CN1966074A - Combined vaccine for anthrax and black death - Google Patents

Abstract

The invention relates to a coupled vaccine for protecting human or animal from bacillus anthracis and Yersinia, wherein it comprises bacillus anthracis PA antigen, Yersinia V antigen, and Yersinia F1, or their protective epitope. The antigen is recombined protein separated and/or purified. The DNA of integral or partial PA antigen, integral or partial F1 antigen, and integral or partial V antigen can be directly used as nucleic acid vaccine.

Description

Anthrax, plague combined vaccine

Technical field

The present invention relates to the combined vaccine of anti-simultaneously Bacillus anthracis and Yersinia pestis infection, belong to medical biotechnology field.

Background technology

Anthrax is the infectious disease of serious threat human health, has that spread speed is fast, case fatality rate is high, easily cause characteristics such as public's fear and social disturbances, and its pathogen is an anthrax bacillus.At present, human anthrax still have in the old epidemic-stricken area of different continents distribute or local area property popular, mainly in developing country, especially western popular comparatively serious with Africa.Estimate that according to World Health Organization (WHO) 20,000～100,000 cases are arranged global every year approximately.

The plague is by the deadly infectious disease due to the bacillus pestis.Put down in writing worldwide being very popular of 3 plagues in history and made huge disaster to the mankind.Up to now, Africa, India, the U.S., Indonesia, Burma and Vietnam constantly take place popular among a small circle.In provinces and regions, state-owned 19 plague areas, the plague is popular fierce between the Mus of some areas, the human world plague happens occasionally.

At present, not only side effect is big for the chemotherapeutic agent of the anthrax and the plague (plague is mainly used gentamycin, streptomycin etc., and anthrax is mainly with penicillin etc.), and along with the appearance of multiple antibiotic resistant strain, curative effect is more and more undesirable.Inoculation anthrax and pestilence vaccine have been proved to be to prevent the effective means of the anthrax and the plague.

Human anthrax vaccine mainly contains two classes both at home and abroad at present, and a class is the living spores Seedling by the preparation of anthrax attenuated strain, mainly uses in China and Russia; Another kind of is by the PA absorption vaccine of anthrax attenuated strain culture supernatant preparation, mainly uses in American-European countries.All there are some defectives in these two kinds of vaccines, only limit to special population at present, use as the stock farmer.In recent years, all stepping up to develop novel anthrax vaccine both at home and abroad, mainly is to be fundamental construction with reorganization PA, and indivedual researchs have entered clinical experimental stage.

Pestilence vaccine has bacterin, purification vaccine and attenuated live vaccine, and countries in the world scholar's research thinks that attenuated live vaccine is more effective than the above two.The EV76 plague attenuated live vaccine strain (also being present Chinese people pestilence vaccine) of using at present, though immunogenicity is good, because its remaining virulence is higher, crowd's reaction of inoculation is big, can not be widely used in inoculation.All stepping up to develop novel pestilence vaccine both at home and abroad, mainly is to be fundamental construction with reorganization F1 and V antigen, and indivedual researchs have entered clinical experimental stage.

The anthrax and the plague are 1 class infectious disease of China's statutory report, and there is anthrax and plague nature epidemic focus ground in a big way in China, is difficult to thorough elimination; Bacillus anthracis and Yersinia pestis are extremely important bio-terrorism agent/biological warfare agents, might be discharged simultaneously; Anthrax and bacillus pestis all are extracellular bacterial parasites, and mainly based on humoral immunization, and the two all has clearer and more definite protective antigen.The research that develops into new generation vaccine of technique for gene engineering provides material base, thereby makes vaccine safer, effective, economical.Based on above consideration, the genetic engineering combined vaccine of development anthrax and bacillus pestis will provide safeguard for the reply public health emergency relevant with the anthrax and the plague.

Summary of the invention

The object of the present invention is to provide a kind of combined vaccine that can protect the human or animal to avoid Bacillus anthracis and Yersinia pestis infringement simultaneously; can reduce immune time, saving health resources, increase vaccinate's compliance; be specially adapted to the urgent inoculation of public health emergency; help population health, national security.

Description of drawings

Fig. 1 is that the PCR of expression plasmid pET-F1 and pET-V identifies collection of illustrative plates

Fig. 2 is that the enzyme action of expression plasmid pET-F1 and pET-V is identified collection of illustrative plates

Fig. 3 is that the antigenic SDS-PAGE of Yersinia pestis F1 and V detects

The specific embodiment

Method among the embodiment is conventional method if no special instructions.

