CN110526959A

CN110526959A - A kind of foot-and-mouth disease virus antigen, the gene for expressing foot-and-mouth disease virus antigen and its recombinant vector and Recombinant Lactococcus lactis

Info

Publication number: CN110526959A
Application number: CN201910806736.0A
Authority: CN
Inventors: 潘丽; 张中旺; 吕建亮; 张富东; 马中元; 刘新生; 周鹏; 王永录; 张永光
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2019-08-29
Filing date: 2019-08-29
Publication date: 2019-12-03

Abstract

The present invention provides a kind of foot-and-mouth disease virus antigen, the gene TB1-Co1 of expression foot-and-mouth disease virus antigen and its recombinant vector and Recombinant Lactococcus lactis, belong to recombinant vaccine technical field, the amino acid sequence of the antigen is as shown in SEQ ID NO:1.It is confirmed through animal immune experiment, foot-and-mouth disease virus antigen of the invention has preferable immunogenicity.The present invention also provides a kind of gene TB1-Co1 of foot-and-mouth disease virus antigen described in expression above scheme, the targeting ligand Co1 of M cell is merged by the C-terminal in multi-epitope albumen, targeting ligand Co1 and C5a receptor interaction can not only deliver the model antigen of linking ligand to M cell, and energy challenging antigen specific immune response includes systemic immune response and local mucosal immune response.

Description

A kind of foot-and-mouth disease virus antigen, the gene for expressing foot-and-mouth disease virus antigen and its recombination Carrier and Recombinant Lactococcus lactis

Technical field

The present invention relates to recombinant vaccine technical field more particularly to a kind of foot-and-mouth disease virus antigens, expression aftosa The gene TB1-Co1 and its recombinant vector and Recombinant Lactococcus lactis of viral antigen.

Background technique

Aftosa (Foot-and-mouth disease, FMD) is by foot and mouth disease virus (Foot-and-mouth Disease virus, FMDV) caused by acute, hot, highly contagious disease, mainly encroach on pig, ox, the domestic animals such as sheep and Other wild artiodactyls.Clinical symptoms is that blister rash occurs in mucous membrane of mouth, hoof and skin of breast.Hostis In Picornaviridae Hostis, viral genome is single-stranded positive RNA, about 8.5kb, there is 7 serotypes, nothing between type Cross protection.Its open reading frame is non-structural by L gene, P1 structural protein gene (including VP4, VP2, VP3, VP1 gene), P2 Protein gene (including 2A, 2B, 2C gene) and P3 nonstructural protein gene (3A, 3B, 3C, 3D gene) composition.VP1 albumen contains The critical epitopes of FMD immune response, including VP1 (21~60aa), VP1 (140~160aa), VP1 (200~212aa) are induced, These small peptides are the major antigenic determinant segments for determining various hypotype FMDV immunogenicities.Wherein, on the VP1 of the surface FMDV It include tripeptides RGD ring in 140~160 amino acids polypeptide chains, it is amino acid necessary to FMDV absorption infection host cell Sequence, the antibody that subsite induction generates can close the corresponding site on the surface FMDV, block the combination of FMDV and host cell, right It is of great significance in prevention FMDV invasion host cell.

The hotter FMD new generation vaccine of research includes that subunit vaccine, polyepitope vaccines, synthetic peptide vaccine, gene lack at present Lose vaccine, live vector vaccine and nucleic acid vaccine etc..Major part developed country by inactivated vaccine and slaughters the policy combined at present, Aftosa is successfully eliminated, but the anti-system of many developing countries and third world countries mainly takes immunity inoculation. Since there is scattered poison and the halfway risk of inactivation, many countries to be forbidden to use aftosa and go out for traditional aftosa vaccine Live seedling or Attenuate vaccine, therefore, the correlative study room of countries in the world all are being dedicated to developing safe and effective novel aftosa epidemic disease Seedling.

The area that mucous membrane surface product is occupied in vivo (including alimentary canal, genital tract and respiratory mucosa) is very big, compares body Skin surface product it is more than 400 times big.Mucosal immune system is first for invading animal body for defending extraneous pathogenic microorganism Barrier.The lymphoid tissue of the system is not only distributed very extensively in vivo, but also quantity is also most.Known mucosa-immune position is immunized The quantity of cell (such as T cell, B cell) and immune molecule (such as SIgA, SIgM) is more than systemic immune system, mucosal immune system The effect played in the generation of defence infection disease will be more and more important.Mucosal vaccine is by oral administration, the approach such as eye droppings or collunarium make For mucous membrane surface, mucosal immune response is induced with this.The great advantage of mucosa-immune is can to simulate natural infection approach, directly It connects lymphoid tissue abundant under stimulation respiratory tract and alimentary canal mucous membrane and generates a large amount of immunocompetent cell and antibody, directly cutting Pathogenic microorganism invades the approach of body, therefore mucosa-immune invades the intracorporal pathogenic microorganism effect of machine most by mucous membrane to prevention It is good.The portal that mucous membrane is in communication with the outside as body has very big power-assisted to act on the intrusion of FMDV.M cell (microfold cell) or theca cell (membranous cell), because there are many gain the name due to microcreping pleat in cell free face.M is thin Born of the same parents are widely present in respiratory tract and gastral mucous membrane.The characteristics of M cell, is the antigen be conducive to absorb in chamber and in time will Antigen presentation induces local immune response to lymphocyte.Therefore, M cell is to absorb the first of antigen in mucosa-immune reaction A cell is the primary effect site of mucosa-associated lymphoid tissue.Intake antigen is a step of induction immune response most critical.Institute It is presented in antigen in pathogenic microorganism from respiratory tract invasion and mucosa-immune with M cell and is played an important role.Research recently Show that the intracavitary antigen of M cellular uptake has 3 kinds of approach: 1. non-specific cell swallows；2. the phagocytosis that specific receptor mediates；③ Dendritic Cells is raised to intracavitary stretching by M cell, and DC has very strong phagocytic activity and offers antigenic capacity.Due to M cell In do not have can protein degradation matter lysosome, M cell to antigen without working process, the function of M cell only by antigen or It is transmitted after pathogenic microorganism intake.M cell utilizes special eucaryotic cell structure, can pass antigen within more than ten min Antigen presenting cell nearby is given, proteantigen is degraded into the more of 10 to 14 amino acid lengths with treated by processing Peptide, then it is presented to helper lymphocyte T after combining with MHC class Ⅱmolecule, the local mucosa-immune reaction for creating antagonism former.

In recent years, with the development of molecular biology and to the intensification that Lactococcus lactis recognizes, Lactococcus lactis has been used more The expression of kind albumen.But relative to escherichia coli prokaryotic expression system, Lactococcus lactis expression system (NICE) has certain Defect, the expression quantity for being mainly manifested in foreign protein is lower, certain genetic backgrounds are not clear enough, operation is comparatively laborious, is difficult reality Existing this technical bottleneck of large-scale production.

Summary of the invention

The purpose of the present invention is to provide a kind of foot-and-mouth disease virus antigens, the gene TB1- of expression foot-and-mouth disease virus antigen Co1 and its recombinant vector and Recombinant Lactococcus lactis provide one the present invention is directed to overcome this problem of mucosal vaccine inefficiency The more efficient mucosal vaccine of kind.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

The present invention provides a kind of foot-and-mouth disease virus antigen, the amino acid sequence of the antigen is as shown in SEQ ID NO:1.

The present invention also provides a kind of gene TB1-Co1 of foot-and-mouth disease virus antigen described in expression above scheme, the bases The nucleotide sequence of cause is as shown in SEQ ID NO:2.

Preferably, the foot and mouth disease virus is foot and mouth disease A-type virus.

The present invention also provides a kind of recombinant vectors comprising gene TB1-Co1 described in above scheme.

Preferably, the recombinant vector is using plasmid pNZ8148 as initial carrier.

Preferably, the gene TB1-Co1 is inserted between the NcoI on plasmid pNZ8148 and Hind III.

The present invention also provides a kind of Recombinant Lactococcus lactis comprising recombinant vector described in above scheme.

The present invention also provides the Recombinant Lactococcus lactis described in above scheme in preparing A type aftosa oral vaccine Using.

The present invention also provides the Recombinant Lactococcus lactis described in above scheme in A type aftosa mucosa-immune live vector Application.

