CN100395341C - Method for expressing anthrax bacteria gamma phage lyase and its special gene - Google Patents
Method for expressing anthrax bacteria gamma phage lyase and its special gene Download PDFInfo
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- CN100395341C CN100395341C CNB2006100572985A CN200610057298A CN100395341C CN 100395341 C CN100395341 C CN 100395341C CN B2006100572985 A CNB2006100572985 A CN B2006100572985A CN 200610057298 A CN200610057298 A CN 200610057298A CN 100395341 C CN100395341 C CN 100395341C
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Abstract
The present invention discloses a method for expressing an anthrax bacillus gamma phage lyase and a special gene thereof. The gene has one of the following nucleotide sequences: 1) the DNA sequence of SEQ ID No. 2 in a sequence list; 2) the polynucleotide of an SEQ ID No. 3 protein sequence in a coding sequence list; 3) a nucleotide sequence capable of hybridizing with the DNA sequence limited by SEQ ID No. 2 in the sequence list under the high strict condition. A coli expression system is adopted by the gene of an anthrax bacillus gamma phage lyase optimized by the method, and the expression level of the gene of the anthrax bacillus gamma phage lyase is high. The expressed anthrax bacillus gamma phage lyase reaches 75% of full mycoprotein, has strong lysis capacity to an anthrax bacillus, and has very high specificity. The method can be used for diagnosing and preventing anthrax, and the method settles material and theoretical foundations for research for treating the anthrax.
Description
Technical field
The present invention relates to a kind of method and special-purpose gene thereof of expressing anthrax bacteria gamma phage lyase.
Background technology
Bacillus anthracis is the biological warfare agent that receives much concern, and Bacillus anthracis can produce the very strong gemma of resistance, can survive more than 40 years in exsiccant soil.911 back U.S. anthrax spore terrorist incidents have caused global extensive concern especially, and emergency schedule was particularly important fast and effectively after anthrax infected.
But quick diagnosis, prevention and the treatment to anthrax all has difficulties in the anthrax terror at present.Because Bacillus anthracis is extremely similar to some bacterial strain of cured shape genus bacillus, bacillus thuringiensis, gives accurately, diagnose fast, specifically and bring difficulty; Existing anthrax immune effect of vaccine is low, guard time short; If but the anthrax spores of use war agent level in terrified the attack, antibiotic therapy is with invalid.
The people such as Raymond Schuch of Rockefeller University in 2002 find the lyase PlyG of the single-minded cracking anthrax bacillus of energy.Anthrax bacteria gamma phage lyase PlyG is made up of 233 amino-acid residues, produce by the anthrax bacteria gamma phage coding, as a kind of Ntn hydrolase, amido linkage in the single-minded catalytic hydrolysis anthrax bacillus cell walls between-acetylmuramic acid and the L-L-Ala, thereby the cracking anthrax bacillus discharges the ripe particle of the phage that is equipped with fast.The special glycosyl of kind on the PlyG identification anthrax bacillus cell walls, therefore the splitting action to anthrax bacillus is special.Behind the lethal dose infection due to Bacillus anthracis animal, PlyG can make 80% animals survived.
Therefore anthrax bacteria gamma phage lyase PlyG can develop the medicine that becomes a kind of diagnosis, prevention, treatment anthrax, but at first is the method that will develop efficient production PlyG.Utilizing the recombination engineering is a large amount of at present, economic main method that obtain the purpose functional protein in the expression in escherichia coli exogenous object protein.Because PlyG is by phage-coded, can not efficiently express in intestinal bacteria.
Summary of the invention
The purpose of this invention is to provide a kind of gene that can in intestinal bacteria, efficiently express anthrax bacteria gamma phage lyase.
Anthrax bacteria gamma phage lyase gene provided by the present invention, name is called BPlyG, has one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 2 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein sequences;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.
The rigorous condition of described height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
The expression vector, engineering bacteria and the clone that contain said gene also belong to protection scope of the present invention.
Another object of the present invention provides a kind of method that efficiently expresses anthrax bacteria gamma phage lyase in intestinal bacteria.
The method of expression anthrax bacteria gamma phage lyase provided by the present invention, be that above-mentioned BPlyG gene is inserted procaryotic cell expression carrier, transformed into escherichia coli, screening obtains expressing the engineering bacteria of anthrax bacteria gamma phage lyase, the culturing engineering bacterium expresses obtaining anthrax bacteria gamma phage lyase.
