CN107177539A - A kind of Schistosoma japonicum and lockjaw bivalent be oral or collunarium vaccine - Google Patents

A kind of Schistosoma japonicum and lockjaw bivalent be oral or collunarium vaccine Download PDF

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CN107177539A
CN107177539A CN201710269904.8A CN201710269904A CN107177539A CN 107177539 A CN107177539 A CN 107177539A CN 201710269904 A CN201710269904 A CN 201710269904A CN 107177539 A CN107177539 A CN 107177539A
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vaccine
gemma
ttfc
sj26gst
expression
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李丽
陆家海
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Sun Yat Sen University
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Abstract

The invention provides a kind of construction method of gemma type probiotics mucosal vaccine and immunity evaluation system, the double swivel base expression vectors and Host Strains of causal organism exogenous antigen can be expressed there is provided a kind of, present invention also offers a kind of amalgamation and expression cell factor IL 2 probiotics vaccine carrier, present invention also offers a kind of Japanese blood fluke vaccine and clostridium tetani bivalent vaccine, and a kind of Schistosoma japonicum univalent vaccine, mucosal delivery carrier of the prebiotic bacteria vaccine using gemma type hay bacillus as oral or collunarium approach.The present invention first organically combines the foreign gene such as Schistosoma japonicum Sj26GST and lockjaw TTFC, cell factor IL 2 and withered grass gemma mucosa immune vaccines carrier three, has complementary advantages.It is model antigen from Sj26GST and TTFC, using gemma vehicle delivery model antigen, the restructuring IL 2 shown by gemma and gemma is used as adjuvant, regulation immunization type and enhancing immune response level.

Description

A kind of Schistosoma japonicum and lockjaw bivalent be oral or collunarium vaccine
Technical field
The present invention relates to a kind of vaccine, and in particular to one kind is using IL-2 as adjuvant, and gemma type probiotics carries for mucosal delivery The bivalent vaccine of body.
Background technology
Using the nanoscale gemma of nano grade biological carrier of new generation, i.e. hay bacillus mouth is researched and developed as mucosal delivery carrier Clothes or collunarium vaccine, it is contemplated that the industrial production of one-month period can meet the demand that people at highest risk is immunized.Given birth to by fermentation technique The gemma vaccine of production, is greatly shortened than traditional chicken embryo vaccine incubation time, it is contemplated that be only conventional method life per dosage unit cost Produce the 1/10 of cost.Therefore, the R＆D cycle is short, production is fast, cost is low, environment interference is strong to external world, easily stored fortune It is defeated, lasting cross-protection, the new generation vaccine with adjuvant effect can be produced, it has also become the whole world prevention of infectious disease control Active demand.These demands can be met by the vaccine constructed by mucosal vaccine delivery vector of bacillus subtilis.
The present invention constructs polyvalent antigen amalgamation and expression system using bacillus subtilis endogenous spore, is inhaled from Japanese blood Worm 26kDa GST (Sj26GST) albumen is model antigen and Tetanus Toxin Fragment C (TTFC) as immunologic adjuvant molecule, Gemma outer surface is shown (with gemma CotC coat protein amalgamation and expression CotC-Sj26GST-TTFC)；And build and gemma appearance Double turns of face displaying IL-2 (with gemma CotB-IL-2 coat protein amalgamation and expression) builds the mode being combined and expressed.Fluorescence immunoassay Learn experiment, laser confocal microscope to detect foreign protein and successfully express in gemma outer surface, also demonstrate polyvalent recombinant The macromolecular structure of antigen does not influence assembling and the sporulation of gemma outer layer albumen, while vaccine can be improved by demonstrating IL-2 Cellular immune level.
The oral or Nasal immunization gemma of synthesis or recombination expression can cause the immune response extensively imitated, but the CD8+T of induction Cellular immune level is relatively low.Therefore the present invention is adjusted immunization type and is strengthened cell and exempted from using interleukin 2 (IL-2) Epidemic disease level.
Interleukins (interleukins, ILs) ILs is between mediated leucocytes and the interphase interaction of other cells Cell factor.The cell factor produced by lymphocyte, monocyte or other non-mononuclearcells, in iuntercellular phase interaction Adjusted with, vaccine immunity, play important regulative in hematopoiesis and inflammatory process.Interleukin 2 (IL-2) is main by CD4+ T cell or CD8+T cells are produced, and effect is played with autocrine and paracrine mode.Principal biological function includes：1. activate CD4+ and CD8+T cells, promote cell factor and produce；2. NK cells propagation, activation are stimulated, induction LAK cells are produced；3. activation is promoted B cell proliferation and generation antibody；4. can activated mononuclear-macrophage.IL-2 is human body self component, can adjust the physiology of body Process and raising immunologic function, very low dosage can be played a role, and evident in efficacy, Small side effects are given birth to using technique for gene engineering The recombinant cytokine IL-2 of production is as biological response modifier (biologicalresponse modifier, BRM) in treatment Infectious disease also achieves certain curative effect, is a kind of brand-new biological agent, it has also become medicine of new generation goes through to produce.
Present invention introduces biological response modifier IL-2, its immunoregulatory adjuvant effect is played, immune response class is adjusted Type, strengthens cellular immune level, to improve the immune protective effect of external source recombinant antigen vaccine.
The Performances of Novel Nano-Porous meter level bio-carrier bacillus subtilis that the present invention is used, belongs to gram-positive bacteria, is caused a disease with non- Property, it is widely distributed, it is typically found in soil, it is " convention security level " that food and drug administration FDA, which ratifies it, (generally recognized as safe, GRAS), is used for food or medicine company frequently as probiotics.Bacillus subtilis exists Scientific research and industrial and agricultural production field are widely used, and with good fermentation basis and production technology, it is in relatively simple training Support in base with regard to very high density can be grown into.Many business enzymes are all the extracellular protein that bacillus produces, such as alphalise starch Enzyme, protease and insecticidal crystal protein of Dipel etc..Bacillus growth is rapid, condition of culture is simple, the heredity back of the body Scape is clearer.The gemma of bacillus is a kind of hypopus (sporulation process shown in Fig. 1), can be protected under the conditions of extraneous resistance The stability of height, including heat endurance are held, rugged environment condition can be resisted.
These characteristics of gemma give the credit to its unique gemma capsid structure, the capsid sandwich construction of gemma kind about more than 70 Capsid protein matter (Cot proteins) constitutes (such as Fig. 2), constitutes inner casing, shell and the outermost cortex crust clothing of gemma Shell.Wherein be located at outermost three capsid proteins CotB, CotC and CotG, be usually used in amalgamation and expression exogenous antigen albumen or Enzyme, is showed in spore surface.
