CN108588093A - Bombina orientalis enteron aisle bacillus, which expresses albumen, has lactating cow excrement the NESS1-A genes of strong deodorizing capability - Google Patents
Bombina orientalis enteron aisle bacillus, which expresses albumen, has lactating cow excrement the NESS1-A genes of strong deodorizing capability Download PDFInfo
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Abstract
Bombina orientalis enteron aisle bacillus, which expresses albumen, has lactating cow excrement the NESS1 A genes of strong deodorizing capability, belong to Animal Biotechnology field, it is characterised in that Bombina orientalis enteron aisle bacillus expresses the nucleotide sequence such as Seq ID No that albumen has lactating cow excrement the NESS1 A genes of strong deodorizing capability：Shown in 1, the lactating cow fresh excreta for being mixed with testing sample solution is sub-packed in 24 porocyte culture plates that 3 have sterilized, 37 DEG C of cultures in illumination box, every 1 hour, take out the mixture in corresponding number, then ammonia nitrogen determination instrument is used to measure the ammonia-nitrogen content in mixture, deodorizing capability of the calculation expression albumen to lactating cow excrement, as a result after 7 hours, ammonia-nitrogen content is zero in mixture, it is seen that the expression albumen of Bombina orientalis enteron aisle bacillus NESS1 A genes has stronger deodorizing capability to lactating cow excrement.
Description
Technical field
The present invention relates to a kind of Bombina orientalis enteron aisle bacillus expression albumen to have strong deodorization energy to lactating cow excrement
The NESS1-A genes of power, belong to Animal Biotechnology field.
Background technology
With the fast development of cattle-raising, fecal pollution has become a great problem, and the excrement that a cow head generates every year is 7
Ton or more.Since various regions are inadequate to the processing most attention of cow dung, the place of concentration is compared in some cowboying, is substantially all no ox
Excrement treatment facility, this results in cow dung to stack everywhere, and especially to summer, stinking smell assaulting one's nostrils, is both made to the normal life of surrounding resident
At harmful effect, meanwhile, cow dung is the source that various bacteria pathogen multiplies breeding again, has serious shadow to cultured population
It rings.In addition, raw cow dung Shangdi, generates heat, oxygen in soil is consumed, causes to burn root burn seedlings, also to the ovum of parasite, the micro- life of cause of disease
Object plays dissemination.Therefore, cow dung processing seems particularly significant.Bombina orientalis (Bombina orientalis) is bell toad
Section, genus bombina Amphibia vertebrate, the long 36-48mm of body is flat, and kiss circle, forward and backward limb is short, pachylosis, pierces black,
Back is in taupe brown, or the back of the body is miscellaneous with irregular black splotch for green, and the outside of belly has piebald, is black and vermilion or orange
Color.Bombina orientalis be carnivorous species, can whole day predation, food include ant, ichneumon wasp, mosquitos and flies class, lepidopterous larvae, stinkbug class,
The higher animal of the protein contents such as beetle, spider, river snail, still, the excrement of Bombina orientalis is almost without stink, wherein bud
Spore bacillus has played important function.How the bacillus that ammonia nitrogen in excrement can be handled is searched out in Bombina orientalis excrement,
Finding expression albumen there is the gene of strong deodorizing capability to become a great problem for being badly in need of solving lactating cow excrementSo hair
Bright Bombina orientalis enteron aisle bacillus expression albumen has lactating cow excrement the NESS1-A genes of strong deodorizing capability, finds
There is the NESS1-A genes of strong deodorizing capability to be necessary lactating cow excrement to an expression albumen.
Invention content
The bacillus that can handle ammonia nitrogen in excrement how is searched out in Bombina orientalis excrement in order to overcome, and finds table
There is the problem of the gene of strong deodorizing capability up to albumen to lactating cow excrement, the present invention provides a Bombina orientalis enteron aisle buds
Spore bacillus, which expresses albumen, has lactating cow excrement the NESS1-A genes of strong deodorizing capability, and experiment is placed in light with mixture
According to 37 DEG C of cultures in incubator, every 1 hour, the mixture in corresponding number is taken out, then uses ammonia nitrogen determination instrument to measure mixed
Close the ammonia-nitrogen content in object, deodorizing capability of the calculation expression albumen to lactating cow excrement；As a result it is found that after 7 hours, mixing
Ammonia-nitrogen content is zero in object, i.e. the expression albumen of the gene all disposes the ammonia nitrogen in lactating cow excrement, it is seen that east
The expression albumen of bell toad enteron aisle bacillus NESS1-A genes has stronger deodorizing capability to lactating cow excrement.
