CN1912106A - Dysentery multivalent genetic engineering vaccine and preparation method thereof - Google Patents

Abstract

The invention discloses dysentery polyvalence gene engineering vaccine. Its active constituent can express not only affecting albumen antigen IpaA, IpaB, IpaC, IpaD, but also fushi and songneishi double-valence LPS-O polysaccharide antigen. Its advantages are that it has good polyvalence immunogenicity to protect immunized mouse and monkey, resist S.F2a and S.sonnei; it can reduce immunizing dose, offer polyvalence protection to effectively defend bacillary dysentery; and it can be produced in large scale.

Description

A kind of dysentery multivalent genetic engineering vaccine and preparation method thereof

Technical field

The present invention relates to a kind of dysentery multivalent genetic engineering vaccine and preparation method thereof in the field of biological pharmacy.

Background technology

Whole world dysentery year, morbidity example surpassed 2.5 hundred million, and wherein 5,000,000 examples need hospital care, and is year dead about 2,000,000, and the report data surplus in the of ten year is growing on and on.Mainly be popular in developing country.Dysentery is common and multiple transmissible disease, and sickness rate is positioned at preceding 3 of 24 kinds of Notifiable diseases all the time, and downtrending is year by year arranged, and at present the annual report case is about about 1,000,000, and the actual case number surpasses several times.Morbidity is based on infant and person between twenty and fifty, and old man and children are more common in death, and main popular type is Fu Shi and Song Nei Shi.Though dysentery has pharmacological agent, very easily transfer to chronicly, carrying disease germs for a long time becomes potential contagium, and the resistance problem is very serious.The oral rehydration that WHO recommends is to being that the dysentery of main pathogenic characteristic is invalid with the invasion and attack intestinal epithelial cell, especially because of influence of dysentery diarrhoea and obstacle infant normal development, influence throughout one's life, and more and more evidences illustrates that dysentery infection is relevant with autoimmune disease.Especially in recent years, China urban floating population increases severely, and is engaged in food and the processing number increases, and this part people's inhabitation and work under bad environment, all to the propagation of dysentery and popularly provide control environment, popular approach and responsive crowd.

Shigella (Shigella) is a class gram negative bacillus, is human bacillary dysentery common pathogenic bacteria the most, the common name dysentery bacterium.K and O antigen are arranged and do not have H antigen.K antigen is the somatic surface antigen from new isolating some bacterial strain of patient, and is thermo-labile, heat 100 ℃ 1 hour destroyed.K antigen is meaningless on serological typing, but can stop O antigen and corresponding sero-fast agglutination reaction.O antigen is divided into group specific antigen and type specific antigen, and the former is everlasting and occurs between several proximate bacterial classifications; The specificity height of type specific antigen is used to distinguish bacterial type.According to the difference of Shigellae antigen construct, can be divided into 48 serotypes of four group (comprising hypotype):

(1) A group: claim shigella dysenteriae (Sh.dysenteriae) again, the common name dysentery bacterium.Nonfermented N.F,USP MANNITOL.12 serotypes are arranged, and wherein 8 types are divided into three hypotypes again.

(2) B group: claim shigella flexneri (Sh.flexneri) again, the common name shigella flexneri.Fermentation N.F,USP MANNITOL.15 serotypes (containing hypotype and mutation) are arranged, and the antigen construct complexity has group antigen and type antigen.According to the antigenic difference of type, be divided into 6 types, the difference according to group antigen is divided into hypotype with type again; X, Y mutation do not have specific antigens, and different group antigens is only arranged.

(3) C group: claim Shigella bogdii (Sh.boydii) again, common name Bao Shi dysentery bacterium.Fermentation N.F,USP MANNITOL has 18 serotypes, various no cross reaction.

(4) D group: claim bacillus ceylonensis A (Sh.sonnei) again, the common name sonne bacillus.Fermentation N.F,USP MANNITOL, and delayed reaction lactose generally need 3～4 days.Has only a serotype.Two variation phases are arranged, promptly I mutually with II mutually; I is the S type mutually, and II is the R type mutually.

Invasin protein antigen (Ipas), be the common invasin protein antigen (Ipas of each group dysentery bacterium, comprise IpaA, IpaB, IpaC, IpaD), IpaA, IpaB, IpaC, IpaD are the protein that has amino acid residue sequence shown in (1,2,3 and 4) in the sequence table respectively, and their encoding gene is respectively shown in sequence in the sequence table (5,6,7 and 8).

The LPS-O polysaccharide antigen, the outside that promptly is present in dysentery bacterium muramyl peptide glycan is the structure of adventitia.It is the same with most of Gram-negative bacterias, and mainly by lipoid A, core polysaccharide and O-specific side chains (O antigen) are formed, and with intestinal bacteria very big similarity are arranged.Chemical analysis shows that the lipoid A of Salmonella LPS is identical with bacillus coli gene in Fu Shi and the Song, and the O specific side chains of LPS is the main position of decision serotype, is the important pathogenic factor and protective antigen of dysentery bacterium.

Countries such as U.S., method, day, Australia, Sweden, English, Soviet Union, India drop into a large amount of manpowers for a long time, financial resources are carried out shigella vaccine and corresponding fundamental research thereof, and dysentery bacterium virulence and morbific recent advances in research of molecular mechanism have promoted the development and the development of shigella vaccine of new generation.The method and the approach of development dysentery bacterium seedling mainly contain at present:

1, make up hybrid strain: how with Salmonellas such as Ty21a, or the E.coli bacterium is the acceptor strain, accepts the hybrid strain that Song Nei Shi or Fu Shi 2a dysentery bacillus plasmid or chromosomal function fragment make up.As in Song-the Ty21a hybrid strain, human body oral 10 ¹⁰Cfu safety; attack to Song Nei Shi wild strain has 40% protection effect, but between its freeze-drying prods batch fluctuation is arranged, its reason be Song Nei Shi LPS-O side chain can not with the cause of the LPS center sugar chain stable bond of Ty21a; improve, but do not see the report of success.