Described procaryotic cell expression carrier can be the carrier that is suitable at the expression in escherichia coli foreign gene, as pET series, pGEX series, pMAL series, pBAD series etc.
Described procaryotic cell expression carrier is pET-22b (+).
Described intestinal bacteria are e. coli bl21 (DE3).
Described BPlyG is inserted between the restriction enzyme site of the Nde I of pET-22b (+) and BamH I.
In the aforesaid method, the described abduction delivering that is expressed as, used inductor are IPTG, and induced concentration 0.1-1.2umol/L is preferably 0.8umol/L.
The culture temperature of described engineering bacteria is 37 ℃, and the culture temperature of abduction delivering is 20-37 ℃, is preferably 23 ℃.
The present invention is according to the bacillus coli gene codon-bias, under the prerequisite that does not change the anthrax bacteria gamma phage lyase protein sequence, the anthrax bacteria gamma phage lyase gene is transformed, method by the reorganization extension PCR obtains the gene BPlyG be suitable for efficiently expressing in intestinal bacteria, the BPlyG gene that obtains can be in e. coli bl21 (DE3) efficient soluble-expression, the expression level height, the content of gamma bacterial virus catenase reaches 75% of whole bacterial protein, and determination of activity shows that the activity of the gamma bacterial virus catenase that the present invention expresses reaches 1000U/mg.The gamma bacterial virus catenase of expressing is by behind the ion exchange chromatography purifying, the reorganization PlyG that obtains has very strong cracking ability to anthrax bacillus, and have very high specificity, can be used for diagnosis, prevention anthrax, and be that material and theoretical basis are laid in the research for the treatment of anthrax.
Description of drawings
Fig. 1 is a pcr amplification BPlyG gene electrophoretogram
Fig. 2 is that NdeI, the BamHI double digestion of plasmid pET-22b (+)-BPlyG identified collection of illustrative plates
Fig. 3 efficiently expresses collection of illustrative plates for PlyG in intestinal bacteria
Fig. 4 is the efficient cracking anthrax bacillus of reorganization PlyG collection of illustrative plates
Fig. 5 is the special cracking anthrax bacillus of reorganization PlyG collection of illustrative plates
Embodiment
Method among the embodiment is ordinary method if no special instructions.
The acquisition of the anthrax bacteria gamma phage lyase encoding gene BPlyG of embodiment 1, optimization
1, design of primers
Utilize the method for overlapping extension PCR, obtain target gene sequences.The design primer is as follows:
P1：(
NdeI)5’＞GGTATA
CCATATGGAAATCCAGAAAAAAC＜3’
P2：5’＞GTACTAAGTGCCCGTACACCATGAAACCGAAATACATTACTGTACACAACACTTACAATG＜3’
P3：5’＞GTATCTTACATGATCTCTAACAATAACGAAGTGTCTTTCCACATCGCTGTAGACGATAAG＜3’
P4：5’＞CTGGAACGTAACGCTTGGGCTTGTGGTGATGGCAACGGCTCTGGTAACCGTCAGTCTATC＜3’
P5：5’＞CTAAATCCGGTGGCGACCGTTACTATAAAGCTGAAGACAACGCCGTGGATGTCGTTCGTC＜3’
P6：5’＞CATCCCGATTGAGAACGTGCGTACCCACCAATCTTGGTCCGGTAAATACTGCCCGCACCG＜3’
P7：5’＞GGGGTGCATTTATCCAAAAAGTTAAGAACGGCAATGTTGCTACCACTTCTCCGACCAAAC＜3’
P8：5’＞CTTTCTCCCCGTACGAAACACCGGACGTGATGGGTGCCCTGACTTCTCTGAAGATGACTG＜3’
P9：5’＞GACGGCCTGACCTACTTCATTTCTAAACCAACTTCCGACGCTCAATTGAAAGCCATGAAG＜3’
P10：5’＞ATGGTGTACGGGCACTTAGTACCGTATTTGGACGGATCAACCAGTTTTTTCTGGATTTCC＜3’
P11：5’＞TAGAGATCATGTAAGATACTTCGTTTTCAGCCGGAGCGTCATTGTAAGTGTTGTGTACAG＜3’
P12：5’＞CCAAGCGTTACGTTCCAGCGGAATGCCCTGGATAGCTTTCTTATCGTCTACAGCGATGTG＜3’
P13：5’＞CGGTCGCCACCGGATTTAGAATAGCAAATTTCCACGCTGATAGACTGACGGTTACCAGAG＜3’
P14：5’＞CGCACGTTCTCAATCGGGATGTTGTACATAGACATCAGTTGACGAACGACATCCACGGCG＜3’
P15：5’＞CTTTTTGGATAAATGCACCCCAACGGCCCTCGGCCAGCATACGGTGCGGGCAGTATTTAC＜3’
P16：5’＞TTTCGTACGGGGAGAAAGCACCGGACTGAATGATGTTCTGTTTGGTCGGAGAAGTGGTAG＜3’
