CN109825531A - The method for removing pXO2 plasmid in Bacillus anthracis - Google Patents

Abstract

The invention discloses the methods of pXO2 plasmid in removal Bacillus anthracis.The method of pXO2 plasmid in removal Bacillus anthracis disclosed by the invention, it is completed using CRISPR/Cas9 system, it include the sgRNA of entitled sgRNA2 in CRISPR/Cas9 system, the target sequence of sgRNA2 identification is that pXO2 plasmid contains and distinguished sequence that Bacillus anthracis does not contain, target sequence are DNA fragmentation shown in 1-20 of sequence 4 in sequence table.The present invention carries out the removal of Bacillus anthracis virulence Large plasmid pXO2 plasmid using CRISPR/Cas9 system, the Bacillus anthracis without containing pXO2 plasmid is successfully obtained, method of the invention is easy to operate, 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 removing pXO2 plasmid in Bacillus anthracis

Technical field

The present invention relates to the methods in field of biotechnology, removing 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 pXO1 The anthrax toxins such as encoding protective antigens, lethal factor and edema factor albumen and their modulin.Plasmid pXO2 coding Participate in the gene that pod membrane is formed and degraded.The two plasmids lose any one for the pathogenic most important of bacillus anthracis Plasmid all can cause the virulence of bacillus anthracis greatly to lower.It therefore is always to grind for the research of bacillus anthracis virulence Large plasmid The hot spot studied carefully.Building removal virulence plasmid mutant strain for research plasmid bacillus anthracis cause a disease in effect and with dyeing The associated regulatory of body is extremely important.

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, i.e., in removal purpose Other plasmids other than purpose plasmid may be removed 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 type CRISPR/Cas9 system it is artificial reconstructed be one chimeric Single stranded RNA, referred to as unidirectionally lead RNA (single guide RNA, sgRNA), by its 5 ' end 20nt (as spacer sequence Column, referred to herein as N20) specificity sequence match to target the site DNA, PAM (the 5 '-NGG- of 3 ' end of target DNA sequence 3 ') it is Cas9 recognition site, cannot be included among sgRNA.Using when only need to change sgRNA 5 ' ends N20 sequence Arrange the target dna sequence that Cas Protein cleavage can be instructed different.The system takes the lead in being applied to the mankind and mice embryonic within 2013 In the gene editing of stem cell, be successfully applied at present mouse, pig, machin, zebra fish, arabidopsis, sorghum, tobacco, In a variety of animals and plants such as rice, nematode, yeast, Escherichia coli and microorganism, becomes biology and each field of medicine is widely applied Gene editing tool.

Summary of the invention

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

In order to solve the above technical problems, present invention firstly provides a kind of sides of pXO2 plasmid in removal Bacillus anthracis Method, the method remove pXO2 plasmid in the Bacillus anthracis that sets out, the CRISPR/Cas9 using CRISPR/Cas9 system It include the sgRNA of entitled sgRNA2 in system, the target sequence of the sgRNA2 identification is that pXO2 plasmid contains and anthrax spores The distinguished sequence that bacillus does not contain.

The Bacillus anthracis that sets out contains pXO2 plasmid.The Bacillus anthracis that sets out can contain pXO2 plasmid, Without containing pXO2 plasmid.

In one embodiment of the invention, the Bacillus anthracis that sets out is A16Q1.

In the above method, the target sequence can be following A1), A2) or A3):

A1) DNA fragmentation shown in 1-20 of sequence 4 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.

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

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.

In the above method, the T in sequence table in sequence 4 is concretely replaced with what U was obtained by the sequence of the sgRNA2 RNA sequence.

The above method may include that the encoding gene for containing the sgRNA2 is imported into the Bacillus anthracis that sets out Expression cassette expresses the sgRNA2, obtains the Bacillus anthracis of removal pXO2 plasmid.

Into the Bacillus anthracis that sets out import containing the sgRNA2 encoding gene expression cassette can pass through by Recombinant vector containing the expression cassette 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 sgRNA2 and the table of the encoding gene containing Cas9 protein It can be by being realized in the Bacillus anthracis that sets out described in the importing of the recombinant vector containing the two expression cassettes up to box.