2, make up the virulence gene attenuation mutant: be primarily aimed at the functional gene of sending out inside and outside dysentery bacillus breeding and the born of the same parents such as the gene of the arobactin on VirG (icsA) and regulatory gene or the karyomit(e), insert sudden change, make this bacterial strain can invade host cell, but can't effectively breed, on animalcule and monkey body, experimentize and do not seen the anthroposcopy effect.

3, make up the auxotroph of dysentery bacillus strain: make Y type dysentery bacillus because aroD gene disruption or disappearance, obtain Fu Shi dysentery attenuated strain, it can invade host cell, but can not effectively breed in born of the same parents, there are 2 strains to carry out volunteer's experiment, do not test on a large scale but be embedded in human body.

Above-mentioned three class vaccines have the human immunology observes except that 2 strain aroD auxotrophy bacterial strains, and other does not see the report of human body II, III phase clinical observation result.Carry out the aroD disappearance and the 2 strain attenuated strains that successfully construct carry out tens routine volunteer's safety respectively and attack experiment from F2a2457 strain and Fu Shi Y type, think oral 10 * 10 ⁸Cfu is safer, and the experimenter 44% finds positive serum antibody, and side reaction is relevant with clothes seedling dosage, it is said that Y type attenuated strain Δ aroD124 is effectively on-the-spot in Vietnam.

4, the U.S. carries out crosslinked being called with dysentery bacillus LPS polysaccharide and albumen such as CT-B and combines seedling in recent years, and it is effective to carry out the animalcule experimental observation, observes also effectively at the on-the-spot human of Vietnam, but does not form goods.

Obtain national a kind new medicine certificate in 1998 and produce the dysentery bivalent gene engineering vaccine (bacterial strain FSM2117) of criticizing code; can express the two valency LPS-O polysaccharide antigens of Fu Shi and Song Nei Shi simultaneously; through different animals models such as mouse, cavy, rabbit and monkey proof dysentery bivalent gene engineering vaccine oral immunity animal safety, two valency immunogenicities, two valency protection activity and stable are arranged.And the on-the-spot on a large scale use of the crowd that is further used for, the oral dysentery bivalent gene engineering vaccine of reference safety has two valency immunogenicities and immune protective effect.And obtain national 1 class new biological product certificate and country trial production code in December, 1998.But before this dysentery bivalent gene engineering vaccine lyophilisate is oral earlier in the oral sodium bicarbonate and hydrochloric acid in gastric juice, and oral dose reaches the very good not enough problem of protection effect greatly, and influences immune protective effect.

The innovation and creation content

The purpose of this invention is to provide a kind of dysentery multivalent genetic engineering vaccine.

Dysentery multivalent genetic engineering vaccine provided by the present invention, its activeconstituents are both to have expressed invasin protein antigen I paA, IpaB, and IpaC and IpaD express the dysentery multivalent genetic engineering vaccine strain of the two valency LPS-O polysaccharide antigens of Fu Shi and Song Nei Shi again.

The production substratum of described dysentery multivalent genetic engineering vaccine is that pH is Hou Shi substratum or the LB substratum of 6.5-7.6.The pH of described production substratum is preferably 7.2-7.5.

Described Hou Shi substratum is prepared as follows: fresh beef 7500g adds 11250ml distilled water and boils 40min, be cooled to 45 ℃ of fresh pig pancreas 1500mg that add homogenate, transfer pH8.0, and adding trichloromethane 200ml, digestion 7d, with preceding 4 times of dilutions, and add sodium-chlor 0.5%, transfer pH6.5-7.6 standby.

The LB substratum is grouped into by following one-tenth: peptone 10g, yeast leach liquor 5g, sodium-chlor 10g, add water to 1000ml, and wherein the pH of institute is preferably 7.2-7.5.

The formulation of described dysentery multivalent genetic engineering vaccine is two kinds of oral capsule formulation or form of nose drops.

The dysentery multivalent genetic engineering vaccine of described formulation can adopt lyophilization to be prepared.

The lyophilized vaccine that is adopted in the dysentery multivalent genetic engineering vaccine process of the described formulation of preparation is for containing 0.5% gelatin, the pH7.0-7.6 phosphoric acid buffer of 5% sucrose, 0.1% vitamins C, 0.1% thiaminogen, 0.1% skim-milk etc.; Described percentage composition is the quality percentage composition.

Second purpose of the present invention provides a kind of method for preparing above-mentioned dysentery multivalent genetic engineering vaccine strain.

The method of the above-mentioned dysentery multivalent genetic engineering vaccine strain of preparation provided by the present invention may further comprise the steps:

1) will contain invasin protein IpaA, IpaB, the I of IpaC and IpaD gene plasmid mutually transfer among the shigella flexneri 2a type II, obtain containing the reorganization shigella flexneri 2a type II of described I phase plasmid;

2) will contain aroD genetically deficient among the reorganization shigella flexneri 2a type II of described I phase plasmid, both expressed invasin protein antigen I paA, IpaB, IpaC and IpaD express the dysentery multivalent genetic engineering vaccine strain of the two valency LPS-O polysaccharide antigens of Fu Shi and Song Nei Shi again.

The described invasin protein IpaA that contains, IpaB, the I of IpaC and IpaD gene plasmid mutually comes from Song Nei Shi dysentery bacterial strains such as Song Nei Shi dysentery bacillus strain 48025-11 or S63.This strain provides the I of 120-140MD mutually big plasmid, and invasin protein and invasion and attack phenotype are all positive (to have four kinds of invasin protein IpaA, IpaB, IpaC and IpaD, the Hela cell is invaded positive, the contact hemolysis positive, the Congo red test positive, binding film test in cavy angle is positive).

