CN101991864B - Leptospira interrogans DNA (Deoxyribose Nucleic Acid) vaccine as well as construction method and application thereof - Google Patents
Leptospira interrogans DNA (Deoxyribose Nucleic Acid) vaccine as well as construction method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of medical biology, in particular to a leptospira interrogans DNA (Deoxyribose Nucleic Acid) vaccine as well as a construction method and application thereof. The leptospira interrogans can rapidly invade the inside of human and various animals to cause infection via the skin or the mucous membranes, and result in leptospirosis with the main symptoms of fever, jaundice and bleeding after humans are infected, the leptospirosis is the common zoonosis with wide world distribution and seriously threat the health of the humans and the production of animal husbandry, and China is one of the main countries with the epidemic leptospirosis. The invention provides the leptospira interrogans DNA vaccine, which comprises a eukaryon expression vector and leptospira interrogans LB018 genes, wherein the nucleotide sequence of the LB018 genes is shown as SEQ ID NO: 1. The leptospira interrogans DNA vaccine can effectively induce the immune response reaction of a piggy, which shows that the leptospira interrogans DNA vaccine has a certain immune protection action on the leptospira interrogans of the animals, and the leptospira interrogans DNA vaccine has the advantages of simple production technology, low cost and stable physical and chemical properties, and is convenient for storage and transportation.
Description
Technical field
The present invention relates to the medical biotechnology field, be specifically related to a kind of leptospira interrogans dna vaccination and construction method and application.
Background technology
Leptospira interrogans (is called for short coupler body; Letospira interrogans) can invade rapidly in people and the multiple animal body through skin or mucosa and cause infection; Cause behind the human infection with heating, jaundice, hemorrhage be that the coupler body of cardinal symptom is sick, symptom is slight or asymptomatic mostly behind the zoogenetic infection.Because question mark coupler body natural reservoir (of bird flu viruses) is numerous, and water source bamboo telegraph through polluting, so being a kind of world, the coupler body disease distributes that wide, serious threat human health and animal husbandry are produced, common Amphixenosis, China is one of main popular country of coupler body disease.
Whole cell leptospira vaccine shows is the sick important means of prevention coupler body, though coupler body inactivated vaccine of using at present and adventitia vaccine on probation have the effect that excellent prevention homotype coupler body infects, the two all can not effectively prevent the infection of multiple different serotypes coupler body; And the immunity persistent period is short, causes protecting effect not satisfactory (Min Lin, Om Suruballi; Klaus Nielsen, et al, Identification of a 35-kilodaltonserovar-cross-reactive flagellar protein; FlaB, from Leptospira interrogans byN-terminal sequencing, gene cloning; And sequence analysis; Infection andImmunity, 1997,4355-4359).
Dna vaccination is the new generation vaccine that rises in recent years, has antibody titer and holds time long and advantage such as cross protection is effective.Dna vaccination also has following outstanding advantage in addition: body fluid and the comprehensive immunne response of cell can be brought out in (1), play preventive effect; (2) can immunological adjuvant CpG sequence be integrated in the dna vaccination, improve the immunne response level of body and need not to add in addition adjuvant; (3) simple, with low cost, good stability of production technology and storing are convenient.
Still the report that does not have at present relevant leptospira interrogans dna vaccination.
Summary of the invention
The object of the present invention is to provide a kind of leptospira interrogans dna vaccination, and construction method and application.
The present invention adopts leptospira to rely type to rely the LB018 gene of strain to make up the leptospira interrogans dna vaccination.LB018 is the gene that is positioned at the encoding proteins on No. 2 chromosomes of leptospira; The prediction of LB018 albumen is positioned at adventitia (Hong-Liang Yang, YongZhang Zhu, et al; In silico andmicroarray-based genomic approaches to identifying potential vaccine candidatesagainst Leptospira interrogans; BMC Genomics, 2006,7:293).What the present invention adopted is that question mark coupler body jaundice hemorrhage crowd relies type 56601 strain coupler bodies, and the clone of the LB018 gene of relevant this type coupler body does not appear in the newspapers always.
