CN110055255A - The method for cutting off pXO1 and pXO2 plasmid in Bacillus anthracis - Google Patents

Abstract

The invention discloses the methods of pXO1 and pXO2 plasmid in excision Bacillus anthracis.The method of pXO1 and pXO2 plasmid is carried out using CRISPR/Cas9 system in excision Bacillus anthracis provided by the invention, be respectively two sgRNA of sgRNA1 and sgRNA2 comprising title in CRISPR/Cas9 system, the target sequence of sgRNA1 identification for pXO1 plasmid contains and distinguished sequence that Bacillus anthracis does not contain；The target sequence of sgRNA2 identification for pXO2 plasmid contains and distinguished sequence that Bacillus anthracis does not contain.The present invention has successfully obtained the Bacillus anthracis without containing pXO1 and pXO2 plasmid, construction method is simple, it cuts off high-efficient, and it can once screen the bacterial strain of three kinds of different plasmid loss phenotypes, for construct new vaccine strain provide it is faster, be more convenient new means, provide new thinking for the prevention and treatment of Bacillus anthracis.

Description

The method for cutting off pXO1 and pXO2 plasmid in Bacillus anthracis

Technical field

The present invention relates to the methods in field of biotechnology, cutting off pXO1 and pXO2 plasmid in Bacillus anthracis.

Background technique

Bacillus anthracis (also referred to as Bacillus anthracis) is a kind of Gram-positive, the aerobic bar that can form brood cell Bacterium, can cause the anthracnose of people and animals, if be not treated in time, the death rate is high, cause very big economic loss, life-threatening peace Entirely.Bacillus anthracis is containing there are two the relevant virulence Large plasmids that causes a disease: pXO1 (181.6kb) and pXO2 (96.2kb).Plasmid The anthrax toxins such as pXO1 encoding protective antigens, lethal factor and edema factor albumen and their modulin.Plasmid pXO2 Coding participates in the gene that pod membrane is formed and degraded.The two plasmids lose the pathogenic most important of Bacillus anthracis Any one plasmid all can cause the virulence of Bacillus anthracis greatly to lower.Therefore matter big for Bacillus anthracis virulence The research of grain has been a hot spot of research.The mutant strain of building excision virulence plasmid is for research plasmid in Bacillus anthracis Effect and extremely important with the associated regulatory of chromosome in causing a disease.

Chemical reagent, such as acridine orange, neomycin, Ethidum Eremide etc. can be used in the Large plasmid of early stage removal bacterium.It is high The methods of temperature culture or ultraviolet irradiation.But these methods have potential problem, first is poor specificity, that is, is driving away purpose Other plasmids other than purpose plasmid may be driven away during plasmid, second is that possible host cell generate during processing A possibility that random mutation.

CRISPR/Cas system is a kind of repetitive structure being distributed widely in bacterium and archaeal genome, it is considered to be former The external acquired immune system biting mattress body, plasmid or other exogenous DNAs and infecting of core biophylaxis, the crRNA in the system exist Under the auxiliary of one trans-activation crRNA (tracrRNA), raises effect protein (Cas albumen) and they are taken to target DNA Sequence, Cas albumen cut exogenous DNA array using the function of its nuclease, DNA double chain are caused to be broken (Double Strand Break,DSB).The system has been widely used carry out gene editing at present.The system is simply utilized in order to more convenient, is ground Study carefully personnel by the crRNA-tracrRNA double-stranded RNA complex in II CRISPR/Cas9 system it is artificial reconstructed be one chimeric Single stranded RNA, referred to as unidirectionally lead RNA (single guide RNA, sgRNA), 5 ' end 20nt (as spacer sequence, Referred to herein as N20) specificity sequence match to target the site DNA, the PAM (5 '-NGG-3 ') of 3 ' end of target DNA sequence is Cas9 recognition site cannot be included among sgRNA.Using when only need to change sgRNA 5 ' ends N20 sequence energy Enough instruct Cas Protein cleavage target dna sequence.The system in 2013 takes the lead in the gene for being applied to the mankind and mouse embryo stem cell In editor, it has been successfully applied to mouse, pig, machin, zebra fish, arabidopsis, sorghum, tobacco, rice, nematode, ferment at present In a variety of animals and plants such as mother, Escherichia coli and microorganism, become the widely applied gene editing work of biology and each field of medicine Tool.

Summary of the invention

The technical problem to be solved by the present invention is to how remove Bacillus anthracis pXO1 and pXO2 plasmid.

In order to solve the above technical problems, present invention firstly provides pXO1 and pXO2 plasmids in excision Bacillus anthracis Method, the method cut off pXO1 and pXO2 plasmid in the Bacillus anthracis that sets out using CRISPR/Cas9 system, are free of Have a Bacillus anthracis of pXO1 and pXO2 plasmid, in the CRISPR/Cas9 system comprising title be respectively sgRNA1 and The target sequence of two sgRNA of sgRNA2, the sgRNA1 identification for pXO1 plasmid contains and what Bacillus anthracis did not contained Distinguished sequence；The target sequence of sgRNA2 identification for pXO2 plasmid contains and distinguished sequence that Bacillus anthracis does not contain.

The Bacillus anthracis that sets out contains pXO1 and pXO2 plasmid.

In one embodiment of the invention, the Bacillus anthracis that sets out is A16.

