CN110327460A

CN110327460A - Streptococcus suis-Haemophilus parasuis bigeminy subunit vaccine and preparation method

Info

Publication number: CN110327460A
Application number: CN201910485142.4A
Authority: CN
Inventors: 王春来; 刘思国; 李刚
Original assignee: Harbin Weike Biotechnology Development Co; Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Weike Biotechnology Development Co; Harbin Veterinary Research Institute of CAAS
Priority date: 2019-06-05
Filing date: 2019-06-05
Publication date: 2019-10-15

Abstract

The present invention relates to Streptococcus suis-Haemophilus parasuis bigeminy subunit vaccines and preparation method thereof.Bigeminy subunit vaccine of the present invention contains haemophilus parasuis antigen protein AfuA, OppA2, CdtB, OppA and Streptococcus suis antigen MRP and SLY；Or contain haemophilus parasuis fusion protein AfuA-OppA2, two kinds of antigens of fusion protein CdtB-OppA and Streptococcus suis antigen protein two kinds of antigens of MRP and SLY.The bigeminy subunit vaccine can excite the strong immune response of mouse; haemophilus parasuis and Streptococcus suis to different serotypes attack malicious mouse and all have good cross-protection; and it is better than traditional inactivated vaccine, basis is provided to develop efficient, wide spectrum, cheap Streptococcus suis-Haemophilus parasuis vaccine research.

Description

Streptococcus suis-Haemophilus parasuis bigeminy subunit vaccine and preparation method

Technical field

The present invention relates to veterinary applications, specially Animal diseases are prevented and treated, and are more particularly to Streptococcus suis-pair pig Haemophilus disease bigeminy subunit vaccine and preparation method thereof.

Background technique

Streptococcus suis is by the caused meningitis with pig of Streptococcus suis (Streptococcus suis, SS), sepsis The bacterial infectious disease that the clinical symptoms such as disease, arthritis are characterized is that one of the global pig breeding industry of puzzlement for a long time mainly asks Topic.Streptococcus suis serotype is numerous, 2 type of Streptococcus suis serum (SS2) therein or a kind of zoonosis cause of disease.People is logical Cause of disease (Zhang Anding, Jin Meilin, Chen Huanchun, the streptococcus suis 2 can be infected by crossing specific route of transmission (such as wound exposure etc.) Type progress (summary) cultivation and feed, 2005,13-18.), if treatment is not in time, septicemia, meningitis can be caused Etc. poor prognosis is caused, public health security is seriously threatened.The prevalence of each once Streptococcus suis in 1998 and 2005, Cause very big loss.But since last time Epidemic outbreak of disease in 2005, China's Mainland only reports 4 sporadic people Class case (Yefei, Zhu, Zhongmin, et al., A Streptococcus suis serotype 2caused streptococcal toxic shock syndrome(STSS)in a patient.Journal of Nanjing Medical University,2008:22,313-316.2008；Yoo JY,HG Song,JH Lee,et al.,A case of overwhelming postsplenectomy infection with purpura fulminans by Streptococcus pneumoniae.2006).Nineteen sixty-eight, Europe report the case of human infection Streptococcus suis for the first time, It is worth noting that, all cases reported before nineteen eighty-three both are from West Europe.Last an example human cases of Britain be 2001 annual reports.It is most of all related with wild boar hunter although few case reports since nineteen ninety-five in France (Watkins EJ,P Brooksby,MS Schweiger,et al.,Septicaemia in a pig-farm worker: The Lancet.Lancet,2001:357,38-38)。

Virulence factors of streptococcus suis mainly includes MRP, SLY, FBP, HP0197, RfeA.Wherein, MRP can be in sick pig body It is found in isolated bacterial strain, the bacterial strain separated in health pig body is simultaneously free of this small molecule.It is specific in pathogenic course Act on it is still not very clear, and in different regions, MRP and it is pathogenic between have no it is determining contact, since these factors lack The bacterial strain of mistake virulence compared with parent strain does not change.Therefore they are tended to be considered the marker of virulence, rather than Virulence factor.Piglet is immunized after adding adjuvant emulsion in the MRP and EF that Wisselink et al. extracts streptococcus suis 2-type, it is found that its is right Streptococcus suis 2-type pathogenic strain has preferable immune protective efficiency (Wisselink HJ, N Stockhofe-Zurwieden, LAT Hilgers,et al.,Assessment of protective efficacy of live and killed vaccines based on a non-encapsulated mutant of Streptococcus suis serotype 2.Veterinary Microbiology, 2002b:84,155-168), it is subunit vaccine that this, which illustrates that MRP and EF has development, Potentiality.Hemolysin (suilysin) is secreted in supernatant, is the perforation toxin family member of mercaptan activation.Have experiments have shown that pig Streptococcus, which generates the amount of hemolysin and the pathogenicity of bacterial strain, certain relationship (Norton PM, C Rolph, PN Ward, et al.,Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin. Pathogens&Disease,2010:26,25- 35.).But there is research to confirm that it is not required in streptococcus pathogenic course.SLY has hemolytic activity and to people O-shaped red blood cell hemolytic activity it is most strong.

Haemophilus parasuis is caused by haemophilus parasuis (Haemophilus parasuis, HPS) with pig The bacterial infectious disease that meningitis, pericarditis, arthritis and polyserositis are characterized.The bacterium was by Germany's science in 1910 Family Glasser is separated to haemophilus parasuis in the slurries sexual secretion of sick pig for the first time and is described, so the disease and quilt Referred to as Ge Lazeshi disease (Glasser ' s disease).Haemophilus parasuis is serious to pig breeding industry harm and in global Generally it is distributed.Haemophilus parasuis serotype is numerous, and existing serotype cannot identify all haemophilus parasuis point From strain.Serotype investigation shows that the serotype of domestic and international prevalence is mainly the stronger serotype of the virulence such as 5 types, 4 types and 13 types (Liu Zhengfei, Cai Xuwang, Chen Huanchun, etc. haemophilus parasuis progress animal medicine progress, 2003:24,17-19.). Currently, the disease is in apparent ascendant trend in Compact Develop, mainly cause suckling pig, weanling pig dead, pig growth Depauperation, efficiency of feed utilization is low, and haemophilus parasuis and the mixed infection phenomenon of other cause of diseases are very universal, secondary pig Haemophilus is up to 20% from the separation rate in clinically ill pig, this also brings great difficulty to the diagnosis and treatment of swine disease (Kim J,HK Chung,T Jung,et al.,Postweaning multisystemic wasting syndrome of pigs in Korea:prevalence,microscopic lesions and coexisting microorganisms.Journal of Veterinary Medical Science,2002:64,57.)