P17：5’＞ATGAAGTAGGTCAGGCCGTCGGACTGCAGGATAAAGTCAGCAGTCATCTTCAGAGAAGTC＜3’
P18：
5′＞GCG
TCACTTGACTTCGTACCACCAGCCTTTACGGTCCAGGTATTCCTTCATGGCTTTCAATTGAG＜3′
2, overlapping extension PCR amplification BPlyG full-length gene
The first round PCR of overlapping extension PCR with primer P1-P18 each other template extend, primer P1-P18 respectively gets 0.5ug, carries out first round pcr amplification, program is as follows: (1) 94 ℃, 5 minutes; (2) 94 ℃, 30 seconds, 52 ℃, 30 seconds, 72 ℃, 30 seconds, 10 circulations; (3) 72 ℃, 7 minutes.
With 2ul first round PCR product is template, adds primer P1 and each 1ug of P18, carries out pcr amplification, and the PCR program is as follows: (1) 94 ℃, and 5 minutes; (2) 94 ℃ 30 seconds, 52 ℃ 1 minute, 72 ℃ 1 minute, 30 circulations; (3) 72 ℃, 7 minutes.The PCR product reclaims purifying after gel electrophoresis, electrophoretogram as shown in Figure 1, swimming lane 1 is Marker D2000 among Fig. 1,2 is BPlyG, about 700bp, cut this gene fragment with NdeI and BamHI enzyme then, be cloned between the multiple clone site NdeI and BamHI of pET-22b (+) (Novagen company) carrier, obtain recombinant plasmid, transformed into escherichia coli DH5 α, select positive colony and extract plasmid, carry out double digestion with NdeI and BamHI and identify, obtain the gene fragment and pET-22b (+) fragment of 702bp size, its enzyme is cut and is identified collection of illustrative plates as shown in Figure 2, swimming lane 1 is Marker D2000 among Fig. 2, and swimming lane 2 is process NdeI, the recombinant plasmid of BamHI double digestion.Double digestion is identified correct recombinant plasmid order-checking, the result shows that the fragment of this 702bp size has the nucleotide sequence of sequence 2 in the sequence table, show that by analysis this gene is consistent with protogene (sequence 1) expressed proteins, with its called after BPlyG, the amino acid residue sequence of sequence 3 (aminoacid sequence of anthrax bacteria gamma phage lyase) in this gene coded sequence table.Correct recombinant plasmid called after pET-22b (+)-BPlyG will be identified.
With recombinant plasmid pET-22b (+)-BPlyG transformed into escherichia coli BL21 (DE3), screening obtains expressing engineering bacteria-BpET-22b (+)-BPlyG of anthrax bacteria gamma phage lyase, the single bacterium colony of inoculation BpET-22b (+)-BPlyG was cultivated 12 hours for 37 ℃ in 10ml LB substratum (containing Pyocianil 100 μ g/ml).By the fresh LB substratum (containing Pyocianil 100 μ g/ml) of 1: 100 volume ratio inoculation, 37 ℃, 250rpm were cultivated 10 hours, were seeded in the fermentor tank of 30L by 1: 50 volume ratio then and were cultured to bacterium liquid A
600=1.4-1.6, be divided into three parts, add IPTG respectively to final concentration 0.2umol/L, 0.4umol/L, 0.8umol/L, continue to cultivate 4 hours at 23 ℃, centrifugal collection thalline, the BL21 (DE3) that changes the BL21 (DE3) of pET-22b (+)-BPlyG and do not change pET-22b (+)-BPlyG with inductive not is contrast, thalline was boiled in boiling water 5 minutes with the resuspended back of 1 * electrophoresis sample-loading buffer, carry out SDS-polyacrylamide gel electrophoresis (SDS-PAGE) then respectively and detect expression, the result as shown in Figure 3, the result shows that at final concentration be under the inducing of 0.8umol/L IPTG, reorganization PlyG is efficiently expressed, the anthrax bacteria gamma phage lyase that shows expression after measured accounts for 75% of bacterial protein, measure the enzyme activity of lyase through the method for cracking anthrax bacillus, enzyme activity unit is defined as: the anthrax bacillus vaccine bacterium A16R of 37 ℃ of cultivations is diluted to A600=0.6 with buffered soln, the PlyG that 1mg/ml the