CN109402032A - A kind of genetic engineering bacterium producing Resuscitation-promoting Factor RpfE and its application - Google Patents
A kind of genetic engineering bacterium producing Resuscitation-promoting Factor RpfE and its application Download PDFInfo
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- CN109402032A CN109402032A CN201811224213.7A CN201811224213A CN109402032A CN 109402032 A CN109402032 A CN 109402032A CN 201811224213 A CN201811224213 A CN 201811224213A CN 109402032 A CN109402032 A CN 109402032A
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
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Abstract
The invention discloses a kind of genetic engineering bacterium for producing Resuscitation-promoting Factor RpfE and its applications.The present invention in host cell by introducing the Resuscitation-promoting Factor rpfE gene of external source and expressing Resuscitation-promoting Factor, so that realizing application project bacterium prepares Resuscitation-promoting Factor RpfE.Resuscitation-promoting Factor RpfE produced can be applied to the exploitation of microbial resources in a dormant state in environmental sample and promote microorganism growth, to improve the degradation efficiency of microorganism to environmental pollutants.
Description
Technical field
The invention belongs to bioengineering fields, and in particular to a kind of production Resuscitation-promoting Factor (Resuscitation
Promoting Factor, Rpf) RpfE genetic engineering bacterium and the side of RpfE albumen is quickly prepared using the genetic engineering bacterium
Method, and in particular to the application of recombinant protein.
Background technique
Resuscitation-promoting Factor (Resuscitation Promoting Factor, Rpf) is by bacterium autocrine or other point
The protein that the one kind secreted can promote suspend mode bacterium to recover and grow under picomolar concentrations.The factor is initially in gamboge coccus
Discovery in (Micrococcus luteus) also has the bacterium of normal growth in addition to the resurrection and growth that can promote suspend mode bacterium
Effect, mechanism may be to take part in intercellular signal transduction.The structural domain prediction of the albumen and nuclear magnetic resonance spectroscopy are shown,
It has a highly conserved structural domain being made of 70 or so amino acid residues, significant to growth promoting function,
That is Rpf spline structure domain.Had found successively in other gram-positive bacterias later it is similar to the effect of this albumen, equally have Rpf
The protein in spline structure domain forms Rpf protein family.
Currently, the research about Rpf albumen focuses primarily upon the context of detection of foodborne bacterial pathogens and doctor in agricultural system
It learns and the latent pathogen discovery in epidemiology field, vaccine development.And rarer research is gone with the visual angle of environmental functional bacterium
Study recovery of the Rpf albumen to microorganism, growth-promoting effect.
Summary of the invention
Present invention solves the problem in that providing a kind of genetic engineering bacterium for producing recombination Resuscitation-promoting Factor RpfE, application
In the growth of promotion microorganism and its degradation to pollutant in the environmental samples such as soil, water body.
The present invention it is specific the technical solution adopted is as follows:
A kind of genetic engineering bacterium producing Resuscitation-promoting Factor RpfE, the genetic engineering bacterium are prokaryotic cells, have packet
Exogenous expression's carrier of the genetic fragment of RpfE containing Resuscitation-promoting Factor；The core of the Resuscitation-promoting Factor RpfE genetic fragment
Nucleotide sequence is as shown in SEQ.ID.NO.1.
Further, the expression vector comprising bacteria resuscitation promotive factor genetic fragment is pEASY Blunt-
rpfE。
The genetic engineering bacterium of production Resuscitation-promoting Factor RpfE, bacterial strain have been preserved in Chinese microorganism strain preservation management
Committee's common micro-organisms center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute, the deposit date is on 08 29th, 2018, deposit number was CGMCC NO.16351.
Another object of the present invention is to provide a kind of recombination RpfE albumen produced by said gene engineering bacteria.
Another object of the present invention is to provide a kind of above-mentioned recombination RpfE albumen to promote microorganism growth or microorganism
Application to the degradation of organic pollutant.
