat ccgcacttcc tgttccatcc ggggccgttc gattctgagg catggcgtcg 2520
tctgcagctg gctctggagg acgatgatgt gctgggtctg accgttcaat acgctctgtt 2580
caacatgagc actccgccgc agccgaacag cccggtgttc catgatatgg tcggtgttgt 2640
tggcctgtgg cgccgtggtg aactggccag ctacccggcc ggccgtctgc tgcgtccgcg 2700
ccagccgggt ctgggtgatc tgacccttcg tgttaacggt ggccgcgtgg ccttgaacct 2760
ggcttgcgcg atcccgttct ctactcgcgc ggctcagccg agcgcaccgg atcgcctgac 2820
cccggatctg ggcgcgaaac tgccgctggg tgatctcctg ctgcgtgatg aagatggtgc 2880
actgctggca cgcgttccgc aggcgctcta ccaggactac tggactaacc acggcattgt 2940
ggatctgccg ctgctgcgtg agccgcgtgg ttccttaacc ctgtcctctg aactggccga 3000
gtggcgtgaa caggactggg tgacgcagag tgatgcttcc aacctgtatc tggaggctcc 3060
ggaccgccgt catggccgtt tcttcccgga atccattgct ctgcgttctt actttcgcgg 3120
cgaagcacgc gctcgcccgg atatcccaca ccgtattgaa ggcatgggtc tggtaggtgt 3180
tgaatcccgc caagacggcg atgcggcgga atggcgtctg accggcctgc gtcctggtcc 3240
tgcacgcatt gtgctcgatg atggtgcgga agcaatcccg ctgcgtgtcc tgccggatga 3300
ttgggcgctg gatgacgcaa cggtggaaga agtagactac gcgttcctgt accgccacgt 3360
gatggcgtac tatgagttgg tctatccgtt catgagcgat aaagttttca gcctggcgga 3420
ccgttgtaaa tgcgaaacct atgctcgtct gatgtggcag atgtgcgatc ctcagaaccg 3480
taataaatct tactatatgc cgtccacgcg cgaactgagc gcgccgaaag cacgcttgtt 3540
tctgaaatac ctggcgcatg tagaaggtca ggcacgcctc caggctccgc cgccggcggg 3600
cccggcccgc atcgaaagca aagctcagct ggccgccgaa ctgcgtaaag cagttgacct 3660
ggaactgtcc gtcatgctgc aatatctcta cgctgcatat agcatcccaa actacgcgca 3720
aggtcagcag cgcgtgcgtg atggcgcgtg gaccgctgaa cagctgcagc tggcatgtgg 3780
tagcggcgat cgccgccgcg acggcggtat ccgcgcggcg ctgctggaga ttgcgcacga 3840
agagatgatc cactatctgg tcgtgaacaa tctgctgatg gccctgggcg aaccgttcta 3900
cgcgggcgtt cctctgatgg gtgaagcggc acgtcaggcg tttggcctgg acactgagtt 3960
cgccctggag ccgttttctg aatctactct ggcgcgcttc gttcgcctgg aatggccgca 4020
tttcatcccg gcgccgggca agtccatcgc tgactgttac gcagcaattc gccaggcatt 4080
cctggacctg ccggacctgt ttggtggcga agccggtaaa cgtggtggcg aacaccactt 4140
gtttctgaat gaactgacca atcgcgcgca tccgggttac cagctggaag tgtttgatcg 4200
tgattccgcc ctgtttggta ttgcgttcgt gaccgatcag ggcgaaggtg gtgctctgga 4260
ttccccgcac tacgaacata gccactttca gcgcctgcgt gaaatgtcgg ctcgtatcat 4320
ggcgcaaagc gctccattcg aaccggcgct gccggccctg cgtaacccgg tcctggatga 4380
gtctccgggc tgccagcgtg ttgcggatgg tcgtgcgcgt gcgctgatgg cgctgtatca 4440
gggtgtttac gaactgatgt tcgcgatgat ggcacagcat tttgctgtta aaccgctggg 4500
ttctctgcgt cgtagccgcc tgatgaacgc tgcaatcgat ctgatgaccg gtctgctgcg 4560
tccgctgagc tgcgccctga tgaatctgcc gagcggtatt gcgggtcgta ccgcgggtcc 4620
gccgctgcct ggcccggttg acactcgttc ctatgatgac tacgcactgg gctgccgtat 4680
gctggcacgt cgctgtgaac gtctgctgga acaggcgtct atgctggaac cgggttggct 4740
gccggacgcg cagatggaac tgctggactt ttaccgtcgt cagatgctgg acctggcgtg 4800
tggtaaactg agccgtgaag cttaattaag gaggtaaaaa aaatgaaacg tgcgatcatc 4860
gttggcggtg gtctggcggg tggcctgacc gcgatctacc tggccaaacg tggctacgaa 4920
gttcatgttg tggaaaaacg tggtgacccg ctgcgtgacc tgagcagcta cgttgacgtt 4980
gtttctagcc gtgcgatcgg tgttagcatg accgttcgtg gtatcaaatc cgttctggct 5040