present invention is expressed is diluted to the solution that final volume is 1ml by different multiples, add the 1ml anthrax bacillus, 37 ℃ of incubations 15 minutes, the extension rate that makes the enzyme solution of anthrax bacillus A600 drop by half is an enzyme activity unit.Detected result shows that 1ul concentration is that the present invention of 1mg/ml PlyG solution dilution of expressing is during to final volume 1ml (extension rate is 1000 times), 37 ℃ of incubations of anthrax bacillus of A600=0.6 that the enzyme solution of this dilution makes 1ml after 15 minutes A600 be reduced to 0.3, so the enzyme activity of the PlyG that the present invention expresses is 1000U/mg.Among Fig. 3, swimming lane M is for dying the molecular weight of albumen standard in advance, and swimming lane 1,2,3 is respectively the expression of final concentration 0.2umol/L, 0.4umol/L, 0.8umol/L IPTG inductive pET-22b (+)-BPlyG; Swimming lane 4 is inductive BpET-22b (+)-BPlyG not; Swimming lane 5 is inductive BL21 (DE3) not.
With the recombinant anthrax bacillus gamma bacterial virus catenase that the method purifying of ion exchange chromatography is expressed, carry out the N terminal amino acid sequence and measure, the sequencing result shows that the N terminal amino acid sequence is MEIQKKLVDPSKYGTKCPYT, with natural consensus amino acid sequence.
Embodiment 3: the efficient special cracking anthrax bacillus of reorganization PlyG
Anthrax bacillus vaccine bacterium A16R (available from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL) is cultured to A600=0.4, bacterium liquid with the resuspended one-tenth of PBS washing A600=1.4, get 1ml, adding 0ug, 2.5ug, 5.0ug, 10.0ug respectively mixes with the reorganization PlyG that the method purifying of ion exchange chromatography efficiently expresses, with ultraviolet spectrophotometer dynamic measurement A16R bacterium liquid A600 value, the decline of A600 value reflection bacterium is cleaved.The result as shown in Figure 4, the result shows that PlyG becomes positive correlation to the splitting action of A16R with the amount of PlyG, the PlyG of 10ug can efficient cracking anthrax bacillus.
With with the closely similar Tribactur of Bacillus anthracis and cured shape genus bacillus in contrast, detect PlyG that the present invention expresses specificity, the PlyG and 5 * 10 that 50ug the present invention is expressed to the Bacillus anthracis splitting action
5The PlyG that cfu anthrax bacillus vaccine bacterium A16R mixes, 50ug the present invention is expressed and 5 strains respectively 10
8The PlyG that the Tribactur of cfu mixes separately respectively, 50ug the present invention is expressed respectively with 5 strains each 2 * 10
8The cured shape genus bacillus of cfu mixes, dynamic observe the value of bacterium liquid A600 with ultraviolet spectrophotometer, 10 minutes total times, every 1 minute reading of data, the basic cracking of A16R is complete after 4 minutes, calculate cleavage rate, (1-A3/A0) (A0 is initial A600 value to %, A3 is 4 minutes A600 values that read), the result as shown in Figure 5, the result shows that the PlyG that the present invention expresses does not have influence substantially to Tribactur and cured each bacterial strain of shape genus bacillus, and anthrax bacillus A16R is had higher kill ratio, and the PlyG that presentation of results the present invention expresses has very strong specificity to the splitting action of anthrax bacillus; 1,2,3,4,5 is Bacillus thuringiensis bacterial strain among Fig. 5, and 6,7,8,9,10 is cured shape Bacillus strain, and 11 is anthrax bacillus vaccine bacterium A16R; Above-mentioned all bacterial strains are all available from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.
