CN106928373A - A kind of porcine mycoplasmal pneumonia multi-epitope mucosal vaccine - Google Patents

Abstract

The present invention relates to a kind of preparation and application of porcine mycoplasmal pneumonia multi-epitope mucosal vaccine.The vaccine is with mycoplasma hyopneumoniae memebrane protein：Attachment proteins P97, lipoprotein P65, specific membrane protein P46 B cells epitope, Th epitopes, CTL epitopes and b subunit of cholera toxin are used as vaccine frame structure, connected by flexible linker, Escherichia coli are converted after being cloned into pRSETA carriers, by fermentation, the technique such as purifying, preparation, obtains the porcine mycoplasmal pneumonia multi-epitope mucosal vaccine with Desirable immunogenic.Production process has used homemade mucosal adjuvant, makes the production of vaccine and uses process eased, convenient.Zoopery shows that not only security is good for porcine mycoplasmal pneumonia multi-epitope mucosal vaccine, and can excite effective mucosal immunity, humoral immunity and cell immune response.

Description

A kind of porcine mycoplasmal pneumonia multi-epitope mucosal vaccine

Technical field

The invention belongs to biotechnology genetic engineering field, a kind of porcine mycoplasmal pneumonia multi-epitope mucosal vaccine is related generally to Preparation and application.Specifically, using gene recombination technology, by the cholera toxin attachment proteins P97 relevant with porcine mycoplasmal pneumonia, Lipoprotein P65 and specific membrane protein P46 epitopes are connected and are cloned into carrier, convert Host Strains, by fermentation, pure The techniques such as change, preparation, obtain a kind of porcine mycoplasmal pneumonia recombinant multi-epitope mucosal vaccine and the vaccine in prevention porcine mycoplasmal Application in pneumonia.

Background technology

Porcine mycoplasmal pneumonia (Mycoplasmal hyopneumoniae, MHP) is widely current in each pig-raising countries in the world, Removed once infecting and being difficult to purification on pig farm.Sick main clinical manifestation heating, cough, the expiratory dyspnea, anorexia, growth resistance Stagnant, feed conversion rate reduction, Time To Market postpones.Therefore, although the death rate after pig-pig infection mycoplasma pneumoniae is not high, but still The old economic benefit for having a strong impact on pig enterprise.Mycoplasma hyopneumoniae can also cause the pulmonary lesion of infected pigs, cause immune suppression System and scabies secondary infection, cause the viral disease such as the disease and swine flu, indigo plant otopathy, pseudoabies and the type smoothing filter operator of pig 2 and The bacteriosises such as pasteurellosis, pleuropneumonia, streptococcosis and Haemophilus parasuis, form respiratory diseases in pigs comprehensive Simulator sickness, increased the difficulty of swine disease diagnosis and prevention and control.

China's serum epidemiological survey sick to this shows that 95% pig farm MHP serum antibodies are presented positive, weanling pig Positive rate of antibody is up to 30%, and boar and growing and fattening pigs positive rate of antibody are up to 100%, and seroprevalence is with age in days Growth constantly raise.At present, to the protective antigens and immunologic mechanism of MHP in terms of research without breakthrough, lead Cause clinically lacks effective vaccine protection and treatment method.

Mycoplasma pneumoniae will be colonized in airway epithelial cell, breed and cause pulmonary lesion, it is necessary to overcome mucociliary The scavenging action of system, through glycosylated mucin layer, the blocking for overcoming the extracellular composition of host to be colonized MHP is escaped host and is adapted to Property immune response, is firmly adhered on the cilium of respiratory apparatus, and cell membrane becomes its pathogenic key.Research finds branch Substance can recruit heparin to after birth surface, after liver fat is combined with extracellular matrix, can increase MHP field planting to host's breathing The ability of tract epithelial cell cilium.MHP and sticking for porcine respiratory cilium are the coefficient results of multiple adhesion factors, are sticked Mechanism is not confirmed also completely, but has identified multiple adhesion factors.At present, the attachment proteins for having obtained mainly has： P97、P102、P107、P116、P146、P116、P159、P216、P271.The expression of attachment proteins is in mycoplasma hyopneumoniae Discrepant, mycoplasma may escape the identification of host immune system by this differential expression of adhesion factor.

Mycoplasma hyopneumoniae is subordinate to mantle body guiding principle, Mycoplasma, be it is a kind of being capable of the fast minimum of self-replacation, evolutionary rate Prokaryotes.MHP simple structures, whole cell is made up of DNA, 3 kinds of organelles of ribosomes and cell membrane, this be carry out it is autologous Breeding and the organelle species of bottom line necessary to synthetic macromolecule.Lack cell membrane, so not fixed form, greatly It is small to differ, under extrusion can be by 0.22 μm of filter.Mycoplasma can synthesize itself macromolecular, but due to its genome It is small, carry information content seldom, and less than 30% more than G+C%, biosynthesis ability is limited, it is necessary to which from external world's intake, some compare Complicated matrix or precursor.The expression of mycoplasma gene codon is sometimes different from the codon implication of general bacterium, pig pneumonia Mycoplasma TGA codes for amino acid tryptophan, and general bacterium is with TGG codes for amino acid tryptophan, TGA is used as terminator codon.

P97 memebrane proteins are studied at most most deep adhesion factors.Young has found that P97 albumen is that MHP is main and is immunized One of original, for 30 days more early than the antibody produced for other major antigens of the antibody that P97 albumen is produced, this proves that it is main Virulence factor, the early detection method of MHP can be set up and new generation vaccine is prepared.The partitioned representation P97 eggs in vitro such as Cheryl In vain, it is found that its C-terminal and N-terminal can be adhered to heparin, wherein C disconnected N1, N2 area is particularly important to adhesive attraction.Liu Maojun etc. The P97 gene orders of different virulence MHP are expanded, by sequencing, comprehensive comparative analysis has been carried out and is found different strains P97 albumen Nucleotide sequence and amino acid sequence and its virulence have it is a certain degree of contact, while also having correlation with other Disease-causing genes. During mycoplasma hyopneumoniae infection host, infected pig produces the IgA and IgM that earliest antibody is directed to P97, and being immunized for causing should Answer much earlier than other antigen proteins.So, P97 can be used for the early diagnosis of MHP infected pigs and vaccine research.

P65 albumen is MHP surface of cell membrane lipoprotein, is a main immunogens albumen of mycoplasma hyopneumoniae, and it is exempted from Epidemic focus domain is placed in cell membrane outside positioned at C-terminal, belongs to the surface antigen of MHP, is the surface for causing early immune to react No cross reaction between lipoprotein, with the pathogen such as pig mycoplasma flocculare, mycoplasma hyorhinis and mycoplasma hyosynoviae, is pig pneumonia The major immunodominant albumen of mycoplasma, it has speciesspecific epitope.The N-terminal of its albumen is similar to lipolytic enzyme, has Esterase active can reduce fat acid.P65 destroys pulmoalveolar surfactant in MHP courses of infection, makes the liquid of lung surface Power increases, so as to mediate damages of the MHP to lungs.Additionally, there is researcher to find that the antibody of rabbit-anti P65 albumen can suppress external The growth of MHP, it is believed that the vinegar enzymatic activity of P65 may play a role during mycoplasma intake aliphatic acid.Meanwhile, P65 is heat One of shock protein HSP70 family members, therefore also there is immunostimulation.So, current P65 is often by as immunodominance Albumen is applied to the exploitation of mycoplasma hyopneumoniae DNA vaccination and subunit vaccine.