The technical solution adopted by the present invention to solve the technical problems is：
Bombina orientalis enteron aisle bacillus, which expresses albumen, has lactating cow excrement the NESS1-A genes of strong deodorizing capability
Nucleic acid sequence be Seq ID No：1, specific structure and verification process are as follows：
1. the PCR amplification of gene：The gemma that will be extracted from the Bombina orientalis enteron aisle that the Heilongjiang Province of China mountains Mao Er are arrested
Bacillus (Bacillus sp.) genomic DNA carries out PCR amplification using Ness-5/Ness-3 primer pairs, and primer sequence is shown in Table 1.
With reference to the N-terminal and C-terminal sequence homology of the gene coding regions NESS1-A announced in GenBank, a pair of of specificity of design synthesis is drawn
Object NESS5/NESS3 introduces NdeI and SalI restriction enzyme sites (shown in arrow), for expanding full-length gene at 5 ' ends respectively.Root
According to different loci design synthesis tetra- primers of NESS51, NESS52, NESS31, NESS32 of full-length gene, introduced at 5 ' ends
BamHI and SalI restriction enzyme sites (shown in arrow), for building missing gene.
2.PCR reaction systems：50 μ L of KOD polymeric enzyme reactions system；1 μ L of KOD enzymes；1 μ L of primer pair (10mmol/L)；Template
1μL；10xPCR Buffer 5μL；MgSO43μL；dNTP 5μL；ddH2O complements to 50 μ L；
3.PCR reaction conditions (table 2)
4.DNA is recycled：(1) gel containing target DNA fragment is cut using sterilizing scalpel, be placed in 1.5mL centrifugations；
(2) the colloidal sol buffer solution A of 3 times of volume colloidal sol is added, is placed in 10min or so in 50 DEG C of water-baths；(3) add after glue is completely dissolved
Enter the B solution of 0.5 A colloidal sol buffer solution volume；(4) recovery column is transferred to after mixing, 12000r/min centrifuges 1min；(5) it removes
Elution buffer W is added in residual liquid1500 μ L, 12000r/min centrifuge 1min；(6) residual liquid, elution buffer are removed
W2700 μ L, elution is twice；(7) the TE solution of 30 μ L is added in recovery column, is stored at room temperature 2min；(8) by upper step recovery column
12000r/min centrifuges 1min, spare.It such as needs that 7-8 steps can be repeated primary.
5. connection reaction：By DNA glue recovery product and pEB carriers according to linked system (4 μ L of target fragment DNA, carrier
5 μ L of 1 μ L of DNA, connection kit Solution I) it is attached reaction.After mixing well, 4h or 4 DEG C of condition of 16 DEG C of connections
Lower connection is overnight.
6. Escherichia coli heat shock converts：Connection product full dose is added to the competent cell of 100 μ L e. coli jm109s
In, mixing, ice bath 30min or more takes out centrifuge tube, 42 DEG C of accurate thermal shock 90s, then ice bath 3min, and 900 μ L LB liquid are added
37 DEG C of culture medium culture 1h or so, takes 200 μ L to be coated in LB solid plates, is added corresponding antibiotic, 40 μ L of X-gal solution,
4 μ L of IPTG solution, 37 DEG C of overnight incubations carry out blue day shift screening, and screening outgoing direction is connected correct positive colony, carried with PEB
The forward primer pEBF of body and the reverse primer of institute's clone gene carry out PCR identifications.The plasmid for extracting positive colony bacterial strain, is transferred to
In Escherichia coli Rosetta bacterial strain competent cells.Again by sequencing after the identifications such as PCR, digestion, what is identified is correct
Recombinant conversion carries out induction and express express target protein.
