CN100548385C - A kind of Brucellosis nucleic acid vaccine - Google Patents
A kind of Brucellosis nucleic acid vaccine Download PDFInfo
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- CN100548385C CN100548385C CNB2007100648175A CN200710064817A CN100548385C CN 100548385 C CN100548385 C CN 100548385C CN B2007100648175 A CNB2007100648175 A CN B2007100648175A CN 200710064817 A CN200710064817 A CN 200710064817A CN 100548385 C CN100548385 C CN 100548385C
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Abstract
The invention discloses a kind of Brucellosis nucleic acid vaccine.This Brucellosis nucleic acid vaccine, its active component is following any mixture: 1) be cloned into BCSP31, sodC and L7/L12 gene in the eukaryotic expression vector respectively, obtain containing the BCSP31 gene recombinant expression carrier, contain the recombinant expression carrier of sodC gene and contain the recombinant expression carrier of L7/L12 gene, described at least two kinds of recombinant expression carriers are mixed the mixture that obtains; 2) three kinds of genes of BCSP31, sodC and L7/L12 are merged with other nucleotide sequence respectively or disappearance partial sequence or part coding mutation after and codified and BCSP31, sodC and L7/L12 have the nucleotide sequence of identical activated protein, be cloned into respectively in the eukaryotic expression vector, at least two kinds of recombinant expression carriers that obtain are mixed the mixture that obtains.Brucellosis nucleic acid vaccine of the present invention can be used for the prevention of people and animals' cloth Lu Shi disease.
Description
Technical field
The present invention relates to a kind of Brucellosis nucleic acid vaccine.
Background technology
Cloth Lu Shi disease is the chronic expendable infectious disease of a kind of infecting both domestic animals and human of global range, due to a kind of facultative born of the same parents' endophyte of brucellar Gram-negative by name, and the mankind, cattle, sheep, Rodents has wide-scale distribution in the mammals such as pig.In the important species of herding, this disease can cause the forfeiture of animal reproductive performance greatly; For the mankind, then the clinical symptoms such as heating of discontinuity can appear, so the ripple heat symptom-complex that is otherwise known as.Vaccine immunity is most important for control cloth Lu Shi disease.The S19 attenuated live vaccine is the earliest and a kind of vaccine of using in cows the most widely.Although this vaccine can produce certain protection power, the serological reaction that it continues causes people can not distinguish immunity and the cattle that infects.This vaccine is morbific to the mankind in addition, and can cause pregnancy period cattle miscarriage.Similarly there are the defective of stability and effectiveness aspect in vaccine such as Rev1 or RB51.
Dna vaccination can be proved on the animal model of a lot of diseases can induce body fluid and cellullar immunologic response; Therefore, dna vaccination is being created a new chance for new brucellar alternative vaccine.At present, people have studied some and have reprinted coding L7/L12, SOD, P39 or the protective effect of the isogenic eukaryotic expression system of pteridine synzyme in model animal.Recently, the unit price dna vaccination of coding sheep brucella 31kDa memebrane protein is proved and can excites special cell killing effect.Yet the neither one protective effect is better than the S19 or the H38 vaccine effect that use at present in the above-described vaccine.
Summary of the invention
The purpose of this invention is to provide a kind of Brucellosis nucleic acid vaccine.
Brucellosis nucleic acid vaccine provided by the present invention, its active component are following any mixture:
1) be cloned into BCSP31, sodC (Copper/Zinc superoxide dismutase) and L7/L12 gene in the eukaryotic expression vector respectively, obtain containing the BCSP31 gene recombinant expression carrier, contain the recombinant expression carrier of sodC gene and contain the recombinant expression carrier of L7/L12 gene, described at least two kinds of recombinant expression carriers are mixed the mixture that obtains;
2) BCSP31, sodC (Copper/Zinc superoxide dismutase) and three kinds of genes of L7/L12 are merged with other nucleotide sequence respectively or disappearance partial sequence or part coding mutation after and codified and BCSP31, sodC (Copper/Zinc superoxide dismutase) and L7/L12 have the nucleotide sequence of identical activated protein, be cloned into respectively in the eukaryotic expression vector, at least two kinds of recombinant expression carriers that obtain are mixed the mixture that obtains.
The active component of described Brucellosis nucleic acid vaccine is preferably is cloned into BCSP31, sodC and L7/L12 gene in the eukaryotic expression vector respectively, obtain containing the BCSP31 gene recombinant expression carrier, contain the recombinant expression carrier of sodC gene and contain the recombinant expression carrier of L7/L12 gene, with described three kinds of mixture that recombinant expression carrier mixes to obtain.
The aminoacid sequence of described BCSP31 such as GenBank Accession Number M20404; Aminoacid sequence such as the GenBank Accession NumberYP_418725 of sodC (Copper/Zinc superoxide dismutase); The aminoacid sequence of L7/L12 such as GenBank Accession Number L19101.
The nucleotide sequence of described BCSP31 gene such as GenBank Accession Number M20404; The nucleotide sequence of sodC gene such as GenBank Accession Number NC_007624; The nucleotide sequence of L7/L12 such as GenBank Accession Number L19101.
The ratio of weight and number of described three kinds of recombinant expression carriers can be 1: 1: 1.
Described carrier for expression of eukaryon can be the expression vector that has the eukaryotic expression promoter, and as cytomegalovirus (CMV) early gene promoter, the promoter of mouse house-keeping gene etc. are preferably mammalian cell expression vector, as pJW4303.
Animal expression carrier pJW4303, its physical map as shown in Figure 7, its sequence is shown in sequence in the sequence table 1.Having cytomegalovirus (CMV) early gene promoter, is the strong promoter that guarantees that genes of interest is efficiently transcribed in muscle cell.This carrier also has the exocytosis signal sequence of tissue plasminogen activator, directed cloning can successfully be secreted into outside the born of the same parents at the exogenous genes products in this signal sequence downstream, increased the touch opportunity of antigen and macrophage, quicken to depend on the degraded of the endogenous synthetic antigen of proteasome, caused the inductive cell-mediated enhancing of replying in the body.In addition; pJW4303 contains 11 CpG sequences; bringing out body at dna vaccination produces in the immunne response and plays an important role; can under the situation that no T cell exists, directly activate the B cell; stimulate immunoglobulin,exocrine and interleukin-6, also directly activated mononuclear cell, macrophage and dendritic cell, the expression of rise costimulatory molecules; help producing multiple Th1 cytokines, improved protective immunity.