The antigenic preparation of embodiment 1 Bacillus anthracis PA

The bacillus anthracis protective antigen that the clone of PA gene delivers with reference to GenBank (PA) gene order (serial number :) and document; the design primer; PA 5 ' holds primer: 5 '-AGGAGAACCGGTTATTAAATGAATC-3; PA 3 ' holds primer: 5 '-CGCrTATCCTATCTCATAGCCTT TTTTAG-3 '; carry out pcr amplification; the a small amount of antibacterial of picking from the agar plate of cultivating the A16R bacterial strain; put in the 100uL pure water; boiled 10 minutes, and got the 5uL supernatant after centrifugal and carry out the PCR reaction as template.The PCR product cloning is carried out sequencing to the pMD-T carrier, it is correct that determined dna sequence is proved conclusively synthetic gene, gene has the nucleotide sequence of sequence 1 in the sequence table, classifies coded sequence as from the 1st-2292 nucleotides sequences of 5 ' end, the aminoacid sequence of sequence 2 in its code sequence tabulation.

The structure PCR product SacI enzyme action of pAS-PA forms 5 ' flat terminal 3 ' sticking end; After carrier pAS22 cut with Stu I and Sac I, carrier was connected the conventional Transformed E .coli DH5a in back with fragment, and PCR (primer and condition are with step 2) screening positive clone, upgrading grain carry out enzyme action to be identified and sequencing.The plasmid called after pAS-PA that sequence is correct.

The expression of recombinant anthrax protective antigen-PA and purification:

1, expresses

The E.coli DH5a engineering bacteria list bacterium colony that picking is transformed by plasmid pAS-PA on the solid LB culture medium inserts in the LB culture medium that contains the 100ug/mL ampicillin 37 ℃ 250 rev/mins and is cultured to OD ₆₀₀=0.7-0.8 adds IPTG to 0.5mmol/L, 28 ℃ of 250 rev/mins of abduction delivering 5h, centrifugal collection bacterial precipitation.

2, the separation of the outer pericentral siphon of antibacterial

Thalline after 1L inducing culture thing is centrifugal, resuspended with 100mL 20% sucrose solution (20mmol/L Tris pH 8.0,1mmol/L EDTA, 1mmol/L PMSF), place 5min on ice, 4 ℃ of centrifugal 20min of 8000rpm.Precipitation 100mL 5mmol/L Mg ₂SO4.(1mmol/L PMSF) is resuspended, places 10min on ice, and 4 ℃ of centrifugal 10min of 10000rpm collect supernatant and are used for purification rPA.

3, ion exchange

The supernatant of collecting in the step 2 is carried out thick purification with Q Sepharose earlier.With level pad (20mmol/L Tris pH 8.0) balance columns, regulate the sample salinity and to 20mmol/L Tris pH8.0, go up sample.Carry out eluting with the level pad that contains 0.5mol/l NaCl.The fraction collection eluent carries out 12% SDS-PAGE analysis.

The pipe that contains rPA merges the back and carries out next step purification with Source 30Q.With level pad (the same) balance columns, go up sample behind 5 times of the diluted samples.With the level pad gradient elution that contains 0～0.25mol/L NaCl.

4, hydrophobic chromatography

The Source 30Q eluent merging back of step 3 is further purified with phenyl sepharose drainage column.

The preparation of embodiment 2 plague candidate vaccine F1 and V

Plague reference culture and Bacillus anthracis bacterial strain are preserved by this research department, and DNA restricted enzyme, T4 dna ligase, Pyrobest archaeal dna polymerase and pMD-T carrier are available from TaKaRa company.

The Omega test kit is adopted in the extraction of E.coli plasmid; The operational approach reference molecule of plasmid construction is cloned a book.

The bacillus pestis k antigen of delivering with reference to GenBank (Fraction 1, F1) gene order (serial number: AF542378.1), design primer, F1up:5 '-GCC CATATGAAAAAAATCAGTTCCG-3 ' and F1 down:5 '-GGG GAATTCTTATTGG TTAGATACGGTTACG '-3 (CATATG and GAATTC are respectively the restriction enzyme sites of Nde I and EcoR I, are used for the PCR fragment cloning to expression vector pET-32a (+)); Carry out pcr amplification, the PCR product cloning is carried out sequencing to the pMD-T carrier, determined dna sequence conclusive evidence cloned genes is correct, gene has the nucleotide sequence of sequence 3 in the sequence table, classify coded sequence as from the 1st the-the 510th nucleotides sequence of 5 ' end, the aminoacid sequence of sequence 4 in its code sequence tabulation.