Beneficial effects of the present invention: the present invention provides a kind of foot-and-mouth disease virus antigen, the amino acid sequence of the antigen As shown in SEQ ID NO:1；The molecular weight of the foot-and-mouth disease virus antigen is 40kD.It is confirmed through animal immune experiment, the present invention Foot-and-mouth disease virus antigen have preferable immunogenicity.The present invention also provides hoof-and-mouth diseases described in a kind of expression above scheme The gene TB1-Co1 of malicious antigen merges the targeting ligand Co1, targeting ligand Co1 of M cell by the C-terminal in multi-epitope albumen The model antigen that can not only deliver linking ligand is interacted with C5a receptor to M cell, and can challenging antigen specificity Immune response includes systemic immune response and local mucosal immune response.It include the gene the present invention also provides one kind The recombinant vector of TB1-Co1 and Recombinant Lactococcus lactis comprising the recombinant vector, although mucosal vaccine have it is a lot of other Advantage not available for vaccine, but it there is also many problems to be solved: in a, drinking-water or the immunologic process of feeding, Degradation of the antigen through alimentary canal by gastric acid and pepsin, required amount of vaccine is big, and secondary immunity is needed to can be only achieved expection Effect；B, it is suitable for the stronger virus of immunogenicity；C, destination protein needs to overcome on intestines during Mucosal Immunity Skin barrier；D, mucosa-immune is resistant to.In oral mucosa-immune, since antigen can degrade in gastric juice, it is possible to capsule or Protectiveness carrier carrys out present antigen, and the present invention, as live vector submission purpose antigen, is protected to the greatest extent using Lactococcus lactis Purpose antigen is protected, enable stimulates body to generate effective immune response at site of action (mouse PP knot).For overcoming The barrier action of enteric epithelium can solve submission by mucosa-immune reinforcing agent, plant oral carrier, targeting M cell and DC The low problem of efficiency.

Detailed description of the invention

Fig. 1 is the building route map of recombinant plasmid pNZ8148-TB1 and pNZ8148-TB1-Co1 in embodiment 2；

Fig. 2 is the digestion qualification figure of recombinant plasmid pNZ8148-TB1 and pNZ8148-TB1-Co1 in embodiment 2；Wherein, M:DNA molecular mass standard；1~2 is product of the recombinant plasmid pNZ8148-TB1 after III double digestion of Nco I/Hind；3~5 For product of the recombinant plasmid pNZ8148-TB1-Co1 after III double digestion of Nco I/Hind；

Fig. 3 is Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 and NZ9000/pNZ8148-TB1- in embodiment 3 Co1 expresses the SDS-PAGE figure of albumen；Wherein, M is protein standard；1: the empty vector control of induction；2: not luring The NZ9000/pNZ8148-TB1 led；3: the NZ9000/pNZ8148-TB1-Co1 not induced；4~5: the NZ9000/ of induction pNZ8148-TB1；6~7: the NZ9000/pNZ8148-TB1-Co1 of induction)；

Fig. 4 is the Western blot detection figure of Recombinant Lactococcus lactis expression albumen in embodiment 3；Wherein, A is warp The destination protein of the Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 expression of Nisin induction；B is the weight induced through Nisin The destination protein of group Lactococcus lactis NZ9000/pNZ8148-TB1-Co1 expression；C is the Lactococcus lactis induced through Nisin The albumen of NZ9000/pNZ8148 expression；

Fig. 5 is the dynamic change figure of sIgA antibody level in mouse intestinal juice in embodiment 4；

Fig. 6 is the dynamic change figure of sIgA antibody level in mouse lung in embodiment 4；

Fig. 7 is the dynamic change figure of IgG antibody level in mice serum in embodiment 4；

Fig. 8 is the dynamic change figure of IgA antibody level in mice serum in embodiment 4；

It is the Flow cytometry figure of CD4+, CD8+T lymphocyte in mouse peripheral blood in Fig. 9 embodiment 4；

It is the accounting figure of CD4+, CD8+T lymphocyte in mouse peripheral blood that Figure 10, which is in embodiment 4,；

Figure 11 is mouse splenic T lymphproliferation response figure in embodiment 4；

Figure 12 is the dynamic change figure of IL-5 level in mice serum in embodiment 4；

Figure 13 is the dynamic change figure of IFN- level in mice serum in embodiment 4；

Figure 14 is the electron microscope that destination protein TB1 and TB1-Co1 enter mouse PP knot in embodiment 4；Wherein a.TB1 is through connecing PPs electron microscope after kind mouse when 20min；PPs electron microscope after b.TB1-Co1 Mice Inoculated when 20min；C.TB1 is through being inoculated with PPs electron microscope after mouse when 40min；PPs electron microscope after d.TB1-Co1 Mice Inoculated when 40min；E.TB1-Co1 inoculation PPs electron microscope after mouse when 60min, each scale bar represent 50 μm.

Specific embodiment

The present invention provides a kind of foot-and-mouth disease virus antigen (recombinant protein), the amino acid sequence of the antigen such as SEQ ID Shown in NO:1, specifically: MGMKKKIISAILMSTVILSAAAPLSGVYAISISEIGKVIVKHIEGIFLLGGGGSGG GGSE TQVQRRYHTDVGFLMDRFVQIKPVGPTHVIDLMQTHQHGGGGGSGGGGSQNRRGDLGPLAARLAAQLPASGGGGSG GGGSHKQKIIAPAKQLLGGGGSGGGGSETQVQRRHHTDVSFIMDRFVQIKPVSPTHVIDLMQTHQHGGGGGSGGGG SATRRGDLGSLAARLAAQLPASGGGGSGGGGSATRRGDLGSLAARLAAQLPASGGGGSGGGGSETQVQRRQHTNVG FIMDRFVKIPSQSPTHVIDLMQTHQHGGGGGSGGGGSNAGRRGDLGSLAARVAAQLPAGGGGSGGGGSRHKQRIIA PAKQLGGGGSGGGGSAAIEFFEGMVHDSIKGGGGSGGGGSSFHQLPARSPLP；The molecule of the foot-and-mouth disease virus antigen Amount is about 40kD；The foot-and-mouth disease virus antigen has preferable immunogenicity.

The present invention also provides a kind of gene TB1-Co1 of foot-and-mouth disease virus antigen described in expression above scheme, the bases The nucleotide sequence of cause is as shown in SEQ ID NO:2, overall length 1256bp, specially (5 ' -3 '): CCATGGGTATGAAAAAG AAAATTATTTCAGCAATTCTTATGTCTACAGTTATTTTAAGTGCTGCAGCTCCACTTTCAGGTGTTTATGCTATTA GTATTTCAGAAATTAAAGGTGTTATTGTTCATAAAATTGAAACAATTCTTTTTGGAGGAGGTGGATCAGGTGGAGG TGGATCTGAAACACAAGTTCAACGTAGATATCATACTGATGTTGGATTTTTAATGGATCGTTTTGTTCAAATTAAA CCAGTTGGACCTACACATGTTATTGATCTTATGCAAACTCATCAACATGGTGGAGGTGGAGGTAGTGGAGGTGGAG GTTCACAAAATCGTAGAGGAGATTTAGGTCCATTAGCAGCTAGATTAGCAGCTCAATTACCTGCATCAGGTGGAGG TGGAAGTGGTGGTGGAGGTTCACATAAACAAAAAATTATTGCACCTGCTAAACAACTTTTGGGTGGTGGAGGTTCT GGAGGTGGAGGTAGTGAAACTCAAGTACAACGTAGACATCATACTGATGTTTCTTTTATTATGGATAGATTTGTAC AAATTAAACCAGTTAGTCCAACTCATGTAATTGATTTAATGCAAACACATCAACACGGTGGAGGAGGAGGTTCTGG AGGAGGTGGATCTGCAACAAGACGTGGAGATTTAGGTTCATTAGCAGCTCGTTTAGCAGCTCAATTACCAGCAAGT GGAGGTGGAGGATCTGGTGGAGGTGGATCTGCTACAAGAAGAGGAGATTTAGGTTCTTTAGCTGCACGATTGGCAG CTCAATTACCTGCTAGTGGAGGTGGAGGAAGTGGAGGTGGTGGTTCAGAAACTCAAGTACAAAGAAGACAACATAC TAATGTTGGTTTTATTATGGATCGTTTTGTTAAAATTCCATCTCAAAGTCCAACTCATGTTATTGATTTAATGCAG ACACATCAACACGGAGGTGGAGGTGGTTCAGGTGGTGGAGGTTCAAATGCAGGACGTCGTGGTGATTTAGGAAGTT TAGCAGCTAGAGTTGCAGCTCAATTACCAGCTGGTGGAGGTGGTTCAGGAGGTGGAGGTAGTCGTCATAAACAACG TATTATTGCTCCTGCTAAACAATTGGGAGGTGGAGGATCTGGAGGAGGTGGTTCTGCAGCTATTGAATTTTTCGAA GGAATGGTTCATGATAGTATTAAAGGAGGTGGAGGATCAGGTGGAGGTGGTTCATCTTTTCATCAATTACCAGCTA GATCTCCATTACCTTAGAAGCTT。