Described mammalian cell expression vector comprises plasmid vector and viral vector two big classes.As not with plasmid-type expression vector pTK2, the pHyg of eucaryon replication initiation sequence and pRSVneo etc., the deutero-expression vector pMSG of plasmid expression vector, SV40, the pSVT7 of band eucaryon virus regulating and controlling sequence element and pMT2 etc., bovine papilloma virus (BPV) derivative vector, the deutero-expression vector of herpes virus hominis (EBV), the deutero-expression vector of adenovirus hominis, the deutero-expression vector of retrovirus retrovirus.
The active component of described Brucellosis nucleic acid vaccine is preferably pJBCSP31, pJsodC and pJL7/L12.
Also contain adjuvant IL-12, IL-15, DDA (two octadecyl dimethyl ammonium bromide) in the above-mentioned Brucellosis nucleic acid vaccine, MPL (monophosphoryl lipid plastid A), Quil-A (the purification thing of Saponin), RIBI adjuvant (U.S. RIBI ImmunoChem research limited company produces) and/or other adjuvant (aluminium adjuvant, Freund adjuvant etc.).In this vaccine, the ratio of weight and number of active component and DDA or MPL can be 5: 6, can be 3: 1 with the ratio of weight and number of other adjuvant; The concentration of active component in saline can be 1mg/ml.
Brucellosis nucleic acid vaccine of the present invention can be used for preventing and/or treating animal cloth Lu Shi disease, particularly prevents and/or treats people, cattle, sheep cloth Lu Shi disease.During use, can divide three times or more times immunity, each immunizing dose can be 50-100mg active component/50kg body weight.
Brucellosis nucleic acid vaccine of the present invention induces BCSP31, the sodC of body generation and the specific IgG antibodies reaction level of L7/L12 to be significantly higher than the S19 vaccine.The mice that the Brucellosis nucleic acid vaccine immunity is crossed can produce the effective and secular immunity of brucella and excite significant Th1 cytokine reaction.The specific IFN-gamma cells quantity of the inductive BCSP31 of Brucellosis nucleic acid vaccine, sodC, L7/L12 and Brucella (Brucella whole protein extract) is apparently higher than the S19 vaccine.The inductive T cellular level of Brucellosis nucleic acid vaccine is high by 40% than the S19 vaccine, wherein with CD8
+The T cell is main.Brucellosis nucleic acid vaccine to the protection efficient of mice apparently higher than the S19 vaccine.Show that Brucellosis nucleic acid vaccine of the present invention can replace the prevention that traditional S19 vaccine is used for cloth Lu Shi disease.
Brucellosis nucleic acid vaccine of the present invention has not only strengthened the body humoral response reaction of immune animal, strengthened cell-mediated immunoreation simultaneously, and the latter is the important prerequisite condition that improves dna vaccination efficient, the more important thing is that Brucellosis nucleic acid vaccine of the present invention has greatly strengthened the brucellar cellular level of specific killing.Not only can improve the cell effect of TH1 type if add adjuvant IL-12 or IL-15, more can prolong the protection period of nucleic acid vaccine.
The present invention uses the combined DNA vaccine strategy of coding BCSP31, sodC, L7/L12 to improve the effectiveness of dna vaccination to cloth Lu Shi disease.BCSP31 albumen is the leading albumen of immunity, and sodC and L7/L12 also have been proved and can cause certain immunoreation.
Brucellosis nucleic acid vaccine of the present invention is produced easily, and stability is strong, and is safer, can infected person or cattle.After the difference of brucella vaccine of the present invention and existing Brucellosis nucleic acid vaccine is Brucellosis nucleic acid vaccine immune animal of the present invention, because the expression of carrier high-efficiency has guaranteed that the high-efficiency continuous expression justacrine of antigen is outside born of the same parents.The present invention can use the protection efficient of raising vaccines such as dda adjuvant.Brucella vaccine of the present invention is tested on the larger animal cattle at present, has obtained and has very obviously protected efficient.If, will significantly reduce the tuberculate probability of cattle, guarantee the output and the quality of beef and milk with this brucella vaccine immunity cattle.If with this Brucellosis nucleic acid vaccine immunity people, not only can allow human body produce, and not have infected possibility, strengthen the safety of vaccine to brucellar immunity.In a word, Brucellosis nucleic acid vaccine of the present invention is to improving animal and human to brucellar immunity, more effectively controls the propagation of cloth Lu Shi disease and popular, prosperity animal husbandry and strengthen the human physique and have important significance for theories and using value.
Description of drawings
Figure 1A identifies BCSP31, sodC and L7/L12 expression of gene result in the Brucellosis nucleic acid vaccine immune mouse for RT-PCR
Figure 1B is the expression of results of BCSP31, sodC and L7/L12 in pJBCSP31GFP, pJsodCGFP or the pJL7/L12GFP immune mouse
Fig. 2 A is the specific IgG level in 0,3,6 and 9 weeks behind the initial immunity
Fig. 2 B is the specific IgG level in 9 weeks behind the initial immunity
Fig. 2 C is the specific IgG 1 and the IgG2a level in 9 weeks behind the initial immunity
Fig. 3 is specific cell factor level bar diagram in the spleen cell culture supernatant all around of immunity back
Fig. 4 is the ELISPOT analysis result of IFN-γ
Fig. 5 is the bar diagram of the immune mouse antigenic specificity killer T cell level of vaccine-induced generation
Fig. 6 is the granzyme ELISPOT qualification result of immune mouse
Fig. 7 is the physical map of animal expression carrier pJW4303
The specific embodiment
Method among the following embodiment is conventional method if no special instructions.