The described invasin protein IpaA that contains, IpaB, the I of IpaC and IpaD gene plasmid mutually transfer among the shigella flexneri 2a type II by luring kinoplaszm grain R386.

The described kinoplaszm grain R386 that lures comes from E.coli.This bacterial strain of E.coli provides and lures kinoplaszm grain R386 and selected marker Kan (kantlex) resistance.

Described shigella flexneri 2a type II can be shigella flexneri 2a type II T32 bacterial strain.

Dysentery multivalent genetic engineering vaccine of the present invention has increased the total invasin protein antigen (Ipas comprises IpaA, IpaB, IpaC, IpaD) of each group dysentery bacterium than dysentery bivalent gene engineering vaccine.Oral or the collunarium approach immunity through different experiments animal (mouse and monkey); result's proof is by the 0th; 3; 6 Wednesdays time oral or collunarium immune programme for children; mouse oral 0.1 hundred million; rhesus monkey oral 0.5 hundred million; human oral 1.0 hundred million or mouse collunarium 0.001 hundred million; rhesus monkey collunarium 0.005 hundred million; the dysentery multivalent genetic engineering vaccine of people's collunarium 0.01 hundred million; can produce good multivalent immunogenic; can protect immune mouse; rhesus monkey; the people effectively resists the attack of S.F2a and S.sonnei strain; and the oral route immune protective effect than dysentery bivalent gene engineering vaccine improves nearly 30%; collunarium approach immune protective effect improves more than 30%; can both effectively induce the two valency specific IgA of mucous membrane; the antibody response of IgG excrement and saliva; the immunoprotection of dysentery bacterium polyvalent antigen can be provided; and carry out pilot scale production and in 5 provinces; the city; people's on-site assessment surplus the area 30,000 can fine effective prevention bacillary dysentery.

Description of drawings

Fig. 1 is the building process synoptic diagram of dysentery multivalent genetic engineering vaccine strain of the present invention

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

Embodiment 1. dysentery multivalent genetic engineering vaccine strains make up, screen and identify

1, the structure of dysentery multivalent genetic engineering vaccine strain and screening

(1) structure of dysentery multivalent genetic engineering vaccine strain

The building process of dysentery multivalent genetic engineering vaccine strain as shown in Figure 1, concrete grammar is as follows:

Select Song Nei Shi dysentery bacillus strain 48025-11 (to purchase in Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL seed culture of viruses storehouse, provide the mutually big plasmid pSS120 of I of 120-140MD), express the IpaA relevant with invasion and attack, B, C, four invasin protein bands of D have the HeLa cell and invade, Congo red test, the invasion and attack phenotype such as contact hemolysis and the cavy angle binding film virulence test positive.Intestinal bacteria E.coli (R386::Tn5, Kan ^r25ug/ml purchases in capital children's hospital), the selected marker that lures kinoplaszm grain R386 and screening kalamycin resistance is provided.At first will lure kinoplaszm grain R386 to forward among the dysentery bacillus strain 48025-11 by the method for conjugal transfer, concrete grammar is as follows: the intestinal bacteria E.coli that providing of getting that glycerin bouillon preserves lures kinoplaszm grain R386 and each 50 μ 1 of dysentery bacillus strain 48025-11 are inoculated in respectively and contain and do not contain in the 5ml LB meat soup of kantlex, 37 ℃ of shaking culture 15 hours, centrifugal 10 minutes of 7000rpm, remove supernatant, lawn suspends with 0.2ml LB meat soup respectively, two bacterium are applied to the LB flat board after mixing, include Kan (kantlex) 50ug/ml, 37 ℃ of incubators were cultivated 30 hours, grow bacterium colony and dysentery Song Nei Shi standard serum is positive, continuously containing on the LB flat board of kantlex purifying 3 times, the gained bacterium colony is both for containing the dysentery bacillus strain 48025-11 that lures kinoplaszm grain R386.

By luring luring of kinoplaszm grain R386, the mutually big plasmid of I is transferred to Fu Shi 2a shigella vaccine T32 (expression type II, group 3,4 antigen Str (Streptomycin sulphate) ^r50ug/ml, Ipa-purchases in Lanzhou Institute of Biological Products) in, concrete grammar is as follows: the 48025-11 that glycerin bouillon is preserved (contains the mutually big plasmid of I and lures kinoplaszm grain R386Kan ^r) and Fu Shi 2a shigella vaccine T32 (Str ^r) respectively get 50 μ l and be inoculated in respectively and contain Kan (kantlex) 50ug/ml and containing in the 5ml LB meat soup of Str (Streptomycin sulphate) 50ug/ml, 37 ℃ of shaking culture 15 hours, 7000rpm removed supernatant in centrifugal 10 minutes, lawn suspends with 0.2ml LB meat soup respectively, two bacterium are applied to the LB flat board after mixing, include Kan (kantlex) 50ug/ml, Streptomycin sulphate 50ug/ml, 37 ℃ of incubators were cultivated 30 hours, grow bacterium colony and dysentery Song Nei Shi and Fu Shi 2a type II and group 3,4 standard serums are positive, continuously containing on the LB flat board of kantlex and Streptomycin sulphate purifying 3 times, the gained bacterium colony both had been the purpose bacterium colony, and this purpose bacterium colony is the Fu Shi 2a shigella vaccine T32 that changes the mutually big plasmid of I over to, called after FS-15 strain.The FS-15 strain is expressed Song's Nei Shi I phase antigen and Fu Shi 2a type II, group's 3,4 antigens, and express the IpaA relevant with invasion and attack, B, C, four invasin protein bands of D, have the intrusion of HeLa cell, the Congo red test positive, invasion and attack such as contact hemolysis phenotype, the cavy angle binding film virulence test positive.