The sgRNA1 target sequence identified is denoted as target sequence 1, the target sequence 1 can be following A1), A2) or A3):

A1) DNA fragmentation shown in 1-20 of sequence 3 in sequence table；

A2 the DNA sequence dna) and A1) limited have 75% or 75% or more identity as A1) derived from DNA fragmentation；

A3) under strict conditions with A1) limit DNA sequence dna hybridize as A1) derived from DNA fragmentation.

The sgRNA2 target sequence identified is denoted as target sequence 2, the target sequence 2 can be following B1), B2) or B3):

B1) DNA fragmentation shown in 1-20 of sequence 4 in sequence table；

B2 the DNA sequence dna) and B1) limited have 75% or 75% or more identity as B1) derived from DNA fragmentation；

B3) under strict conditions with B1) limit DNA sequence dna hybridize as B1) derived from DNA fragmentation.

Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair 1-20 of 1-20 of bright sequence 3 or sequence 4 have 75% or higher or 85% or higher or 90% or higher, Or 95% or higher identity nucleotide sequence.Identity can with the naked eye or computer software is evaluated.Use calculating Machine software, the identity between two or more sequences can be indicated with percentage (%), can be used to evaluate correlated series Between identity.

Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

The stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film 2 times, every time 5min, but in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and washes film 2 times, each 15min；Or, 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution in, hybridize under the conditions of 65 DEG C and wash film.

The sequence of the sgRNA1 can be that the T in sequence table in sequence 3 is replaced with the RNA sequence that U is obtained.

The sequence of the sgRNA2 can be that the T in sequence table in sequence 4 is replaced with the RNA sequence that U is obtained.

The method may include importing to contain the sgRNA1 and sgRNA2 into the Bacillus anthracis that sets out Encoding gene expression cassette, express the sgRNA1 and the sgRNA2, obtain excision pXO1 and pXO2 plasmid Bacillus anthracis (is free of the Bacillus anthracis of pXO1 and pXO2 plasmid).

Into the Bacillus anthracis that sets out import containing the sgRNA1 encoding gene expression cassette can pass through by The recombinant vector of the expression cassette of encoding gene containing the sgRNA1 sets out and realizes in Bacillus anthracis described in importing.

Into the Bacillus anthracis that sets out import containing the sgRNA2 encoding gene expression cassette can pass through by The recombinant vector of the expression cassette of encoding gene containing the sgRNA2 sets out and realizes in Bacillus anthracis described in importing.

In the above method, the CRISPR/Cas9 system may also include Cas9 protein.The sequence of Cas9 protein can be Sequence 2 in sequence table.

The above method may also include the anthrax bud that sets out described in the expression cassette importing by the encoding gene containing Cas9 protein In born of the same parents bacillus, express Cas9 protein.

The encoding gene of the Cas9 protein can be DNA molecular shown in sequence 1 in sequence table.

In the above method, set out anthrax spores described in the expression cassette importing by the encoding gene containing Cas9 protein It can be by will be realized in the Bacillus anthracis that sets out described in the recombinant vector for containing expression cassette importing in bacillus.

Specifically, the expression cassette of the encoding gene containing the sgRNA1 and the table of the encoding gene containing Cas9 protein It can be by the Bacillus anthracis that sets out described in the importing of the recombinant vector (being denoted as recombinant vector 1) containing the two expression cassettes up to box It realizes.Concretely pJO1T, the pJO1T are to be inserted into the target between the multiple cloning sites for the carrier that sets out to the recombinant vector 1 The recombinant vector that can express sgRNA1 the and Cas9 protein that sequence obtains.The carrier that sets out can be temperature sensitive type carrier, Such as pJOE8999.

The expression cassette of the expression cassette of encoding gene containing the sgRNA2 and the encoding gene containing Cas9 protein can By being realized in the Bacillus anthracis that sets out described in recombinant vector (the being denoted as recombinant vector 2) importing containing the two expression cassettes. Concretely pJO2T, the pJO2T are to be inserted into the target sequence between the multiple cloning sites for the carrier that sets out to the recombinant vector 2 What is obtained can express the recombinant vector of the sgRNA2 and Cas9 protein.The carrier that sets out can be temperature sensitive type carrier, such as pJOE8999。

The method, which may additionally include, imports the recombinant vector 1 and the recombination into the Bacillus anthracis that sets out Demethylation is carried out to the recombinant vector 1 and the recombinant vector 2 before carrier 2.The demethylation can be by will be described heavy Group carrier 1 or the recombinant vector 2, which import in Escherichia coli SCS110, to be realized.

The method cuts off the recombinant vector 1 again after may additionally include excision pXO1 and pXO2 plasmid and the recombination carries Body 2.

The recombinant vector 1 and the recombinant vector 2 of cutting off can will import the recombinant vector 1 and recombination load It body 2 and has cut off the recombinant anthrax bacillus after pXO1 and pXO2 plasmid and has been trained at a temperature of the carrier that sets out is sensitive It supports, obtains the purpose anthrax spores bar that the recombinant vector 1 and the recombinant vector 2 and pXO1 and pXO2 plasmid are cut off Bacterium.The temperature of the carrier sensitivity of setting out can be 37-42 DEG C.

The present invention also provides a kind of complete sgRNA, the complete sgRNA is by the sgRNA1 and the sgRNA2 group At.