The preparation of embodiment 1, brucella vaccine
1, Construction of eukaryotic:
With PCR method increase brucellar BCSP31, sodC and L7/L12 gene and be cloned on the carrier for expression of eukaryon pJW4303 and be used in eukaryotic cell, expressing.The GenBank number of including is respectively: BCSP31:M20404; SodC:NC_007624; L7/L12:L19101.Cattle brucella (Brucellaabortus 2308) (available from the Nat'l Pharmaceutical ﹠ Biological Products Control Institute) genomic DNA that extracts with conventional method is a template, with pcr amplification BCSP31, sodC and L7/L12 gene and directed cloning tissue plasminogen activator (TPA) the signal sequence downstream to the pJW4303 carrier, form fusion rotein.The used primer sequence of BCSP31 that increases is as follows: 5 '-GCTAGCCATATGAAATTCGGAAGCAAAATCCGTC-3 ' and 5 '-CGGGATCCTTATTTCAGCACGCCCGCTTCC-3 '; The used primer sequence of sodC that increases is as follows: 5 '-GCTAGCCATATGAAAAAACGGCTT-3 ' and 5 '-CGGGATCCTTACTTTTTCTTCATATC-3 '; The used primer sequence of L7/L12 that increases is as follows:: 5 '-GCTAGCCATATGATGGCTGATCTCGCAAAGATCG-3 ' and 5 '-CGGGATCCTTACTTGAGTTCAACCTTGGCGCCAGC-3 '.5 ' end primer all contains Nhe I site, and 3 ' end primer all contains BamH I site.The PCR product is pressed the operation of QIAquick GelExtraction Kit description and is reclaimed behind agarose gel electrophoresis, obtain genes of interest.Use Nhe I and BamH I double digestion genes of interest product and pJW4303 carrier DNA respectively, use T through gel-purified
4Dna ligase is handled 16h (16 ℃), gets 5 μ l and connects product (cumulative volume 20 μ l) transformed competence colibacillus e.colistraindh5.Whether picking transformed bacteria list bacterium colony extracting plasmid, it is correct to confirm inserting fragment sequence to carry out sequence analysis after Nhe I and BamH I double digestion are identified.Choose 3 bacterial strains that contain the various objectives expression vector respectively and carry out dna sequence analysis, to obtain the expression vector that has correct reading frame, containing BCSP31 expression carrier called after pJBCSP31, containing sodC expression carrier called after pJsodC, containing L7/L12 expression carrier called after pJL7/L12.PJBCSP31, pJsodC and pJL7/L12 are transformed in the TOP10 escherichia coli, in the LB culture medium that contains 100 μ g/ml ampicillin (Amp), screen recombinant bacterial strain once more, prepare plasmid DNA in a large number with QIAGEN PlasmidMaxi Kit and Mega Kit, being diluted to concentration with sterile saline is 1-2 μ g/ μ l, and ultraviolet spectrophotometer is quantitative.
2, the preparation of brucella vaccine:,, make Brucellosis nucleic acid vaccine with normal saline or phosphate buffer (PBS) dissolving with pJBCSP31, pJsodC and pJL7/L12 equivalent mixing.
The evaluation that embodiment 2, Brucellosis nucleic acid vaccine are expressed in vivo
1, RT-PCR identifies BCSP31, sodC and L7/L12 expression of gene
20 C57BL/6 mices are divided into 2 groups, carry out following processing respectively: first group is non-immune group (contrast), and every intramuscular injection contains the 150 μ l normal saline of 150 μ g pJW4303; Second group is the Brucellosis nucleic acid vaccine group, gets plasmid pJBCSP31, pJsodC and each 50 μ g of pJL7/L12 of preparation among the embodiment 1, is dissolved in jointly in the 150 μ l normal saline, and mixing obtains Brucellosis nucleic acid vaccine.With this Brucellosis nucleic acid vaccine intramuscular injection immunity, immunizing dose is 150 μ l/.Around after immunity is finished, get 10 mouse spleens for every group, use Trizol reagent (U.S. Gibco company) to extract its total RNA.Whether reverse transcription then becomes cDNA, as template, uses the Auele Specific Primer of BCSP31, sodC and three kinds of antigen genes of L7/L12 to carry out pcr amplification with this, express to identify three kinds of genes.Wherein, the used primer sequence of amplification BCSP31 is as follows: 5 '-GCTAGCCATATGAAATTCGGAAGCAAAATCCGTC-3 ' and 5 '-CGGGATCCTTATTTCAGCACGCCCGCTTCC-3 '; The used primer sequence of sodC that increases is as follows: 5 '-GCTAGCCATATGAAGTCCTTATTT-3 ' and 5 '-CGGGATCCTTATTCGATCACGCC-3 '; The used primer sequence of L7/L12 that increases is as follows:: 5 '-GCTAGCCATATGATGGCTGATCTCGCAAAGATCG-3 ' and 5 '-CGGGATCCTTACTTGAGTTCAACCTTGGCGCCAGC-3 '.
The result shows immunity 4 weeks of back, and the mice of 10 immune Brucellosis nucleic acid vaccines that detected is all expressed BCSP31, sodC and L7/L12 gene in spleen; And 10 mices of immune pJW4303 do not have BCSP31, sodC and L7/L12 expression of gene (Figure 1A) in spleen.Among Figure 1A, increase after "+R " expression reverse transcription, " R " expression does not have just amplification of reverse transcription.
