CN101798565B - Bacillus anthracis capable of displaying PA20 protein on the surface of spores and its application - Google Patents

Abstract

The invention discloses an anthracis bacillus strain capable of displaying PA ₂₀ protein on the surface of spores and an application thereof. The bacterial strain is obtained by inserting the pagA ₂₀ gene into the terminus of the bclA gene of Bacillus anthracis. Specifically, it can be Bacillus anthracis CGMCC No. 3403. The bacterial strain can be used as a host for preparing immune preparations, and the immune preparations are preferably inactivated vaccines, live attenuated vaccines, subunit vaccines or genetically engineered vaccines.

Description

Can be at the spore surface showing PA 20The Bacillus anthracis of albumen and application thereof

Technical field

The present invention relates to a strain can be at the spore surface showing PA ₂₀The Bacillus anthracis of albumen and application thereof.

Anthrax spore attack after 911 events has caused the fear of people to biological weapon.It is long that the gemma that anthrax bacillus forms has the survival time, is easy to propagate, and characteristics such as the fast and mortality ratio height of the sick disease time of the anthrax bacillus that causes are listed in Category B notifiable disease in China, are one of terrorist's first-selection weapons.

The anthrax bacillus toxic strain contains two big plasmids, is respectively the pXO1 plasmid (184.5kbp) of toxin-encoding base, the pXO2 plasmid (95.3kbp) of synthetic pod membrane genes involved.Virulent strain needs the existence of two kinds of plasmids, lacks pXO1 and does not then produce toxin, is weak toadstool; Lack pXO2 and then do not form pod membrane; than low hundred times of wild-type bacteria virulence; therefore the anthrax bacillus virulence factor mainly is pod membrane and two aspects of anthrax toxin; and can cause body to its composition that produces immune protection response modern the discovery mainly be protective antigen (PA) in the anthrax toxin, the gemma composition of anthrax bacillus, edema factor (EF) and lethal gene (LF) all have the effect of enhancing immunity protection in addition.The A16R strain that China is currently applied to vaccine is the low virulent strain that only has the big plasmid of pXO1, but there are certain shortcomings such as side effect in residual certain toxicity.We have eliminated pXO1 toxicity plasmid, set up the anthrax bacillus gemma mutant strain of " nontoxic ", and the patented (patent No.: ZL200610007229.3).

According to the crystalline structure of PA albumen, can be divided into 4 structural domains: structural domain 1 (PAD1) is made up of 1～258 amino-acid residue, wherein PA ₂₀The maturation protein sequence is the 20th～186 amino-acid residue, and PAD1 contains the cleavage site of furin (furin) and the binding site of LF and EF; Structural domain 2 (PAD2) is made up of 259～487 amino-acid residues, participates in the formation of albumen heptamer and the formation that LF, EF enter the tenuigenin duct; Structural domain 3 (PAD3) is made up of 488～595 amino-acid residues, participates in the formation of heptamer; Structural domain 4 (PAD4) is made up of 596～735 amino-acid residues, is the cell receptor binding domains.PAD1 comprises the action site of furin proteolytic enzyme and the binding site of EF/LF, anti-PA ₂₀Antibody capable stops LF or EF to be combined with PA, thereby stops LF or EF to enter in the cell.

The purpose of this invention is to provide a strain can be at the spore surface showing PA ₂₀Anthrax bacillus mutant strain and the application thereof of albumen.

Provided by the present invention can be at the spore surface showing PA ₂₀The anthrax bacillus strain of albumen is with pagA ₂₀Gene is inserted into Bacillus anthracis (Bacillus anthracis) gemma albumen bclA gene and does not hold the bacterial strain of acquisition.

Described pagA ₂₀5 ＇ that the albumen of genes encoding has from GeneID:2820165 hold 20-186 amino acids sequence; 5 ＇ that described bclA gene has from GeneID:62823670 hold 1-389 position nucleotide sequence.

Described can be at the spore surface showing PA ₂₀The Bacillus anthracis strain of albumen is also through removing the antibiotic marker of introducing in the building process.