The complete sgRNA can be used for cutting off pXO1 and/or pXO2 plasmid in Bacillus anthracis, it can also be used to which preparation is cut Except pXO1 and/or pXO2 plasmid product in Bacillus anthracis, it may also be used for preparation prevention and/or treatment Bacillus anthracis institute Cause disease vaccine and/or drug, it can also be used to prevention and/or treatment Bacillus anthracis associated diseases.

The present invention also provides a kind of complete biomaterial, the complete biomaterial is respectively biomaterial 1 by title It is formed with two kinds of biomaterials of biomaterial 2；

The biomaterial 1 is any one of following C1) to C4):

C1 the nucleic acid molecules of the sgRNA1) are encoded；

C2) contain C1) expression cassettes of the nucleic acid molecules；

C3) contain C1) recombinant vectors of the nucleic acid molecules or contain C2) recombinant vector of the expression cassette；

C4) contain C1) recombinant microorganisms of the nucleic acid molecules or contain C2) recombinant microorganism of the expression cassette or Contain C3) recombinant microorganism of the recombinant vector；

The biomaterial 2 is any one of following D1) to D4):

D1 the nucleic acid molecules of the sgRNA1) are encoded；

D2) contain D1) expression cassettes of the nucleic acid molecules；

D3) contain D1) recombinant vectors of the nucleic acid molecules or contain D2) recombinant vector of the expression cassette；

D4) contain D1) recombinant microorganisms of the nucleic acid molecules or contain D2) recombinant microorganism of the expression cassette or Contain D3) recombinant microorganism of the recombinant vector.

The complete biomaterial can be used for cutting off pXO1 and/or pXO2 plasmid in Bacillus anthracis, it can also be used to make PXO1 and/or pXO2 plasmid product in standby excision Bacillus anthracis, it may also be used for preparation prevention and/or treatment anthrax spores bar Bacterium associated diseases vaccine and/or drug, it can also be used to prevention and/or treatment Bacillus anthracis associated diseases.

The present invention also provides a kind of complete sets of products, the complete sets of products is following X1) or X2):

X1) complete sets of products is made of the complete sgRNA and Cas9 protein；

X2) complete sets of products is made of the complete biomaterial and biomaterial relevant to Cas9 protein；It is described with The relevant biomaterial of Cas9 protein is any one of following E1) to E4):

E1 the nucleic acid molecules of Cas9 protein) are encoded；

E2) contain E1) expression cassettes of the nucleic acid molecules；

E3) contain E1) recombinant vectors of the nucleic acid molecules or contain E2) recombinant vector of the expression cassette；

E4) contain E1) recombinant microorganisms of the nucleic acid molecules or contain E2) recombinant microorganism of the expression cassette or Contain E3) recombinant microorganism of the recombinant vector.

The complete sets of products can be used for cutting off pXO1 and/or pXO2 plasmid in Bacillus anthracis, it can also be used to which preparation is cut Except pXO1 and/or pXO2 plasmid product in Bacillus anthracis, it may also be used for preparation prevention and/or treatment Bacillus anthracis institute Cause disease vaccine and/or drug, it can also be used to prevention and/or treatment Bacillus anthracis associated diseases.

The method of pXO1 and pXO2 plasmid or the complete sgRNA or described are complete in the excision Bacillus anthracis Following any applications of biomaterial or the complete sets of products, also belong to protection scope of the present invention:

Y1) the application in excision Bacillus anthracis in pXO1 and/or pXO2 plasmid；

Y2) the application in preparation excision Bacillus anthracis in pXO1 and/or pXO2 plasmid product；

Y3) the application in preparation prevention and/or treatment Bacillus anthracis associated diseases vaccine and/or drug；

Y4) preventing and/or treating the application in Bacillus anthracis associated diseases.

The present invention carries out cutting for Bacillus anthracis virulence Large plasmid pXO1 and pXO2 plasmid using CRISPR/Cas9 system It removes, has successfully obtained the Bacillus anthracis without containing pXO1 and pXO2 plasmid, method of the invention has the advantage that

1. construction method is simple: one section of specific N20 oligonucleotide sequence only being needed to be inserted into skeleton plasmid In pJOE8999 sgRNA 5 ' end can, do not need to know this section of sequence on earth to be what function；

2. cutting off high-efficient: method of the invention is to know that nuclease Cas9 cuts target using sgRNA specificity DNA, specificity is good, and cutting efficiency is high.After inducing Cas9 expression, it is only necessary to which transferring 1-2 times, which can be obtained by, has cut off target matter The bacterial strain of grain, and from the point of view of the result that PCR is identified, at least 50% or more clone has centainly lost target plasmid, thus cuts Cut ultrahigh in efficiency；

3. method of the invention mixes two scissors plasmid equivalent and imports in wild strain, can be according to different experiment mesh , mutant strain of the primary screening to three kinds of different plasmid loss phenotypes.

The present invention for construct new vaccine strain provide it is faster, be more convenient new means, be the anti-of Bacillus anthracis It controls and provides new thinking.

Detailed description of the invention

Fig. 1 is skeleton plasmid pJOE8999 map.

The PCR that Fig. 2 is scissors plasmid pJO1T is identified.M is DNA molecular amount standard.

The PCR that Fig. 3 is scissors plasmid pJO2T is identified.M is DNA molecular amount standard.