Concretely pJO2T, the pJO2T are to described in insertion between the multiple cloning sites for the carrier that sets out to the recombinant vector The recombinant vector that can express sgRNA2 the and Cas9 protein that target sequence obtains.The carrier that sets out can carry for temperature sensitive type Body, such as pJOE8999.

The method, which may additionally include, to be imported before the recombinant vector into the Bacillus anthracis that sets out to described heavy Group carrier carries out demethylation.The demethylation can be realized by importing the recombinant vector in Escherichia coli SCS110.

The method removes the recombinant vector after may additionally include removal pXO2 plasmid again.

The removal recombinant vector can will import the recombinant vector and remove the recombinant anthrax after pXO2 plasmid Bacillus is cultivated at a temperature of the recombinant vector is sensitive, obtains the recombinant vector and pXO2 plasmid removes Purpose Bacillus anthracis.The temperature of the recombinant vector sensitivity can be 37-42 DEG C.

The present invention also provides a kind of sgRNA, the sgRNA is the sgRNA2；

The present invention also provides biomaterial (being denoted as biomaterial 1) relevant to the sgRNA2, the biomaterials 1 For any one of following B1) to B4):

B1 the nucleic acid molecules of the sgRNA2) are encoded；

B2) contain B1) expression cassettes of the nucleic acid molecules；

B3) contain B1) recombinant vectors of the nucleic acid molecules or contain B2) recombinant vector of the expression cassette；

B4) contain B1) recombinant microorganisms of the nucleic acid molecules or contain B2) recombinant microorganism of the expression cassette or Contain B3) recombinant microorganism of the recombinant vector.

B1) nucleic acid molecules can be DNA molecular shown in sequence 4 in sequence table.

B2 the expression cassette (sgRNA2 expression casette) containing the nucleic acid molecules for encoding the sgRNA2 described in), refers to The DNA of sgRNA2 can be expressed in host cell, which not only may include the promoter for starting sgRNA2 genetic transcription, also It may include the terminator for terminating sgRNA2 genetic transcription.Further, the expression cassette may also include enhancer sequence.

The recombinant vector of the sgRNA2 expression casette can be contained with existing expression vector establishment.The carrier can be Plasmid, sticking grain, bacteriophage or viral vectors.The plasmid concretely pJOE8999.

B3) recombinant vector can be the pJO2T.

The microorganism can be yeast, bacterium, algae or fungi.

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

X1) complete sets of products is made of the sgRNA2 and Cas9 protein；

X2) complete sets of products (is denoted as biomaterial by the biomaterial 1 and biomaterial relevant to Cas9 protein 2) it forms；The biomaterial 2 is any one of following C1) to C4):

C1 the nucleic acid molecules of Cas9 protein) 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 complete sets of products has following any purposes:

X1 pXO2 plasmid in Bacillus anthracis) is removed；

X2) pXO2 plasmid product in preparation removal Bacillus anthracis；

X3) preparation prevention and/or treatment Bacillus anthracis associated diseases vaccine and/or drug；

X4) prevent and/or treat Bacillus anthracis associated diseases.

C1) nucleic acid molecules can be DNA molecular shown in sequence 1 in sequence table.

C2 the expression cassette (Cas9 expression casette) of the nucleic acid molecules containing coding Cas9 protein described in), refers to energy Enough DNA that Cas9 is expressed in host cell, the DNA may include not only the promoter for starting Cas9 genetic transcription, may also include Terminate the terminator of Cas9 genetic transcription.Further, the expression cassette may also include enhancer sequence.

The recombinant vector of the Cas9 expression casette can be contained with existing expression vector establishment.The carrier can be matter Grain, sticking grain, bacteriophage or viral vectors.

C3) recombinant vector can be the pJO2T.

The microorganism can be yeast, bacterium, algae or fungi.