Sequence table
<160>3
<210>1
<211>702
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
atggaaatcc?aaaaaaaatt?agttgatcca?agtaagtatg?gtacaaagtg?tccgtataca 60
atgaagccta?aatatatcac?tgttcacaac?acatataatg?atgctccagc?tgaaaatgaa 120
gtgagttaca?tgattagtaa?caataatgag?gtgtcgtttc?atattgcagt?agatgacaag 180
aaagcgattc?aaggtattcc?gttggaacgt?aatgcatggg?cttgcggaga?cggcaatggt 240
tcggggaatc?gtcaatccat?ttctgtagaa?atctgttatt?caaaatcagg?aggagataga 300
tactataaag?ctgaggataa?tgctgttgat?gttgtacgac?aacttatgtc?tatgtacaat 360
attccgattg?aaaatgttcg?aactcatcaa?tcctggtcag?gtaaatattg?tccgcataga 420
atgttagctg?agggaaggtg?gggagcattc?attcagaagg?ttaagaatgg?gaatgtggcg 480
actacttcac?caacaaaaca?aaacatcatc?caatcagggg?ctttctcacc?gtatgaaacc 540
cctgatgtta?tgggagcatt?aacgtcactt?aaaatgacag?ctgattttat?cttacaatcg 600
gatggattaa?cttattttat?ttccaaaccg?acttcagatg?cacaactaaa?agcaatgaaa 660
gaataccttg?accgtaaagg?ttggtggtat?gaagttaaat?aa 702
<210>2
<211>702
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
atggaaatcc?agaaaaaact?ggttgatccg?tccaaatacg?gtactaagtg?cccgtacacc 60
atgaaaccga?aatacattac?tgtacacaac?acttacaatg?acgctccggc?tgaaaacgaa 120
gtatcttaca?tgatctctaa?caataacgaa?gtgtctttcc?acatcgctgt?agacgataag 180
aaagctatcc?agggcattcc?gctggaacgt?aacgcttggg?cttgtggtga?tggcaacggc 240
tctggtaacc?gtcagtctat?cagcgtggaa?atttgctatt?ctaaatccgg?tggcgaccgt 300
tactataaag?ctgaagacaa?cgccgtggat?gtcgttcgtc?aactgatgtc?tatgtacaac 360
atcccgattg?agaacgtgcg?tacccaccaa?tcttggtccg?gtaaatactg?cccgcaccgt 420
atgctggccg?agggccgttg?gggtgcattt?atccaaaaag?ttaagaacgg?caatgttgct 480
accacttctc?cgaccaaaca?gaacatcatt?cagtccggtg?ctttctcccc?gtacgaaaca 540
ccggacgtga?tgggtgccct?gacttctctg?aagatgactg?ctgactttat?cctgcagtcc 600
gacggcctga?cctacttcat?ttctaaacca?acttccgacg?ctcaattgaa?agccatgaag 660
gaatacctgg?accgtaaagg?ctggtggtac?gaagtcaagt?ga 702
<210>3
<211>233
<212>PRT
＜213〉anthrax bacteria gamma phage
<400>3
Met?Glu?Ile?Gln?Lys?Lys?Leu?Val?Asp?Pro?Ser?Lys?Tyr?Gly?Thr?Lys
1 5 10 15
Cys?Pro?Tyr?Thr?Met?Lys?Pro?Lys?Tyr?Ile?Thr?Val?His?Asn?Thr?Tyr
20 25 30
Asn?Asp?Ala?Pro?Ala?Glu?Asn?Glu?Val?Ser?Tyr?Met?Ile?Ser?Asn?Asn
35 40 45
Asn?Glu?Val?Ser?Phe?His?Ile?Ala?Val?Asp?Asp?Lys?Lys?Ala?Ile?Gln
50 55 60
Gly?Ile?Pro?Leu?Glu?Arg?Asn?Ala?Trp?Ala?Cys?Gly?Asp?Gly?Asn?Gly
65 70 75 80
Ser?Gly?Asn?Arg?Gln?Ser?Ile?Ser?Val?Glu?Ile?Cys?Tyr?Ser?Lys?Ser
85 90 95
Gly?Gly?Asp?Arg?Tyr?Tyr?Lys?Ala?Glu?Asp?Asn?Ala?Val?Asp?Val?Val
100 105 110
Arg?Gln?Leu?Met?Ser?Met?Tyr?Asn?Ile?Pro?Ile?Glu?Asn?Val?Arg?Thr
115 120 125
His?Gln?Ser?Trp?Ser?Gly?Lys?Tyr?Cys?Pro?His?Arg?Met?Leu?Ala?Glu
130 135 140
Gly?Arg?Trp?Gly?Ala?Phe?Ile?Gln?Lys?Val?Lys?Asn?Gly?Asn?Val?Ala
145 150 155 160
Thr?Thr?Ser?Pro?Thr?Lys?Gln?Asn?Ile?Ile?Gln?Ser?Gly?Ala?Phe?Ser
165 170 175
Pro?Tyr?Glu?Thr?Pro?Asp?Val?Met?Gly?Ala?Leu?Thr?Ser?Leu?Lys?Met
180 185 190
Thr?Ala?Asp?Phe?Ile?Leu?Gln?Ser?Asp?Gly?Leu?Thr?Tyr?Phe?Ile?Ser
195 200 205
Lys?Pro?Thr?Ser?Asp?Ala?Gln?Leu?Lys?Ala?Met?Lys?Glu?Tyr?Leu?Asp
210 215 220
Arg?Lys?Gly?Trp?Trp?Tyr?Glu?Val?Lys
225 230
Claims (13)
1. anthrax bacteria gamma phage lyase gene, its base sequence is shown in SEQ ID NO:2.