Described Bacillus anthracis (Bacillus anthracis) is the attenuated live vaccine bacterial strain, is specially Bacillus anthracis (Bacillus anthracis) AP422CGMCC № .1569.

Described can be at the spore surface showing PA ₂₀The Bacillus anthracis strain of albumen is preferably Bacillus anthracis (Bacillus anthracis) AP423CGMCC № .3403.Bacillus anthracis (Bacillus anthracis) AP423CGMCC № .3403 is with pagA by homologous recombination ₂₀Gene is inserted into Bacillus anthracis (Bacillus anthracis) gemma albumen bclA gene and does not hold, and that resistant gene of card that adopts recombinase to introduce knocks out, can be at the spore surface showing PA by a strain of a large amount of evaluation and screenings acquisitions ₂₀Albumen, and the Bacillus anthracis strain of antibiotic-free mark.

Described Bacillus anthracis (Bacillus anthracis) AP423, be preserved in Chinese microorganism strain preservation board of trustee reason person on November 05th, 2009 and understand common micro-organisms center (abbreviation CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC № .3403.

The present invention also provide a kind of can be at the spore surface showing PA ₂₀The preparation method of the Bacillus anthracis strain of albumen is pagA just ₂₀Gene is inserted into Bacillus anthracis (Bacillus anthracis) gemma albumen bclA gene and does not hold.

Described pagA ₂₀The albumen of genes encoding has from 5 ＇ of GeneID:2820165 holds the 20th～186 amino acids sequence; 5 ＇ that described bclA gene has from GeneID:62823670 hold 1-389 position nucleotide sequence; Described pagA ₂₀Gene is knocked in the described Bacillus anthracis (Bacillus anthracis) by the method for homologous recombination.Described Bacillus anthracis (Bacillus anthracis) is preferably Bacillus anthracis (Bacillus anthracis) AP422 CGMCC № .1569.

Above-mentioned can be at the spore surface showing PA ₂₀The Bacillus anthracis strain of albumen can be used for preparing attenuated live vaccine.

Bacillus anthracis of the present invention (Bacillus anthracis) AP423 applying gene target practice technology is successfully with pagA ₂₀Gene is inserted into anthrax bacillus gemma albumen bclA gene and does not hold, and detects PA in this mutant gemma albumen ₂₀Existence, this lays the first stone in the future developing novel anthrax bacillus gemma vaccine, as inactivated vaccine, attenuated live vaccine, subunit vaccine or recombinant vaccine.Bacillus anthracis (Bacillus anthracis) AP423 bacterial strain can be used for preparing attenuated live vaccine, can produce PA after using this attenuated live vaccine immunity humans and animals ₂₀Immunne response with relevant gemma albumen.

Description of drawings

Fig. 1 cuts for the recombinant vectors enzyme and identifies 1% agarose electrophoresis figure; Among the figure, swimming lane M:DNA Marker; Swimming lane 1:pT-pagA ₂₀Result with Pst I and Sac II double digestion; Swimming lane 2:pT-pagA ₂₀Result with Pst I and Sac II double digestion; The swimming lane 3:pT-da result of EcoR I and Xho I double digestion; The swimming lane 4:pT-lpka result of Sac II and EcoR I double digestion.

Fig. 2 is pagA ₂₀Gene is knocked in the method synoptic diagram that adopts.

Fig. 3 is pagA ₂₀Gene is knocked in positive transformant reorganization bacterium PCR qualification result.

Fig. 4 successfully removes the sub-PCR qualification result of recombinant conversion for the kana gene.