(purchase the Research in Walter Reed Army Institute of, America contains the P1 phage, Kan with intestinal bacteria NK5131 ^r50ug/ml, Tet ^r12.5ug/ml Tn10 inserts the aroD gene of deactivation) preparation P1 lysate, tiring needs greater than 10 ¹⁰Can use, mix with P1 lysate and FS-15 strain bacterium liquid, mode by transduction changes the aroD gene in the recipient bacterium over to, concrete grammar is as follows: the FS-1550 μ l that glycerin bouillon is preserved is inoculated in 5ml and contains in the LB meat soup of Kan50ug/ml, 37 ℃ shaking culture 8-9 hour, its whole transferred speciess are contained in the LB meat soup of Kan 50ug/ml in 200ml, it is centrifugal to get 60ml bacterium liquid, centrifugal 10 minutes of 4000rpm is suspended from 40mlCa, Mg damping fluid (5mM Cacl ₂, 10mM MgSO ₄) in, 37 ℃ of water-baths 30 minutes.Every 2ml bacterium liquid adds 2ml P1 lysate, and 37 ℃ adsorbed centrifugal 10 minutes of 4000rpm 30 minutes, every pipe is suspended from the 0.5ml LB meat soup, be laid on 3 LB flat boards that contain 50ug/ml kantlex and 12.5ug/ml Tet (tsiklomitsin), cultivated 55 hours for 42 ℃, have transduttant to occur.Continuous purifying 3 times on the flat board that contains kan 50ug/ml and Tet 12.5ug/ml.The transduttant dibbling on the M9 flat board that adds and do not add die aromatischen Aminosaeuren, is selected the bacterium colony of die aromatischen Aminosaeuren defective and preserved.Pass through Bochner substratum (peptone 10 grams, yeast 5 grams, 50 milligrams of duomycin .HCl, NaCl 10 grams, NaH again ₂PO ₄11.3 gram, agar 15 grams, water 1000ml, PH6.0-6.5), screening sensitive tetracycline mutant.Again with sensitive tetracycline strain dibbling on the NC film, after the cracking with a- ³²The aroD probe of the 1.0Kb of P mark is done bacterium colony in situ hybridization, and (conventional alkaline denaturation extracts the pKD201 plasmid and (purchases the HarborLaboratory in Cold Spring, America), with ClaI and EcoRV double digestion, reclaim the 1.0kb fragment, this fragment of random primering mark is as the aroD probe, colony hybridization method carries out routinely), the person of being negative is the aroD gene-deleted strain.The aroD gene-deleted strain is being contained nad (nicotinamide), VitB1 (VITMAIN B1), asp (aspartic acid), phe (phenylalanine), try (tryptophane), tyr (tyrosine), PABA (para-amino benzoic acid), make the auxotrophy of die aromatischen Aminosaeuren on the M9 substratum of DHB (2.3-resorcylic acid) (being 40ug/ml) and identify, and with NK5131 as positive control, filter out the bacterial strain (FS) that 4 strains can not be grown on above-mentioned M9 substratum, this 4 strain bacterium had both been expressed two valency LPS-O polysaccharide antigens, each group of stably express dysentery bacterium common Ipas proteantigen again, aroD genetically deficient, and have invasive ability.This 4 strain bacterium is dysentery multivalent genetic engineering vaccine strain (inv ⁺Ipas ⁺Δ aroD) FS.

(2) screening of dysentery multivalent genetic engineering vaccine strain

According to the IpaA that GeneBank has announced, IpaB, IpaC, IpaD gene order design PCR primer is a template with Salmonellas or intestinal bacteria, obtains IpaA, IpaB, IpaC, IpaD gene respectively by gene amplification, is implemented in pQE ₃₀On (purchasing invitrogen company) prokaryotic expression carrier, import DH5 α recipient bacterium and obtain IpaA, IpaB, IpaC, IpaD Recombinant Protein Expression, prepared IpaA, IpaB, IpaC, the IpaD recombinant protein antigen of purifying through ni-sepharose purification in the U.S..Add subcutaneous multiple spot immunity BALB/c mouse behind the incomplete Fu Shi adjuvant emulsion of equivalent respectively with IpaA, IpaB, IpaC, the IpaD recombinant protein antigen of purifying, three times antibody titer is surveyed in the immunity back, get its immune BALB/c mouse spleen, preparation splenocyte and myeloma cell (SP2/0) are merged, the hybridoma cell strain of screening IpaA, IpaB, IpaC, IpaD antibody positive.Adopt limiting dilution to obtain to produce the hybridoma cell strain of antibody; and further identify that with ELISA IpaA, IpaB, IpaC, IpaD hybridoma cell strain secretory antibody are at IpaA; IpaB; IpaC; IpaD protective epitope's monoclonal cell strain (being respectively: 3G11,2E8,5F2,5D3) is respectively IgG2a, IgG1b, IgG1a, IgG2b through the antibody subtype Analysis and Identification.

Adopt Western blotting hybridization to detect the dysentery multivalent genetic engineering vaccine strain and express IpaA, IpaB, IpaC, the antigenic situation of IpaD invasin protein, concrete grammar is: sample on dysentery multivalent genetic engineering vaccine strain and the SDS-PAGE sample-loading buffer heated and boiled, carry out 10%SDS-PAGE, respectively with above-mentioned at IpaA, IpaB, IpaC, IpaD protective epitope's antibody is one anti-, sheep anti-mouse igg (available from Britain Amersham company) with the HRP mark is the two anti-Western blotting that carry out, and results of hybridization shows the IpaA hybridization band that has obtained 70kDa in the dysentery multivalent genetic engineering vaccine strain sample, the IpaB hybridization band of 62kDa, the IpaC hybridization band of 42kDa, the IpaD of 36kDa; Illustrate that 4 strain dysentery multivalent genetic engineering vaccine strains of the present invention can express IpaA, IpaB, IpaC, IpaD.