The invention provides a kind of leptospira interrogans dna vaccination, this dna vaccination is by carrier for expression of eukaryon and leptospira interrogans LB018 genomic constitution, and the nucleotide sequence of described LB018 gene is shown in SEQ ID NO:1.
Question mark coupler body jaundice hemorrhage crowd relies the LB018 gene of type 56601 strain coupler bodies:
A) sequence signature
Length: 984bp
Type: nucleic acid
Chain number: two strands
Geometry: linearity
B) molecule type: DNA
C) source: question mark coupler body bacterial strain, the jaundice hemorrhage crowd relies type, and middle medicine inspection code name 56601 is available from Nat'l Pharmaceutical & Biological Products Control Institute's vaccine Room 2.
D) sequence description: shown in SEQ ID NO:1.
Above-mentioned leptospira interrogans dna vaccination, wherein said carrier for expression of eukaryon are the pVAX plasmid.The present invention clones the LB018 gene, analyze, and is inserted into carrier for expression of eukaryon pVAX, is built into Leptospira DNA vaccine pVAX-LB018.
Above-mentioned leptospira interrogans dna vaccination, wherein said carrier for expression of eukaryon also can be pQE31 plasmid, pcDNA3.1 plasmid or pGEX-1 λ T plasmid.
The present invention also provides the construction method of above-mentioned leptospira interrogans dna vaccination, and this method comprises the steps:
A) design forward primer: 5 '-CTG
GGATCC 
GGAAGGTTCGTTTT-3 ' (shown in SEQ ID NO:2),
Downstream primer: 5 '-ACA
GAATTC 
GAATTGGTATCTATACC-3 ' (shown in SEQID NO:3),
Carry out PCR, the DNA sequence of the leptospira interrogans LB018 gene that obtains encoding is shown in SEQID NO:1;
B) DNA sequence with above-mentioned coding leptospira interrogans LB018 gene is cloned into carrier for expression of eukaryon.
Above-mentioned PCR primer is synthetic by Shanghai biological engineering company limited.The LB0018 gene clone scheme of coupler body adopts the method for PCR to carry out; The roughly flow process of this method is: according to the two ends sequential design PCR primer of this gene; Genomic DNA with 56601 strain coupler bodies is a template then, and the corresponding genetic fragment of pcr amplification gets final product the extension amplification outcome order-checking again.Concrete grammar and reaction system thereof are referring to " molecular cloning " (Science Press published in 1992) related Sections.
Carrier for expression of eukaryon in the said method is the pVAX plasmid.PVAX (available from Invitrogen company, sequence and physical map are seen the catalogue of the said firm); In building process and the preparation process, the used host bacterium of plasmid amplification is DH5 (available from a GIBCO company).
Amplification leptospira genomic DNA, the pcr amplification product of 1092bp exposes sticky end with BamH I and EcoR I enzyme action.With BamH I/EcoR I double digestion carrier pVAX, electrophoresis reclaims the big fragment of carrier simultaneously.After above two fragments are connected into the pVAX-LB018 plasmid, reuse CaCl
2Method transformed into escherichia coli DH5 α, screening obtains positive recombinant, promptly obtains the engineering bacteria DH5 α (pVAX-LB018) that contains recombiant plasmid pVAX-LB018.
The mechanism of action of dna vaccination of the present invention is: clone's leptospira gene (LB018) is recombined into eukaryon expression plasmid; Be injected in muscular tissue; Make it in the myocyte, express coupler body antigen; Through the angtigen presentation process, can activate intravital immunity system and cell immune system.Activated immunity system can produce special antibody, eliminates and is free in the virus in the blood, prevents leptospiral invasion; And activated cell immune system can produce cellulotoxic lymphocyte (CTL), attacks infected cells, eliminates the leptospira of hiding.