The virulence factor of haemophilus parasuis mainly includes AfuA, CDT, OppA and OppA2.AfuA gene coding iron from Sub- abc transport Binding Capacity albumen, is primarily involved in transmembrane transport Fe³⁺, have important work to the utilization of transferrins and lactoferrin With play an important role (Wei X, S Cao, L Zhang, et al., Comparative during bacterium infection body proteome analysis of the extracellular proteins of two Haemophilus parasuis strains Nagasaki and SW114.Biochemical& Biophysical Research Communications, 2014:446,997-1001).Charland's studies have shown that with the presence of turning iron-binding protein receptor in the serum of pig (Charland N,CG D'Silva,RA Dumont,et al.,Contact-dependent acquisition of transferrin-bound iron by two strains of Haemophilus parasuis.Canadian Journal of Microbiology,1995:41,70).Research finds turning for the afuA gene for the 5 type reference strains for having virulence The horizontal reference strain for being higher than avirulent 3 type of record.Cell-lethal expands toxin (cytolethal distending Toxin, CDT) it is the proteotoxin that a variety of Gram-negative bacterias generate, belong to the exotoxin of thermally labile, eukaryon can be caused thin The expansion and death of born of the same parents.Its holotoxin is made of three kinds of subunits, i.e. CdtA, CdtB, CdtC.Studies have shown that individually CdtB has There is DNase active, can enter cell, the double chain breaks of inducing cell nuclear dna, CDTA, CDTC can enhance individually or together The DNase I activity of CDTB, and CDT holotoxin is most strong to the toxicity of gene, and can induce PK-15 cell and p53 dependence occurs Apoptosis (Niu Hui, haemophilus parasuis cell-lethal expand toxin cytotoxic mechanism study the Chinese Academy of Agricultural Sciences, 2015.).Oligopeptides belongs to a member of ABC binding cassette transporters family, ABC binding cassette transporter egg through enzyme OppA and OppA2 White superfamily participates in the transdermal delivery of machine vivo protein, polypeptide and various ions.The study found that abc transport albumen and bacterium Virulence is closely related, participates in intake of the pathogenic bacteria to external nourishment and metal ion and the suction-operated to host cell.The family Race's albumen has good immunogenicity, can be used as candidate antigens albumen (P L, B M, the dW L, et of recombinant subunit vaccine al.,Three different putative phosphate transport receptors are encoded by the Mycobacteium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG.Journal of Bacteriology,1997:179,2900-2906)。

Streptococcus suis and haemophilus parasuis are the bacterial infectious disease of clinically common pig, and belong to conditionity cause The generation of germ, two kinds of bacterial diseases is closely related with the immune state, environmental factor, the virulence of pathogenic strain of pig.Therefore, add Strong feeding management keeps colony house health, maintains reasonable stocking density, reducing the stress reaction of pig to be that prevention and treatment swinery passes The key of infectious diseases.Once there are typical clinical symptoms and answer after making a definite diagnosis and treated simultaneously using enough antibiotic Preventive administration (Chang Tianxing, Ji Hongxing, Chen Lanlan, etc. Streptococcus suis and haemophilus parasuis are carried out to the pig not fallen ill The prevention and treatment China animal and veterinary digest 2013 of sick mixed infection).

Streptococcus suis and Haemophilus parasuis have similar clinical symptoms, and are all common porcine respiratory diseases Disease.Carrying out immunoprophylaxis using Combined vaccine can reach the anti-effect of a needle two, and can greatly reduce immunity inoculation number, subtract Light manpower and material resources consumption, is the research direction for preventing and treating the vaccine of both bacterial diseases.For traditional bigeminy epidemic disease of two kinds of bacterial diseases Seedling is mostly that inactivated vaccine targetedly is made using the more popular clinical strains in a certain area.Biology is limited before the section of Wuhan Streptococcus suis-Haemophilus parasuis bigeminy vaccine of company's granted three classes novel chiral synthon in 2017 is with domestic popular LT plants of streptococcus suis 2-type, MD0322 plants and SH0165 plants of 4 type of haemophilus parasuis inactivation after made of, China major part Area can generate good preventive effect to Streptococcus suis and Haemophilus parasuis.Recently a kind of new beast of granted country The Streptococcus suis and Haemophilus parasuis bigeminy genetic engineering subunit vaccine of medicine certificate be in the world first be directed to this The genetic engineering subunit vaccine of two kinds of bacterial diseases predominantly solves the more serotype common senses of encountered pathogenic in two kinds of clinical productions The problem of dye.But its practical application effect still needs to further verify.Finally, vaccine effect lacks unified evaluation criterion, together One candidate antigens can be generated in different laboratories opposite result (Youjun F, P Xiuzhen, S Wen, et al., Streptococcus suis enolase functions as a protective antigen displayed on the bacterial cell surface.Journal of Infectious Diseases,2009:200,1583-1592.).Cause This, the mode of associated antigen protein screening and criterion should also attract people's attention.

There is a plurality of types of albumen, the presence of these surface proteins is that bacterium carries out cell on all microorganism surfaces Between the biological functions such as signal identification, signal transduction, surface adhesion, field planting and immune response molecular basis.In these life In activity, interaction or bridge joint mechanism composition between protein and albumen, between protein and carbohydrate dominate these The interaction of process.From the team where Freudl and Charbit in 1986 delivered respectively by by external source peptide fragment and carry Body protein blend and be showed in the work of bacterium surface since (Freudl R, S Macintyre, M Degen, et al., Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12.Journal of Molecular Biology, 1986:188,491-494.), bacterium surface displaying technology is constantly sent out It opens up and includes as fields such as vaccine delivery vehicles, protein library building, cell adsorbent and biocatalysts by application (Desvaux M,E Dumas,I Chafsey,M,Protein cell surface display in Gram-positive bacteria:from single protein to macromolecular protein structure.Fems Microbiology Letters,2010:256,1-15)。

Epitope screening is the important prerequisite that recombinant bacterial strain is constructed by genetic engineering means, can be from antigen or antibody Start with and is screened.Start with from the primary structure of antigen, the surface texture by analyzing antigen predicts possible antigen table Position or sessile antibody analysis obtain its corresponding epitope with small peptide that it specifically binds indirectly.Rely on antigen into Capable Antigen Epitope Prediction needs optimize the segment of acquisition, than relatively time-consuming (in Dong Lin, Wang Yanping, Miao Li, etc. epiposition vaccine Design and optimization strategy and application herding and the animal doctor in animal vaccine research, 2011:43,95-98), in actual operation Common method is amino acid sequence (And GPS, VA of the screening in conjunction with specific antibodies in random peptide library Petrenko,Phage Display.Chemical Reviews,1997:97,391-410)。

The polypeptide sequence of random coded is showed in bacterium surface building bacterium rondom polypeptide library, it is anti-with known solid phase After body and random peptide library effect, is repeatedly eluriated, the small peptide fragment in conjunction with antibody specificity is obtained, by the monoclonal of acquisition The sequence of you can get it after the sequencing peptide fragment, or else this breaks optimal speed than traditional prediction epitope faster, only by several Wheel is incubated for and eluriates the peptide fragment that can be obtained the specificity for a certain antigen.And by bacteria display random peptide library The screening epitope of specificity that can also obtain being not present in nature, it might even be possible to by it directly as emergency epidemic disease Seedling come using.

Killed bacterial vaccine based on bacterium surface displaying technology is said relative to traditional inactivated vaccine, can will be passed through artificial The epitope of the specificity of selection optimization is largely showed in bacterium surface, and body is made more easily to go to identify.Meanwhile bacterium Thalli granule is big, and the lipopolysaccharides on outer membrane itself is exactly good immunopotentiator in one, it is easier to stimulate the immune anti-of body It answers, promotes body to efficient identification (Peng W, W Si, L Yin, the et al., Salmonella of the antigen shown enteritidis ghost vaccine induces effective protection against lethal challenge in specific-pathogen-free chicks.Immunobiology,2011:216, 558-565.)。 There is research to confirm, the antigen protein for being showed in BCG vaccine surface is pierced than the albumen that same dosage is obtained through Prokaryotic expression, purification Swash body generate antibody level it is 100 to 1000 times high (Stover CK, GP Bansal, MS Hanson, et al., Protective immunity elicited by recombinant bacille Calmette-Guerin(BCG) expressing outer surface protein A (OspA)lipoprotein:a candidate Lyme disease vaccine.Journal of Experimental Medicine,1993: 178,197-209).Although bacterium living is one Very promising platform can provide heterologous antigen for the common various bacteriosis of human and animal, but should Suitable host strain and expression vector are selected, with caution to avoid the security risk being likely to occur.Developing the same of bacterial vaccine living When, it the safety of these genetically modified organisms and should also be taken seriously to the potential threat of external environment and the mankind.