P46 albumen is a kind of with species specific MHP memebrane proteins, and gene size is 1260bp, there is 3 codings in sequence The codon TGA of tryptophan, plants inner height and guards.P46 albumen is for detecting most in known mycoplasma hyopneumoniae antigen protein It is preferable albumen.Having proven to P46 at present can cause acute or infection mycoplasma hyopneumoniae first weanling pig and grower pigs Early immune response.Ma Fengying etc. by main immunogens albumen P36, p46, p65 of the mycoplasma hyopneumoniae of prokaryotic expression and P97R1-NrdF is combined, and is prepared into a kind of subunit vaccine, and wherein P36, p46 and p65 is mixed into one group, P36, p46, p65 One group is mixed into P97R1-NrdF, immune mouse, it is found that both subunit vaccines are exempted from induction of body fluid higher respectively Epidemic disease and cellular immunity, wherein, the addition of P97R1 and NrdF enhances immune effect.Another Research Thinking of subunit vaccine It is to stimulate body to produce with the heterologous protein expression for having immunoadjuvant function the immunogen protein of mycoplasma hyopneumoniae Humoral immunity, cellular immunity and mucosa-immune.

Foreign countries will be developing based on inactivated vaccine.Early in the nineties in last century, porcine mycoplasmal pneumonia inactivated vaccine product is just It is existing to be used in U.S.'s registration listing, in the market using most inactivated vaccines be also based on external import product, its In be most widely used with auspicious times of suitable inactivated vaccine of Pfizer Animal Health Care Products Corporation development ＆ production.In addition, the vigorous woods of Germany The FLEX series mattress lattice hairs that Ge Yingehan animal healths Co., Ltd releasesSingle needle that inactivated vaccine is created due to it is immune with Preferable immune effect also enjoys market to praise highly.The pig gram of French Cimmeria animal health Co., Ltd production breathes heavily (HYORESP) Happiness Yifeng (MYPRAVACSUIS) inactivated vaccine of the biological big pharmaceutical factory production of inactivated vaccine, Spain Hai Bolai, U.S. richness road are moved Rui Fute (Suvaxyn Respifend MH) inactivated vaccine of thing Healthcare production etc. occupies certain market share.State Inside also there is the inactivated vaccine product of porcine mycoplasmal pneumonia, there is certain protective effect to the preventing and treating of the disease.Mainly with research, life Produce based on attenuated vaccine, cause the weak weak toadstool seedling of internal organs by using intraperitoneal injection using allosome, run into popularization and application very big Resistance.The vaccine of this use produced in conventional processes encounters many problems：(1) because MHP is slow-growing, bacterium number titre is not It is very high, causes the live vaccine production cycle long, it is relatively costly；(2) MHP is susceptible to antigenicity in culture medium succeeding generations Change, cause immune effect bad.

There are two important defects in the vaccine for using at present：1. immune protective effect is not good, after vaccine inoculation, is exempted from The pig of epidemic disease protection is compared less than half with other diseases immune effect, and gap is too big, unsatisfactory；2. after inoculation live vaccine, pig Only remain able to carry disease germs, and can mutually propagate, the pig morbidity for causing not to be immunized and being partly immunized, production performance reduction, It is experimentally confirmed that being immunized and infection rate between pig of the MHP that causes without immune pig is without significant difference, illustrate on the market Vaccine do not play expected immunoprotection and make effectively suppress propagation of the MHP between swinery.Therefore, in order to prevent The generation of MHP and its propagation of cause of disease prepare the vaccine with immune protective efficiency high, it is necessary to restudy.Accordingly it is desirable to On the basis of in terms of the MHP major protein antigen researchs, using bioengineering means, by great expression MHP major antigens come Produce efficient MHP vaccines.In terms of using MHP major antigens albumen research immune effect, external scholar has been carried out Attempt.

Mucosa-immune be immunity of organism defence the first line of defence, the pathogen such as defense against bacterial, virus cause enteron aisle and Played a major role in respiratory tract infection, but the special physicochemical property of mucous membrane surface, the intensity of mucosal immune response is reduced, or even Cause immune tolerance.Good immunologic adjuvant can make a small amount of antigen have induction body to produce early stage, efficient and lasting immune Response.Cholera toxin (cholera toxin, CT) has good adjuvanticity, wherein nontoxicity B subunits are quite by research people Member's pro-gaze.CTB can specifically bind with the cell surface Ganglioside GM1 of most of mammals, inducing cell film configuration Change.The main mucosa-immune reaction of induction in the following manner：(1) permeability of epithelial cell is increased, enhancement antigen absorbs； (2) Antigen-presenting role of different antigen presenting cells is strengthened；(3) differentiation of B cell is adjusted, making the formation of IgA increases；(4) The generation of the propagation and cell factor of regulatory T-cell.When the antigen of it and chemical coupling or Gene Fusion enter simultaneously in vivo it Afterwards, the immunogenicity of fusion epitope antigen can be evoked, body is produced strong immune response, made so as to reach immunoprotection With.CTB subunits are non-toxic, with very strong immunogenicity, can stimulate and produce mucous membrane IgA and serum IgG antibody, with can add The characteristics of strong antigen epitope antigen, therefore B subunits are often selected as the adjuvant of peptide vaccine.It is anti-for strengthening polysaccharide, small peptide etc. half It is former.CTB albumen is a kind of ideal protein for oral vaccine delivery, because it can not only improve absorption of the enteron aisle to protein Rate, and the antibody titer that body is produced can be improved as immunologic adjuvant.CTB subunit vaccines are except by ganglioside Fat GM1 is combined and is caused that its assistance oral vaccine enters extracellular, and it can also specifically influence the intercellular connection of small intestinal mucosa Or small band closedown structure, increase permeability, prevent oral vaccine that decomposition is digested at mucous membrane of small intestine, its antigenicity is kept, make Body produces good immune response.

Summary of the invention

The present invention is with the relevant major surfaces memebrane protein of porcine mycoplasmal pneumonia：Adhesion protein (P97), lipoprotein (P65)/spy The B cell epitope of specific membrane protein (P46), helper cell (Th) epitope, killer T cell (CTL) epitope and cholera toxin Used as vaccine construct, after expression in escherichia coli, by techniques such as protein purification, preparations, acquisition is exempted from ideal for B subunits The porcine mycoplasmal pneumonia multi-epitope mucosal vaccine of epidemic focus.Effective mucosa-immune, body are can induce after this vaccine immunity target animals Liquid is immunized and cell immune response.

An object of the present invention there are provided and a kind of new can excite mucosal immunity, humoral immunity, cellular immunity Porcine mycoplasmal pneumonia multi-epitope mucosal vaccine polypeptide and its vaccine combination, vaccine combination can be used for prevent porcine mycoplasmal draw The pneumonia of hair；The second object of the present invention there are provided structure and the acquisition of the porcine mycoplasmal pneumonia multi-epitope mucosal vaccine Method；The third object of the present invention there are provided the gene work that can express the porcine mycoplasmal pneumonia multi-epitope mucosal vaccine Journey bacterial strain；The fourth object of the present invention there are provided the preparation method of the porcine mycoplasmal pneumonia multi-epitope mucosal vaccine；This The fifth purpose of invention there are provided purposes of the multi-epitope mucosal vaccine in porcine mycoplasmal pneumonia is prevented.