Withered grass gemma has unique immunological characteristic, is often used as the nano grade biological of restructuring or non-recombinant mucosal vaccine Carrier：1. about 0.6-1.0 μm of gemma diameter is oral or Nasal immunization, can be by gut-associated lymphoid in animal intestinal tract or mucous membrane of nasopharynx (GALT) or nasal-associated lymphoid tissue (NALT) identification are organized, immunoadjuvant function is played, activates the associated lymphatic of intestinal mucosa Tissue, strengthens the secretion of secreting type (SIgA) antibody, improves Immune discrimination ability, and induce T, bone-marrow-derived lymphocyte and macrophage Cell factor is produced, systemic immune system is activated by lymphocyte recirculation, so as to strengthen the non-specific and spy of body Specific immunological system.2. gemma has immunoregulatory adjuvant effect, can induce being immunized for the Th1/Th2 mixed types for producing balance Reaction, can not only activate specific humoral immune response, can also activate specific cellullar immunologic response.Gemma capsid is shown System is widely used in expressing the restructuring mucous membrane vaccine research of exogenous antigen albumen；Further, since its bioadhesive, also by with In the research of absorbent-type vaccine carrier.Gemma has immunologic adjuvant effect simultaneously, available for enhancing DNA vaccination, polypeptide vaccine, base Because of engineered protein vaccine, the immune effect of inactivated virus particle vaccine, so as to save the single immunization dosage of foreign protein, simultaneously Improve immune protective effect.Therefore, bacillus subtilis is considered as most promising mucosal vaccine carrier, is widely used In the development of pathogenic microorganism vaccine.
At present, there is not yet the report of Sj26GST-TTFC/IL-2- bacillus subtilises restructuring mucosal vaccine application.If logical Gemma support C otB capsid protein amalgamation and expression cell factor IL-2 are crossed, it is showed in gemma capsid surface, with exogenous two Valency antigen gene merges double expression in spore surface.So, IL-2 immunoregulation effect can be both played, enhancing Th1 is immune should Answer, improve specific C D8+T cellular immune levels, the adjuvant effect of gemma can be played again, strengthen the intensity of immune response.
According to vaccine development technology, vaccine can be divided into traditional vaccine and new generation vaccine.Used in the research of new generation vaccine In, it is found that the vaccine that the antigen only obtained with gene clone technology is prepared is not enough to obtain effective immanoprotection action.Carry The antigen with immanoprotection action can be induced by showing that real effective vaccine depends not only upon to clone, and also depend on structure excellent Carrier system, and research and assess antigen induced immanoprotection action mechanism and approach.In recent years, new generation vaccine is studied Carrier, to obtain safe and stable and effective new generation vaccine as focus of concern.Carrier for preparing new generation vaccine Including recombinant bacteria, recombinant virus, DNA vector, RNA carriers, BMDC, T cell and polypeptide.Novel vaccine carrier is put down The variation of platform also enriches immunisation route so that can produce and hold in infection site there is provided a variety of antigen means of conveyance Long immunity, and preferable immunoprotection type can be induced as needed.Thus, the application of novel antigens submission platform has Induction is hoped extensively to imitate lasting immune protective efficiency, while shortening vaccine development and production time.
Bacillus subtilis has unique advantage as novel vaccine carrier, it is considered to be most promising mucosal vaccine Carrier, and start the development applied to pathogenic microorganism vaccine.Isticato and Mauriello etc. uses Tetanus Toxin Fragment C (TTFC, 51.8kDa) and escherichia coli enterotoxin B subunits (LTB, 12kDa) gene tetC and eltB and encoding B bud Spore capsid protein CotB or CotC Gene Fusion, utilize CotB or CotC promoter expressed fusion proteins.Fusion protein CotB- TTFC, CotC-TTFC and CotC-LTB are in spore surface stably successful expression, and research shows that amalgamation and expression does not influence antigen Protectiveness, do not destroy the characteristic of gemma yet.Recombinant spore oral immunity mouse can cause the system for TTFC or LTB to be exempted from Epidemic disease and local immune response.Vaccine evaluation research shows that after CotB-TTFC gemma oral immunity mouse, serum IgG is significantly higher than Control group, can resist 20 times of TTFC challenge infection.Hinc etc. is used as immunologic adjuvant by amalgamation and expression IL-2 gemma, can adjust The immune response based on Th1 is saved, by recombination, amalgamation and expression or absorption exogenous antigen, specific cellular immunity is produced anti- Should, there is certain immune protective effect to helicobacter pylori.In recent years with the understanding to gemma adhesiveness, withered grass gemma carrier exists There is desk study in influenza and avian influenza vaccine development：Song with inactivate withered grass gemma as mucosal immunity adjuvant and antigen The HA mixing and absorptions of protein carrier and H5N1 virus, show significant immune modulator adjuvant effect, can strengthen immune response water It is flat, and the immune response of the Th1/Th2 mixed types based on Th1 is adjusted, there is protecting effect to avian influenza virus attack. Guangyu Zhao etc. can be produced by force by the M2e albumen of gemma capsid CotB protein fusion expression influenza virus As, oral immunity The lasting specific system of effect is immunized and mucosal immunity, fully against influenza virus H1N1 attack.
Traditional vaccine development technology can not meet the demand of current vaccine, it is necessary to by a variety of new ideas, new skill Art, dynamical vaccine platform are combined with each other and realize target jointly.The present invention passes through model antigen, IL-2 recombinant spores, withered grass The comprehensive study of gemma mucosal vaccine platform, is that Japanese blood fluke vaccine and tetanus vaccine research and development have lasting immunity protection Oral or collunarium vaccine technical support and theoretical foundation are provided.
R＆D cycle is short, and production cost is low, it is easy to which storage transport, immunization wayses are not required to injection, can produce lasting intersection Protecting effect, while activating humoral immunity and cellular immunity, itself has the new generation vaccine of adjuvant effect, it has also become compel in the whole world The demand cut.
The content of the invention
It is of the invention to go back it is an object of the invention to overcome the weak point of prior art presence there is provided a kind of Host Strains There is provided a kind of Schistosoma japonicum and lockjaw bivalent be oral or collunarium vaccine, the bivalent vaccine using gemma type probiotics be viscous Film delivery vector.
To achieve the above object, the technical scheme taken：The invention provides a kind of Host Strains, the Host Strains pass through Following methods are prepared from：
(1) by CotB gene promoter sequences, CotB coded sequences, the nucleotide sequence of connector and IL-2 nucleotides Sequence is consecutively connected to the first expression vector, obtains expressing the first recombinant expression carrier of cotB-linker-IL2 fusion proteins；
(2) the outer single endonuclease digestion of the first recombinant expression carrier Insert Fragment causes the first restructuring table in the step (1) Up to vector linearization, the first bacillus competent cell is then transformed into, screening obtains expression CotB-linker-IL-2 and melted First positive strain of hop protein；
(3) by the nucleotides sequence of CotC gene promoter sequences, CotC coded sequences and Sj26GST-TTFC described above Row are consecutively connected to the second expression vector, obtain expressing the second recombinant expression carrier of CotC-Sj26GST-TTFC fusion proteins；
(4) the outer single endonuclease digestion of the second recombinant expression carrier Insert Fragment causes the second restructuring table in the step (3) Up to vector linearization, the second bacillus competent cell is then transformed into, screening obtains expression CotC-Sj26GST-TTFC and melted Second positive strain of hop protein；
(5) dyeing of the second positive strain of expression CotC-Sj26GST-TTFC fusion proteins in the step (4) is extracted Body, is transformed into sense prepared by the first positive strain of the expression IL-2CotB-linker-IL-2 fusion proteins in the step (2) By state cell, the positive colony of CotB-IL2 fusion proteins and CotC-Sj26GST-TTFC fusion proteins is expressed in screening simultaneously, i.e., Obtain the Host Strains.
