CN109825531B - Method for removing pXO2 plasmid in Bacillus anthracis - Google Patents

Abstract

The invention discloses a method for removing pXO2 plasmid in Bacillus anthracis. The method for removing the pXO2 plasmid in the bacillus anthracis is completed by adopting a CRISPR/Cas9 system, the CRISPR/Cas9 system comprises sgRNA with the name of sgRNA2, a target sequence recognized by the sgRNA2 is a specific sequence contained in pXO2 plasmid and not contained in the bacillus anthracis, and the target sequence is a DNA fragment shown in the 1 st to 20 th sites of the sequence 4 in a sequence table. The method adopts the CRISPR/Cas9 system to remove the bacillus anthracis virulence large plasmid pXO2 plasmid, successfully obtains the bacillus anthracis without pXO2 plasmid, has simple operation, provides a faster, more convenient and new means for constructing a new vaccine strain, and provides a new thought for the prevention and the treatment of the bacillus anthracis.

Description

Method for removing pXO2 plasmid in Bacillus anthracis

Technical Field

The invention relates to a method for removing pXO2 plasmid in Bacillus anthracis in the field of biotechnology.

Background

Bacillus anthracis (also called as Bacillus anthracis) is a gram-positive aerobic bacillus capable of forming spores, can cause anthracnose of people and livestock, has extremely high death rate if not treated in time, causes great economic loss and threatens life safety. Bacillus anthracis contains two virulence large plasmids associated with pathogenesis: pXO1(181.6kb) and pXO2(96.2 kb). Plasmid pXO1 encodes anthrax toxin proteins such as protective antigens, lethal factors and edema factors, and their regulatory proteins. Plasmid pXO2 encodes a gene involved in capsule formation and degradation. The pathogenicity of the two plasmids is crucial to the pathogenicity of the bacillus anthracis, and the loss of any one plasmid can cause the virulence of the bacillus anthracis to be greatly reduced. Therefore, the research on the bacillus anthracis virulence large plasmid is always a hot spot of research. The construction of a mutant strain for removing virulence plasmids is very important for researching the role of the plasmids in the pathogenic bacteria of the bacillus anthracis and the related regulation and control of chromosomes.

Early removal of large plasmids of bacteria may use chemical reagents such as acridine orange, neomycin, ethidium bromide, etc. High temperature culture or ultraviolet irradiation. However, these methods have potential problems, firstly, poor specificity, i.e., the possibility of removing plasmids other than the desired plasmid during the process, and secondly, the possibility of random mutation of the host cell during the process.

CRISPR/Cas system is a repetitive structure widely distributed in bacterial and archaeal genomes, considered as an acquired immune system for prokaryotes to defend against foreign phage, plasmid or other foreign DNA infection, in which system crRNA, with the aid of a transactivating crRNA (tracrrna), recruits effector proteins (Cas proteins) and brings them to the target DNA sequence, which cleaves the foreign DNA sequence using its nuclease function, causing DNA Double Strand Breaks (DSB). This system is widely used for gene editing. In order to make the use of the system more convenient and simpler, researchers have artificially transformed the crRNA-tracrRNA double-stranded RNA complex in the type II CRISPR/Cas9 system into a chimeric single-stranded RNA, called single guide RNA (sgRNA), by virtue of its specific sequence pairing of 20nt at the 5 'end (i.e. the spacer sequence, here called N20) to target a DNA site, and PAM (5' -NGG-3 ') at the 3' end of the target DNA sequence is a Cas9 recognition site, which cannot be included in the sgRNA. When the method is used, only the N20 sequence at the 5' end of the sgRNA needs to be replaced to guide the Cas protein to cut different target DNA sequences. The system is firstly applied to gene editing of human and mouse embryonic stem cells in 2013, is successfully applied to various animals, plants and microorganisms such as mice, pigs, cynomolgus monkeys, zebrafish, arabidopsis thaliana, sorghum, tobacco, rice, nematodes, yeasts, escherichia coli and the like at present, and becomes a gene editing tool widely applied to various fields of biology and medicine.

Disclosure of Invention

The invention aims to solve the technical problem of how to remove the plasmid pXO2 of the bacillus anthracis.