In a first aspect, many invention provides a kind of restructuring porcine mycoplasmal pneumonia multi-epitope mucosal vaccine for prevention Peptide and combinations thereof.It contains major membrane protein：Adhesion protein (P97), lipoprotein (P65), the B of specific membrane protein (P46) Cell epitope, Th epitopes, CTL epitopes and choleratoxin B subunit.B cell epitope, helper T cell epitope, lethal T are thin Born of the same parents refer to a part of polypeptide in the major outer membrane protein with immunogenicity or its there is essentially identical immunogenicity function Equivalent.The helper cell epitope is the epitope for referring to strengthen Thelper cytoactives.The multi-epitope Mucosal vaccine refers to that multiple epitopes are connected in series together formed albumen or polypeptide.Series connection can be by genetic engineering method Or it is artificial synthesized carry out, except containing major membrane protein P97, P65 and P46 epitope, also including in multi-epitope mucosal vaccine Nonimmune active material.The nonimmune active material is the coupling part of each polypeptide, without P97, P65 and P46 antigen table The immunogenicity of position, also without any adjuvanticity, mainly has joint peptide, chemical modification part, N-terminal signal peptide and C-terminal many Polyadenylic acid etc..The pharmaceutically acceptable salt refers to non-toxic, stimulates and allergy, it is adaptable to human or animal tissues Salt.Inert matter and pharmaceutically acceptable salt are well known to those skilled in the art.

It is preferred that the major membrane protein epitope sequence described in the multi-epitope mucosal vaccine polypeptide of first aspect present invention Row are respectively selected from adhesion protein P97B cell antigen epitopes, and amino acid sequence is located at the bit amino of PROTEIN C end the 487th~502 respectively Acid, the 124th~155 amino acids, in the 382nd~407 amino acids and the 293rd~308 amino acids peptide fragment；Lipoprotein P65B cell antigen epitopes, amino acid sequence is located at the amino acids of PROTEIN C end the 17th~41, the 192nd~248 bit amino respectively Acid, in the 269th~308 amino acids peptide fragment；Specific membrane protein P46B cell antigen epitopes, amino acid sequence is located at egg respectively The white amino acids of C-terminal the 74th~100, in the 143rd~165 amino acids and the 396th~411 amino acids peptide fragment；Lipoprotein P65Th epitopes, amino acid sequence is located at the amino acids of PROTEIN C end the 323rd~343, the 151st~183 amino acids respectively In peptide fragment；Specific membrane protein P46Th epitopes, amino acid sequence is located at the amino acids of PROTEIN C end the 9th~31 respectively, the In 160~192 amino acids peptide fragments；Adhesion protein P97CTL cell epitopes, amino acid sequence respectively be located at PROTEIN C end the 17th~ 33 amino acids, in the 930th~951 amino acids peptide fragment；Lipoprotein P65CTL cell epitopes, amino acid sequence is located at egg respectively The white amino acids of C-terminal the 353rd~369, in the 536th~547 amino acids peptide fragment；

In addition preferably, the amino acid sequence of multi-epitope mucosal vaccine polypeptide is as follows described in first aspect present invention：

(1) the amino acids sequence of P97B cell antigen epitopes 487~502 is Be1 (SEQ ID No.8)： GKEEQAKLDYGNILNP；

(2) the amino acids sequence of P97B cell antigen epitopes 124~155 is Be2 (SEQ ID No.10)： PDDVNQNFKVKFQALQKLHNGDIAKSDIYEQT；

(3) the amino acids sequence of P97B cell antigen epitopes 382~407 is Be3 (SEQ ID No.12)： KENIYDFGKYNGKFNDRLNSPNLEYS；

(4) the amino acids sequence of P97B cell antigen epitopes 293~308 is Be-Tc4 (SEQ ID No.14)： AREILASPDEVQPVIN；

(5) the amino acids sequence of P65B cell antigen epitopes 17~41 is Be5 (SEQ ID No.16)： VVAIIENQKPVVLENPNGKRTTPSV；

(6) the amino acids sequence of P65B cellular antigens table 192~248 is Be6 (SEQ ID No.18)：

STSGDNHLGGDDWDNEIVNWLVKKIKEVYDFDPKSDKMALTRL KEEAEKTKINLSNQ

(7) the amino acids sequence of P65B cellular antigens table 269~308 is Be7 (SEQ ID No.18)： ELELKRSEFEKMTAHLIDRTRKPIVDALKQAKIEASDLDE；

(8) the amino acids sequence of P46B cellular antigens table 74~100 is Be8 (SEQ ID No.18)： QKDIISYVDETEAATSTITKNQDAQNN；

(9) the amino acids sequence of P46B cellular antigens table 143~165 is Be9 (SEQ ID No.18)： DRLITGSDKYDWYVSFDNEKVGE；

(10) the amino acids sequence of P46B cellular antigens table 396~411 is Be10 (SEQ ID No.18)： GKNINTILVSPVIVTK；

(11) the amino acids sequence of P65 helper cell epitope 323~343 is The1 (SEQ ID No.18)： SMIEHTLNKKPNRSINPDEVV；

(12) the amino acids sequence of P65 helper cell epitope 151~183 is The2 (SEQ ID No.18)： AALAFGLDKTEKEMKVLVYDLGGGTFDVSVLEL；

(13) the amino acids sequence of P46 helper cell epitope 9~31 is The3 (SEQ ID No.18)： FLYSSAIYATSLASIIAFVAAGC；

(14) the amino acids sequence of P46 helper cell epitope 160~192 is The4 (SEQ ID No.18)： NEKVGELQGLSLAAGLLGKEDGAFDSIDQMNEY；

(15) the amino acids sequence of P97 killer T cells epitope 17~33 is Tce1 (SEQ ID No.18)： KIGLTAGIVGLGVFGLT；

(16) the amino acids sequence of P97 killer T cells epitope 930~951 is Tce2 (SEQ ID No.22)： KLSLKTPEINVFLELVHQSEYE；

(17) the amino acids sequence of P65 killer T cells epitope 353~369 is Tce3 (SEQ ID No.22)： VLAGEIGDVLLLDVTPL；

(18) the amino acids sequence of P65 killer T cells epitope 536~547 is Tce4 (SEQ ID No.22)： LLEKQIQELKDL；

In addition preferably, 3~10 main advantage memebrane protein B cell antigen tables are included in the polypeptide fragment described in first aspect Position, 2~4 killer T cell epitopes and 2~4 helper cell epitopes.Above-mentioned different memebrane protein B cell and The permutation and combination method of killer T cell epitope and T cell helper antigen epitope has hundreds of thousands kind in theory, is based on Biochemical characteristic considers in itself for vaccine challenge mechanism and vaccine, is preferably as follows order：

It is preferred that polypeptide fragment combination order is：The1-The2-The3-The4-Be1-Be2-Be3-Be4-Be5- Be6-Be7-Be8-Be9-Be10-Tc4-Be5-Be6-Tce1-Tce2-Tce3-Tce4, Be1-Be2-Be3-Be4-Be5-Be6- Be7-Be8-Be9-Be10-The1-The2-The3-The4-Tce1-Tce2-Tce3-Tce4, Be1-Be2-Be3-Be4-Be5- Be6-Be7-Be8-Be9-Be 10-Tce 1-Tce2-Tce3-Tce4-The 1-The2-The3-The4, Tce1-Tce2- Tce3-Tce4-Be1-Be2-Be3-Be4-Be5-Be6-Be7-Be8-Be9-Be10-The1- The2-The3-The4 etc., more preferably , polypeptide fragment combination order is：Be1-Be2-Be3-Be4-Be5-Be6-Be7-Be8-Be9-Be10-The1-The2- The3-The4-Tce1-Tce2-Tce3-Tce4。

Additionally, optimal polypeptide fragment combination order is CTB-Be1-Be2-Be3- with the optimum combination of b subunit of cholera toxin Be4-Be5-Be6-Be7-Be8-Be9-Be10-The1-The2-The3-The4-Tce1-Tce2-Tce3-Tce4.