Preferably, the amino acid sequence of the connector is as shown in SEQ ID NO.1.
Preferably, in the step (3) Sj26GST-TTFC nucleotide sequence as shown in SEQ ID NO.6
Preferably, the first expression vector is plasmid pDG364 in the step (1), and the second expression is carried in the step (3) Body is plasmid pDG1664.
Preferably, the first bacillus competent cell is that bacillus subtilis PY79 competence is thin in the step (2) The second bacillus competent cell is bacillus subtilis PY79 competent cells in born of the same parents, the step (4).
The invention provides Host Strains described above, Schistosoma japonicum and lockjaw bivalent be oral or collunarium vaccine preparing In purposes.
The invention provides a kind of Schistosoma japonicum and lockjaw bivalent be oral or collunarium vaccine, the Schistosoma japonicum and Lockjaw bivalent is oral or collunarium vaccine contains gemma obtained by Host Strains culture described above.
The present invention is on the probiotic type bacillus subtilis mucosal vaccine platform built, in order to induce with specificity Immune response type based on CD8+T cells, by cell factor IL-2 regulation enhancing Th1 immune responses, so as to improve special The immune response level of property CD8+T cells.Shown by the CotB capsid protein amalgamation and expression cell factors IL-2 of gemma carrier In gemma capsid surface, it is played adjuvant effect, adjust the type of immune response, strengthen cellular immunity, prevent and thoroughly clear Except the pathogenic microorganism of infection body；Using gemma capsid protein CotB amalgamation and expression IL-2, acquisition being capable of efficient activation CD8+T The strain of the gemma with adjuvant effect of cell, makes up the deficiency of simple gene vaccine；Given birth to using immune bioinformatics and structure Thing Technology design fusion mode gene Sj26GST-TTFC, and by capsid protein CotC by Sj26GST-TTFC amalgamation and expressions In the IL-2 gemma strain built, the double swivel base recombinant bacillus gemma epidemic diseases of CotB-IL-2/CotC-Sj26GST-TTFC epitopes are built Miao Zhu.Restructuring bivalent-IL-2- withered grass gemma the vaccine strains of structure, production is fast, and cost is low, can be sprayed by collunarium or nasopharynx viscous Film is immunized, and technology and theoretical foundation have been established to research and develop the bivalent vaccine with lasting immunity protection.
The beneficial effects of the present invention are：
The present invention is based on invention group in immunology, Pathogen Biology, bioinformatics, molecular biology, epidemiology With Research foundation good in terms of nanometer biotechnology, integration project group membership carries in bacillus subtilis mucosal delivery vaccine Body and adjuvant research, model antigen research, bivalent vaccine study the impressive progress obtained, and system is carried out bacillus subtilis and glued The technology of film delivery vector is applied to the basic research of bivalent vaccine.Amalgamation and expression bivalent exogenous antigen basis proposed by the present invention On, by the vaccine research technology and bivalent amalgamation and expression pattern of the gemma mucosal immunity delivery vector of international ripe hay bacillus Antigen gene technology is combined, and quotes cell factor IL-2 as immune modulator adjuvant molecule, is realized and is invaded door in pathogenic microorganism Family is mucocutaneous to wait local generation mucosal immunity, especially further activation system immune response, specific C D8+T cellular immunities Reaction, reaches the effect of Cross immunogenicity.Hay bacillus is easy to genetic modification, and working condition and equipment are simple, gemma vaccine Be adapted to it is safe efficient, economically produce in enormous quantities, also with fabulous heat endurance, long half time is particularly suitable for live conduct The feed addition vaccine of animal, and people at highest risk's collunarium or oral immunity, show good advantage.
With following both sides novelty and advantage：
(1) Sj26GST-TTFC bivalents amalgamation and expression antigen sequence, Cytokine adjuvant IL-2 and the gemma of hay bacillus The joint of the multinomial technology such as mucosal vaccine carrier technology is integrated with innovating.Gemma vaccine carrier technology realizes nano grade biological load Body realizes oral or collunarium mucosal immunity mode to the adjuvant effect of delivery of antigens, has ensured the efficient of follow up vaccine production Rate and the low cost for preserving transport；Cytokine adjuvant IL-2 introducing make it possible adjust immunization type, promote spy The enhancing of different in nature CD8+T cell immune responses.This project introduces the joint integrated innovation of multiple technologies, forms mutual supplement with each other's advantages Organic whole, the new technology of perfect mucosa immune vaccines is theoretical, and the research of this aspect falls within theoretic innovation.
(2) innovative design of adjuvant.This project plays the immunity enhancement adjuvant effect and cell of novel microbial carrier Factor IL-2 immune modulator adjuvant effect, the advantage of the two is combined with each other, and can improve the immune protective effect of vaccine, is shown Write the dosage of reduction vaccine inoculation and reduce immune time, the immunity inoculation for feed addition type vaccine and special population is provided Ensure.
The present invention has following three aspects characteristic：
(1) towards sanitarian active demand, efficient probiotic oral type Japanese blood fluke vaccine and broken wound are researched and developed Wind vaccine, is to prevent Japanese schistosomiasis and lockjaw to propagate and popular effective way.Introduce a variety of Development Techniques of vaccine It is the application basic research that prominent scientific theory is combined with practical application with the integrated innovation of adjuvant new technology, can is state Family's solving practical problems；
(2) bivalent is oral or collunarium vaccine can improve the immunity of colony, meets One Health theories.Vaccine researches and develops skill Art, may be used on influenza or avian influenza vaccine, immune with nasopharynx spray mucosa for people at highest risk, realize that people at highest risk wraps The occupational protection for including Human avian influenza is immunized, and the immunity for improving colony is also to block new hair people beast bird flu to general population The effective means of propagation.
(3) gemma of hay bacillus is the mucosal vaccine of carrier, and heat endurance is good, and the building mode of recombinant spore can be protected Exogenous antigen, extends the half-life period of vaccine, it is easy to storage and normal temperature transport, suitable developing country and under-developed area Actual demand, is a kind of breakthrough compared with traditional vaccine development method, there is huge Prospect of R ＆ D and commercial application value.