The present invention also provides the application of above-mentioned leptospira interrogans dna vaccination in prevention and treatment coupler body medicine.
1, vivoexpression of this dna vaccination and detection; Be through the electroporation rotaring redyeing COS 7 cell with constructed dna vaccination; Harvesting after 72 hours; Detect LB018 expression of gene level in culture supernatant and the cell breakage liquid with indirect elisa method, the result shows has expression, explains that dna vaccination of the present invention really can express in mammalian cell.(seeing the experiment 1 of embodiment 2 for details)
2, expression in vivo of this dna vaccination and detection are with behind the pure article immune mouse of dna vaccination that obtains, and detect the antigen levels (as shown in Figure 3) in the serum with the ELISA method.The result can express the LB018 gene after showing this DNA vaccine immunity animal.(seeing the experiment 2 of embodiment 2 for details)
3, this dna vaccination brings out the detection that body produces the HI level, is with behind the pure article immune mouse of dna vaccination that obtains, and detects the level of anti-coupler body antibody in the serum with the ELISA method.The dna vaccination group can be kept the antibody titer of higher level in for a long time as a result; And as the inactivated vaccine of matched group (with cultured coupler body through 56 ℃; After the deactivation in 60 minutes, after ultrasonication, the insoluble composition of centrifugal removal; Supernatant is measured protein concentration with Coomassie brilliant blue G-250 staining, protein concentration is adjusted into 5mg/mL.) antibody titer then descends from March, subunit vaccine group antibody titer is higher than dna vaccination, but antibody titer promptly descended since March (as shown in Figure 4).(seeing the experiment 3 of embodiment 2 for details)
4, the intersecting protective of this dna vaccination: in order to observe vaccine, with leptospira 56601 strains and 56609 common strains, the Cavia porcellus of 56608 strains (available from Nat'l Pharmaceutical & Biological Products Control Institute's vaccine Room 2) challenge infection immunity pVAX-LB018 vaccine to the cross immunity of three kinds of different type coupler bodies protection situation.Gather painstaking effort and carry out In vitro culture.The result finds that dna vaccination pVAX-LB018 compares with traditional inactivated vaccine, and the challenge infection with coupler body is all had good protection effect, but dna vaccination and inactivated vaccine are relatively, and cross-protection is superior to inactivated vaccine, and the result is as shown in Figure 5.(seeing the experiment 4 of embodiment 2 for details)
5, this dna vaccination is to the immanoprotection action of piglet coupler body infection; Be with the immune piglet of the pure article of dna vaccination that obtains; Booster immunization once after 10 days; 56601 strains of challenge infection coupler body are got blood from ear vein after 2 days and are carried out the coupler body cultivation, and the 20th day aseptic collection kidney BIAO and BEN after attack cultivated afterwards.Reflect the protectiveness of dna vaccination through the infection conditions of observing coupler body to pig challenge infection coupler body.As a result, the immune group pig is not compared with immune swine, relative lassitude behind the coupler body challenge infection,, not gloss fluffy and disorderly by hair.The result of coupler body In vitro culture blood and renal tissue is consistent.This vaccine infects the piglet coupler body and has the certain protection effect.(seeing the experiment 5 of embodiment 2 for details)
The present invention can bring out the immune response of piglet effectively, explains that its infection due to Leptospira interrogans to animal has certain immanoprotection action; And production technology is simple, and is with low cost, and physicochemical property is stablized, is convenient to store and transportation.
Description of drawings
Fig. 1 contains the plasmid map of LB018 gene
The structure flow chart of Fig. 2 dna vaccination pVAX-LB018
Fig. 3 dna vaccination pVAX-LBO18 is at the intravital expression curve of BALB/c mouse
Fig. 4 dna vaccination pVAX-LBO18 brings out the time graph of mice serum antibody titer
The cross-protection that Fig. 5 dna vaccination pVAX-LBO18 attacks Cavia porcellus different shaped coupler body
The specific embodiment:
Below in conjunction with accompanying drawing and embodiment the present invention is further described, but enforcement of the present invention is not limited only to this.