Therefore, restructuring porcine mycoplasmal pneumonia multi-epitope mucosal vaccine polypeptid acid sequence is as follows：

MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQ HIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANGDIPGCKGKEEQAKLDYGNILNPGSGPDDV NQNFKVKFQALQKLHNGDIAKSDIYEQTGSGKENIYDFGKYNGKFNDRLNSPNLEYSGSGAREILASPDEVQPVING SGVVAIIENQKPVVLENPNGKRTTPSVGSGSTSGDNHLGGDDWDNEIVNWLVKKIKEVYDFDPKSDKMALTRLKEEA EKTKINLSNQGSGELELKRSEFEKMTAHLIDRTRKPIVDALKQAKIEASDLDEGSGQKDIISYVDETEAATSTITKN QDAQNNGSGDRLITGSDKYDWYVSFDNEKVGEGSGGKNINTILVSPVIVTKCAAYSMIEHTLNKKPNRSINPDEVVG SGAALAFGLDKTEKEMKVLVYDLGGGTFDVSVLELGSGFLYSSAIYATSLASIIAFVAAGCGSGNEKVGELQGLSLA AGLLGKEDGAFDSIDQMNEYCAAYKIGLTAGIVGLGVFGLTGSGKLSLKTPEINVFLELVHQSEYEGSGVLAGEIGD VLLLDVTPLGSGLLEKQIQELKDL.

In second aspect, the invention provides a kind of nucleic acid molecule, the pig branch described in its coding first aspect present invention Pneumonias multi-epitope mucosal vaccine polypeptide.Nucleotide of the present invention can be rna form, DNA form, by artificial synthesized side Formula synthesizes many epitope tandem sequences, then enters carrier by genetic engineering operation connection rear clone, is transformed into Escherichia coli, Screening, after purification fermentation, acquisition porcine mycoplasmal pneumonia multi-epitope mucosal vaccine polypeptide.The nucleic acid can be carried out in the present invention Conventional molecular biology manipulations, such as：PCR, digestion with restriction enzyme, connection etc., the end of nucleic acid design 5 ' and 3 ' ends add Restriction enzyme site.It is preferred that the nucleotide sequence in the present invention is as follows：

ATG ATT AAA TTA AAA TTT GGT GTT TTT TTT ACA GTT TTA CTA TCT TCA GCA TAT GCA CAT GGA ACA CCT CAA AATATT ACT GAT TTG TGT GCA GAA TAC CAC AAC ACA CAA ATA CAT ACG CTA AAT GAT AAG ATA TTT TCG TAT ACA GAA TCT CTA GCT GGA AAA AGA GAG ATG GCT ATC ATT ACT TTT AAG AAT GGT GCA ACT TTT CAA GTA GAA GTA CCA GGT AGT CAA CAT ATA GAT TCA CAA AAA AAA GCG ATT GAA AGG ATG AAG GAT ACC CTG AGG ATT GCA TAT CTT ACT GAA GCT AAA GTC GAA AAG TTA TGT GTA TGG AAT AAT AAA ACG CCT CAT GCG ATT GCC GCA ATT AGT ATG GCA AAT GGT GAT ATC CCA GGT TGC AAG GGT AAA GAA GAA CAA GCA AAA TTA GAC TAT GGT AAT ATC TTA AAT CCA GGT TCT GGT CCT GAT GAT GTC AAT CAA AAT TTT AAG GTA AAA TTT CAG GCA TTA CAA AAA CTT CAT AAT GGT GAT ATT GCC AAA TCT GAT ATT TAT GAG CAA ACA GGT TCT GGT AAA GAA AAT ATT TAT GAC TTT GGT AAA TAC AAT GGA AAA TTC AAT GAC CGT CTT AAC TCG CCA AAT TTA GAA TAT AGC GGT TCT GGT GCC CGT GAA ATT TTA GCT AGC CCA GAT GAA GTT CAG CCA GTT ATT AAC GGT TCT GGT GTT GTT GCA ATT ATT GAA AAT CAA AAA CCT GTC GTT CTC GAA AAT CCC AAC GGA AAA AGA ACA ACT CCA TCC GTT GGT TCT GGT TCA ACA AGT GGT GAT AAT CAT TTA GGT GGG GAT GAC TGG GAT AAT GAA ATT GTA AAT TGG CTT GTT AAA AAA ATC AAA GAA GTA TAT GAT TTT GAT CCA AAA AGT GAT AAA ATG GCG CTT ACA AGA CTT AAA GAA GAG GCT GAA AAA ACC AAA ATT AAT CTT TCA AAT CAA GGT TCT GGT GAA CTT GAA CTT AAA AGA TCA GAA TTT GAA AAA ATG ACT GCC CAT TTA ATC GAT AGA ACT CGC AAA CCA ATT GTT GAT GCT CTA AAA CAA GCA AAA ATT GAG GCT TCA GAT CTT GAT GAA GGT TCT GGT CAA AAA GAT ATT ATT TCT TAT GTT GAT GAA ACA GAG GCA GCA ACT TCA ACA ATT ACA AAA AAC CAG GAT GCA CAA AAT AAC GGT TCT GGT GAT CGA CTA ATT ACT GGA TCT GAT AAA TAT GAT TGG TAT GTT TCT TTT GAT AAT GAA AAA GTT GGC GAA GGT TCT GGT GGT AAA AAT ATT AAT ACA ATT TTA GTA AGT CCA GTA ATT GTT ACA AAA TGT GCA GCA TAC TCA ATG ATT GAG CAT ACT TTA AAT AAA AAG CCA AAT CGT TCA ATT AAT CCT GAT GAG GTA GTC GGT TCT GGT GCC GCA CTT GCT TTT GGC CTT GAT AAA ACT GAA AAA GAA ATG AAA GTT CTT GTC TAT GAC TTA GGT GGG GGA ACT TTT GAT GTC TCA GTT TTA GAA TTA GGT TCT GGT TTT TTG TAT TCA TCA GCT ATT TAT GCA ACT TCG CTT GCA TCA ATT ATT GCA TTT GTT GCA GCA GGT TGT GGT TCT GGT AAT GAA AAA GTT GGC GAA TTA CAA GGT CTT TCA CTT GCT GCG GGT CTA TTA GGA AAA GAA GAT GGT GCT TTT GAT TCA ATT GAT CAA ATG AAT GAA TAT TGT GCA GCA TAC AAA ATT GGT TTG ACT GCC GGG ATT GTT GGT CTT GGA GTT TTT GGT CTA ACT GGT TCT GGT AAA TTA AGC CTA AAA ACA CCG GAA ATT AAT GTA TTT TTA GAA CTA GTT CAT CAA AGC GAG TAT GAA GGT TCT GGT GTT CTA GCT GGA GAG ATC GGC GAT GTT CTA CTT TTA GAT GTT ACT CCT TTA GGT TCT GGT TTA CTT GAA AAA CAA ATT CAA GAA TTA AAA GAT CTT TAA

In the third aspect, the invention provides a kind of carrier, it is except containing the coding pig described in second aspect present invention Eaton agent pneumonia multi-epitope mucosal vaccine nucleic acid molecule, also containing with the exercisable connection of the nucleotide sequence, it is thin in protokaryon Expression control element needed for cellular expression (transcription and translation).Most basic expression control element includes promoter, tanscription termination Son, enhancer, selected marker etc., these controlling elements are known in the art.Preferred e. coli bl21 in the present invention (DE3, Plys) is used as destination protein expression Host Strains.

In fourth aspect, the invention provides a kind of host cell, it contains the carrier described in third aspect present invention.Place Chief cell is inverted or transfects the gene order containing encoding proteins of the present invention, then has good heredity after testing After expression stability, the porcine mycoplasmal pneumonia multi-epitope mucosal vaccine antigen needed for can be used for fermentation expression production.

