CN102643762A - Brevibacillus parabrevis as well as method and application of protein series prepared thereby - Google Patents

Brevibacillus parabrevis as well as method and application of protein series prepared thereby Download PDF

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CN102643762A
CN102643762A CN201110441893XA CN201110441893A CN102643762A CN 102643762 A CN102643762 A CN 102643762A CN 201110441893X A CN201110441893X A CN 201110441893XA CN 201110441893 A CN201110441893 A CN 201110441893A CN 102643762 A CN102643762 A CN 102643762A
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genus bacillus
gyzc01
thrombus
short genus
protein
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CN102643762B (en
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杨再昌
杨小生
宋丹丹
陆伦维
杜润孜
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Guizhou University
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Abstract

The invention discloses brevibacillus parabrevis as well as a method and application of a protein series prepared thereby. The classification is named as brevibacillus parabrevis GYZC01, CCTCC NO:M2011461; 16SrRNA of the brevibacillus parabrevis has a sequence of SEQ ID NO:1; the protein series prepared by the brevibacillu sparabrevis GYZC01 strain can be used for preparing thrombolytic medicine, can be used for slowly dissolving thrombus and has durability (EC50 being 200), and can be used for effectively dissolving fresh thrombus (EC50 being 400); and aiming at old thrombus, the activity of GYZC01 strain crude protein is obviously stronger than that of urokinase (EC50 being 800).