Streptococcus suis and two kinds of Haemophilus parasuis sick pathogen Streptococcus suis (Streptococcus suis, SS), haemophilus parasuis (Haemophilus parasuis, HPS) serotype is numerous, and can divide in same illness pig body From the bacterial strain for arriving different serotypes.Cross-protection and bad between different serotypes bacterial strain.Current vaccine research is more prone to In the exploitation of subunit vaccine, the hair of multiple HPS and the relevant immunogenicity of SS in recent years good pathogenesis related protein molecule Now make the research strategy of the subunit vaccine for both bacterial diseases more attractive.The present inventor's early period is directed to pig hammer The related antigen of bacterium disease and haemophilus parasuis is studied, and obtain multinomial patent such as 201310268347.X, 201310268250.9,201310268863.2,201310268810.0 etc., in addition, also directed to Haemophilus parasuis development Subunit vaccine composition, it includes seven kinds of haemophilus parasuis immune protective antigen, that is, HbpA, afuA, oppA, OppA2, D15, Hps06257 and nqrA.But need the exploitation subunit vaccine good for both sick cross-protections.

Summary of the invention

The object of the present invention is to provide the joint epidemic diseases good to Streptococcus suis and Haemophilus parasuis cross-protection Seedling.Specifically be to provide Streptococcus suis-Haemophilus parasuis bigeminy subunit vaccine, especially by multiple Streptococcus suis and The good antigen molecule of the immunogenicity of haemophilus parasuis is showed in surface of E. coli, sets up based on submission expression-form Streptococcus suis-Haemophilus parasuis bigeminy subunit vaccine.

The present invention provides a kind of Streptococcus suis-Haemophilus parasuis bigeminy subunit vaccine, bloodthirsty containing secondary pig Bacteroides antigen albumin A fuA, OppA2, CdtB, OppA and Streptococcus suis antigen MRP and SLY.

Preferably each antigen protein is expressed in the form of surface display respectively, more specifically by respectively By the gene constructed at surface submission expression vector of encoding said proteins, and then it is prepared into the recombinant bacterium of surface submission expression, training It supports mixing after obtaining bacterium solution and obtains the vaccine.Abbreviation surface display bigeminy subunit vaccine II below.

In one embodiment, the recombinant bacterium is Escherichia coli, more specifically BL21 (DE3) bacterium.

Also need under normal conditions addition adjuvant, adjuvant is selected from, but is not limited to following type, oil-in-water adjuvant, polymer and Water adjuvant, water-in-oil adjuvant, aluminum hydroxide adjuvant, vitamin E adjuvant, preferably ISA201VG are mixed in the ratio of equal mass ratioes It is emulsified to obtain the vaccine after conjunction.

The wherein concentration for each antigen protein when in use can be respectively 0.1-0.5mg/mL, preferably 0.2- 0.4mg/mL, most preferably 0.3mg/mL.

In a preferred embodiment, it is sub- single to provide a kind of Streptococcus suis-Haemophilus parasuis bigeminy by the present invention Position vaccine, contains haemophilus parasuis fusion protein AfuA-OppA2, two kinds of antigens of fusion protein CdtB-OppA and pig Two kinds of antigens of streptococcal antigenic proteins MRP and SLY.

Preferably, each albumen exists by surface submission expression-form, more specifically by will encode institute respectively The gene constructed at surface submission expression vector of albumen is stated, and then is prepared into the recombinant bacterium of surface submission expression, culture obtains bacterium Mixing obtains the vaccine after liquid.Abbreviation surface display bigeminy subunit vaccine I below.

Wherein the mode of surface submission expression can choose suitable approach, can choose gram-positive bacteria surface display System is also possible to Gram-negative bacteria surface exhibition system.Wherein gram-positive bacteria cell display systems can choose cell Wall anchorin is shown, cell surface associated proteins are shown；For negative bacterium surface display system, outer membrane protein exhibition can be Show, shown from transhipment body display, flagellum displaying and ice nucleation protein INP.And each antigen protein can use it is identical or different Surface display system carrys out submission expression.In one embodiment, the recombinant bacterium is Escherichia coli, more specifically BL21 (DE3) bacterium.

It can be respectively 0.1-0.5mg/mL, preferably 0.2-0.4mg/mL wherein for the concentration of each albumen, most preferably For 0.3 mg/mL.

Surface display bigeminy subunit vaccine of the invention can be by intramuscular, intradermal or subcutaneous administration, thus can prepare At adaptable dosage form, the preferably dosage form of subcutaneous administration.Vaccine can be applied by prime-boost immunization protocol.For example, After pig carries out first time inoculation, it can receive to reinforce exempting from for second after a period of time (for example, about 7,14,21 or 28 days) Epidemic disease.In general, the dosage of booster immunization exempts from applied dose equal or lower than first.Reinforce exempting from addition it is also possible to carry out third time Epidemic disease, such as immune latter 2-3 months, 6 months or 1 year.

The present invention also provides above-mentioned Streptococcus suis-Haemophilus parasuis bigeminy subunit vaccine preparation method, It is characterized in that, respectively by the gene constructed at surface submission expression vector of encoding said proteins, and then be prepared into containing described The recombinant bacterium of expression vector, mixing obtains the vaccine after culture obtains bacterium solution.In the preferred embodiment, preferred adjuvant is After ISA201VG is mixed by equal mass ratioes with the bacterium solution, emulsified to obtain the vaccine.

Preferably, in the vaccine of preparation each albumen concentration, can be respectively 0.1-0.5mg/mL, preferably 0.2- 0.4mg/mL, most preferably 0.3mg/mL.

Further, the vaccine preparation is at by intramuscular, intradermal or subcutaneous administration dosage form, preferably subcutaneous administration Dosage form.

Present invention technical effect obtained is illustrated by analysis and Binding experiment further below.

In terms of Streptococcus suis antigen selection, FBP, RfeA, HP0197 are the good immunogenicities reported nearly ten years Albumen.But from the point of view of the present inventor carries out immunoprotection challenge test, the immunogenicity of classical antigen MRP and SLY are bright It is aobvious to be better than FBP, RfeA, HP0197.Therefore, select antigen MRP and SLY as the antigen in combined vaccine of the present invention.Into one Step selects aspect for the antigen of haemophilus parasuis, and the present invention has selected these four antigens of AfuA, OppA2, CdtB, OppA Albumen, combined effect is more advantageous (will also address below), while in order to simplify technique and reduce production cost, also into one Step explores two kinds of fusion proteins of AfuA-OppA2, CdtB-OppA, and having reached not only can be reduced process complexity but also can improve The purpose of immune effect, and then bigeminy subunit vaccine contains haemophilus parasuis fusion protein AfuA-OppA2, fusion protein Two kinds of antigens of CdtB-OppA and Streptococcus suis antigen protein two kinds of antigens of MRP and SLY.Further importantly, this hair The bright recombinant expression mode for taking surface submission to express, further improves immune effect.

The present invention has rated surface display bigeminy subunit vaccine and attacks poison respectively to haemophilus parasuis and Streptococcus suis The protecting effect of mouse, while setting up purifying protein subunit vaccine group, HPS-SS inactivated vaccine group and the control of GAP-associated protein GAP Group.Every group of mouse uses HN10 plants of the Serotype 5 of HPS, ZD12 plants of 13 type of serum and M126 plants of 2 type of streptococcic serum, blood respectively GZ2 plants of clear 9 type attacks poison.As can be seen that surface display bigeminy subunit vaccine I is respectively to the HPS protective rate for attacking malicious mouse 80%, 80%, surface display bigeminy subunit vaccine II is respectively 90%, 60% to the protective rate of mouse, and the two effect is basic Quite.Illustrate that fusion protein shows the energy for showing stimulation mouse body generation corresponding antibodies respectively with single albumen in the present invention Power is identical, but expression strain class needed for amalgamation and expression is less, and production operation is more simple.The protection of mouse after poison is attacked to HPS Effect and purifying protein subunit vaccine and HPS-SS dyad inactivated vaccine are quite (respectively 90%, 60% and 80%, 60%). The protecting effect for attacking mouse after poison to M126 plants of 2 type of Streptococcus suis serum is 50%, is maintained an equal level with inactivated vaccine group.