With reference to the bacillus pestis V gene order (serial number: AF 074612.1) that GenBank delivers, design primer, V up:5 '-GCG CATATGATTAGAGCCTACGAAC-3 ' and V down:5 '-CCC GAATTCTTATTTACCAGACGTGTCA TC-3 ', (CATATG and GAATTC are respectively the restriction enzyme sites of NdeI and EcoR I, be used for the PCR fragment cloning to expression vector pET-32a (+)), carry out pcr amplification, the PCR product cloning is carried out sequencing to the pMD-T carrier, and determined dna sequence conclusive evidence cloned genes is correct, and gene has the nucleotide sequence of sequence 5 in the preface sequence table, classify coded sequence as from the 1st the-the 978th nucleotides sequence of 5 ' end, the aminoacid sequence of sequence 6 in its code sequence tabulation.

F1 that determined dna sequence is correct and V gene downcut from the pMD-T carrier with Nde I and Eco RI, and gel reclaims purification, the F1 of purification is connected with the fragment of V gene with the 5400bp that (novagen) obtains through Nde I and Eco RI enzyme action pET-32a (+), transformed into escherichia coli DH5a, select ampicillin (100 μ g/mL) resistance clone, utilizing primer V up, V down and F1 UP, F1down (sequence is the same) to carry out PCR respectively identifies, screening obtains positive colony, and (swimming lane 1 among Fig. 1,2, the band of about respectively 500bp and 1000bp).The PCR positive colony is extracted plasmid, the expression vector called after pET-V and the pET-F1 of structure.Further carry out double digestion and evaluation, obtain the band of 1000bp and 5400bp as a result behind the pET-V enzyme action respectively, obtain the band of 500bp and 5400bp behind the pET-F1 enzyme action respectively, ( swimming lane 1,2 among Fig. 2) with Nde I/Eco RI.Select PCR and enzyme action and identify that correct clone send Bo Ya biotech firm to carry out determined dna sequence.

The expression vector that the abduction delivering of V and F1 antigen protein and fermentation culture will check order correct transforms BL21 (DE3) competence, the clone who grows, the expression bacterium that needs exactly on the LB flat board that contains 100 μ g/ml Carbenicillins.The single colony inoculation of picking in the LB culture medium that contains antibiotic (Carbenicillin 100 μ g/ml), 37 ℃ of overnight incubation; Next day, by 1: 100 amplification culture, 37 ℃ are cultured to the nectar degree was A ₆₀₀During=0.8-1.0, add IPTG to final concentration be 1mmol/L, continue training 5h at 37 ℃, get the centrifugal collection of 1.0ml bacterium, 100 μ l aqueous suspensions precipitation adds 100 μ l, 2 * sds gel sample loading buffer, boils 5min, getting 10 μ l after centrifugal carries out the SDS-PAGE analysis.Inoculate single bacterium colony in 10ml LB culture medium (containing Carbenicillin 100 μ g/ml), cultivated 10 hours for 37 ℃.By 1: 100 fresh LB culture medium (containing Carbenicillin 100 μ g/ml) of inoculation, 37 ℃, 250rpm overnight incubation, next day is by cultivating in the fermentation tank that is seeded to 30L at 1: 50 to bacterium liquid A ₆₀₀=0.8-1.0 adds IPTG to final concentration 1mmol/L, continues training 5h at 37 ℃, centrifugal collection bacterium, and-20 ℃ are frozen.Frozen antibacterial room temperature is melted, and with the phosphate buffer thalline (10ml/g wet thallus) that suspends, after the ultrasonic grinding (200W, ultrasonic 5s, intermittently 5s, 10min altogether), the centrifugal 20min of 14000rpm, supernatant are soluble protein, and precipitation is occlusion body.

The purification of the purification F1 antigen protein of V and F1 antigen protein: with the precipitation after the ultrasonic grinding respectively after the carbamide washing with 1% Triton X100 and 0.5M, the carbamide washing of precipitation reuse 2.0M, the centrifugal 15min of 14000rpm, supernatant is thick pure F1 antigen protein, phosphate buffer dialysis with 20mM, last hydrophobic chromatography post carries out purification, collects the destination protein peak.The purification of V antigen protein, the supernatant after the ultrasonication directly carries out purification with the hydrophobic chromatography post, collects the destination protein peak, and with the phosphate buffer dialysis of 20mM, last DEAE chromatographic column is further purified.