Fig. 4 is that scissors plasmid converts Escherichia coli and bacillus anthracis PCR identifies electrophoretogram.Scissors plasmid pJO1T and pJO2T Bacillus coli DH 5 alpha, SCS110 (figure A and figure B) are converted respectively, and mixed in equal amounts plasmid pJMix (pJO1T+pJO2T) converts anthrax Bacillus A16 (figure C), carries out PCR identification with a pair of of special primer pJOE8999-F/R on skeleton plasmid pJOE8999, just It walks and determines that scissors plasmid converts successfully.

Fig. 5 is Bacillus anthracis A16Mix (pXO1⁺pXO2⁺pJO1T⁺pJO2T⁺) in pXO1 and pXO2 excision situation Preliminary screening PCR identifies electrophoretogram.M swimming lane is nucleic acid molecular weight marker, and A is pag gene magnification (pag-F/R) as a result, B It is capA gene magnification (capA-F/R) as a result, 1-12 is the 12 A16Mix (pXO1 selected⁺pXO2⁺pJO1T⁺pJO2T⁺) Monoclonal.

Fig. 6 is that different clones cut off Large plasmid situation PCR identification electrophoretogram.M is nucleic acid molecular weight marker, A clone 1, B It is clone 6 for 7, C of clone.

Fig. 7 is the detection of expression result of PA.M is albumen marker.

Fig. 8 is (100 times) observation results of thallus pod membrane india ink stain optical microscopy.A figure is A16MD1p, and B figure is A16MD2p, C figure are A16MDDp.

Fig. 9 is that the PCR that external source " scissors plasmid " is cut off from Bacillus anthracis identifies electrophoretogram.

Specific embodiment

The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified. Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.In following embodiments, such as without special Illustrate, the 1st of each nucleotide sequence is the 5 ' terminal nucleotides of corresponding DNA/RNA in sequence table, and last bit is corresponding 3 ' the terminal nucleotides of DNA/RNA.

Embodiment 1 cuts off Bacillus anthracis pXO1 plasmid and pXO2 plasmid using CRISPR/Cas9 system

1. the plasmid (" scissors plasmid ") of system containing CRISPR/Cas9 and target sequence constructs

1.1 pXO1 plasmids and pXO2 plasmid target sequence (N20) sequence design

With the Duan Xu on Bacillus anthracis virulence Large plasmid pXO1 (GenBank accession no.AF065404) Arranging (ORF16) is target sequence, with Bacillus anthracis virulence Large plasmid pXO2 (GenBank accession NO.NC_ 007323) Duan Xulie (GBAA_pXO2_0038) on is target sequence, using software sgRNAcas9_V3.0_GUI (or its His software) the special target sequence of N20 in design sgRNA, the target sequence on pXO1 is denoted as O1T (sequence 3 in sequence table 1-20), the target sequence on pXO2 is denoted as O2T (in sequence table 1-20 of sequence 4), sequence is as shown in table 1. The chromosome sequence (GenBank accession NO.NC_003997) of this two sections of target sequences and Bacillus anthracis Ames It is compared, this two sections of target sequences are not present on chromosome.

The external source " scissors plasmid " of 1.2 building cutting pXO1 and pXO2

1.2.1 O1T is inserted into responsive to temperature type shuttle plasmid pJOE8999 (7.8Kb) (Josef Altenbuchner.Editing of the Bacillus subtilis Genome by the CRISPR-Cas9System [J] .Applied and Environmental Microbiology, 2016,82 (17): 5421-5427.) (see Fig. 1) two Between the site Bsal, the correct recombinant plasmid of obtained sequence is named as pJO1T, pJOE8999 contains the encoding gene of Cas9 Expression cassette, the coding gene sequence of Cas9 are sequence 1 in sequence table, Cas9 shown in energy coded sequence 2.Steps are as follows:

(1) it synthesizes N20 oligonucleotides: being connect respectively at the both ends of O1T sequence and O1T reverse complementary sequence plus 4nt protrusion Head (TACG；AAAC), synthetic oligonucleotide (being shown in Table 1):

Forward OT1(FOT1):5′-TACGATAACTTGTAATAGCCCTTT-3′；

Reverse OT1(ROT1):5′-AAAC AAAGGGCTATTACAAGTTAT-3′。

(2) double-strand N20 is obtained: FOT1 and ROT1 first anneals fusion, obtains double-strand N20 (containing two prominent connectors).

(3) skeleton plasmid linearizes: utilizing I digestion pJOE8999 of Bsa, recycles carrier framework (i.e. linearization plasmid).

(4) double-strand N20 is connect with linearized plasmid vector: the double-strand N20 that Connection Step (2) obtains is obtained with step (3) Carrier framework, obtain connection product.

(5) screening and identification of recombinant plasmid: the connection product of step (4) is turned into DH5 α, blue hickie screening (applies 4 μ L IPTG, 40 μ L X-gal), hickie is selected, (spacer_F/R is shown in Table and 1) carries out using a pair of of special primer on pJOE8999 PCR identification, using pJOE8999 as control.O1T is successfully inserted into skeleton plasmid pJOE8999 (figure as the result is shown 2), this recombinant plasmid is sequenced by commercial company, reaffirms construction of recombinant plasmid success, sequence is correct, this recombinant plasmid It is denoted as pJO1T, as cuts off " the scissors plasmid " of pXO1.PJO1T is the DNA identified two Bsa I of pJOE8999 between sequence Segment replaces with the recombinant plasmid that O1T is obtained, and pJO1T can turn containing DNA fragmentation shown in sequence 3 in ordered list, the DNA fragmentation The sgRNA (being denoted as sgRNA1) of record targeting O1T.