Antibody level detection in, surface display bigeminy subunit vaccine stimulate mouse generate AfuA, CdtB, OppA, The antibody titer of OppA2 maintains an equal level with purifying protein subunit vaccine substantially, is superior to inactivated vaccine.This is because microorganism is anti- Former complicated, the antibody level generated for specific protein is limited, albumen such as AfuA, OppA involved in the present invention, OppA2 is not the surface protein of thallus, and it is also not high to be directed to the antibody level that these types of antigen generates for body after inactivated vaccine is immune.

In mice serum in terms of cytokines measurement, IL-4 is generated by the Th2 subgroup of mouse, related to humoral immunity. The horizontal a little higher than purifying protein subunit vaccine of the IL-4 that surface display bigeminy subunit vaccine stimulates mouse to generate, this be by In other than the foreign protein for being showed in phage surface, the thallus of the E.coil BL21 (DE3) as display platform can also swash Send out the humoral immunity of mouse.IL-4 in HPS-SS inactivated vaccine group mice serum is higher than other two groups, shows that HPS-SS bigeminy is gone out Live seedling can preferably excite humoral immune reaction strong in Mice Body.This is because the thallus in HPS-SS inactivated vaccine will not Cell is actively invaded, mainly excites mouse humoral immune as exogenous antigen.Surface display bigeminy subunit vaccine is by removing institute Outside the albumen of submission, also containing the thallus inactivated, therefore body can be also stimulated to generate more compared to purifying protein immune group IFN-γ.In short, the Streptococcus suis based on surface display-Haemophilus parasuis bigeminy subunit vaccine can excite mouse Strong immune response, haemophilus parasuis and Streptococcus suis to different serotypes attack malicious mouse and all have good friendship Protecting effect is pitched, and is better than traditional inactivated vaccine.Absolutely prove the surface display bigeminy subunit vaccine pair prepared in the present invention Haemophilus parasuis and Streptococcus suis have good protecting effect, are the novel recombination of Streptococcus suis-Haemophilus parasuis The research and development of subunit vaccine provide new approaches, to develop efficient, wide spectrum, cheap Streptococcus suis-Haemophilus parasuis Vaccine research provides basis.

Detailed description of the invention

The clone of Fig. 1 fbp, hp0197, mrp, rfeA, sly gene

M:DL2000；1:fbp；2:hp0197；3:mrp；4:rfeA；5:sly

The verifying of Fig. 2 recombinant plasmid double digestion

M:DL15000；1:pET-28a-FBP；2:pET-28a-HP0197；3:pET-28a-MRP；4:pET-28a- RfeA；5:pET -28a-SLY

The verifying of Fig. 3 protein expression

M: protein standard 26616；1:BL21 (DE3)-(pET-28a)；2:BL21 (DE3)-(pET-28a-FBP)；3: BL21(DE3)- (pET-28a-HP0197)；4:BL21 (DE3)-(pET-28a-MRP)；5:BL21 (DE3)-(pET-28a- RfeA)；6:BL21 (DE3)-(pET-28a-SLY)

The SDS-PAGE of Fig. 4 purifying protein is detected

M: protein standard 26616；1:rFBP；2:rHP0197；3:rRfeA；4:rMRP；5:rSLY.

The Western-blot of each protein expression of Fig. 5 is verified

Negative control:BL21 (DE3) (pMD-INP-His)；A:BL21 (DE3) (pMD-INP-AfuA)；B: BL21(DE3) (pMD-INP-OppA)；C:BL21 (DE3) (pMD-INP-OppA2)；D:BL21 (DE3) (pMD-INP-SLY)； E:BL21 (DE3) (pMD-INP-MRP)；F:BL21 (DE3) (pMD-INP-CdtB)；G:BL21 (DE3) (pMD-INP-CdtB- OppA)；H:BL21 (DE3) (pMD-INP-AfuA-OppA2)

The positioning of Fig. 6 subcellular components

Positioning scenarios of each albumen of Fig. 7 on film

A:BL21 (DE3) (pMD-INP-His)；B:BL21 (DE3) (pMD-INP-AfuA-OppA2)；C:BL21 (DE3) (pMD- INP-CdtB-OppA)；D:BL21 (DE3) (pMD-INP-AfuA)；E:BL21 (DE3) (pMD-INP-OppA2)；F: BL21 (DE3)(pMD-INP-CdtB)；G:BL21 (DE3) (pMD-INP-OppA)；H:BL21 (DE3) (pMD-INP-MRP)； I:BL21 (DE3) (pMD-INP-SLY)

Fig. 8 difference immune group serum cytokines testing result

The ELISA antibody titer of Fig. 9 difference albumen detects

Figure 10 challenge test result

A: vaccine attacks malicious result to HPS HN10；B: vaccine attacks malicious result to HPS ZD12；C: vaccine attacks poison to SS M126 As a result；D: vaccine attacks malicious result to SS GZ2.

Specific embodiment

Below by specific embodiment, the present invention is further illustrated, and to better understand the present invention, but it is not It is construed as limiting the invention.

The screening of one Streptococcus suis antigen protein of embodiment

1 Streptococcus suis genome extracts

The Streptococcus suis 05ZYH33 glycerol stock that laboratory is saved drew lines in THA culture medium (containing 10% horse serum), then It is placed in 37 DEG C of constant incubators overnight, picking single colonie is forwarded to quiet in incubator in THB culture medium (containing 10% horse serum) Set culture, then expand culture into 10mL THB culture medium (containing 10% horse serum), with bacterial genomes extracts kit according to Specification extracts bacterial genomes.The genome of extraction be placed in -20 refrigerators freeze it is spare.

2 construction of recombinant plasmid and verifying

According to gene order (accession number CP000407.1) design primer (table 2-1) of 05ZYH33 bacterial strain in GenBank. Using type strain 05ZYH33 genome as template, by table 2-2 response procedures carry out PCR, respectively amplifying target genes fbp, Hp0197, mrp, rfeA, sly, since PrimerSTAR Max DNA Polymerase has specificity height, reaction sensitivity Feature high, amplification efficiency is high, the characteristic that itself very high annealing efficiency shortens annealing and extension of time substantially, according to Specification, 95 DEG C of 10s of denaturation can be used to the PCR of target gene in this；Anneal 55 DEG C of 10s；Extend 72 DEG C of 30s, 30 are followed The response procedures of ring.Nucleic acid electrophoresis is carried out after the completion of PCR, as the result is shown the stripe size of fbp, hp0197, mrp, rfeA, sly Respectively 1668bp, 1693bp, 2267 bp, 566bp, 1497bp, meet expection.

DNA recycling is carried out according still further to plastic recovery kit specification after electrophoresis, by the system of table 2-3 by pET-28a plasmid With fbp, mrp, rfeA, sly NdeI, BamHI double digestion of recycling, hp0197 is subjected to double digestion, enzyme with NotI, NdeI It cuts after product carries out glue recycling and is attached by the system of table 2-4.Product is transformed into E.coli DH5 α competent cell, then Thallus is coated on the plate of LB (containing 50 μ g/ μ L Kan) and carries out resistance screening.The monoclonal of acquisition is seeded to LB respectively In (containing 50 μ g/ μ L Kan) fluid nutrient medium, after 37 DEG C of shaking table shake culture 12h, upgrading grain carries out sequencing identification.