The preparation and the immunity evaluation of embodiment 3 anthraxs and plague combined vaccine

The preparation of anthrax and plague combined vaccine

Immune programme for children and dosage design independent V, F1 and PA immune group and F+V, and the F+V+PA immune group is totally 5 groups.With the antigen protein of purification immunity Balb/c mice, 2 week the back survey the titre of antibody, booster immunization simultaneously, 5 week the back survey antibody titers, the 3rd immunity also merged the preparation monoclonal antibody with SP2/O.Provide the group and the dosage of immunity below.

1, immune F1 antigen, the about 4-5mg/mL of original protein concentration, 13 μ L/, totally 3.Inhale 40ulAg+460ul normal saline+0.5ml Fu Shi Freund's complete adjuvant, 3 Balb/c of immunity.

2, immune V antigen, original Ag concentration 4-5mg/ml, 3 μ L/, totally 3.Inhale 40ulAg+460ul normal saline+3 Balb/c of 0.5ml Fu Shi Freund's complete adjuvant immunity.

3, immune F+V antigen, divide two dosage to carry out immunity:

(1) F1 25ul+V 25ul+ normal saline 450ul+0.5ml Freund's complete adjuvant is exempted from 3 altogether.

(2) F1 12.5ul+V 12.5ul+ normal saline 475ul+0.5ml Freund's complete adjuvant is exempted from 2 altogether.

4, immune F1+V+PA

(1) F1 26ul+V 26ul+PA 34ul+457ul normal saline+0.5ml Fu Shi Freund's complete adjuvant is exempted from 2 altogether.

(2) F1 8ul+V 8ul+PA 12ul+472ul normal saline+0.5ml Fu Shi Freund's complete adjuvant is exempted from 2 altogether.

5, immune PA Ag (3mg/ml) 17ul/ only inhales 34ulAg+466ul normal saline+0.5ml Fu Shi Freund's complete adjuvant altogether, exempts from 2.More than equal stirring and evenly mixings, subcutaneous multiple spot immunity Balb/C ♀ mice.

Get the Balb/C mouse tail vein blood of immunity for the first time after 2 weeks, tire (the seeing Table 1-3) of measuring serum antibody got immunity the 2nd pin behind the blood, and dosage redeterminates tire (the seeing Table 4-6) of antibody with first after 6 weeks.

The animal trial test is the result confirm substantially, and the combined vaccine that this present invention is prepared has good safety and immunogenicity.

Table is annotated: the mouse of a left side and tail expressive notation in the table.

Data are all got the tail hematometry in the table.

Data in the table ++ ++ refer to the OD that measures ₆₀₀Value is about 1.9, +++about 1.0, ++ about 0.5.

1. 50ug dosage group, 2. 25ug dosage group

The table 1 antibody titer situation of the different immune group of detection of the antigen coated elisa plate of F1:

	1∶20	1∶40	1∶80	1∶160	1∶320	1∶640	1∶1280	1∶2560
									The right F1 tail of the left F1 of F1 F1+V is 1. 2. 2. 1. 1. 2. 2. tail of left F+V+PA of tail F+V+PA of left F+V+PA of tail F+V+PA of left F1+V of tail F1+V of left F1+V 1.	++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++	++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++	++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++	++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++	++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++	++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++	++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ +++ +++	++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ ++ ++ ++

The table 2 antibody titer situation of the different immune group of detection of the antigen coated elisa plate of V:

Group	1∶20	1∶40	1∶80	1∶160	1∶320	1∶640	1∶1280	1∶2560
									The right V tail of the left V of V F+V is 1. 2. 2. 1. 1. 2. 2. tail of left F+V+PA of tail F+V+PA of left F+V+PA of tail F+V+PA of left F+V of tail F+V of left F+V 1.	++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++	++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++	++++ ++++ ++++ ++++ ++++ ++ ++++ +++ ++++ ++++ ++++	++++ ++++ ++++ ++++ ++++ ++ +++ +++ +++ +++ +++	++++ ++++ ++++ ++++ ++++ ++ +++ ++ ++ ++ ++	+++ +++ +++ +++ +++ ++ +++ ++ ++ ++ ++	++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++	+ + + + + + + + + + +

Table 3 detects the antibody titer situation of different immune group with the antigen coated elisa plate of PA:

	1∶20	1∶40	1∶80	1∶160	1∶320	1∶640	1∶1280	1∶2560
									The 1. left F+V+PA of PA left side PA tail F+V+PA is 2. tail of the 2. left F+V+PA of tail F+V+PA 1.	+ + ± ± ± ±	+ +	+ +	+ +	± ±	± ±	± ±	- -