7. e. coli plasmid dna extracts：(1) picking positive transformant overnight incubation in LB liquid medium, takes 1-
4ml bacterium solutions centrifuge 1min in 12000r/min, discard supernatant；(2) S that 250 μ L contain 50mg/mlRNANase is added1Solution is outstanding
It drifts along shallow lake；(3) 250 μ L bacterial lysates S2 are added and fully slowly leniently spin upside down 4-6 times, until solution transparent clear
Until, the step for should not be more than 5 minutes；(4) 350 μ L S are added3Neutralizer is simultaneously fully leniently spun upside down 6-8 times,
It is centrifuged 10 minutes under 12000r/min；(5) supernatant is then drawn, is then transferred into and prepares in pipe, under the conditions of 12000r/min
It centrifuges 1 minute and discards filtrate；(6) 500 μ L cleaning solutions W are then added1, centrifuge 1 minute and discard under the conditions of 12000r/min
Filtrate；(7) 700 μ L, W are then added2, excess salt is washed away, centrifuges 1 minute under the conditions of 12000r/min, is repeated once later；
(8) column will be collected to be transferred on new 1.5ml centrifuge tubes, collect column center be added dropwise 60-80 μ L be preheated to 65 DEG C TE buffer solutions or
Aqua sterilisa static 5 minutes, centrifuges 1 minute under the conditions of 12000r/min, air-dry and be stored in -20 DEG C it is spare.
8. the induced expression of gene：(1) single bacterium colony of Escherichia coli is inoculated under the conditions of 37 DEG C of LB liquid medium
220rpm activates 12-16h；(2) 1% inoculum concentration is used to be inoculated with positive expression bacterial strain in the conical flask of 200mL LB liquid mediums
In, under the conditions of 37 DEG C, 220rpm shaken cultivations about 2h to OD600=0.6 or so；The 200 μ L of IPTG of 1M are added later；It places
Induce about 4-16h, expression bacterial strain different condition also different under 150rpm low speed culture on shaking table, 16-30 DEG C of cryogenic conditions；
(3) culture solution of induction is centrifuged to 5min under conditions of 6000rpm and collects thalline, with the 10mmol/L Tris of precooling
Cl (pH is adjusted to 8.0 or so) suspension thalline 2-3 times；(4) ultrasonic disruption thalline in mixture of ice and water, super 3s stop 3s, 50ml
Suspension bacteria liquid about ultrasound 10min；(5) centrifugation 10min collects supernatant precipitation respectively under the conditions of 4 DEG C and 12000rpm, sinks
Shallow lake component still uses 10mmol/L TrisCl to suspend.It is SDS-PAGE to carry out protein electrophoresis detection respectively later.
9. protein electrophoresis detects：Electrophoresis：Crude protein sample and 3x loadings Buffer are pressed into 2: 1 mixings, boiling water bath processing
10min, taking analyte sample fluid 10, (120V prerunning 10-15min are arranged in L loadings, electrophoresis apparatus, then carry out 150V constant pressure electrophoresis
1h.Dyeing：Protein gel is taken out after electrophoresis, is put into 50mLSI solution, and microwave stove heat 30s, 60rpm oscillation is set
5min；It is put into after glue is taken out in the mixed liquor of SII and SIII and (adds 200 (L SIII), then microwave stove heat per 50mL SII
Shaking table 60rpm is taken out after 30s vibrates, gel imaging system imaging high-visible to protein adhesive band.
10. determination and analysis of sequence：Sequencing is completed by LC SCIENCES Chinese companies of the U.S., uses Clone
The softwares analytical sequence such as Manager, Omiga, NCBI BLAST, DNAMAN, result are Seq ID No：1.