Description

The method and the purposes of the protein series of short genus bacillus of one kind and preparation thereof
Technical field
The present invention relates to the method and the purposes of the protein series of short genus bacillus of a kind and preparation thereof.
Background technology
The mortality ratio of cardiovascular and cerebrovascular diseases occupy second always, and wherein thrombotic diseases is that sickness rate, disability rate and mortality ratio are the highest.Thrombotic diseases be meant have in the blood circulation thrombosis and thrombus from local shedding with in the blood vessel of blood flow to the place ahead, block or the partial blocking lumen of vessels, cause one type of disease of thromboembolism.To thrombotic diseases, the clinical treatment of existing employing has three kinds: i.e. surgical operation, anti-bolt therapy and thrombolytic therapy.The position of the necessary clear and definite embolism of operative treatment implements comparison difficulty and dangerous property; Anti-bolt therapy is that utilization medicine control thrombus forms again, and invalid to the thrombus that has formed; The thrombus that thromboembolism treatment is to use medicine dissolution to form is dredged the blood vessel that gets clogged again.Thromboembolism treatment is the emphasis of research at present.
Thrombolytic therapy is to utilize the direct thrombus of thrombolytics or plasmin in the blood is former to be converted into the main matrix thrombus scleroproein hydrolysis that Tryptase comes the catalysis thrombus through activating.The thrombolytics of official approval use roughly is divided into the three generations, the first-generation clinically: streptokinase (Streptokinase), urokinase (Urokinase); The s-generation: Eminase (Anistreplase), recombinant type tissue plasminogen activator (Alteplase, recombinant human tissue-type plasminogen activator); The third generation: reteplase (Reteplase), TNK-tPA (Tenecteplase), lanoteplase (Lanoteplase), staphylokinase (Staphylokinase).
Said medicine has obtained significant achievement on the treatment thrombus disease, but also has certain distance from the ideal thrombolytic drug, and its shortcoming is that the thrombolysis specificity is not high, and the transformation period is short, causes internal hemorrhage etc. easily.The ideal thrombolytic drug should have safety, effectively, convenient drug administration, high specificity, long half time, can dissolve outmoded thrombus, low, the no characteristics such as side reaction, reasonable price such as hemorrhage of recurrence rate.
Summary of the invention
The technical problem that the present invention will solve is: the thrombolysis specificity that overcomes existing antithrombotic reagent existence is not high; Transformation period is short; Cause shortcomings such as internal hemorrhage easily; The present invention provides a kind short genus bacillus GYZC01, can be used for preparing thrombolytic agent with the molecular weight of its preparation greater than the protein series of 7000D, and is respond well.
The short genus bacillus of another object of the present invention type of providing prepares the method for protein series.
The short genus bacillus of a kind of the present invention, the short genus bacillus of its classification called after class ( Brevibacillus parabrevis) GYZC01, be deposited in Chinese typical culture collection center, the preservation address: Chinese Wuhan City Wuhan University, deposit number is: CCTCC NO:M 2011461, preservation date is: on December 11st, 2011.Of the present invention type of short genus bacillus (Brevibacillus parabrevis) GYZC01 is a kind of gram-positive microorganism that is present in the soil, is from the soil of school district, the Cai Jia of Guizhou University pass, to be separated to a strain bacterium, in the plain agar flat board, can grow; The righttest culture temperature is 30 ℃, and bacterium colony is lark, the long 3-4 micron of thalline, wide 0.5-0.8 micron; Aerobic, can utilize Citrate trianion, ammonium salt, can not utilize nitrate salt; Ability caseinhydrolysate, gelatin; Can utilize glucose, fructose, SANMALT-S to produce acid, aerogenesis, can not reduce lactose, its protein series has the effect of thrombus dissolving; Through identifying the short genus bacillus of bacterial strain GYZC01 type of being (Brevibacillus parabrevis).
The short genus bacillus of described class ( Brevibacillus parabrevis) the 16S rRNA of GYZC01 has the sequence of SEQ ID NO:1, institute's calling sequence is compared through Blast program and GenBank amplifying nucleic acid data, its series with Brevibacillus parabrevisM3; The AB215101 bacterial strain has 99% similarity, with other Brevibacillus parabrevisThe similarity of bacterial strain is between 97-98.5%.Judge that thus GYZC01 belongs to new bacterial strain.Further set up systematic evolution tree, prove that also GYZC01 is new bacterial strain with MEGA5.0.
The short genus bacillus of a described kind prepares the method for protein series, and ferment product freeze overnight that will the short genus bacillus GYZC01 of type of containing is melted in room temperature; Obtain freezing and separate liquid, freeze and separate liquid to put whizzer centrifugal, collect supernatant; Supernatant adds the ammonium sulfate powder gradually, and until saturated, collecting precipitation spends the night; Throw out is dialysed with dialysis tubing, obtains protein series.
The short genus bacillus of a described kind prepares the method for protein series, and the molecular weight of protein series is greater than 7000D.
Described protein series is in the application of thrombolytic agent.