gcgggtatcc cgcgtgcgga actggatgct tgcggtgaac cgatcgttgc gatggcgttc 5100
agcgttggtg gtcagtatcg tatgcgtgaa ctgaaaccgc tggaagattt ccgtccgctg 5160
tccttgaacc gtgctgcgtt ccagaaactg ctgaacaaat acgcgaacct ggcgggcgtt 5220
cgttactact tcgaacataa atgcctggac gtggacctgg atggtaaaag cgtgctgatc 5280
cagggtaaag atggccagcc gcagcgtctg cagggcgata tgattatcgg tgcagatggt 5340
gcgcactctg cggtgcgtca ggctatgcag agcggcctgc gtcgttttga atttcagcag 5400
accttcttcc gtcacggtta taaaaccctg gttctgccgg atgcgcaggc gctgggctac 5460
cgtaaagata ccctgtattt cttcggtatg gattccggtg gcctgttcgc gggccgtgcg 5520
gcgaccattc cggatggttc tgtttctatc gccgtgtgcc tgccgtacag cggttctccg 5580
tccctgacca ccaccgacga accgaccatg cgtgcgttct ttgatcgtta ctttggcggt 5640
ctgccgcgtg acgcgcgtga cgaaatgctg cgtcagttcc tggctaaacc gtccaacgat 5700
ctgattaacg ttcgttctag caccttccac tataaaggta acgttctgct gctgggtgat 5760
gcggcgcacg cgaccgcccc gttcctgggc cagggtatga acatggcgct ggaagatgcg 5820
cgcacctttg tggaactgct ggatcgccac cagggtgatc aggataaagc cttcccggaa 5880
tttaccgaac tgcgtaaagt tcaggcggat gcaatgcagg atatggcgcg tgcgaactac 5940
gacgttctga gctgctctaa cccgatcttc tttatgcgcg cgcgctacac ccgttacatg 6000
cactctaaat ttccgggcct gtacccgccg gatatggcgg aaaaactgta cttcacctcc 6060
gaaccgtacg atcgtctgca gcagatccag cgtaaacaga acgtttggta taaaatcggt 6120
cgtgttaaca cctctggtga ttataaagat gatgatgata aataattaag gaggtaaaaa 6180
aaatgaaaat cctggttatc ggtgctggtc cggcaggtct ggttttcgct tcccagctga 6240
aacaggcgcg tccgctgtgg gcgatcgata tcgttgaaaa gaacgatgaa caggaagttc 6300
tgggctgggg cgttgttctg ccgggccgtc caggtcagca cccggccaac ccgctgagct 6360
atctggatgc tccggaacgt ctgaacccgc agttcctgga agatttcaaa ctggtgcacc 6420
acaacgaacc gagcctgatg agcaccggcg ttctgctgtg cggtgttgaa cgtcgtggcc 6480
tggtgcacgc gctgcgtgat aaatgccgta gccagggtat cgctatccgt ttcgaaagcc 6540
cgctgctgga acacggcgaa ctgccgctgg ctgactacga tctggttgtt ctggcaaacg 6600
gtgttaacca caaaaccgcg cacttcactg aagcgctggt tccgcaggtt gattacggtc 6660
gtaacaaata catctggtac ggtactagcc agctgttcga tcagatgaac ctggttttcc 6720
gtacccacgg caaagatatc ttcatcgctc acgcgtacaa atacagcgat accatgtcca 6780
cctttatcgt tgaatgctct gaagaaacct acgcgcgtgc gcgtctgggt gaaatgagcg 6840
aagaagcatc tgcggaatac gtggcgaaag ttttccaggc tgaactgggc ggtcacggtc 6900
tggttagcca gccgggtctg ggttggcgta acttcatgac cctgagccac gatcgttgcc 6960
acgatggtaa actggttctg ctgggcgatg ctctccagtc tggtcacttc tctatcggcc 7020
acggcaccac tatggcggtt gttgttgcgc agctgctggt taaagcgctg tgcaccgaag 7080
atggtgttcc ggcggcgctg aaacgtttcg aagaacgtgc gctgccgctg gttcagctgt 7140
tccgtggtca cgctgataac agccgtgttt ggttcgaaac cgttgaagaa cgtatgcacc 7200
tgagcagcgc ggaatttgtt cagagcttcg acgcgcgtcg taaaagcctg ccgccgatgc 7260
cggaagctct ggcgcagaac ctgcgttacg cgctgcagcg tacctctggt gattacaaag 7320
atgatgatga ttaattaagg aggtaaaaaa aatggaaaac cgtgaaccgc cgctgctgcc 7380
ggctcgttgg agcagcgcgt acgttagcta ctggagcccg atgctgccgg atgatcagct 7440
gaccagcggt tactgctggt tcgattacga acgtgatatc tgccgtatcg atggtctgtt 7500
caacccgtgg agcgaacgtg ataccggcta ccgtctgtgg atgtctgaag ttggcaacgc 7560
ggcgtccggc cgtacctgga aacagaaagt tgcgtacggt cgtgaacgca ccgcgctggg 7620