2, the evaluation of 4 strain dysentery multivalent genetic engineering vaccine strains

1. invade phenotypic evaluation

Song Nei Shi, Fu Shi 2a type II, group's 3,4 reference cultures (purchasing) in the national drug biological products assay institute.

The ordinary flat line is carried out in dilution after the dysentery multivalent genetic engineering vaccine strain activation culture, cultivate 20-22h for 37 ℃, choose single bacterium colony and Fu Shi type II, group 3,4 and Song Nei Shi I phase factor and invasin protein IpaA, IpaB, IpaC, IpaD antibody do slide agglutination test, observe the invasion phenotype.Concrete grammar is: respectively Song Nei Shi, Fu Shi 2a type II, group's 3,4 reference cultures (purchasing in Nat'l Pharmaceutical ﹠ Biological Products Control Institute) are dripped in slide, scrape and get an amount of lawn, add corresponding invasin protein IpaA, IpaB, IpaC, the agglutination reaction of IpaD antibody respectively, the result shows, strong agglutination reaction all takes place with IpaA, IpaB, IpaC, IpaD antibody in multivalence Song Shi, Fu Shi and invasin protein antigen, performance Song Nei Shi ⁺⁺⁺, Fu Shi 2a type II ⁺⁺⁺, group 3,4 ⁺⁺⁺And IpaA ⁺⁺⁺, IpaB ⁺⁺⁺, IpaC ⁺⁺⁺, IpaD ⁺⁺⁺Be the strong agglutination reaction of multivalence (" ⁺⁺⁺" represent strong agglutination reaction).

The evaluation that A, invasin protein are expressed:

Select for use the Hayes broth culture to cultivate strain of 18h dysentery multivalent genetic engineering vaccine and dysentery bivalent gene engineering vaccine FSM2117 (doing contrast) respectively, abandoning supernatant after centrifugal washes once with physiological saline, the recovery original volume adds equivalent sample preparation liquid (1% tetrabromophenol sulfonphthalein) and boils, application of sample carries out the 10%SDS-PAGE electrophoresis, seal with bovine serum albumin-PBS on the transfer film, add corresponding mouse-anti IpaA then, IpaB, IpaC, the IpaD monoclonal antibody is one anti-, ambient temperature overnight PBS washes several times, and the 37 ℃ of temperature of sheep anti-mouse igg (available from Britain Amersham company) that add again with the HRP mark are bathed, and PBS washes back DAB colour developing several times.WB (western blot test), ELISA, Immunoblot result show, the antibody of dysentery multivalent genetic engineering vaccine strain and Song Nei Shi, Fu Shi 2a type II, group 3,4 and invasin protein IpaA, IpaB, IpaC, IpaD is the polyvalent antigen positive reaction, and dysentery bivalent gene engineering vaccine FSM2117 and Song Nei Shi, Fu Shi 2a type II, group's 3,4 LPS-O is positive, but with the reaction that is negative of the monoclonal antibody of invasin protein IpaA, IpaB, IpaC, IpaD.

The evaluation of B, Congo red adsorption experiment:

Dysentery multivalent genetic engineering vaccine strain, dysentery bivalent gene engineering vaccine FSM2117, T32 attenuated strain (negative control), F2a-12 virulent strain (positive control) are inoculated in the LB broth culture respectively, 37 ℃ of joltings are spent the night, be seeded in respectively to contain on the 0.01% Congo red LB plate and rule, again 37 ℃ of overnight incubation.The result shows, it is translucent being creamy white that dysentery bivalent gene engineering vaccine FSM2117, T32 attenuated strain do not absorb Congo red lawn, the F2a-12 virulent strain absorbs Congo red lawn and is strong redness, and the dysentery multivalent genetic engineering vaccine strain absorbs Congo red lawn for red, but degree is lower than the F2a-12 virulent strain, its degree and between the feminine gender and the positive.

The evaluation of C, contact hemolysis experiment:

Dysentery multivalent genetic engineering vaccine strain, dysentery bivalent gene engineering vaccine FSM2117, T32 attenuated strain (negative control), F2a-12 virulent strain (positive control) are inoculated in the Hayes broth culture respectively, 37 ℃ of joltings are spent the night, centrifugal washing twice, to preserve in the liquid guinea pig blood cell centrifugation simultaneously washes 4 times, and with guinea pig blood cell and 37 ℃ of 2h of testing sample mixing, centrifuging and taking supernatant then, ultraviolet detection 570nm OD value.The result shows that dysentery multivalent genetic engineering vaccine strain OD value is significantly less than the F2a-12 virulent strain, and apparently higher than Non-Invasive dysentery bivalent gene engineering vaccine FSM2117.

The evaluation of D, Hela cell invasion experiment:

With 4 * 10 of cultivation ⁴The Hela cell of individual cell/ml is put into the bottle that little cover glass is housed and is cultivated 24h, is 5 * 10 with the bacterial strain dilution to be checked of newly cultivating ⁸CFU/ml, equivalent adds Hela cell bottle and infects 2h, takes out this slide PBS and washes 4 times, fix with methyl alcohol, Giemsa dyeing, mirror is 1000 Hela cells of meter down, calculate the invasion number of bacterial strain to the Hela cell.The result shows that it is 80% that the F2a-12 virulent strain is invaded the Hela cell count, and it is 36% that the dysentery multivalent genetic engineering vaccine bacterial strain is invaded the Hela cell count, and dysentery bivalent gene engineering vaccine FSM2117 intrusion Hela cell count is 2%.