At the 5th aspect, the invention provides a kind of preparation method of porcine mycoplasmal pneumonia multi-epitope mucosal vaccine, its bag Include following steps：Engineering bacterium fermentation expresses porcine mycoplasmal pneumonia vaccine polypeptide, by thick purifying and polishing purification technique and after It is continuous to match somebody with somebody seedling technique, the polypeptide required for obtaining.The method being directed to including but not limited to bacterial cell disruption, inclusion body washing, Centrifugation, denaturation, affinity chromatography, hydrophobic chromatography, anion-exchange chromatography, reverse-phase chromatography, renaturation etc..

At the 6th aspect, the invention provides a kind of recombinant multi-epitope mucosal vaccine for preventing porcine mycoplasmal pneumonia, It includes the polypeptide and pharmaceutically acceptable carrier described in first aspect present invention.The multi-epitope mucosal vaccine can be pre- The outburst of anti-porcine mycoplasmal pneumonia.Pharmaceutically acceptable carrier of the present invention be immunopotentiator or immunologic adjuvant, it is excellent Select immunologic adjuvant for the special adjuvant of mucous membrane, component is：0.2% sodium hyaluronate, 0.4% sodium alginate, 0.2% arginine, 0.5mM EDTA, 0.1% skimmed milk power, PH8.0, adjuvant: antigen=1: 1 proportions, the speed of 80~100r/min is slowly stirred, Mix and can be used to be immunized.

At the 7th aspect, the invention provides the restructuring porcine mycoplasmal pneumonia multi-epitope mucosal vaccine described in the 6th aspect Using.Vaccine can necessarily effective dose aerosol inoculation or intramuscular injection, intracutaneous or inoculated with subcutaneous injections animal, preferably spraying exempt from Epidemic disease can produce mucosa-immune effective enough, humoral immunity and cell immune response (see embodiment five, six, seven, eight, nine, Tenth, 11,10 two), stimulate the generation of IgA and IgG, inducing peripheral blood T lymphopoiesis, while antiviral activity is provided, Lesion reaction is reduced, protection animal keeps the normal speed of growth from the attack of mycoplasma hyopneumoniae.Additionally, in the present invention Embodiment in, carry out laboratory safety experiment by vaccine, show porcine mycoplasmal pneumonia multilist of the present invention Position mucosal vaccine is safe (see example IV).

In addition, it is necessary to, it is noted that on the basis of the disclosure of the context of the application, of the invention other have The aspect of substantive distinguishing features is obvious for the ordinary skill people of this area.Additionally, the present invention which also uses disclosure Document, their entire contents are included and referred to herein.

Brief description of the drawings

Drawings below is used to illustrate specific embodiments of the present invention, rather than limits what is be defined by the claims The scope of the invention.Fig. 1 porcine mycoplasmal pneumonia multi-epitope mucosal vaccine expression plasmid pRSETA-CTB-Mhp (P97/P65/P46) structure Build figure；Fig. 2 pRSETA-CTB-Mhp (P97/P65/P46) vector plasmid cleavage map, wherein swimming lane 1 are DNA marker, from upper 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp are followed successively by lower molecular weight, swimming lane 2 is complete plasmid, and swimming lane 3 is Negative control, swimming lane 4 is digested plasmid；Fig. 3 SDS-PAGE detection figures, wherein swimming lane 1 are not induce control sample, swimming lane 2 Albumen Marker, is followed successively by 116KD, 66KD, 45KD, 35KD, 25KD, 18.4KD, 14.4KD from top to bottom, and swimming lane 3 is induction Sample, it is the destination protein of expression that arrow is signified；Fig. 4 Western-blot detection figures, wherein swimming lane 1 are pre-dyed marker, from 97.4KD, 66.2KD, 43KD, 31KD, 22KD, 14.4KD are up to followed successively by down, and swimming lane 2 is purpose albumen, and swimming lane 3 is blank pair According to.Fig. 5 schemes for fermented sample SDS-PAGE：Wherein swimming lane 1 be albumen Marker, be followed successively by from top to bottom 116KD, 66KD, 45KD, 35KD, 25KD, 18.4KD, 14.4KD, swimming lane 2 are positive control, and swimming lane 3 is fermentation inducement sample, and swimming lane 4 is not lure Lead sample；Fig. 6 is that fermented sample Western-blot detects figure, and wherein swimming lane 1 is positive control lane, and 2 is pre-dyed marker, 97.4KD, 66.2KD, 43KD, 31KD, 22KD, 14.4KD are followed successively by from top to bottom, and swimming lane 3 is fermentation purification of samples, and swimming lane 4 is Negative control.Fig. 7 is that ELISA method detects vaccine IgG antibody result；Fig. 8 is IFN-gamma detection knots in immune serum Really；Fig. 9 is peripheral blood specific lymphoproliferation testing result；Respiratory tract specificity SIgA testing results after Figure 10 is immune

Specific embodiment

Specific test method description described in embodiment is only exemplary description, for elaborating the present invention, but simultaneously It is not meant to limit the scope of the invention.

The mentality of designing of the porcine mycoplasmal pneumonia multi-epitope mucosal vaccine albumen of embodiment one

The present invention is according to major membrane protein in country's porcine mycoplasmal pneumonia Major Epidemic pnca gene group at present：Adhesion protein (P97), lipoprotein (P65), specific membrane protein (P46) amino acid sequence, are carried out using relevant biological information software to it Th epitopes, CTL epitopes and B cell epitope analysis.In expression in escherichia coli after designed epitope is connected, by fermentation, The techniques such as purifying, preparation, obtain the porcine mycoplasmal pneumonia multi-epitope mucosal vaccine with Desirable immunogenic.Made using the present invention Standby vaccine can effectively prevent porcine mycoplasmal pneumonia.

The domestic porcine mycoplasmal pneumonia epidemic strain genome sequence of comprehensive analysis, antigenic structure, Advance of Epidemiological Research, it is right Porcine mycoplasmal pneumonia multi-epitope mucosal vaccine is optimized design, final to determine 10 sections of B cell epitope, Thelper antigen tables 4 sections of the polypeptide in position, 4 sections of CTL antigen epitope polypeptides, 3 sections of B cell antigen epi-position polypeptide forms epidemic disease after all epitopes are connected with CTB The skeleton structure of seedling.The general structure of the vaccine is：CTB-Be1-Be2-Be3-Be4-Be5-Be6-Be7-Be8-Be9-Be10- The1-The2-The3-The4-Tce1-Tce2-Tce3-Tce4。

The coli expression carrier of embodiment two and the structure for expressing bacterial strain

1 polypeptide-coding nucleotide that will be designed serves extra large handsome biotech company's synthesis, nucleotide fragments two ends point BamHI (5 ' end) and HindIII (3 ' end) restriction enzyme site are not devised, are cloned into respectively after this fragment is synthesized On pMD18T carriers, sequencing confirms that insertion genetic fragment is consistent with implementation sequence (see sequence table).Recombinant plasmid is distinguished It is named as pMD18T-CTB-Mhp (P97/P65/P46).Plasmid is carried out into digestion treatment, large intestine with corresponding restriction enzyme Bacillus expression vector also uses identical restriction enzyme ferment treatment, digestion from the pRSETA plasmids of Invitrogen companies Condition：2 μ l plasmids are added in 10 μ l reaction systems, system, restriction enzyme is 5 active unit (New England Biolabs), the μ l of 10 × buffer solution 1, deionized water polishing, 37 DEG C of digestions 1.5 hours are added.Digestion adds 1 μ l after terminating 200mM EDTA terminating reactions.In 1% agarose gel electrophoresis, electrophoresis 30 minutes.By 2.86kb pRSETA matter under uviol lamp Grain and 2573bp CTB-Mhp (P97/P65/P46) fragment cut, and enter according to Qiagen companies gel reclaims kit specification Row glue reclaim.According to carrier：The ratio of fragment 1: 2~3 individually mixes multi-epitope nucleotide fragments with expression vector, reactant It is 15 μ l, is attached by T4DNA ligases, 16 DEG C of connections overnight, obtains recombinant plasmid and are named as pRSETA-CTB-Mhp (P97/P65/P46), (see Fig. 1), transformed competence colibacillus e. coli bl21 (DE3) pLysS.

