CN101509000A - Bacteria copper resistant gene, recombinant vector containing the same, preparation and expression thereof - Google Patents
Bacteria copper resistant gene, recombinant vector containing the same, preparation and expression thereof Download PDFInfo
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- 229910052802 copper Inorganic materials 0.000 title claims abstract description 63
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Abstract
The invention discloses a gene encoding copper resistance of Serratia marcescens. The gene is generated by a Serratia sp. KMR-3 strain; the whole length of the gene sequence is 1,117bp; derived copper resistance protein is encoded by 194 amino acids, is CutF protein with molecular weight of about 22KDa and theoretical pI value of 5.30; and the gene has highest homology with Yersinia mollaretii ATCC 43969 copper resistance lipoprotein N1pE, and reaches 70 percent. The invention also provides a recombinant plasmid containing the gene and a recombinant strain expressing the resistance gene. The method for expressing resistance protein heterology of the gene comprises the following steps: copper resistance related gene can be cloned through constructing a genome DNA library; according to comparison of amino acid sequences, the cloned protein is found to be CutF protein, and the protein is further expressed in colibacillus successfully. The technology of cloning the copper resistance gene establishes foundation for bacterium copper resistance mechanism and biological restoration technology of copper ion polluted region.
Description
Technical field
The invention belongs to the genetically engineered field, particularly, the gene that relates to Serratia coding copper resistant, dna sequence dna and deduced amino acid, and the heterogenous expression of resistance protein, relate to recombinant plasmid that contains this gene and the recombinant bacterial strain of expressing this resistant gene, and their preparation and expression method.
Background technology
Though heavy metal appears in some ecosystem naturally, but its industrial application causes many serious environmental problem, therefore heavy metal comes into one's own day by day to the pollution and the improvement of environment, and effective utilization of metal resistance bacterium then can be removed metal from the environment that pollutes.Therefore for the understanding of heavy metal resistance regulation and control, will help to handle bio-waste and assess the influence of industrial activity natural ecosystems.
Copper is a kind of important trace metal, plays the part of important role in cells physiological, and it also is the important prothetic group of a series of enzymes in the respiration.But, when surpassing finite concentration then pair cell have toxicity, the cupric ion of high density is mainly by producing highly active radical damage DNA, destroy membrane structure and making enzyme deactivation.Therefore; avoid the high density heavy metallic poison in order to protect cell; bacterium utilizes a series of resistance mechanisms: self avoid the murder by poisoning of copper as reaching the outer sequester of born of the same parents, outflow pump, enzyme detoxification and restoring system protection in permeability barrier, the born of the same parents, and guarantee to satisfy its nutritional requirement.
Clone's resistant gene is the prerequisite to the research of heavy metal resistance mechanism, simultaneously, the resistant gene of being cloned into is transformed, not only can provide certain theoretical foundation, can also provide theoretical investigation and application foundation for the development and application of the bioremediation technology of heavy metal contamination region for inquiring into bacterium heavy metal resistance mechanism.
Summary of the invention
The present invention aims to provide the dna sequence dna of a kind of copper resistant gene and this gene and the amino acid (Fig. 1) of derivation, and the proteic heterogenous expression of copper resistant (Fig. 2).
Another object of the present invention is to provide a kind of recombinant expression plasmid and recombinant bacterial strain of copper resistant gene.
Contain the recombinant plasmid pUC118-Cu1 and the pET28a-Cu1 of above-mentioned dna sequence dna, and recombination bacillus coli BL21 (the DE3)-pET28a-Cu1 that utilizes carrier pET28a-Cu1 to transform.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
The copper resistant gene, its dna sequence dna as shown in Figure 1.
The copper resistant gene, its aminoacid sequence as shown in Figure 1.
Described copper resistant gene, it is produced by thunder Bordetella (Serratia sp.) KMR-3 bacterial strain, gene order total length 1,117bp, the anti-cuproprotein of derivation is encoded by 194 amino acid, is CutF albumen, and molecular weight is about 22KDa, and theoretical pI value is 5.30; The highest with Mohs Yersinia (Yersinia mollaretii) ATCC 43969 copper resistant lipoprotein NlpE homologys, reach 70%.
The recombinant vectors pUC118-Cu1 that comprises the dna sequence dna of claim 1.
The recombinant vectors pET28a-Cu1 that comprises the dna sequence dna of claim 1.
Recombination bacillus coli BL21 (the DE3)-pET28a-Cu1 that utilizes recombinant vectors pET28a-Cu1 to transform.
