CN106636138A

CN106636138A - DNA (Deoxyribonucleic Acid) vaccine aiming at sheep breeding type 3 brucella infection and preparation method thereof

Info

Publication number: CN106636138A
Application number: CN201710022380.2A
Authority: CN
Inventors: 李增魁
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-01-12
Filing date: 2017-01-12
Publication date: 2017-05-10

Abstract

The invention provides a separated DNA (Deoxyribonucleic Acid) segment, a DNA vaccine aiming at sheep breeding type 3 brucella infection, a vaccine preparation, a method for preparing the DNA vaccine and a method for preparing the vaccine preparation. The separated DNA segment provided by the invention is an Omp31 gene obtained by cloning from sheep breeding type 3 brucella; the DNA vaccine and the vaccine preparation, which are prepared according to the gene, have pertinence on the sheep breeding type 3 brucella which is widely popular in China; the vaccine and the vaccine preparation have the advantages of safety and no toxin, high immune efficiency, long lasting time, convenience for production and transportation and the like.

Description

A kind of DNA vaccination for the type Infected with Brucella of sheep kind 3 and preparation method thereof

Technical field

The present invention relates to DNA vaccination field, in particular to a kind of detached DNA fragmentation, for the type cloth Shandong of sheep kind 3 The preparation method of DNA vaccination, bacterin preparation, the preparation method of DNA vaccination and bacterin preparation that Salmonella infects.

Background technology

Brucellosis (Brucellosis), also known as brucellosiss, abbreviation brucellosis.Brucellosis are by Brucella Antibacterial invades the infectious disease for causing infection-allergy infecting both domestic animals and human after body.The mankind, domestic animal and animal are generally susceptible, people Infection is mainly shown as the symptoms such as heating, shiver with cold, night sweat, general malaise, and animal infected clinical symptoms are mainly miscarriage, no Pregnant, genitals are encroached on.Brucellosis are classified as B class animal epidemics by OIE (OIE), and China is also classified as Two class animal epidemics.In recent years, there is rebound momentum in China and other countries of the world and area in people and animals' epidemic situation of brucellosis.China The as shown by data of prevention and control of diseases information system, after 2004, China's brucellosis sickness rate constantly rose, to China in 2009 Human world brucellosis report 35816 people of morbidity.Medical expense that China produces every year because of brucellosis, delay work loss and to animal husbandry Direct, indirect economic loss is caused to have become an astronomical figure.Therefore, brucellosis how to be prevented and treated and has become the task of top priority.

The cause of disease of brucellosis is brucella (Brucella), belongs to gram negative bacteria, and plasmid-free, spore and pod membrane are simultaneous Property is parasitized in mononuclear phagocyte.The bacterium produces virose endotoxin, and endogenous toxin have pyrogenicity, lethal effect and skin mistake Quick originality.The main Jing respiratory systems of pathogenic bacteria, digestive system, reproductive system etc. enter body, and with lymph fluid lymph node is reached, and are swallowed Cell swallows.As phagocyte fails killing bacteria, then antibacterial forms local primary lesion, therewith in intracellular growth and breeding A large amount of antibacterials enter lymph fluid and blood circulation forms bacteremia, and with blood flow band to whole body, in liver, spleen, lymph node, bone marrow etc. Breeding in the monokaryon-mononuclear phagocyte system at place, forms multiple focus.

According to the different structure of host's difference, biochemical reaction feature and phage surface, brucella can be divided into 6 differences Kind, including brucella melitensis (B.melitensis), B. abortus (B.abortus), pig kind brucella (B.suis), Br.ovis (B.ovis), sarin Mus kind brucella (B.neotomae) and Br. cants (B.canis)；Wherein, brucella melitensis include 1 type, 2 types and 3 types again.In recent years, the popular brucella of China is mainly The type brucella of sheep kind 3, the brucella has very strong virulence and aggressivity to people, animal, and pathogenicity is also very strong, human poultry infection Afterwards symptom is more serious, easily causes outburst and the prevalence of brucellosis.Therefore, preventing and treating sheep kind 3 of adopting an effective measure is drawn by type brucella The brucellosis for rising are most important to animal husbandry and the development of publilc health cause.

At present, it is to slaughter and immunity combination that the sick main method is eliminated in worldwide, and is occurred in brucellosis relative Serious China, due to the high cost slaughtered, vaccine prevention becomes primary disease major control means.Existing brucella disease vaccine Attenuated live vaccine and inactivated vaccine are concentrated mainly on, wherein, mainly there are attenuated live vaccine S19, Rev.1 in wide variety of vaccine Vaccine, brucella melitensis M5 attenuated live vaccines.But above-mentioned vaccine belongs to traditional attenuated live vaccine, certain poison is still kept , easily there is virus virulence reply in power, i.e., " atavism ", there is a problem of safety.In addition, live vaccine must be in cryogenic conditions Lower preservation and transport, the requirement to storage and transport is higher.