In the present invention, the gene TB1-Co1 is by having merged M cell in the C-terminal of aftosa multi-epitope TB1 sequence The sequence of targeting ligand Co1 obtains；The TB1 sequence is by connecting several multi-epitope genes according to concatenated strategy is repeated To obtaining together；The nucleotide sequence of the TB1 sequence is as shown in SEQ ID NO:3, overall length 1190bp, specially (5 '- 3 '): CCATGGGTATGAAAAAGAAAATTATTTCAGCAATTCTTATGTCTACAGTTATTTTA AGTGCTGCAGCTCCAC TTTCAGGTGTTTATGCTATTAGTATTTCAGAAATTAAAGGTGTTATTGTTCATAAAATTGAAACAATTCTTTTTGG AGGAGGTGGATCAGGTGGAGGTGGATCTGAAACACAAGTTCAACGTAGATATCATACTGATGTTGGATTTTTAATG GATCGTTTTGTTCAAATTAAACCAGTTGGACCTACACATGTTATTGATCTTATGCAAACTCATCAACATGGTGGAG GTGGAGGTAGTGGAGGTGGAGGTTCACAAAATCGTAGAGGAGATTTAGGTCCATTAGCAGCTAGATTAGCAGCTCA ATTACCTGCATCAGGTGGAGGTGGAAGTGGTGGTGGAGGTTCACATAAACAAAAAATTATTGCACCTGCTAAACAA CTTTTGGGTGGTGGAGGTTCTGGAGGTGGAGGTAGTGAAACTCAAGTACAACGTAGACATCATACTGATGTTTCTT TTATTATGGATAGATTTGTACAAATTAAACCAGTTAGTCCAACTCATGTAATTGATTTAATGCAAACACATCAACA CGGTGGAGGAGGAGGTTCTGGAGGAGGTGGATCTGCAACAAGACGTGGAGATTTAGGTTCATTAGCAGCTCGTTTA GCAGCTCAATTACCAGCAAGTGGAGGTGGAGGATCTGGTGGAGGTGGATCTGCTACAAGAAGAGGAGATTTAGGTT CTTTAGCTGCACGATTGGCAGCTCAATTACCTGCTAGTGGAGGTGGAGGAAGTGGAGGTGGTGGTTCAGAAACTCA AGTACAAAGAAGACAACATACTAATGTTGGTTTTATTATGGATCGTTTTGTTAAAATTCCATCTCAAAGTCCAACT CATGTTATTGATTTAATGCAGACACATCAACACGGAGGTGGAGGTGGTTCAGGTGGTGGAGGTTCAAATGCAGGAC GTCGTGGTGATTTAGGAAGTTTAGCAGCTAGAGTTGCAGCTCAATTACCAGCTGGTGGAGGTGGTTCAGGAGGTGG AGGTAGTCGTCATAAACAACGTATTATTGCTCCTGCTAAACAATTGGGAGGTGGAGGATCTGGAGGAGGTGGTTCT GCAGCTATTGAATTTTTCGAAGGAATGGTTCATGATAGTATTAAATAGAAGCTT；The albumen of the TB1 sequential coding The amino acid sequence of matter as shown in SEQ ID NO:4, specifically: MGMKKKIISAILMSTVILSAAAPLSGVYAISISEIGK VIVKHIEGIFLLGGGGSGGGGSETQVQRRYHTDVGFLMDRFVQIKPVGPTHVIDLMQTHQHGGGGGSGGGGSQNRR GDLGPLAARLAAQLPASGGGGSGGGGSHKQKIIAPAKQLLGGGGSGGGGSETQVQRRHHTDVSFIMDRFVQIKPVS PTHVIDLMQTHQHGGGGGSGGGGSATRRGDLGSLAARLAAQLPASGGGGSGGGGSATRRGDLGSLAARLAAQLPAS GGGGSGGGGSETQVQRRQHTNVGFIMDRFVKIPSQSPTHVIDLMQTHQHGGGGGSGGGGSNAGRRGDLGSLAARVA AQLPAGGGGSGGGGSRHKQRIIAPAKQLGGGGSGGGGSAAIEFFEGMVHDSIK；TB1 the sequence (+generation specific as follows Table Linker:GGGGSGGGGS is that structure is simple or the amino acid with specific function, such as shown in SEQ ID NO:5 Gly, Ser, Pro, Ala and Thr, especially Gly and Ser are the most commonly used, they can provide enough spaces and flexibility): TB1 { Usp45 signal peptide/universal helper T lymphocyte epitope+FMDV/A/MM [VP1 (21~60aa)+VP1 (140~160aa) + VP1 (200~212aa)]+FMDV/A/WHHP [VP1 (21~60aa)+VP1 (140~160aa)+VP1 (140~160aa)]+ FMDV/A/AF72 [VP1 (21~60aa)+VP1 (140~160aa)+VP1 (200~212aa)]+3A (21~35aa) }.

In the present invention, the gene TB1-Co1 it is specific as follows (+represent Linker:GGGGSGGGGS, such as SEQ ID NO:4 It is shown): TB1-Co1 { Usp45 signal peptide/universal helper T lymphocyte epitope+FMDV/A/MM [VP1 (21~60aa)+VP1 (140~160aa)+VP1 (200~212aa)]+FMDV/A/WHHP [VP1 (21~60aa)+VP1 (140~160aa)+VP1 (140~160aa)]+FMDV/A/AF72 [VP1 (21~60aa)+VP1 (140~160aa)+VP1 (200~212aa)]+3A (21~35aa)+Co1 }.Gene TB1-Co1 of the present invention is excellent to fusion progress according to lactic acid bacteria codon preference Change obtains；In specific implementation process of the present invention, the gene TB1-Co1 is by Nanjing Jin Sirui Bioisystech Co., Ltd full genome Synthesis.

In the present invention, the targeting ligand Co1 is screened from phage display library, by with complement 5a by The interaction of body.

In the present invention, the foot and mouth disease virus is preferably foot and mouth disease A-type virus.

The present invention also provides a kind of recombinant vectors comprising gene TB1-Co1 described in above scheme；The recombinant vector Preferably using plasmid pNZ8148 as initial carrier；The gene TB1-Co1 is preferably inserted in the Nco on plasmid pNZ8148 Between I and Hind III；The present invention is not particularly limited the method for building recombinant vector, using conventional method in that art.

The present invention also provides a kind of Recombinant Lactococcus lactis comprising recombinant vector described in above scheme；The recombination cream The original strain of yogurt coccus is preferably Lactococcus lactis NZ9000；The Recombinant Lactococcus lactis preferably uses following methods It prepares: the recombinant vector being imported into Lactococcus lactis, obtains Recombinant Lactococcus lactis；The mode of the importing is preferred For electrotransformation；The voltage of the electrotransformation is preferably 2500V, and capacitor is preferably 25 μ F, and resistance is preferably 200, and the burst length is excellent It is selected as 1.5~5ms.

After obtaining Recombinant Lactococcus lactis, currently preferred further includes being inoculated in Recombinant Lactococcus lactis containing 10 μ g/ In the GM17 fluid nutrient medium of mL chloramphenicol, 30 DEG C of cultures to OD value are 0.4, and with concentration for 5ng/mL streptococcus lactis peptide It (Nisin) is inducer, inducing expression 4h obtains foot-and-mouth disease virus antigen.

Lactococcus lactis expression system (NICE) used in the present invention has lot of advantages: a, Nisin are food-grade corrosion-resistants Agent, minimal amount of Nisin can induce the great expression of foreign protein, and Lactococcus lactis has immune defense function to it, Growth will not be suppressed；B, expression regulation system is harsh, and destination protein is in undetectable level when no inducer, does not consume Or the few cellular resources of consumption and the normal growth for not influencing bacterium, this characteristic also help the expression of toxic protein；c, Protein expression level can be controlled by the amount of inducer, inducer concentration and protein yield linear pass in a certain range System；D, the foreign protein of expression is soluble, no inclusion body；E, without gemma etc..Therefore we can obtain the outer of solubility Source protein expression product directly can carry out oral immunity with Recombinant Lactococcus lactis, thus eliminate conventional prokaryotic expression Complicated purification process and renaturation process after system expression.

Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood For limiting the scope of the present invention.

The acquisition of 1 TB1 and TB1-Co1 gene of embodiment

1 materials and methods

1.1 materials and source

Lactococcus lactis NZ9000 and expression plasmid pNZ8148 are purchased from German MoBiTec, competent E.coli Top10 and cloning vector pUC57 are purchased from Nanjing Genscript Biotechnology Co., Ltd..

1.2 test method

1.2.1 the transformation and synthesis of gene

In eukaryon or prokaryotes, protein is after translation synthesis, or secrets out of extracellular or storage in the cell.Letter Number peptide is that protein secretion goes out extracellular determinant.Signal peptide is one section of amino acid sequence positioned at secretory protein N-terminal, usually Be made of 21~30 amino acid residues, when synthesis together with secretion peptide transcription and translation, collectively form propetide, propetide is correct After positioning, signal peptide is hydrolyzed by signal peptidase, and propetide is processed into mature protein and realizes transdermal delivery.The choosing of signal peptide One of an important factor for being protein efficient secretion is selected, the not stringent specificity of signal peptide, Usp45 signal peptide is that one kind comes from The endogenous signal peptide of Lactococcus lactis guides the highest signal of heterologous protein secretion efficiency to be currently known in Lactococcus lactis Peptide, common a kind of signal peptide when being foreign protein secreting, expressing in Lactococcus lactis.