Brief description of the drawings
Fig. 1 is that bacillus subtilis B.subtilis cell divisions and sporulation process (are sprouted in F, forespore Spore；MC, mother cell mother cells；S, spore gemma；VC, vegetative cell breed body cell)；
Fig. 2 is spores of bacillus subtilin B.subtilis Spore capsid proteins electron microscopes and structure distribution schematic diagram (Daisuke etc,Yakugaku zasshi,2012)；
Fig. 3 is cotB-Peptide linker-IL-2 Fusion gene construction schematic diagrames in the embodiment of the present invention 1；
Fig. 4 is cotC-Sj26GST-TTFC Fusion gene construction schematic diagrames in the embodiment of the present invention 1；
Fig. 5 is CotB-IL-2 amalgamation and expressions in the embodiment of the present invention 1 in gemma capsid outermost layer schematic diagram；
Fig. 6 shows for CotC-Sj26GST-TTFC epitope peptides amalgamation and expression in the embodiment of the present invention 1 in gemma capsid outermost layer It is intended to；
Fig. 7 is by the schematic diagram of two sections of foreign gene swivel bases to bacillus subtilis chromosome in the embodiment of the present invention 1；
Fig. 8 is CotB-IL-2 in the embodiment of the present invention 1 with CotC-Sj26GST-TTFC epitope peptides while double amalgamation and expressions In gemma capsid outermost layer schematic diagram；
Fig. 9 is Sj26GST, TTFC and Sj26GST-TTFC positive colony PCR identifications in the embodiment of the present invention 1,1-4 swimming Road corresponds to Marker DL10000, Sj26GST, TTFC and Sj26GST-TTFC PCR primer respectively；
Figure 10 is recombinant spore positive colony PCR identifications in the embodiment of the present invention 1；
Figure 11 is SDS-PAGE and Western blot analyses SjGST, TTFC and SjGST-TTFC in the embodiment of the present invention 1 Protein expression figure；Scheme A, 12%SDS-PAGE. analyzes three protein expression (lane after IPTG induced expressions 3), target protein (SjGST, TTFC and SjGST-TTFC) has obvious expression, and non-induced group is used as control (lane 4) .Lane 2 egg is proved for SjGST, TTFC and SjGST-TTFC protein (arrow is signified) the figures B, Western blot of purifying The expression of white matter.Sj26GST, TTFC, Sj26GST-TTFC be incubated primary antibody be respectively the anti-Sj26GST serum of mouse, mouse resist TTFC serum, the anti-Sj26GST-TTFC serum of mouse.Secondary antibody is with the signified protein size of HRP goat anti-mouse antibody arrows；
Figure 12 is that immunofluorescence in the embodiment of the present invention 1 and laser confocal microscope detect Sj26GST-TTFC in gemma The expression of shell.Scheme the expression of CotC-Sj26GST-TTFC recombinant spores strain shown in A.Sample and the rabbit-anti TTFC serum of purifying It is incubated, secondary antibody anti-rabit.IgG-Fluoresceinisothiocyanate (green, FITC；sigma)；Scheme shown in B Recombinant C otC-Sj26GST-TTFC gemma and the anti-Sj26GST antibody incubations of mouse, secondary antibody anti-mouse IgG- tetramethyl rhodamine isothiocyanate conjugate(red,TRITC；Sigma).Scheme non-recombinant shown in C Gemma wild strain PY79 is compareed；
Figure 13 is that immunofluorescence in the embodiment of the present invention 1 and laser confocal microscope detect IL-12 in recombinant spore table The expression in face；Sample and the anti-IL-2 sera incubations of mouse of purifying, secondary antibody anti-mouse anti-mouseIgG-tetramethyl rhodamine isothiocyanate conjugate(red,TRITC；Sigma).Scheme the cotB-IL- of recombinant spore strain shown in a 2 spore surface expression；Non-recombinant wild strain PY79 shown in figure b is compared；
Figure 14 changes for the level of anti-Sj26GST-TTFC specific serums IgG after mucosal immunity in the embodiment of the present invention 1；
Full splenocyte IFN-γ detection in Figure 15 embodiment of the present invention 1；
Figure 16 is immunofluorescence and flow cytomery in the embodiment of the present invention 1；
Expression of the pUS186-CotC-Sj26GST in spore surface；
Figure 17 is serum after recombinant spore WB600/pUS186-CotC-GST intragastric mouse in the embodiment of the present invention 1 Sj26GST specific IgG levels, IgG1/IgG2a ratios and excrement sIgA levels (OD450)。
Embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention It is described further.
Embodiment 1
Present inventor in early-stage Study using Schistosoma japonicum 26kDa GST (Sj26GST) albumen as model antigen, Using hay bacillus expression plasmid pUS186 by Sj26GST it is gene constructed in spores of bacillus subtilin capsid gene CotC promoters and The downstream of its coded sequence, realizes foreign protein Sj26GST high in gemma capsid surface in the extracellular enzyme defect bacterial strains of WB600 Expression.
Using withered grass bar shuttle integrated plasmid pDG1664, CotC-Sj26GST-TTFC is replaced by dual crossing and integrated In bacillus subtilis chromosome, with CotC amalgamation and expressions in gemma capsid outer surface；Hay bacillus shuttle integrated plasmid is utilized simultaneously PDG364, is integrated in bacillus subtilis chromosome by dual crossing displacement by CotB-peptide linker-IL-2, melts with CotB Conjunction is expressed in gemma capsid outer surface, realizes and constructs polyvalent antigen amalgamation and expression body using bacillus subtilis endogenous spore System.Fluorescence immunoassay experiment, laser confocal microscope detect foreign protein and successfully express (Figure 13) in gemma outer surface, Also demonstrating the macromolecular structure of multivalent recombinant antigens simultaneously does not influence assembling and the sporulation of gemma outer layer albumen.
The immunologic adjuvant effect of gemma is have evaluated, we are analyzed using molecular information and experimentally have detected The physicochemical characteristics of bacillus different genera, selection bacillus subtilis PY79, HU58 and Bacillus clausii O/C make Carrier for non-recombinant gemma-protein vaccine is mixed with albumen, it was confirmed that suction-operated of the gemma to albumen, with adhesion protein Gemma Nasal immunization mouse can cause strong mucosal immunoreaction, and high level can be detected in lung tissue, excrement, saliva Sj26GST specificity sIgA secretion.Meanwhile, mouse system immune response can be also activated, IgG subclass prompts for accounting for Th1 Leading Th1/Th2 mixed type immune responses.Gemma is confirmed as novel mucous membrane vaccine adjuvant, can improve body specificity and Innate immune activity, and adjust immune response type.
Research shows that spores of bacillus subtilin vaccine platform is anti-for specified pathogen physical efficiency inducing mucosal and systemic immunity Should, include the immune response of specific C D8+T lymphocytes, can effectively prevent the infection of causal organism, be mucosal vaccine Research and development provide fundamental basis.
1st, goal in research
Polyvalent antigen amalgamation and expression system is constructed using hay bacillus endogenous spore herein, from Schistosoma japonicum 26kDa GST (Sj26GST) albumen be model antigen and Tetanus Toxin Fragment C (TTFC) as immunologic adjuvant molecule, in bud Spore outer surface is shown (with gemma CotC coat protein amalgamation and expression CotC-Sj26GST-TTFC)；And build and gemma outer surface Double turns of displaying IL-2 (with gemma CotB-IL-2 coat protein amalgamation and expression) builds the mode being combined and expressed.Fluorescence immunoassay Experiment, laser confocal microscope detect foreign protein and successfully expressed in gemma outer surface, also demonstrate polyvalent recombinant and resist Former macromolecular structure does not influence assembling and the sporulation of gemma outer layer albumen, while the thin of vaccine can be improved by demonstrating IL-2 Born of the same parents' immune level.