8 weeks after the first immunisation, the mensuration of 2 immunity back 6 all antibody titers:

Table 4 detects the antibody titer situation of different immune group with the antigen coated elisa plate of F:

Group	1∶160	1∶320	1∶640	1∶1280	1∶2560	1∶5120	1∶10240	1∶20480
									The 1. left F1+V+PA of F1+V+PA is 2. tail of the 2. left F1+V+PA of tail F1+V+PA 1.	++++ ++++ ++++ ++++	++++ ++++ ++++ ++++	++++ ++++ ++++ ++++	++++ ++++ ++++ ++++	++++ ++++ ++++ ++++	++++ ++++ ++++ ++++	+++ +++ +++ +++	+++ +++ +++ +++

Table 5 detects the antibody titer situation of different immune group with the antigen coated elisa plate of V:

Group	1∶160	1∶320	1∶640	1∶1280	1∶2560	1∶5120	1∶10240	1∶20480
									The 1. left F1+V+PA of F1+V+PA is 2. tail of the 2. left F1+V+PA of tail F1+V+PA 1.	++++ ++++ ++++ ++++	++++ ++++ ++++ ++++	++++ ++++ ++++ ++++	+++ +++ +++ +++	+++ +++ +++ +++	++ ++ ++ ++	++ ++ ++ ++	++ ++ ++ ++

Table 6 detects the antibody titer situation of different immune group with the antigen coated elisa plate of PA:

Group	1∶160	1∶320	1∶640	1∶1280	1∶2560	1∶5120	1∶10240	1∶20480
									The 1. left F+V+PA of PA left side PA tail F+V+PA is 2. tail of the 2. left F+V+PA of tail F+V+PA 1.	++++ ++++ ++++ ++++ ++++ ++++	++++ ++++ ++++ ++ ++++ ++	++++ ++++ ++++ ++ ++++ ++	++++ ++++ ++++ ++ ++++ ++	++++ ++++ ++++ ++ ++++ ++	++++ ++++ +++ - +++ -	++++ ++++ +++ - +++ -	+++ + +++ + +++ - +++ -