There is the defects such as residual virulence to overcome conventional vaccine, scientific and technical personnel are also carried out to brucellosis DNA vaccination Research.DNA vaccination refers to the recombinant eukaryon expression vector by certain proteantigen is encoded also known as gene vaccine or nucleic acid vaccine It is injected directly in animal body, exogenous gene is in vivo expressed, the immune system of the antigenic activation body of generation, so as to lure Lead the humoral immunization and cellullar immunologic response of specificity.DNA vaccination has inoculation carrier (such as plasmid) simple structure, production cost It is low, be suitable to produce in enormous quantities, be readily transported and preserve, it is more safer than traditional vaccine the advantages of.Successfully make in prior art It is standby go out the DNA vaccination such as L7/L12, P39, but there is many defects in above-mentioned DNA vaccination, and for example, what pCDNA3-L7/L12 stimulated exempts from Epidemic disease reaction can only keep shorter time (4 weeks), P39 eucaryon vaccines there was only relatively low Vaccine effectiveness, and the immunity after counteracting toxic substances 8 weeks should Just substantially (log0.73), but compared with traditional S19 attenuated vaccines, also there is certain gap in the effect answered.Sum it up, existing A kind of safe and stable, antibody titer is high, immune efficacy time length brucellosis DNA vaccination to have technology to lack, and particularly lacks A kind of few DNA vaccination for the type Infected with Brucella of sheep kind 3.

In view of this, it is special to propose the present invention.

The content of the invention

The first object of the present invention is to provide a kind of detached DNA fragmentation, and the DNA fragmentation is to be isolated from the type cloth of sheep kind 3 The Omp31 genes of Shandong Salmonella.

The second object of the present invention is to provide a kind of DNA vaccination for the type Infected with Brucella of sheep kind 3, described DNA vaccination is directed to the brucellar infection of the type of sheep kind 3, with safety non-toxic, immune efficacy strong, persistent period length, is readily transported The advantages of with preserving.

The third object of the present invention is to provide a kind of bacterin preparation.

The fourth object of the present invention is to provide a kind of preparation side of the DNA vaccination for the type Infected with Brucella of sheep kind 3 Method, the preparation method is simple, is easy to large-scale production.

The fifth object of the present invention is to provide a kind of preparation method of bacterin preparation.

In order to realize the above-mentioned purpose of the present invention, spy employs the following technical solutions：

A kind of detached DNA fragmentation, the nucleotide sequence such as SEQ ID NO of the DNA fragmentation：Shown in 3.

DNA fragmentation of the present invention is to be isolated from the brucellar Omp31 genes of the type of sheep kind 3, the Omp31 gene codes Omp31 albumen with immunization.Therefore, DNA fragmentation of the present invention can be used for preparing for the type brucella of sheep kind 3 Vaccine.

A kind of DNA vaccination for the type Infected with Brucella of sheep kind 3, the DNA vaccination is comprising the true of above-mentioned DNA fragmentation Nuclear expression carrier.

The antigen gene of DNA vaccination of the present invention is cloned from the brucellar genes of Omp 31 of the type of sheep kind 3.Therefore, originally The DNA vaccination is invented to there is specific aim in the pandemic type brucella of sheep kind 3 of China, is conducive to preventing and treating by sheep kind 3 The brucellosis that type brucella causes.On the other hand, DNA vaccination of the present invention with tradition attenuation M5 vaccines compared with, with higher Lymphopoiesis index and higher cell kill power.And with regard to antibody expression and for the persistent period, institute of the present invention State DNA vaccination substantially suitable with the antibody expression of attenuation M5 vaccines and antibody persistent period.Therefore, DNA of the present invention Vaccine has immune effect more more preferable than traditional attenuated vaccine, with the antibody titer substantially suitable with traditional attenuated vaccine and holds The continuous time, the immunity for overcoming brucella DNA vaccination in prior art is weak, potency is low, the defect that the antibody persistent period is short.

DNA vaccination as described above, it is preferable that carrier for expression of eukaryon is selected from pVAX1, pCI, pcDNA3.1 and pJW4303.

A kind of bacterin preparation, the preparation includes above-mentioned DNA vaccination and adjuvant.

Bacterin preparation as above, it is preferable that the adjuvant is exempted from selected from cytokine gene adjuvant and/or function vector One or more in epidemic disease adjuvant.

Bacterin preparation as above, it is preferable that the cytokine gene adjuvant selected from IL-2, IL-12, IFN-γ, One or more in GM-CSF, the function vector immunological adjuvant is selected from liposome, immunostimulating complex (Immune Stimulating complexes, ISCOMs) and attenuation aggressive intracellular bacteria in one or more.

Bacterin preparation as above, it is preferable that the mass ratio of the DNA vaccination and the adjuvant is 1：1-5.