Fig. 5 is Bacillus anthracis (Bacillus anthracis) AP423 gemma protein electrophoresis figure

Fig. 6 is Bacillus anthracis (Bacillus anthracis) AP423 gemma protein immunoblot figure

Fig. 7 is the experiment of Bacillus anthracis (Bacillus anthracis) AP423 gemma protein immunization effect

Embodiment

Method among the following embodiment if no special instructions, is ordinary method.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

Embodiment 1, a strain can be at the spore surface showing PA ₂₀The acquisition of the anthrax bacillus mutant strain of albumen

1, the structure of targeting vector

Extract anthrax bacillus A16R strain (the proteome analysis Dong Mei of Bacillus anthracis A16R strain sterile culture filtrate, Zhuan Hanlan, institute of the good Military Medical Science Institute of Wang Xi periodical the 33rd the 1st phase of volume of February in 2009, preserved by Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A) interior plasmid pXO1 is that template obtains pagA by the PCR reaction with plasmid pXO1 ₂₀Gene, primer is PA20F (5 ' CCAATGCATGAAGTTAAACAGGAGAA3 ' (sequence 1 in the sequence table)) and PA20R (5 ' TCCCCGCGGTTATCGCTTTTTTCT 3 ' (sequence 2 in the sequence table)), the pcr amplification condition is 94 ℃ of pre-sex change 5min, 30 circulations of increasing then: 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min, last 72 ℃ are extended 10min, purifying PCR fragment also is connected into pEASY-T1 carrier (full Shi Jin biotech company), obtains recombinant plasmid pT1-pagA ₂₀, and it is carried out enzyme cut and identify and sequencing analysis shows that vector construction is correct.Its enzyme is cut the swimming lane 1 that qualification result is seen Fig. 1, and the result shows, recombinant plasmid pT1-pagA ₂₀The pagA that contains this 501bp ₂₀Gene, and identify through order-checking and not undergo mutation.

(be preserved in Chinese microorganism strain preservation board of trustee reason person and understand the common micro-organisms center, deposit number is CGMCC № .1569 with anthrax bacillus AP422 for upstream target practice fragment and downstream target practice fragment; The patent No. is ZL200610007229.3, Granted publication number is CN 100362094C) genome is that template is carried out the PCR reaction, upstream target practice fragment primer is ARMBclAF (5 ' CCGGATCCACTAGGACTTCCAGCAGGAC 3 ' (sequence 3 in the sequence table)) and ARMBclAR (5 ' CGCTGCAGACCTGGAGCAACTTTTTCAATAATAATG 3 ' (sequence 4 in the sequence table)), and downstream target practice fragment primer is DAFF (5 ' GACGAATTCACTTAGCAGTAAAACTGATATC 3 ' (sequence 5 in the sequence table)) and DAFR (5 ' TCGAGCTCTTTCCTTGACTACAGTTTCC 3 ' (sequence 17 in the sequence table).The pcr amplification condition is 94 ℃ of pre-sex change 5min, 30 circulations of increasing then: 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min, last 72 ℃ are extended 10min, purifying PCR fragment also is connected into the recombinant plasmid pT1-da that pEASY-T1 carrier (full Shi Jin biotech company) obtains containing the recombinant plasmid pT1-ua of upstream target practice fragment and contains downstream target practice fragment respectively, and it is carried out enzyme cut evaluation and sequencing analysis.The recombinant plasmid pT1-ua that contains upstream target practice fragment cuts qualification result respectively shown in swimming lane among Fig. 12 and swimming lane 3 with the enzyme that contains the recombinant plasmid pT1-da of downstream target practice fragment, the result shows that pT1-ua contains the upstream target practice fragment of 1218bp, the downstream target practice fragment that contains 851bp among the pT1-da, and identify through order-checking and not undergo mutation.