cgaacagctg tgcgaacgtc cgctggatga cgaaaccggc ccgttcgcgg aactgttcct 7680
gccgcgtgat gttctgcgcc gtctgggcgc gcgtcacatt ggtcgtcgtg tggttctggg 7740
ccgtgaagcg gatggttggc gttaccagcg tccgggcaaa ggtccgagca ccctgtacct 7800
ggatgcggcg agcggtactc cgctgcgtat ggttaccggc gatgaagcga gccgtgcgtc 7860
cctgcgtgat ttcccgaacg ttagcgaagc ggaaatcccg gatgcggttt tcgcggcgaa 7920
acgtgagctc 7930
<210> 2
<211> 144
<212> PRT
<213> Artificial sequence
<400> 2
Met Glu Asn Asn Leu Glu Asn Leu Thr Ile Gly Val Phe Ala Lys Ala
1 5 10 15
Ala Gly Val Asn Val Glu Thr Ile Arg Phe Tyr Gln Arg Lys Gly Leu
20 25 30
Leu Arg Glu Pro Asp Lys Pro Tyr Gly Ser Ile Arg Arg Tyr Gly Glu
35 40 45
Ala Asp Val Val Arg Val Lys Phe Val Lys Ser Ala Gln Arg Leu Gly
50 55 60
Phe Ser Leu Asp Glu Ile Ala Glu Leu Leu Arg Leu Asp Asp Gly Thr
65 70 75 80
His Cys Glu Glu Ala Ser Ser Leu Ala Glu His Lys Leu Lys Asp Val
85 90 95
Arg Glu Lys Met Ala Asp Leu Ala Arg Met Glu Thr Val Leu Ser Glu
100 105 110
Leu Val Cys Ala Cys His Ala Arg Lys Gly Asn Val Ser Cys Pro Leu
115 120 125
Ile Ala Ser Leu Gln Gly Glu Ala Gly Leu Ala Arg Ser Ala Met Pro
130 135 140
<210> 3
<211> 418
<212> PRT
<213> Artificial sequence
<400> 3
Met Lys His Ser Ser Asp Ile Cys Ile Val Gly Ala Gly Ile Ser Gly
1 5 10 15
Leu Thr Cys Ala Ser His Leu Leu Asp Ser Pro Ala Cys Arg Gly Leu
20 25 30
Ser Leu Arg Ile Phe Asp Met Gln Gln Glu Ala Gly Gly Arg Ile Arg
35 40 45
Ser Lys Met Leu Asp Gly Lys Ala Ser Ile Glu Leu Gly Ala Gly Arg
50 55 60
Tyr Ser Pro Gln Leu His Pro His Phe Gln Ser Ala Met Gln His Tyr
65 70 75 80
Ser Gln Lys Ser Glu Val Tyr Pro Phe Thr Gln Leu Lys Phe Lys Ser
85 90 95
His Val Gln Gln Lys Leu Lys Arg Ala Met Asn Glu Leu Ser Pro Arg
100 105 110
Leu Lys Glu His Gly Lys Glu Ser Phe Leu Gln Phe Val Ser Arg Tyr
115 120 125
Gln Gly His Asp Ser Ala Val Gly Met Ile Arg Ser Met Gly Tyr Asp
130 135 140
Ala Leu Phe Leu Pro Asp Ile Ser Ala Glu Met Ala Tyr Asp Ile Val
145 150 155 160
Gly Lys His Pro Glu Ile Gln Ser Val Thr Asp Asn Asp Ala Asn Gln
165 170 175
Trp Phe Ala Ala Glu Thr Gly Phe Ala Gly Leu Ile Gln Gly Ile Lys
180 185 190
Ala Lys Val Lys Ala Ala Gly Ala Arg Phe Ser Leu Gly Tyr Arg Leu
195 200 205
Leu Ser Val Arg Thr Asp Gly Asp Gly Tyr Leu Leu Gln Leu Ala Gly
210 215 220
Asp Asp Gly Trp Lys Leu Glu His Arg Thr Arg His Leu Ile Leu Ala
225 230 235 240
Ile Pro Pro Ser Ala Met Ala Gly Leu Asn Val Asp Phe Pro Glu Ala
245 250 255
Trp Ser Gly Ala Arg Tyr Gly Ser Leu Pro Leu Phe Lys Gly Phe Leu
260 265 270
Thr Tyr Gly Glu Pro Trp Trp Leu Asp Tyr Lys Leu Asp Asp Gln Val
275 280 285
Leu Ile Val Asp Asn Pro Leu Arg Lys Ile Tyr Phe Lys Gly Asp Lys
290 295 300
Tyr Leu Phe Phe Tyr Thr Asp Ser Glu Met Ala Asn Tyr Trp Arg Gly
305 310 315 320
Cys Val Ala Glu Gly Glu Asp Gly Tyr Leu Glu Gln Ile Arg Thr His
325 330 335
Leu Ala Ser Ala Leu Gly Ile Val Arg Glu Arg Ile Pro Gln Pro Leu
340 345 350
Ala His Val His Lys Tyr Trp Ala His Gly Val Glu Phe Cys Arg Asp
355 360 365
Ser Asp Ile Asp His Pro Ser Ala Leu Ser His Arg Asp Ser Gly Ile
370 375 380