The preparation method of above-mentioned copper resistant gene, recombinant vectors, recombinant bacterial strain, extract the chromosomal DNA of serratia marcescens KMR-3 bacterial strain, carry out partially digested with restriction enzyme Sau3A I, glue reclaims enzyme and cuts product, and be connected with pUC118 BamH I/BAP carrier, connect product through ethanol sedimentation, electric Transformed E .coliDH5, coating contains the LB flat board of penbritin, successfully makes up genome dna library; Screen recon containing on the selectivity flat board of penbritin and cupric ion, from recombinant plasmid pUC118-Cu1, isolate the dna fragmentation of cupric resistant gene,, obtain DNA as shown in Figure 1 through digestion with restriction enzyme; Design primer amplification copper resistant gene is connected and transformed into escherichia coli BL21 (DE3) bacterial strain with the pET28a carrier, obtains recombinant plasmid pET28a-Cu1 and recombinant bacterial strain e. coli bl21 (the DE3)-pET28a-Cu1 that contains recombinant expression plasmid.
Serratia (Serratia sp.) the KMR-3 bacterial strain that the strain that utilization of the present invention is separated to the waste water around the smeltery, Kunming has high resistance to cupric ion, its preserving number is: CGMCC No.2636.By in bacillus coli DH 5, making up the genome dna library of this bacterium, be cloned into the gene relevant with copper resistant.The albumen of the copper resistant coded by said gene of being cloned into belongs to CutF albumen, and is the highest with Mohs Yersinia (Yersinia mollaretii) ATCC 43969 copper resistant lipoprotein NlpE homologys by 194 amino acid codings, reaches 70%.Obtain to contain the recombinant plasmid of copper resistant gene by ordinary method, transformed into escherichia coli bacterial strain successful expression the albumen of coding copper resistant gene.
In addition,, the copper resistant gene clone that the present invention relates to can be applied in the bacterial strain of sewage disposal to other, thereby improve it to the tolerance of copper and then reach the purpose that heavy metal ion are removed in enrichment, conversion etc. by molecular biology method.
The serratia that the present invention relates to (Serratia sp.) KMR-3 bacterial strain on August 26th, 2008 in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (BeiJing, China) preservation, preserving number is: CGMCC No.2636.
Concrete technological step of the present invention is as follows: extract serratia KMR-3 strain chromosome DNA, carry out partially digested with restriction enzyme Sau3A I.Glue reclaims enzyme and cuts product, and is connected with pUC118 BamH I/BAP carrier.Connect product through ethanol sedimentation, electric Transformed E .coli DH5, coating contains the LB flat board of penbritin, successfully makes up genome dna library.Screen recon containing on the selectivity flat board of penbritin and cupric ion.From recombinant plasmid pUC118-Cu1, isolate the dna fragmentation of cupric resistant gene,, obtain DNA as shown in Figure 1 through digestion with restriction enzyme.Screen the gene relevant with copper resistant, behind the extraction plasmid Transformed E .coli, its tolerance to copper has been brought up to 300mg/L by 160mg/L, and therefore the copper resistant gene of being cloned into has the potentiality of the sewage disposal of being applied to.Order-checking shows: the copper resistant gene of being cloned into is encoded by 194 amino acid, and molecular weight is about 22KDa.Design primer amplification copper resistant gene is connected and transformed into escherichia coli BL21 (DE3) bacterial strain with the pET28a carrier, obtains recombinant plasmid pET28a-Cu1 and recombinant bacterial strain e. coli bl21 (the DE3)-pET28a-Cu1 that contains recombinant expression plasmid.Induce down at IPTG, in intestinal bacteria, carried out efficiently expressing (Fig. 2).
The copper resistant gene that the present invention is cloned into, the highest with Mohs Yersinia (Yersinia mollaretii) ATCC43969 copper resistant lipoprotein NlpE homology, reach 70%.Utilize software to carry out positioning analysis in this proteic cell.Find that by analyzing this anti-cuproprotein is evenly distributed in outside kytoplasm, plasma membrane, pericentral siphon, adventitia and the born of the same parents.And known bibliographical information shows, the CutF albumen of E.coli is outer membrane lipoprotein, participates in the outflow of cupric ion and transports cupric ion to the dependent enzyme of copper.Studies show that NlpE (CutF) is a kind of new outer membrane lipoprotein E, and NlpE is first example that relevant outer membrane lipoprotein participates in signal transduction and activation lysophospholipids.
At present, much studies show that: NlpE participation competent cell combines with abiotic surface, and transmits signal by activating the double-deck component system of Cpx; The adventitia loss that NlpE took place in the time of may participating in competent cell and contact with hydrophobic surface, and transmit signal and give the Cpx approach; NlpE also monitors the integrity of coating; Eliminate paraprotein in the pericentral siphon, repair the amino acid in the N hunger; The pressure of induction adventitia, the transmitter kinase c pxA of mediation signal to the cytoplasmic membrane.Though the CutF albumen that the present invention is cloned into has very high homology with NlpE, but much still different to some extent with NlpE in nature, especially from distributing, NlpE is distributed in outer after birth, the effect of mainly taking on the signal conduction, and CutF do not have and significantly strides the film district, and it more resembles a cytoplasmic protein, but how it has participated in the copper resistant of bacterium and still need do further research.