In the preparation of polyepitope vaccines, universal helper T lymphocyte epitope (promiscuous T helper is added Cell epitope) it is beneficial to induction body generation immune response, and universal complementary T is added in epitope antigen Cell epitope can overcome the MHC of various animals and Different Individual to limit, and can induce more effective immune response.

By consulting pertinent literature, if choosing representative strain FMDV/A/MM, FMDV/A/WHHP and FMDV/A/AF72 Dry epitope, respectively VP1 (21~60aa), VP1 (140~160aa), VP1 (200~212aa) and 3A (21~35aa), These small peptides are the major antigenic determinant segments for determining various hypotype FMDV immunogenicities, in addition, on the VP1 of the surface FMDV It include tripeptides RGD ring in 140~160 amino acids polypeptide chains, it is amino acid necessary to FMDV absorption infection host cell Sequence, the antibody that subsite induction generates can close the corresponding site on the surface FMDV, block the combination of FMDV and host cell, right It is of great significance in prevention FMDV invasion host cell.

M cell-targeting ligand Co1 is screened from phage display library, by mutual with C5a receptor Effect, by the interaction with C5a receptor, not only transmissibility and the model antigen of ligand binding enter M cell, and And energy challenging antigen specific immune response, the mucosal immune response including systemic immune response and part.In mucosa-immune In, targeting purpose antigen is capable of the intake of enhancement antigen, this is the factor of a significant in the design of polyepitope vaccines.

Based on described above, this experiment has obtained the sequence of multi-epitope TB1 and TB1-Co1, specific as follows (+represent Linker:GGGGSGGGGS):

TB1 { Usp45 signal peptide/universal helper T lymphocyte epitope+FMDV/A/MM [VP1 (21~60aa)+VP1 (140 ~160aa)+VP1 (200~212aa)]+FMDV/A/WHHP [VP1 (21~60aa)+VP1 (140~160aa)+VP1 (140~ 160aa)]+FMDV/A/AF72 [VP1 (21~60aa)+VP1 (140~160aa)+VP1 (200~212aa)]+3A (21~ 35aa)}。

TB1-Co1 { Usp45 signal peptide/universal helper T lymphocyte epitope+FMDV/A/MM [VP1 (21~60aa)+VP1 (140~160aa)+VP1 (200~212aa)]+FMDV/A/WHHP [VP1 (21~60aa)+VP1 (140~160aa)+VP1 (140~160aa)]+FMDV/A/AF72 [VP1 (21~60aa)+VP1 (140~160aa)+VP1 (200~212aa)]+3A (21~35aa)+Co1 }.The fusion is optimized according to lactic acid bacteria codon preference, after transformation sequence by Nanjing gold this Auspicious Bioisystech Co., Ltd's full genome synthesis, is cloned on carrier pUC57, recipient bacterium is Escherichia coli Top10, by what is obtained Two plants of recombinant bacteriums are respectively designated as Top10/pUC57-TB1 and Top10/pUC57-TB1-Co1, and it is public that optimization serves extra large Sani Department carries out sequencing.

2 test results

The base sequence of the TB1-Co1 gene of acquisition is as shown in SEQ ID NO:2.

Sequencing result shows that artificial synthesized TB1 and TB1-Co1 are identical with intrinsic DNA sequence dna, total 1190bp And 1256bp.Link peptide Linker is 10 amino acid sequences (GGGGSGGGGS) of coding, is all structure simply or with special The amino acid of function, if Gly, Ser, Pro, Ala and Thr, especially Gly and Ser are the most commonly used, they can provide enough Space and flexibility.

The acquisition of embodiment 2 recombinant expression carrier pNZ8148-TB1 and pNZ8148-TB1-Co1

1 materials and methods

1.1 materials and source

Restriction enzyme Nco I and Hind III, T4 DNA ligase are purchased from NEB company；DNA Marker DL5, 000 is purchased from Dalian treasured biotech firm；Lysozyme is purchased from U.S. Amresco company；M17 dehydrated medium, agar powder, glucose are equal Purchased from OXOID company, Britain；Small (Gram-negative bacteria) kit and the DNA gel QIAquick Gel Extraction Kit of mentioning of plasmid is purchased from the U.S. OmegaBio-Tek company；Small (gram-positive bacteria) kit that mentions of plasmid is purchased from Beijing Suo Laibao Science and Technology Ltd；Clone Carrier pUC57 is provided by Nanjing Genscript Biotechnology Co., Ltd..

1.2 test method

The recombinant plasmid pUC57-TB1 and pUC57-TB1-Co1 of embodiment 1 respectively with III double digestion of Nco I/Hind after Small fragment, connect that (Fig. 1), conversion, lysozyme splits with the Lactococcus lactis expression vector pNZ8148 large fragment through same double digestion Solution upgrading grain, with electrophoresis, enzymatic cleavage methods identify transformant, obtain positive recombinant plasmid be named as pNZ8148-TB1 and pNZ8148-TB1-Co1。

2 test results

Segment of the recombinant plasmid pNZ8148-TB1 and pNZ8148-TB1-Co1 through double digestion with it is expected that size is consistent, explanation Construction of recombinant plasmid success (Fig. 2).

The preparation and detection of expression A type foot-and-mouth disease virus antigen TB1 and the TB1-Co1 recombinant bacterium of embodiment 3

1 materials and methods

1.1 materials and source

(1) inducer: streptococcus lactis peptide (Nisin) is purchased from Sigma Co., USA；

(2) antibiotic: ammonia benzyl mycin (Amr), chloramphenicol (Chr) are purchased from U.S. Amresco company.

1.2 test method

1.2.1 the screening of electrotransformation and resistant strain of the target gene in Lactococcus lactis NZ9000: after electrotransformation Lactococcus lactis NZ9000 is placed in rejuvenation 3h in 30 DEG C of thermostat water bath, and the bacterium solution of 1mL is then condensed into 200 μ L and is coated on On GM17 solid medium containing 10 μ g/mL chloramphenicol (sealing), 48h is cultivated in 30 DEG C of constant incubators.It will be on plate Bacterium colony is incubated overnight after choosing spot respectively in the GM17 fluid nutrient medium containing 10 μ g/mL chloramphenicol.

1.2.2 influence of the various concentration inducer concentration to target gene inducing expression: by above-mentioned bacterium solution connecing with 1:100 Kind rate is inoculated in the GM17 fluid nutrient medium containing 10 μ g/mL chloramphenicol, and 30 DEG C of cultures to OD value are 0.4 (about 3h), is being trained Support base in be added inducer Nisin, the final concentration of inducer be respectively 1ng/mL, 2ng/mL, 3ng/mL, 4ng/mL, 5ng/mL, 6ng/mL, 4h takes bacterium solution after determining the concentration of inducer.Same Lactococcus lactis of the induction containing empty carrier pNZ8148, as Control group, handles bacterial sediment respectively after induction and culture solution supernatant does electrophoretic analysis, is obtained most by Westernblot Good inducer concentration.

1.2.3 the SDS-PAGE analysis of expression product: bacterial sediment is collected.It is washed 3 times with the PBS of pre-cooling, use is suitable PBS carries out ultrasonication, power 250W after bacterial sediment is resuspended, and each cycle operation 2 seconds is spaced 2 seconds, total 30min.Take 30 μ L is crushed liquid and 10 μ L4 × SDS sample-loading buffers is added, and piping and druming mixes.Boiling water bath 10min, after cooling, 4 DEG C, 12,000 × g, from Heart 5min takes supernatant 10ul loading to carry out SDS-PAGE electrophoretic analysis.

1.2.4 the Western blot detection of Recombinant Lactococcus lactis: the recombination lactic acid milk-globule for taking 10mL to induce through Nisin Bacterium ultrasonication supernatant, is added the sample-loading buffer of certain volume, boils 10min, routinely method electrophoresis, transfer, closing, drift It washes, FMD positive Swine serum is incubated at room temperature 3h, the anti-pig IgG room temperature vibration of the goat marked after rinsing with horseradish peroxidase (HRP) It swings and is incubated for 2h, ECL colour developing is carried out after rinsing.

2 test results

The acquisition of 2.1 Recombinant Lactococcus lactis: the bacterium colony of the chief is cultivated in liquid medium after electrotransformation coated plate, through mentioning Double digestion after plasmid is taken to identify that stripe size is suitable；Experiments have shown that the voltage of electrotransformation be 2500V, 25 μ F of capacitor, resistance 200, Burst length is that 1.5~5ms parameter is most suitable, electrotransformation efficiency highest.