The method of pXO2 plasmid or the sgRNA2 or the biomaterial 1 in the removal Bacillus anthracis, or Following any applications of the complete sets of products, also belong to protection scope of the present invention:

Y1) the application in removal Bacillus anthracis in pXO2 plasmid；

Y2) the application in preparation removal Bacillus anthracis in 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 the removal of Bacillus anthracis virulence Large plasmid pXO2 plasmid using CRISPR/Cas9 system, at Function has obtained the Bacillus anthracis without containing pXO2 plasmid, and method of the invention is easy to operate, and the vaccine strain to construct new mentions Supplied it is faster, be more convenient new means, provide new thinking for the prevention and treatment of Bacillus anthracis.

Detailed description of the invention

Fig. 1 is skeleton plasmid pJOE8999 map.

Fig. 2 is the PCR qualification result of scissors plasmid pJO1T.M swimming lane is marker.

Fig. 3 is the PCR qualification result of scissors plasmid pJO2T.M swimming lane is marker.

Fig. 4 is to identify that scissors plasmid converts the electrophoretogram of Escherichia coli and bacillus anthracis PCR using pJOE8999-F/R.Its In, each swimming lane is followed successively by DNA molecular amount standard (M, marker), DH5 α containing pJO1T, containing pJO1T's from left to right SCS110, the A16PI2 containing pJO1T, DH5 α containing pJO2T, the SCS110 containing pJO2T and the A16Q1 containing pJO2T.

Fig. 5 is that scissors plasmid pJO1T cuts off pXO1 preliminary screening PCR identification electrophoretogram in bacillus anthracis.Wherein, M swimming lane For marker ,+swimming lane is A16PI2 (pXO1⁺pXO2^-) compare, 1-12 swimming lane is 12 monoclonals selected.

Fig. 6 is that scissors plasmid pJO1T drives away the multipair primer PCR identification electrophoretogram of pXO1 in bacillus anthracis.M swimming lane is It is template as a result, b is using A16PI2TC as the result of template that marker, a, which are using A16PI2,.

Fig. 7 is that scissors plasmid pJO2T drives away pXO2 preliminary screening PCR identification electrophoretogram in bacillus anthracis.M swimming lane is Marker ,+swimming lane compare for A16Q1, and 1-18 swimming lane is 18 monoclonals selected.

Fig. 8 is that scissors plasmid pJO2T drives away the multipair primer PCR identification electrophoretogram of pXO2 in bacillus anthracis.M swimming lane is It is template as a result, b is using A16Q1TC as the result of template that marker, a, which are using A16Q1,.

Fig. 9 is PA and LF detection of expression result in A16PI2 and A16PI2TC.

Figure 10 is (100 times) observation results of thallus capsule stain optical microscopy.A is A16Q1's as a result, B is The result of A16PI2TC.

Figure 11 is that the PCR that external source " scissors plasmid " pJO1T and pJO2T drives away from bacillus anthracis identifies electrophoretogram.M swimming lane It is the testing result that pJO1T drives away for marker, A, B is the testing result that pJO2T drives away.

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 in sequence table, and last bit is the 3 ' of corresponding DNA Terminal nucleotide.

A16Q1 (Liu X, Wang D, Wang H, Feng E, Zhu L, Wang H.Curing of in following embodiments Plasmid pXO1from Bacillus anthracis Using Plasmid Incompatibility.PLoS One, 2012,7 (1): e29875) public can obtain the biomaterial from applicant, and which only attaches most importance to the phase of duplicate invention It closes used in experiment, not can be used as other purposes and use.

A16PI2 (Wang H, Liu X, Feng E, Zhu L, Wang D, Liao X, Wang in following embodiments H.Curing the plasmid pXO2from Bacillus anthracis A16using plasmid Incompatibility.Curr Microbiol, 2011,62 (3): 703-709.) public can obtain the biology from applicant Material, the biomaterial are only attached most importance to used in the related experiment of duplicate invention, not can be used as other purposes and are used.