Bacterin preparation of the present invention uses cooperatively DNA vaccination with adjuvant, it is found that adjuvant can significantly increase DNA vaccination Immunogenicity so as to improving the immune effect of DNA vaccination.Especially by the carrier for expression of eukaryon of DNA vaccination and cytokine base Because of adjuvant, preferred IL12, IL2 gene adjuvant is used cooperatively, and can be greatly enhanced the effect of immune formulation of the present invention. The present invention is optimized to the amount ratio of DNA vaccination and adjuvant, it is found that carrier for expression of eukaryon is 1 with the mass ratio of adjuvant：1-5 When, the immune effect of DNA vaccination of the present invention is especially good.Carrier for expression of eukaryon can also be 1 with the mass ratio of adjuvant:2-4 Or 1:3 etc..

A kind of method for preparing above-mentioned DNA vaccination, methods described includes：Build and include DNA fragmentation described in claim 1 Carrier for expression of eukaryon.

Preparation method of the present invention relates generally to the structure of carrier for expression of eukaryon, it is not necessary to directly contact pathogenic bacterium, behaviour Make simple and safe, it is easy to large-scale production.

Method as above, it is preferable that methods described specifically includes following steps：(1) obtained by PCR method amplification Above-mentioned DNA fragmentation；(2) with EcoR I and BamH II difference enzyme action PCR amplifications gained DNA fragmentation and pVAX1 carriers；(3) ligase DNA fragmentation and pVAX1 carriers after cutting, transformed competence colibacillus cell；(4) picking positive colony, by way of double digestion and sequencing Positive colony is confirmed；(5) plasmid of positive colony is extracted.

As mentioned above the method for bacterin preparation, the DNA vaccination is mixed with the adjuvant.

The preparation method of bacterin preparation of the present invention mixes DNA vaccination with adjuvant, can significantly increase vaccine system The immune efficacy of agent.

A kind of method of the type Infected with Brucella of prevention sheep kind 3, including：By DNA vaccination as above or the vaccine Preparation is imported in sheep body, allows its antigen expressed albumen in host cell, induction body to produce immunne response.

Preferably, the sheep includes argali (Argali), urial (Urial), sheep, family sheep, mouflon (Mouflon), bighorn (Bighorn sheep), white bighorn (Dall sheep), snow mountain argali (Snow sheep) or red Argali.

Compared with prior art, beneficial effects of the present invention are：

1), DNA fragmentation of the present invention is clone from the brucellar Omp31 genes of the type of sheep kind 3, and the gene can be used for Specificity is prepared for the brucellar vaccine of the type of sheep kind 3.

2) DNA vaccination of the present invention is directed to the type brucella of sheep kind 3, can effectively prevent and treat and extensively be flowed in China Row ground causes brucellosis by the type brucella of sheep kind 3, the brucellar vaccine of the type of sheep kind 3 is not also directed in prior art, especially It is DNA vaccination.Compared with traditional attenuated vaccine, DNA vaccination of the present invention has substantially quite, or even slightly strong immunity effect Really, but there is no the danger of virulence reply, its safety is higher, and is easy to transport and preserves.And with existing for brucella DNA vaccination compare, DNA vaccination of the present invention has higher immune effect and longer antibody persistent period.

3) DNA vaccination preparation of the present invention uses cooperatively DNA vaccination with adjuvant, and the species to adjuvant, DNA vaccination It is optimized with the consumption proportion of adjuvant, the bacterin preparation for being obtained has more preferable immune effect.

4) preparation method of DNA vaccination of the present invention and the preparation method of bacterin preparation relate generally to vector construction, matter Grain extracts equimolecular biologic operation, it is not necessary to contact with poisonous pathogenic bacterium, safer, and the method for the invention is operated Simply, it is easy to large-scale production.

Description of the drawings

In order to be illustrated more clearly that the specific embodiment of the invention or technical scheme of the prior art, below will be to concrete The accompanying drawing to be used needed for embodiment or description of the prior art is briefly described, it should be apparent that, in describing below Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid Put, can be with according to these other accompanying drawings of accompanying drawings acquisition.

Fig. 1 is the Gram’s staining result by inspection bacterium；

Fig. 2 is Ke Ziluo Paderewskis dyeing (Ke's Albert'stain Albert) result by inspection bacterium；

Fig. 3 is the AMOS-PCR qualification results by inspection bacterium, and wherein M represents Marker, and 1 represents pig kind vaccine strain S2, and 2 represent Sheep kind vaccine strain M5,3 represent by inspection bacterium, and 4 represent the uncommon bacterium of escherichia coli angstrom；

Fig. 4 is the electrophoretogram of Omp31 gene PCR amplified productions in embodiment 2, and wherein M represents Marker, and 1～3 represents Pcr amplification product；

Fig. 5 is the electrophoretogram of double digestion confirmatory experiment in embodiment 2, and wherein M represents Marker, and 1 represents positive colony；

Fig. 6 is the coomassie brilliant blue staining result of SDS-PAGE in embodiment 2, and wherein M represents Marker, and 1 represents control, 2nd, 3 positive colony is represented；

Fig. 7 is WB testing results in embodiment 2, and wherein M represents Marker, and 1 represents positive colony；