2. security is identified

5,000,000,000 CFU dysentery multivalent genetic engineering vaccine strains are put into the conjunctival sac of the eyes of cavy, and the another eyes are not done any processing as blank, carry out dysentery multivalent genetic engineering vaccine cavy angle conjunctival butter according to a conventional method, the result shows that the dysentery multivalent genetic engineering vaccine strain is to the cavy no pathogenicity; The mouse virulence experiment reaches the vaccine safety requirements, and (mouse peritoneal injects 5 * 10 ⁹Cfu does not have dead).The oral 20,000,000,000 CFU dysentery multivalent genetic engineering vaccine strains of monkey body, mouse peritoneal inject 1,000,000,000 CFU dysentery multivalent genetic engineering vaccine strains and all have no adverse reaction.Dysentery multivalent genetic engineering vaccine strain mouse LD ₅₀Detect, the result proves the mouse LD of dysentery multivalent genetic engineering vaccine strain ₅₀Be worth suitable with dysentery bivalent gene engineering vaccine FSM2117.So far finish toxicity and the virulence experiment of dysentery polyvalent vaccine and all identify, prove that this dysentery multivalent genetic engineering vaccine strain is safe in utilization.

3. stability is identified

Divide supplementary biography 60 generations the seed bank and the work storehouse bacterial strain of dysentery multivalent genetic engineering vaccine strain; by the Congo red absorption of the biological phenotype of difference; contact hemolysis; Hela cell invasion identification invasin protein antigen monoclonal antibody (3G11; 2E8; 5F2; 5D3); with full bacterium WB (western blot test); ELISA; methods such as Immunoblot are carried out two valency LPS-O protective antigens; the observation of Ipas invasin protein antigen and biological phenotypic stability; experimental result shows; the dysentery multivalent genetic engineering vaccine strain passed for 60 generations and preserves 5 years energy stably express protectiveness LPS-O antigen and invasin protein IpaA; IpaB; IpaC; IpaD, and biological phenotype such as invasion is stable.

To the oral capsule or the collunarium freeze-dried preparation of the different batches that lasts 5 years and nearly 200 routine volunteers are got rid of vaccine, the system that carries out biochemistry, genetics and antigen presentation one by one detects, proof is got rid of bacterium and oral capsule vaccine and collunarium vaccine at aspect indifferences such as plasmid size, biochemical characteristic, antigen presentation and cavy corneal tests, consistent with a large amount of animal test results in laboratory, prove that the dysentery multivalent genetic engineering vaccine strain has good stability.

Preserve the finished product preparation in 1 year,, all do not have change, prove that thus the dysentery multivalent genetic engineering vaccine strain has good stability at aspects such as genetics, biochemical, antigen presentations except that dysentery multivalent genetic engineering vaccine strain viable count has the decline of 5-8%.

4. immunogenicity is identified

A) with dysentery multivalent genetic engineering vaccine strain oral immunity mouse 3 times, each 0.1 hundred million CFU, use dysentery bivalent gene engineering vaccine FSM2117 oral immunity mouse 3 times simultaneously, each 1,000,000,000 CFU, each 14d at interval, 7d gathers the production of blood, lung-douching fluid and intestines, reproductive tract washing fluid detection IgA, IgG specific antibody after the last immunity.ELISA result shows, the anti-Fu Shi of dysentery multivalent genetic engineering vaccine strain, anti-Song Nei Shi and IpaA, IpaB, IpaC, it is respectively (1280,640 and 640,640,640,320) that IpaD serum IgA specific antibody is tired, and IgG is respectively (2560,1280 and 640,640,640,640); It is respectively (2560,2560 and 1280,1280,1280,12800) that lung-douching fluid sIgA specific antibody is tired, and IgG is respectively (2560,1280 and 1280,1280,1280,1280); It is respectively (1280,1280 and 640,640,640,640) that intestines washing lotion sIgA specific antibody is tired, and IgG is respectively (1280,640 and 320,640,320,640); It is respectively (1280,640 and 640,640,640,640) that reproductive tract washing fluid sIgA specific antibody is tired, and IgG is respectively (1280,640 and 320,320,320,320).Simultaneously ELISA result shows, the anti-Fu Shi of dysentery bivalent gene engineering vaccine strain FSM2117, anti-Song Nei Shi serum IgA specific antibody is tired is respectively (640,640), and IgG is respectively (1280,1280); It is respectively (1280,1280) that lung-douching fluid sIgA specific antibody is tired, and IgG is respectively (1280,1280); It is respectively (1280,640) that intestines washing lotion sIgA specific antibody is tired, and IgG is respectively (640,320); It is respectively (640,320) that reproductive tract washing fluid sIgA specific antibody is tired, and IgG is respectively (640,320).The result shows that special IgG of dysentery multivalent genetic engineering vaccine strain oral immunity mouse and IgA antibody are higher than the dysentery bivalent gene engineering vaccine.Especially more obvious with the multivalence specific antibody reaction of blood and lung-douching fluid, and can induce reproductive tract and the interior antibody response of intestines.