2, the evaluation of protein expression in the body
(1) cattle brucella (Brucella abortus 2308) (available from the Nat'l Pharmaceutical ﹠ Biological Products Control Institute) genomic DNA that extracts with conventional method is a template, uses the Auele Specific Primer of BCSP31, sodC and three kinds of antigen genes of L7/L12 to carry out pcr amplification.Wherein, the used primer sequence of amplification BCSP31 is as follows: 5 '-GCTAGCCATATGAAATTCGGAAGCAAAATCCGTC-3 ' and 5 '-CGGGATCCTTATTTCAGCACGCCCGCTTCC-3 '; The used primer sequence of sodC that increases is as follows: 5 '-GCTAGCCATATGAAGTCCTTATTT-3 ' and 5 '-CGGGATCCTTATTCGATCACGCC-3 '; The used primer sequence of L7/L12 that increases is as follows:: 5 '-GCTAGCCATATGATGGCTGATCTCGCAAAGATCG-3 ' and 5 '-CGGGATCCTTACTTGAGTTCAACCTTGGCGCCAGC-3 '.BCSP31, sodC and three kinds of antigen genes of L7/L12 that PCR obtains are cloned into respectively between the restricted enzyme NheI and BamHI site of (available from U.S. Clontech Laboratories company) among the mammal carrier for expression of eukaryon pEGFP-N1, obtain containing GFP protein gene and BCSP31 fusion gene recombinant expression carrier pJBCSP31GFP, contain GFP protein gene and sodC fusion gene recombinant expression carrier pJsodCGFP, contain the recombinant expression carrier pJL7/L12GFP of GFP protein gene and L7/L12 fusion gene.
(2) immune mouse
According to document Cai et al., 2005, Vaccine 23, the method among the 4167-4174,50 μ gpJBCSP31GFP, 50 μ g pJsodCGFP or 50 μ g pJL7/L12GFP respectively with after 150 μ l india inks mix, are injected in C57BL/6 mice mid calf respectively.Simultaneously with 50 μ g pJW4303 with after 150 μ l india inks mix, be injected in C57BL/6 mice mid calf, in contrast.Handle each 10 mice for every kind.After two weeks, injection site muscle is separated, after fixing, the about 10 μ m of slice thick observe down in fluorescence microscope, and the result shows immunity 2 weeks of back, in the mouse muscle tissue of 10 immune pJBCSP31GFP that detected, all expresses BCSP31; In the mouse muscle tissue of 10 immune pJsodCGFP that detected, all express sodC; In the mouse muscle tissue of 10 immune pJ L7/L12GFP that detected, all express L7/L12; In the mouse muscle tissue of 10 immune pJW4303 that detected, all do not have BCSP31, sodC and L7/L12 and express (Figure 1B).Among Figure 1B, DNA represents to inject the mouse muscle tissue of pJBCSP31GFP, pJsodCGFP or pJL7/L12GFP, and Vector represents to inject the mouse muscle tissue of pJW4303.
The immune effect experiment of embodiment 3, Brucellosis nucleic acid vaccine:
60 C57BL/6 mices are divided into 4 groups, carry out following processing respectively: first group is the Brucellosis nucleic acid vaccine group, each 50 μ g of plasmid pJBCSP31, the pJsodC of preparation and pJL7/L12 among the embodiment 1, be dissolved in jointly in the 150 μ l normal saline, mixing obtains Brucellosis nucleic acid vaccine.Mice is carried out three intramuscular injection immunity, and immunizing dose is 150 μ l//times, and time point was the 0th week, the 3rd week, the 6th week.Second group is the S19 vaccine group, 1 * 10
9CFU S19 lyophilized protein (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, numbering 040611, authentication code: new crude drug word (2001) 287710), obtain the S19 vaccine with 150 μ l physiological saline solutions; Mice is carried out three intramuscular injection immunity, and immunizing dose is 150 μ l//times, and time point was the 0th week, the 3rd week, the 6th week.The 3rd group is the empty carrier group, and 50 μ g pJW4303 are dissolved in the 150 μ l normal saline, obtains the empty carrier vaccine; Mice is carried out three intramuscular injection immunity, and immunizing dose is 150 μ l//times, and time point was the 0th week, the 3rd week, the 6th week.The 4th group is the normal saline group, and every in the 0th week injection 150 μ l normal saline.The mice that these four groups immunity are handled carries out following experiment.
1, the evaluation of immunity back humoral immune reaction:
(1) L7/L12, sodC, the antigenic preparation of BCSP31
Cattle brucella (Brucella abortus 2308) (available from the Nat'l Pharmaceutical ﹠ Biological Products Control Institute) genomic DNA that extracts with conventional method is a template, with pcr amplification BCSP31, sodC and L7/L12 gene.Wherein, the used primer sequence of amplification BCSP31 is as follows: 5 '-GCTAGCCATATGAAATTCGGAAGCAAAATCCGTC-3 ' and 5 '-CAGCTCGAGTTTCAGCACGCCCGCTTCCTTGTAAAAGC-3 ' (XhoI site); The used primer sequence of sodC that increases is as follows: 5 '-CAGCTCGAGTTACTTTTTCTTCATATC-3 ' (XhoI site) and 5 '-CGGGATCCTTACTTTTTCTTCATATC-3 '; The used primer sequence of L7/L12 that increases is as follows:: 5 '-GCTAGCCATATGATGGCTGATCTCGCAAAGATCG-3 ' and 5 '-CAGCTCGAGCTTGAGTTCAACCTTGGCGCCAGCAGC-3 ' (XhoI site).
Pcr amplification BCSP31, sodC and L7/L12 gene are cloned into respectively between restricted enzyme NdeI and XhoI site among the pET22b (+) (available from Novagen company), obtain containing the recombinant vector pET-BCSP31 of BCSP31, the recombinant vector pET-sodC that contains sodC contains the recombinant vector pET-L7/L12 of L7/L12.After changing recombinant vector pET-BCSP31, pET-sodC or pET-L7/L12 over to escherichia coli DE3BL21 (plySs), express and induced 4 hours for 37 ℃ with 0.6 μ M IPTG.Obtain L7/L12, sodC, BCSP31 antigen according to pET22b (+) operation instructions purification.