2 conversions：PRSETA-CTB-Mhp (P97/P65/P46) is put into thawed on ice, 2 μ l coupled reaction liquid is added, again Mix, ice-water bath 30 minutes, 42 DEG C 30 seconds, ice bath is then put back to rapidly 1.5 minutes, add 1mL LB nutrient solutions, it is 37 DEG C, quiet Culture 1 hour is put, 4000g low-temperature centrifugations abandon supernatant in 10 seconds, with the 200 μ resuspended thalline of lLB culture mediums；By bacterium solution even spread Culture 12~16 hours in 37 DEG C of insulating boxs on the LB agar plates containing 100 μ g/mL ampicillins, are inverted in, directly To Clone formation.

3 identifications：In monoclonal to LB culture mediums on picking flat board, 37 DEG C, 200rpm concussion and cultivates 12 hours extract matter Grain, carries out double digestion using restriction endonuclease BamH I and HindIII respectively, can cut out corresponding porcine mycoplasmal pneumonia vaccine gene big The clone of small fragment, 2573bp can primarily determine that to be positive colony (see Fig. 2)；It is further that positive colony carries out determined dna sequence Verify its correctness (see sequence table).

4 induced expressions.Will positive colony incubated overnight, morning next day by 1: 100 switching, culture 3 hours after, add 0.1mM IPTG, continue to cultivate 4 hours, prepare sample；Conventional SDS-PAGE testing goals protein expression situation --- in 73KD (see Fig. 3), it is correct clone to see specific band；Correctly clone is taken, amplifies culture, after SDS-PAGE verification tables reach correctly, It is further characterized by using conventional Western-blot and expresses accuracy (see Fig. 4)；By after above-mentioned structure and evaluation program, can The positive colony that will be selected carries out the foundation of original species word bank, strain name pRSETA-CTB-Mhp (P97/ as engineering bacteria P65/P46)/BL21 (DE3, Plys).

The fermentation of the engineering bacteria of embodiment three, purifying and preparation

1 fermentation takes production strain pRSETA-CTB-Mhp (P97/P65/P46)/BL21 (DE3, Plys), is inoculated in 2mL (contain 100 μ g/mL ampicillins) in LB fluid nutrient mediums, 37 DEG C, 12 hours activated spawns of 200rpm shaken cultivations.Again with 1: 100 inoculum concentration accesses shaking flask, and 37 DEG C of shaken cultivations can be inoculated with into fermentation tank to OD600=3 in 10% ratio.Fermentation is trained It is semisynthetic medium to support base, is prepared with distilled water.Correction dissolved oxygen and pH value electrode, open tank body stirring, and revolution is 300rpm, Tank body sterilizes online, when culture-liquid temp in tank is down to 37.0 DEG C, demarcates pH and dissolved oxygen (OD) zero point.Fermentation temperature is 37.0 ± 0.1 DEG C, stream adds and mends when thalline OD600=1.0~1.2 are cultivated in dissolved oxygen control in 40% or so, pH controls 7.0, after inoculation Material 500mL, adds IPTG (final concentration of 0.2mM) induced expression in 1 hour after feed supplement, and 5 hour after fermentation of continuous induction terminate, Sampling is SDS-PAGE and identifies expression (see Fig. 5).

The thalline that 2 purifying will be collected into, with occlusion body washing lotion I (1%Triton X-100,20mMTris-cl PH8.0) Ultrasound, 2000W ultrasonic degradations 1 hour are carried out after suspension.4 DEG C, 12000rpm is collected by centrifugation occlusion body, and with occlusion body washing lotion II (1%DOC, 4M urea, 20mMTris-cl PHS.0) suspension twice ultrasonic washs occlusion body, and secondary low-temperature centrifugation is collected and included Body.Occlusion body precipitation 8M urea, 0.3% β-ME, 20mM Tris-cl (pH=8.00) is mixed, is stirred at room temperature 4 hours, 8000rpm low-temperature centrifugation 30min, discard precipitation.Albuminate 1: 100 dilutes, renaturation solution Tris (PHS.0) buffer system, 0.3M arginine is added, 4 DEG C are stirred renaturation 24 hours.The renaturation solution 20mM phosphate buffers of pH=8.0,0.5M sodium chloride, 20mM imidazoles, affinity column in balance, with the 20mM phosphate buffers of pH=8.0,0.5M sodium chloride, 0.5M imidazoles wash-out； Obtain final product restructuring porcine mycoplasmal pneumonia multi-epitope mucosal vaccine semi-finished product stoste, take semi-finished product stoste be westernblot identification (see Fig. 6).

The PBS that the semi-finished product of purifying sterilize is diluted to 200 μ g/mL, mucosa-immune special adjuvant (0.2% glass by 3 preparations Uric acid, 0.4% sodium alginate, 0.2% arginine, 0.5mM EDTA, 0.1% skimmed milk power, PH8.0) by 121 DEG C, sterilizing It is 15 minutes, standby.By adjuvant: vaccine=1: 1 proportions, the speed of 80~100r/min is slowly stirred, mix i.e. available In immune.

Example IV porcine mycoplasmal pneumonia multi-epitope mucosal vaccine safety testing

1 material

1.1 vaccines：Mycoplasma hyopneumoniae multi-epitope mucous membrane immunization vaccine is provided by the bright diligent biology research and development centre in Qingdao, lot number It is 201601,21602,201603.

1.2 experimental animals：18-22g Balb/C small white mouses are purchased from Beijing China Fukang.20~30 age in days sodium selenite (pig lungs Scorching mycoplasma negative antibody) provided by the pharmacy of Guangdong Yongshun.

2 methods

Security of 2.1 vaccines to small white mouse

18-22g Balb/C small white mouses, every batch of vaccine immunity 5, totally three batches, are immunized 15 small white mouses, and side is immunized Method is：Aerosol immunization, droplet particle is 0.1~5 μm, is sprayed at 0.5~0.8m of immunized mice overhead, people's distance spraying Place more than 2m, forms nebulization region, closes spray booth after spraying immediately, and the shut-in time is 2h, and immunizing dose is 2ml/, while 2 negative controls of setting, aerosol immunization adjuvant, Continuous Observation 10 days observes the health status of small white mouse.

Security of 2.2 vaccines to piglet

20~30 age in days sodium selenites of selection, aerosol immunization mycoplasma pneumoniae multi-epitope mucous membrane immunization vaccine, per batch 5 Head, totally three batches, are immunized 15 piglets.Immunization method is：Aerosol machine is sprayed, and droplet particle is 0.1~5 μm, distance of spraying At 0.5~0.8m of immune pig overhead, people more than 2m at spraying forms nebulization region, closes spray after spraying immediately Between mist, the shut-in time is 2h, and immunizing dose is 4ml/ heads.Set up negative control 2, aerosol immunization adjuvant, clinical observation simultaneously 14 days.

3 results

Safety testing of 3.1 vaccines to small white mouse

Result such as table 1, after being immunized, the appetite of all mouse, spiritual and health status are without exception, nothing consistent with control group Death occurs, it is seen that mycoplasma hyopneumoniae multi-epitope mucous membrane vaccine is safe to small white mouse, is shown in Table 1.