Sequence table

＜110〉Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL

＜120〉anthrax, plague combined vaccine

<130>

<160>6

<210>1

<211>2295

<212>DNA

＜213〉Bacillus anthracis

<400>1

atgaaaaaac gaaaagtgtt aataccatta atggcattgt ctacgatatt agtttcaagc 60

acaggtaatt tagaggtgat tcaggcagaa gttaaacagg agaaccggtt attaaatgaa 120

tcagaatcaa gttcccaggg gttactagga tactatttta gtgatttgaa ttttcaagca 180

cccatggtgg ttacctcttc tactacaggg gatttatcta ttcctagttc tgagttagaa 240

aatattccat cggaaaacca atattttcaa tctgctattt ggtcaggatt tatcaaagtt 300

aagaagagtg atgaatatac atttgctact tccgctgata atcatgtaac aatgtgggta 360

gatgaccaag aagtgattaa taaagcttct aattctaaca aaatcagatt agaaaaagga 420

agattatatc aaataaaaat tcaatatcaa cgagaaaatc ctactgaaaa aggattggat 480

ttcaagttgt actggaccga ttctcaaaat aaaaaagaag tgatttctag tgataactta 540

caattgccag aattaaaaca aaaatcttcg aactcaagaa aaaagcgaag tacaagtgct 600

ggacctacgg ttccagaccg tgacaatgat ggaatccctg attcattaga ggtagaagga 660

tatacggttg atgtcaaaaa taaaagaact tttctttcac catggatttc taatattcat 720

gaaaagaaag gattaaccaa atataaatca tctcctgaaa aatggagcac ggcttctgat 780

ccgtacagtg atttcgaaaa ggttacagga cggattgata agaatgtatc accagaggca 840

agacaccccc ttgtggcagc ttatccgatt gtacatgtag atatggagaa tattattctc 900

tcaaaaaatg aggatcaatc cacacagaat actgatagtc aaacgagaac aataagtaaa 960

aatacttcta caagtaggac acatactagt gaagtacatg gaaatgcaga agtgcatgcg 1020

tcgttctttg atattggtgg gagtgtatct gcaggattta gtaattcgaa ttcaagtacg 1080

gtcgcaattg atcattcact atctctagca ggggaaagaa cttgggctga aacaatgggt 1140

ttaaataccg ctgatacagc aagattaaat gccaatatta gatatgtaaa tactgggacg 1200

gctccaatct acaacgtgtt accaacgact tcgttagtgt taggaaaaaa tcaaacactc 1260

gcgacaatta aagctaagga aaaccaatta agtcaaatac ttgcacctaa taattattat 1320

ccttctaaaa acttggcgcc aatcgcatta aatgcacaag acgatttcag ttctactcca 1380

attacaatga attacaatca atttcttgag ttagaaaaaa cgaaacaatt aagattagat 1440

acggatcaag tatatgggaa tatagcaaca tacaattttg aaaatggaag agtgagggtg 1500

gatacaggct cgaactggag tgaagtgtta ccgcaaattc aagaaacaac tgcacgtatc 1560

atttttaatg gaaaagattt aaatctggta gaaaggcgga tagcggcggt taatcctagt 1620

gatccattag aaacgactaa accggatatg acattaaaag aagcccttaa aatagcattt 1680

ggatttaacg aaccgaatgg aaacttacaa tatcaaggga aagacataac cgaatttgat 1740

tttaatttcg atcaacaaac atctcaaaat atcaagaatc agttagcgga attaaacgca 1800

actaacatat atactgtatt agataaaatc aaattaaatg caaaaatgaa tattttaata 1860

agagataaac gttttcatta tgatagaaat aacatagcag ttggggcgga tgagtcagta 1920

gttaaggagg ctcatagaga agtaattaat tcgtcaacag agggattatt gttaaatatt 1980

gataaggata taagaaaaat attatcaggt tatattgtag aaattgaaga tactgaaggg 2040

cttaaagaag ttataaatga cagatatgat atgttgaata tttctagttt acggcaagat 2100

ggaaaaacat ttatagattt taaaaaatat aatgataaat taccgttata tataagtaat 2160

cccaattata aggtaaatgt atatgctgtt actaaagaaa acactattat taatcctagt 2220

gagaatgggg atactagtac caacgggatc aagaaaattt taatcttttc taaaaaaggc 2280

tatgagatag gataa 2295

<210>2

<211>764

<212>PRT

＜213〉Bacillus anthracis

<400>2

Met Lys Lys Arg Lys Val Leu Ile Pro Leu Met Ala Leu Ser Thr

1 5 10 15

Ile Leu Val Ser Ser Thr Gly Asn Leu Glu Val Ile Gln Ala Glu

20 25 30

Val Lys Gln Glu Asn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser

35 40 45

Gln Gly Leu Leu Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala

50 55 60

Pro Met Val Val Thr Ser Ser Thr Thr Gly Asp Leu Ser Ile Pro

65 70 75

Ser Ser Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr Phe Gln

80 85 90

Ser Ala Ile Trp Ser Gly Phe Ile Lys Val Lys Lys Ser Asp Glu

95 100 105

Tyr Thr Phe Ala Thr Ser Ala Asp Asn His Val Thr Met Trp Val

110 115 120

Asp Asp Gln Glu Val Ile Asn Lys Ala Ser Asn Ser Asn Lys Ile

125 130 135

Arg Leu Glu Lys Gly Arg Leu Tyr Gln Ile Lys Ile Gln Tyr Gln

140 145 150

Arg Glu Asn Pro Thr Glu Lys Gly Leu Asp Phe Lys Leu Tyr Trp

155 160 165

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

170 175 180

Gln Leu Pro Glu Leu Lys Gln Lys Ser Ser Asn Ser Arg Lys Lys

185 190 195

Arg Ser Thr Ser Ala Gly Pro Thr Val Pro Asp Arg Asp Asn Asp

200 205 210

Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr Thr Val Asp Val

215 220 225

Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser Asn Ile His

230 235 240

Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu Lys Trp

245 250 255

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

260 265 270

Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val