Ile Ala Cys Ser Asp Ala Tyr Thr Glu His Cys Gly Trp Met Glu Gly
385 390 395 400
Gly Leu Leu Ser Ala Arg Glu Ala Ser Arg Leu Leu Leu Gln Arg Ile
405 410 415
Ala Ala
<210> 4
<211> 998
<212> PRT
<213> Artificial sequence
<400> 4
Met Ser Ile Leu Asp Phe Pro Arg Ile His Phe Arg Gly Trp Ala Arg
1 5 10 15
Val Asn Ala Pro Thr Ala Asn Arg Asp Pro His Gly His Ile Asp Met
20 25 30
Ala Ser Asn Thr Val Ala Met Ala Gly Glu Pro Phe Asp Leu Ala Arg
35 40 45
His Pro Thr Glu Phe His Arg His Leu Arg Ser Leu Gly Pro Arg Phe
50 55 60
Gly Leu Asp Gly Arg Ala Asp Pro Glu Gly Pro Phe Ser Leu Ala Glu
65 70 75 80
Gly Tyr Asn Ala Ala Gly Asn Asn His Phe Ser Trp Glu Ser Ala Thr
85 90 95
Val Ser His Val Gln Trp Asp Gly Gly Glu Ala Asp Arg Gly Asp Gly
100 105 110
Leu Val Gly Ala Arg Leu Ala Leu Trp Gly His Tyr Asn Asp Tyr Leu
115 120 125
Arg Thr Thr Phe Asn Arg Ala Arg Trp Val Asp Ser Asp Pro Thr Arg
130 135 140
Arg Asp Ala Ala Gln Ile Tyr Ala Gly Gln Phe Thr Ile Ser Pro Ala
145 150 155 160
Gly Ala Gly Pro Gly Thr Pro Trp Leu Phe Thr Ala Asp Ile Asp Asp
165 170 175
Ser His Gly Ala Arg Trp Thr Arg Gly Gly His Ile Ala Glu Arg Gly
180 185 190
Gly His Phe Leu Asp Glu Glu Phe Gly Leu Ala Arg Leu Phe Gln Phe
195 200 205
Ser Val Pro Lys Asp His Pro His Phe Leu Phe His Pro Gly Pro Phe
210 215 220
Asp Ser Glu Ala Trp Arg Arg Leu Gln Leu Ala Leu Glu Asp Asp Asp
225 230 235 240
Val Leu Gly Leu Thr Val Gln Tyr Ala Leu Phe Asn Met Ser Thr Pro
245 250 255
Pro Gln Pro Asn Ser Pro Val Phe His Asp Met Val Gly Val Val Gly
260 265 270
Leu Trp Arg Arg Gly Glu Leu Ala Ser Tyr Pro Ala Gly Arg Leu Leu
275 280 285
Arg Pro Arg Gln Pro Gly Leu Gly Asp Leu Thr Leu Arg Val Asn Gly
290 295 300
Gly Arg Val Ala Leu Asn Leu Ala Cys Ala Ile Pro Phe Ser Thr Arg
305 310 315 320
Ala Ala Gln Pro Ser Ala Pro Asp Arg Leu Thr Pro Asp Leu Gly Ala
325 330 335
Lys Leu Pro Leu Gly Asp Leu Leu Leu Arg Asp Glu Asp Gly Ala Leu
340 345 350
Leu Ala Arg Val Pro Gln Ala Leu Tyr Gln Asp Tyr Trp Thr Asn His
355 360 365
Gly Ile Val Asp Leu Pro Leu Leu Arg Glu Pro Arg Gly Ser Leu Thr
370 375 380
Leu Ser Ser Glu Leu Ala Glu Trp Arg Glu Gln Asp Trp Val Thr Gln
385 390 395 400
Ser Asp Ala Ser Asn Leu Tyr Leu Glu Ala Pro Asp Arg Arg His Gly
405 410 415
Arg Phe Phe Pro Glu Ser Ile Ala Leu Arg Ser Tyr Phe Arg Gly Glu
420 425 430
Ala Arg Ala Arg Pro Asp Ile Pro His Arg Ile Glu Gly Met Gly Leu
435 440 445
Val Gly Val Glu Ser Arg Gln Asp Gly Asp Ala Ala Glu Trp Arg Leu
450 455 460
Thr Gly Leu Arg Pro Gly Pro Ala Arg Ile Val Leu Asp Asp Gly Ala
465 470 475 480
Glu Ala Ile Pro Leu Arg Val Leu Pro Asp Asp Trp Ala Leu Asp Asp
485 490 495
Ala Thr Val Glu Glu Val Asp Tyr Ala Phe Leu Tyr Arg His Val Met
500 505 510
Ala Tyr Tyr Glu Leu Val Tyr Pro Phe Met Ser Asp Lys Val Phe Ser
515 520 525
Leu Ala Asp Arg Cys Lys Cys Glu Thr Tyr Ala Arg Leu Met Trp Gln
530 535 540
Met Cys Asp Pro Gln Asn Arg Asn Lys Ser Tyr Tyr Met Pro Ser Thr
545 550 555 560
Arg Glu Leu Ser Ala Pro Lys Ala Arg Leu Phe Leu Lys Tyr Leu Ala
565 570 575
His Val Glu Gly Gln Ala Arg Leu Gln Ala Pro Pro Pro Ala Gly Pro
580 585 590