Influence of the 2.2 various concentration inducer concentration to target gene inducing expression: the kind daughter bacteria of activation is with 1% rate of vaccination It is inoculated in the GM17 fluid nutrient medium containing 10 μ g/mL chloramphenicol, 30 DEG C of cultures to OD value are 0.4, are added lure in the medium Lead object Nisin, inducer concentration induction expression protein amount highest in 5ng/mL.

2.3 soluble expression products: the thallus supernatant after ultrasonication passes through SDS-PAGE electrophoresis and Western Blot detects that destination protein is expressed, and Fig. 3 preliminary proof destination protein can express and be that (wherein, M is egg to soluble protein White matter molecular mass standard；1: the empty vector control of induction；2: the NZ9000/pNZ8148-TB1 not induced；3: not inducing NZ9000/pNZ8148-TB1-Co1；4~5: the NZ9000/pNZ8148-TB1 of induction；6~7: the NZ9000/ of induction pNZ8148-TB1-Co1).Complicated purification process and renaturation mistake after thus eliminating conventional prokaryotic expression Journey, test are more preferably to utilize the system table by the exploration of the ideal expression condition to Lactococcus lactis expression system (NICE) It lays a good foundation up to destination protein.

The Western blot of 2.4 Recombinant Lactococcus lactis is detected: the thallus supernatant of Recombinant Lactococcus lactis through electrophoresis and After transferring film, as the result is shown: expression albumen can identify that the recombinant bacterial strain through inducing is about by foot and mouth disease virus positive Swine serum antibody Visible apparent specific immune response band at 40kD, and the empty carrier bacterial strain not induced has very faint blanking bar (figure 4).Illustration purpose gene has been expressed in Recombinant Lactococcus lactis, and expressed albumen has reactionogenicity.

4 animal immune experiment of embodiment

1 materials and methods

1.1 materials and source

The Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 of induction, the Recombinant Lactococcus lactis NZ9000/ of induction PNZ8148-TB1-Co1 and Lactococcus lactis NZ9000/pNZ8148；Test is Balb/c female mice with mouse, 137 Only, 7 week old, weight are purchased from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's Experimental Animal Center in 18~20g or so；A type mouth Fever aphthous inactivated vaccine is purchased from middle peasant Witter Biotechnology Ltd.；Rat anti-mouse CD3 antibody, the RPE label of APC label Rat anti-mouse CD4 antibody, FITC label rat anti-mouse CD8 antibody, FITC label sheep anti-mouse igg be purchased from AbD Serotec company；Erythrocyte cracked liquid is purchased from U.S. BD biosciences company；Lymphocyte separation medium is purchased from the U.S. MPbiomedicals company；ConA, CFSE, DMSO are purchased from sigma company, the U.S.；HANK ' S, RPMI-1640 culture medium；1M HEPES buffer solution be purchased from Gibio company, the U.S.；The rabbit-anti sheep IgG antibody of HRP label, sheep anti-Mouse IgA antibody, HRP mark The goat anti-mouse igg antibody of note, the sheep anti-Mouse IgA antibody of HRP label are purchased from abcam company, the U.S.；Paraformaldehyde is purchased from Sinopharm Chemical Reagent Co., Ltd.；DAPI is purchased from biology Co., Ltd, Beijing Zhong Shan Golden Bridge.

1.2 test method

1.2.1 animal packet and immunity inoculation

Balb/c mouse is randomly divided into 5 groups, every group 25, respectively PBS blank control group (A group), Lactococcus lactis NZ9000/pNZ8148 empty carrier negative control group (B group), recombinant bacterial strain NZ9000/pNZ8148-TB1 group (C group), recombinant bacterium Strain NZ9000/pNZ8148-TB1-Co1 group (D group), A type FMDV inactivated vaccine positive controls (E group).Preceding 4 groups of mouse carry out Intragastric, immunization interval are 7 days, each continuous immunity 3 days, 1 time a day.A type FMD inactivated vaccine positive controls mouse It is immune using the method for intramuscular injection on the inside of hind leg, it is immunized when head exempts from once, is then immunized 1 time again after 7 days.In the test It, need to 6h fasting for solids and liquids to experimental animal before beginning.Specific grouping is shown in Table 1.It is immunized altogether three times, each continuous immunity three days, every two The secondary immunization interval time is 7d, and immunization time point is respectively 1/2/3d；11/12/13d and 21/22/23d.

1 animal immune of table is grouped situation

1.2.2 in mucous membrane sample (intestinal juice and lung liquid) sIgA detection

Intragastric administration on mice inoculation after, from five groups of mouse every group randomly select different time sections (10d, 20d, 30d, 37d, 44d, 51d) each 3 intestines mucus for acquiring immune mouse of mouse and lung liquid sample to detect sIgA level.Method is as follows:

(1) antigen coat: according to the ratio antigen coat diluted A type FMDV inactivation antigen of 1:20, elisa plate The antigenic dilution of 100 μ L is added in every hole, and 4 DEG C of coatings are incubated overnight.

(2) it washs: taking out coated elisa plate overnight, to falling coating buffer and pat dry, the PBST that 200 μ L are added in every hole exists Oscillation washing 5min on shaking table, outwells to pat dry after PBST and continuously adds PBST board-washing 5 times.

(3) close: with containing 5% skimmed milk power PBST, every hole is added 200 μ L, is placed in 37 DEG C of insulating boxs and closes 2h, closes 5 times are washed with PBST after the completion and are patted dry.

(4) loading: the mouse measuring samples of 100 μ L are added in every hole, and each sample setting multiple holes compare, 37 DEG C of incubation 1h, 5 times are washed with PBST and are patted dry.

(5) be incubated for secondary antibody: the sheep anti-Mouse IgA secondary antibody (1:10,000 dilution) of 100 μ L HRP label is added in every hole, is placed in It is incubated for 1h in 37 DEG C of constant incubators, wash 5 times with PBST and is patted dry.

(6) develop the color: the TMB developing solution of 100 μ L, 37 DEG C of incubation 15min are added in every hole；

(7) it terminates and saves: terminating reaction to the terminate liquid that 50 μ L are added in every hole rapidly, terminate in reaction 15min in enzyme The OD value under 450nm wavelength is read on mark instrument and saves result.

1.2.3 in blood IgA, IgG detection

Every group randomly select it is immune after experiment mice each 3 of different time sections (10d, 20d, 30d, 37d, 44d, 51d) Orbital vein blood sampling is carried out, the method for detecting IgA is same as above.IgG detection method is as follows:

(1) antigen coat: according to the ratio antigen coat diluted A type FMDV inactivation antigen of 1:20,96 orifice plates The antigenic dilution of 100 μ L is added in every hole, and 4 DEG C of coatings are incubated overnight.

(2) it washs: taking out coated 96 orifice plate overnight, to falling coating buffer and pat dry, the PBST board-washing of 200 μ L is added in every hole It 5 times and pats dry.

(4) loading: the mouse blood serum sample to be checked (1:50 dilution) of 100 μ L is added in every hole, and multiple holes pair are arranged in each sample According to 37 DEG C of incubation 1h wash 5 times with PBST and pat dry.

(5) be incubated for secondary antibody: the rabbit anti-mouse IgG secondary antibody (1:1,000 dilution) of 100 μ L HRP label is added in every hole, is placed in It is incubated for 1h in 37 DEG C of constant incubators, wash 5 times with PBST and is patted dry.

(7) it terminates and saves: terminating reaction to the terminate liquid that 50 μ L are added in every hole rapidly, terminate in reaction 15min in enzyme Mark the OD value read under 450nm wavelength on instrument.

1.2.4 the detection of peripheral blood CD4+, CD8+T cell subsets

Acquisition head exempts from the 500 μ L of anticoagulation of rear 30d mouse, carries out the detection of CD4+, CD8+T lymphocyte subgroup, tool Steps are as follows for gymnastics work:

(1) mouse anticoagulation is acquired by eyeball veniplex, the ratio of 1:1 and Sample dilution mix by volume.

(2) centrifuge tube is taken, the separating liquid with sample equivalent after dilution is first added, after carefully drawing dilution with suction pipe Blood sample, slowly soft to be added on separating liquid liquid level, 4 DEG C of 500 × g are centrifuged 30min.

(3) after being centrifuged, centrifuge tube is divided into plasma layer, buffy coat, separation liquid layer and red blood cell layer from top to bottom, The lymphocyte of the second layer is carefully drawn with suction pipe into the centrifuge tube marked, and the cleaning solution of 500 μ L is added and blows and beats mixing, 4 DEG C of 250 × g are centrifuged 10min, and light and slow discards supernatant.

(4) centrifuge washing cell is resuspended with the cleaning solution of 500 μ L, 4 DEG C of 250 × g are centrifuged 10min, and light and slow discards supernatant.

(5) step (4) are repeated.