With plasmid pBE2 (reference: the structure of the multi-functional shuttle vectors of Bacillus subtilus one intestinal bacteria, Guo Xinghua, bear accounts for, Zhou Min, Jia Shifang, Xu Yi, biotechnology journal (70): 224～229,1991, preserved by Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A) carry out the PCR reaction for template and obtain LoxP-kana gene fragment (carrying that resistant gene fragment of card of recombinase recognition site LoxP), primer is lpkaF(5 ' TCCCCGCGGATAACTTCGTATAGCATACATTATACGAAGTTATGTCGACAATTAGA CTATGATCCAA3 ' (sequence 6 in the sequence table)) and lpkaR(5 ' GCCGAATTCGTATGACAAGGTGAGATCTATAACTTCGTATAATGTATGCTATACGA AGTTATGTCGACTCAAAATGGTATGCGT3 ' (sequence 7 in the sequence table)), the pcr amplification condition is 94 ℃ of pre-sex change 5min, 30 circulations of increasing then: 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min, last 72 ℃ are extended 10min, purifying PCR fragment also is connected into pEASY-T1 carrier (full Shi Jin biotech company) and obtains recombinant plasmid pT1-lpka, and it is carried out enzyme cut and identify and sequencing analysis shows that vector construction is correct, the LoxP-kana gene fragment that contains 1400bp among the pT1-lpka, and identify through order-checking and not undergo mutation.Wherein, enzyme is cut the swimming lane 4 that qualification result is seen Fig. 1.

With Pst I and Sac II double digestion pT1-PA ₂₀Reclaim pagA ₂₀Fragment (encoded protein have from 5 ＇ of GENBANKGeneID:2820165 hold the 20th～186 amino acids sequence) is with pagA ₂₀Fragment is inserted between the Pst I and Sac II enzyme recognition site of pT1-ua, obtains recombinant vectors, and it is carried out the enzyme evaluation of cutting and check order, and evaluation is shown correct recombinant plasmid called after pT1-pagA ₂₀Ua.With BamH I and Sac II double digestion pT1-pagA ₂₀Ua reclaims pagA ₂₀Ua upstream arm fragment is inserted into this fragment between the BamH I and Sac II restriction enzyme site of pT1-lpka, obtains recombinant vectors, and recombinant vectors is carried out the enzyme evaluation of cutting and check order, and will identify correct recombinant plasmid called after pT1-pagA ₂₀Ualpka.

With EcoR I and the pT1-pagA of Xho I ₂₀Ualpka and pT1-da carry out double digestion, reclaim pT1-pagA ₂₀Ualpka carrier segments and da fragment, the T4DNA ligase enzyme connects, and obtains recombinant vectors, and recombinant vectors is carried out the enzyme evaluation of cutting and check order, and will identify correct recombinant plasmid called after pT1-pagA ₂₀Ualpkada.

With BamH I and Xho I double digestion plasmid pT1-pagA ₂₀Ualpkada and pMAD reclaim pMAD carrier segments and target practice fragment pagA ₂₀Ualpkada obtains recombinant vectors, and recombinant vectors is carried out the enzyme evaluation of cutting and check order, and will identify correct recombinant plasmid called after pMAD-pagA ₂₀Ualpkada is with pMAD-pagA ₂₀Ualpkada electricity Transformed E .coli SCS110(is available from Stratagene company), extract plasmid and obtain non-methylated plasmid pMAD-pagA ₂₀Ualpkada is used for transforming B.anthracis.

2, Cre recombinase expression plasmid makes up

With Bacillus subtilus № 168(available from American type culture collection, be called for short ATCC, deposit number is ATCCNo.23857) be template, utilize primer amyrF and amyrR amplification to obtain amylase promotor AmyR1, and introduce BamH I and Hind III site respectively at two ends.Transformed E .coli Top10 after the fragment that amplification is reclaimed is handled the back and plasmid pHY304 through same enzymes double zyme cutting is connected with BamH I and Hind III enzyme double digestion, the recombinant plasmid that obtains, the enzyme evaluation of cutting and check order will be identified correct plasmid called after pHY-AmyR.Being template with plasmid pBS185 (available from GibcoBRL company) then, is primer with creF and creR, and amplification Cre recombinase gene is introduced Hind III and Xho I site respectively by primer sequence at two ends.The Cre recombinase gene fragment that pcr amplification is obtained is with being connected Transformed E .coli Top10 with plasmid pHY-AmyR through same double digestion behind Hind III and the Xho I double digestion, the recombinant plasmid that obtains, the enzyme evaluation of cutting and check order, to identify correct plasmid Transformed E .coliSCS110 again, finally obtain can be used for transforming the plasmid pHY-Cre that goes firstization of anthrax bacillus.Above-mentioned primer sequence is: amyrF:GCGGATCCGACGACAGGGGGATTC (sequence 8 in the sequence table), amyrR:CCCAAGCTTTCTTGACTCCTTAT (sequence 9 in the sequence table); CreF:CCCAAGCTTATGTCCAATTTACTG (sequence 10 in the sequence table), creR:CCGCTCGAGCTAATCGCCATCTTCC (sequence 11 in the sequence table).