Ala Arg Ile Glu Ser Lys Ala Gln Leu Ala Ala Glu Leu Arg Lys Ala
595 600 605
Val Asp Leu Glu Leu Ser Val Met Leu Gln Tyr Leu Tyr Ala Ala Tyr
610 615 620
Ser Ile Pro Asn Tyr Ala Gln Gly Gln Gln Arg Val Arg Asp Gly Ala
625 630 635 640
Trp Thr Ala Glu Gln Leu Gln Leu Ala Cys Gly Ser Gly Asp Arg Arg
645 650 655
Arg Asp Gly Gly Ile Arg Ala Ala Leu Leu Glu Ile Ala His Glu Glu
660 665 670
Met Ile His Tyr Leu Val Val Asn Asn Leu Leu Met Ala Leu Gly Glu
675 680 685
Pro Phe Tyr Ala Gly Val Pro Leu Met Gly Glu Ala Ala Arg Gln Ala
690 695 700
Phe Gly Leu Asp Thr Glu Phe Ala Leu Glu Pro Phe Ser Glu Ser Thr
705 710 715 720
Leu Ala Arg Phe Val Arg Leu Glu Trp Pro His Phe Ile Pro Ala Pro
725 730 735
Gly Lys Ser Ile Ala Asp Cys Tyr Ala Ala Ile Arg Gln Ala Phe Leu
740 745 750
Asp Leu Pro Asp Leu Phe Gly Gly Glu Ala Gly Lys Arg Gly Gly Glu
755 760 765
His His Leu Phe Leu Asn Glu Leu Thr Asn Arg Ala His Pro Gly Tyr
770 775 780
Gln Leu Glu Val Phe Asp Arg Asp Ser Ala Leu Phe Gly Ile Ala Phe
785 790 795 800
Val Thr Asp Gln Gly Glu Gly Gly Ala Leu Asp Ser Pro His Tyr Glu
805 810 815
His Ser His Phe Gln Arg Leu Arg Glu Met Ser Ala Arg Ile Met Ala
820 825 830
Gln Ser Ala Pro Phe Glu Pro Ala Leu Pro Ala Leu Arg Asn Pro Val
835 840 845
Leu Asp Glu Ser Pro Gly Cys Gln Arg Val Ala Asp Gly Arg Ala Arg
850 855 860
Ala Leu Met Ala Leu Tyr Gln Gly Val Tyr Glu Leu Met Phe Ala Met
865 870 875 880
Met Ala Gln His Phe Ala Val Lys Pro Leu Gly Ser Leu Arg Arg Ser
885 890 895
Arg Leu Met Asn Ala Ala Ile Asp Leu Met Thr Gly Leu Leu Arg Pro
900 905 910
Leu Ser Cys Ala Leu Met Asn Leu Pro Ser Gly Ile Ala Gly Arg Thr
915 920 925
Ala Gly Pro Pro Leu Pro Gly Pro Val Asp Thr Arg Ser Tyr Asp Asp
930 935 940
Tyr Ala Leu Gly Cys Arg Met Leu Ala Arg Arg Cys Glu Arg Leu Leu
945 950 955 960
Glu Gln Ala Ser Met Leu Glu Pro Gly Trp Leu Pro Asp Ala Gln Met
965 970 975
Glu Leu Leu Asp Phe Tyr Arg Arg Gln Met Leu Asp Leu Ala Cys Gly
980 985 990
Lys Leu Ser Arg Glu Ala
995
<210> 5
<211> 440
<212> PRT
<213> Artificial sequence
<400> 5
Met Lys Arg Ala Ile Ile Val Gly Gly Gly Leu Ala Gly Gly Leu Thr
1 5 10 15
Ala Ile Tyr Leu Ala Lys Arg Gly Tyr Glu Val His Val Val Glu Lys
20 25 30
Arg Gly Asp Pro Leu Arg Asp Leu Ser Ser Tyr Val Asp Val Val Ser
35 40 45
Ser Arg Ala Ile Gly Val Ser Met Thr Val Arg Gly Ile Lys Ser Val
50 55 60
Leu Ala Ala Gly Ile Pro Arg Ala Glu Leu Asp Ala Cys Gly Glu Pro
65 70 75 80
Ile Val Ala Met Ala Phe Ser Val Gly Gly Gln Tyr Arg Met Arg Glu
85 90 95
Leu Lys Pro Leu Glu Asp Phe Arg Pro Leu Ser Leu Asn Arg Ala Ala
100 105 110
Phe Gln Lys Leu Leu Asn Lys Tyr Ala Asn Leu Ala Gly Val Arg Tyr
115 120 125
Tyr Phe Glu His Lys Cys Leu Asp Val Asp Leu Asp Gly Lys Ser Val
130 135 140
Leu Ile Gln Gly Lys Asp Gly Gln Pro Gln Arg Leu Gln Gly Asp Met
145 150 155 160
Ile Ile Gly Ala Asp Gly Ala His Ser Ala Val Arg Gln Ala Met Gln
165 170 175
Ser Gly Leu Arg Arg Phe Glu Phe Gln Gln Thr Phe Phe Arg His Gly
180 185 190
Tyr Lys Thr Leu Val Leu Pro Asp Ala Gln Ala Leu Gly Tyr Arg Lys
195 200 205
Asp Thr Leu Tyr Phe Phe Gly Met Asp Ser Gly Gly Leu Phe Ala Gly