Fig. 8 is the electrophoretogram that double digestion identifies eukaryon recombinant plasmid in embodiment 3, and wherein M represents Marker, and 1 represents quilt The eukaryon recombinant plasmid of enzyme action；

Fig. 9 is the immunocytochemistry qualification result of Omp31 in COS-7 cells；

Figure 10 is Omp31 secondary structure prediction results, wherein most long vertical line represents alpha-helix, vice-minister's vertical line represents extension Chain；Vice-minister's vertical line represents β-corner, and most short vertical line represents random coil；

Figure 11 is Omp31 Tertiary structure predictions results；

Figure 12 is Omp31 molecular surface probability analysiss results；

Figure 13 is Omp31 molecular hydrophylic analysis results；

Figure 14 is Omp31 molecular flexibility analysis results；

Figure 15 is Omp31 molecular antigen index analysis results；

Figure 16 for immune mouse lymphopoiesis index analysis result, wherein * VS.saline group, p< 0.05；#Vs.M5group,p<0.05；

Figure 17 is immune mouse cell in vitro killing experiments result；

Figure 18 is immune serum antibody horizontal, and wherein M represents M5 vaccines, and S represents Omp31DNA vaccines, and P represents phosphorus Phthalate buffer.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted concrete in embodiment Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are The conventional products that commercially available purchase is obtained can be passed through.

Embodiment 1

The brucellar identification of the type of sheep kind 3

The type brucella of sheep kind 3 is isolated from Qinghai Province Haiyan County miscarriage lamb, and it is identified.

1st, strain culturing and dyeing microscopic examination

Miscarriage lamb is placed in Biohazard Safety Equipment carries out sample of tissue, and grinding takes tissue sample, takes grinding suspension TSA culture medium is inoculated in, is put into after incubator growth 24h and is taken out, single bacterium colony is inoculated in into TSB culture medium culturings.Observation bacterium The form that falls simultaneously carries out as follows Gram’s staining and Ke Ziluo Paderewskis dyeing (Ke's Albert'stain Albert).

Gram staining method：Hooked with inoculating loop and take a small amount of bacterium solution uniform application in dry microscope slide central authorities, flame is fixed, Contaminate 1min at the beginning of Deca ammonium oxalate crystal violet, dye liquor washed away with distilled water, plus iodine solution cover painting face dye about 1min, with water rinse, Absorbent paper sucks moisture, plus 95% ethanol few drops, and is shaken gently for being decolourized, and washes after 20s, sucks moisture, Huang red dyeing After (dilute) the dye 30s of liquid, distilled water flushing is dried, microscopy.

Ke Ziluo Paderewskis dye (Ke's Albert'stain Albert)：Hooked with inoculating loop and take a small amount of bacterium solution uniform application in dry microscope slide Central authorities, flame is fixed, Deca husky of common dye solution, alcohol burner heating 2min or so, till there is bubble, Deca peafowl after washing Green to be redyed, distilled water flushing is dried, microscopy.

By inspection bacterium Jing after 37 DEG C of constant temperature culture 2d～4d, the visible colourless, translucent colony on liver infusion solid medium, Neat in edge, refractive power is more apparent, drop-wise of revealing.Thalline is less after Gram's staining, circular or club shape, takes on a red color, without spore, nothing Flagellum, without pod membrane；Kozlovsky differential staining is visible to have rhodobacterium to be distributed.Gram’s staining and Kozlovsky contaminate The coloration result of color is as shown in Fig. 1～Fig. 2.

2nd, biochemical identification, specificity serum A and M agglutination test and Phage display peptide library

By biochemical identification, specificity serum A and M agglutination test and Phage display peptide library to being entered to advance by inspection bacterium One step is identified.Qualification result is as shown in table 1.

The biochemical identification of table 1, specificity serum A and M agglutination test are Phage display peptide library

3rd, AMOS-PCR qualification results

It is as shown in Figure 3 to being entered kind of a type identification of advancing, the testing result of AMOS-PCR by inspection bacterium using AMOS-PCR methods.Root Understand according to Fig. 3, the specific fragment with sheep kind immunity strain M5 formed objects is obtained by inspection bacterium Jing PCR amplifications.

Tried according to bacterium colony observation, dyeing, biochemical identification, specificity serum A and M agglutination test and phage splitting Test and the qualification result of AMOS-PCR tests can determine that the bacterial strain isolated from Hai Sheng Haiyan Counties miscarriage lamb is sheep kind 3 type brucella.