11. express albumen has strong deodorizing capability determination of activity to lactating cow excrement
It weighs 30g lactating cow fresh excretas to set in sterilizes culture dish, 3mL testing sample solutions is added, it is abundant with spoon
It stirs evenly, is placed at room temperature for.It is thin that the lactating cow fresh excreta for being mixed with testing sample solution is sub-packed in 24 holes that 3 have sterilized
In born of the same parents' culture plate, each hole is numbered, number is 1-24 respectively, and 0.2g mixtures are put into per hole.By 3 24 hole cell trainings
Foster plate is placed in 37 DEG C of cultures in illumination box, every 1 hour, takes out the mixture in corresponding number, is then surveyed with ammonia nitrogen
Determine the ammonia-nitrogen content in instrument measurement mixture, deodorizing capability of the calculation expression albumen to lactating cow excrement.By 7 hours places
Reason, substantially can dispose the ammonia nitrogen in mixture, the deodorizing capability of mixture is stronger.
Beneficial effects of the present invention are：Bombina orientalis enteron aisle bacillus expression albumen of the present invention has lactating cow excrement
The lactating cow fresh excreta for being mixed with testing sample solution is sub-packed in 3 and sterilized by the NESS1-A genes for having strong deodorizing capability
24 porocyte culture plates in, each hole is numbered, number is 1-24 respectively, is put into 0.2g mixtures per hole.By 3 24
Porocyte culture plates are placed in 37 DEG C of cultures in illumination box, every 1 hour, take out the mixture in corresponding number, then
The ammonia-nitrogen content in mixture, deodorizing capability of the calculation expression albumen to lactating cow excrement are measured with ammonia nitrogen determination instrument；As a result
It is found that after 7 hours, ammonia-nitrogen content is zero in mixture, i.e. the expression albumen of the gene is by the ammonia nitrogen in lactating cow excrement
All dispose, it is seen that the expression albumen of Bombina orientalis enteron aisle bacillus NESS1-A genes to lactating cow excrement have compared with
Strong deodorizing capability.
Description of the drawings
The following further describes the present invention with reference to the drawings.
Fig. 1 is that Bombina orientalis enteron aisle bacillus expresses albumen to NESS1- of the lactating cow excrement with strong deodorizing capability
Nucleic acid sequence (the Seq ID No of A genes：1).
Specific implementation mode
Embodiment one
1. the PCR amplification of gene：
Bacillus (the Bacillus that will be extracted from the Bombina orientalis enteron aisle that the Heilongjiang Province of China mountains Mao Er are arrested
Sp.) subtilis genomic dna extracted carries out PCR amplification using Ness-5/Ness-3 primer pairs, and primer sequence is shown in Table 1.
With reference to the N-terminal and C-terminal sequence homology of the gene coding regions Ness3A announced in GenBank, a pair of of specificity of design synthesis is drawn
Object NESS5/NESS3 introduces NdeI and SalI restriction enzyme sites (shown in arrow), for expanding full-length gene at 5 ' ends respectively.Root
According to different loci design synthesis tetra- primers of NESS51, NESS52, NESS31, NESS32 of full-length gene, introduced at 5 ' ends
BamHI and SalI restriction enzyme sites (shown in arrow), for building missing gene.
Table 1：Primer sequence
2.PCR reaction systems
3.PCR reaction conditions
Table 2：PCR reaction conditions
4.DNA is recycled
(1) gel containing target DNA fragment is cut using sterilizing scalpel, be placed in 1.5mL centrifuge tubes；
(2) the colloidal sol buffer solution A of 3 times of volume colloidal sol is added, is placed in water-bath 10min or so in 50 DEG C of water-baths；
(3) B solution of 0.5 A colloidal sol buffer solution volume is added after colloidal sol is completely dissolved；
(4) recovery column is transferred to after mixing, 12000r/min centrifuges 1min；
(5) residual liquid is removed, elution buffer W is added1500 μ L, 12000r/min centrifuge 1min；
(6) residual liquid, elution buffer W are removed2700 μ L, elution is twice；
(7) the TE solution of 30 μ L is added in recovery column, is stored at room temperature 2min；
(8) upper step recovery column 12000r/min is centrifuged into 1min, it is spare.It such as needs that 7-8 steps can be repeated primary.
5. connection reaction
DNA glue recovery product is attached with pEB carriers according to following linked system and is reacted.After mixing well, 16 DEG C
It is connected overnight under the conditions of 4h or 4 DEG C of connection.