Embodiment 1: the structure of dna vaccination pVAX-LB018 of the present invention, structure and preparation:
The structure of dna vaccination of the present invention is with carrier for expression of eukaryon---pVAX is the recombinant plasmid dna molecule (available from Invitrogen company) of skeleton, and general structure is as shown in Figure 1.The about 4.0kb of total length (kilobase to) except that containing elements such as pMB 1ori plasmid replication initiation site, kalamycin resistance gene Kanamycin, has also comprised a complete eukaryotic transcription translation unit.The eukaryotic transcription translation unit contains eukaryotic transcription translates necessary element: eucaryon cytomegalovirus promoter P
CMV, respective coding gene LB018 and 3 ' end polyadenylic acid BGH pA.On the whole, but this vaccine is the recombinant plasmid dna that can in eukaryotic cell, express LB018 and activated lymphocyte.
1, makes up engineering bacteria DH5 α (pVAX-LB018) (as shown in Figure 2)
Leptospira rely type rely strain (56601 strain) in the EMJH culture medium 28 ℃ to be cultured to cell density be 10
8During/ml, the centrifugal 10min of 10000g collects thalline.Extract leptospira genomic DNA (Shanghai China Shun biotech firm) with bacterial genomes DNA extraction agent box.The gene order (the GenBank number of landing is AAN51577.1) that provides according to GenBank designs the LB061 primer.
Forward primer: 5 '-CTG
GGATCCATGGGAAGGTTCGTTTT-3 ' (shown in SEQ ID NO:2),
Downstream primer: 5 '-ACA
GAATTC 
GAATTGGTATCTATACC-3 ' (shown in SEQ ID NO:3).
Primer is synthetic by Shanghai biological engineering company limited.Amplification leptospira genomic DNA, the pcr amplification product of 1092bp exposes sticky end with BamH I and EcoR I enzyme action.With BamH I/EcoR I double digestion carrier pVAX, electrophoresis reclaims the big fragment of carrier simultaneously.After above two fragments are connected into the pVAX-LB018 plasmid, reuse CaCl
2Method transformed into escherichia coli DH5 α (available from GIBCO company), screening obtains positive recombinant, promptly obtains the engineering bacteria DH5 α (pVAX-LB018) that contains recombiant plasmid pVAX-LB018.Concrete grammar and reaction system thereof are referring to the related content of " molecular cloning " (Science Press published in 1992).
2, preparation dna vaccination pVAX-LB018
Dna vaccination of the present invention prepares with above-mentioned engineered strain DH5 α (pVAX-LB018).The cultivation amplification reaches to make with extra care can carry out (seeing aforementioned " molecular cloning " related content for details) through antibacterial culturing, plasmid amplification and the purification process of routine.
Training method is generally liquid culture.Available air shaking table shaken cultivation during a small amount of the amplification; When producing, industrialness need use the capable high density suspension of bacterial fermentation jar aerobic culture.Cultivation source in the culture medium does not have special requirement; The general LB fluid medium of using; Like the need High Density Cultivation; Then can select for use 2 * YT (contain 1.6% tryptone, 1% yeast extract and 0.5% sodium chloride, PH7.0) or the SOC fluid medium (contain 2% tryptone, 0.5% yeast extract, 0.05% sodium chloride, 20mmol/L glucose, 2.5mmol/LKCl, 10mmol/L MgCl
2, PH7.0).Also can add in case of necessity moving, plant, the mineral wet goods is as defoamer.Cultivation temperature is generally 37 ℃, the setting that is decided by culture medium, training method and ventilation condition opportunity of incubation time and collection antibacterial.