Embodiment 1 removes 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

1.1pXO1 plasmid 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-Cas9 System [J] .Applied and Environmental Microbiology, 2016,82 (17): 5421-5427.) (see figure 1) between two sites Bsal, the correct recombinant plasmid of obtained sequence is named as pJO1T, pJOE8999 contains the coding of Cas9 The expression cassette of gene, the coding gene sequence of Cas9 are sequence 1 in sequence table, Cas9 shown in energy coded sequence 2.Step is such as Under:

(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), following oligonucleotides (being shown in Table 1) is synthesized:

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 linearization plasmid: the load that the double-strand N20 and step (3) that Connection Step (2) obtains are obtained Body skeleton, obtains 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 As pJO1T as removes " 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 removes " 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′-TACGATAACTTGTAATAGCCCTTT-3′；

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

2. the removal 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.For ease of operation, the present invention is tested using the less-virulent strain of Bacillus anthracis. PJO1T is imported into Bacillus anthracis A16PI2 (pXO1⁺pXO2^-) (bacterial strain contains pXO1, does not contain pXO2, hereinafter referred to as A16PI2 in), the pXO1 in the bacterial strain is removed, pJO2T is imported into Bacillus anthracis A16Q1 (pXO1^-pXO2⁺) (bacterial strain contains There is pXO2 without containing in pXO1, hereinafter referred to as A16Q1), removes the pXO2 in the bacterial strain.

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, goes in SCS110 (Fig. 4), The obtained recombinant bacterium containing pJO1T is denoted as SCS110-pJO1T, the obtained recombinant bacterium containing pJO2T is denoted as SCS110-pJO2T。

Electricity is transferred to A16PI2 and A16Q1 respectively by scissors plasmid pJO1T and pJO2T: respectively from SCS110-pJO1T and SCS110-pJO2T extracts pJO1T and pJO2T, and scissors the plasmid pJO1T and pJO2T of extraction is taken to be added separately to the anthrax of 40 μ L (preparation of competent cell, reference literature: Gao Meiqin, Liu Xiankai, Feng in bacillus A16PI2 and A16Q1 competent cell That tinkling of pieces of jade, Tang Hengming, Zhu Li, Chen Fusheng, A16R plants of eag gene deletion mutants of Wang Hengliang Bacillus anthracis construct microorganisms Journal, 2009,49 (1): 23-31.), ice bath 2min, shock by electricity (condition: voltage, 0.6kv；Resistance, 500；Capacitor, 25 μ F；It is used Electric shock instrument are as follows: originate from II electroporator of Bio-RAD GenePulser in the U.S.；Electric shock cup is 0.1cm.), electricity is added Turn resuscitation fluid 1mL, is placed in the LB solid plate of 30 DEG C of shaking table recovery 3h, Tu Hanyou Kan (25 μ g/ml) resistances, places 30 DEG C of trainings It supports and is cultivated in case, next day grows apparent monoclonal.Toothpick picking monoclonal is made bacteria suspension and makees template, special with pJOE8999 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).It will Positive colony is sequenced with primer pair Spacer-F/R and (is shown in Table 1), and confirmation scissors plasmid is correctly transformed into Bacillus anthracis In, the A16PI2 containing pJO1T is named as A16PI2T (pXO1⁺ pJO1T⁺) (referred to as A16PI2T), it will contain pJO2T's A16Q1 is named as A16Q1T (pXO2⁺ pJO2T⁺) (referred to as A16Q1T).

2.2 screening processes:

2.2.1 induction Cas9 expression: A16PI2T (pXO1 is picked them separately⁺ pJO1T⁺) and A16Q1T (pXO2⁺ pJO2T⁺) Monoclonal is inoculated into 5ml LB liquid medium (containing 25 μ g/ml Kan), 30 DEG C 220rpm shaking table culture 3 hours, add 0.4% mannose turns 25 DEG C 220rpm shaking table Fiber differentiation 10 hours, and 1%, which connects bacterium amount switching 5ml LB liquid medium, passes Generation, the same terms induced for 1 generation again, obtain culture solution.

2.2.2PCR virulence Large plasmid loss situation is verified: the culture solution dilution 10 that step 2.2.1 is obtained⁵LB is applied after times 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.Tooth Picking monoclonal is signed, whether the removal of PCR identification pXO1 and pXO2 succeeds.