Further, which includes the following steps: the recombination RpfE albumen and the target organic contamination that can degrade
The microorganism of object is added to together in the environmental system containing target organic pollutant.
Further, the microorganism is thermophilic biphenyl Rhodococcus sp TG9.
Further, the organic pollutant is biphenyl (biphenyls, BP).
In the present invention, RpfE is produced by genetic engineering bacterium and promotes microorganism growth or microorganism to organic pollutant
The method of degradation is to be achieved through the following technical solutions:
(1) using the genomic DNA of thermophilic biphenyl Rhodococcus sp TG9 as template, pass through PCR amplification rpfE gene；By PCR product with
Recombinant plasmid is then transferred to greatly by expression vector pEASY-Blunt E1 connection, construction recombination plasmid pEASY-Blunt-rpfE
Enterobacteria obtains genetic engineering bacterium.
(2) the RpfE protein expression strain of TG9 is induced, the Rpf recombinant protein for making its expression obtain TG9 slightly mentions
Liquid.Resulting TG9Rpf protein crude extract is purified using nickel affinity chromatographic column, and is examined with SDS-PAGE and obtains recombination
Albumen determines the size of recombinant protein.
(3) Rpf recombinant protein crude extract is added in TG9 bacterium solution, measures the growth and biphenyl degradation performance change of TG9,
To probe into Rpf albumen to TG9 growth and the influence of biphenyl degradation performance.
Compared with prior art, advantage possessed by the present invention:
(1) since the Resuscitation-promoting Factor content of thermophilic biphenyl Rhodococcus sp itself secretion is low, it is difficult to extract, it is unfavorable for straight
It connects using thermophilic biphenyl Rhodococcus sp and produces Resuscitation-promoting Factor.Resuscitation-promoting Factor gene is transferred to by the present invention can be quickly numerous
It grows, in the simple escherichia coli host of condition of culture, can effectively solve these problems.
(2) the engineering bacterium expression product that the present invention constructs is intracellular soluble protein, passes through cell ultrasonication, nickel parent
After column purification and dialysis treatment, the target protein of high-purity can be obtained.
(3) using recombination RpfE albumen produced by the invention, for promoting, microorganism grows and microorganism is to organic pollutant
Degradation application, this to enhancing microorganism in environment remediation field using significant.
Detailed description of the invention
Fig. 1 PCR amplification, which is detected from the rpfE gene of thermophilic biphenyl Rhodococcus sp TG9 through 1% agarose gel electrophoresis, to be schemed.M
Swimming lane is molecular weight standard, and 1-7 swimming lane is the parallel loading of rpfE gene PCR product, and 8 swimming lanes are sterile water negative control.
The schematic diagram of Fig. 2 recombinant plasmid pEASY Blunt-rpfE.
Fig. 3 recombinates RpfE albumen through 15%SDA-PAGE analysis chart.M swimming lane is protein molecular weight standard；1st, 2 swimming lane
For protein crude extract；3rd, 4 swimming lane is ni-sepharose purification component (recombination RPF albumen).
Fig. 4 RpfE recombinant protein influences thalli growth.
Facilitation of Fig. 5 RpfE recombinant protein to TG9 degradation biphenyl.
Biomaterial preservation
Produce Resuscitation-promoting Factor RpfE genetic engineering bacterium, it is proposed that classification naming be escherichia coli
Escherichia coli, bacterial strain are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation
Location is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and Institute of Microorganism, Academia Sinica, the deposit date is 08 month 2018
29, deposit number was CGMCC NO.16351.
Specific embodiment
Method used in following examples is conventional method unless otherwise specified.
Thermophilic biphenyl Rhodococcus sp TG9 employed in the following embodiments of the present invention has been preserved in Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro- life of the Chinese Academy of Sciences
Object research institute, the deposit date is on October 08th, 2014, deposit number was CGMCC No.1.12975, hereinafter referred to as TG9.
The present invention is described in further detail with reference to the accompanying drawing, described to be explanation of the invention rather than limit
It is fixed.