2. contain the described expression carrier of claim 1.
3. the engineering bacteria that contains the described gene of claim 1.
4. the clone that contains the described gene of claim 1.
5. method of expressing anthrax bacteria gamma phage lyase, be that the described anthrax bacteria gamma phage lyase gene of claim 1 is inserted procaryotic cell expression carrier, transformed into escherichia coli, screening obtains expressing the engineering bacteria of anthrax bacteria gamma phage lyase, the culturing engineering bacterium expresses obtaining anthrax bacteria gamma phage lyase.
6. method according to claim 5 is characterized in that: described procaryotic cell expression carrier is pET-22b (+).
7. method according to claim 6 is characterized in that: described anthrax bacteria gamma phage lyase gene is inserted between the restriction enzyme site of the Nde I of pET-22b (+) and BamH I.
8. according to claim 5 or 6 or 7 described methods, it is characterized in that: described intestinal bacteria are e. coli bl21 (DE3).
9. method according to claim 5 is characterized in that: the described abduction delivering that is expressed as, used inductor are IPTG, induced concentration 0.1-1.2umol/L.
10. method according to claim 9 is characterized in that: the concentration of described IPTG is 0.8umol/L.
11. according to claim 5 or 6 or 7 described methods, it is characterized in that: the culture temperature of described engineering bacteria is 37 ℃.
12. according to claim 9 or 10 described methods, it is characterized in that: the culture temperature of described abduction delivering is 20-37 ℃.
13. method according to claim 12 is characterized in that: the culture temperature of described abduction delivering is 23 ℃.
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| CN101003569B (en) * | 2007-01-19 | 2010-06-23 | 中国人民解放军军事医学科学院生物工程研究所 | Antigen epitope and mutant of lyase in gamma bacteriophage of anthrax bacillus, and application |
| US8962297B2 (en) * | 2010-09-01 | 2015-02-24 | The United States Of America, As Represented By The Secretary Of Agriculture | Bacteriophage lytic enzymes as alternative antimicrobials |
| CN115181732A (en) * | 2022-07-21 | 2022-10-14 | 宜兴市天石饲料有限公司 | Preparation method of phage lyase source feed additive capable of effectively removing impurities |
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| US20050004030A1 (en) * | 2002-05-17 | 2005-01-06 | Fischetti Vincent A. | Phage-associated lytic enzymes for treatment of Bacillus anthracis and related conditions |
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| US20050004030A1 (en) * | 2002-05-17 | 2005-01-06 | Fischetti Vincent A. | Phage-associated lytic enzymes for treatment of Bacillus anthracis and related conditions |
Non-Patent Citations (4)
| Title |
|---|
| Bacteriolytic agent that detects and kills Bacillus anthracis. Schuch R et al.Nature,Vol.418 No.8. 2002 |
| Bacteriolytic agent that detects and kills Bacillus anthracis. Schuch R et al.Nature,Vol.418 No.8. 2002 * |
| 炭疽杆菌噬菌体裂解酶基因在大肠杆菌中的表达及鉴定. 李晓静等.生物工程学报，第21卷第2期. 2005 |
| 炭疽杆菌噬菌体裂解酶基因在大肠杆菌中的表达及鉴定. 李晓静等.生物工程学报，第21卷第2期. 2005 * |
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