(1) preliminary screening of the removal of pXO1

It a use of specific gene (protective antigens pag) on Bacillus anthracis pXO1 is that target progress PCR is preliminary Whether screening, verifying pXO1 remove.It is control with A16PI2 (+swimming lane), picks 12 clones's (1-12 swimming lane), use primer PCR verifying is carried out to pag-F and pag-R (table 1), as the result is shown 6 no PCR specific amplified bands of clone (3,5,7,8,9, 10 swimming lanes), show that the pXO1 of these clones may lose (Fig. 5).

(2) confirmation of the excision of pXO1

Obtain 1 of selecting step (1) may eliminate the Bacillus anthracis of pXO1, use 17 pairs be located on pXO1 This clone of primer pair further verifying confirmation, is control with A16PI2, A16PI2 has PCR specific amplified item as the result is shown Band, all special primers, all without PCR specific amplified band, show this clone's in the Bacillus anthracis of picking PXO1 has been removed (Fig. 6), and naming this clone is A16PI2TC (pXO1^-pJO1T⁺) (referred to as A16PI2TC) (be shown in Table 2)。

17 pairs of primers used are as follows: pXO1-7 (pXO1-7F and pXO1-7R), pXO1-13 (pXO1-13F and pXO1- 13R), pXO1-16 (pXO1-16F and pXO1-16R), pXO1-23 (pXO1-23F and pXO1-23R), pXO1-32 (pXO1-32F With pXO1-32R), pXO1-42 (pXO1-42F and pXO1-42R), pXO1-51 (pXO1-51F and pXO1-51R), pXO1-55 (pXO1-55F and pXO1-55R), pXO1-59 (pXO1-59F and pXO1-59R), pXO1-67 (pXO1-67F and pXO1-67R), PXO1-70 (pXO1-70F and pXO1-70R), pXO1-90 (pXO1-90F and pXO1-90R), pXO1-95 (pXO1-95F with PXO1-95R), pXO1-98 (pXO1-98F and pXO1-98R), pXO1-116 (pXO1-116F and pXO1-116R), pXO1-133 (pXO1-133F and pXO1-133R), pXO1-142 (pXO1-142F and pXO1-142R), sequence is shown in Table 1.

(3) preliminary screening of the excision of pXO2

It a use of specific gene (capsule gene capA) on Bacillus anthracis pXO2 is that target progress PCR is tentatively sieved Whether choosing, verifying pXO2 remove.It is control with A16Q1 (+swimming lane), picks 18 clones's (1-18 swimming lane), use primer CapA-F and capA-R carry out PCR verifying (being shown in Table 1), as the result is shown 10 no PCR specific amplified bands of clone (1,2,5,6, 8,9,10,13,14,16 swimming lanes), show that the pXO2 of these clones may lose (Fig. 7).

(4) confirmation of the excision of pXO2

Obtain 1 of selecting step (3) may eliminate the Bacillus anthracis of pXO2, use 12 pairs be located on pXO2 This clone of primer pair further verifying confirmation, is control with A16Q1, A16Q1 has PCR specific amplified item as the result is shown Band, all special primers, all without PCR specific amplified band, show this clone's in the Bacillus anthracis of picking PXO2 has lost (Fig. 8), and naming this clone is A16Q1TC (pXO2^-pJO2T⁺) (referred to as A16Q1TC).

The primer is as follows: pXO2-007 (pXO2-007F and pXO2-007R), pXO2-016 (pXO2-016F and pXO2- 016R), pXO2-023 (pXO2-023F and pXO2-023R), pXO2-027 (pXO2-027F and pXO2-027R), pXO2-039 (pXO2-039F and pXO2-039R), pXO2-060 (pXO2-060F and pXO2-060R), pXO2-084 (pXO2-084F with PXO2-084R), pXO2-089 (pXO2-089F and pXO2-089R), pXO2-094 (pXO2-094F and pXO2-094R), PXO2-097 (pXO2-097F and pXO2-097R), pXO2-107 (pXO2-107F and pXO2-107R), pXO2-111 (pXO2- 111F and pXO2-111R), primer sequence is as shown in table 1.