(2) evaluation of immunity back humoral immune reaction:
Special IgG of BCSP31, sodC, L7/L12 and Brucella whole protein and the mensuration of hypotype IgG1, IgG2a thereof use the method for indirect ELISA to measure in the serum of each time of mice immunity back.The serum of immune mouse was used for behind 2 times of serial dilutions since 1: 100 respectively detecting, L7/L12, sodC, BCSP31 antigen and PPD antigen (total protein of cattle brucella (Brucella abortus 2308)) are diluted to 10 μ g/ml with coating buffer (0.05mol/LpH9.6 carbonate buffer solution), add 96 hole ELISA Plate (NUNC) respectively, the every hole of 100 μ l/, 4 ℃ are spent the night, with cleaning mixture (PBS/Tween0.05%) washing 3 times.Every hole adds 200 μ l confining liquids (5% defatted milk powder PBS-Tween 0.05%), puts 37 ℃ of calorstat sealing 60min, by last method washing 3 times.Add test serum, put 37 ℃ of calorstat 2h, cyclic washing 3 times.The rabbit anti-mouse igg that adds the anti antibody-enzyme labelled antibody HRP labelling of dilution in 1: 2000, the every hole of IgG1 or IgG2a (available from Britain Serotec company) adds 100 μ l, puts 37 ℃ of incubation 2h, washs 3 times, after TMB (tetramethyl benzidine two hydrochloric acid) colour developing, survey wavelength 450nm absorbance (A).As negative control, absorbance more than 0.05, experimental group absorbance A value/4th group sample absorbance A value 〉=2.0 are positive with the corresponding dilution factor of normal serum.
The result is shown in Fig. 2 A, Fig. 2 B and Fig. 2 C, and Fig. 2 A shows that the antibody of antigenic specificity is induced in Brucellosis nucleic acid vaccine, and just to exempt from 9 weeks of back, the antibody of anti-BCSP31 is for the highest.Fig. 2 A is the meansigma methods of every group of 5 mices, and Brucella represents PPD antigen.
Fig. 2 B shows, compares with the S19 vaccine group, and all there is the difference that significantly arrives utmost point significant level in three kinds of antigenic specific antibodies.Fig. 2 B is the meansigma methods of every group of 5 mices, and five black circles are represented this 5 mices, and Brucella represents PPD antigen.
Fig. 2 C shows that the Brucellosis nucleic acid vaccine group has been induced high-caliber IgG2a, and the average antibody titre ratio of itself and IgG1 reaches 8 times, makes that immunoreation has tangible TH1 type tendentiousness in the mice body.Fig. 2 C is the meansigma methods of every group of 5 mices, and five black circles are represented this 5 mices, and Brucella represents PPD antigen.
2, the evaluation of the various cytokines in immunity back: around after the last immunity, get 5 mouse spleen cells for every group, stimulate with L7/L12, sodC or the BCSP31 antigen of 5 μ g/ml, the while is with 10
8(heat-inactivated cattle brucella (Brucella abortus 2308) stimulates as positive control the HKBA of CFU/ml.Hatched after the stimulation 3 days.Get cells and supernatant, measure five kinds of cytokines that comprise IFN-γ, IL-4, IL-5, IL-2 and TNF-α with TH1/TH2 cytokines magnetic bead kit (CBA, U.S. company BD).The sensitivity of this detection method is 7 μ g/ml.The mensuration of IL-10 is that antibody (U.S. eBioscience company) uses sandwich ELISA method to carry out with anti-mouse IL-10 then in addition.
The result shows that the Brucellosis nucleic acid vaccine group induced high-caliber IFN-γ shown in A to D among Fig. 3, IL-2 and TNF-α, and these have all reached nanogram level level as the immunoreactive cytokine of TH1 type, are significantly higher than other immune group; The cytokine of TH2 type such as IL-4 and IL-10 then only about 100pg, do not have notable difference with normal saline and empty carrier group.Among Fig. 3, A is specific cell factor level bar diagram in the spleen cell culture supernatant that stimulates of BCSP31, B is specific cell factor level bar diagram in the spleen cell culture supernatant that stimulates of sodC, C is specific cell factor level bar diagram in the spleen cell culture supernatant that stimulates of L7/L12, and D is specific cell factor level bar diagram in the spleen cell culture supernatant that stimulates of HKBA; Saline represents the normal saline group, and Vector DNA represents the empty carrier group, and S19 Vaccine represents the S19 vaccine group, and DNA Vaccine represents the Brucellosis nucleic acid vaccine group.
3, the ELISPOT of IFN-γ analyzes: the part splenocyte shop of separating in the step 2 is distributed on the ELISPOT plate, with specific antigen L7/L12, sodC or BCSP31 (final concentration 5 μ g/ml) or HKBA (10
8CFU/ml) behind the irritation cell, hatched 48 hours in 37 ℃, remove the cell of reaction after, with the link coupled IFN-gamma antibodies of biotin (available from U.S. eBioscience company) in conjunction with and further colour developing.Count in the hole and be the frequency of the secretion interferon cell in the cloth splenocyte of spreading.The result as shown in Figure 4, the Brucellosis nucleic acid vaccine group has induced BCSP31, sodC, L7/L12 and Brucella whole protein (after cattle brucella (the Brucella abortus 2308) ultrasonication, the total protein of being carried) specific IFN-gamma cells, the experiment record its speckle amount generally about 90/2 * 10
5Individual splenocyte.The S19 vaccine group then generally about 40, significantly is lower than the Brucellosis nucleic acid vaccine group.Among Fig. 4, numerical value is 2 * 10
5Speckle number in the individual splenocyte, unit are individual; RBCSP31 represents that result, rSOD that BCSP31 stimulates represent that result, rL7/L12 that sodC stimulates represent that result, HKBA that L7/L12 stimulates represent the result that HKBA stimulates; Medium represents not add the result that antigen only stimulates with culture medium, as negative control.
4, the evaluation of lymphocyte subtype: in 6 time-of-weeks after last immunity, peripheral blood lymphocytes is separated with the Ficoll-Hypaque density, and use then contains BCSP31, the 5 μ g/mlsodC of 5 μ g/ml and the antigenic solution of 5 μ g/ml L7/L12 stimulates these cells.After isolating lymphocyte and rat anti-mouse CD3-RPE, CD19-FITC, CD4-FITC, CD8-RPE (available from U.S. eBioscience company) are hatched jointly from leukocyte, on flow cytometer, measure, thus the situation of change of the various cell subsets of difference after immunity.