Sequencing result shows recombinant plasmid pET-28a-FBP, pET-28a-HP0197, pET-28a-MRP, pET-28a- Gene order accords in segment fbp, hp0197, mrp, rfeA, sly sequence and GenBank being inserted into RfeA, pET-28a-SLY Conjunction rate is 100%, without any base mutation, it was demonstrated that genetic fragment is successively inserted on pET-28a carrier.By recombinant plasmid PET-28a-FBP, pET-28a-MRP, pET-28a-RfeA, pET-28a-SLY carry out double digestion with BamHI, NdeI, will weigh Nucleic acid electrophoresis is carried out respectively after group plasmid pET-28a-HP0197 NotI, NdeI double digestion, is separated after double digestion as the result is shown Segment and genetic fragment it is in the same size, further prove that this five genetic fragments are correctly cloned into pET-28a carrier respectively On.

The primer sequence of table 2-1 fbp, hp0197, mrp, rfeA, sly

Table 2-2

PCR reaction system

Table 2-3 endonuclease reaction system

Table 2-4 linked system

The expression and purification of 3 restructuring destination proteins

The correct recombinant plasmid of sequence verification is transferred in E.coli BL21 (DE3) competent cell, then thallus is coated with In carrying out resistance screening, picking recombinant bacterial strain pET-28a-FBP-BL21 (DE3), pET- on LB (contain 50 μ g/ μ L Kan) plate 28a-HP0197- BL21(DE3)、pET-28a-MRP-BL21(DE3)、pET-28a-RfeA-BL21(DE3)、pET-28a- The monoclonal of SLY-BL21 (DE3) is seeded to respectively in LB (containing 50 μ g/ μ L Kan) fluid nutrient medium, then is switched to three In pipe 10mL LB (containing 50 μ g/ μ L Kan) fluid nutrient medium, to bacterium solution OD₆₀₀The IPTG of final concentration of 1mM is added when ≈ 0.7, By each recombinant bacterium respectively 37 DEG C, 28 DEG C, 16 DEG C of progress it is protein induced, after 6h collect and by thallus ultrasonication and pass through Centrifuge separation precipitating and supernatant, have determined that each recombinant protein most by the expression of SDS-PAGE testing goal albumen Good inductive condition.

Thallus is collected after the recombinant bacterium of above-mentioned building is induced, appropriate progress SDS-PAGE is taken after resuspension and is walked around to PVDF Film carries out Western-blot by primary antibody of the His antibody of the mouse of commercialization, as a result as Fig. 3 is shown, it was demonstrated that albumen can be in weight It is expressed in group bacterium.

After inducing expression condition determines, recombinant bacterial strain pET-28a-FBP-BL21 (DE3), the pET-28a- that will newly draw lines HP0197- BL21(DE3)、pET-28a-MRP-BL21(DE3)、pET-28a-RfeA-BL21(DE3)、pET-28a-SLY- BL21 (DE3) is inoculated with and transfers expansion culture into 1L LB (containing 50 μ g/ μ L Kan) fluid nutrient medium, and 37 DEG C, 200rpm shakes To OD600 be 0.7 when, the IPTG of final concentration of 1mM is added, each recombinant bacterium is induced under its most suitable inductive condition.To After the completion of induction, thallus is collected, centrifuge separation bacterium supernatant and precipitating after ultrasonication is resuspended.Affine layer is carried out with Ni resin again Analyse purifying protein.

Being determined by experiment recombinant protein rFBP, rHP0197, rRfeA can be expressed in the form of soluble, rMRP, rSLY with The form of inclusion body is centrifuged in resulting precipitating after being present in ultrasound, the operation of repeatability after being denaturalized before purification.Benefit SDS-PAGE detection is carried out with Ni resin affinitive layer purification recombinant protein, and by recombinant protein, the results show that purifying RFBP, rHP0197, rMRP, rRfeA, rSLY size are respectively 63kDa, 54kDa, 110kDa, 19kDa, 58kDa, with expection It is consistent.

4 mouse susceptible strains and the selection for attacking toxic bacterial strain

Greater wax moth (Galleria mellonella) is one kind of galleria mellonella waxmoth, parasitizes honeycomb, is the evil of bee raising industry Worm.Some researches show that greater wax moth can be used as the model (Velikova, 2016) of SS separation strains virulence.Referring to the document, use Concrete operation step of the greater wax moth as animal experimental model evaluation SS2 separation strains virulence are as follows: by the Streptococcus suis of fresh cultured 2 type separation strains JMS74, M15, SW1206, MD15, YM23, M126, NM167, OG47, OG3, SU07 are diluted to bacterium solution OD₆₀₀= 0.5, bacterial concentration is 1 × 10 at this time⁸Cfu/mL draws 20 μ L bacterium solutions with micro syringe, greater wax moth is fixed back up, From first pair of right side, abdominal foot inserting needle rapidly injects bacterium solution.Constant incubator is put it by group after the completion of all injections, is seen Examine the survival state of greater wax moth in 72h.Normal greater wax moth is ecru, becomes sepia after dead.

ICR, C57BL/6, KM each 9 are taken, the mouse of each strain is further divided into 3 groups, every group 3.Divide three and attacks toxic dose Gradient, respectively 1 × 10⁸cfu/mL、2×10⁸cfu/mL、3×10⁸Cfu/mL attacks toxic bacterial strain and selects to greater wax moth toxicity most Strong bacterial strain.The survival state that mouse in one week is observed and recorded after attacking poison is injected intraperitoneally.

For the virulence for further verifying this 10 plants of separation strains, take virulence most strong to greater wax moth toxicity, intermediate, toxicity most weak The setting of three plants of bacterium, which attacks malicious gradient and carries out intraperitoneal injection to 8 week old KM mouse, attacks poison, counts the survival condition of mouse.

It is evaluated by the death rate of greater wax moth after the bacterium solution of observation injection 10 plants of streptococcus suis 2-types, 10 plants of clinical separation strains The virulence of this 10 plants of clinical separation strains show that this several plants of virulence are descending and is followed successively by M126 > JMS74 > M15 > NM167 > MD15 > SW1206 > SU07 > OG3 > OG47 > YM23.

Therefore selecting M126 is to attack toxic bacterial strain and screen to mouse species, from table 2-6 as can be seen that ICR relative to Other two kinds of mouse are to streptococcus relative tolerance.Compared to C57BL/c mouse, KM mouse weight is bigger, it is believed that KM mouse The toxic bacterial strain M126 that attacks against each other is more sensitive, and therefore, this test intended uses experimental animal of the KM mouse as protest test.

The best Screening of Strains Against result of table 2-6

5 immunoprotection challenge tests

For the immunogenicity for verifying rFBP, rHP0197, rMRP, rRfeA, rSLY, need to carry out protest test.It will The albumen of purifying emulsifies after mixing respectively with oil adjuvant ISA201VG by equal mass ratioes, makes weight in the vaccine after the completion of emulsifying Histone content is 500 μ g/mL.4 week old female KM mice (weight 21-24g) 60 is taken again, is randomly divided into 6 groups, every group 10, respectively rFBP+ISA201VG group, rHP0197+ISA201VG group, rMRP+ISA201VG group, rRfeA+ISA201VG Group, rSLY+ISA201VG group totally five immune groups and control group PBS+ISA201VG group, dorsal sc multi-point injection, 200 μ L/ Only.14d carries out secondary immunity, immunizing dose and mode after first time is immune and first time immunological phase is same.

Docking blood sampling is carried out to each group mouse when secondary immunity 14d, separation serum is spare.Simultaneously with streptococcus suis 2-type point It carries out attacking poison from strain, attacking toxic dose is 3 × 10⁸Cfu/ is only.Observe and count the Survival of mouse in one week.