In order to solve the technical problems, the invention firstly provides a method for removing pXO2 plasmid in Bacillus anthracis, the method adopts a CRISPR/Cas9 system to remove pXO2 plasmid in original Bacillus anthracis, the CRISPR/Cas9 system comprises sgRNA named sgRNA2, and the target sequence recognized by the sgRNA2 is a specific sequence which is contained in pXO2 plasmid and not contained in the Bacillus anthracis.

The starting B.anthracis contains the pXO2 plasmid. The starting Bacillus anthracis may contain the pXO2 plasmid and not the pXO2 plasmid.

In one embodiment of the invention, the Bacillus anthracis starting bacterium is A16Q 1.

In the above method, the target sequence may be a1), a2), or A3) as follows:

A1) a DNA fragment shown in 1 st to 20 th sites of a sequence 4 in a sequence table;

A2) a DNA fragment derived from a1) having 75% or more than 75% identity to the DNA sequence defined in a 1);

A3) a DNA fragment derived from A1) which hybridizes under stringent conditions with the DNA sequence defined in A1).

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to positions 1-20 of the sequence 4 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In the above method, the sequence of the sgRNA2 may specifically be an RNA sequence obtained by replacing T in sequence 4 in the sequence table with U.

The method can comprise the steps of introducing an expression cassette containing a coding gene of the sgRNA2 into the starting bacillus anthracis, and expressing the sgRNA2 to obtain the bacillus anthracis with the pXO2 plasmid removed.

Introduction of an expression cassette containing the sgRNA2 encoding gene into the b.anthracis starter can be achieved by introducing a recombinant vector containing the expression cassette into the b.anthracis starter.

In the above method, the CRISPR/Cas9 system may further comprise a Cas9 protein. The sequence of the Cas9 protein may be sequence 2 in the sequence listing.

The method can also comprise the step of introducing an expression cassette containing a coding gene of the Cas9 protein into the starting Bacillus anthracis to express the Cas9 protein.

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

In the method, the introduction of the expression cassette containing the encoding gene of the Cas9 protein into the starting Bacillus anthracis can be realized by introducing a recombinant vector containing the expression cassette into the starting Bacillus anthracis.

Specifically, the expression cassette containing the encoding gene of sgRNA2 and the expression cassette containing the encoding gene of Cas9 protein can be realized by introducing a recombinant vector containing the two expression cassettes into the starting Bacillus anthracis.

The recombinant vector can be specifically pJO2T, and pJO2T is a recombinant vector which is obtained by inserting the target sequence between multiple cloning sites of an initial vector and can express the sgRNA2 and the Cas9 protein. The starting vector can be a temperature-sensitive vector such as pJOE 8999.

The method may further comprise demethylating the recombinant vector prior to introduction of the recombinant vector into the Bacillus anthracis producer. The demethylation can be achieved by introducing the recombinant vector into the E.coli SCS 110.

The method may further comprise removing the recombinant vector after removing the pXO2 plasmid.

The recombinant vector can be removed, so that the recombinant bacillus anthracis which is introduced into the recombinant vector and from which the pXO2 plasmid is removed can be cultured at a temperature sensitive to the recombinant vector, and the target bacillus anthracis from which both the recombinant vector and the pXO2 plasmid are removed is obtained. The temperature to which the recombinant vector is sensitive may be 37-42 ℃.

The invention also provides an sgRNA which is 2;

the invention also provides a biomaterial (denoted as biomaterial 1) related to the sgRNA2, wherein the biomaterial 1 is any one of the following B1) to B4):

B1) a nucleic acid molecule encoding the sgRNA 2;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

B1) The nucleic acid molecule can be a DNA molecule shown in a sequence 4 in a sequence table.

B2) The expression cassette containing a nucleic acid molecule encoding the sgRNA2 (sgRNA2 gene expression cassette) refers to a DNA capable of expressing the sgRNA2 in a host cell, and the DNA may include not only a promoter for initiating transcription of the sgRNA2 gene, but also a terminator for terminating transcription of the sgRNA2 gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the sgRNA2 gene expression cassette can be constructed by using an existing expression vector. The vector may be a plasmid, cosmid, phage or viral vector. The plasmid may be pJOE 8999.

B3) The recombinant vector may be the pJO 2T.

The microorganism may be a yeast, bacterium, algae or fungus.

The invention also provides a kit of parts, the product being the following X1) or X2):

x1) kit consisting of the sgRNA2 and Cas9 protein;

x2) kit consisting of the biomaterial 1 and a biomaterial related to Cas9 protein (denoted as biomaterial 2); the biological material 2 is any one of the following C1) to C4):

C1) a nucleic acid molecule encoding a Cas9 protein;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2);

C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector.