Embodiment 2

The clone of Omp31 genes, in expression in escherichia coli, purification and identification

1st, the clone of Omp31 genes

Omp31 genes are cloned as follows：1) separate from embodiment 1 and extracted in the type brucella of sheep kind 3 for obtaining Genomic DNA；2) with reference to Genebank coding sequences, the type brucella outer membrane protein Omp31 genes of sheep kind 3 are designed Primer, it is contemplated that expand purpose fragment length for 685bp, synthetic primer, upstream primer sequence such as SEQ ID NO：Shown in 1, downstream Primer sequence such as SEQ ID NO：Shown in 2；3) using step 1) extract genomic DNA and step 2) design primer carry out PCR is expanded, and obtains purpose fragment；4) agarose gel electrophoresiies separate amplification gained PCR primer；5) glue is cut, target DNA bar is reclaimed Band；6) PCR primer for reclaiming is connected with pMD19-T carriers, connection product is transformed into competent cell, and transformed bacteria is uniformly applied It is distributed on the LB ampicillin solid mediums containing IPTG and X-gal overnight, next day chooses white colony, is enlarged culture； 7) plasmid is extracted from the bacterium solution of amplification culture, with BamH I and Xho I double digestion identification is carried out, choose positive colony, served Extra large Hua Da Gene Tech. Company Limited is sequenced, and positive colony is named as pMD19-T-Omp31.Wherein, PCR amplifications and Enzyme action result is as shown in Figures 4 and 5.Jing sequencings confirm that the gene that PCR reaction amplifications are obtained is Omp31, its gene order such as SEQ ID No：Shown in 3, the aminoacid sequence such as SEQ ID NO of the DNA encoding the protein：Shown in 4.

2nd, expression of the Omp31 genes in escherichia coli, purification and identification

Double digestion Omp31 genes, enter afterwards row agarose gel electrophoresis, reclaim purpose fragment, reconnect purpose fragment with Also pass through the prokaryotic expression carrier of double digestion.Connection product is transformed in competent cell, subsequently coated containing On the solid medium of antibiotic, picking single bacterium colony extracts plasmid, to Omp31 by way of PCR and double digestion and sequencing Prokaryotic expression carrier is identified.The identified positive monoclonal bacterium colony containing Omp31 prokaryotic expression carriers is enlarged into training Support, add derivant to induce the expression of Omp31 albumen.After culture terminates, culture fluid is collected, thalline is collected by centrifugation, extracted and pure Change albumen, carry out SDS-PAGE electrophoresis.After electrophoresis terminates, dyeed with Coomassie brilliant blue, observation Omp31 genes are in large intestine bar Expression in bacterium.Coloration result is as shown in Figure 6.Using electroporation by SDS-PAGE glue surfaces protein delivery to NC films, carry out Western-blot is detected.Testing result is as shown in Figure 7.It can be seen from Fig. 6～7, present invention successful expression in escherichia coli Go out Omp31 albumen.

Embodiment 3

The clone of Omp31 genes, in eukaryotic cell expression and identification

1st, the structure of eukaryon recombinant plasmid and enzyme action are identified

With EcoR I and BamH II difference enzyme action Omp31 genes and pVAX1 carriers, connect the Omp31 genes after enzyme action with PVAX1 genes, build Omp31-pVAX1 carrier for expression of eukaryon.By the carrier for expression of eukaryon conversion escherichia coli, with the Hes of EcoR I BamH II carries out double digestion identification, screens positive recombinant plasmid, and is sequenced.Concrete enzyme action testing result is as shown in Figure 8.According to figure 8 understand, the present invention successfully constructs Omp31-pVAX1 carrier for expression of eukaryon.

2nd, COS-7 cells and immunocytochemistry identification are transfected

Carried out according to Invitrogen companies liposome description, respectively transfect in Omp31-pVAX1, pVAX1 carrier COS-7 cells, are subsequently placed in 37 DEG C, 5% CO₂Cultivate in incubator.After transfection 24h, COS-7 cells are collected, by immunity Expression of the cytochemical method identification Omp31 genes in eukaryotic cell COS-7.Concrete testing result is as shown in Figure 9. It can be seen from Fig. 9, Omp31 genes can the high expression in the cell of eucaryon mammal.

Embodiment 4

Omp31 protein molecular Epitope prediction results

1st, the physicochemical property of protein molecular

Using the type brucella adventitia of ProtParam analysis sheep kind 3 of biological software DNAstar and ExPASy servers The physicochemical property of albumen Omp31 molecules, as a result as shown in table 2.

The physicochemical property result of the outer membrane protein molecule of table 2

2nd, two grades, tertiary structure of protein molecular

By the secondary structure of the SOPMA and DNAstar software prediction Omp31 molecules of NPS@IBCP servers, prediction knot Fruit is as shown in Figure 10.Visible according to Figure 10, Omp31 molecules ɑ-spiral accounts for 14.54%, β-corner and accounts for 13.22%, and random coil is accounted for 36.12%, extended chain accounts for 36.12%.

The tertiary structure of Omp31 protein moleculars is set up by homologous Modeling Server SWISS-MODEL.Omp31 three-levels are tied Structure predicts the outcome as shown in figure 11.

3rd, the probability on protein molecular surface

Using the probability on the Emini methods analysis Omp31 protein moleculars surface of DNAstar, predict the outcome as shown in figure 12. As a result show：46aa～the 53aa of the amino acid residue of Omp31 molecules, 107aa～112aa, 126aa～129aa, 160aa～ 165aa, 181aa～197aa sections, the probability that they are distributed in molecular surface is larger, therefore becomes the epitope of molecule Probability it is also larger.