2nd, research contents
(1) bioinformatic analysis and Gene Fusion of divalence model antigen gene order
1) it is model antigen and Tetanus Toxin Fragment C (TTFC) by Schistosoma japonicum 26kDa GST (Sj26GST) albumen As immunologic adjuvant molecule, its gene order is analyzed, and recombinate fusion Sj26GST-TTFC gene orders
2) recombination sequence sequencing detection
3) recombination sequence Sj26GST-TTFC is built into the C-terminal in gemma capsid protein CotC genes
4) identification of spores of bacillus subtilin capsid surface amalgamation and expression CotC-Sj26GST-TTFC and amalgamation and expression
(2) IL-2 with adjuvant effect expresses the structure of gemma
1) cell factor IL-2 and gemma capsid CotB sequence is analyzed
2) by connect segment by IL-2 build gemma capsid protein CotB genes C-terminal
3) identification of spores of bacillus subtilin capsid surface amalgamation and expression CotB-IL-2 and amalgamation and expression
(3) structure of recombinant bacillus gemma vaccine IL-2 and Sj26GST-TTFC double expression vaccine strains
1) gemma capsid CotC sequences are analyzed, recombinant fusion gene Sj26GST-TTFC sequences are analyzed
2) C-terminal of the Sj26GST-TTFC sequence constructs in CotC genes will be merged
3) on the basis of IL-2 expression gemma, the strain of Sj26GST-TTFC gemma capsid fusions double expression, i.e. CotB- are built IL-2/CotC-Sj26GST-TTFC
(4) identification of its biological activity of spores of bacillus subtilin vaccine and protectiveness evaluation
1) Security test of restructuring or non-recombinant vaccine
2) the immunological characteristic research of vaccine, immune level are determined
3) the protection sexual assault experimental study of vaccine
3rd, research method and laboratory facilities
(1) structure of recombined bacillus subtilis vaccine candidate strain
1)amyE:The structure of cotB-IL-2 recombined bacillus subtilis shuttle integrated plasmids
From the 1091bp DNA fragmentations of B.subtilis PY79 chromosome amplification cotB promoters and coded sequence and insert Plasmid pDG364.PDG364 carries resistance cat genes and multiple cloning sites, and can lead in amylase amylase (amyE) site Cross amyE genes and the cat of carrying and insertion exogenous sequences are incorporated into B.subtilis chromosomes.Under pDG364-cotB C-terminal is swum, common 457bp artificial synthesized sequences are cloned into：Including the peptide linkage sequence with powerful αhelix GGGEAAAKGGG (peptide linker) (SEQ ID NO.1) sequences and the human IL-2's sequence for coming from Homo sapiens, institute The plasmid of structure is pDG364-cotB-linker-IL2, and whether sequencing identification is built succeeds (such as Fig. 3).
The gene order of the peptide linkage of αhelix, is directly synthesized in primer:
Linker-gggeaaakggg-F (band Eco RI)：
GCC TGT TAG GAA TTC CGC TCC AAT CTC TTT TTA CAA TAG AAT ATA TGG AAC CGA AAA TCA TGG CGA TGT ATG AAC GGA TTA GGC C
Linker-gggeaaakggg-R (band Bam HI)：
CGC GGA TCC TCC TCC ACC TTT CGC TGC TGC TTC TCC TCC ACC GGA TGA TTG ATC ATC TGA AG
In cotB-linker-IL2 sequences, IL2 primer sequence:
IL2link-F (band Pcil)：
5'-GCT TCA CAT GTT TAC GTC AGT GTA GAG ATG ATA GAT TGG C-3'
IL2link-R (band BamHI)：
5'-CAT ATG GAT CCG GTG GAG GAG AAG CAG CAG CG-3'.
2)thrC:CotC-Sj26GST-TTFC recombined bacillus subtilis shuttles integrated plasmid is built
From B.subtilis PY79 chromosome amplification cotC promoters and coded sequence insertion pDG1664.PDG1664 exists ThrC gene locis, carry ErmR genes and multiple cloning sites, and can be by thrC genes by the ErmR of carrying and insertion Exogenous sequences are incorporated into B.subtilis chromosomes.Second step, expands the Sj26GST-TTFC sequences designed and synthesized, insertion PDG1664-CotC downstreams C-terminal, constructed recombinant plasmid is pDG1664-cotC-Sj26GST-TTFC, and sequencing identification, which is built, is No success (such as Fig. 4).
The gene magnification primer of table 1
Sj26GST contains a 657bp most long ORFs, and initiation codon is that ATG, termination codon are TGA, coding 218 amino acid.Its theoretical molecular and isoelectric point are respectively 25.63kDa and 6.09.Its nucleotide sequence such as SEQ ID NO: 2, amino acid sequence such as SEQ ID NO:Shown in 3.
TTFC contains a 1359bp most long ORFs, and initiation codon is that ATG, termination codon are TAA, coding 452 amino acid.Its theoretical molecular and isoelectric point are respectively 51.77kDa and 6.7.Its nucleotide sequence such as SEQ ID NO: 4, amino acid sequence such as SEQ ID NO:Shown in 5.
Sj26GST-TTFC contains a 2019bp most long ORFs, and initiation codon is that ATG, termination codon are TAA, encodes 672 amino acid.Its theoretical molecular and isoelectric point are respectively 77.4kDa and 6.27.Its nucleotide sequence such as SEQ ID NO:6, amino acid sequence such as SEQ ID NO:Shown in 7.
The Sj26GST sequences that Sj26GST-P1 and Sj26GST-P5PCR comes out can be with TTFC-P3 and TTFC-P4 primer PCRs TTFC sequences out are linked into Sj26GST-TTFC sequences, and corresponding restriction enzyme site just may be inserted under cotC sequences Swim
The Sj26GST sequences that Sj26GST-P1 and Sj26GST-P2PCR comes out are used to be inserted separately into pet vector, express GST Albumen is used to prepare antibody.
3) IL-2 and withered grass gemma capsid CotB amalgamation and expressions structure
Single endonuclease digestion outside recombinant plasmid pDG364-cotB-linker-IL2 Insert Fragments is linearized, and converts PY79 impressions State cell, tests negative transformant by amylase flat board and selects positive colony with CmR (5 μ g/ml) LB flat screens, be named as IL-2 withered grass gemma strain, this strain will express CotB-linker-IL-2 fusion proteins.Pass through flow cytometer, Western Blot With Dot Blot to amalgamation and expression Identification of Fusion Protein and quantitative analysis, merged by immunofluorescence and laser confocal microscope analysis Protein expression is in gemma outermost layer (such as Fig. 5).
4) Sj26GST-TTFC and withered grass gemma capsid CotC amalgamation and expressions structure
Single endonuclease digestion outside recombinant plasmid pDG1664-cotC-Sj26GST-TTFC Insert Fragments is linearized and PY79 senses are converted By state cell, ThrC- negative transformants are selected into positive colony with ErmR (1 μ g/ml) LB flat screens, Sj26GST- is named as TTFC withered grass gemma strain, this strain will express CotC-Sj26GST-TTFC fusion proteins.Pass through flow cytometer, Western Blot and Dot Blot are analyzed by immunofluorescence and laser confocal microscope amalgamation and expression Identification of Fusion Protein and quantitative analysis Expressing fusion protein is in gemma outermost layer (such as Fig. 6).