Attacking malicious result from table 2-7 mouse can be seen that M126 plants of virulence is apparently higher than other two kinds, and three plants of bacterium really Virulence size it is consistent with the virulence evaluation result of bacterial strain in 2.3.5.This also demonstrate to a certain extent use greater wax moth as Evaluate the feasibility and reliability of pig streptococcus bacterial strain virulence.

Table 2-7 attacks toxic bacterial strain virulence verification result

It can be seen that the traditional immunoprotection type antigen MRP and SLY of Streptococcus suis for pig hammer from the result of table 2-8 2 type M126 bacterial strain of bacterium has excellent protecting effect, better than newfound protection type antigen FBP, RfeA in recent years, HP0197。

Table 2-8 challenge test result

The building and verifying of two surface display vector of embodiment

According to aforementioned experimental results, pig hammer is shown respectively in E.coli BL21 (DE3) phage surface in the present embodiment Bacterium antigen two foreign proteins of MRP and SLY, and using Haemophilus parasuis AfuA, CdtB, OppA, OppA2 by its It is showed in surface of E. coli and evaluates its immunogenicity by animal experiment.

The design of 1 relevant primer

Connector Linker is the 3 duplicate sequences for 5 amino acid GGGGS for separating two protein sequences, both ends Connector needs the base sequence complementary with albumen both ends.Linearized vector for clone will be with pMD-28a-INP-CAP-His Plasmid is that template reversely expands, and two terminal sequences are also required to complementary with the terminal base sequence of the connected albumen in two sides.With In GenBank based on HPS gene order (NC_011852.1) and the gene order (LS483418.1) of SS, designs and be used for Construct pMD-INP-CdtB-OppA, pMD-INP-AfuA-OppA2, pMD-INP-MRP, pMD-INP-AfuA, pMD-INP- Primer sequence (table 3-1) needed for OppA2, pMD-INP-CdtB, pMD-INP- OppA.Primer is by Jilin Ku Mei biotech firm Synthesis.

Table 3-1 list of primers

The building of 2 surface display vectors

With pET-28a-AfuA, pET-28a-OppA, pET-28a-OppA2, pET- of building early period being disclosed The pUC57- that 22b-CdtB, pET-28a-MRP, pET-28a-SLY of the building of preceding part and general biotech firm construct as required Linker plasmid is template, respectively in table 3-1 respective primer PCR amplifying target genes afuA, oppA2, cdtB, oppA, Mrp, sly and Linker for afuA-oppA2, Linker for cdtB-oppA, after gel extraction with afuA, oppA2, CdtB, oppA, Linker are that template carries out fusion DNA vaccine, construct fusion segment (its of afuA-oppA2, cdtB-oppA respectively In link peptide be pass through the better GGGGS of research effect, i.e. 4 glycine, a serine).Again with pMD-28a-INP- CAP-His is that template is reversely expanded for connecting afuA-oppA2, cdtB- respectively according to 14 to 28 primers listed in table 3-1 The linearized vector sequence of oppA, mrp, sly, afuA, oppA2, cdtB, oppA simultaneously carry out product recycling with kit.According to The PrimerSTAR Max DNA Polymerase operation instruction of manufacturer, denaturation can be used in all PCR in this test 95℃10s；Anneal 55 DEG C of 10s；Extend the response procedures of 72 DEG C of 30s.Then with a step connect kit by afuA-oppA2, CdtB-oppA, mrp, sly, afuA, oppA2, cdtB, oppA are attached with respective carrier referring to specification respectively to react, And it is transferred in E.coli DH5 α competent cell, then be coated on progress resistance sieve on LB (the 100 μ g/mL containing Amp) plate Choosing.Next day picking monoclonal is inoculated into 37 DEG C of shake cultures in LB (the 100 μ g/mL containing Amp) fluid nutrient medium, extracts after 12h Plasmid send to Jilin Ku Mei biotech firm and is sequenced.

The results show that Insert Fragment in 8 recombinant vectors and afuA-oppA2, cdtB-oppA, mrp, sly, afuA, The sequence matching degree of oppA2, cdtB, oppA are 100%, abasic site mutation, it was demonstrated that afuA-oppA2, cdtB- OppA, mrp, sly, afuA, oppA2, cdtB, oppA are correctly cloned into surface showing plasmid.

Correct 8 recombinant expression carriers of sequence verification are transferred to respectively in E.coli BL21 (DE3) competent cell, Resistance screening is carried out, it is spare to select positive colony guarantor bacterium.

The expression of 3 surface display bacterial strains is verified

Take the BL21 (DE3) (pMD-INP-His) newly to draw lines, BL21 (DE3) (pMD-INP-CdtB-OppA), BL21 (DE3)(pMD-INP- AfuA-OppA2)、BL21(DE3)(pMD-INP-MRP)、BL21(DE3)(pMD-INP-SLY)、BL21 (DE3)(pMD-INP-AfuA)、BL21 (DE3)(pMD-INP-OppA2)、BL21(DE3)(pMD-INP-CdtB)、BL21 (DE3) (pMD-INP-OppA) recombinates the monoclonal of surface display bacterium, expands culture to 10mL LB (containing Amp final concentration of 100 μ g/mL) in fluid nutrient medium, 37 DEG C of 200r/min shake to OD₆₀₀The inducer that final concentration of 1mM IPTG is added when ≈ 0.6 exists 6000r/min is centrifuged 10min and collects thallus after 37 DEG C of induction 4h, freezes spare.

The appropriate PBS of the thallus being collected by centrifugation after inducing expression is hanged, loading carries out after adding loading-buffer SDS-PAGE is then switched into and is carried out Western-blot on pvdf membrane, and the BL21 (DE3) for adding empty carrier is used as negative control, It is anti-with the Mouse Polyclonal of haemophilus parasuis antigen protein AfuA, CdtB, OppA2, OppA of laboratory pre-production respectively The Mouse Polyclonal Antibody of MRP, SLY for being obtained when Streptococcus suis antigen protein screens in body and this research and the mouse of commercialization Anti- His antibody is primary antibody, mountain sheep anti-mouse igg (marking containing Dylight 680) is that secondary antibody carries out Western-blot detection correlation The expression of albumen.

The results show that being showed in foreign protein AfuA-OppA2, CdtB- of E.coli BL21 (DE3) phage surface OppA, MRP, SLY, AfuA, CdtB, OppA2, OppA can be with its more anti-bindings.Wherein the source of mouse of MRP and CdtB mostly it is anti-also with Bacteria suspension after empty carrier induction reacts.The source of mouse His antibody of commercialization is sent out with the His label on corresponding albumen Raw reaction, and the bacteria suspension with BL21 containing empty carrier (DE3) (pMD-INP-His) does not react.Therefore, proposed adoption commodity The source of mouse His antibody of change carries out protein subcellular positioning and immuno-electron microscope test as primary antibody.

The separation of 4 subcellular components

Western-blot verification experimental verification E.coli BL21 (DE3) can express recombinant protein, but only be showed in thallus The foreign protein on surface just can produce best immune effect.Therefore, foreign protein is in the expression portion of E.coli BL21 (DE3) Position needs further verifying.

The monoclonal of each bacterium newly to draw lines is taken, expands culture to 200mL LB (the final concentration of 100 μ g/mL containing AMP) liquid In culture medium, to OD₆₀₀IPTG is added when ≈ 0.6 in 37 DEG C of induction 4h, bacterium subcellular separating step is as follows:

(1) thallus is washed 3 times with PBS, removes culture solution ingredient, is resuspended in the 0.1M PMSF solution of 10mL, ice-water bath Ultrasound is carried out after 15min, effective ultrasonic time is 8-10min.

(2) it is centrifuged 10min in 4 DEG C of 3000g after ultrasound, discards precipitating, take supernatant, 4 DEG C of 27000g are centrifuged 1h.In collection Clearly.