2.2.3 phenotypic evaluation:

(1) Western blot is analyzed to identify PA and LF and expresses in Bacillus anthracis A16PI2TC

The obtained A16PI2TC of A16PI2 and building is inoculated into solid LB media respectively, is placed into CO₂Incubator 37 DEG C culture 13h.Lawn is scraped with oese, is added in 2ml concussion pipe (added with bead), preparing Urea Lysis liquid, (this is molten Liquid is that 1%DTT is added into 8M aqueous solution of urea, 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 divides Dress, -80 DEG C freeze.40 μ l protein solutions are taken, isometric 2 × SDS-PAGE sample-loading buffer are added, boiling 10min makes albumen Denaturation.The pre-prepared colloid for taking 12%, the egg obtained according to albumen Marker (Beijing Quanshijin Biotechnology Co., Ltd), A16PI2 The every 10 μ l of hole loading of the sequence for the protein solution that white solution, A16PI2TC are obtained repeats three parts of loading, carries out SDS-PAGE albumen Electrophoresis.After protein electrophorese, portion is used to Coomassie brilliant blue and dyes, and is in addition utilized respectively PA antibody and LF antibody work for two parts For primary antibody carry out Western blot analysis, detect PA used in PA antibody be rabbit polyclonal antibody (abcam company, http: // Www.abcom.cn, ab21268), secondary antibody is the goat antirabbit of HRP label.It is anti-for mouse monoclonal to detect LF antibody used in LF Body (abcam company, http://www.abcom.cn, ab69486), secondary antibody are the mountain sheep anti mouse of HRP label.

The result of Coomassie brilliant blue dyeing shines glue using scanner, and the result of Western blot analysis uses low temperature gel Imager photograph.The results show that A16PI2 has PA (83kDa) and LF (90kDa) expression, A16PI2TC is without PA and LF expression (figure 9), show A16PI2TC without containing pJO1T.Wherein, PA and LF is the specifically expressed protein of pXO1.

(2) india ink stain confirms Bacillus anthracis A16Q1TC 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: A16Q1 and A16Q1TC are inoculated into LB solid medium (containing 8 ‰ (mass percents) NaHCO₃, 5% (percent by volume) horse serum), it is then placed into logical 5%CO₂37 DEG C of culture 48h of constant incubator or so, choose Take Bacillus anthracis that bacteria suspension is made, backward bacteria suspension in plus 1 drop prepared Chinese ink mix well, one is then added dropwise on glass slide Drop, covered compress, and wiping redundant solution using lens wiping paper can microscopy.100 times of microscopic examination results, background grey black, bacterium Body is darker, and A16Q1 has pod membrane, and pod membrane permeability is high, has one layer of colorless and transparent circle (A in Figure 10) outside thallus, A16Q1TC is without pod Film, thallus without colorless and transparent circle (B in Figure 10), show A16Q1TC without containing pJO2T outside.

3. the process that external source " scissors plasmid " pJO1T and pJO2T is removed from Bacillus anthracis

Because external source " scissors plasmid " pJO1T and pJO2T that the present invention constructs is Thermo-sensitive plasmid, lost at 37-42 DEG C It loses, and 37 DEG C of optimum growth temps for Bacillus anthracis, in order to avoid Bacillus anthracis passes in excessively high temperature growth Generation, so external source recombinant plasmid pJO1T and pJO2T are lost in selection at 37 DEG C.Steps are as follows:

The monoclonal of A16PI2TC or A16Q1TC that picking step 2 obtains are connected to 5mL LB liquid medium at 37 DEG C 10-12h is cultivated on shaking table, is then transferred in another 5mL LB liquid medium in cultivating 12h on 37 DEG C of shaking tables, instead by 1% After transferring 3 times by 1% again, gradient dilution is carried out to bacterium solution, takes 10⁴、10⁵The bacterium solution of two dilutions again, coating nonreactive are flat Plate.It is placed in 30 DEG C of incubators and cultivates, the monoclonal on next day picking plate carries out contact plate, each monoclonal distinguishes contact plate In LB nonreactive plate and LB (containing 25 μ g/ml Kan) agar plate, the plate after contact plate is placed in 30 DEG C of incubators and is cultivated.It is secondary Day, picking grown on non-resistant plate and on Kan (containing 25 μ g/ml Kan) resistance agar plate non-growing monoclonal into Row bacterium colony PCR identification and Kan resistance LB liquid medium switching culture (30 DEG C of shaking table cultures), determine it in Kan resistance LB liquid It is not grown in body culture medium, which is positive colony.The positive colony selected is inoculated in non-resistant LB liquid medium Culture, then genome, does template with genome, using the special primer pJOE8999-F/R (being shown in Table 1) on vector plasmid into Row PCR, identification external source recombinant plasmid are removed situation, and using pJOE8999 as control.

The results show that positive colony does not have PCR specific amplified band, show that the pJO1T of these clones and pJO2T have lost It loses (Figure 11).So far, having obtained pXO1 or pXO2 plasmid missing, the Bacillus anthracis without other exogenous plasmids is prominent again simultaneously The bacterial strain of no pXO1 and pXO2 is respectively designated as A16PI2C (pXO1 by mutant^-pJO1T^-) (referred to as A16PI2C) and A16Q1C (pXO2^-pJO2T^-) (referred to as A16Q1C).

Oligonucleotide sequence and primer used in 1. present invention of table

Bacterial strain and characteristic in 2. present invention of table

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 pXO2 plasmid in Bacillus anthracis is removed

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 4104

<212> DNA

<213>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

<400> 2

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

245 250 255

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

260 265 270

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

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

435 440 445

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

<400> 3

ataacttgta atagcccttt gctagaaata gcaagttaaa ataaggctag tccgttatca 60

acttgaaaaa gtggcaccga gtcggtgctt ttt 93

<210> 4

<211> 93

<212> DNA

<213>artificial sequence

<400> 4

acacaaagtg atagcctaga gctagaaata gcaagttaaa ataaggctag tccgttatca 60

acttgaaaaa gtggcaccga gtcggtgctt ttt 93

Claims (10)

1. the method for removing pXO2 plasmid in Bacillus anthracis, it is characterised in that: the method uses CRISPR/Cas9 system PXO2 plasmid in the Bacillus anthracis that sets out is removed, includes the sgRNA of entitled sgRNA2 in the CRISPR/Cas9 system, The target sequence of sgRNA2 identification for pXO2 plasmid contains and distinguished sequence that Bacillus anthracis does not contain.

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

A1) DNA fragmentation shown in 1-20 of sequence 4 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.

3. method according to claim 1 or 2, it is characterised in that: the sequence of the sgRNA2 is by sequence 4 in sequence table In T replace 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 sgRNA2 is imported in born of the same parents bacillus, expresses the sgRNA2, is removed The Bacillus anthracis of pXO2 plasmid.

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.sgRNA is any sgRNA2 in claim 1-3.

8. biomaterial relevant to the sgRNA2 any in claim 1-3, is following B1) any one of to B4):

B1 the nucleic acid molecules of any sgRNA2 in claim 1-3) are encoded；

B2) contain B1) expression cassettes of the nucleic acid molecules；

B3) contain B1) recombinant vectors of the nucleic acid molecules or contain B2) recombinant vector of the expression cassette；

B4) contain B1) recombinant microorganisms of the nucleic acid molecules or contain B2) recombinant microorganism of the expression cassette or contain B3) the recombinant microorganism of the recombinant vector.

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

X1) complete sets of products is made of the sgRNA2 and Cas9 protein any in claim 1-3；

X2) complete sets of products, the biomaterial described in claim 8 and biomaterial relevant to Cas9 protein form；It is described Biomaterial relevant to Cas9 protein is any one of following C1) to C4):

C1 the nucleic acid molecules of Cas9 protein) 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.

10. any sgRNA2 or claim 8 institute in any the method or claim 1-3 in claim 1-6 State following any applications of complete sets of products described in biomaterial or claim 9:

Y1) the application in removal Bacillus anthracis in pXO2 plasmid；

Y2) the application in preparation removal Bacillus anthracis in 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.