The result shows among the A that it is the T of labelling and B cell subsets situation of change in six weeks and the relative scale (little figure) around the time thereof after immunity with CD3 and CD19 as shown in Figure 5.Then show with CD4 and CD8 to be ratio variation in six weeks after immunity of the T4 and the T8 cell subsets of surface molecular labelling among the B.It seems that totally after the immunity, Brucellosis nucleic acid vaccine group group has been induced more intensive T cellular level, and is totally high by 40% than the S19 vaccine group, wherein with CD8
+The T cell is main, and Brucellosis nucleic acid vaccine has been induced the remarkable rising of this type of lethal cell.Fig. 5 is the average result of every group of 5 mices.
5, granzyme ELISPOT identifies: the evaluation of the granzyme ELISPOT R﹠amp of U.S. Millipore company; The mice granzyme ELISPOT detection kit of D company is identified.Earlier with the special monoclonal antibody (R﹠amp of U.S. Millipore company at granzyme B; The mice granzyme ELISPOT detection kit of D company carries) wrap by to pvdf membrane.
The 6th week after the last immunity, each is organized the mice peripheral blood lymphocytes and separates with the Ficoll-Hypaque density, then stimulates these cells with BCSP31, the 5 μ g/ml sodC of 5 μ g/ml or the antigenic solution of 5 μ g/ml L7/L12 respectively.After stimulating back and rat anti-mouse CD8-RPE (available from U.S. eBioscience company) to hatch jointly, with flow cytometer separation of C D8+ cell.
EL4 cell (U.S. sigma-aldrich company, production code member: 85023105) hatch 20h together with isolating respectively organized CD8+ cell and transfection in advance pJBCSP31, pJsodC and pJL7/L12.With the EL4 cell that does not have transfection as blank.Target cell and EL4 can produce granzyme after interacting, so with film on antibody response, with behind the chromogenic reagent promptly visible its on film, stay speckle.
The result as shown in Figure 6, the mice of immune S19 vaccine has comparatively faint speckle to show about 40/2 * 10
4Individual CD8
+Cell, compare immunity Brucellosis nucleic acid vaccine of the present invention mice produced about 100/2 * 10
4Individual CD8
+The speckle of cell has significant difference.This experiment has verified that also the cell killing effect is that cell is removed a brucellar important channel.Among Fig. 6, rBCSP31 represents that result, rSOD that BCSP31 stimulates represent that result, rL7/L12 that sodC stimulates represent that result, the Medium of L7/L12 stimulation represent not have the EL4 cell of transfection, as negative control.Fig. 6 is the average experimental result of every group of 5 mices.
6, counteracting toxic substances experiment
The 6th week after the last immunity is with 5 * 10
6CFU cattle brucella (Brucella abortus 2308)/dosage only is to each immune group mice enforcement vein counteracting toxic substances, and mouse anesthesia is put to death around the infection back, gets spleen, coats after the grinding on the culture medium, and 37 spend cultivation after three days, count of bacteria.
The result is as shown in table 1, shows that Brucellosis nucleic acid vaccine of the present invention obtained protection up to 3.58 units to mice, apparently higher than normal saline and empty carrier group.The S19 vaccine then has only 2.87 units to the protection of mice in the S19 vaccine group, is starkly lower than the Brucellosis nucleic acid vaccine group.That is to say that the bacteria reducing amount of Brucellosis nucleic acid vaccine group is 5 times of S19 vaccine group, and mice is had significant protective effect.
The protective effect of table 1. Brucellosis nucleic acid vaccine after to C57BL/6 mouse infection brucella.
Data are got variance by 10 mices of each group after average and get in the table 1, and the experiment repetition is carried out for twice.
* P＜0.01, * * P＜0.001, the significance of difference is added up according to one-way ANOVA method, compares succeeded by Tukey ' s check.
Sequence table
<160>1
<210>1
<211>5130
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
gacgtcgcgg?ccgctctagg?cctccaaaaa?agcctcctca?ctacttctgg?aatagctcag 60
aggccgaggc?ggcctcggcc?tctgcataaa?taaaaaaaat?tagtcagcca?tgcatggggc 120
ggagaatggg?cggaactggg?cggagttagg?ggcgggatgg?gcggagttag?gggcgggact 180
atggttgctg?actaattgag?atgcatgctt?tgcatacttc?tgcctgctgg?ggagcctggg 240
gactttccac?acctggttgc?tgactaattg?agatgcatgc?tttgcatact?tctgcctgct 300
ggggagcctg?gggactttcc?acaccctaac?tgacacacat?tccacagaat?taattcccgg 360
ggatcgatcc?ggtcgacaat?attggctatt?ggccattgca?tacgttgtat?ctatatcata 420