4 μ L of target fragment DNA
1 μ L of carrier DNA
Connect 5 μ L of kit Solution I
6. Escherichia coli heat shock converts
Connection product full dose is added in the competent cell of 100 μ L e. coli jm109s, mixing, ice bath 30min with
On, centrifuge tube, 42 DEG C of accurate thermal shock 90s, then ice bath 3min are taken out, it is left that 900 μ L LB liquid mediums, 37 DEG C of culture 1h are added
The right side takes 200 μ L to be coated in LB solid plates, and corresponding antibiotic, 40 μ L, IPTG solution of X-gal solution, 4 μ L, 37 DEG C of trainings are added
Support overnight, carry out blue day shift screening, screening outgoing direction connects correct positive colony, with the forward primer pEBF of PEB carriers and
The reverse primer of institute's clone gene carries out PCR identifications.The plasmid for extracting positive colony bacterial strain, is transferred to Escherichia coli Rosetta bacterium
In strain competent cell.Again by sequencing after the identifications such as PCR, digestion, correct recombinant conversion identified is lured
It leads and express express target protein.
7. e. coli plasmid dna extracts
(1) picking positive transformant overnight incubation in LB liquid medium takes 1-4ml bacterium solutions to be centrifuged in 12000r/min
1min is discarded supernatant；
(2) S that 250 μ L contain 50mg/mlRNANase is added1Solution suspension precipitates；
(3) 250 μ L bacterial lysates S are added2And fully slowly leniently spin upside down 4-6 times, until solution is transparent clear
Until bright, the step for should not be more than 5 minutes；
(4) 350 μ L S are added3Neutralizer is simultaneously fully leniently spun upside down 6-8 times, and 10 points are centrifuged under 12000r/min
Clock；
(5) supernatant is then drawn, is then transferred into and prepares in pipe, centrifuged 1 minute under the conditions of 12000r/min and is discarded
Filtrate；
(6) 500 μ L cleaning solutions W are then added1, centrifuge 1 minute under the conditions of 12000r/min and discard filtrate；
(7) 700 μ L, W are then added2, excess salt is washed away, centrifuges 1 minute under the conditions of 12000r/min, later repeatedly one
It is secondary；
(8) column will be collected to be transferred on new 1.5ml centrifuge tubes, collects column center and the TE that 60-80 μ L are preheated to 65 DEG C is added dropwise
Buffer solution or aqua sterilisa static 5 minutes, centrifuge 1 minute under the conditions of 12000r/min, air-dry and be stored in -20 DEG C it is spare.
8. the induced expression of gene
(1) 220rpm activates 12-16h under the conditions of the single bacterium colony of Escherichia coli being inoculated in 37 DEG C of LB liquid medium；
(2) 1% inoculum concentration is used to be inoculated with positive expression bacterial strain in the conical flask of 200mL LB liquid mediums, 37 DEG C of items
Under part, 220rpm shaken cultivations about 2h to OD600=0.6 or so；The IPTG200 μ L of 1M are added later；It is positioned on shaking table
150rpm low speed cultures induce about 4-16h, strain different condition also different under 16-30 DEG C of cryogenic conditions；
(3) culture solution of induction is centrifuged to 5min under conditions of 6000rpm and collects thalline, with the 10mmol/L of precooling
TrisCl (pH is adjusted to 8.0 or so) suspension thalline 2-3 times；
(4) ultrasonic disruption thalline in mixture of ice and water, super 3s stop 3s, 50ml suspension bacteria liquids about ultrasound 10min；
(5) centrifugation 10min collects supernatant precipitation respectively under the conditions of 4 DEG C and 12000rpm, and deposited components are still used
10mmol/L TrisCl suspend.It is SDS-PAGE to carry out protein electrophoresis detection respectively later.
9. protein electrophoresis detects
Electrophoresis：Crude protein sample and 3x loadings Buffer are pressed into 2: 1 mixings, boiling water bath handles 10min, takes analyte sample fluid
10 (120V prerunning 10-15min are arranged in mL loadings, electrophoresis apparatus, then carry out 150V constant pressure electrophoresis 1h.