1.2.2 according to the method for step 1.2.1, O1T is replaced with into O2T, other steps are constant, obtain containing O2T's Recombinant plasmid pJO2T as cuts off " the scissors plasmid " of pXO2.Use a pair of of special primer (spacer_F/ on pJOE8999 R is shown in Table 1) progress PCR identification, and qualification result is as shown in figure 3, correlated series are shown in Table 1.O2T is successfully inserted into as the result is shown In skeleton plasmid pJOE8999.PJO2T obtains for the DNA fragmentation between two Bsa I of pJOE8999 identification sequence is replaced with O2T The recombinant plasmid arrived, for pJO2T containing DNA fragmentation shown in sequence 4 in ordered list, which can transcribe targeting O2T's SgRNA (is denoted as sgRNA2).

N20 oligonucleotides used is following (being shown in Table 1):

Forward OT2(FOT2):5′-TACGACACAAAGTGATAGCCTAGA-3′；

Reverse OT2(ROT2):5′-AAAC TCTAGGCTATCACTTTGTGT-3′。

2. the excision of Bacillus anthracis virulence Large plasmid pXO1/pXO2

2.1 conversions: step 1 is constructed into correct " scissors plasmid " pJO1T and pJO2T before importing Bacillus anthracis It to be first transferred to Escherichia coli SCS110 (Beijing Quanshijin Biotechnology Co., Ltd) demethylation, then its electricity is transferred to charcoal respectively In subcutaneous ulcer bacillus competent cell.

PJO1T and pJO2T changes turn SCS110 respectively: each 5 μ L of solution of scissors plasmid pJO1T and pJO2T are added separately to In the Escherichia coli SCS110 Competent cell of 50 μ L, ice bath 2min again after ice bath 30min after mixing, thermal shock 90s, then Appropriate LB liquid medium is added, is placed in 30 DEG C of shaking table recovery 1h, takes 150 μ L coating containing kanamycins (Kan, 25 μ g/ml) LB agar plate places overnight incubation in 30 DEG C of incubators.Toothpick picking monoclonal is made bacteria suspension and makees template, with pJOE8999 Upper primer pair pJOE8999-F/R carries out PCR verifying (being shown in Table 1), and confirmation scissors plasmid, which has been changed, to be gone to SCS110 (Fig. 4), will The obtained recombinant bacterium containing pJO1T is denoted as SCS110-pJO1T, and the obtained recombinant bacterium containing pJO2T is denoted as SCS110- pJO2T。

Electricity after the mixed in equal amounts of scissors plasmid pJO1T and pJO2T is transferred to Bacillus anthracis wild strain A16 (to contain PXO1 and pXO2, A16 (pXO1⁺pXO2⁺)): pJO1T and pJO2T is extracted from SCS110-pJO1T and SCS110-pJO2T respectively, The mass mixings such as scissors the plasmid pJO1T and pJO2T of extraction are taken, turns upside down and shakes up, obtain mixing scissors plasmid, take 5 μ L mixed Close the Bacillus anthracis A16 sense that scissors plasmid (pJO1T+pJO2T, the abbreviation pJMix in later description) is added to 40 μ L By in state cell (preparation of competent cell, reference literature: Gao Meiqin, Liu Xiankai, Feng Erling, Tang Hengming, Zhu Li, Chen Fusheng, A16R plants of eag gene deletion mutants of Wang Hengliang Bacillus anthracis construct microorganism journal, 2009,49 (1): 23-31.), Ice bath 2min, shock by electricity (condition: voltage, 0.6kv；Resistance, 500；Capacitor, 25 μ F；Electric shock instrument used are as follows: originate from the Bio- in the U.S. RAD GenePulserⅡelectroporator；Electric shock cup is 0.1cm.), electrical conversion Soviet Union liquid 1mL is added, is placed in 30 DEG C of shaking tables The LB solid plate of recovery 3h, Tu Hanyou Kan (25 μ g/ml) resistance, places and cultivates in 30 DEG C of incubators, next day grows significantly Monoclonal.Wherein 4 monoclonals are respectively prepared bacteria suspension and make template toothpick picking, with pJOE8999 special primer pJOE8999- F/R carries out PCR verifying (being shown in Table 1), and confirmation scissors plasmid, which has been changed, goes in Bacillus anthracis (Fig. 4).Positive colony primer Spacer-F/R is sequenced and (is shown in Table 1), confirms that two scissors plasmids are correctly transformed into Bacillus anthracis, will contain The positive colony of two scissors plasmids is denoted as A16Mix (pXO1⁺pXO2⁺pJO1T⁺pJO2T⁺) (referred to as A16Mix).

2.2 screening processes:

2.2.1 induction Cas9 expression: the A16Mix (pXO1 of picking step 2.1⁺pXO2⁺pJO1T⁺pJO2T⁺) monoclonal connects Kind by 30 DEG C of 5ml LB liquid medium (contain 25 μ g/ml Kan) 220rpm shaking table culture 3 hours, 0.4% mannose is added to turn 25 DEG C 220rpm shaking table Fiber differentiation 10 hours, 1% connects bacterium amount switching 5ml LB liquid medium passage, and the same terms lure again It led for 1 generation, obtains culture solution.