The kit has any one of the following uses:

x1) removing pXO2 plasmid in the bacillus anthracis;

x2) preparing a pXO2 plasmid product for removing the plasmid in the bacillus anthracis;

x3) preparing vaccine and/or medicament for preventing and/or treating diseases caused by bacillus anthracis;

x4) for the prevention and/or treatment of diseases caused by bacillus anthracis.

C1) The nucleic acid molecule can be a DNA molecule shown as a sequence 1 in a sequence table.

C2) The expression cassette containing a nucleic acid molecule encoding a Cas9 protein (Cas9 gene expression cassette) refers to a DNA capable of expressing Cas9 in a host cell, and the DNA can comprise a promoter for starting the transcription of the Cas9 gene and a terminator for stopping the transcription of the Cas9 gene. Further, the expression cassette may also include an enhancer sequence.

Existing expression vectors can be used to construct recombinant vectors containing the Cas9 gene expression cassette. The vector may be a plasmid, cosmid, phage or viral vector.

C3) The recombinant vector may be the pJO 2T.

The microorganism may be a yeast, bacterium, algae or fungus.

The method for removing the pXO2 plasmid in the bacillus anthracis, or the sgRNA2, or the biological material 1, or any one of the following applications of the complete set of products also belong to the protection scope of the invention:

y1) in removing pXO2 plasmid in Bacillus anthracis;

y2) in the preparation of pXO2 plasmid products for removing Bacillus anthracis;

y3) in the preparation of vaccines and/or medicaments for preventing and/or treating diseases caused by the Bacillus anthracis;

y4) in the prevention and/or treatment of diseases caused by Bacillus anthracis.

The method adopts the CRISPR/Cas9 system to remove the bacillus anthracis virulence large plasmid pXO2 plasmid, successfully obtains the bacillus anthracis without pXO2 plasmid, has simple operation, provides a faster, more convenient and new means for constructing a new vaccine strain, and provides a new thought for the prevention and the treatment of the bacillus anthracis.

Drawings

FIG. 1 is a map of backbone plasmid pJOE 8999.

FIG. 2 shows the result of PCR identification of scissor plasmid pJO 1T. And the lane M is marker.

FIG. 3 shows the result of PCR identification of scissor plasmid pJO 2T. And the lane M is marker.

FIG. 4 is an electrophoretogram of PCR for identifying Scissors plasmid transformed E.coli and B.anthracis using pJOE 8999-F/R. From left to right, the lanes include DNA molecular weight standards (M, marker), DH 5. alpha. containing pJO1T, SCS110 containing pJO1T, A16PI2 containing pJO1T, DH 5. alpha. containing pJO2T, SCS110 containing pJO2T, and A16Q1 containing pJO 2T.

FIG. 5 is the electrophoresis diagram of the pXO1 preliminary screening PCR of Bacillus anthracis excised by scissor plasmid pJO 1T. Wherein Lane M is marker and Lane + is A16PI2(pXO 1)⁺pXO2^-) Comparison was madeLanes 1-12 are selected 12 single clones.

FIG. 6 is a PCR identification electrophoretogram of Scissors plasmid pJO1T for expelling pXO1 pairs of primers from Bacillus anthracis. Lane M is marker, a is the result using a16PI2 as a template, and b is the result using a16PI2TC as a template.

FIG. 7 shows the electrophoresis of the PCR identification of pXO2 in Scissors plasmid pJO2T for expelling Bacillus anthracis. Marker in lane M, A16Q1 in lane + for control, and 18 single clones selected in lanes 1-18.

FIG. 8 is a PCR identification electrophoretogram of Scissors plasmid pJO2T for expelling pXO2 pairs of primers from Bacillus anthracis. Lane M is marker, a is the result using a16Q1 as a template, and b is the result using a16Q1TC as a template.

FIG. 9 shows the results of PA and LF expression assays in A16PI2 and A16PI2 TC.

FIG. 10 shows the results of observation of bacterial capsule staining by an optical microscope (100-fold magnification). A is the result of A16Q1, B is the result of A16PI2 TC.