Separation and purification DNA from the engineering bacteria after the amplification can adopt some conventional methods.For example available detergent SDS cracking thalline utilizes DNA and chromosomal DNA to become the difference of renaturation characteristic, through to the degeneration of DNA and the processing of renaturation, DNA and chromosomal DNA is separated; Utilize DNA and impurity to carry out further purification again, and remove endotoxin in the difference of aspects such as dissolubility, ions bind power, absorption affinity.Specifically; After being present in the DNA process desalting processing in the renaturation solution; Available anion exchange resin (like DEAE Sepharose) exchanges; Use TE (containing 10mmol/L Tris and 1mmol/L EDTA) buffer solution elution then, can make highly purified DNA (specifically seeing embodiment 2).Through PBS dilution packing, can obtain the pure article of transparent clarifying dna vaccination of the present invention.These article physicochemical property is very stable, can preserve down and transportation in room temperature, before the use, gets final product with the water for injection dissolving.
Embodiment 2: the Function detection of dna vaccination pVAX-LB018 of the present invention:
1, obtain highly purified dna vaccination of the present invention:
From-80 ℃ of refrigerators, take out frozen engineered strain---DH5 α (pVAX-LB018), after room temperature is melted, dip in inoculating loop and to take a morsel the streak inoculation of bacterium liquid on 1.5% LB agar plate (containing kanamycin 50ug/ml); After 37 ℃ of overnight incubation; Get monoclonal be inoculated in 50ml LB fluid medium (contain 1% tryptone, 0.5% yeast extract and 1% sodium chloride, kanamycin 50ug/ml, PH7.0) in; 37 ℃ of shaken cultivation 40 hours are as seed liquor.In 1: 20 ratio seed liquor is inoculated in (on the basis of aforementioned composition, other contains kanamycin 50 μ g/ml) in 1 liter of SOC fluid medium, cultivated 25 hours in 37 ℃ of shaken; Adding chloromycetin to final concentration is 170 μ g/ml, continues to cultivate 16 hours in 37 ℃ of shaken.With bacterium liquid in 4 ℃ with 6000rpm centrifugal 10 minutes; Remove supernatant; Thalline is resuspended in the solution I (containing 50mmol/L glucose, 25mmol/L Tris.Cl, 10mmol/L EDTA PH8.0) of the alkaline lysis of 30ml and adds lysozyme 40mg; Add 60ml solution II (0.2mol/L NaOH and 1%SDS), slowly put upside down mixing 5 times, room temperature was placed 5 minutes.The solution III (containing potassium ion 3mol/L, acetate 5mol/L) that adds the pre-cooling of 45ml ice is slowly put upside down mixing for several times, ice bath 10 minutes; In 4 ℃ with 10000r/m centrifugal 10 minutes, get supernatant, cross 500ml G25 post desalination after, last 20mlDEAE Sepharose anion-exchange column, wait to combine to adsorb finish after, press salt ion (Cl
-) (0～1mol/L) progressive linear gradient elution method is slowly cleaned anion-exchange column with the PBS (PH8.0) and the sodium chloride solution of 10 column volumes to concentration, collects the eluent of 260nm place absworption peak, obtains the bullion of dna vaccination from low to high.Be further purified then, add the dehydrated alcohol deposition of 2 times of volumes, 12000r/m4 ℃ centrifugal 10 minutes, remove supernatant, behind 70% ethanol rinsing airing, be dissolved in PBS (PH8.0) solution, promptly get the pure article of dna vaccination of the present invention; With ultraviolet spectrophotometry quantitatively after, process finished product by every part 200 μ g/ml packing again.
2, the prophylactic immunization scheme of dna vaccination of the present invention is:
Get final product with finished product 1ml intramuscular injection, dosage is every piglet 200 μ g.Supplementary immunization once can obtain to react than strong immune response after 10 days.As then effect is better with the subunit vaccine therapeutic alliance.