5) IL-2 and Sj26GST-TTFC double expression bacterial strains structure (amyE::cotB-IL2；thrC::cotC- Sj26GST-TTFC)
Extract the chromosome of the recombinant strain Sj26GST-TTFC withered grass gemma strain of above-mentioned structure, conversion IL-2 recombinant spore strains The competent cell of making, and with ErmR (1 μ g/ml) screening positive clone.This pair of swivel base recombinant spore strain will be simultaneously in gemma Capsid outermost layer expression two kinds of fusion proteins of CotB-IL2 and CotC-Sj26GST-TTFC (such as Fig. 7,8).Pass through fluidic cell Instrument, Western Blot and Dot Blot pass through immunofluorescence and laser copolymerization to amalgamation and expression Identification of Fusion Protein and quantitative analysis Focusing microscope identifies expressing fusion protein in gemma outermost layer.
(4) Security test of recombinant spore vaccine
Experimental animal and packet：Sterile Balb/c mouse, the 6-8w of male and female half and half, per group 20, sets up recombinant spore time Select vaccine A groups, non-recombinant gemma candidate vaccine B groups, blank control group, totally three groups.Tested mouse is inoculated with gemma candidate through collunarium Vaccine, blank control group is without any processing.Close observation and record the Temperature changing of each group mouse, activity in 48h afterwards, take the photograph Food and drinking times.3d, 7d and 21d after immune, put to death 3 mouse at random respectively, take injection site muscle, the heart, liver, Spleen, lung, kidney and brain etc. are organized, and 4% paraformaldehyde makes paraffin section after fixing, and whether each organs and tissues of Microscopic observation disease occur Reason changes.Tested mouse is in after immune 8w, and male and female are raised together with, and make its natural mating.It is young that the young number of young mouse, deformity are counted and observed after birth Number, post-natal lethality number and development condition.
(5) the immunological characteristic research of vaccine
1) recombinant spore vaccine mucosal immunity effect assessment
Experimental animal and packet：Sterile Balb/c mouse, female 6-8w, sets up Naive groups, PY79, CotB- by every group 6 IL-2 gemma group, CotC-Sj26GST-TTFC gemma group, CotB-IL-2/CotC-Sj26GST-TTFC double expression gemma group, 2 μ 7 groups of g IL-2+2 μ g Sj26GST-TTFC collunariums group, 2 μ gSj26GST-TTFC collunarium groups etc..Mouse is through Anesthesia machine halothane (halothane) after light anesthesia, in the 1st, 22,43d collunariums collunarium (Gilson pipette tip, 40 μ l) immunoprophylaxis 2 ×109Gemma.The -1st, 20,41,62d collections serum, saliva, excrement be stored in -20 DEG C it is standby.I.e. the after final immunization 62d, mouse is put to death with CO2, and acupuncture treatment takes blood outside heart thoracic cavity, is opened chest and is taken lung and open abdomen and takes spleen.ELISA detects serological specificity IgG and subclass and saliva, excrement, the specific sIgA levels of lung tissue extract；After spleen cell culture, cellular immunity is carried out Horizontal analysis：Determine splenocyte CD4+ and CD8+ cell percentages, detection antigenic stimulus after, spleen cell secretion of gamma-IFN and The cytokine levels such as TNF-α, MTT detection lymphopoiesis situations.
3rd, result of study
(1) identification of pET-28b-SjGST-TTFC plasmids is recombinated
Monoclonal after picking conversion extracts plasmid, and using recombinant plasmid as masterplate, with SjGST, TTFC, SjGST-TTFC Special primer enters performing PCR, all obtains and target gene fragment of the same size (Fig. 9).Positive colony sequencing result is shown, is inserted Fragment and SjGST, TTFC, SjGST-TTFC gene order are completely the same, show that recombinant protein and antigen-4 fusion protein gene are cloned into Work(.
(2) recombinant spore positive colony PCR is identified
Figure 10 is that, get, tetC, this three groups of gene clonings of gst-tetC have entered after bacillus subtilis gene, the weight turned out Whether group spores of bacillus subtilin, direct row bacterium colony PCR Testing and appraisals clone successfully international practices, and small letter represents Gene Name, example As tetC represents Gene Name, TTFC capitalization is correspondence protein literary style.
(3) WB analyses restructuring Sj26GST, TTFC, SjGST-TTFC protein immunogenic
Use the anti-Sj26GST, anti-TTFC, anti-SjGST-TTFC of preparation mouse specific antisera, Western Blot analyses show that three kinds of recombinant proteins can be recognized by specific antisera, show Sj26GST, TTFC, SjGST-TTFC weight Histone has immunogenicity and immunoreactivity (Figure 11).
(4) immunofluorescence and laser confocal microscope analyze displayings of the Sj26GST-TTFC on recombinant spore surface
As shown in figure 12, immunofluorescence and laser confocal microscope show that CotC-Sj26GST-TTFC recombinant spores lead to Cross gemma capsid protein CotC successful presentations and spore surface
(5) immunofluorescence and laser confocal microscope analyze displayings of the IL-2 on recombinant spore CotB-IL-2 surfaces
As shown in figure 13, immunofluorescence and laser confocal microscope show successful tables of the CotB-IL-2 in spore surface Reach.
(6) anti-Sj26GST-TTFC specific serums IgG level changes after mucosal immunity
Nasal immunization each group mouse after mouse light anesthesia.Clearly show such as Figure 14 results, CotB-IL2/CotC- Sj26GST-TTFC recombinant spore groups, were significantly higher than control group rSj26GST- in the 21st day, the 42nd day detection Serological IgG level TTFC groups, PY79 groups and Naive groups (P<0.05),
(7) the IFN-γ level of splenocyte secretion
To assess the cell immune response induced after recombinant spore vaccine immune mouse, we detect splenocyte secretion IFN-γ level.Such as Figure 15 data display CotB-IL2/CotC-Sj26GST-TTFC recombinant spore collunarium groups (P<0.05)IFN- γ levels are significantly higher than control group, with statistical significance.
(8) expression of immunofluorescence and flow cytomery pUS186-CotC-Sj26GST in spore surface
The CotC-Sj26GST that pUS186 carrier successful expressions are used in gemma strain WB600 cited by the present invention, with stream Formula cell instrument and immunofluorescence microscopy detect GST protein expressions in gemma outer surface (as shown in figure 16).