It is 1392 bp that type short genus bacillus (Brevibacillus parabrevis) GYZC01 strain of the present invention uses 27f and 1492r to carry out the 16S rRNA base size that pcr amplification goes out bacterium as primer, and sequence is seen Nucleotide or the aminoacid sequence table that specification sheets is last.
The beneficial effect that the present invention reaches:
The molecular weight that the present invention obtains is greater than the thrombus dissolving effect of class short genus bacillus (Brevibacillus parabrevis) the series of protein matter of 7000D: adopt external thrombus dissolving TP; And can produce through fermentation mode; This protein series is thrombus slowly; Persistent, EC 50Be 200 g/ml.In vivo tests shows that this protein series can effectively be dissolved fresh thrombus (EC 50Be 400 g/ kg), but to the old thrombus, the activity of GYZC01 bacterial strain crude protein obviously is better than urokinase, EC 50Be 800 g/ml, urokinase EC 50Be 1600 g/ml.Research shows; Molecular weight is a metalloprotease greater than the thrombus dissolving activity composition of class short genus bacillus (Brevibacillus parabrevis) the series of protein matter of 7000D; Ability is thrombus directly; Can not plasminogen activation, so can not cause spinoffs such as hemorrhage after using in the body, have earth shaking using value.
Figure of description
Fig. 1 sets up systematic evolution tree for MEGA5.0.
Fig. 2 is the short genus bacillus GYZC01 of a class of the present invention genome electrophoretic analysis collection of illustrative plates.
Fig. 3 is the short genus bacillus GYZC01 of a class of the present invention PCR product electrophoretogram.
When Fig. 4 is GYZC01 crude protein thrombus dissolving-and the effect graphic representation, wherein the concentration of GYZC01 crude protein () is 800 g/ml, urokinase (◆) concentration is 200 g/ml.
Fig. 5 is thrombus dissolving (fresh thrombus) chicken embryo test in the body, wherein ▲ and be urokinase, ◆ be GYZC01, ■ is a saline water.
Fig. 6 is thrombus dissolving (outmoded thrombus) chicken embryo test in the body.▲ be GYZC01, ◆ be urokinase, ■ is a saline water.
Embodiment
Embodiment 1: type short genus bacillus GYZC01 16S rRNA sequencing
One, genome extracts and electrophoresis detection
1. genome leaching process:
Extract according to giving birth to worker SK1201-UNIQ-10 pillar bacterial genomes DNA extraction agent box specification sheets.
2. genome electrophoretic analysis collection of illustrative plates: see Figure of description 2
Two, PCR reaction
1. the PCR system is set up (50ul):
Template (genome) 10pmol
Primer?up (10uM) 1ul
Primer?down?(10?uM) 1ul
dNTP?mix?(10Mm?each) 1ul
10*Taq?reaction?Buffer 5ul
Taq?(5u/ul) 0.25?ul
Add water to 50ul
The PCR program setting
Preparatory 98 ℃ of 5mim of sex change;
The 95 ℃ of 35S that circulate, 55 ℃ of 35S, 72 ℃ of 1min 30s, 8min is extended in 35 circulations.
3. PCR product electrophoretogram
4. primer sequence:
27f 5’AGAGTTTGATCCTGGCTCAG?3’ 20bp
1492r 5’?GGTTACCTTGTTACGACTT?3’ 19bp
Three, the DNA agarose is cut glue purification
Cut required DNA purpose band by PCR product electrophoresis result.
Four, DNA order-checking
TTTTAGGACCCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTTTCAGACCGGGATAACATAGGGAAACTTATGCTAATACCGGATAGGTTTTTGGATTGCATGATCCGAAAAGAAAAGATGGCTTCGGCTATCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCTGTTGTCAGGGACGAACACGTGCCGTTCGAATAGGGCGGTACCTTGACGGTACCTGACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGCGCGCAGGCGGCTATGTAAGTCTGGTGTTAAAGCCCGGAGCTCAACTCCGGTTCGCATCGGAAACTGTGTAGCTTGAGTGCAGAAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGGTTTCAATACCCTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCTGACCGCTCTGGAGACAGAGCTTCCCTTCGGGGCAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGAGACTGCCGTCGACAAGACGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGGGATGCTACCTCGCGAGGGGACGCCAATCTCTGAAAACCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCGCAAGGAGCCAGCCGCCGAAA
Embodiment 2: type short genus bacillus GYZC01 strain molecule amount is greater than the preparation of 7000D protein series
1. experiment material
The GYZC01 strain of class short genus bacillus (Brevibacillus parabrevis), peptone, sodium-chlor, glucose, dialysis tubing, ammonium sulfate.
2. experimental technique
2.1 microbial culture
The composition of bacteria culture medium: peptone 1g, sodium-chlor 0.5 g, glucose 1.5 g, zero(ppm) water 100ml, subsequent use behind the autoclaving.Microbionation in the nutrient solution of 100ml, is cultivated 48h, obtained seed for 30 ℃.Ratio according to 1 to 100 will be planted daughter bacteria liquid and added in the nutrient solution, cultivate 5L altogether, and 30 ℃ leave standstill that to cultivate 96-120h subsequent use.
Ferment product (5L) freeze overnight is melted in room temperature, freeze overnight again, 3 times so repeatedly; Obtain freezing and separate liquid, freeze and separate liquid and put the centrifugal 10-15min of whizzer 5000-10000r/min, collect supernatant, add the ammonium sulfate powder gradually; Until saturated, hold over night, collecting precipitation (20g).Throw out is dialysed with the dialysis tubing of 7000D repeatedly, obtains the protein series of molecular weight greater than 7000D, with the dialysis tubing desalination of 3500D, collects crude protein solution, lyophilize, and dry thing (5g) is stored in refrigerator (4 ℃).
Bacterium GYZC01 strain is at 30 ℃ of well-growns, and when being cultured to 120h, the bacterium number reaches the peak; Previous experiments is verified; Protein with thrombus dissolving activity both had been present in born of the same parents and also had been present in outward in the born of the same parents, therefore adopted freeze thawing mode cracking bacterium, through oversalting; After dialysis desalination and the lyophilize, obtain molecular weight greater than the common 5g of the crude protein of 7000D.