The vivoexpression and the detection of experiment 1DNA vaccine
Method with electroporation changes plasmid over to the COS7 cell, and the perforation condition is: electric capacity 960 μ F, and voltage is 250V, plasmid 40 μ g, the COS7 cell concentration is 1 * 10
7/ ml, the time is 16.7s.Establish the contrast of empty carrier pVAX simultaneously, harvesting behind the 72h.Detect the expression of LB018 gene in COS7 cell conditioned medium and cell pyrolysis liquid with indirect elisa method; Cells and supernatant and cell pyrolysis liquid are encapsulated 96 hole elisa plates respectively; One anti-is the anti-coupler body serum of rabbit (self-control), two anti-be goat-anti rabbit-HRP (available from magnificent biological engineering company limited).After the colour developing, positive greater than 1.8 with P/N ratio, the LB018 gene has obtained expression in the COS7 cell as a result, and is all positive with cell pyrolysis liquid in the supernatant.Explain that this dna vaccination can express in the COS7 cell.
Intravital antigen levels detects test after testing 2 immunity inoculations
20 of the female BALB/c mouses (average weight 20g) in 6～8 ages in week are divided into 2 groups at random, and 10 every group, first group of immunity dna vaccination (every inoculation 100 μ g), the 2nd group is the PBS negative control.Plasmid 100 μ g are injected to 1 group of mice through tibialis anterior, and 2 groups of mices then inoculate equal-volume PBS.Before injection same day injection with after the injection, got blood through the tail vein on the 2nd, 4,6,8,10 day, detect plasmid behind the separation of serum in the lump in the expression of serum situation.
Detect the level of LB018 in the serum with indirect elisa method, with encapsulating 96 hole elisa plates after the mice serum dilution in 1: 20, one anti-ly is the anti-coupler body serum of rabbit (self-control), two anti-be goat-anti rabbit-HRP (available from magnificent biological engineering company limited).Negative control is the blank mice serum of vaccinate not.After the colour developing, to detect the OD of hole and negative control hole
450Ratio P/N＞1.5 are positive.The result is as shown in Figure 3.
As can be seen from Figure 3, vaccine detected antigen levels in the mice body is the highest at second day, descends gradually later on, and during by the 10th day, content is atomic.
Experiment 3DNA vaccine brings out the detection that body produces the HI level
For holding time of comparison dna vaccine pVAX-LB018 HI, BALB/c mouse is divided into 4 groups at random, 10 every group; The 1st group is the dna vaccination group, every injected in mice pVAX-LB018 vaccine 100 μ g, and the 2nd group is the subunit vaccine group; Every injected in mice protein 10 0 μ g (the GST-LB018 subunit vaccine is this chamber self-control: contain the strain of fusion expression plasmid, and ultrasonication after thermal induction, inclusion body is through 1%Triton X-100; 2M, 4M carbamide washs step by step, dissolves with 8M carbamide again; 3M carbamide renaturation, saturated ammonium sulphate precipitation is dissolved in PBS.), the 3rd group be the inactivated vaccine group (with cultured coupler body through 56 ℃, deactivation in 60 minutes, after ultrasonication, the insoluble composition of centrifugal removal is measured protein concentration with supernatant with Coomassie brilliant blue G-250 staining, and protein concentration is adjusted into 5mg/mL.), every mouse subcutaneous injection deactivation coupler body 10
8Bar, the 4th group is the PBS matched group.Every first quarter moon is got blood 1 time in two months after the same day of injecting and injection, gets blood once in later every month, and getting the blood position is the tail vein.Dna vaccination group and deactivation group were observed to 1 year, subunit vaccine group June.Detect the level of anti-coupler body antibody in the serum with indirect elisa method; GST-LB018 with purification encapsulates elisa plate; After adding serum to be checked; The anti-mice IgG two of rabbit anti-(available from magnificent biological engineering company limited) that adds the HRP labelling again measures OD value under the 450nm wavelength after the chromogenic reaction, positive greater than 2 with P/N.