Embodiment 1: the clone of Resuscitation-promoting Factor gene and construction of genetic engineering
(1) culture of thermophilic biphenyl Rhodococcus sp Rhodococcus biphenylivorans TG9 and its genomic DNA mention
It takes
TG9 is seeded in LB culture medium, 30 DEG C of resurrection cultures.
LB culture medium prescription: it is molten that distilled water 1000mL is added in 10.0g tryptone, 5.0g yeast extract, 10.0g sodium chloride
Solution adjusts pH=7, autoclave sterilization.
Using gram-positive bacteria genome DNA extracting reagent kit carry out extracting genome DNA, by mentioned DNA solution in-
20 DEG C freeze.
(2) clone of Resuscitation-promoting Factor gene and engineered strain building
Contain Resuscitation-promoting Factor encoding gene rpfE in thermophilic biphenyl Rhodococcus sp TG9 genome, nucleotide sequence is such as
Shown in SEQ.ID.NO.1.RpfE gene is to expand to obtain from the genomic DNA of above-mentioned bacterial strains by PCR method.According to rpfE
The sequence design upstream and downstream primer of gene, the synthesis of commission Shanghai Sheng Gong bio-engineering corporation.
Upstream primer P1:5 '-ATGGCAAAGTTCACGATCAAGC-3 '；
Downstream primer P2:5 '-TCAGAGGTACTGACCGCAGACC-3 '.
PCR reaction system are as follows: 2 × TransStart FastPfu PCR SuperMix 12.5 μ L, 2 μ L of DNA profiling,
Each 0.5 μ L of P1, P2 primer, adds water to 25 μ L.
PCR reaction condition: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, annealing temperature and time is respectively 50.5 DEG C,
30s, elongating temperature and time are respectively 72 DEG C, 30s, are recycled 30 times；72 DEG C of extension 5min.And pass through 1% Ago-Gel
Electrophoresis detection PCR product.Molecular weight and expected (330bp) are consistent (see Fig. 1；2nd swimming lane).
PCR product is connect with pEASY-Blunt E1Expression Vector, connection product is transferred to Escherichia coli
In Trans1-T1 competent cell, it is coated on 37 DEG C of overnight incubations on the LB plate containing 100 μ g/mL ampicillins, picking sun
Property clone.
The positive colony filtered out is subjected to sequencing analysis, sequencing result and known sequence are completely the same, it was demonstrated that
The recombinant plasmid containing Resuscitation-promoting Factor gene has been arrived, pEASY-Blunt-rpfE is named as.
Embodiment 2: Resuscitation-promoting Factor construction of genetic engineering and its expression, purifying
Recombinant plasmid is extracted from the clone strain of embodiment 1 using plasmid extraction kit, mentioned recombinant plasmid is turned
Enter in e. coli bl21 (DE3), is coated on 37 DEG C of overnight incubations on the LB plate containing 100 μ g/mL ampicillins, picking sun
Property clone, 37 DEG C of shaken cultivations are stayed overnight in the LB culture medium of 100 μ g/mL ampicillin of Yu Han.Pass through PCR amplification and agarose
Gel electrophoresis verifying expression strain construction success.Bacterial strain preservation is commonly micro- to China Committee for Culture Collection of Microorganisms
Bio-Centers, the i.e. genetic engineering bacterium of the production Resuscitation-promoting Factor RpfE of deposit number CGMCC NO.16351.
The bacterium solution being proved to be successful is seeded in the culture medium of the identical resistance of 200mL by 1% inoculum concentration, 37 DEG C of shaken cultivations
Isopropylthio β-D- the galactoside (IPTG) of 100 μ L 50mg/mL, 20 DEG C of 100rpm trainings are added until bacterium solution is muddy in 2-3h
It supports overnight, it is induced to express Rpf albumen.Cultured bacterium solution is sub-packed in centrifuge tube, thalline were collected by centrifugation.