(3) 27000g centrifuged deposit is resuspended in TE buffer, 200r/min shakes in 37 DEG C of constant-temperature tables It is centrifuged 1h in 4 DEG C of 27000g after 40min, gained precipitating is cell wall constituent, is resuspended in the NH of 10mM₄HCO₃In solution It saves.

(4) supernatant that the centrifugation of the 2nd, 3 steps obtains is mixed, 4 DEG C of 100,000g are centrifuged 4h, gained supernatant be cytoplasm at Point, sediment fraction is cell membrane component, is resuspended in the NH of 10mM₄HCO₃It is saved backup in solution.

(5) cytoplasm of 8 plants of recombinant bacteriums separation, cell wall, cell membrane component sampling are subjected to SDS-PAGE, it is anti-with His Body as primary antibody, secondary antibody with the mountain sheep anti-mouse igg that Dylight 680 is marked carry out it is infrared sweep film, observe recombinant protein in thallus Positioning on subcellular structure.

Each recombinant bacterium separation after inducing expression is obtained into cytoplasm, 3 kinds of subcellular components of cell membrane and cell wall carry out Western-blot analysis, as a result (Fig. 6) is shown, different foreign proteins table on the cytoplasm of thallus, cell membrane and cell wall The relative abundance reached is different, can navigate on mantle and cell membrane.

5 immuno-electron microscopes

The monoclonal of each bacterium newly to draw lines is taken, expands culture to 10mL LB (the final concentration of 100 μ g/mL containing Amp) liquid and trains It supports in base, to OD₆₀₀IPTG is added when ≈ 0.6 induces 4h in 37 DEG C of 180r/min.Sample is sent to Harbin animal doctor after receiving bacterium Research institute's Electron Microscope Laboratory carries out immuno-electron microscope microsection manufacture and observation.

The immuno-electron microscope experimental result of Fig. 7 is shown: BL21 (DE3) (pMD-INP) phage surface containing empty carrier does not occur There is colloid gold particle shown in red arrow (Fig. 7) in colloid gold particle, other each phage surfaces.Immuno-electron microscope result is simultaneously It not can determine that gene expression abundance of the albumen on film, can only intuitively show that the recombination surface display vector of this test building can be with E.coli BL21 (DE3) be carrier by foreign protein AfuA-OppA2, CdtB-OppA, MRP, SLY, AfuA, CdtB, This 8 albumen of OppA2, OppA are showed in its surface respectively.

Three animal experiment of embodiment

1 vaccine preparation

1.1 surface display bigeminy subunit vaccines

(1) it shakes bacterium: taking the glycerol stock of the surface display recombinant bacterial strain built flat respectively at LB (the 100 μ g/mL containing Amp) It draws lines on plate, is statically placed in 8h in 37 DEG C of constant temperature incubators.Next day picks them separately monoclonal and is inoculated in LB (the 100 μ g/mL containing Amp) liquid In body culture medium, respectively at 37 DEG C, 180r/min shake culture, then 500mL phase is forwarded to respectively with the ratio of 1:100 respectively It answers in culture medium, 180r/min shakes in 37 DEG C of constant-temperature tables.

(2) receive bacterium, inactivation: after aseptically receiving bacterium and washing 3 times with PBS plus appropriate PBS is resuspended, and working concentration is added Inactivate thallus for 1.5 ‰ formaldehyde, coated plate checks whether that inactivation is complete after 48h.

(3) quantitative: transferring film carries out Western-blot after taking 8 kinds of bacterium solutions after being resuspended to carry out SDS-PAGE, with commercialization Source of mouse His antibody is primary antibody, and mountain sheep anti-mouse igg (label of Dylight 680) is secondary antibody, with known concentration with His label Albumen is that standard items carry out western-blot.Image gray analysis software I mageJ is in resuspended bacterium solution after film will be swept The albumen of rAfuA-OppA2, rCdtB-OppA, rAfuA, rOppA2, rCdtB, rOppA, rMRP, rSLY are quantified.

(4) prepared by vaccine I: taking BL21 (DE3) (pMD-INP-CdtB-OppA), BL21 (DE3) (pMD- after being resuspended INP-AfuA- OppA2), BL21 (DE3) (pMD-INP-MRP), BL21 (DE3) (pMD-INP-SLY) bacteria suspension mixing, then It is emulsified after mixed bacterium solution and ISA201VG are mixed in the ratio of equal mass ratioes, makes after emulsification rAfuA- in vaccine The content of OppA2, rCdtB-OppA, rMRP, rSLY are respectively 0.3mg/mL.This is surface display bigeminy subunit vaccine I, Contain fusion protein rAfuA-OppA2, rCdtB-OppA and rMRP, rSLY.

(5) prepared by vaccine II: taking BL21 (DE3) (pMD-INP-MRP), BL21 (DE3) (pMD-INP- after being resuspended SLY)、 BL21(DE3)(pMD-INP-AfuA)、BL21(DE3)(pMD-INP-OppA2)、BL21(DE3)(pMD-INP- CdtB), the bacteria suspension mixing of BL21 (DE3) (pMD- INP-OppA), then by mixed bacterium solution and ISA201VG by etc. quality It is emulsified after the ratio mixing of ratio, makes after emulsification containing for rAfuA, rOppA2, rCdtB, rOppA, rMRP, rSLY in vaccine Amount is respectively 0.3mg/mL.This is surface display bigeminy subunit vaccine II, contains rAfuA, rOppA2, rCdtB, rOppA With rMRP, rSLY.

1.2 purifying protein bigeminy subunit vaccines

Purified albumen rAfuA, rOppA2, rCdtB, rOppA, rMRP, rSLY are taken, is mixed, by mixed albumen Solution emulsifies after mixing with mass ratioes such as ISA201VG, make emulsification after vaccine in rAfuA, rOppA2, rCdtB, rOppA, The concentration of rMRP, rSLY are 0.3mg/mL.

1.3 dyad inactivated vaccine

Haemophilus parasuis Serotype 5 HN10 bacterial strain and 13 type ZD12 bacterial strain of serum is taken to draw lines in TSA (containing 10 μ g/mL NAD+5% horse serum) in solid medium, take 2 type M126 bacterial strain of Streptococcus suis serum and 9 type GZ2 bacterial strain of serum draw lines in In THA (containing 10% horse serum) solid medium, it is placed in 37 DEG C of constant temperature incubators overnight.Picking HN10 plants, ZD12 plants of next day Monoclonal is inoculated in TSB (containing 10 μ g/mL NAD+5% horse serums) fluid nutrient medium, HN10 plants, ZD12 plants monoclonals of picking Be inoculated in THA (containing 10% horse serum) fluid nutrient medium, respectively after 37 DEG C, 200r/min shake culture 12h, respectively with The ratio of 1:100 is successively forwarded in the corresponding culture medium of 500mL, and 200r/min shakes in 37 DEG C of constant-temperature tables.The secondary bloodthirsty bar of pig Bacterium overnight incubation, Streptococcus suis cultivate 4h.4 kinds of bacterium solutions take 100 μ L gradient dilutions to be coated on respective plate respectively, to 4 kinds Bacterium carries out bacterium colony counting.After aseptically receiving bacterium and washing 3 times with PBS plus appropriate PBS is resuspended, and working concentration, which is added, is 1.5 ‰ formaldehyde inactivates thallus, and coated plate checks whether that inactivation is complete after 48h.4 kinds of bacterium solutions and adjuvant are pressed again and wait mass ratioes Mode mix after emulsify, make after emulsification containing the concentration of 4 kinds of bacterium to be respectively 1 × 10¹⁰cfu/mL。

2 mouse immuning tests

4 week old female KM mice 200 is taken, is randomly divided into five groups, every group 40, according to the Immunization programme of table 4-1 to small Mouse is immunized.Carried out after 14d is immunized in first time second immune, immunizing dose and approach and first time immunological phase are same. 14d carries out all mouse to cut tail blood sampling after immune for the second time, and separation serum and packing freeze spare.