275 280 285

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

290 295 300

Ser Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Gln Thr

305 310 315

Arg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr Ser

320 325 330

Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp Ile

335 340 345

Gly Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser Ser Thr

350 355 360

Val Ala Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp

365 370 375

Ala Glu Thr Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn

380 385 390

Ala Asn Ile Arg Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn

395 400 405

Val Leu Pro Thr Thr Ser Leu Val Leu Gly Lys Asn Gln Thr Leu

410 415 420

Ala Thr Ile Lys Ala Lys Glu Asn Gln Leu Ser Gln Ile Leu Ala

425 430 435

Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu Ala Pro Ile Ala Leu

440 445 450

Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile Thr Met Asn Tyr

455 460 465

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

470 475 480

Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe Glu Asn

485 490 495

Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val Leu

500 505 510

Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile Phe Asn Gly Lys

515 520 525

Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala Val Asn Pro Ser

530 535 540

Asp Pro Leu Glu Thr Thr Lys Pro Asp Met Thr Leu Lys Glu Ala

545 550 555

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

560 565 570

Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp Gln

575 580 585

Gln Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala

590 595 600

Thr Asn Ile Tyr Thr Val Leu Asp Lys Ile Lys Leu Asn Ala Lys

605 610 615

Met Asn Ile Leu Ile Arg Asp Lys Arg Phe His Tyr Asp Arg Asn

620 625 630

Asn Ile Ala Val Gly Ala Asp Glu Ser Val Val Lys Glu Ala His

635 640 645

Arg Glu Val Ile Asn Ser Ser Thr Glu Gly Leu Leu Leu Asn Ile

650 655 660

Asp Lys Asp Ile Arg Lys Ile Leu Ser Gly Tyr Ile Val Glu Ile

665 670 675

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

680 685 690

Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly Lys Thr Phe Ile

695 700 705

Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser Asn

710 715 720

Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu Asn Thr

725 730 735

Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr Asn Gly Ile

740 745 750

Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly

755 760

<210>3

<211>513

<212>DNA

＜213〉Yersinia pestis

<400>1

atgaaaaaaa tcagttccgt tatcgccatt gcattatttg gaactattgc aactgctaat 60

gcggcagatt taactgcaag caccactgca acggcaactc ttgtcgaacc agcccgcatc 120

actcttacat ataaggaagg cgctccaatt acaattatgg acaatggaaa catcgataca 180

gaattacttg ttggtacgct tactcttggc ggctataaaa caggaaccac tagcacatct 240

gttaacttta cagatgccgc gggtgatccc atgtacttaa catttacttc tcaggatgga 300

aataaccacc aattcactac aaaagtgatt ggcaaggatt ctagagattt tgatatctct 360

cctaaggtaa acggtgagaa ccttgtgggg gatgacgtcg tcttggctac gggcagccag 420

gatttctttg ttcgctcaat tggttccaaa ggcggtaaac ttgcagcagg taaatacact 480

gatgctgtaa ccgtaaccgt atctaaccaa taa 513

<210>4

<211>170

<212>PRT

＜213〉Yersinia pestis

<400>2

Met Lys Lys Ile Ser Ser Val Ile Ala Ile Ala Leu Phe Gly Thr

1 5 10 15

Ile Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser Thr Thr Ala

20 25 30

Thr Ala Thr Leu Val Glu Pro Ala Arg Ile Thr Leu Thr Tyr Lys

35 40 45

Glu Gly Ala Pro Ile Thr Ile Met Asp Asn Gly Asn Ile Asp Thr

50 55 60

Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly

65 70 75

Thr Thr Ser Thr Ser Val Asn Phe Thr Asp Ala Ala Gly Asp Pro

80 85 90

Met Tyr Leu Thr Phe Thr Ser Gln Asp Gly Asn Asn His Gln Phe

95 100 105

Thr Thr Lys Val Ile Gly Lys Asp Ser Arg Asp Phe Asp Ile Ser

110 115 120

Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val Val Leu

125 130 135

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

140 145 150

Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val Thr Val

155 160 165

Thr Val Ser Asn Gln

170

<210>5

<211>981

<212>DNA

＜213〉Yersinia pestis

<400>1

atgattagag cctacgaaca aaacccacaa cattttattg aggatctaga aaaagttagg 60

gtggaacaac ttactggtca tggttcttca gttttagaag aattggttca gttagtcaaa 120

gataaaaata tagatatttc cattaaatat gatcccagaa aagattcgga ggtttttgcc 180

aatagagtaa ttactgatga tatcgaattg ctcaagaaaa tcctagctta ttttctaccc 240

gaggatgcca ttcttaaagg cggtcattat gacaaccaac tgcaaaatgg catcaagcga 300