Precipitating is reset to suspension with 25mM Tris-HCl solution, is collected in the centrifuge tube of ice bath；Ultrasonication
45min.(12000rpm, 20min) will be centrifuged by liquid after ultrasonication, collect supernatant (albumen containing Rpf) and obtain RpfE egg
White crude extract.
The recombinant protein in RpfE protein crude extract is purified using nickel affinity column, with the elution of 100mM imidazoles and column
Expect the destination protein combined, elution volume 5mL.Eluent is added in the bag filter after distilled water boils cleaning, in
In 25mM Tris-HCl solution, it is stirred overnight.Followed by the purity of SDS-PAGE electrophoresis detection target protein, as a result such as Fig. 3
It is shown: the 3rd, 4 swimming lane visible uniform protein band near 12kDa, it is consistent with theoretical estimated molecular weight.
Embodiment 3: Resuscitation-promoting Factor is grown to microorganism and the facilitation of organic matter degradation performance
(1) preparation of TG9 bacteria suspension
TG9 is seeded in LB culture solution, 30 DEG C of shaken cultivations are used as seed culture fluid afterwards for 24 hours.Seed liquor is pressed 1%
(v/v) inoculum concentration is seeded in the LB liquid medium of certain volume, 30 DEG C of shaken cultivations to logarithmic growth phase (OD600=
0.98) it after, taking bacterium solution centrifugation at this time, is washed twice with 0.85% sterile saline, the inorganic salts culture solution of sterilizing is added, adjusted
Save Fungal biodiversity OD600=1.00 or so, obtain TG9 bacteria suspension.
Minimal medium formula: KH2PO41g, K2HPO4·3H2O 3g, MgSO40.2g, FeSO4·7H2O 0.02g,
NaCl 1g, (NH4)2SO40.5g, CaCl20.01g, Trace salts solution (mg/L:MoO34mg, ZnSO4·5H2O 28mg,
CuSO4·5H2O 0.02g, H3BO34mg, MnSO4·5H2O 4mg, CoCl2·6H2O 4mg) 1mL, add distilled water 1000mL molten
Solution adjusts pH=7.3, autoclave sterilization.
(2) facilitation of the Resuscitation-promoting Factor to TG9 growth and biphenyl degradation ability
The BP solution that 1mL concentration is 25g/L is added into 50mL glass centrifuge tube, is volatilized completely to solvent (n-hexane)
Afterwards, the TG9 bacteria suspension of 5mL preparation is added, makes BP concentration 5000mg/L in system.And a certain amount of embodiment is added as requested
The RpfE protein crude extract (20%, v/v) prepared in 2, specific additive amount are shown in Table 1.One layer of aluminium-foil paper is covered in glass cap,
Cover tightly bottle cap, 30 DEG C, 180r/min constant-temperature table tested.This experiment is sampled since 0 moment, primary every 12h sampling,
Total sampling 6 times.Each experimental group all has 24 samples, and each sampling moment all has four in parallel.
Table 1 probes into the experimental design that RpfE crude extract influences bacterial strain TG9
Note: "-" expression is not added；" inactivation " indicates the high pressure sterilization 20min at 121 DEG C.
Since the solubility of BP in water is smaller, therefore this experiment is using destructive sampling.When every sub-sampling, sample is first measured
OD600, line chart is drawn according to test result, as a result as shown in Figure 4: the thalli growth situation of Rpf experimental group is better than inactivation
TG9 control group and Tris-HCl control group.This shows that recombination RpfE albumen of the invention can promote thalli growth.Then to from
The HCl that 100 μ L concentration are 2mol/L is added in heart pipe, is then placed into 4 DEG C of refrigerators, waits the extraction and measurement of BP.It utilizes
GC-MS measures the BP concentration of sample, and calculates its biphenyl degradation rate, histogram is drawn according to calculated result, as a result such as Fig. 5 institute
Show: the biphenyl degradation rate of Rpf experimental group is significantly higher than other control groups.These results illustrate that Rpf promotes the growth of TG9, from
And promote degradation of the TG9 to biphenyl, therefore the recombination RpfE albumen protected of the present invention can be used for promoting microorganism to grow and micro-
Degradation of the biology to organic pollutant.