The dna vaccination group can be kept the antibody titer of higher level in for a long time as a result, and the inactivated vaccine antibody titer then descended from March, and subunit vaccine group antibody titer is higher than dna vaccination, but antibody titer promptly descended since March.(as shown in Figure 4)
Experiment 4DNA vaccine brings out the test that body produces intersecting protective
In order to observe the protection situation of dna vaccination to Cavia porcellus challenge infection coupler body, Cavia porcellus is divided into 3 groups, the 1st group 10 is the PBS matched group, and the 2nd group 30 is the inactivated vaccine group, and through subcutaneous approach injection, the 3rd group 30 are the pVAX-LB018 group.Wherein 2,3 groups are divided into three groups again, challenge infection coupler body 56601 strains respectively, 56609 strains and 56608 strains (available from Nat'l Pharmaceutical & Biological Products Control Institute's vaccine Room 2).Immunizing dose is 100 μ g/200 μ l, and the 5th day booster immunization is 1 time in first immunisation inoculation back, attacks the coupler body pathogen at postvaccinal the 21st day first, and inoculation position is a Cavia porcellus rear legs muscle.After attack, gathered cardiac blood on the 2nd day, killed Cavia porcellus on the 7th day, the aseptic collection kidney is seeded to respectively in the Qie Shi culture medium, and every kind of BIAO and BEN is all inoculated two-tube.28 ℃ of painstaking effort cultivate that to have coupler body growth explanation to cultivate in 2 weeks positive, and it is effective that vaccine group is judged to be vaccine with 2/3 Cavia porcellus negative culture results, and matched group all should be negative.Kidney is cultivated and to be observed March Wugou bulk-growth always and be judged to feminine gender.
The result finds that dna vaccination pVAX-LB018 compares with traditional inactivated vaccine, and the challenge infection of homotype coupler body is all had good protection effect, but dna vaccination and inactivated vaccine comparison, cross-protection is superior to inactivated vaccine (x
2Check, p＜0.05).The result is as shown in Figure 5.
Experiment 5DNA vaccine detects the immanoprotection action that the piglet coupler body infects
Piglet is divided into 2 groups at random, 10 every group, the 1st group of intramuscular injection dna vaccination pVAX-LB018 200 μ g/ml/ heads, booster immunization is once after 10 days; The 2nd group is the PBS matched group.Challenge infection coupler body 56601 strains in the 20th day of initial immunity, every 10
9Bar coupler body, challenge infection are got blood from ear vein after 2 days and are carried out the coupler body cultivation, and the 20th day aseptic collection kidney BIAO and BEN after attack cultivated afterwards.Vaccine group has the certain protection effect to the coupler body infection of piglet as a result, and statistical discrepancy (P＜0.05) is arranged between two groups.The immune group pig is not compared with immune group, and lassitude behind the coupler body challenge infection is fluffy and disorderly by hair, not gloss.
Above experimental result can prove that dna vaccination pVAX-LB018 of the present invention can bring out the immune response of body effectively, and piglet prevention leptospiral infection is played good immanoprotection action.
Claims (5)
1. a leptospira interrogans dna vaccination is characterized in that this dna vaccination by carrier for expression of eukaryon and leptospira interrogans LB018 genomic constitution, and the nucleotide sequence of described LB018 gene is shown in SEQ ID NO:1.
2. leptospira interrogans dna vaccination according to claim 1 is characterized in that wherein said carrier for expression of eukaryon is the pVAX plasmid.
3. the construction method of a leptospira interrogans dna vaccination as claimed in claim 1 is characterized in that this method comprises the steps:
A) the design forward primer is shown in SEQ ID NO:2, and downstream primer carries out PCR shown in SEQ ID NO:3, and the DNA sequence that obtains leptospira interrogans LB018 gene is shown in SEQ ID NO:1;
B) DNA sequence with above-mentioned leptospira interrogans LB018 gene is cloned into carrier for expression of eukaryon.
4. the construction method of leptospira interrogans dna vaccination according to claim 3 is characterized in that wherein said carrier for expression of eukaryon is the pVAX plasmid.
5. the application of leptospira interrogans dna vaccination according to claim 1 or claim 2 in preparation prevention and treatment coupler body medicine.
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