(9) serum Sj26GST specific IgG water after recombinant spore WB600/pUS186-CotC-GST intragastrics mouse Flat, IgG1/IgG2a ratios and excrement sIgA levels (OD450)
After recombinant spore CotC-GST oral immunity mouse, excrement extracts the secretion that liquid energy detects specific anti-SjGST B in type IgA (sIgA), such as figure 17 above.As a result show, specific sIgA levels in CotC-SjGST recombinant spore immune group excrement It is significantly higher than control group (CotC gemma group and naive groups, P<0.05).It can detect that sIgA had notable rise at the 16th day, arrive Peak within 33rd day, but the 51st day detection excrement sIgA significantly reduced (P compared with the 33rd day<0.05).Oral immunity Mouse, the detection anti-SjGST total IgGs antibody level of serological specificity (a in Figure 17), after as a result display second is immune 33rd day and 51st day after third time is immune, CotC-SjGST recombinant spore immune group specific IgGs are significantly higher than control group, P<0.05 (CotC gemma immune group and naive groups).CotC gemma immune group and naive groups are without significant difference, P>0.05.As a result show Sj26GST can stimulate body to produce system in hay bacillus WB600 capsid surfaces and CotC amalgamation and expressions, and with immunogenicity System immune response.For further analysis IgG hypotype levels, detection Sj26GST specific IgGs 1 and IgG2a results are shown, are immunized The 33rd day afterwards, IgG1 and IgG2a had rise, and were persistently increased to the 51st day (c in Figure 17).Detected at the 16th day, IgG1 Antibody level is higher by 1.19 ± 0.19 times of IgG2a antibody levels, but has no significant difference (P>0.05), after immune for the second time, The rise of IgG2a levels is higher by 1.28 ± 0.16 times of (P than IgG1 antibody level>0.05), after final immunization, the 51st day, detect IgG2a is persistently raised as 1.52 ± 0.23 times of (P of IgG1 levels>0.05).Show after being immunized for the first time, Th2 type immune responses are It is main, and second, third time it is immune after, based on Th1 type immune responses, but have no notable significant difference, point out CotC- Sj26GST recombinant spores oral vaccine delivery is immunized after mouse, it is possible to create Th1/Th2 mixed type immune responses.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention And scope.
Sequence table
<110>Zhongshan University
<120>A kind of Schistosoma japonicum and lockjaw bivalent be oral or collunarium vaccine
<160> 16
<170> PatentIn version 3.3
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ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatga 657
<210> 3
<211> 218
<212> PRT
<213>Artificial sequence
<400> 3
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys
210 215
<210> 4
<211> 1359
<212> DNA
<213>Artificial sequence
<400> 4
atgaaaaatc tggattgttg ggttgataat gaagaagata tagatgttat attaaaaaag 60
agtacaattt taaatttaga tattaataat gatattatat cagatatatc tgggtttaat 120
tcatctgtaa taacatatcc agatgctcaa ttggtgcccg gaataaatgg caaagcaata 180
catttagtaa acaatgaatc ttctgaagtt atagtgcata aagctatgga tattgaatat 240
aatgatatgt ttaataattt taccgttagc ttttggttga gggttcctaa agtatctgct 300
agtcatttag aacaatatgg cacaaatgag tattcaataa ttagctctat gaaaaaacat 360
agtctatcaa taggatctgg ttggagtgta tcacttaaag gtaataactt aatatggact 420
ttaaaagatt ccgcgggaga agttagacaa ataactttta gggatttacc tgataaattt 480
aatgcttatt tagcaaataa atgggttttt ataactatta ctaatgatag attatcttct 540
gctaatttgt atataaatgg agtacttatg ggaagtgcag aaattactgg tttaggagct 600
attagagagg ataataatat aacattaaaa ctagatagat gtaataataa taatcaatac 660
gtttctattg ataaatttag gatattttgc aaagcattaa atccaaaaga gattgaaaaa 720
ttatacacaa gttatttatc tataaccttt ttaagagact tctggggaaa ccctttacga 780
tatgatacag aatattattt aataccagta gcttctagtt ctaaagatgt tcaattgaaa 840
aatataacag attatatgta tttgacaaat gcgccatcgt atactaacgg aaaattgaat 900
atatattata gaaggttata taatggacta aaatttatta taaaaagata tacacctaat 960
aatgaaatag attcttttgt taaatcaggt gattttatta aattatatgt atcatataac 1020
aataatgagc acattgtagg ttatccgaaa gatggaaatg cctttaataa tcttgataga 1080
attctaagag taggttataa tgccccaggt atccctcttt ataaaaaaat ggaagcagta 1140
aaattgcgtg atttaaaaac ctattctgta caacttaaat tatatgatga taaaaatgca 1200
tctttaggac tagtaggtac ccataatggt caaataggca acgatccaaa tagggatata 1260
ttaattgcaa gcaactggta ctttaatcat ttaaaagata aaattttagg atgtgattgg 1320
tactttgtac ctacagatga aggatggaca aatgattaa 1359
<210> 5
<211> 452
<212> PRT
<213>Artificial sequence
<400> 5
Met Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val
1 5 10 15
Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile
20 25 30
Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp
35 40 45
Ala Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn
50 55 60
Asn Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr
65 70 75 80
Asn Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro
85 90 95
Lys Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser
100 105 110
Ile Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp
115 120 125
Ser Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser
130 135 140
Ala Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe
145 150 155 160
Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp
165 170 175
Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser
180 185 190
Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr
195 200 205
Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp
210 215 220
Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys
225 230 235 240
Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly
245 250 255
Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser
260 265 270
Ser Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu
275 280 285
Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg
290 295 300
Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn
305 310 315 320
Asn Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr
325 330 335
Val Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly
340 345 350
Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala
355 360 365
Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp
370 375 380
Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala
385 390 395 400
Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro
405 410 415
Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys
420 425 430
Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly
435 440 445
Trp Thr Asn Asp
450
<210> 6
<211> 2019
<212> DNA
<213>Artificial sequence
<400> 6
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaaggatcc 660
atgaaaaatc tggattgttg ggttgataat gaagaagata tagatgttat attaaaaaag 720
agtacaattt taaatttaga tattaataat gatattatat cagatatatc tgggtttaat 780
tcatctgtaa taacatatcc agatgctcaa ttggtgcccg gaataaatgg caaagcaata 840
catttagtaa acaatgaatc ttctgaagtt atagtgcata aagctatgga tattgaatat 900
aatgatatgt ttaataattt taccgttagc ttttggttga gggttcctaa agtatctgct 960
agtcatttag aacaatatgg cacaaatgag tattcaataa ttagctctat gaaaaaacat 1020
agtctatcaa taggatctgg ttggagtgta tcacttaaag gtaataactt aatatggact 1080
ttaaaagatt ccgcgggaga agttagacaa ataactttta gggatttacc tgataaattt 1140
aatgcttatt tagcaaataa atgggttttt ataactatta ctaatgatag attatcttct 1200
gctaatttgt atataaatgg agtacttatg ggaagtgcag aaattactgg tttaggagct 1260
attagagagg ataataatat aacattaaaa ctagatagat gtaataataa taatcaatac 1320
gtttctattg ataaatttag gatattttgc aaagcattaa atccaaaaga gattgaaaaa 1380
ttatacacaa gttatttatc tataaccttt ttaagagact tctggggaaa ccctttacga 1440
tatgatacag aatattattt aataccagta gcttctagtt ctaaagatgt tcaattgaaa 1500
aatataacag attatatgta tttgacaaat gcgccatcgt atactaacgg aaaattgaat 1560