FIG. 11 is a PCR identification electropherogram of the exclusion of exogenous "scissors plasmids" pJO1T and pJO2T from Bacillus anthracis. The M lane is marker, A is the detection result of the expulsion of pJO1T, and B is the detection result of the expulsion of pJO 2T.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

A16Q1(Liu X, Wang D, Wang H, Feng E, Zhu L, Wang H. curing of Plasmid pXO1from Bacillus antrhacis Using Plasmid Incompatibility. PLoS One,2012,7(1): E29875) in the following examples was publicly available from the applicant, and was used only for repeating the experiments related to the present invention, and was not usable for other purposes.

A16PI2(Wang H, Liu X, Feng E, Zhu L, Wang D, Liao X, Wang H. Current the plasmid pXO2from Bacillus anthrocus A16using plasmid compatibility. Curr Microbiol,2011,62(3):703 and 709.) in the following examples was publicly available from the applicant, and was used only for repeating the experiments related to the present invention, and was not usable for other purposes.

Example 1 removal of Bacillus anthracis pXO1 plasmid and pXO2 plasmid using CRISPR/Cas9 System

1. Construction of plasmid containing CRISPR/Cas9 system and target sequence (scissor plasmid)

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

Taking a section of sequence (ORF16) on a bacillus anthracis virulence large plasmid pXO1(GenBank accession No. AF065404) as a target sequence; a sequence (GBAA _ pXO2_0038) on a bacillus anthracis virulence large plasmid pXO2(GenBank accession NO. NC-007323) is used as a target sequence, software sgRNAcas9_ V3.0_ GUI (or other software) is used for designing an N20 specific target sequence in the sgRNA, the target sequence on pXO1 is marked as O1T (1 st to 20 th positions of a sequence 3 in a sequence table), and the target sequence on pXO2 is marked as O2T (1 st to 20 th positions of a sequence 4 in the sequence table), and the sequences are shown in Table 1. The two target sequences are aligned with the chromosome sequence (GenBank access NO. NC-003997) of the Bacillus anthracis Ames, and the two target sequences do not exist on the chromosome.

1.2 construction of exogenous "Scissors plasmids" that cleave pXO1 and pXO2 "

1.2.1 inserting O1T into temperature-sensitive shuttle plasmid pJOE8999(7.8Kb) (Josef Altenbuchner. edition of the Bacillus subtilis Genome by the CRISPR-Cas9 System [ J ]. Applied and Environmental Microbiology,2016,82(17):5421-5427.) (see FIG. 1) between two Bsal sites, the obtained recombinant plasmid with correct sequence is named as pJO1T, pJOE8999 contains expression cassette of coding gene of Cas9, the coding gene sequence of Cas9 is sequence 1 in the sequence table, and Cas9 shown in the coding sequence 2. The method comprises the following steps:

(1) synthesis of N20 oligonucleotide: the following oligonucleotides (see Table 1) were synthesized by adding 4nt protruding linkers (TACG; AAAC) to both ends of the O1T sequence and the reverse complement of O1T, respectively:

Forward OT1(FOT1):5′-TACG ATAACTTGTAATAGCCCTTT-3′；

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

(2) double strand N20 gave: FOT1 and ROT1 were annealed and fused to obtain double-stranded N20 (containing two protruding linkers).

(3) Backbone plasmid linearization: the vector backbone (i.e., linearized plasmid) was recovered by digesting pJOE8999 with Bsa I.

(4) Double strand N20 was attached to the linearized plasmid: and (3) connecting the double-chain N20 obtained in the step (2) with the carrier skeleton obtained in the step (3) to obtain a connecting product.

(5) Screening and identifying recombinant plasmids: the ligation product from step (4) was transformed into DH 5. alpha. and screened for white spots (coated with 4. mu.L IPTG, 40. mu. L X-gal), white spots were selected and identified by PCR using a pair of specific primers (spacer _ F/R, see Table 1) on pJOE8999, using pJOE8999 as a control. The results showed that O1T was successfully inserted into the backbone plasmid pJOE8999 (fig. 2), and the recombinant plasmid was sequenced by commercial companies, and the sequence was confirmed to be correct again, and the recombinant plasmid was pJO1T, which is the "scissors plasmid" with pXO1 removed. pJO1T is a recombinant plasmid obtained by replacing a DNA fragment between two Bsa I recognition sequences of pJOE8999 with O1T, and pJO1T contains a DNA fragment shown in sequence 3 of the sequence table, and the DNA fragment can transcribe sgRNA (denoted as sgRNA1) targeting O1T.