210 215 220
Arg Ala Ala Thr Ile Pro Asp Gly Ser Val Ser Ile Ala Val Cys Leu
225 230 235 240
Pro Tyr Ser Gly Ser Pro Ser Leu Thr Thr Thr Asp Glu Pro Thr Met
245 250 255
Arg Ala Phe Phe Asp Arg Tyr Phe Gly Gly Leu Pro Arg Asp Ala Arg
260 265 270
Asp Glu Met Leu Arg Gln Phe Leu Ala Lys Pro Ser Asn Asp Leu Ile
275 280 285
Asn Val Arg Ser Ser Thr Phe His Tyr Lys Gly Asn Val Leu Leu Leu
290 295 300
Gly Asp Ala Ala His Ala Thr Ala Pro Phe Leu Gly Gln Gly Met Asn
305 310 315 320
Met Ala Leu Glu Asp Ala Arg Thr Phe Val Glu Leu Leu Asp Arg His
325 330 335
Gln Gly Asp Gln Asp Lys Ala Phe Pro Glu Phe Thr Glu Leu Arg Lys
340 345 350
Val Gln Ala Asp Ala Met Gln Asp Met Ala Arg Ala Asn Tyr Asp Val
355 360 365
Leu Ser Cys Ser Asn Pro Ile Phe Phe Met Arg Ala Arg Tyr Thr Arg
370 375 380
Tyr Met His Ser Lys Phe Pro Gly Leu Tyr Pro Pro Asp Met Ala Glu
385 390 395 400
Lys Leu Tyr Phe Thr Ser Glu Pro Tyr Asp Arg Leu Gln Gln Ile Gln
405 410 415
Arg Lys Gln Asn Val Trp Tyr Lys Ile Gly Arg Val Asn Thr Ser Gly
420 425 430
Asp Tyr Lys Asp Asp Asp Asp Lys
435 440
<210> 6
<211> 383
<212> PRT
<213> Artificial sequence
<400> 6
Met Lys Ile Leu Val Ile Gly Ala Gly Pro Ala Gly Leu Val Phe Ala
1 5 10 15
Ser Gln Leu Lys Gln Ala Arg Pro Leu Trp Ala Ile Asp Ile Val Glu
20 25 30
Lys Asn Asp Glu Gln Glu Val Leu Gly Trp Gly Val Val Leu Pro Gly
35 40 45
Arg Pro Gly Gln His Pro Ala Asn Pro Leu Ser Tyr Leu Asp Ala Pro
50 55 60
Glu Arg Leu Asn Pro Gln Phe Leu Glu Asp Phe Lys Leu Val His His
65 70 75 80
Asn Glu Pro Ser Leu Met Ser Thr Gly Val Leu Leu Cys Gly Val Glu
85 90 95
Arg Arg Gly Leu Val His Ala Leu Arg Asp Lys Cys Arg Ser Gln Gly
100 105 110
Ile Ala Ile Arg Phe Glu Ser Pro Leu Leu Glu His Gly Glu Leu Pro
115 120 125
Leu Ala Asp Tyr Asp Leu Val Val Leu Ala Asn Gly Val Asn His Lys
130 135 140
Thr Ala His Phe Thr Glu Ala Leu Val Pro Gln Val Asp Tyr Gly Arg
145 150 155 160
Asn Lys Tyr Ile Trp Tyr Gly Thr Ser Gln Leu Phe Asp Gln Met Asn
165 170 175
Leu Val Phe Arg Thr His Gly Lys Asp Ile Phe Ile Ala His Ala Tyr
180 185 190
Lys Tyr Ser Asp Thr Met Ser Thr Phe Ile Val Glu Cys Ser Glu Glu
195 200 205
Thr Tyr Ala Arg Ala Arg Leu Gly Glu Met Ser Glu Glu Ala Ser Ala
210 215 220
Glu Tyr Val Ala Lys Val Phe Gln Ala Glu Leu Gly Gly His Gly Leu
225 230 235 240
Val Ser Gln Pro Gly Leu Gly Trp Arg Asn Phe Met Thr Leu Ser His
245 250 255
Asp Arg Cys His Asp Gly Lys Leu Val Leu Leu Gly Asp Ala Leu Gln
260 265 270
Ser Gly His Phe Ser Ile Gly His Gly Thr Thr Met Ala Val Val Val
275 280 285
Ala Gln Leu Leu Val Lys Ala Leu Cys Thr Glu Asp Gly Val Pro Ala
290 295 300
Ala Leu Lys Arg Phe Glu Glu Arg Ala Leu Pro Leu Val Gln Leu Phe
305 310 315 320
Arg Gly His Ala Asp Asn Ser Arg Val Trp Phe Glu Thr Val Glu Glu
325 330 335
Arg Met His Leu Ser Ser Ala Glu Phe Val Gln Ser Phe Asp Ala Arg
340 345 350
Arg Lys Ser Leu Pro Pro Met Pro Glu Ala Leu Ala Gln Asn Leu Arg
355 360 365
Tyr Ala Leu Gln Arg Thr Ser Gly Asp Tyr Lys Asp Asp Asp Asp
370 375 380
<210> 7
<211> 191
<212> PRT
<213> Artificial sequence
<400> 7