3, pagA ₂₀Gene is knocked in

PagA ₂₀Gene is knocked in the strategy that adopts as shown in Figure 2.Concrete grammar is as described below:

Line separates single bacterium colony from the attenuated live vaccine B.anthracis AP422 CGMCC № .1569 bacterial classification of-70 ℃ of preservations, be inoculated in the 5mL LB substratum, 37 ℃ of incubated overnight. overnight culture is inoculated in the new LB substratum of 100mL in 1% ratio, and 37 ℃ of thermal agitations are cultivated. work as OD ₆₀₀Took out from shaking table (about 2 hours) when value reached 0.5-0.6, the preparation competent cell.

Per 40 μ L competent cells add 3 μ L (about 0.9 μ g) plasmid pMAD-pagA ₂₀Ualpkada mixes back ice bath 5 minutes in 0.1cm electric shock cup, carries out the electricity conversion with the Bio-Rad electroporation then, and condition is: 1.8kV, 200 Ω and 25 μ F.Add the ice-cold BH substratum of 1mL (the 3.7%Brain-Heart substratum is available from BD company) immediately after electric shock finishes, 30 degree were hatched 2.5 hours.200 μ L coat on the LB flat board that contains that enzyme element of card (25 μ g/mL) respectively, cultivate 24 hours for 30 ℃.

Picking list bacterium colony is inoculated into and contains in that enzyme element of card (25 μ g/mL) 5mL LB substratum, cultivates 12 hours for 30 ℃, and get 10 μ L cultures and continue inoculation and contain in that enzyme element of card (25 μ g/mL) 5mL LB substratum, after passing for 5 generations continuously, get 5 μ L culture gradient dilutions, get 10 ^-2, 10 ^-3With 10 ^-4Each 100 μ L of diluent coat on the LB flat board that contains that enzyme element of card (25 μ g/mL) 37 ℃ of incubated overnight.Owing to contain beta-galactosidase gene on the plasmid pMAD, therefore under the situation that X-gal exists, can show blue, so if plasmid and genome are not recombinated, bacterium colony is blue so, and having only bacterium colony is that white bacterium just might be that homologous recombination has taken place.Therefore according to behind the aforesaid operations, the single bacterium colony of picking white is identified with carrying out PCR at random.Get single bacterium colony that 10 37 ℃ of incubation growth go out, be inoculated in the LB substratum that contains that enzyme element of card (25 μ g/mL) 37 ℃ of overnight incubation.get overnight culture 5 μ L and carry out full bacterium PCR evaluation, primer GP1 (5 ' AACGGGCTATTGTCTTCTCCTCATC 3 ' (in sequence table, sequence 12)) and primer PA20R (5 ' TCCCCGCGGTTATCGCTTTTTTCT 3 ' (in sequence table, sequence 13)) with 100bp place, the BclA upstream region of gene homology arm outside, 100bp left and right, the downstream homology arm outside primer GP2 (5 ' ATCATCGATTTGAGTCATAGGAGT 3 ' (in sequence table, sequence 14)) and two pairs of primer pair recombinant conversion of primer KP1 (5 ' AACAGAGGAAGCAGAGTTCAGCC 3 ' (in sequence table, sequence 15)) carry out the PCR evaluation.Simultaneously do contrast with normal B.anthracis AP422 bacterial strain.Primer GP1 and primer PA20R amplification obtain the fragment of 1939bp, and when primer GP2 and primer KP1 amplification obtained 1588bp, then screening obtained positive recombinant conversion.The result that part bacterium colony PCR identifies as shown in Figure 3, the result shows that screening obtains positive recombinant conversion of 1 strain, two pairs of primers designed of positive recombinant conversion all can successfully amplify corresponding dna fragmentation, the fragment of DNA amplification is carried out sequential analysis show the gene recombination success, the fragment of DNA amplification is carried out sequential analysis show pagA ₂₀The not end of successful insertion bclA gene, this does not hold BclA albumen and expresses PA ₂₀Albumen is significant, and with this bacterial strain called after AP423K, AP423K is under 42 ℃ of conditions, and recombinant bacterial strain makes recombinant plasmid lose, with the possible erythromycin resistance of thorough removal in 3 generations of LB liquid training substratum continuous passage.Among Fig. 3, M:DNA Marker; 1:GP1 and PA ₂₀R amplification PCR fragment; 2:GP2 and KP1 amplification PCR fragment.