4th, the hydrophilic of protein molecular

The hydrophilic of Omp31 protein moleculars is analyzed using the Hydropathy-Kyte-Doolittle methods of DNAstar, point Analysis result is as shown in figure 13.It is visible according to Figure 13：The hydrophilic area distribution of above-mentioned protein molecular is far longer than hydrophobic region, so as to Be conducive to the expression of albumen.34aa～57aa in the primary structure of Omp31 protein moleculars, 71aa～79aa, 86aa～93aa, 104aa～129aa, 149aa～167aa, 176aa～228aa section is high hydrophilic area；These regions are exposed to molecular surface Probability is larger, and the probability for becoming the epitope of molecule is also maximum.

5th, the pliability in protein molecular polypeptide chain backbone area

Using the pliability in the Karplus-Schulz methods prediction Omp31 protein molecule polypeptide chain backbones area of DNAstar, Analysis result is as shown in figure 14.Result shows shown in Figure 14：8aa～the 26aa of Omp31 molecules, 36aa～63aa, 79aa～ 112aa, 127aa～166aa, 183aa～211aa section, the probability height that they are distorted, fold in space, can form Abundant secondary structure.So the probability that these peptide fragments of protein molecular form molecular antigen epi-position is larger.

6th, protein molecular antigenic index analysis

Omp31 protein molecular antigenic indexs are carried out using the Antigenic Index-Jameson-Wolf methods of DNAStar Analysis, analysis result is as shown in figure 15.Result shows shown in Figure 15：Antigenic index shows in their primary structure aminoacid sequence The section of work is 34aa～57aa, 71aa～79aa, 86aa～93aa, 104aa～129aa, the 149aa～167aa of Omp31, 176aa～228aa sections.The probability that above-mentioned section forms the epitope of molecule is very big, is also the corresponding albumen point of research The main object of the antigenic determinant of son.

7th, protein molecular B cell antigen epi-position analysis

The B cell antigen table of Omp31 protein moleculars is predicted using the BepiPred in CBS Prediction Servers Position, the output after arrangement by marginal value more than 0.5, it is seen that Omp31 albumen contains 9 potential epitopes, predict highest Region be located at 7aa～23aa sections.Analysis result is as shown in table 3.

The Omp31 protein molecular B cell antigen epi-position analysis results of table 3

This test expands outer membrane protein Omp31 molecular gene sequences, after sequencing, exists with some to it with DNAMAN softwares The homology of the gram negative bacteria of cross reaction is analyzed, and finds the outer membrane protein of the type of sheep kind brucella 3 Omp31 and small intestinal yersinia intestinal etc. may occur the antibacterial sequence of cross reaction without homology with brucella, so as to keep away Exempt from the cross reaction with other gram negative bacteria.

Most of antibody is only combined with the part of protein surface, and most of antigenic determinant is hydrophilic, is separately permitted Many known antigenic determinants be in moving havens domain, therefore it is logical can to brucella outer membrane protein Omp31 molecular surfaces Energy property, hydrophilic, pliability, antigenic index and B cell antigen epi-position carry out comprehensive analysis：The most strong position of hydrophilic is considered as It is the position combined with monoclonal antibody, is conducive to and antibodies.According to diagram Omp31 molecular predictions, its epitope is likely located at 6 Individual most hydrophilic position.The probability that these regions are exposed to molecular surface is larger, becomes the probability of the epitope of molecule Maximum, so that this is combined with the antigen receptor of corresponding lymphocyte, activated lymphocyte causes immunne response to antigen.

Antibody generally specifically recognizes the specific region on antigen protein surface, and these regions are referred to as B cell epi-position. From from space structure, the space conformation epi-position of the discontinuity of B cell epi-position is close on space structure by those, But the table of sequentially discontinuous aminoacid composition, the spatial dependence with height, Secondary structure and protein Bit distribution has larger relation.The study find that Omp31 protein epitopes are rich in circulus, and lack spiral and foldable structure. Because circulus is more flexible than the secondary structure of other forms, beneficial to the combination of antibody.

Embodiment 5

The immunological characteristic research of brucella Omp31DNA vaccines

The DNA vaccination of the acquisition of embodiment 3, i.e. Omp31-pVAX1 carrier for expression of eukaryon (Omp31 groups), tradition is respectively adopted M5 attenuated vaccines (M5 groups) and normal saline (physiological saline group) immune mouse simultaneously pass through lymphocyte proliferation assay (MTS), CTL Cell killing tests the immunological characteristic with ELISA method research Omp31 groups, M5 groups and physiological saline group.

1st, lymphocyte proliferation assay (MTS)

Using conventional lymphocyte proliferation assay (MTS) the detection Omp31 groups in this area, M5 groups and physiological saline group immunity The lymphopoiesis index of mice, testing result is as shown in figure 16.As can be seen from Figure 16, Omp31 groups are put down Jing after stimulating Stimulation index is 1.61, hence it is evident that higher than M5 groups (1.46, P ＜ 0.05) and physiological saline group (0.92, P ＜ 0.05).