Embodiment 3: type short genus bacillus GYZC01 strain molecule amount is greater than the external thrombus dissolving test of 7000D protein series
1, experiment material
The short genus bacillus GYZC01 bacterial strain molecular weight of class is greater than crude protein, kapillary, urokinase, the plate of 7000D; 2, experimental technique
Alcohol disinfecting is nameless, and aculeus blood-taking sucks blood with kapillary, horizontal positioned, approximately 10min blood coagulation.The short genus bacillus GYZC01 of class bacterial strain crude protein is processed the sample solution of series concentration with physiological saline solution, and urokinase is done positive control, saline water is done negative control, and above-mentioned sample, positive control and negative control solution are inserted respectively in the plate of 60mm; It is long to be cut into 5mm to the kapillary of band blood clot, puts into plate, 5 of every plates; Make it immerse liquid; Plate is added a cover, and puts in the room temperature, picks up counting.Observe, write down the dissolved situation of blood clot in the kapillary behind the 24h;
3, result
Table 1 is external thrombus dissolving test-results, and visible urokinase thrombus dissolving activity is the strongest, and the GYZC01 crude protein is 800 in concentration
Figure 201110441893X100002DEST_PATH_IMAGE001
The time can 100% ground thrombus, EC 50Be 200
Figure 843212DEST_PATH_IMAGE001
Figure 760353DEST_PATH_IMAGE002
The GYZC01 crude protein is relatively gentleer to the dissolving of thrombus, in the 24h thrombus is all dissolved, and can thrombus all be dissolved in the urokinase 12h.
Experimental result can find out that there is good external thrombus dissolving activity in GYZC01 protein series, and can produce through fermentation mode, being characterized as of thrombus dissolving: gentle dissolving, persistent.
Embodiment 4: type short genus bacillus GYZC01 strain molecule amount is greater than the chicken embryo thrombus dissolving test of 7000D protein series
1. experiment material
Type short genus bacillus GYZC01 bacterial strain molecular weight is greater than the crude protein of 7000D, urokinase, instar chicken embryo on the 8th, saline water, iron trichloride, qualitative filter paper.
2. experimental technique
Open chorion from 8 age in days chick embryo air sac ends, expose chorioallantoic membrane, use the crochet hook separating blood vessel, with the filter paper bar contact chicken embryo blood vessel 15-20s that contains liquor ferri trichloridi, promptly form thrombus in the contact position again, naked eyes are visible.Behind the about 2h of thrombosis behind (fresh thrombus) or 6 h (outmoded thrombus); Beginning administration (urokinase is done positive control, saline water is done negative control); Be about to medicine liquid droplet and be added in the thrombosis place, or soup is injected blood vessel or allantoic cavity, with sterile transparent adhesive plaster sealing openning.2,8,16,20,24 h observe the thrombolysis situation after the administration.
3, result
Find out that from experimental result GYZC01 bacterial strain molecular weight is active in urokinase greater than thrombus dissolving (fresh thrombus) in the crude protein body of 7000D, EC 50Be 400
Figure 866323DEST_PATH_IMAGE001
, urokinase EC 50Be 100
Figure 261532DEST_PATH_IMAGE001
, but to the old thrombus, the activity of GYZC01 bacterial strain crude protein is better than urokinase, EC 50Be 800
Figure 138221DEST_PATH_IMAGE001
, urokinase EC 50Be 1600
Figure 960684DEST_PATH_IMAGE001
Nucleotide or aminoacid sequence table SEQ ID NO:1
< 110>Guizhou University
The method and the purposes of the protein series of short genus bacillus of < 120>one kinds and preparation thereof
<160>?1
<170>?Patent?In?Version BLAST?2.1
<210>?1
<211>?1392
<212>?RNA
<213>The short genus bacillus of class ( Brevibacillusparabrevis) GYZC01
<400>?1
TTTTAGGACC?CTAGCGGCGG?ACGGGTGAGT?AACACGTAGG?CAACCTGCCT TTCAGACCGG 60
GATAACATAG?GGAAACTTAT GCTAATACCG?GATAGGTTTT?TGGATTGCAT?GATCCGAAAA 120
GAAAAGATGG?CTTCGGCTAT?CACTGGGAGA TGGGCCTGCG?GCGCATTAGC?TAGTTGGTGG 180
GGTAACGGCC?TACCAAGGCG?ACGATGCGTA?GCCGACCTGA?GAGGGTGACC?GGCCACACTG 240
GGACTGAGAC?ACGGCCCAGA?CTCCTACGGG?AGGCAGCAGT?AGGGAATTTT?CCACAATGGA 300
CGAAAGTCTG?ATGGAGCAAC?GCCGCGTGAA?CGATGAAGGT?CTTCGGATTG?TAAAGTTCTG 360
TTGTCAGGGA?CGAACACGTG?CCGTTCGAAT?AGGGCGGTAC?CTTGACGGTA?CCTGACGAGA 420
AAGCCACGGC?TAACTACGTG?CCAGCAGCCG?CGGTAATACG?TAGGTGGCAA?GCGTTGTCCG 480
GATTTATTGG?GCGTAAAGCG?CGCGCAGGCG?GCTATGTAAG?TCTGGTGTTA?A?AGCCCGGAG 540
CTCAACTCCG?GTTCGCATCG?GAAACTGTGT?AGCTTGAGTG?CAGAAGAGGA?AAGCGGTATT 600
CCACGTGTAG?CGGTGAAATG?CGTAGAGATG?TGGAGGAACA?CCAGTGGCGA?AGGCGGCTTT 661
CTGGTCTGTA?ACTGACGCTG?AGGCGCGAAA?GCGTGGGGAG?CAAACAGGAT?TAGATACCCT 720
GGTAGTCCAC?GCCGTAAACG?ATGAGTGCTA?GGTGTTGGGG?GTTTCAATAC?CCTCAGTGCC 780
GCAGCTAACG?CAATAAGCAC?TCCGCCTGGG?GAGTACGCTC?GCAAGAGTGA?AACTCAAAGG 840
AATTGACGGG?GGCCCGCACA?AGCGGTGGAG?CATGTGGTTT?AATTCGAAGC?AACGCGAAGA 900
ACCTTACCAG?GTCTTGACAT?CCCGCTGACC?GCTCTGGAGA?CAGAGCTTCC?CTTCGGGGCA 960
GCGGTGACAG?GTGGTGCATG?GTTGTCGTCA?GCTCGTGTCG?TGAGATGTTG?GGTTAAGTCC 1020
CGCAACGAGC?GCAACCCTTA?TCTTTAGTTG?CCAGCATTCA?GTTGGGCACT?CTAGAGAGAC 1080
TGCCGTCGAC?AAGACGGAGG?AAGGCGGGGA?TGACGTCAAA?TCATCATGCC?CCTTATGACC 1140
TGGGCTACAC?ACGTGCTACA?ATGGTTGGTA?CAACGGGATG?CTACCTCGCG?AGGGGACGCC 1200
AATCTCTGAA?AACCAATCTC?AGTTCGGATT?GTAGGCTGCA?ACTCGCCTAC?ATGAAGTCGG 1260
AATCGCTAGT?AATCGCGGAT?CAGCATGCCG?CGGTGAATAC?GTTCCCGGGC?CTTGTACACA 1320
CCGCCCGTCA?CACCACGGGA?GTTTGCAACA?CCCGAAGTCG?GTGAGGTAAC?CGCAAGGAGC 1380
CAGCCGCCGA?AA 1392