4, the removal of reorganization bacterium kana gene

With plasmid pHY-Cre step of converting 3 obtain go down to posterity after strains A P423K, method adopts electricity to transform equally, the same method described in 3 set by step of condition adds the ice-cold BH substratum of 1mL immediately according to the described method of step 3 after electric shock finishes, and 30 degree were hatched 2.5 hours.Getting 50 μ L coats on the LB flat board that contains erythromycin (5 μ g/mL), 30 ℃ of incubated overnight, get single bacterium colony in the LB liquid nutrient medium that contains erythromycin (5 μ g/mL) 30 ℃ passed for two generations after, (LB substratum) passes the three generations continuously under the condition that 37 ℃ of antibiotic-frees exist, get the LB flat board that the dilution of bacterium liquid is coated with antibiotic-free then, single bacterium colony reference numerals mark on this flat board, getting each single bacterium colony inoculates respectively and contains antibiotic-free, in the LB liquid nutrient medium of kantlex and erythromycin, obtain at the not long bacterial strain of the LB of kantlex and erythromycin liquid nutrient medium, with its called after Bacillus anthracis (Bacillus anthracis) AP423.

Described Bacillus anthracis (Bacillus anthracis) AP423, be preserved in Chinese microorganism strain preservation board of trustee reason person on November 05th, 2009 and understand common micro-organisms center (abbreviation CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC № .3403.

With the genomes of band kalamycin resistance reorganization bacterium AP423K and AP423 two strain bacterium as template, carry out PCR with primer PA20F (5 ' CCAATGCATGAAGTTAAACAGGAGAA 3 ' (sequence 16 in the sequence table)) and DAFR (5 ' TCGAGCTCTTTCCTTGACTACAGTTTCC 3 ' (sequence 17 in the sequence table)) and identify, the results are shown in Figure 4.From figure we as can be seen, reorganization bacterium AP423 genome expands the about 1.3kb of the clip size that with above-mentioned primer, the AP423K genome expands the about 2.7kb of the clip size that with above-mentioned primer, and AP423 fails to amplify the PCR fragment, this is suitable with theoretical value.Check order after the fragment that obtains of amplification is connected to the T carrier, the result shows kanamycin gene disappearance fully, and corresponding site is surplus next LoxP site only, and this has just confirmed that at gene level kanamycin gene successfully removes.M:DNA Marker among Fig. 4; 1:AP423; 2:AP423K.

The gemma analysis of protein of embodiment 2, Bacillus anthracis (Bacillus anthracis) AP423

AP423 is inoculated in the 100mL LB liquid nutrient medium with Bacillus anthracis (Bacillus anthracis), 37 ℃ of cultured continuously 5 days, and be coated with bacterium dyeing and observe gemma and form situation.

Get all nutrient solutions, centrifugal 5 minutes of 17000g abandons supernatant, again with the washing of equal-volume sterilized water once, use 100 μ L gemma protein lysates (0.1M DTT, 0.5%(quality percentage composition) SDS at last, 0.1M NaCl, pH10.0) resuspended thalline, hatched 2 hours 30 minutes for 37 ℃, centrifugal 10 minutes of 17000g abandons supernatant, precipitation is dissolved the bacterium whole bacterial protein that obtains recombinating with 100 μ L PBS, carries out the SDS-PAGE electrophoretic analysis with 10% gel.