1.2.2 according to the method of step 1.2.1, replacing O1T with O2T, and keeping the other steps unchanged to obtain a recombinant plasmid pJO2T containing O2T, namely a 'scissor plasmid' with pXO2 removed. PCR was performed using a pair of specific primers (spacer _ F/R, see Table 1) on pJOE8999, and the results are shown in FIG. 3, and the sequences are shown in Table 1. The results showed that O2T was successfully inserted into the backbone plasmid pJOE 8999. pJO2T is a recombinant plasmid obtained by replacing a DNA fragment between two Bsa I recognition sequences of pJOE8999 with O2T, and pJO2T contains a DNA fragment shown by a sequence 4 in a sequence table, and the DNA fragment can transcribe sgRNA (denoted as sgRNA2) targeting O2T.

The N20 oligonucleotides used were as follows (see table 1):

Forward OT2(FOT2):5′-TACG ATAACTTGTAATAGCCCTTT-3′；

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

2. removal of B.anthracis virulence large plasmid pXO1/pXO2

2.1 conversion: before introducing the bacillus anthracis into the correct scissors plasmids pJO1T and pJO2T constructed in the step 1, the bacillus anthracis is transferred into escherichia coli SCS110 (Beijing Quanyu Biotechnology Co., Ltd.) for demethylation, and then the bacillus anthracis is respectively transferred into bacillus anthracis competent cells by electricity. For convenience of operation, the invention adopts a weakly toxic strain of Bacillus anthracis to carry out the test. Introduction of pJO1T into Bacillus anthracis A16PI2(pXO 1)⁺pXO2^-) (the strain contains pXO1 and does not contain pXO2, hereinafter referred to as A16PI2), pXO1 in the strain is removed, and pJO2T is introduced into Bacillus anthracis A16Q1(pXO 1)^-pXO2⁺) (the strain contains pXO2 but not pXO1, hereinafter referred to as A16Q1), pXO2 in the strain was removed.

pJO1T and pJO2T were transformed into SCS 110: mu.L of each solution of scissor plasmids pJO1T and pJO2T was added to 50. mu.L of chemically competent cells of Escherichia coli SCS110, mixed uniformly, then ice-cooled for 30min, hot shocked for 90s, ice-cooled for 2min, then added with a proper amount of LB liquid medium, put in a 30 ℃ shaking table for 1h recovery, 150. mu.L of LB agar plate coated with kanamycin (Kan, 25. mu.g/ml) was taken, and put in a 30 ℃ incubator for overnight culture. Picking a single clone of toothpick to prepare a bacterial suspension as a template, carrying out PCR verification on pJOE8999-F/R by using a specific primer pair pJOE8999 (see Table 1), confirming that a scissor plasmid is transformed into SCS110 (figure 4), marking the obtained recombinant bacterium containing pJO1T as SCS110-pJO1T, and marking the obtained recombinant bacterium containing pJO2T as SCS110-pJO 2T.

Scissor plasmids pJO1T and pJO2T were electroporated into A16PI2 and A16Q1, respectively: respectively extracting pJO1T and pJO2T from SCS110-pJO1T and SCS110-pJO2T, respectively, and adding the extracted scissor plasmids pJO1T and pJO2T into 40 mu L of competent cells of Bacillus anthracis A16PI2 and A16Q1 (susceptible cells)Preparation of competent cells, reference is made to the literature: gaomei, Liumikai, Von Erling, Tangchangming, Zhuli, Chenfusheng, Wang Hengliang, Bacillus anthracis A16R strain eag gene deletion mutant strain construction, microbiological report, 2009,49(1):23-31.), ice bath for 2min, electric shock (condition: voltage, 0.6 kv; resistance, 500; capacitance, 25 μ F; the electric shock instrument is as follows: Bio-RAD GenePulser II electroporator available from USA; the electric shock cup is 0.1 cm. ) Adding 1mL of electrotransformation tempering liquid, putting the mixture in a shaker at 30 ℃ for 3h for resuscitation, coating an LB solid plate containing Kan (25 mu g/mL) resistance, putting the mixture in an incubator at 30 ℃ for culture, and growing obvious monoclonal on the next day. Single clones were picked and made into bacterial suspension as a template, and PCR was performed with pJOE8999 specific primer pJOE8999-F/R (see Table 1) to confirm that the scissor plasmid was transformed into Bacillus anthracis (FIG. 4). The primer pair for positive clones Spacer-F/R was sequenced (see Table 1), confirming the correct transformation of the Scissors plasmid into B.anthracis, and A16PI2 containing pJO1T was designated A16PI2T (pXO 1)⁺ pJO1T⁺) (abbreviated as A16PI2T), A16Q1 containing pJO2T was named A16Q1T (pXO 2)⁺ pJO2T⁺) (abbreviated as A16Q 1T).