2nd, CTL cell killings experiment

Using the CytoTox of Promega companiesNonRadioactive Cytotoxicity Assay carry out CTL Cell killing experiment (concrete grammar refers to its description), testing result is represented with killing activity, and computing formula is as follows：

The testing result that CTL cells kill experiment is as shown in figure 17.As can be seen from Figure 17, Omp31 groups are in different effects Target is than can induce the specific killing action that T cell is mediated, and CTL effects strengthen with the rising of effect target ratio；Omp31 Under identical effect target ratio, its CTL effect value is higher than M5 groups to group.Experimental result illustrates that DNA vaccination antigen of the present invention is not only able to lure Specific lymphoproliferation effect is led while the non-specific Proliferation of lymphocytes of induction can also be strengthened, can be obvious Ground specifically stimulates lymphopoiesis, and DNA vaccination of the present invention has the CTL more higher than traditional M5 attenuated vaccines Killing functions of immunocytes.

3rd, ELISA method detection immune mouse serum antibody titer

The polyclonal antibody potency of immune mouse is detected using indirect ELISA method, by after immunity the 2nd, 3,4,5,6,7,8 The mice docking blood sampling in week, separating serum insulation liquid carries out doubling dilution, indirect detection serum titer, testing result such as Figure 18 It is shown.Result shows shown in Figure 18, and after with DNA vaccination immune mouse, serum titer started to begin to ramp up from first week, until Reach summit within the 4th week after immunity, serum titer is up to 1:1560000, immunizing potency is lasted till the 7th week after immunity, the Eight weeks start to be gradually reduced.The antibody titer level of DNA vaccination of the present invention and antibody persistent period and M5 attenuated vaccine bases This is quite.

Embodiment 6

The compatibility test of DNA vaccination and adjuvant

The DNA vaccination that embodiment 3 is obtained is used cooperatively with the adjuvant of different type, different amounts, and immune mouse enters Row lymphocyte proliferation assay, detects its immune efficacy, and experimental result is as described in table 4-6.

The lymphocyte proliferation assay result of table 4

Wherein, PI represents lymphopoiesis index, and 1 represents the DNA vaccination prepared by embodiment 3, IL2, IL12, IFN γ, GM-CSF are cytokine gene adjuvant, and ISCOMs represents immunostimulating complex.

It can be seen from the experimental result of table 4, DNA vaccination is used cooperatively with adjuvant can effectively strengthen its immune efficacy.Its In, DNA vaccination is used cooperatively with cytokine gene adjuvant, particularly IL2 with the cytokine gene adjuvants of IL 12, Neng Goute The immune efficacy of DNA vaccination is not effectively strengthened.

The lymphocyte proliferation assay result of table 5

PI represents lymphopoiesis index, and 1 represents the DNA vaccination prepared by embodiment 3, and IL2 is cytokine gene Adjuvant, listed ratio is mass ratio in table 5.

The lymphocyte proliferation assay result of table 6

PI represents lymphopoiesis index, and 1 represents the DNA vaccination prepared by embodiment 3, and IL12 is cytokine gene Adjuvant, listed ratio is mass ratio in table 6.

Quality it can be seen from the experimental result of table 5-6, between DNA vaccination and IL 2, IL12 cytokine gene adjuvants Than 1：Immune effect can be preferably improved between 1-5.

Finally it should be noted that：Various embodiments above only to illustrate technical scheme, rather than a limitation；To the greatest extent Pipe has been described in detail with reference to foregoing embodiments to the present invention, but it will be understood by those within the art that：Its Still the technical scheme described in foregoing embodiments can be modified, either to which part or all technical characteristic Carry out equivalent；And these modifications or replacement, do not make the essence disengaging various embodiments of the present invention skill of appropriate technical solution The scope of art scheme.

SEQUENCE LISTING

<110>Qinghai University

<120>One kind is for brucellar DNA vaccination of the type of sheep kind 3 and preparation method thereof