On the basis that SDS-PAGE analyzes, carry out immunoblotting assay.With AP422(whole bacterial protein preparation method with above-mentioned Bacillus anthracis (Bacillus anthracis) AP423) and reorganization bacterium whole bacterial protein carry out the SDS-PAGE electrophoresis with 10% gel, transfer on the nitrocellulose filter then.The nitrocellulose filter film sealed 2 hours with 5% skim-milk room temperature; Again with anti-PA ₂₀Antiserum(antisera) (1:5000 dilution) hatched altogether 2 hours for 37 ℃; With PBST washing 3 times, each 5 minutes, hatched altogether 1 hour with the goat anti-rabbit igg antibody of HRP mark (this antibody is available from middle China fir company, 1:5000 dilution) again, at last film is developed the color with HRP colouring reagents box (available from sky root company) after with the PBST wash clean.

Above-mentioned anti-PA ₂₀Sero-fast preparation method be: according to bacillus anthracis protective antigen PA20 gene mature polypeptide coding sequence design primer (described pagA ₂₀5 ＇ that the albumen of genes encoding has from GeneID:2820165 hold 20-186 amino acids sequence) (PA20F:CGAGGATCCAGAAGTTAAACAGGAGAAC, PA20R:GCACTCGAGTCGCTTTTTTCTTGAGTTC).Extract anthrax bacillus A16R strain (the proteome analysis Dong Mei of Bacillus anthracis A16R strain sterile culture filtrate, Zhuan Hanlan, institute of the good Military Medical Science Institute of Wang Xi periodical the 33rd the 1st phase of volume of February in 2009, preserved by Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A) interior plasmid pXO1, be template with plasmid pXO1, amplify pagA ₂₀Gene fragment is inserted into prokaryotic expression carrier pET-28a(available from Novagen company with this fragment orientation) in (restriction enzyme site BamH I and Xho I), obtained pET-PA ₂₀The prokaryotic expression recombinant plasmid obtains to express PA with this recombinant plasmid transformed e. coli bl21 (DE3) (available from Invitrogen company) ₂₀The reorganization bacterium, in the picking reorganization bacterium inoculation LB substratum, when bacterium liquid OD600 value reaches 0.8, add IPTG(available from Promega company) carried out abduction delivering (the IPTG final concentration is 1mmol/L) 4 hours, centrifugal collection thalline and to its ultrasonication, to the PA that expresses ₂₀Albumen carries out affinitive layer purification with nickel post (available from GE company), with the protein immunization New Zealand large ear rabbit (immunizing dose is 0.5mg/, immunity three times, each two weeks at interval) of purifying, gets rabbit anteserum after immune two weeks for the third time and obtains PA ₂₀Polyclonal antibody serum.Serum is carried out antibody titers detect, the result shows that antibody titers can reach 1:10000.

Identify correct pagA for gene level ₂₀Gene inserts the mutant strain bacterial strain and carries out the SDS-PAGE electrophoretic analysis with 10% gel, and the result as shown in Figure 5.With anti-PA ₂₀The rabbit anti-serum of albumen carries out reorganization bacterium Bacillus anthracis (Bacillus anthracis) AP423 is carried out immunoblotting assay proof PA as primary antibodie ₂₀Albumen has obtained expression, the results are shown in Figure 6.Among Fig. 5 and Fig. 6, M: albumen Marker; 1:AP422; 2:AP423.

The gemma immune effect checking of embodiment 3, Bacillus anthracis (Bacillus anthracis) AP423

With AP423 brood cell's immunity Balb/c mouse, every little groin injects about 10 ⁹Individual brood cell, immunity is three times altogether, twice immunity two week of the timed interval, get serum after immune two weeks for the third time and carry out the ELISA reaction detection, the results are shown in Figure 7, the result shows that serum titer can reach 1600 times (p＜0.01).Ck is immune AP422 brood cell's immunity Balb/c mouse among Fig. 7.