2.2.2PCR virulence Large plasmid loss situation is verified: the culture solution dilution 10 that step 2.2.1 is obtained⁵It is flat that LB is applied again Plate (contains 25 μ g/ml Kan), and then plate is placed in 30 DEG C of incubators and is cultivated, grows apparent clone within about 1 day.Toothpick Picking monoclonal, PCR identify the Large plasmid excision situation of different clones, including pXO1 excision, pXO2 excision, two plasmids (pXO1 and pXO2) cuts off three kinds of situations simultaneously.

(1) preliminary screening

Using a specific gene (protective antigens pag) on Bacillus anthracis pXO1, one on pXO2 is special Gene (pod membrane encoding gene capA) is that target carries out PCR preliminary screening, preliminary screening verify in different clones pXO1, pXO2 and The clone that double-mass model (pXO1pXO2) is cut off simultaneously.Pick 12 clones, using two couples of primers pag-F/R, capA-F/R into Row PCR verifies (being shown in Table 1), and cloning 1-5 as the result is shown does not have the amplified band of pag gene, shows that the pXO1 of this clone may Excision, clone 7-11 do not have the amplified band of capA gene, show that the pXO2 of this clone may be cut off, pag in clone 6,12 Gene and capA gene show that two plasmids of pXO1 and pXO2 of this clone may be cut off simultaneously all without amplified band (Fig. 5)

(2) confirmation of Large plasmid situation is cut off to different clones using polygenes PCR amplification

The Bacillus anthracis (clone 1, A i.e. in step (1)) of pXO1,1 possibility may have been cut off by selecting 1 respectively The Bacillus anthracis (clone 7, B i.e. in step (1)) of pXO2 is cut off, 1 may cut off two plasmid (pXO1 simultaneously And pXO2) Bacillus anthracis (clone 6, C i.e. in step (1)), use be located at pXO1 on 5 pairs of special primers and position In 5 pairs of special primers (being shown in Table 2) on pXO2, to this 3 clones further verifying confirmation.

5 pairs of special primers used on pXO1 are as follows: pXO1-70F and pXO1-70R, pXO1-90F and pXO1- 90R, pXO1-95F and pXO1-95R, pXO1-98F and pXO1-98R, pXO1-116F and pXO1-116R.

5 pairs of special primers used on pXO2 are as follows: pXO2-89F and pXO2-89R, pXO2-94F and pXO2- 94R, pXO2-97F and pXO2-97R, pXO2-107F and pXO2-107R, pXO2-111F and pXO2-111R.The results show that institute There is the special primer being located on pXO1 in clone 1 all without PCR amplification to specific band (Fig. 6), shows this clone's PXO1 is already cut off, and naming this clone is A16MD1p (pXO1^-pXO2⁺pJMix⁺) (referred to as A16MD1p) (being shown in Table 2).

All special primers on pXO2, all without PCR amplification to specific band (Fig. 6), show this in clone 7 The pXO2 of a clone is already cut off, and naming this clone is A16MD2p (pXO1⁺pXO2^-pJMix⁺) (referred to as A16MD2p) (being shown in Table 2).

All special primers on the pXO1 and pXO2 in clone 6 all without PCR amplification to specific band (Fig. 6), Show that the pXO1 and pXO2 of this clone are cut off simultaneously, naming this clone is A16MDDp (pXO1^-pXO2^-pJMix⁺) (referred to as A16MDDp) (is shown in Table 2).

2.2.3 phenotypic evaluation:

(1) Western blot is analyzed to identify PA and expresses in Bacillus anthracis A16MD1p and A16MDDp

A16MD1p (pXO1^-pXO2⁺pJMix⁺)、A16MD2p(pXO1⁺pXO2^-pJMix⁺) and A16MDDp (pXO1^- pXO2^-pJMix⁺) it is inoculated into solid LB media, it is placed into CO₂37 DEG C of culture 13h of incubator.Lawn is scraped with oese, is added Enter to 2ml and shake in pipe (added with bead), preparing Urea Lysis liquid, (solution is to be added 1% into 8M aqueous solution of urea DTT, ready-to-use), 200 μ l of Urea Lysis liquid is added into oscillating tube and mixes.Then it is shaken 10-15 times in homogenizer 5500, 16 DEG C, 1300rpm is centrifuged 20min, collects supernatant (i.e. protein solution) and dispenses, -80 DEG C freeze.40 μ l protein solutions are taken, are added Enter isometric 2 × SDS-PAGE sample-loading buffer, boiling 10min makes albuminous degeneration.The pre-prepared colloid for taking 12%, according to albumen Every 10 μ of hole loading of sequence of Marker (Beijing Quanshijin Biotechnology Co., Ltd), A16MD1p, A16MD2p and A16MDDp L repeats two parts of loading, carries out SDS-PAGE protein electrophoresis.After protein electrophorese, portion is used to Coomassie brilliant blue and dyes, In addition portion is utilized respectively PA antibody as primary antibody and carries out Western blot analysis, and PA antibody used is rabbit polyclonal antibody (abcam company,http://www.abcom.cn, ab21268), secondary antibody is the goat antirabbit of HRP label.

The result of Coomassie brilliant blue dyeing utilizes scanner scanning image, and the result of Western blot analysis uses low temperature Gel imager photograph.The results show that A16MD2p have PA (83kDa) expression, cut off pXO1 after A16MD1p and A16MDDp without PA expresses (Fig. 7).