Safety testing result of the vaccine of table 1 to small white mouse

Group

Size of animal

Body temperature

Appetite

Spirit

Health status

The dead quantity

201601

5

Normally

Normally

Normally

It is in a good state of health

0

201602

5

Normally

Normally

Normally

It is in a good state of health

0

201603

5

Normally

Normally

Normally

It is in a good state of health

0

Control

2

Normally

Normally

Normally

It is in a good state of health

0

Safety testing of 3.2 vaccines to piglet

Result such as table 2, in whole 14 days experimental observation phases, all immune piglets, body temperature, spirit and appetite are normal, Without there is any clinical anomaly, after off-test, 15 piglets are good for and live.Compare adjuvant group 2 piglets also without times What adverse reaction.This explanation, mycoplasma hyopneumoniae multi-epitope mucous membrane immunization vaccine is safe to piglet.

Safety testing result of the vaccine of table 2 to piglet

Group

Size of animal

Body temperature

Appetite

Spirit

Unusual condition

The dead quantity

201601

5

Normally

Normally

Normally

Nothing

0

201602

5

Normally

Normally

Normally

Nothing

0

201603

5

Normally

Normally

Normally

Nothing

0

Control

2

Normally

Normally

Normally

Nothing

0

The porcine mycoplasmal pneumonia multi-epitope mucosal vaccine potency test animal packet of embodiment five, immune, evaluation method

For the efficiency evaluation parameter for obtaining vaccine as much as possible, the present invention have detected the development and change feelings after attacking poison Condition, exempts from including clinical observation (appetite, the back of a bow, cough, activity, the state of mind, fur etc.), measurement anus temperature, mucosa-immune, body fluid Epidemic disease, cellular immunity immune level and cut open inspection lungs pathological change.

1 experimental animal

36 first 20~30 age in days sodium selenites (mycoplasma hyopneumoniae negative antibody), test pig is fitted starting to test preceding environment Ying Yizhou.

2 vaccines

Porcine mycoplasmal pneumonia multi-epitope mucosal vaccine is provided by the bright diligent biology research and development centre in Qingdao, lot number is 201601, 201602nd, 201603, porcine mycoplasmal pneumonia live vaccine (168 plants) lot number is 169109.

3 experimental designs and method

3.1 animal feedings animal immune, are attacked poison, sampling and are carried out according to experiment schedule (being shown in Table 3) in BSL-2 isolation wards. Animal is divided into random 6 groups, 4 groups of vaccine immunity, non-to exempt to attack malicious control group and non-exempt from non-to attack control group, 5/group.Head exempts from rear 14 My god, once, immunization method is booster immunization：Aerosol machine is sprayed, and droplet particle is 0.1~5 μm, the immune pig crown of spraying distance At 0.5~0.8m of top, people more than 2m at spraying forms nebulization region, closes spray booth, shut-in time after spraying immediately It is 2h, immunizing dose is 2ml/ heads.Porcine mycoplasmal inactivated vaccine is immunized with reference to specification.42 days after immune, immune group and non-exempt to attack The malicious control group strong poison JS of tissue, after 1: 100 dilution, intratracheal injection 5ml attacks poison.

3.2 anus temperature are attacked poison and a few days ago record anus temperature daily within 14 days to after attacking poison；

3.3 antibody and IFN γ detection collection immune group and control group are 0 day after immune, 28 days 14 days, the serum of 42 days, By IDEXX mycoplasma hyopneumoniae antibody assay kits, detection and the IFN γ Concentration Testing of immune antiboidy IgG are carried out；

3.4 lymphocyte proliferation assays gather 0 day, 14 days, 28 days, 42 days immune groups and control group blood sample, after test tube of hepari Lymphocyte proliferation assay is carried out, t subset lymphocyte count is carried out；

3.5SIgA antibody tests gather immune group and control group 0 day, 7 days, 14 days, 21 days, 28 days, 35 days, the nose of 42 days Swab, is detected using mycoplasma hyopneumoniae SIgA-ELISA antibody kits.

3.6 attack cut open inspection record lung and scoring after poison

Attack 28 days after poison, slaughter test pig, scoring is made in the pathological change to the various pieces of lungs respectively, by lesion model Enclose and reach (0-25) % and be chosen as 1 point, (26-50) % is chosen as 2 points, and (51-75) % is chosen as 3 points, (76-100) % is chosen as 4 points, system Count each experiment swine disease tuberculosis varying index (up to 28 points) experiment pig lesion index and morbidity is judged to more than 10 points, be less than 4 points Health (excludes porcine mycoplasmal pneumonia infection), according to tuberculosis varying index, test pig is calculated as follows and attacks malicious protective index：

Experiment pig attacks that malicious protective index=(the change of immunized controls group tuberculosis varying index average value-experiment thumps does not refer to attack poison Number/(attacking poison not immunized controls group tuberculosis varying index average value-do not attack poison not immunized controls group tuberculosis varying index average value) × 100%.

3.7 daily gains

Experimental animal is weighed before attacking before poison and slaughtering, and calculates average daily gain.

3.8 attack malicious protective rate

It is unprotect that experiment pig attacks malicious protective index less than 60%, is have protection more than or equal to 60%, then by following public affairs Formula calculates test pig and attacks malicious protective rate：Experiment pig attacks malicious protective rate=have protection test pig number/test pig number；

Experiment is effective in positive controls all morbidity negative control group at least all health

The vaccine potency search time table of table 3

Note：After exempting from headed by time described in this column is equal number of days (my god).

Embodiment six attacks clinical observation and anus temperature measurement after poison

Self tapping poison a few days ago measured the change of anus temperature to 14 days to after attacking poison.

Vaccine immunity target animals attack poison after 42 days, two kinds of all experimental animals of vaccine immunity group do not go out during entirely attacking poison Existing obvious clinical symptoms, it is nonimmune attack malicious control group gradually showed since 3 days after attacking poison by the thick unrest of hair, anorexia, One's spirits are drooping, the movable clinical apparent symptom such as not good, while all exempt to attack poison according to group animal heat detection more than 40 DEG C to non-.Attack Second week after poison, it is non-exempt to attack malicious control group have 2, body temperature is more than 40 DEG C.4 immune groups it is whole attack poison during body temperature all the time Keep normal.

The serum evaluation of embodiment seven：IgG antibody is detected

Serum is detected with commercialization ELISA (IDEXX, porland, ME), the generation of Mhp antibody is analyzed.By to experiment The serum antibody IgG testing results of 42 days 28 days 14 days 0 day are shown in Fig. 7 after vaccine immunity of animals, and live vaccine (168 plants) intrapulmonary is immunized Approach is immunized, and in peripheral blood and is not detected by the immune antiboidy IgG on porcine mycoplasmal pneumonia, illustrate live vaccine simultaneously un-activation Humoral immune reaction.Three batches of vaccine aerosol routes prepared by the present invention are immunized, and 28 days after immune, can detect in peripheral blood The immune antiboidy IgG of the porcine mycoplasmal pneumonia containing higher concentration, exempts from non-to attack control group and live vaccine immune group, difference compared to non- Significantly (p ＜ 0.05).

Embodiment eight is immunized animal IFN γ Concentration Testing

0 day, 14 days, 28 days, 42 days experimental animal serum after conventional method collection is immune, according to goat-anti pig IFN γ ELISA Detection kit specification carries out IFN γ Concentration Testing to the serum for gathering.Result shows, after vaccine immunity, with it is immune when Between increase, IFN γ concentration persistently rises, and continues in higher concentration within 28 days~42 days after being immunized；Rather than exempt from the non-examination for attacking control group Test the relatively low concentration of the weightless holding of pig IFN γ concentration (see Fig. 8).