<130> 11111

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 36

<212> DNA

<213>Artificial sequence

<400> 1

gatgacacca tatggccgac gtggttgttt ctgaac 36

<210> 2

<211> 35

<212> DNA

<213>Artificial sequence

<400> 2

gacacctcga gttagaactt gtagttcaga ccgac 35

<210> 3

<211> 666

<212> DNA

<213> Brucella melitensis

<400> 3

gccgacgtgg ttgtttctga accttccgcc cctactgctg ctcctgttga caccttctcg 60

tggaccggcg gctatatcgg tatcaacgcc ggttacgcag gcggcaagtt caagcatcca 120

ttttctagct ttgacaagga agacaacgaa caggtttccg gttcgctcga cgtaacagct 180

ggcggcttcg tcggtggtgt tcaggccggt tacaactggc agctcgacaa cggcgtcgtg 240

ctcggcgcgg aaaccgactt ccagggatcg agcgttacgg gttcgattta cgccggtgcc 300

agcggtctcg aaggcaaagc tgaaaccaag gtcgagtggt tcggcacagt tcgtgcccgt 360

cttggctaca cggctaccga acgcctcatg gtttatggta ccggcggtct ggcctatggt 420

aaggtcaagt ctgcgttcaa cctgggtgat gatgcaagtg ccctgcacac gtggtccgac 480

aagacgaaag ctggctggac cctcggcgct ggtgctgaat atgccatcaa caacaactgg 540

acgctcaagt cggaatacct ctacaccgac ctcggcaagc gcaacctcgt cgacgttgac 600

aatagcttcc ttgagagcaa ggtcaatttc cacactgttc gcgtcggtct gaactacaag 660

ttctaa 666

<210> 4

<211> 221

<212> PRT

<213> Brucella melitensis

<400> 4

Ala Asp Val Val Val Ser Glu Pro Ser Ala Pro Thr Ala Ala Pro Val

1 5 10 15

Asp Thr Phe Ser Trp Thr Gly Gly Tyr Ile Gly Ile Asn Ala Gly Tyr

20 25 30

Ala Gly Gly Lys Phe Lys His Pro Phe Ser Ser Phe Asp Lys Glu Asp

35 40 45

Asn Glu Gln Val Ser Gly Ser Leu Asp Val Thr Ala Gly Gly Phe Val

50 55 60

Gly Gly Val Gln Ala Gly Tyr Asn Trp Gln Leu Asp Asn Gly Val Val

65 70 75 80

Leu Gly Ala Glu Thr Asp Phe Gln Gly Ser Ser Val Thr Gly Ser Ile

85 90 95

Ser Ala Gly Ala Ser Gly Leu Glu Gly Lys Ala Glu Thr Lys Val Glu

100 105 110

Trp Phe Gly Thr Val Arg Ala Arg Leu Gly Tyr Thr Ala Thr Glu Arg

115 120 125

Leu Met Val Tyr Gly Thr Gly Gly Leu Ala Tyr Gly Lys Val Lys Ser

130 135 140

Ala Phe Asn Leu Gly Asp Asp Ala Ser Ala Leu His Thr Trp Ser Asp

145 150 155 160

Lys Thr Lys Ala Gly Trp Thr Leu Gly Ala Gly Ala Glu Tyr Ala Ile

165 170 175

Asn Asn Asn Trp Thr Leu Lys Ser Glu Tyr Leu Tyr Thr Asp Leu Gly

180 185 190

Lys Arg Asn Leu Val Asp Val Asp Asn Ser Phe Leu Glu Ser Lys Val

195 200 205

Asn Phe His Thr Val Arg Val Gly Leu Asn Tyr Lys Phe

210 215 220

Claims

1. a kind of detached DNA fragmentation, it is characterised in that the nucleotide sequence of the DNA fragmentation such as SEQ ID NO：Shown in 3.

2. a kind of DNA vaccination for the type Infected with Brucella of sheep kind 3, it is characterised in that the DNA vaccination is will comprising right Seek the carrier for expression of eukaryon of DNA fragmentation described in 1.

3. DNA vaccination as claimed in claim 2, it is characterised in that the carrier for expression of eukaryon selected from pVAX1, pCI, PcDNA3.1 and pJW4303.

4. a kind of bacterin preparation, it is characterised in that the preparation includes DNA vaccination and adjuvant described in Claims 2 or 3.

5. bacterin preparation as claimed in claim 4, it is characterised in that the adjuvant selected from cytokine gene adjuvant and/or One or more in function vector immunological adjuvant.

6. bacterin preparation as claimed in claim 5, it is characterised in that the cytokine gene adjuvant is selected from IL-2, IL- 12nd, one or more in IFN-γ, GM-CSF, the function vector immunological adjuvant is selected from liposome, immunostimulating complex One or more in the aggressive intracellular bacteria of (Immune stimulating complexes, ISCOMs) and attenuation.

7. bacterin preparation as claimed in claim 4, it is characterised in that the mass ratio of the DNA vaccination and the adjuvant is 1： 1-5。

8. a kind of method for preparing DNA vaccination described in Claims 2 or 3, it is characterised in that methods described includes：Structure is included The carrier for expression of eukaryon of DNA fragmentation described in claim 1.

9. method as claimed in claim 8, it is characterised in that methods described specifically includes following steps：(1) by PCR side Method amplification obtains DNA fragmentation described in claim 1；(2) with EcoR I and BamH II difference enzyme action PCR amplification gained DNA fragmentation and PVAX1 carriers；(3) DNA fragmentation and pVAX1 carriers after enzyme action, transformed competence colibacillus cell are connected；(4) picking positive colony, leads to Cross double digestion and sequencing mode confirms to positive colony；(5) plasmid of positive colony is extracted.

10. a kind of method for preparing bacterin preparation described in any one of claim 4-7, it is characterised in that by the DNA vaccination with The adjuvant mixing.