Met Glu Asn Arg Glu Pro Pro Leu Leu Pro Ala Arg Trp Ser Ser Ala
1 5 10 15
Tyr Val Ser Tyr Trp Ser Pro Met Leu Pro Asp Asp Gln Leu Thr Ser
20 25 30
Gly Tyr Cys Trp Phe Asp Tyr Glu Arg Asp Ile Cys Arg Ile Asp Gly
35 40 45
Leu Phe Asn Pro Trp Ser Glu Arg Asp Thr Gly Tyr Arg Leu Trp Met
50 55 60
Ser Glu Val Gly Asn Ala Ala Ser Gly Arg Thr Trp Lys Gln Lys Val
65 70 75 80
Ala Tyr Gly Arg Glu Arg Thr Ala Leu Gly Glu Gln Leu Cys Glu Arg
85 90 95
Pro Leu Asp Asp Glu Thr Gly Pro Phe Ala Glu Leu Phe Leu Pro Arg
100 105 110
Asp Val Leu Arg Arg Leu Gly Ala Arg His Ile Gly Arg Arg Val Val
115 120 125
Leu Gly Arg Glu Ala Asp Gly Trp Arg Tyr Gln Arg Pro Gly Lys Gly
130 135 140
Pro Ser Thr Leu Tyr Leu Asp Ala Ala Ser Gly Thr Pro Leu Arg Met
145 150 155 160
Val Thr Gly Asp Glu Ala Ser Arg Ala Ser Leu Arg Asp Phe Pro Asn
165 170 175
Val Ser Glu Ala Glu Ile Pro Asp Ala Val Phe Ala Ala Lys Arg
180 185 190

Claims (10)

1. The recombinant bacterium is obtained by introducing a recombinant vector A into a recipient bacterium;
the recombinant vector A contains a DNA fragment A;
and the DNA fragment A contains a mer bidirectional promoter, one side of the mer bidirectional promoter is a MerR protein coding gene, and the other side of the mer bidirectional promoter is a violacein synthetic gene module.
2. The recombinant bacterium according to claim 1, wherein: the violacein synthetic gene modules are assembled in polycistronic form and encode a VioA protein, a VioB protein, a VioC protein, a VioD protein and a VioE protein.
3. The recombinant bacterium according to claim 1 or 2, wherein: the nucleotide sequence of the mer bidirectional promoter is 442-512 th site of SEQ ID No. 1; and/or
The amino acid sequence of the MerR protein is shown as SEQ ID No. 2; and/or
The amino acid sequence of the VioA protein is shown as SEQ ID No. 3; and/or
The amino acid sequence of the VioB protein is shown as SEQ ID No. 4; and/or
The amino acid sequence of the VioC protein is shown as SEQ ID No. 5; and/or
The amino acid sequence of the VioD protein is shown as SEQ ID No. 6; and/or
The amino acid sequence of the VioE protein is shown as SEQ ID No. 7.
4. The recombinant bacterium according to any one of claims 1 to 3, wherein: the nucleotide sequence of the encoding gene of the MerR protein is a reverse complementary sequence of the 7 th to 441 th positions of SEQ ID No. 1; and/or
The nucleotide sequence of the VioA protein coding gene is the 555-friendly 1811 site of SEQ ID No. 1; and/or
The nucleotide sequence of the coding gene of the VioB protein is 1829-4825 th site of SEQ ID No. 1; and/or
The nucleotide sequence of the VioC protein coding gene is 4843-6165 site of SEQ ID No. 1; and/or
The nucleotide sequence of the coding gene of the VioD protein is 6183-7334 of SEQ ID No. 1; and/or
The nucleotide sequence of the coding gene of the VioE protein is 7352-7924 of SEQ ID No. 1;
further, the nucleotide sequence of the violacein synthetic gene module is 555-7924 of SEQ ID No. 1;
further, the nucleotide sequence of the DNA fragment A is SEQ ID No. 1.