As mentioned above, the copper resistant gene and the encoded protein principal character thereof that the present invention relates to are as follows:
(1) serratia (Serratia sp.) KMR-3 bacterial strain produces.
(2) by 194 amino acid codings, molecular weight is about 22KDa.The highest with Mohs Yersinia (Yersiniamollare tii) ATCC43969 copper resistant lipoprotein NlpE homology, reach 70%.
(3) IPTG induces down, efficiently expresses in intestinal bacteria.
Description of drawings:
Fig. 1 clone's copper resistant gene order and deduced amino acid;
Fig. 2 resistance protein is at expression in escherichia coli figure.
M---albumen Marker wherein; Before 1---BL21 (DE3)-pET28a control strain IPTG induces; After 2---BL21 (DE3)-pET28a control strain IPTG induces; Before 3---BL21 (DE3)-pET28a-Cu1 recombinant bacterial strain IPTG induces; After 4---BL21 (DE3)-pET28a-Cu1 recombinant bacterial strain IPTG induces.
Embodiment:
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with embodiment, but do not limit the present invention with this.
Embodiment 1:
Serratia (Serra tiasp.) KMR-3 bacterial strain copper resistant gene clone and evaluation:
A strain that is separated to the waste water around the smeltery, Kunming has high resistance to cupric ion serratia (Serratia sp.) KMR-3 bacterial strain.This serratia (Serratia sp.) KMR-3 bacterial strain on August 26th, 2008 in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (BeiJing, China) preservation, preserving number is: CGMCC No.2636.
The structure of genome dna library: the extraction (F. Ao Sibai etc., fine works molecular biology experiment guide, Beijing: Science Press, 1999) of the total DNA of serratia (Serratia sp.) KMR-3 strain gene group.With the partially digested genome DNA of restriction enzyme Sau3A I, glue reclaims enzyme and cuts product, is connected with pUC118 BamHI/BAP carrier, connects product Transformed E .coli DH5.Coating contains the LB flat board of penbritin, and the fraction of coverage of this genome dna library reaches 99.9%, is effective library, has successfully made up the genome dna library of this bacterium.Screen recon containing on the selectivity flat board of penbritin and cupric ion.
The evaluation of copper resistant gene: utilize pcr amplification and double digestion method to identify recon.To CaCl
2Cultivate in the LB liquid nutrient medium at the bacterium colony of growing on the selectivity flat board behind the method Transformed E .coli DH5, with vast Tyke test kit extracting plasmid.With M
13(RV/M4) be primer, detect converted product by pcr amplification, primer sequence is as follows: M
13(M
4): 5 '-GTTTTCCCAGTCACGACGTTGTAAA-3 ', M
13(RV): 5 '-ATTTCACACAGGAAACAGCTATGAC-3 '.Further detect and insert pulsating size by double digestion.The pcr amplification result is consistent with the double digestion result, inserts pulsating size and is about 1.2kb.
Screen the gene relevant with copper resistant, behind the extraction plasmid Transformed E .coli, its tolerance to copper has been brought up to 300mg/L by 160mg/L, and therefore the copper resistant gene of being cloned into has the potential of the sewage disposal of being applied to; Also the copper resistant gene clone that the present invention relates to can be applied in the bacterial strain of sewage disposal to other, thereby improve it to the tolerance of copper and then reach the purpose that heavy metal ion are removed in enrichment, conversion etc.
Embodiment 2:
The proteic heterogenous expression of copper resistant:
The copper resistant genetic analysis: order-checking shows copper resistant gene order total length 1,117bp, and the anti-cuproprotein of derivation is encoded by 194 amino acid, is CutF albumen.This proteic reckoning molecular weight is about 22KDa, and theoretical pI value is 5.30.Find that by analyzing this anti-cuproprotein is evenly distributed in outside kytoplasm, plasma membrane, pericentral siphon, adventitia and the born of the same parents.And known bibliographical information shows, the CutF albumen of E.coli is outer membrane lipoprotein, participates in the outflow of copper and transports cupric ion to the dependent enzyme of copper.Utilize Blast (Ver2.2.14) sequence homology comparison to show: itself and Mohs Yersinia (Yersinia mollare tii) ATCC 43969 copper resistant lipoprotein NlpE homologys are the highest, are 70%.