Claims (5)

1. the short genus bacillus of a kind is characterized in that: the short genus bacillus of its classification called after class ( Brevibacillus parabrevis) GYZC01, being deposited in Chinese typical culture collection center, deposit number is: CCTCC NO:M 2011461, preservation date is: on December 11st, 2011.
2. the short genus bacillus of a kind according to claim 1 is characterized in that: the 16S rRNA of described type of short genus bacillus GYZC01 has the sequence of SEQ ID NO:1.
3. the short genus bacillus of a kind according to claim 1 and 2 prepares the method for protein series, it is characterized in that: ferment product freeze overnight that will the short genus bacillus GYZC01 of type of containing, melt in room temperature; Obtain freezing and separate liquid, freeze and separate liquid to put whizzer centrifugal, collect supernatant; Supernatant adds the ammonium sulfate powder gradually, and until saturated, collecting precipitation spends the night; Throw out is dialysed with dialysis tubing, obtains protein series.
4. the short genus bacillus of a kind according to claim 3 prepares the method for protein series, it is characterized in that: the molecular weight of described protein series is greater than 7000D.
5. the protein series described in claim 4 is in the application of thrombolytic agent.
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Cited By (3)

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CN107561233A (en) * 2017-09-25 2018-01-09 中国标准化研究院 A kind of bird egg freshness detection device
CN108996862A (en) * 2018-09-05 2018-12-14 中国石油化工股份有限公司 A kind of water base oil removal treatment method and device of Sludge in Oilfields
CN109097309A (en) * 2018-09-05 2018-12-28 中国石油化工股份有限公司 A kind of petroleum hydrocarbon degradation bacterium and its fermentation process

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107561233A (en) * 2017-09-25 2018-01-09 中国标准化研究院 A kind of bird egg freshness detection device
CN107561233B (en) * 2017-09-25 2023-11-10 中国标准化研究院 Poultry egg freshness detection device
CN108996862A (en) * 2018-09-05 2018-12-14 中国石油化工股份有限公司 A kind of water base oil removal treatment method and device of Sludge in Oilfields
CN109097309A (en) * 2018-09-05 2018-12-28 中国石油化工股份有限公司 A kind of petroleum hydrocarbon degradation bacterium and its fermentation process
CN109097309B (en) * 2018-09-05 2022-02-11 中国石油化工股份有限公司 Petroleum hydrocarbon degrading bacteria and fermentation method thereof

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