5. The recombinant bacterium according to any one of claims 1 to 4, wherein: the recombinant vector A is obtained by replacing a small fragment between the enzyme cutting sites BglII and SacI of the pET-21a (+) plasmid with the DNA fragment A.
6. The recombinant bacterium according to any one of claims 1 to 5, wherein: the recipient bacterium is escherichia coli;
further, the Escherichia coli is Escherichia coli TOP 10.
7. The use of the recombinant bacterium of any one of claims 1-6 as or in the preparation of a mercury ion microbial whole cell biosensor that uses violacein as an output signal.
8. Any one of the following products or applications:
p1, the recombinant vector A of any one of claims 1 to 6;
p2, the DNA fragment A of any one of claims 1 to 6;
use of the recombinant vector A of P3 or P1 or the DNA fragment A of P2 in the preparation of the recombinant bacteria of any one of claims 1-6;
p4, kit I, comprising the recombinant bacterium of any one of claims 1 to 6 and a water-insoluble alcoholic solution;
p5, kit II, containing the recombinant bacterium of any one of claims 1 to 6, a water-insoluble alcohol solution and mercury ions as a standard;
use of the kit II of the kit I of the recombinant vector A of P6 or P1 or the kit II of the kit I of P5 or the recombinant bacterium of any one of claims 1 to 6 for detecting mercury ions, wherein the kit A of the recombinant vector A of P2 or the DNA fragment A of P4 of P6 or P1 is used for detecting mercury ions;
further, the detection of the mercury ions is the qualitative and/or quantitative detection of the mercury ions on the liquid sample.
9. Any one of the following methods:
the method comprises the following steps: a method for detecting whether a liquid sample contains mercury ions is a method A or a method B or a method C:
the method A comprises the following steps: the thallus observation method comprises the following steps: placing the recombinant bacterium of any one of claims 1-6 in the liquid sample to be tested, shake-culturing at 37 ℃ for 5-12h, collecting the bacterium, observing the color change of the bacterium, and if the color of violacein is presented, determining that the liquid sample to be tested contains or is candidate to contain mercury ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain mercury ions;
the method B comprises the following steps: the bacterial liquid observation method comprises the following steps: placing the recombinant bacterium of any one of claims 1-6 in the liquid sample to be tested, shake-culturing at 37 ℃ for 5-12h, collecting the bacterium, resuspending with SDS aqueous solution, adding water-insoluble alcohol solution, shaking for 5min, standing for layering, observing the color change of the upper organic phase, and if the chromogenic reaction of violacein is observed, determining that the liquid sample to be tested contains or is candidate to contain mercury ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain mercury ions;
the method C comprises the following steps: an A490 value assay comprising the steps of: placing the recombinant bacterium of any one of claims 1-6 in the liquid sample to be tested, shake-culturing for 5-12h at 37 ℃, collecting the thallus, resuspending with SDS aqueous solution, adding water-insoluble alcohol solution, shaking for 5min, centrifuging, taking the supernatant, and determining the A490 value, referred to as the A490 value of the liquid sample group to be tested; if the A490 value of the liquid sample group to be detected is larger than the A490 value of the control group, the liquid sample to be detected contains or is candidate to contain mercury ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain mercury ions; wherein the A490 value of the control group is determined by replacing the liquid sample to be tested with a liquid sample not containing mercury ions compared with the A490 value of the liquid sample group to be tested;
the second method comprises the following steps: a method for detecting the content of mercury ions in a liquid sample comprises the following steps:
(A1) placing the recombinant strain of any one of claims 1-6 in a series of mercury ion liquid samples with known concentration, performing shake culture at 37 ℃ for 5-12h, collecting thalli, adding a water-insoluble alcohol solution, performing shake for 5min, detecting the A490 value of the series of mercury ion liquid samples with known concentration, and then drawing a standard curve according to the mercury ion concentration and the A490 value;
(A2) and (D) replacing the series of mercury ion liquid samples with known concentrations in the step (A1) with the liquid sample to be detected, repeating the step (A1) to obtain the A490 value of the liquid sample to be detected, and substituting the A490 value into the standard curve to obtain the mercury ion content in the liquid sample to be detected.
10. A recombinant bacterium or product or use or method according to any one of claims 1 to 9, wherein: the mercury ions are divalent mercury ions; and/or
The water-insoluble alcohol solution is n-butanol.
CN202110345979.6A 2021-03-31 2021-03-31 Construction and application of mercury ion microorganism whole-cell biosensor taking violacein as output signal Pending CN113234651A (en)

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