The subclone of copper resistant gene: according to sequencing result, design primer (restriction enzyme site that contains Nde I and Xho I respectively) is by pcr amplification copper resistant gene.The purpose clip size is about 600bp, detects segmental size by 1% agarose gel electrophoresis, and glue reclaims the purpose fragment.The purpose fragment of the copper resistant gene that pET28a plasmid and glue reclaim is used two kinds of restriction enzymes (Nde I and Xho I) to carry out enzyme simultaneously and is cut.Glue reclaims enzyme and cuts product, and uses T
4Dna ligase connects.Connect product transformed into escherichia coli BL21 (DE3) competent cell, correspondingly obtain a series of recombinant escherichia coli strains.Identify recon by pcr amplification and double digestion.Consistent as a result showing: the successful subclone of copper resistant gene is to the pET28a plasmid.The recombination bacillus coli that contains recombinant plasmid pET28a-Cu1 is called e. coli bl21 (DE3)-Cu1 bacterial strain.
The proteic heterogenous expression of copper resistant: will cut recombination bacillus coli BL21 (the DE3)-pET28a-Cu1 bacterial strain of evaluation and e. coli bl21 (DE3)-pET28a that control strain promptly contains the pET28a plasmid by pcr amplification and enzyme, be inoculated in the LB liquid nutrient medium that contains kantlex, 37 ℃, 150rpm cultivates 12h.Bottle is shaken in inoculum size switching with 1%, is cultured to thalline OD
600Reach 0.6-0.8, take out the thalline of 1ml, the centrifugal 5min of 5000rpm abandons supernatant.Adding final concentration in remaining liquid nutrient medium is the IPTG of 1mM, induces 4h for 37 ℃.Carry out centrifugal to the inductive thalline equally.With sterilized water suspend induce preceding and induce after thalline, and add isopyknic 2 * sample-loading buffer simultaneously, mixing, 95 ℃ of thermo-cracking 15min in the centrifugal 5min of 12000rpm, draw the last all product of 101 supernatants as SDS-PAGE.The resolving gel concentration of SDS-PAGE is 12.5%, and concentrated gum concentration is 5%.SDS-PAGE result shows: the albumen of the copper resistant genes encoding of subclone is great expression (Fig. 2) in e. coli bl21 (DE3).
Claims (7)
1, copper resistant gene, its dna sequence dna as shown in Figure 1.
2, copper resistant gene, its encoded protein matter have the described aminoacid sequence of accompanying drawing 1.
3, copper resistant gene as claimed in claim 1 produces gene order total length 1 by husky Bordetella (Serratia sp.) the KMR-3 bacterial strain of thunder, 117bp, the anti-cuproprotein of derivation is encoded by 194 amino acid, is CutF albumen, molecular weight is about 22KDa, and theoretical pI value is 5.30; The highest with Mohs Yersinia (Yersiniamollare tii) ATCC 43969 copper resistant lipoprotein NlpE homologys, reach 70%.
4, the recombinant vectors pUC118-Cu1 that comprises the dna sequence dna of claim 1.
5, the recombinant vectors pET28a-Cu1 that comprises the dna sequence dna of claim 1.
6, recombination bacillus coli BL21 (the DE3)-pET28a-Cu1 that utilizes recombinant vectors pET28a-Cu1 to transform.
7, the preparation method of the described material of claim 1 to 6, extract the chromosomal DNA of serratia marcescens KMR-3 bacterial strain, carry out partially digested with restriction enzyme Sau3A I, glue reclaims enzyme and cuts product, and be connected with pUC118 BamH I/BAP carrier, connect product through ethanol sedimentation, electric Transformed E .coli DH5, coating contains the LB flat board of penbritin, makes up genome dna library; Screen recon containing on the selectivity flat board of penbritin and cupric ion, from recombinant plasmid pUC118-Cu1, isolate the dna fragmentation of cupric resistant gene,, obtain DNA as shown in Figure 1 through digestion with restriction enzyme; Design primer amplification copper resistant gene is connected and transformed into escherichia coli BL21 (DE3) bacterial strain with the pET28a carrier, obtains recombinant plasmid pET28a-Cu1 and recombinant bacterial strain e. coli bl21 (the DE3)-pET28a-Cu1 that contains recombinant expression plasmid.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101717789B (en) * | 2009-12-14 | 2012-06-20 | 昆明理工大学 | Method for preparing culture medium for efficiently producing haematochrome |
CN103937703A (en) * | 2014-03-10 | 2014-07-23 | 赵晗 | Serratia marcescens M7a and application of Serratia marcescens M7a in heavy metal ion removing |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101717789B (en) * | 2009-12-14 | 2012-06-20 | 昆明理工大学 | Method for preparing culture medium for efficiently producing haematochrome |
CN103937703A (en) * | 2014-03-10 | 2014-07-23 | 赵晗 | Serratia marcescens M7a and application of Serratia marcescens M7a in heavy metal ion removing |
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