Table 4-1 immunization protocol

Cytokines measurement in 3 mice serums

The step of according to the specification of mice serum IL-2, IL-4 and IFN-γ reagent box for detecting content, two are detected respectively The content of three kinds of IL-2, IL-4 in mice serum after secondary immune 14d, IFN-γ cell factors.

The IL-4 that II group of stimulation mouse of the vaccine I group of surface display as the result is shown and surface display vaccine of Fig. 8 generates is to go out The 1/4 of live seedling group, it is little with purifying protein subunit group gap；The amount of IFN-γ is higher than purifying protein subunit group, but is lower than Inactivated vaccine group；The content of IL-2 is not high in each group mice serum.

Antibody level detects in 4 mice serums

By six kinds of albumen rAfuA, rOppA2, rCdtB, rOppA, rMRP, rSLY of purifying according to the concentration of every 0.1 μ g of hole It is coated in elisa plate, carries out indirect ELISA test with the elisa plate being coated with.Each immune group mice serum gradient is dilute Detect the antibody level of AfuA, OppA2, CdtB, OppA, MRP, SLY in each immune group mice serum after releasing as primary antibody, two The anti-goat anti-mouse IgG antibodies marked with HRP, are developed the color with TMB developing solution, read OD with microplate reader₄₅₀Numerical value.With sample OD₄₅₀Value >=negative control OD₄₅₀Value+0.2 is Positive judgement standards.The highest extension rate that serum reaches positive value is the blood Clearly to the potency of a certain antibody.

Surface display subunit vaccine I group and the II group of generation of surface display vaccine resist each albumen to Fig. 9 as the result is shown Body is on close level, with protein purification subunit vaccine group quite and be superior to inactivated vaccine group.

5 challenge tests

After second immune 14d, the mouse of each immune group is randomly divided into 4 groups again, every group 10, respectively with fresh HN10 plants of the haemophilus parasuis Serotype 5 of culture, ZD12 plants of 13 type of serum and M126 plants of 2 type of Streptococcus suis serum, serum 9 GZ2 plants of bacterium solutions of type carry out intraperitoneal injection to mouse in isolator and attack poison, attack toxic dose are as follows: HN10 strain 4 × 10⁹Cfu/, ZD12 strain 1 × 10⁹Cfu/, M126 strain 1.5 × 10⁸Cfu/, GZ2 strain 5 × 10⁷Cfu/ is only.It then observes and counts and attack poison The survival condition of mouse in latter week.

The results are shown in Figure 10, the control group of immune PBS+ISA201VG with after attacking malicious HN10, ZD12, M126, GZ2 Dead in 2d, 3d, 2d, 4d, surface display subunit vaccine I attacks the protective rate of malicious mouse to HN10, ZD12, M126, GZ2 It is 80%, 80%, 50%, 100%；Surface display vaccine II is to HN10, ZD12, M126, GZ2 protective rate for attacking malicious mouse 90%, 60%, 20%, 80%；Protein purification subunit vaccine is to HN10, ZD12, M126, GZ2 protective rate for attacking malicious mouse 90%, 60%, 100%, 80%；Inactivated vaccine to HN10, ZD12, M126, GZ2 attack malicious mouse protective rate be 80%, 60%, 50%, 60%.

Sequence table

<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture is (in China Animal Health and Epidemiology Center Harbin point The heart), Harbin Weike Biologic Technology Ltd.

<120>Streptococcus suis-Haemophilus parasuis bigeminy subunit vaccine and preparation method

<160>38

<170> PatentIn Version 3.1

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<211>25

<212>DNA

<213>artificial sequence

<400> 32

acgggtaaag gttgtcattt gggtc 25

<210>33

<211>22

<212>DNA

<213>artificial sequence

<400> 33

tttacggctt actcactcga gc 22

<210>34

<211>26

<212>DNA

<213>artificial sequence

<400> 34

acctgcaact aatttcattt gggtct 26

<210>35

<211>25

<212>DNA

<213>artificial sequence

<400> 35

cgtcgtaacg aagattcact cgagc 25

<210>36

<211>27

<212>DNA

<213>artificial sequence

<400>36

actttccttg aaaaccattt gggtctg 27

<210>37

<211>26

<212>DNA

<213>artificial sequence

<400> 37

gatgaggtga tagagtcact cgagca 26

<210>38

<211>27

<212>DNA

<213>artificial sequence

<400>38

aatatcttgt ttggacattt gggtctg 27

Claims

1. a kind of Streptococcus suis-Haemophilus parasuis bigeminy subunit vaccine, which is characterized in that it contains the bloodthirsty bar of secondary pig Bacterium antigen protein AfuA, OppA2, CdtB, OppA and Streptococcus suis antigen MRP and SLY；Preferably containing the bloodthirsty bar of secondary pig Bacterium fusion protein AfuA-OppA2, two kinds of antigens of fusion protein CdtB-OppA and Streptococcus suis antigen protein MRP and SLY two Kind antigen.

2. Streptococcus suis according to claim 1-Haemophilus parasuis bigeminy subunit vaccine, which is characterized in that Each antigen protein is respectively submission expression-form.

3. Streptococcus suis according to claim 2-Haemophilus parasuis bigeminy subunit vaccine, which is characterized in that Respectively by the gene constructed at surface submission expression vector of encoding said proteins, and then it is prepared into the recombination of surface submission expression Bacterium, mixing obtains the vaccine after culture obtains bacterium solution, and further preferred surface submission expression vector is that pMD is load of setting out Body.

4. Streptococcus suis according to any one of claims 1 to 3-Haemophilus parasuis bigeminy subunit vaccine, It is characterized in that, wherein the concentration for each antigen protein when in use, respectively 0.1-0.5mg/mL, preferably 0.2-0.4mg/ ML, most preferably 0.3mg/mL.

5. Streptococcus suis according to any one of claims 1 to 3-Haemophilus parasuis bigeminy subunit vaccine, It is characterized in that, further includes adjuvant, preferably oil-in-water adjuvant, polymer and water adjuvant, water-in-oil adjuvant, aluminium hydroxide assistant Agent, vitamin E adjuvant, more preferably ISA201VG.

6. Streptococcus suis according to claim 3-Haemophilus parasuis bigeminy subunit vaccine, which is characterized in that The recombinant bacterium is gram-positive bacteria or Gram-negative bacteria, more preferably BL21 (DE3) bacterium.

7. Streptococcus suis according to claim 3-Haemophilus parasuis bigeminy subunit vaccine, which is characterized in that It is prepared into and is suitable for through intramuscular, intradermal or subcutaneous administration dosage form, the preferably dosage form of subcutaneous administration.

8. Streptococcus suis according to any one of claims 1 to 3-Haemophilus parasuis bigeminy subunit vaccine, It is characterized in that, it is therein for two kinds of haemophilus parasuis fusion protein AfuA-OppA2, fusion protein CdtB-OppA antigens Link peptide is GGGGS.

9. Streptococcus suis according to any one of claims 1 to 8-Haemophilus parasuis bigeminy subunit vaccine system Preparation Method, which is characterized in that respectively by the gene constructed at surface submission expression vector of encoding said proteins, and then be prepared into and contain Mixing obtains the vaccine after having the recombinant bacterium of the expression vector, culture to obtain bacterium solution.

10. a kind of Streptococcus suis according to claim 9-Haemophilus parasuis bigeminy subunit vaccine preparation side Method, which is characterized in that after to be preferably ISA201VG mixed in the ratio of equal mass ratioes with the bacterium solution, emulsify by adjuvant To the vaccine.