B) with dysentery multivalent genetic engineering vaccine strain collunarium immune mouse 3 times, each 0.001 hundred million CFU, use dysentery bivalent gene engineering vaccine FSM2117 collunarium immune mouse 3 times simultaneously, each 0.1 hundred million CFU, each 14d at interval, 7d gathers the reaction of blood, lung-douching fluid and intestines, reproductive tract washing fluid IgA, IgG specific antibody after the last immunity.ELISA result shows, the anti-Fu Shi of dysentery multivalent genetic engineering vaccine strain, anti-Song Nei Shi and IpaA, IpaB, IpaC, it is respectively (2560,1280 and 1280,640,640,640) that IpaD serum IgA specific antibody is tired, and IgG is respectively (5120,2560 and 1280,1280,1280,12800); It is respectively (2560,1280 and 1280,1280,1280,12800) that lung-douching fluid sIgA specific antibody is tired, and IgG is respectively (5120,2560 and 1280,1280,640,1280); It is respectively (2560,1280 and 1280,640,640,640) that intestines washing lotion sIgA specific antibody is tired, and IgG is respectively (1280,640 and 640,640,640,640); It is respectively (2560,1280 and 1280,6400,640,640) that reproductive tract washing fluid sIgA specific antibody is tired, and IgG is respectively (1280,640 and 640,640,320,320).Simultaneously ELISA result shows, the anti-Fu Shi of dysentery bivalent gene engineering vaccine FSM2117, anti-Song Nei Shi serum IgA specific antibody is tired is respectively (1280,640), and IgG is respectively (1280,1280); It is respectively (1280,1280) that lung-douching fluid sIgA specific antibody is tired, and IgG is respectively (2560,1280); It is respectively (1280,640) that intestines washing lotion sIgA specific antibody is tired, and IgG is respectively (640,640); It is respectively (640,320) that reproductive tract washing fluid sIgA specific antibody is tired, and IgG is respectively (640,320).The result shows that special IgG of dysentery multivalent genetic engineering vaccine strain collunarium immune mouse and IgA antibody are higher than dysentery bivalent gene engineering vaccine FSM2117.Especially more obvious with the multivalence specific antibody reaction of blood and lung-douching fluid, and can induce reproductive tract and the interior antibody response of intestines.

C) with dysentery multivalent genetic engineering vaccine strain oral immunity rhesus monkey 3 times, each 0.2 hundred million CFU, use dysentery bivalent gene engineering vaccine FSM2117 oral immunity rhesus monkey 3 times simultaneously, each 5.0 hundred million CFU, each 14d at interval, 7d gathers the reaction of blood, lung-douching fluid and intestines, reproductive tract washing fluid IgA, IgG specific antibody after the last immunity.ELISA result shows, the anti-Fu Shi of dysentery multivalent genetic engineering vaccine strain, anti-Song Nei Shi and IpaA, IpaB, IpaC, it is respectively (2560,640 and 640,640,640,640) that IpaD serum IgA specific antibody is tired, and IgG is respectively (2560,1280 and 1280,1280,640,640); It is respectively (6400,2560 and 2560,1280,2560,12800) that lung-douching fluid sIgA specific antibody is tired, and IgG is respectively (5120,1280 and 1280,1280,2560,1280); It is respectively (2560,1280 and 1280,640,640,640) that intestines washing lotion sIgA specific antibody is tired, and IgG is respectively (2560,1280 and 640,640,640,640); It is respectively (2560,1280 and 640,1280,640,640) that reproductive tract washing fluid sIgA specific antibody is tired, and IgG is respectively (1280,640 and 640,640,640,640).Simultaneously ELISA result shows, the anti-Fu Shi of dysentery bivalent gene engineering vaccine FSM2117, anti-Song Nei Shi serum IgA specific antibody is tired is respectively (1280,640), and IgG is respectively (1280,640); It is respectively (2560,1280) that lung-douching fluid sIgA specific antibody is tired, and IgG is respectively (1280,1280); It is respectively (640,640) that intestines washing lotion sIgA specific antibody is tired, and IgG is respectively (640,640); It is respectively (320,160) that reproductive tract washing fluid sIgA specific antibody is tired, and IgG is respectively (640,160).The result shows that special IgG of dysentery multivalent genetic engineering vaccine strain oral immunity rhesus monkey and IgA antibody are higher than dysentery bivalent gene engineering vaccine FSM2117.Especially more obvious with the multivalence specific antibody reaction of blood and lung-douching fluid, and can induce reproductive tract and the interior antibody response of intestines.

D) with dysentery multivalent genetic engineering vaccine strain collunarium immunity rhesus monkey 3 times, each 0.005 hundred million CFU, use dysentery bivalent gene engineering vaccine FSM2117 collunarium immunity rhesus monkey 3 times simultaneously, each 0.5 hundred million CFU, each 14d at interval, 7d gathers the reaction of blood, lung-douching fluid and intestines, reproductive tract washing fluid IgA, IgG specific antibody after the last immunity.ELISA result shows, the anti-Fu Shi of dysentery multivalent genetic engineering vaccine strain, anti-Song Nei Shi and IpaA, IpaB, IpaC, it is respectively (2560,1280 and 1280,1280,1280,640) that IpaD serum IgA specific antibody is tired, and IgG is respectively (6400,2560 and 1280,1280,640,1280); It is respectively (6400,5120 and 2560,2560,2560,25600) that lung-douching fluid sIgA specific antibody is tired, and IgG is respectively (5120,5120 and 2560,2560,2560,1280); It is respectively (2560,1280 and 1280,1280,1280,1280) that intestines washing lotion sIgA specific antibody is tired, and IgG is respectively (2560,1280 and 1280,1280,640,1280); It is respectively (2560,1280 and 1280,1280,1280,640) that reproductive tract washing fluid sIgA specific antibody is tired, and IgG is respectively (1280,1280 and 1280,640,12800,640).Simultaneously ELISA result shows, the anti-Fu Shi of dysentery bivalent gene engineering vaccine FSM2117, anti-Song Nei Shi serum IgA specific antibody is tired is respectively (1280,1280), and IgG is respectively (1280,1280); It is respectively (5120,2560) that lung-douching fluid sIgA specific antibody is tired, and IgG is respectively (1280,1280); It is respectively (640,320) that intestines washing lotion sIgA specific antibody is tired, and IgG is respectively (640,320); It is respectively (640,320) that reproductive tract washing fluid sIgA specific antibody is tired, and IgG is respectively (640,320).The result shows that dysentery multivalent genetic engineering vaccine strain collunarium immunity special IgG of rhesus monkey and IgA antibody are higher than dysentery bivalent gene engineering vaccine FSM2117.Especially more obvious with the multivalence specific antibody reaction of blood and lung-douching fluid, and can induce reproductive tract and the interior antibody response of intestines.