atatattata gaaggttata taatggacta aaatttatta taaaaagata tacacctaat 1620
aatgaaatag attcttttgt taaatcaggt gattttatta aattatatgt atcatataac 1680
aataatgagc acattgtagg ttatccgaaa gatggaaatg cctttaataa tcttgataga 1740
attctaagag taggttataa tgccccaggt atccctcttt ataaaaaaat ggaagcagta 1800
aaattgcgtg atttaaaaac ctattctgta caacttaaat tatatgatga taaaaatgca 1860
tctttaggac tagtaggtac ccataatggt caaataggca acgatccaaa tagggatata 1920
ttaattgcaa gcaactggta ctttaatcat ttaaaagata aaattttagg atgtgattgg 1980
tactttgtac ctacagatga aggatggaca aatgattaa 2019
<210> 7
<211> 672
<212> PRT
<213>Artificial sequence
<400> 7
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Gly Ser Met Lys Asn Leu
210 215 220
Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu Lys Lys
225 230 235 240
Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile
245 250 255
Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln Leu Val
260 265 270
Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu Ser Ser
275 280 285
Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp Met Phe
290 295 300
Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala
305 310 315 320
Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser
325 330 335
Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val Ser Leu
340 345 350
Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly Glu Val
355 360 365
Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn Ala Tyr Leu
370 375 380
Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg Leu Ser Ser
385 390 395 400
Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala Glu Ile Thr
405 410 415
Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp
420 425 430
Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys Phe Arg Ile
435 440 445
Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr Thr Ser
450 455 460
Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg
465 470 475 480
Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp
485 490 495
Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro
500 505 510
Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn
515 520 525
Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp
530 535 540
Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn
545 550 555 560
Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn
565 570 575
Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro
580 585 590
Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys Thr Tyr
595 600 605
Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser Leu Gly Leu
610 615 620
Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn Arg Asp Ile
625 630 635 640
Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu
645 650 655
Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp
660 665 670
<210> 8
<211> 85
<212> DNA
<213>Artificial sequence
<400> 8
gcctgttagg aattccgctc caatctcttt ttacaataga atatatggaa ccgaaaatca 60
tggcgatgta tgaacggatt aggcc 85
<210> 9
<211> 62
<212> DNA
<213>Artificial sequence
<400> 9
cgcggatcct cctccacctt tcgctgctgc ttctcctcca ccggatgatt gatcatctga 60
ag 62
<210> 10
<211> 40
<212> DNA
<213>Artificial sequence
<400> 10
gcttcacatg tttacgtcag tgtagagatg atagattggc 40
<210> 11
<211> 32
<212> DNA
<213>Artificial sequence
<400> 11
catatggatc cggtggagga gaagcagcag cg 32
<210> 12
<211> 24
<212> DNA
<213>Artificial sequence
<400> 12
cggctagcat gtcccctata ctag 24
<210> 13
<211> 24
<212> DNA
<213>Artificial sequence
<400> 13
cccaagcttt cattttggag gatg 24
<210> 14
<211> 28
<212> DNA
<213>Artificial sequence
<400> 14
cgcggatcca tgaaaaatct ggattgtt 28
<210> 15
<211> 29
<212> DNA
<213>Artificial sequence
<400> 15
cccaagcttt taatcatttg tccatcctt 29
<210> 16
<211> 23
<212> DNA
<213>Artificial sequence
<400> 16
cgcggatcct tttggaggat ggt 23

Claims (7)

1. a kind of Host Strains, it is characterised in that the Host Strains, which are prepared by the following method, to be formed：
(1) by CotB gene promoter sequences, CotB coded sequences, the nucleotide sequence of connector and IL-2 nucleotide sequence The first expression vector is consecutively connected to, obtains expressing the first recombinant expression carrier of cotB-linker-IL2 fusion proteins；
(2) the outer single endonuclease digestion of the first recombinant expression carrier Insert Fragment causes first recombination expression to carry in the step (1) Body is linearized, and is then transformed into the first bacillus competent cell, and screening obtains expression CotB-linker-IL-2 fusion eggs The first white positive strain；
(3) by the nucleotides of CotC gene promoter sequences, CotC coded sequences and external source sexual norm gene Sj26GST-TTFC Sequence is consecutively connected to the second expression vector, and the second recombination expression for obtaining expressing CotC-Sj26GST-TTFC fusion proteins is carried Body；
(4) the outer single endonuclease digestion of the second recombinant expression carrier Insert Fragment causes second recombination expression to carry in the step (3) Body is linearized, and is then transformed into the second bacillus competent cell, and screening obtains expression CotC-Sj26GST-TTFC fusion eggs The second white positive strain；
(5) chromosome of the second positive strain of expression CotC-Sj26GST-TTFC fusion proteins in the step (4) is extracted, is turned The competence for dissolving into the first positive strain preparation of the expression IL-2CotB-linker-IL-2 fusion proteins in the step (2) is thin The positive colony of CotB-IL2 fusion proteins and CotC-Sj26GST-TTFC fusion proteins is expressed in born of the same parents, screening simultaneously, is produced described Host Strains.
2. Host Strains according to claim 1, it is characterised in that the amino acid sequence of the connector such as SEQ ID Shown in NO.1.
3. Host Strains according to claim 1, it is characterised in that Sj26GST-TTFC nucleotides in the step (3) Sequence is as shown in SEQ ID NO.6.
4. Host Strains according to claim 1, it is characterised in that the first expression vector is plasmid in the step (1) The second expression vector is plasmid pDG1664 in pDG364, the step (3).
5. Host Strains according to claim 1, it is characterised in that the first bacillus competence is thin in the step (2) Born of the same parents are bacillus subtilis PY79 competent cells, and the second bacillus competent cell is withered grass gemma in the step (4) Bacillus PY79 competent cells.
6. Host Strains as claimed in claim 1 are preparing the use of Schistosoma japonicum and lockjaw bivalent orally or in collunarium vaccine On the way.
7. a kind of Schistosoma japonicum and lockjaw bivalent be oral or collunarium vaccine, it is characterised in that the Schistosoma japonicum and broken Cold bivalent is oral or collunarium vaccine contains the gemma as obtained by claim 1-4 any described Host Strains cultures.
CN201710269904.8A 2016-04-25 2017-04-24 A kind of Schistosoma japonicum and lockjaw bivalent be oral or collunarium vaccine Pending CN107177539A (en)

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Publication number Priority date Publication date Assignee Title
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JEONG JIN LEE: "Tetanus toxin fragment C expressed in live Salmonella vaccines enhances antibody responses to its fusion partner Schistosoma haematobium glutathione S-transferase", 《INFECTION AND IMMUNITY》 *
LI LI: "Immunogenicity of self-adjuvanticity oral vaccine candidate Based on use of Bacillus subtilis spore displaying Schistosoma japonicum 26 KDa GST protein", 《PARASITOL RES》 *
言慧: "常用基因表达系统的特点及其在寄生虫抗原制备中的应用", 《寄生虫与医学昆虫学报》 *

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Application publication date: 20170919