2.2 screening process:

2.2.1 induction of Cas9 expression: separately pick A16PI2T (pXO 1)⁺ pJO1T⁺) And A16Q1T (pXO 2)⁺ pJO2T⁺) Inoculating the single clone into 5ml LB liquid culture medium (containing 25 mug/ml Kan), shake culturing at 30 deg.C and 220rpm for 3 hours, adding 0.4% mannose, shake culturing at 25 deg.C and 220rpm for 10 hours, transferring 1% inoculum size to 5ml LB liquid culture medium for passage, and inducing again for 1 generation under the same conditions to obtain the culture solution.

2.2.2PCR verification of virulence large plasmid loss: diluting 10 the culture broth obtained in step 2.2.1⁵After doubling, LB plates (containing 25. mu.g/ml Kan) were plated and then the plates were incubated in an incubator at 30 ℃ to allow for about 1 day for obvious colonies to develop. Single clones were picked and PCR verified whether the removal of pXO1 and pXO2 was successful.

(1) preliminary screening for removal of pXO1

A specific gene (protective antigen pag) on the bacillus anthracis pXO1 is used as a target to carry out PCR primary screening to verify whether pXO1 is removed. Using A16PI2(+ lanes) as a control, 12 clones were selected (lanes 1-12), and PCR-verified using primers for pag-F and pag-R (Table 1), indicating that 6 clones had no PCR-specific amplified bands ( lanes 3,5,7,8,9, 10), indicating that pXO1 of these clones may be lost (FIG. 5).

(2) Confirmation of excision of pXO1

Selecting 1 Bacillus anthracis possibly removed with pXO1 obtained in the step (1), further verifying and confirming the clone by using 17 pairs of specific primers on pXO1, taking A16PI2 as a control, showing that the A16PI2 has a PCR specific amplification band, and all the specific primers have no PCR specific amplification band in the picked Bacillus anthracis, indicating that the pXO1 of the clone is removed (figure 6), and naming the clone as A16PI2TC (pXO 1)^-pJO1T⁺) (abbreviated as A16PI2TC) (see Table 2).

The 17 primer pairs used were 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 and 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 pXO 1-1R), pXO1-1 (pXO 1-1F and pXO 1-1R), pXO1-1 and pXO1-1 (pXO 1-1F and pXO 1-1R), pXO1F and pXO 36, pXO1-98(pXO1-98F and pXO1-98R), pXO1-116(pXO1-116F and pXO1-116R), pXO1-133(pXO1-133F and pXO1-133R), and pXO1-142(pXO1-142F and pXO1-142R), the sequences are shown in Table 1.

(3) preliminary screening for excision of pXO2

A specific gene (capsular gene capA) on the bacillus anthracis pXO2 is used as a target for PCR primary screening to verify whether pXO2 is removed. Using a16Q1(+ lanes) as a control, 18 clones were selected (lanes 1-18), and PCR verification was performed using the primers capA-F and capA-R (see table 1), which indicated that 10 clones had no PCR-specific amplified bands ( lanes 1,2,5,6,8,9,10,13,14, 16), indicating that these clones may lose pXO2 (fig. 7).

(4) Confirmation of excision of pXO2

Selecting 1 Bacillus anthracis strain obtained in the step (3) and possibly removing pXO2, and using the strain located in pXO2The clone was further verified and confirmed by the above 12 pairs of specific primers, and the result showed that A16Q1 has a PCR-specific amplification band and none of the specific primers has a PCR-specific amplification band in the picked Bacillus anthracis, indicating that pXO2 of the clone is lost (FIG. 8), and the clone was named A16Q1TC (pXO 2)^-pJO2T⁺) (abbreviated as A16Q1 TC).

The primers used were 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 and 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), and the primer sequences are shown in Table 1.