gtaaaagagt tccttgaatc atcgccgaat acacaatggg aattgcgggc gttcatggca 360

gtaatgcatt tctctttaac cgccgatcgt atcgatgatg atattttgaa agtgattgtt 420

gattcaatga atcatcatgg tgatgcccgt agcaagttgc gtgaagaatt agctgagctt 480

accgccgaat taaagattta ttcagttatt caagccgaaa ttaataagca tctgtctagt 540

agtggcacca taaatatcca tgataaatcc attaatctca tggataaaaa tttatatggt 600

tatacagatg aagagatttt taaagccagc gcagagtaca aaattctcga gaaaatgcct 660

caaaccacca ttcaggtgga tgggagcgag aaaaaaatag tctcgataaa ggactttctt 720

ggaagtgaga ataaaagaac cggggcgttg ggtaatctga aaaactcata ctcttataat 780

aaagataata atgaattatc tcactttgcc accacctgct cggataagtc caggccgctc 840

aacgacttgg ttagccaaaa aacaactcag ctgtctgata ttacatcacg ttttaattca 900

gctattgaag cactgaaccg tttcattcag aaatatgatt cagtgatgca acgtctgcta 960

gatgacacgt ctggtaaata a 981

<210>6

<211>326

<212>PRT

＜213〉Yersinia pestis

<400>2

Met Ile Arg Ala Tyr Glu Gln Asn Pro Gln His Phe Ile Glu Asp

1 5 10 15

Leu Glu Lys Val Arg Val Glu Gln Leu Thr Gly His Gly Ser Ser

20 25 30

Val Leu Glu Glu Leu Val Gln Leu Val Lys Asp Lys Asn Ile Asp

35 40 45

Ile Ser Ile Lys Tyr Asp Pro Arg Lys Asp Ser Glu Val Phe Ala

50 55 60

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

65 70 75

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

80 85 90

Asp Asn Gln Leu Gln Asn Gly Ile Lys Arg Val Lys Glu Phe Leu

95 100 105

Glu Ser Ser Pro Asn Thr Gln Trp Glu Leu Arg Ala Phe Met Ala

110 115 120

Val Met His Phe Ser Leu Thr Ala Asp Arg Ile Asp Asp Asp Ile

125 130 135

Leu Lys Val Ile Val Asp Ser Met Asn His His Gly Asp Ala Arg

140 145 150

Ser Lys Leu Arg Glu Glu Leu Ala Glu Leu Thr Ala Glu Leu Lys

155 160 165

Ile Tyr Ser Val Ile Gln Ala Glu Ile Asn Lys His Leu Ser Ser

170 175 180

Ser Gly Thr Ile Asn Ile His Asp Lys Ser Ile Asn Leu Met Asp

185 190 195

Lys Asn Leu Tyr Gly Tyr Thr Asp Glu Glu Ile Phe Lys Ala Ser

200 205 210

Ala Glu Tyr Lys Ile Leu Glu Lys Met Pro Gln Thr Thr Ile Gln

215 220 225

Val Asp Gly Ser Glu Lys Lys Ile Val Ser Ile Lys Asp Phe Leu

230 235 240

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

245 250 255

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

260 265 270

Thr Thr Cys Ser Asp Lys Ser Arg Pro Leu Asn Asp Leu Val Ser

275 280 285

Gln Lys Thr Thr Gln Leu Ser Asp Ile Thr Ser Arg Phe Asn Ser

290 295 300

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

305 310 315

Met Gln Arg Leu Leu Asp Asp Thr Ser Gly Lys

320 325

Claims (8)

1. combined vaccine of protecting humans and animals to avoid Bacillus anthracis and Yersinia pestis infection; wherein contain bacillus anthracis protective antigen PA, Yersinia pestis V antigen and Yersinia pestis F1 antigen, or these 3 kinds antigenic protective epitope's parts.

2. the combined vaccine of claim 1,3 kinds of antigen that wherein contains or protective epitope separate and/or the recombiant protein of purification.

3, the gene of coding recombiant protein comprises:

Bacillus anthracis protective antigen (PA) gene has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 DNA sequence;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 DNA sequence hybridization that limit.

Yersinia pestis F1 antigen gene has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 3 DNA sequence;

2) polynucleotide of SEQ ID № .4 protein sequence in the code sequence tabulation;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 4 DNA sequence hybridization that limit.

Yersinia pestis V antigen gene has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 5 DNA sequence;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 6 protein sequences;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 6 DNA sequence hybridization that limit.

4. contain the described expression carrier of claim 3, engineering bacteria.

5. the described expression vector of claim 4 comprises procaryotic cell expression carrier and yeast cell to express carrier.

6. claim 4 described engineering bacteria comprises and is used to express, and is fit to the escherichia coli and the yeast of fermentation and scale.

7. the route of inoculation of claim 1 combined vaccine comprises subcutaneous or intramuscular injection, and Transdermal absorption is oral, and the intranasal mucosa drips/sprays.

8. the dosage form of claim 1 combined vaccine comprises nasal spray, freeze dried powder, liquid.

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