(2) india ink stain confirms Bacillus anthracis A16MD2p and A16MDDp without pod membrane structure

Generally make to form a clear area between background and thallus using negative staining method, thallus is set off out convenient for observation It differentiates.The step of capsule stain: by A16MD1p (pXO1^-pXO2⁺pJMix⁺)、A16MD2p(pXO1⁺pXO2^-pJMix⁺) and A16MDDp(pXO1^-pXO2^-pJMix⁺) LB solid medium is inoculated into (containing 8 ‰ NaHCO₃, 5% horse serum), it is placed into logical 5% CO₂37 DEG C of culture 48h of constant incubator or so, bacteria suspension is made in picking Bacillus anthracis, add 1 drop prepared Chinese ink to mix well, Then a drop is added dropwise on glass slide, covered compresses, and wiping redundant solution using lens wiping paper can microscopy.100 times of mirrors As a result, background grey black, thallus is darker, encapsulated simple substance grain cuts off strain A16MD1p for inspection, have outside thallus one layer it is colorless and transparent It encloses (A in Fig. 8), acapsular simple substance grain excision strain A16MD2p and double-mass model excision its thallus of strain A16MDDp are outside without colourless Bright circle (B, C in Fig. 8).Show that A16MD1p contains pJO2T, A16MD2p and A16MDDp and do not contain pJO2T.

3. the process that external source " scissors plasmid " pJMix is cut off from Bacillus anthracis

Because external source " scissors plasmid " pJMix that the present invention constructs is Thermo-sensitive plasmid, lost at 37-42 DEG C, and 37 It DEG C is the optimum growth temp of Bacillus anthracis, in order to avoid Bacillus anthracis is passed in excessively high temperature growth, so Selection loses external source recombinant plasmid at 37 DEG C.Steps are as follows:

The monoclonal of A16MD1p, A16MD2p or A16MDDp that picking step 2 obtains connect 5mL non-resistant LB Liquid Culture Base cultivates 10-12h on 37 DEG C of shaking table, is then transferred in another 5mL non-resistant LB Liquid Culture by 1% in 37 DEG C of shaking tables Upper culture 12h after transferring 3 times by 1% repeatedly, carries out gradient dilution to bacterium solution, takes 10⁴、10⁵The bacterium solution of two dilutions again, It is coated with nonreactive plate.It is placed in 30 DEG C of incubators and cultivates, the monoclonal on next day picking plate carries out contact plate, each Dan Ke Grand contact plate respectively sets the plate after contact plate in 30 DEG C of incubators in LB nonreactive plate and LB (containing 25 μ g/ml Kan) agar plate Culture.Next day, picking are grown and non-growing on Kan (containing 25 μ g/ml Kan) resistance agar plate on non-resistant plate Monoclonal carries out bacterium colony PCR identification and Kan resistance test tube switching culture (30 DEG C of shaking table cultures), determines it in Kan resistance test tube In do not grow, thus select positive colony.Non-resistant Tube propagation is inoculated in the positive colony selected, then extracts its gene Group carries out PCR using the special primer pJOE8999-F/R (being shown in Table 1) on vector plasmid, and identification external source recombinant plasmid is driven Except situation, using pJOE8999 as positive control.

A16MD1p, A16MD2p and A16MDDp show these clones' without PCR specific amplified band as the result is shown External source scissors plasmid has lost (Fig. 9).So far, pXO1 excision, pXO2 excision and double-mass model excision have been obtained, and has been free of external source The mutant strain of plasmid is respectively designated as A16MD1 (pXO2⁺) (referred to as A16MD1), A16MD2 (pXO1⁺) (referred to as A16MD2) And A16MDD.

The oligonucleotide sequence and primer that 1. present invention of table uses

The bacterial strain and characteristic that 2. present invention of table obtains

Note: in table 2, "-" indicates not containing corresponding plasmid, and "+" indicates to contain corresponding plasmid.

<110>PLA Academy of Military Sciences's military medical research institute

<120>method of pXO1 and pXO2 plasmid in Bacillus anthracis is cut off

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 4104

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 1

atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60

atcactgatg attataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120

cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180

gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240

tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300

cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360

aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420

aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480

atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540

gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600

attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660

cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720

ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780

gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840

caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900

ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960

atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020

caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080

ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140

gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200

aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260

gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320

gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380

cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440

gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500

aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560

tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620

tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680

gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740

tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800

attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860

ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920

cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980

cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040

gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100

agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160

catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220

gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280

attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340

atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400

gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460

gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac 2520

attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580

gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640

aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700

acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760

ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820

actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880

aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940

taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000

tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060

atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120

aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180

cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240

gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300

cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360

gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420

tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480

aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540

tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600

tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660

caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720

cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780

cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840

attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900

ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960

cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020

gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080

gatttgagtc agctaggagg ttga 4104

<210> 2

<211> 1367

<212> PRT

<213>artificial sequence (Artificial sequence)

<400> 2

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Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

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Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

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Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

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Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

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Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

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His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

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Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

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Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

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Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

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Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

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Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

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Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

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Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

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Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly

1355 1360 1365

<210> 3

<211> 93

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 3

ataacttgta atagcccttt gctagaaata gcaagttaaa ataaggctag tccgttatca 60

acttgaaaaa gtggcaccga gtcggtgctt ttt 93

<210> 4

<211> 93

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 4

acacaaagtg atagcctaga gctagaaata gcaagttaaa ataaggctag tccgttatca 60

acttgaaaaa gtggcaccga gtcggtgctt ttt 93

Claims (10)

1. the method for cutting off pXO1 and pXO2 plasmid in Bacillus anthracis, it is characterised in that: the method uses CRISPR/ Cas9 system cuts off pXO1 and pXO2 plasmid in the Bacillus anthracis that sets out, and includes title point in the CRISPR/Cas9 system Not Wei sgRNA1 and sgRNA2 two sgRNA, the target sequence of sgRNA1 identification is that pXO1 plasmid contains and anthrax spores The distinguished sequence that bacillus does not contain；The target sequence of sgRNA2 identification for pXO2 plasmid contains and Bacillus anthracis does not contain Distinguished sequence.

2. according to the method described in claim 1, it is characterized by: the sgRNA1 target sequence identified is denoted as target sequence 1, The target sequence 1 is following A1), A2) or A3):

A1) DNA fragmentation shown in 1-20 of sequence 3 in sequence table；

A2 the DNA sequence dna) and A1) limited have 75% or 75% or more identity as A1) derived from DNA fragmentation；

A3) under strict conditions with A1) limit DNA sequence dna hybridize as A1) derived from DNA fragmentation；

And/or the sgRNA2 target sequence identified is denoted as target sequence 2, the target sequence 2 is following B1), B2) or B3):

B1) DNA fragmentation shown in 1-20 of sequence 4 in sequence table；

B2 the DNA sequence dna) and B1) limited have 75% or 75% or more identity as B1) derived from DNA fragmentation；

B3) under strict conditions with B1) limit DNA sequence dna hybridize as B1) derived from DNA fragmentation.

3. method according to claim 1 or 2, it is characterised in that: the sequence of the sgRNA1 is by sequence 3 in sequence table In T replace with the RNA sequence that U is obtained；

And/or the sequence of the sgRNA2 is that the T in sequence table in sequence 4 is replaced with the RNA sequence that U is obtained.

4. method according to claim 1 to 3, it is characterised in that: the method includes to the anthrax bud that sets out The expression cassette that the encoding gene containing the sgRNA1 and sgRNA2 is imported in born of the same parents bacillus, makes the sgRNA1 and described SgRNA2 is expressed, and the Bacillus anthracis of excision pXO1 and pXO2 plasmid is obtained.

5. method according to any one of claims 1-4, it is characterised in that: the CRISPR/Cas9 system further includes Cas9 protein.

6. according to the method described in claim 5, it is characterized by: the method also includes containing the coding of Cas9 protein It is set out in Bacillus anthracis described in the expression cassette importing of gene, expresses Cas9 protein.

7. complete sgRNA is made of the sgRNA1 and sgRNA2 any in claim 1-3.

8. complete biomaterial, it is made of two kinds of biomaterials that title is respectively biomaterial 1 and biomaterial 2；

The biomaterial 1 is any one of following C1) to C4):

C1 the nucleic acid molecules of any sgRNA1 in claim 1-3) are encoded；

C2) contain C1) expression cassettes of the nucleic acid molecules；

C3) contain C1) recombinant vectors of the nucleic acid molecules or contain C2) recombinant vector of the expression cassette；

C4) contain C1) recombinant microorganisms of the nucleic acid molecules or contain C2) recombinant microorganism of the expression cassette or contain C3) the recombinant microorganism of the recombinant vector；

The biomaterial 2 is any one of following D1) to D4):

D1 the nucleic acid molecules of any sgRNA1 in claim 1-3) are encoded；

D2) contain D1) expression cassettes of the nucleic acid molecules；

D3) contain D1) recombinant vectors of the nucleic acid molecules or contain D2) recombinant vector of the expression cassette；

D4) contain D1) recombinant microorganisms of the nucleic acid molecules or contain D2) recombinant microorganism of the expression cassette or contain D3) the recombinant microorganism of the recombinant vector.

9. complete sets of products is following X1) or X2):

X1) complete sets of products, the complete sgRNA and Cas9 protein described in claim 7 form；

X2) complete sets of products, the complete biomaterial described in claim 8 and biomaterial relevant to Cas9 protein form； The biomaterial relevant to Cas9 protein is any one of following E1) to E4):

E1 the nucleic acid molecules of Cas9 protein) are encoded；

E2) contain E1) expression cassettes of the nucleic acid molecules；

E3) contain E1) recombinant vectors of the nucleic acid molecules or contain E2) recombinant vector of the expression cassette；

E4) contain E1) recombinant microorganisms of the nucleic acid molecules or contain E2) recombinant microorganism of the expression cassette or contain E3) the recombinant microorganism of the recombinant vector.

10. complete described in complete sgRNA described in any the method or claim 7 or claim 8 in claim 1-6 Following any applications of complete sets of products described in biomaterial or claim 9:

Y1) the application in excision Bacillus anthracis in pXO1 and/or pXO2 plasmid；

Y2) the application in preparation excision Bacillus anthracis in pXO1 and/or pXO2 plasmid product；

Y3) the application in preparation prevention and/or treatment Bacillus anthracis associated diseases vaccine and/or drug；

Y4) preventing and/or treating the application in Bacillus anthracis associated diseases.