Dyeing：Protein gel is taken out after electrophoresis, is put into 50mLSI solution, microwave stove heat 30s is set, and 60rpm shakes
Swing 5min；It is put into after glue is taken out in the mixed liquor of SII and SIII and (adds 200 (L SIII), then microwave stove heat per 50mL SII
Shaking table 60rpm is taken out after 30s vibrates, gel imaging system imaging high-visible to protein adhesive band.
3 SDS polyacrylamide gels of table prepare table
10. determination and analysis of sequence
Sequencing is completed by LC SCIENCES Chinese companies of the U.S., with Clone Manager, Omiga, NCBI
The softwares analytical sequence such as BLAST, DNAMAN, result are Seq ID No：1.
11. express albumen has strong deodorizing capability determination of activity to lactating cow excrement
It weighs 30g lactating cow fresh excretas to set in sterilizes culture dish, 3mL testing sample solutions is added, it is abundant with spoon
It stirs evenly, is placed at room temperature for.It is thin that the lactating cow fresh excreta for being mixed with testing sample solution is sub-packed in 24 holes that 3 have sterilized
In born of the same parents' culture plate, each hole is numbered, number is 1-24 respectively, and 0.2g mixtures are put into per hole.By 3 24 hole cell trainings
Foster plate is placed in 37 DEG C of cultures in illumination box, every 1 hour, takes out the mixture in corresponding number, is then surveyed with ammonia nitrogen
Determine the ammonia-nitrogen content in instrument measurement mixture, deodorizing capability of the calculation expression albumen to lactating cow excrement.By 7 hours places
Reason, substantially can dispose the ammonia nitrogen in mixture, the deodorizing capability of mixture is stronger.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it will be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this hairs
Bright principle, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these variations
It all fall within the protetion scope of the claimed invention with improvement, its is equivalent by appended claims for the claimed scope of the invention
Object defines.
Claims (1)
1. Bombina orientalis enteron aisle bacillus, which expresses albumen, has lactating cow excrement the NESS1-A genes of strong deodorizing capability,
It is characterized in that Bombina orientalis enteron aisle bacillus, which expresses albumen, has lactating cow excrement in the NESS1-A bases of strong deodorizing capability
The nucleotide sequence of cause such as Seq ID No：Shown in 1.
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| CN201810358520.8A CN108588093A (en) | 2018-04-11 | 2018-04-11 | Bombina orientalis enteron aisle bacillus, which expresses albumen, has lactating cow excrement the NESS1-A genes of strong deodorizing capability |
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| CN201810358520.8A CN108588093A (en) | 2018-04-11 | 2018-04-11 | Bombina orientalis enteron aisle bacillus, which expresses albumen, has lactating cow excrement the NESS1-A genes of strong deodorizing capability |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1255063A (en) * | 1997-03-06 | 2000-05-31 | 综合医院有限公司 | Photosensitizer conjugates for pathogen targeting |
| US20090047546A1 (en) * | 2007-08-17 | 2009-02-19 | Bowman Mark P | Process for forming multilayer coating with radiation curable polyene/polythiol coating compositions |
| CN104152375A (en) * | 2014-07-18 | 2014-11-19 | 齐齐哈尔大学 | Deamination and deodorization bacterial strain QDN01 and application thereof in biological deodorization |
| CN104878026A (en) * | 2015-06-18 | 2015-09-02 | 浙江师范大学 | Application of LRK2 gene in improvement of drought resisting capacity of rice |
-
2018
- 2018-04-11 CN CN201810358520.8A patent/CN108588093A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1255063A (en) * | 1997-03-06 | 2000-05-31 | 综合医院有限公司 | Photosensitizer conjugates for pathogen targeting |
| US20090047546A1 (en) * | 2007-08-17 | 2009-02-19 | Bowman Mark P | Process for forming multilayer coating with radiation curable polyene/polythiol coating compositions |
| CN104152375A (en) * | 2014-07-18 | 2014-11-19 | 齐齐哈尔大学 | Deamination and deodorization bacterial strain QDN01 and application thereof in biological deodorization |
| CN104878026A (en) * | 2015-06-18 | 2015-09-02 | 浙江师范大学 | Application of LRK2 gene in improvement of drought resisting capacity of rice |
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Application publication date: 20180928 |