Sequence table

<160>17

<210>1

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

ccaatgcatg aagttaaaca ggagaa 26

<210>2

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>2

tccccgcggt tatcgctttt ttct 24

<210>3

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

ccggatccac taggacttcc agcaggac 28

<210>4

<211>36

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>4

cgctgcagac ctggagcaac tttttcaata ataatg 36

<210>5

<211>31

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>5

gacgaattca cttagcagta aaactgatat c 31

<210>6

<211>67

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>6

tccccgcgga taacttcgta tagcatacat tatacgaagt tatgtcgaca attagactat 60

gatccaa 67

<210>7

<211>84

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>7

gccgaattcg tatgacaagg tgagatctat aacttcgtat aatgtatgct atacgaagtt 60

atgtcgactc aaaatggtat gcgt 84

<210>8

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>8

gcggatccga cgacaggggg attc 24

<210>9

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>9

cccaagcttt cttgactcct tat 23

<210>10

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>10

cccaagctta tgtccaattt actg 24

<210>11

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>11

ccgctcgagc taatcgccat cttcc 25

<210>12

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>12

aacgggctat tgtcttctcc tcatc 25

<210>13

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>13

tccccgcggt tatcgctttt ttct 24

<210>14

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>14

atcatcgatt tgagtcatag gagt 24

<210>15

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>15

aacagaggaa gcagagttca gcc 23

<210>16

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>16

ccaatgcatg aagttaaaca ggagaa 26

<210>17

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>17

tcgagctctt tccttgacta cagtttcc 28

Claims (10)

1. a strain can be at the spore surface showing PA ₂₀The anthrax bacillus strain of albumen is with pagA ₂₀Gene is inserted into the terminal bacterial strain that obtains of Bacillus anthracis (Bacillus anthracis) gemma albumen bclA gene.

2. anthrax bacillus strain according to claim 1 is characterized in that: described pagA ₂₀The sequence of the albumen of genes encoding is for to hold the 20th～186 amino acids sequence from 5 ＇ of GeneID:2820165; The sequence of described bclA gene is for to hold 1-369 position nucleotide sequence from 5 ＇ of GeneID:62823670.

3. anthrax bacillus strain according to claim 1 and 2 is characterized in that: described can be at the spore surface showing PA ₂₀The anthrax bacillus strain of albumen is also through removing the antibiotic marker of introducing in the building process.

4. anthrax bacillus strain according to claim 1 and 2 is characterized in that: described Bacillus anthracis (Bacillus anthracis) is the attenuated live vaccine bacterial strain.

5. anthrax bacillus strain according to claim 4 is characterized in that: described can be at the spore surface showing PA ₂₀The anthrax bacillus strain of albumen is Bacillus anthracis (Bacillus anthracis) CGMCC № .3403.

One kind make up that claim 1 is described can be at the spore surface showing PA ₂₀The method of the anthrax bacillus strain of albumen is pagA just ₂₀Gene is inserted into Bacillus anthracis (Bacillus anthracis) gemma albumen bclA gene and does not hold.

7. method according to claim 6 is characterized in that: described pagA ₂₀The sequence of the albumen of genes encoding is for to hold the 20th～186 amino acids sequence from 5 ＇ of GeneID:2820165; The sequence of described bclA gene is for to hold 1-369 position nucleotide sequence from 5 ＇ of GeneID:62823670; Described pagA ₂₀Gene is knocked in the described Bacillus anthracis (Bacillus anthracis) by the method for homologous recombination.

8. according to claim 6 or 7 described methods, it is characterized in that: described Bacillus anthracis (Bacillus anthracis) is Bacillus anthracis (Bacillus anthracis) AP422 CGMC № .1569.

9. any one described application that can prepare in the anthrax bacillus strain of spore surface showing PA 20 albumen in the immunological reagent among the claim 1-5.

10. application according to claim 9 is characterized in that: described immunological reagent is inactivated vaccine, attenuated live vaccine, subunit vaccine or recombinant vaccine.

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