(6) 100 μ L PBS (containing 0.5%FBS) are added, cell precipitation is resuspended, fluorescence antibody (4 is incubated under conditions of being protected from light DEG C 30min): it is respectively the rat anti-mouse CD3 antibody of 10 μ LAPC label, the rat anti-mouse CD8 of 10 μ L FITC label is anti- The rat anti-mouse CD4 antibody of body and 10 μ LRPE label, is gently vortexed and mixes.Blank control is set.

(7) centrifuge tube is filled it up with the PBS containing 0.5%FBS, 4 DEG C of 250 × g are centrifuged 10min, discard supernatant.

(8) 100 μ L PBS (containing 0.5%FBS) are added, cell precipitation is resuspended, after the filtering of 300 mesh nylon screens, use stream Formula cell instrument is detected.

1.2.5 spleen lymphocyte proliferation is tested

30d mouse is put to death after head is exempted from, and in 75% alcohol after of short duration soaking disinfection, aseptically dissects mouse And after taking its spleen, shredded after being rinsed with suitable RPMI-1640 culture medium (FBS, 1% dual anti-containing 10%), then It is placed on sieve, is gently ground mouse spleen with the piston in asepsis injector.RPMI- is constantly added between during the grinding process The total 5mL of cell suspending liquid is made in Tissue Culture Dish in 1640 culture mediums.The mice spleen of 5mL is added into the centrifuge tube of 15mL Dirty lymphocyte separation medium, then 5mL cell suspending liquid is slowly gently added to lymphocyte separation medium along inclined tube wall On, 4 DEG C of 500 × g are centrifuged 30min.After absorption tunica albuginea confluent monolayer cells (spleen lymphocyte layer) careful along tube wall, and shift In the 15mL centrifuge tube sterile to another, the cleaning solution of 5mL is added and blows and beats mixing, 4 DEG C of 500 × g are centrifuged 10min, light and slow Discard supernatant, be repeated 3 times.

Spleen lymphocyte precipitating is resuspended with RPMI-1640 culture medium, adjustment cell number is 1.0 × 10⁷A/mL is added Into 96 porocyte culture plates, every 100 μ L of hole.Every group is set up three experimental groups, i.e. negative control group: 100 μ L are only added in every hole RPMI-1640 culture medium；Positive controls: (RPMI-1640 culture medium is diluted to end to the ConA solution of 100 μ L of every hole addition Concentration is 10 μ g/mL)；Antigenic stimulus group: A type FMDV inactivation antigen (the RPMI-1640 culture medium dilution of 100 μ L is added in every hole To final concentration of 10 μ g/mL), it is placed in 37 DEG C, 5%CO₂72h is cultivated in cell constant temperature incubator, it is molten that 10 μ L MTS are added in every hole Liquid reads the absorption photometric value at 490nm in microplate reader.

1.2.6 the detection of cell factor

By the cell factor ELISA kit of the enzyme-linked commercialization in Shanghai detect different time sections (10d, 20d, 30d, 37d, 44d and 51d) cell factor (IFN-γ, IL-5) in serum mass concentration.Specific step is as follows:

(1) sample-adding of standard items: setting standard sample wells and sample aperture, standard sample wells respectively add 50 μ of standard items of various concentration L。

(2) be loaded: set respectively blank well (sample and enzyme marking reagent is not added in blank control wells, remaining each step operation is identical), Sample to be tested hole.First add 40 μ L of sample diluting liquid in sample to be tested hole on enzyme mark coating plate, then adds 10 μ L of sample to be tested again (the final dilution of sample is 5 times).Sample is added on ELISA Plate hole bottom by sample-adding, is not touched hole wall as far as possible, is shaked gently mixing.

(3) enzyme: every hole is added 100 μ L of enzyme marking reagent, except blank well.

(4) it incubates: using 37 DEG C of incubation 60min of sealing plate film sealing plate postposition.

(5) match liquid: will be spare after 20 times of concentrated cleaning solutions, 20 times of distilled water dilutions.

(6) it washs: carefully taking sealing plate film off, discard liquid, pat dry, cleaning solution is filled it up in every hole, is discarded after standing 1min, such as This is repeated 5 times, and pats dry.

(7) develop the color: 50 μ L color developing agent A are first added in every hole, add 50 μ L color developing agent B, and gently oscillation mixes, and 37 DEG C are protected from light Develop the color 15min.

(8) terminate: every hole adds 50 μ L of terminate liquid, terminates reaction, and the color in well becomes yellow from blue at once at this time Color.

(9) it measures: being returned to zero with blank well, 450nm wavelength sequentially measures the OD value in each hole, after measuring Ying Jia terminate liquid It is carried out within 15min.

1.2.7 the compliance test result test of M cell is targeted

12 mouse are randomly divided into two groups, every group 6, one group is used as negative control, the recombination cream induced with inducer Every stomach-filling of yogurt coccus NZ9000/pNZ8148-TB1 bacterium solution (109CFU/mL) is inoculated with 200 μ L, and another group is then inoculated with inducer Every stomach-filling of Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1-Co1 bacterium solution (109CFU/mL) of induction is inoculated with 200 μ L.From Inoculation starts every 20min after randomly selecting 2 mouse execution in each group, and fresh small intestine is taken to do following processing:

(1) fixed: fresh small intestine being cut into small pieces, 4% paraformaldehyde is fixed on and stays overnight.Substance in solidified structure Ingredient keeps structure when its living body as far as possible.It can make the hardening of tissue simultaneously, be conducive to the progress of slice.

(2) be dehydrated: the sharply contraction in order to reduce organization material, should use from low concentration to high concentration be incremented by sequence into 70%, 85%, 95% is passed through until absolute alcohol (dehydrated alcohol), dewatering time is respectively 3h, 2h and 1h.Tissue material after fixation Material need to remove the fixer and its crystalline deposit stayed within the organization, otherwise will affect the dyeing effect in later period.

(3) transparent: clarifier, which is added, dimethylbenzene 10min, and dimethylbenzene is the solvent of paraffin.Absolute alcohol cannot be with paraffin phase It is molten, it also needs to replace out the alcohol in tissue with the solvent that can be mixed with alcohol and paraffin.Material block claims in clarifier dipping process It is transparent.

(4) equivalent mixed liquor of paraffin and dimethylbenzene that organization material block is placed on fusing first waxdip: is impregnated 1h.First move back Enter in the paraffin liquid of 2 fusings and impregnates 1h.

(5) it embeds: its bottom being attached at rapidly in embedded box with a little hot wax liquid, is subsequently placed in quick-frozen, allow stone Wax is fixed.

(6) be sliced: slicer it is sharp keen whether, wax stone hardness whether suitably all directly affect chipping qualities, can be by wax stone As for change wax stone hardness in cold water.Normal slice, which gently holds in the palm to put down gently with a thickness of 4 μm, with writing brush, opens up piece in 37 degree of water-baths.

(7) patch and roasting piece: generally using thermostat water bath, and temperature is controlled at 37 DEG C, is conducive to the expansion of paraffin section, The slice posted is placed in 60 DEG C of insulating boxs 2 hours dry.

(8) slice dewaxing and aquation: the slice after dry, which is placed in dimethylbenzene, carries out dewaxing 10min, then successively uses The sequence successively decreased from high concentration to low concentration carries out carrying out aquation through absolute alcohol, 95%, 85%, 70%.

(9) it dyes: the slice after dewaxing is put into staining jar；PBS is washed 3 times, each 5min；Slice is immersed into 5% rupture of membranes Agent, soaking at room temperature 10min；PBS is washed 3 times, each 5min；10% lowlenthal serum confining liquid, room temperature closing slice 1h is added dropwise；Simultaneously It is incubated for primary antibody, 4 DEG C overnight；PBS is washed 3 times, each 5min；The IgG secondary antibody of FITC label, 37 DEG C of 1h are added dropwise；DAPI, room temperature is added dropwise It is incubated for 10min；PBS is washed 3 times, each 5min.Use anti-fluorescence decay mountant mounting.

(10) photo acquisition: being scanned using laser confocal microscope, and light source uses 488nm and 561nm wavelength respectively Laser excitation green and red fluorescence, Image Acquisition carried out to slice, every slice prior to 100 times under observe whole groups It knits, then chooses 3 visuals field and acquire 200 times of micro-images.

2. test result

The testing result of (intestinal juice and lung liquid) sIgA in 2.1 mucous membrane samples

Testing result referring to figs. 5 and 6, as the result is shown: the level of PBS group kept stable；Lactococcus lactis NZ9000/pNZ8148 group is slightly higher compared with the value of PBS group, illustrates NZ9000/pNZ8148 as the live vector of antigen submission to mouse Mucosal immune response have certain promotion and humidification；Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 group and again Group Lactococcus lactis NZ9000/pNZ8148-TB1-Co1 group all lures that the mucosal immune system of mouse generates a large amount of sIgA into, but From data it can be seen that as time increases, the level of antibody sIgA is continuously increased, reaches peak value in 30d, illustrate multilist Position TB1 can induce stronger mucosa-immune.Pass through difference analysis, Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1-Co1 Group induction antibody level be substantially higher than what Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 was organized, difference it is extremely significant (P < 0.01).Therefore can tentatively illustrate that the presence of targeting proteins Co1 can increase intake of the body to destination protein, so that excitation is more Strong mucosal immune response；The numerical value of FMD inactivated vaccine group changes less in entire experimental period, illustrates inactivated vaccine hardly Effective mucosal immune response can be induced.Because sIgA is the important symbol of mucosa-immune, the level of detection sIgA has Important meaning.