Sequence table
<110>Zhejiang University
<120>a kind of genetic engineering bacterium for producing Resuscitation-promoting Factor RpfE and its application
<141> 2018-10-18
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 330
<212> DNA
<213>thermophilic biphenyl Rhodococcus sp TG9 (Rhodococcus biphenylivorans)
<400> 1
atggcaaagt tcacgatcaa gcgcgccctg ggcaccctcg ctgcctccac tgcactcgtt 60
gcgaccccgt tcgctctggg tgccggtacc gccaatgccg catccggtaa ctgggatgcc 120
gtcgcacagt gcgagagcgg tggtaactgg gcgatcaaca ccggtaacgg ctattacggc 180
ggtctgcagt tctcgcagag cacctgggag gcctacggtg gctcgggaac cgcgaacaac 240
gcgtcgcgcg aggaacagat ccgcgtcgcc gagaacgtcc tcgccggaca gggcgtcggt 300
gcatggccgg tctgcggtca gtacctctga 330
Claims (7)
1. a kind of genetic engineering bacterium for producing Resuscitation-promoting Factor RpfE, which is characterized in that the genetic engineering bacterium is prokaryotic cell,
It has exogenous expression's carrier comprising Resuscitation-promoting Factor RpfE genetic fragment；The Resuscitation-promoting Factor RpfE gene
The nucleotide sequence of segment is as shown in SEQ.ID.NO.1.
2. a kind of genetic engineering bacterium for producing Resuscitation-promoting Factor RpfE, which is characterized in that bacterial strain is preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese section
Institute of microbiology, institute, the deposit date is on 08 29th, 2018, deposit number was CGMCC NO.16351.
3. a kind of recombination RpfE albumen produced by genetic engineering bacterium as claimed in claim 1 or 2.
4. a kind of recombination RpfE albumen as claimed in claim 3 promote microorganism growth or microorganism to the drop of organic pollutant
The application of solution.
5. application as claimed in claim 4, which comprises the steps of: by the recombination RpfE albumen and can
The microorganism of degradation target organic pollutant, is added to together in the environmental system containing target organic pollutant.
6. applying according to claim 4, which is characterized in that the microorganism is thermophilic biphenyl Rhodococcus sp TG9.
7. applying according to claim 4, which is characterized in that the organic pollutant is biphenyl.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109868244A (en) * | 2019-03-26 | 2019-06-11 | 湖北大学 | A kind of phenolic comp ' ds pollution degradation bacteria and its application |
| CN109970183A (en) * | 2019-03-31 | 2019-07-05 | 浙江大学 | The application and processing method of a kind of Resuscitation-promoting Factor in treatment of dyeing wastewater |
| CN114181876A (en) * | 2021-12-01 | 2022-03-15 | 广西民族大学 | Genetic engineering bacterium for producing resuscitation promoting factor RpfB and application thereof |
-
2018
- 2018-10-19 CN CN201811224213.7A patent/CN109402032B/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109868244A (en) * | 2019-03-26 | 2019-06-11 | 湖北大学 | A kind of phenolic comp ' ds pollution degradation bacteria and its application |
| CN109868244B (en) * | 2019-03-26 | 2021-11-02 | 湖北大学 | Phenol pollutant degrading bacterium and application thereof |
| CN109970183A (en) * | 2019-03-31 | 2019-07-05 | 浙江大学 | The application and processing method of a kind of Resuscitation-promoting Factor in treatment of dyeing wastewater |
| CN109970183B (en) * | 2019-03-31 | 2021-01-15 | 浙江大学 | Application of resuscitation promoting factor in treatment of printing and dyeing wastewater and treatment method |
| CN114181876A (en) * | 2021-12-01 | 2022-03-15 | 广西民族大学 | Genetic engineering bacterium for producing resuscitation promoting factor RpfB and application thereof |
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