atatgtacat?ttatattggc?tcatgtccaa?tatgaccgcc?atgttgacat?tgattattga 480
ctagttatta?atagtaatca?attacggggt?cattagttca?tagcccatat?atggagttcc 540
gcgttacata?acttacggta?aatggcccgc?ctcgtgaccg?cccaacgacc?cccgcccatt 600
gacgtcaata?atgacgtatg?ttcccatagt?aacgccaata?gggactttcc?attgacgtca 660
atgggtggag?tatttacggt?aaactgccca?cttggcagta?catcaagtgt?atcatatgcc 720
aagtccggcc?ccctattgac?gtcaatgacg?gtaaatggcc?cgcctggcat?tatgcccagt 780
acatgacctt?acgggacttt?cctacttggc?agtacatcta?cgtattagtc?atcgctatta 840
ccatggtgat?gcggttttgg?cagtacacca?atgggcgtgg?atagcggttt?gactcacggg 900
gatttccaag?tctccacccc?attgacgtca?atgggagttt?gttttggcac?caaaatcaac 960
gggactttcc?aaaatgtcgt?aataaccccg?ccccgttgac?gcaaatgggc?ggtaggcgtg 1020
tacggtggga?ggtctatata?agcagagctc?gtttagtgaa?ccgtcagatc?gcctggagac 1080
gccatccacg?ctgttttgac?ctccatagaa?gacaccggga?ccgatccagc?ctccgcggcc 1140
gggaacggtg?cattggaacg?cggattcccc?gtgccaagag?tgacgtaagt?accgcctata 1200
gactctatag?gcacacccct?ttggctctta?tgcatgctat?actgtttttg?gcttggggcc 1260
tatacacccc?cgctccttat?gctataggtg?atggtatagc?ttagcctata?ggtgtgggtt 1320
attgaccatt?attgaccact?cccctattgg?tgacgatact?ttccattact?aatccataac 1380
atggctcttt?gccacaacta?tctctattgg?ctatatgcca?atactctgtc?cttcagagac 1440
tgacacggac?tctgtatttt?tacaggatgg?ggtcccattt?attatttaca?aattcacata 1500
tacaacaacg?ccgtcccccg?tgcccgcagt?ttttattaaa?catagcgtgg?gatctccacg 1560
cgaatctcgg?gtacgtgttc?cggacatggg?ctcttctccg?gtagcggcgg?agcttccaca 1620
tccgagccct?ggtcccatgc?ctccagcggc?tcatggtcgc?tcggcagctc?cttgctccta 1680
acagtggagg?ccagacttag?gcacagcaca?atgcccacca?ccaccagtgt?gccgcacaag 1740
gccgtggcgg?tagggtatgt?gtctgaaaat?gagctcggag?attgggctcg?caccgtgacg 1800
cagatggaag?acttaaggca?gcggcagaag?aagatgcagg?cagctgagtt?gttgtattct 1860
gataagagtc?agaggtaact?cccgttgcgg?tgctgttaac?ggtggagggc?agtgtagtct 1920
gagcagtact?cgttgctgcc?gcgcgcgcca?ccagacataa?tagctgacag?actaacagac 1980
tgttcctttc?catgggtctt?ttctgcagtc?accgtccaag?cttgcaatca?tggatgcaat 2040
gaagagaggg?ctctgctgtg?tgctgctgct?gtgtggagca?gtcttcgttt?cggctagccc 2100
gggacgcgtg?gatcctcgca?atccctagga?ggattaggca?agggcttgag?ctcacgctct 2160
tgtgagggac?agaaatacaa?tcaggggcag?tatatgaata?ctccatggag?aaacccagat 2220
ctacgtatga?tcagcctcga?ctgtgccttc?tagttgccag?ccatctgttg?tttgcccctc 2280
ccccgtgcct?tccttgaccc?tggaaggtgc?cactcccact?gtcctttcct?aataaaatga 2340
ggaaattgca?tcgcattgtc?tgagtaggtg?tcattctatt?ctggggggtg?gggtggggca 2400
ggacagcaag?ggggaggatt?gggaagacaa?tagcaggcat?gctggggatg?cggtgggctc 2460
tatggaacca?gctggggctc?gacaggaccg?ctatcaggac?atagcgttgg?ctacccgtga 2520
tattgctgaa?gagcttggcg?gcgaatgggc?tgaccgcttc?ctcgtgcttt?acggtatcgc 2580
cgctcccgat?tcgcagcgca?tcgccttcta?tcgccttctt?gacgagttct?tctgagcggg 2640
actctggggt?tcgaaatgac?cgaccaagcg?acgcccaacc?tgccatcacg?agatttcgat 2700
tccaccgccg?ccttctatga?aaggttgggc?ttcggaatcg?ttttccggga?cgccggctgg 2760
atgatcctcc?agcgcgggga?tctcatgctg?gagttcttcg?cccaccccaa?cttgtttatt 2820
gcagcttata?atggttacaa?ataaagcaat?agcatcacaa?atttcacaaa?taaagcattt 2880
ttttcactgc?attctagttg?tggtttgtcc?aaactcatca?atgtatctta?tcatgtctgg 2940
atcgcggccg?cgatcccgtc?gagagcttgg?cgtaatcatg?gtcatagctg?tttcctgtgt 3000
gaaattgtta?tccgctcaca?attccacaca?acatacgagc?cggaagcata?aagtgtaaag 3060
cctggggtgc?ctaatgagtg?agctaactca?cattaattgc?gttgcgctca?ctgcccgctt 3120
tccagtcggg?aaacctgtcg?tgccagctgc?attaatgaat?cggccaacgc?gcggggagag 3180
gcggtttgcg?tattgggcgc?tcttccgctt?cctcgctcac?tgactcgctg?cgctcggtcg 3240
ttcggctgcg?gcgagcggta?tcagctcact?caaaggcggt?aatacggtta?tccacagaat 3300
caggggataa?cgcaggaaag?aacatgtgag?caaaaggcca?gcaaaaggcc?aggaaccgta 3360
aaaaggccgc?gttgctggcg?tttttccata?ggctccgccc?ccctgacgag?catcacaaaa 3420
atcgacgctc?aagtcagagg?tggcgaaacc?cgacaggact?ataaagatac?caggcgtttc 3480
cccctggaag?ctccctcgtg?cgctctcctg?ttccgaccct?gccgcttacc?ggatacctgt 3540
ccgcctttct?cccttcggga?agcgtggcgc?tttctcaatg?ctcacgctgt?aggtatctca 3600
gttcggtgta?ggtcgttcgc?tccaagctgg?gctgtgtgca?cgaacccccc?gttcagcccg 3660
accgctgcgc?cttatccggt?aactatcgtc?ttgagtccaa?cccggtaaga?cacgacttat 3720
cgccactggc?agcagccact?ggtaacagga?ttagcagagc?gaggtatgta?ggcggtgcta 3780
cagagttctt?gaagtggtgg?cctaactacg?gctacactag?aaggacagta?tttggtatct 3840
gcgctctgct?gaagccagtt?accttcggaa?aaagagttgg?tagctcttga?tccggcaaac 3900
aaaccaccgc?tggtagcggt?ggtttttttg?tttgcaagca?gcagattacg?cgcagaaaaa 3960
aaggatctca?agaagatcct?ttgatctttt?ctacggggtc?tgacgctcag?tggaacgaaa 4020
actcacgtta?agggattttg?gtcatgagat?tatcaaaaag?gatcttcacc?tagatccttt 4080
taaattaaaa?atgaagtttt?aatcagcgat?ctagtatata?tgagtaaact?tggtctgaca 4140
gttaccaatg?cttaatcagt?gaggcaccta?tctcagcgat?ctgtctattt?cgttcatcca 4200
tagttgcctg?actccccgtc?gtgtagataa?ctacgatacg?ggagggctta?ccatctggcc 4260
ccagtgctgc?aatgataccg?cgagacccac?gctcaccggc?tccagattta?tcagcaataa 4320