2.2.3 phenotypic characterization:

(1) western blot analysis confirmed that PA and LF are expressed in Bacillus anthracis A16PI2TC

A16PI2 and the constructed A16PI2TC were inoculated into a solid LB medium and placed in CO₂The culture was carried out in an incubator at 37 ℃ for 13 hours. The lawn was scraped off with an inoculating loop, and the scraped lawn was put into a 2ml shaking tube (containing glass beads) to prepare a urea lysis buffer (1% DTT was added to an 8M urea aqueous solution and used as it was)), and 200. mu.l of the urea lysis buffer was added to the shaking tube and mixed. Then shaking in homogenizer 5500 for 10-15 times, centrifuging at 16 deg.C and 1300rpm for 20min, collecting supernatant (protein solution) and subpackaging, and freezing at-80 deg.C. Mu.l of protein solution was taken, added to an equal volume of 2 XSDS-PAGE loading buffer and boiled for 10min to denature the protein. Taking 12% prefabricated gel, loading 10 μ l of protein solution obtained from protein Marker (Beijing all-type gold Biotechnology Co., Ltd.), A16PI2 and A16PI2TC in sequence, repeating the loading three parts, and performing SDS-PAGE protein electrophoresis. After the completion of protein electrophoresis, one part was stained with Coomassie blue, and the other two parts were subjected to Western blot analysis using PA antibody and LF antibody as primary antibodies, respectively, and PA antibodies for PA detection were rabbit polyclonal antibodies (abcam, http:// www.abcom.cn, ab21268), and two antibodiesThe anti-HRP-labeled goat anti-rabbit antibody. LF antibody used for detecting LF is mouse monoclonal antibody (abcam company, http:// www.abcom.cn, ab69486), and the secondary antibody is goat anti-mouse marked by HRP.

Coomassie blue staining was performed using a scanner and Western blot analysis was performed using a cryogel imager. The results showed that a16PI2 had PA (83kDa) and LF (90kDa) expression, and a16PI2TC had no PA and LF expression (fig. 9), indicating that a16PI2TC did not contain pJO 1T. Wherein, PA and LF are both proteins specifically expressed by pXO 1.

(2) Indian ink staining confirmed the acapsular structure of bacillus anthracis a16Q1TC

Generally, a negative staining method is adopted to form a transparent area between the background and the thalli, and the thalli are set off to be convenient for observation and resolution. Capsular staining step: inoculating A16Q1 and A16Q1TC to LB solid medium (containing 8 ‰ (mass%) NaHCO ₃5% (volume percent) horse serum), then placed under 5% CO₂The constant-temperature incubator is used for culturing at 37 ℃ for about 48 hours, bacillus anthracis is picked to prepare bacterial suspension, then 1 drop of Chinese ink is added into the bacterial suspension to be fully mixed, then one drop of Chinese ink is dropped on a glass slide, a cover glass is covered to be compressed, and the redundant solution is wiped off by using a piece of lens wiping paper to carry out microscopic examination. The 100-fold microscopic examination result shows that the background is gray black, the thallus is dark, A16Q1 is capsulated, the permeability of capsule is high, a colorless transparent ring (A in figure 10) is arranged outside the thallus, A16Q1TC is not capsulated, and a colorless transparent ring (B in figure 10) is not arranged outside the thallus, which indicates that A16Q1TC does not contain pJO 2T.

3. Process for removing exogenous "scissors plasmids" pJO1T and pJO2T from B.anthracis

Because the exogenous 'scissors plasmids' pJO1T and pJO2T constructed by the invention are temperature-sensitive plasmids which are lost at 37-42 ℃, and 37 ℃ is the optimal growth temperature of the bacillus anthracis, in order to avoid the growth and the passage of the bacillus anthracis at an overhigh temperature, the exogenous recombinant plasmids pJO1T and pJO2T are lost at 37 ℃. The method comprises the following steps:

picking the single clone of A16PI2TC or A16Q1TC obtained in step 2, inoculating to 5mL LB liquid medium, culturing at 37 deg.C for 10-12h, and transferring to another 5mL LB liquid medium at 1%Culturing on shaking table at 37 deg.C for 12h, repeating 1% transferring for 3 times, diluting the bacterial solution in gradient, and collecting 10⁴、10⁵Duplicate dilutions of the bacterial solution were coated on non-resistant plates. Placing the plate in an incubator at 30 ℃ for culture, picking the monoclones on the plate for spotting, respectively spotting each monoclone on an LB nonreactive plate and an LB (containing 25 mu g/ml Kan) agar plate, and placing the plate after spotting in the incubator at 30 ℃ for culture. The next day, the single clone that grew on the non-resistant plate but not on the Kan (containing 25. mu.g/ml Kan) resistant agar plate was picked for colony PCR identification and Kan-resistant LB liquid medium transfer culture (shake culture at 30 ℃) to confirm that it did not grow in the Kan-resistant LB liquid medium, and the clone was a positive clone. The selected positive clones were inoculated in non-resistant LB liquid medium and cultured, then the genome was subjected to PCR using the genome as a template and the specific primer pJOE8999-F/R (see Table 1) on the vector plasmid, to identify the removal of the foreign recombinant plasmid, and pJOE8999 was used as a control.

The results showed that the positive clones had no PCR-specific amplified bands, indicating that pJO1T and pJO2T of these clones had been lost (FIG. 11). Thus, a Bacillus anthracis mutant strain with pXO1 or pXO2 plasmid deletion and no other exogenous plasmid is obtained, and strains without pXO1 and pXO2 are named as A16PI2C (pXO 1)^-pJO1T^-) (abbreviated as A16PI2C) and A16Q1C (pXO 2)^-pJO2T^-) (abbreviated as A16Q 1C).

TABLE 1 oligonucleotide sequences and primers used in the present invention

TABLE 2 strains and characteristics of the invention

Note: in Table 2, "-" indicates that no corresponding plasmid was contained, and "+" indicates that a corresponding plasmid was contained.

<110> military medical research institute of military science institute of people's liberation force of China

<120> method for removing pXO2 plasmid in Bacillus anthracis

<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

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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

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

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

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

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

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

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

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

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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

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

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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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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

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

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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

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<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 (9)

1. The method for removing the pXO2 plasmid in the bacillus anthracis is characterized by comprising the following steps: the method adopts a CRISPR/Cas9 system to remove pXO2 plasmid in the original Bacillus anthracis, the CRISPR/Cas9 system comprises sgRNA named sgRNA2, and a target sequence recognized by the sgRNA2 is a DNA fragment shown in 1-20 th position of a sequence 4 in a sequence table.

2. The method of claim 1, wherein: the sequence of the sgRNA2 is an RNA sequence obtained by replacing T in a sequence 4 in a sequence table with U.

3. The method of claim 1, wherein: the method comprises the steps of introducing an expression cassette containing a coding gene of the sgRNA2 into the starting bacillus anthracis, so that the sgRNA2 is expressed, and the bacillus anthracis with the pXO2 plasmid removed is obtained.

4. The method of claim 1, wherein: the CRISPR/Cas9 system also includes a Cas9 protein.

5. The method of claim 4, wherein: the method also comprises the step of introducing an expression cassette containing a coding gene of the Cas9 protein into the Bacillus anthracis starting, so that the Cas9 protein is expressed.

An sgRNA2 according to claim 1 or 2.

7. The biomaterial associated with the sgRNA2 of claim 1 or 2, being any one of the following B1) to B4):

B1) a nucleic acid molecule encoding the sgRNA2 of claim 1 or 2;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

8. A kit of parts, X1) or X2) as follows:

x1) kit consisting of the sgRNA2 and the Cas9 protein of claim 1 or 2;

x2) kit consisting of the biomaterial of claim 7 and a biomaterial related to Cas9 protein; the biological material related to the Cas9 protein is any one of the following C1) to C4):

C1) a nucleic acid molecule encoding a Cas9 protein;

C2) an expression cassette comprising the nucleic acid molecule of C1);

C3) a recombinant vector comprising the nucleic acid molecule of C1), or a recombinant vector comprising the expression cassette of C2);

C4) a recombinant microorganism containing C1) the nucleic acid molecule, or a recombinant microorganism containing C2) the expression cassette, or a recombinant microorganism containing C3) the recombinant vector.

9. The method of any one of claims 1-5, or the sgRNA of claim 6, or the biomaterial of claim 7, or the kit of claim 8, for any one of the following uses:

y1) in the preparation of pXO2 plasmid products for removing Bacillus anthracis;

y2) in the preparation of vaccines and/or medicaments for preventing and/or treating diseases caused by the Bacillus anthracis.

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