The testing result of IgG, IgA in 2.2 blood

The experimental result of IgG is such as shown in (Fig. 7) in serum, and Lactococcus lactis NZ9000/pNZ8148 group and PBS group are almost Body cannot be stimulated to generate humoral immune response；Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 group and recombination lactic acid milk-globule Bacterium NZ9000/pNZ8148-TB1-Co1 group all lures that body generates humoral immune response into, and as time increases, IgG's Level also increases, and reaches peak value in 30d, since the latter is increased faster than the former, passes through difference analysis, this two groups of productions Raw antibody level difference is extremely significant (P < 0.01), so can further illustrate that targeting proteins Co1 can increase body to purpose The intake and presentation of albumen, induction body generate significantly more efficient body fluid and exempt from response.Although Recombinant Lactococcus lactis NZ9000/ PNZ8148-TB1-Co1 group can induce stronger humoral immune response, but the IgG generated without inactivated vaccine group is horizontal high. The experimental result of IgA is such as shown in (Fig. 8) in serum, the too big work of generation nothing of PBS group and inactivated vaccine group to IgA in blood With；Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 group and Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1-Co1 group All lure that body generates mucosal immune response into, after reaching peak value through difference analysis discovery after being immunized and to 30d gradually It falls after rise, two groups of the antibody level significant difference (P < 0.05) in 44d, 51d, this illustrates that Recombinant Lactococcus lactis can not only Inducing mouse generates mucosal immune response, and can induce body and generate humoral immune response.

The testing result of 2.3 peripheral blood CD4+, CD8+T cell subsets

PBS group is compared with the CD4+ content of Lactococcus lactis NZ9000/pNZ8148 group, difference it is extremely significant (P < 0.0001) find differences significant (P < 0.05), and compared with the CD8+ content of the two, illustrates to answer in humoral immunity and cellular immunity It answers in reaction, Lactococcus lactis NZ9000/pNZ8148 plays important immunological enhancement.With PBS group and Lactococcus lactis NZ9000/pNZ8148 group is compared, Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 group and Recombinant Lactococcus lactis There is different degrees of increase and with recombination lactic acid in CD4+, CD8+T lymphocyte of NZ9000/pNZ8148-TB1-Co1 group The growth highest of galactococcus NZ9000/pNZ8148-TB1-Co1 group, extremely significant (P < 0.001) illustrates more after difference analysis Neoepitope Western has good immunogenicity, and is best with TB1-Co1.Inactivated vaccine can cause stronger humoral immunity to be answered It answers, but apparent cellullar immunologic response cannot be induced.Due to Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 group and again The content of the CD8+T lymphocyte of group Lactococcus lactis NZ9000/pNZ8148-TB1-Co1 group has certain increasing than other groups Width illustrates them also and can induce body generation cellullar immunologic response.In summary experimental result can obtain: Recombinant Lactococcus lactis Can not only inducing mouse body generate humoral immune response, also can induce cellullar immunologic response.From CD4+, CD8+T lymphocyte Percentage composition can be seen that because the content of CD4+T lymphocyte is apparently higher than CD8+T lymphocyte, with lactic acid Galactococcus be live vector mucosal vaccine induce immune response mainly based on humoral immunity (Fig. 9, Figure 10).

2.4 spleen lymphocyte proliferation test results

The proliferative capacity that mouse spleen lymphocyte is surveyed using MTS, by difference analysis, in negative control group (RPMI- 1640 culture mediums) in NZ9000/pNZ8148-TB1 and NZ9000/pNZ8148-TB1-Co1 spleen lymphocyte proliferation without significant Also there was no significant difference for the spleen lymphocyte proliferation of sex differernce, PBS and inactivated vaccine；In antigenic stimulus group, (A type FMDV inactivation is anti- It is former) in NZ9000/pNZ8148-TB1-Co1 spleen lymph of the spleen lymphocyte proliferation obviously than NZ9000/pNZ8148-TB1 it is thin Born of the same parents are proliferated height, and difference is extremely significant (P < 0.01), and the inactivated vaccine group difference that is apparently higher than of NZ9000/pNZ8148-TB1 is extremely shown It writes (P < 0.001)；The NZ9000/pNZ8148-TB1 and NZ9000/pNZ8148-TB1-Co1 in positive controls (ConA) There was no significant difference for spleen lymphocyte proliferation.Illustrate that the mouse spleen lymphocyte after Recombinant Lactococcus lactis stomach-filling is encountering When FMDV have stronger proliferative capacity, and with NZ9000/pNZ8148-TB1-Co1 be it is optimal, this for cause body produce Raw immune response plays a significant role (Figure 11) to resist FMDV.

The testing result of 2.5 cell factors

Mice serum in different time periods is subjected to cell factor quantitative detection, in Figure 12 in 10d after immune There was no significant difference for the IL-5 content and NZ9000/pNZ8148-TB1 of NZ9000/pNZ8148-TB1-Co1, but other times All there were significant differences (P < 0.05) for section；From the above analysis it can be concluded that Recombinant Lactococcus lactis can induce body generation effectively Humoral immune response and with NZ9000/pNZ8148-TB1-Co1 cause effect it is best.

Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 group and Recombinant Lactococcus lactis NZ9000/ as can be known from Fig. 13 The level of the IFN-γ of pNZ8148-TB1-Co1 group reaches highest after head exempts from 30d, gradually decreases later；PBS group and inactivation epidemic disease The horizontal variation of the IFN-γ of seedling group is little；But the level of the IFN-γ of Lactococcus lactis NZ9000/pNZ8148 group has different width The variation of degree, without Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1 group and Recombinant Lactococcus lactis NZ9000/ The variation of pNZ8148-TB1-Co1 group is obvious.Difference analysis can be seen that the NZ9000/ in 20d, 44d and 51d The IFN-γ content of pNZ8148-TB1-Co1 is apparently higher than the content of NZ9000/pNZ8148-TB1, significant difference (P < 0.05)； From above-mentioned analysis it follows that Recombinant Lactococcus lactis NZ9000/pNZ8148-TB1-Co1 can preferably induce machine after stomach-filling Body generates cellullar immunologic response, and and live vector Lactococcus lactis NZ9000/pNZ8148 have to cellular immunity it is certain Booster action.

The compliance test result result of 2.6 targeting M cells

By the M cell in destination protein combination Figure 14 mouse small intestine PP knot, M cell is tentatively probed into destination protein TB1 With the similarities and differences of TB1-Co1 intake degree.The result shows that when destination protein has targeting ligand Co1, it can be seen that red fluorescence and The combination of green fluorescence is more, this is because the M cell-targeting that Co1 is mediated enhances M in conjunction with the C5aR on M cell, with this Intake of the cell to destination protein.By the presentation of M cell, more powerful mucosal immune response and systematicness is finally caused to be exempted from Epidemic disease responsing reaction.In the 1h of inoculation, for Recombinant Lactococcus lactis there is no transfer on a large scale occurs, this stage should be M Cell carries out digestion intake to multi-epitope albumen, to obtain a process of Antigenic Peptide.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120>a kind of foot-and-mouth disease virus antigen, the gene of expression foot-and-mouth disease virus antigen and its recombinant vector and recombination lactic acid cream Coccus

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 416

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

Ile Leu Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala Ile Ser Ile

20 25 30

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

35 40 45

Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Thr Gln Val Gln

50 55 60

Arg Arg Tyr His Thr Asp Val Gly Phe Leu Met Asp Arg Phe Val Gln

65 70 75 80

Ile Lys Pro Val Gly Pro Thr His Val Ile Asp Leu Met Gln Thr His

85 90 95

Gln His Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asn Arg

100 105 110

Arg Gly Asp Leu Gly Pro Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro

115 120 125

Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Lys Gln Lys

130 135 140

Ile Ile Ala Pro Ala Lys Gln Leu Leu Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Glu Thr Gln Val Gln Arg Arg His His Thr Asp Val Ser

165 170 175

Phe Ile Met Asp Arg Phe Val Gln Ile Lys Pro Val Ser Pro Thr His

180 185 190

Val Ile Asp Leu Met Gln Thr His Gln His Gly Gly Gly Gly Gly Ser

195 200 205

Gly Gly Gly Gly Ser Ala Thr Arg Arg Gly Asp Leu Gly Ser Leu Ala

210 215 220

Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Ala Thr Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala

245 250 255

Arg Leu Ala Ala Gln Leu Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly

260 265 270

Gly Gly Ser Glu Thr Gln Val Gln Arg Arg Gln His Thr Asn Val Gly

275 280 285

Phe Ile Met Asp Arg Phe Val Lys Ile Pro Ser Gln Ser Pro Thr His

290 295 300

Val Ile Asp Leu Met Gln Thr His Gln His Gly Gly Gly Gly Gly Ser

305 310 315 320

Gly Gly Gly Gly Ser Asn Ala Gly Arg Arg Gly Asp Leu Gly Ser Leu

325 330 335

Ala Ala Arg Val Ala Ala Gln Leu Pro Ala Gly Gly Gly Gly Ser Gly

340 345 350

Gly Gly Gly Ser Arg His Lys Gln Arg Ile Ile Ala Pro Ala Lys Gln

355 360 365

Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ile Glu Phe

370 375 380

Phe Glu Gly Met Val His Asp Ser Ile Lys Gly Gly Gly Gly Ser Gly

385 390 395 400

Gly Gly Gly Ser Ser Phe His Gln Leu Pro Ala Arg Ser Pro Leu Pro

405 410 415

<210> 2

<211> 1256

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

ccatgggtat gaaaaagaaa attatttcag caattcttat gtctacagtt attttaagtg 60

ctgcagctcc actttcaggt gtttatgcta ttagtatttc agaaattaaa ggtgttattg 120

ttcataaaat tgaaacaatt ctttttggag gaggtggatc aggtggaggt ggatctgaaa 180

cacaagttca acgtagatat catactgatg ttggattttt aatggatcgt tttgttcaaa 240

ttaaaccagt tggacctaca catgttattg atcttatgca aactcatcaa catggtggag 300

gtggaggtag tggaggtgga ggttcacaaa atcgtagagg agatttaggt ccattagcag 360

ctagattagc agctcaatta cctgcatcag gtggaggtgg aagtggtggt ggaggttcac 420

ataaacaaaa aattattgca cctgctaaac aacttttggg tggtggaggt tctggaggtg 480

gaggtagtga aactcaagta caacgtagac atcatactga tgtttctttt attatggata 540

gatttgtaca aattaaacca gttagtccaa ctcatgtaat tgatttaatg caaacacatc 600

aacacggtgg aggaggaggt tctggaggag gtggatctgc aacaagacgt ggagatttag 660

gttcattagc agctcgttta gcagctcaat taccagcaag tggaggtgga ggatctggtg 720

gaggtggatc tgctacaaga agaggagatt taggttcttt agctgcacga ttggcagctc 780

aattacctgc tagtggaggt ggaggaagtg gaggtggtgg ttcagaaact caagtacaaa 840

gaagacaaca tactaatgtt ggttttatta tggatcgttt tgttaaaatt ccatctcaaa 900

gtccaactca tgttattgat ttaatgcaga cacatcaaca cggaggtgga ggtggttcag 960

gtggtggagg ttcaaatgca ggacgtcgtg gtgatttagg aagtttagca gctagagttg 1020

cagctcaatt accagctggt ggaggtggtt caggaggtgg aggtagtcgt cataaacaac 1080

gtattattgc tcctgctaaa caattgggag gtggaggatc tggaggaggt ggttctgcag 1140

ctattgaatt tttcgaagga atggttcatg atagtattaa aggaggtgga ggatcaggtg 1200

gaggtggttc atcttttcat caattaccag ctagatctcc attaccttag aagctt 1256

<210> 3

<211> 1190

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

ccatgggtat gaaaaagaaa attatttcag caattcttat gtctacagtt attttaagtg 60

ctgcagctcc actttcaggt gtttatgcta ttagtatttc agaaattaaa ggtgttattg 120

ttcataaaat tgaaacaatt ctttttggag gaggtggatc aggtggaggt ggatctgaaa 180

cacaagttca acgtagatat catactgatg ttggattttt aatggatcgt tttgttcaaa 240

ttaaaccagt tggacctaca catgttattg atcttatgca aactcatcaa catggtggag 300

gtggaggtag tggaggtgga ggttcacaaa atcgtagagg agatttaggt ccattagcag 360

ctagattagc agctcaatta cctgcatcag gtggaggtgg aagtggtggt ggaggttcac 420

ataaacaaaa aattattgca cctgctaaac aacttttggg tggtggaggt tctggaggtg 480

gaggtagtga aactcaagta caacgtagac atcatactga tgtttctttt attatggata 540

gatttgtaca aattaaacca gttagtccaa ctcatgtaat tgatttaatg caaacacatc 600

aacacggtgg aggaggaggt tctggaggag gtggatctgc aacaagacgt ggagatttag 660

gttcattagc agctcgttta gcagctcaat taccagcaag tggaggtgga ggatctggtg 720

gaggtggatc tgctacaaga agaggagatt taggttcttt agctgcacga ttggcagctc 780

aattacctgc tagtggaggt ggaggaagtg gaggtggtgg ttcagaaact caagtacaaa 840

gaagacaaca tactaatgtt ggttttatta tggatcgttt tgttaaaatt ccatctcaaa 900

gtccaactca tgttattgat ttaatgcaga cacatcaaca cggaggtgga ggtggttcag 960

gtggtggagg ttcaaatgca ggacgtcgtg gtgatttagg aagtttagca gctagagttg 1020

cagctcaatt accagctggt ggaggtggtt caggaggtgg aggtagtcgt cataaacaac 1080

gtattattgc tcctgctaaa caattgggag gtggaggatc tggaggaggt ggttctgcag 1140

ctattgaatt tttcgaagga atggttcatg atagtattaa atagaagctt 1190

<210> 4

<211> 394

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Ile Leu Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala Ile Ser Ile

20 25 30

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

35 40 45

Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Thr Gln Val Gln

50 55 60

Arg Arg Tyr His Thr Asp Val Gly Phe Leu Met Asp Arg Phe Val Gln

65 70 75 80

Ile Lys Pro Val Gly Pro Thr His Val Ile Asp Leu Met Gln Thr His

85 90 95

Gln His Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asn Arg

100 105 110

Arg Gly Asp Leu Gly Pro Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro

115 120 125

Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Lys Gln Lys

130 135 140

Ile Ile Ala Pro Ala Lys Gln Leu Leu Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Glu Thr Gln Val Gln Arg Arg His His Thr Asp Val Ser

165 170 175

Phe Ile Met Asp Arg Phe Val Gln Ile Lys Pro Val Ser Pro Thr His

180 185 190

Val Ile Asp Leu Met Gln Thr His Gln His Gly Gly Gly Gly Gly Ser

195 200 205

Gly Gly Gly Gly Ser Ala Thr Arg Arg Gly Asp Leu Gly Ser Leu Ala

210 215 220

Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Ala Thr Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala

245 250 255

Arg Leu Ala Ala Gln Leu Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly

260 265 270

Gly Gly Ser Glu Thr Gln Val Gln Arg Arg Gln His Thr Asn Val Gly

275 280 285

Phe Ile Met Asp Arg Phe Val Lys Ile Pro Ser Gln Ser Pro Thr His

290 295 300

Val Ile Asp Leu Met Gln Thr His Gln His Gly Gly Gly Gly Gly Ser

305 310 315 320

Gly Gly Gly Gly Ser Asn Ala Gly Arg Arg Gly Asp Leu Gly Ser Leu

325 330 335

Ala Ala Arg Val Ala Ala Gln Leu Pro Ala Gly Gly Gly Gly Ser Gly

340 345 350

Gly Gly Gly Ser Arg His Lys Gln Arg Ile Ile Ala Pro Ala Lys Gln

355 360 365

Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ile Glu Phe

370 375 380

Phe Glu Gly Met Val His Asp Ser Ile Lys

385 390

<210> 5

<211> 10

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

Claims

1. a kind of foot-and-mouth disease virus antigen, the amino acid sequence of the antigen is as shown in SEQ ID NO:1.

2. a kind of gene TB1-Co1 of foot-and-mouth disease virus antigen described in expression claim 1, the nucleotide sequence of the gene is such as Shown in SEQ ID NO:2.

3. gene TB1-Co1 according to claim 2, which is characterized in that the foot and mouth disease virus is foot-and-mouth disease a type disease Poison.

4. a kind of recombinant vector comprising gene TB1-Co1 described in Claims 2 or 3.

5. recombinant vector according to claim 4, which is characterized in that the recombinant vector is using plasmid pNZ8148 as original Beginning carrier.

6. recombinant vector according to claim 5, which is characterized in that the gene TB1-Co1 is inserted in plasmid pNZ8148 On NcoI and Hind III between.

7. a kind of Recombinant Lactococcus lactis comprising recombinant vector described in claim 4~6 any one.

8. Recombinant Lactococcus lactis as claimed in claim 7 is preparing the application in A type aftosa oral vaccine.

9. application of the Recombinant Lactococcus lactis as claimed in claim 7 in A type aftosa mucosa-immune live vector.