accagccagc?cggaagggcc?gagcgcagaa?gtggtcctgc?aactttatcc?gcctccatcc 4380
agtctattaa?ttgttgccgg?gaagctagag?taagtagttc?gccagttaat?agtttgcgca 4440
acgttgttgc?cattgctaca?ggcatcgtgg?tgtcacgctc?gtcgtttggt?atggcttcat 4500
tcagctccgg?ttcccaacga?tcaaggcgag?ttacatgatc?ccccatgttg?tgcaaaaaag 4560
cggttagctc?cttcggtcct?ccgatcgttg?tcagaagtaa?gttggccgca?gtgttatcac 4620
tcatggttat?ggcagcactg?cataattctc?ttactgtcat?gccatccgta?agatgctttt 4680
ctgtgactgg?tgagtactca?accaagtcat?tctgagaata?gtgtatgcgg?cgaccgagtt 4740
gctcttgccc?ggcgtcaata?cgggataata?ccgcgccaca?tagcagaact?ttaaaagtgc 4800
tcatcattgg?aaaacgttct?tcggggcgaa?aactctcaag?gatcttaccg?ctgttgagat 4860
ccagttcgat?gtaacccact?cgtgcaccca?actgatcttc?agcatctttt?actttcacca 4920
gcgtttctgg?gtgagcaaaa?acaggaaggc?aaaatgccgc?aaaaaaggga?ataagggcga 4980
cacggaaatg?ttgaatactc?atactcttcc?tttttcaata?ttattgaagc?atttatcagg 5040
gttattgtct?catgagcgga?tacatatttg?aatgtattta?gaaaaataaa?caaatagggg 5100
ttccgcgcac?atttccccga?aaagtgccac 5130
Claims (5)
1, a kind of Brucellosis nucleic acid vaccine, its active component are following any mixture:
1) BCSP31, sodC and L7/L12 gene are cloned into respectively among the eukaryotic expression vector pJW4303, obtain containing the BCSP31 gene recombinant expression carrier, contain the recombinant expression carrier of sodC gene and contain the recombinant expression carrier of L7/L12 gene, with described three kinds of mixture that recombinant expression carrier mixes to obtain;
2) with three kinds of genes of BCSP31, sodC and L7/L12 respectively with after other nucleotide sequence merges and codified and BCSP31, sodC and L7/L12 have the nucleotide sequence of identical activated protein, be cloned into respectively among the eukaryotic expression vector pJW4303, with described three kinds of mixture that recombinant expression carrier mixes to obtain;
The aminoacid sequence of described BCSP31 is shown in GenBank Accession Number M20404; The aminoacid sequence of sodC is shown in GenBank Accession Number YP_418725; The aminoacid sequence of L7/L12 is shown in GenBank Accession Number L19101.
2, nucleic acid vaccine according to claim 1 is characterized in that: the nucleotide sequence of described BCSP31 gene is shown in GenBank Accession Number M20404; The nucleotide sequence of sodC gene is shown in GenBankAccession Number NC_007624; The nucleotide sequence of L7/L12 is shown in GenBank Accession NumberL19101.
3, nucleic acid vaccine according to claim 1 and 2 is characterized in that: the ratio of weight and number of described three kinds of recombinant expression carriers is 1: 1: 1.
4, nucleic acid vaccine according to claim 1 and 2 is characterized in that: also contain adjuvant IL-12, IL-15, DDA, MPL, Quil-A, RIBI adjuvant, aluminium adjuvant and/or Freund adjuvant in the described Brucellosis nucleic acid vaccine.
5, nucleic acid vaccine according to claim 4, it is characterized in that: in the described Brucellosis nucleic acid vaccine, the ratio of weight and number of described active component and DDA or MPL is 5: 6, and the ratio of weight and number of described active component and IL-2, IFN-γ, IL-12 or IL-15 is 3: 1.
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| CN101270160B (en) * | 2008-05-05 | 2012-05-02 | 中国人民解放军第四军医大学 | Monoclone antibody 1F1, uses thereof and hybridoma cell line BCSP31-1F1 excreting the antibody |
| CN102198269A (en) * | 2011-04-13 | 2011-09-28 | 中国人民解放军疾病预防控制所 | Application of Brucella GroEL protein and method for recombinant expression of Brucella GroEL protein |
| CN102584957B (en) * | 2012-02-27 | 2014-02-19 | 江苏出入境检验检疫局动植物与食品检测中心 | Specific antibody of Brucella specificity multi-epitope artificial polypeptide, immunomagnetic beads coated with specific bodies, and application of beads |
| CN102703505A (en) * | 2012-05-18 | 2012-10-03 | 中国农业科学院兰州兽医研究所 | Co-expressed brucella recombinant adenovirus as well as preparation method and use thererof |
| CN106854654B (en) * | 2017-02-16 | 2020-06-23 | 内蒙古医科大学 | rBCG for expressing sheep brookfield strain L7/L12 gene and construction method and application thereof |
| CN111796091B (en) * | 2020-07-20 | 2023-09-08 | 天康生物制药有限公司 | Kit for distinguishing brucella or brucella ghost vaccine infected by animals |
| CN113637703B (en) * | 2021-08-06 | 2023-08-25 | 河北科技师范学院 | Construction method and application of Brucella L7/L12 and GroES eukaryotic expression vector |
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| Whole-Genome Analyses of Speciation Events in PathogenicBrucellae. Patrick S. G. Chain et al.INFECTION AND IMMUNITY,Vol.73 No.12. 2005 |
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