The specific lymphoproliferation of embodiment nine is detected

Attack before and after poison, the immune cell-mediated immune reaction for Mhp for being excited is detected by ELISPOT methods (Mateu de Antonio et al., 1998；Meier et al., 2003), to count PBMC crowds of (peripheral Blood mononuclear cells) in virus-specific IFN γ-SC, method is as follows：

1st, the separation of PBMC

Swine PBMC is isolated from fresh venous blood, uses 5mM heparin anti-coagulatings.1500r/min, 37 DEG C of centrifugation 30min, collects Leukocytic cream.PBMC 15ml Hanks balance salt and wash twice, and RPMI culture mediums are suspended, while adding 5% calf serum (Life Technologies, Gaithersburg, MD), 100U/ml penicillin (Sigma), 100mg/ml streptomysins (Sigma), 100U/ml gentamicins (Sigma), 4mM Pidolidones salt (Sigma), 1mM Sodium Pyruvates (Life Technologies), 10mM MEM nonessential amino acids (Life Technologies), and250mM 2 mercapto ethanols (Sigma)。

2nd, ELISPOT determines IFN γ-SC frequency detectings in PBMC

Different immune groups and the degree of control animals antigen-specific cellular immune response are quantitative with IFN γ ELISPOT (method is shown in Zuckermann et al., 1999).96 hole Immulon II TM plates 50ml/ holes 10mg/ml monoclonal antibodies Coating, buffer system is 0.1M carbonate buffer solutions (PH9.6).4 DEG C of incubated overnights, wash three times with aseptic PBS, then per hole 5% calf serum is added in RPMI suspensions, in 37 DEG C of CO₂It is incubated 2 hours in incubator.The PBMC of control group will reach often Hole 5 × 10⁵Individual visible cell.Confirm that in all samples 98% PBMC is living with the active somatic cell colorimetric method of exclusion.External pin To the anamnestic reaction of Mhp by adding the antigenic activation of Mhp (168 plants) form living.Titration PBMC stimulates relevant time Excellent dosage come determine remember viral antigen dosage.This quantity is determined in the range of multiple infection (MOI)：0.1-1.0.

PBMC is in 37 DEG C, CO₂Exposed to 2 hours in antigen in environment.PBST (0.05%Tween20) washs 6 times will be thin Born of the same parents remove.The mAb P97 (Ren Bailiang, 2012), PBST (0.05% of 0.5mg/ml biotin labelings are added in 50 titration holes Tween20) buffer system, titer plate is incubated 1 hour at 37 DEG C, after washing, adds HRP (Sreptavidin- Horseradish peroxidase) mark antibody, by washing, unnecessary labelled antibody is removed, add TMB film peroxides Compound zymolyte (Kirkegaard＆Perry Laboratories Gaithersburg, MD), per the μ l of hole 50, the compound water Bluepoint is caused to occur after solution, the amount that its size and density are combined with IFN γ is directly proportional.Specific IFN γ produces cell to pass through The quantity of Bluepoint is counted to determine.

The hole of PBMC in antigen as negative control group will be not exposed to.In typical Mhp specific reactions, each Kong Fei Background dot caused by Mhp stimulation cells should not be more than 5, and the value for being obtained is subtracted from cell value is each stimulated. Equally, in order to determine the ability that each PBMC sample produces IFN γ, 5 × 10⁴PBMC/ holes are placed in 10mg/ml mitosis unit and plant Cultivated in thing hemagglutinin, in all cases, observe positive reaction.

As a result

In order to evaluate the development of specificity regulation immune response caused by Mhp vaccine immunities, before and after immune, by reality Testing the next PBMC of animal separation carries out IFN γ generation detection.In this detection, do not stimulate in culture occur IFN γ-SC represent it is non- Specific cytokines spontaneous background product.The frequency of IFN γ-SC caused by the PBMC cultures stimulated with recall antigen Higher than the generation that background products show virus-specific IFN γ-SC.(0~42 day) (memory per group-specific during whole experiment Antigenic stimulus) produce frequency and the average value of spontaneous (background) IFN γ-SC to see Fig. 9.Attenuate vaccine group and recombinant protein seedling group are exempted from Epidemic disease group animal detects division stimulin phytohemagglutin phytolectin and reduces virus-specific IFN γ reaction (statistics 42 days after immune Show apparently higher than background).Control group detects negligible IFN γ and produces cell, ignores note in cell culture Recall the appearance of antigen.Comparatively, 28 days all vaccine groups generate high-caliber cell Autocrine IFN γ (model after being immunized Enclose：20~40IFN γ-SC/10⁶pBMC).Pole significant difference (P ＜ 0.01) is formed with group and control group is not stimulated

Respiratory tract specificity SIgA antibody tests after embodiment ten is immune

Collection immune group and control group 0 day, 7 days, 14 days, 21 days, 28 days, 35 days, the nose swab of 42 days, use pig pneumonia Mycoplasma SIgA-ELISA antibody kits are detected.Found by detecting, multi-epitope mucosa-immune vaccine group and live vaccine are immune Group can just detect porcine mycoplasmal pneumonia mucosal antibodies SIgA on 7th day after immune, compared to control group difference extremely significantly (p ＜ 0.01), and in the whole experimental stage remain high-caliber SIgA (see Figure 10).

The Gong Duhou lungs cut open inspection of embodiment 11 with attack malicious protective rate

The result discovery of cut open inspection record pulmonary lesion, three batches of multi-epitope mucosal vaccines and live vaccine (168 plants) after attacking poison, Can protection be produced to immune group experimental animal, from the point of view of lesion score value, without significant difference between immune group, be marked according to judgement Standard, 100% protection, rather than exempt to attack malicious control group protective rate for 0 (being shown in Table 4).

The pulmonary lesion of table 4 scores

Increase day by day re-detection after the second tap of embodiment ten poison

Daily gain result shows after attacking poison, the experimental animal of porcine mycoplasmal pneumonia vaccine is not immunized, in artificial challenge's pig lung After scorching mycoplasma, feed intake greatly reduces, and weightening is substantially reduced, or even negative growth occurs.And immune group is in artificial challenge's pig lung After scorching mycoplasma, there is not obvious body weight reduction phenomenon.With it is non-exempt to attack malicious control group weightening result compared with, all Mian Yi Group increase Weight result is presented pole significant difference (being shown in Table 5).

Table 5 attacks after poison the re-detection that increases day by day

Claims (9)

1. a kind of porcine mycoplasmal pneumonia multi-epitope mucosal vaccine fusion protein, its amino acid sequence is SEQ ID No.2.

2. a kind of nucleic acid molecules, its coding described fusion protein of claim 1.

3. a kind of carrier, it contains the nucleic acid molecules described in claim 2.

4. a kind of host cell, it contains the carrier described in claim 3.

5. nucleic acid molecules described in claim 2, its particular sequence is SEQ ID No.1.

6. a kind of vaccine for preventing porcine mycoplasmal pneumonia, it includes fusion protein described in claim 1 and pharmaceutically Acceptable carrier.

7. the carrier described in claim 6 is the special adjuvant of mucosa-immune, and preferred ingredient is：0.2% sodium hyaluronate, 0.4% marine alga Sour sodium, 0.2% arginine, 0.5mM EDTA, 0.1% skimmed milk power, PH8.0.

8. the vaccine described in claim 6, the ratio of the special adjuvant of antigen and mucous membrane after dilution is 1: 1.

9. the vaccine immunity method described in claim 6 is aerosol immunization：Droplet particle is 0.1~5 μm, and spraying distance is immunized and is At 0.5~0.8m of pig overhead, people more than 2m at spraying forms nebulization region, closes spray booth after spraying immediately, Shut-in time is 2h.