More than in the intravital result of mouse, rhesus monkey simultaneously as seen, collunarium is nearly 1% consumption of oral immunity dosage, all produced better immunogenicity mouse, rhesus monkey, and the collunarium immunity is better than the effect of oral immunity approach.

5. immune protective is identified

On, two approach immune mouses of collunarium oral, the rhesus monkey animal model, use 100LD respectively at existing dysentery multivalent genetic engineered vaccine, dysentery BIVALENT VACCINE FOR NEONTAL ₅₀Fu Shi bacillus, Song Nei Shi virulent strain (purchasing in Nat'l Pharmaceutical ﹠ Biological Products Control Institute) collunarium, oral challenge are observed the immune protective effect of dysentery multivalent genetic engineered vaccine.The result shows, dysentery multivalent genetic engineered vaccine oral immunity mouse is 90%, is 84% to the immune protective effect of Song Nei Shi strain the immunoprotection of Fu Shi strain, and the collunarium immune mouse is 95%, is 88% to the immune protective effect of Song Nei Shi strain the immunoprotection of Fu Shi strain; Dysentery multivalent genetic engineered vaccine oral immunity rhesus monkey is 92%, is 88% to the immune protective effect of Song Nei Shi strain the immunoprotection of Fu Shi strain, and the collunarium immune mouse is 98%, is 92% to the immune protective effect of Song Nei Shi strain the immunoprotection of Fu Shi strain.And dysentery BIVALENT VACCINE FOR NEONTAL FSM2117 oral immunity mouse is 50%, is 41% to the immune protective effect of Song Nei Shi strain the immunoprotection of Fu Shi strain, and the collunarium immune mouse is 56%, is 48% to the immune protective effect of Song Nei Shi strain the immunoprotection of Fu Shi strain; Dysentery BIVALENT VACCINE FOR NEONTAL FSM2117 oral immunity rhesus monkey is 46%, is 40% to the immune protective effect of Song Nei Shi strain the immunoprotection of Fu Shi strain, and the collunarium immune mouse is 55%, is 48% to the immune protective effect of Song Nei Shi strain the immunoprotection of Fu Shi strain.

More than in mouse, the intravital immune protective effect result of rhesus monkey as seen; the collunarium immunizing dose consumes more than 100 times than oral reduction, and the dysentery multivalent genetic engineering vaccine strain has improved nearly more than 30% than dysentery bivalent gene engineering vaccine FSM2117 immune protective effect.Illustrate that dysentery multivalent genetic engineering vaccine strain immunogenicity and immune protective are better than dysentery bivalent gene engineering vaccine FSM2117.

6. human safety, immunogenicity and protectiveness are identified

Finish dysentery multivalent genetic engineering vaccine strain laboratory study, on the safe and effective basis of proof dysentery multivalent genetic engineering vaccine strain laboratory animal, continuous 5 years in five different provinces, city, area, 5-59 year is amounted to 20,000 surplus the people carry out 3 oral dysentery multivalent genetic engineering vaccine strains of whole process.Security, immunogenicity and the protection effect of dysentery multivalent genetic engineering vaccine strain have been carried out strict examination and evaluation result shows:

A) dysentery multivalent genetic engineering vaccine strain people takes safety: the dysentery multivalent genetic engineering vaccine strain is to 10634 routine volunteer oral's immunizing dose 10,000,000,000 CFU, to 10000 routine volunteer's collunarium immunizing dose 5,000,000,000 CFU, and its side reaction rate is respectively 0.136%, 0.108%; Dysentery bivalent gene engineering vaccine FSM2117 is to 5000 routine volunteer oral's immunizing dose 10,000,000,000 CFU, to 3800 routine volunteer's collunarium immunizing dose 5,000,000,000 CFU, and its side reaction rate is respectively 0.114%, 0.110%; Use 0.5% gelatin, oral, the form of nose drops of 5% sucrose, 0.2% vitamins C, 0.1% thiaminogen, 0.1% skim-milk preparation dressing preparation is as placebo, to 5000 routine volunteer oral's immunity placebo 100g, to 5000 routine volunteer's collunarium immunity placebo 10g, placebo side reaction rate is respectively 0.086%, 0.119%.There is not difference from side reaction between above dysentery multivalent genetic engineering vaccine strain, dysentery bivalent gene engineering vaccine FSM2117 and placebo.Oral, collunarium dysentery multivalent genetic engineering vaccine strain safety that the reference uses.

B) dysentery multivalent genetic engineering vaccine strain oral immunity has good immunogenicity and immune protective: by at random, balanced, double blinding principle; successively 5353 routine volunteers are carried out oral immunity 1.0 hundred million CFU; immunity three times; immunity was respectively for 0,2,4 weeks pitch time; and successively 1100 routine volunteers are carried out oral immunity 4,000,000,000 CFU with dysentery bivalent gene engineering vaccine FSM2117; immunity three times, immunity was respectively for 0,2,4 weeks pitch time.180d after both immunity are finished gathers serum, ight soil and saliva, and the ELISA that carries out anti-Fu Shi, anti-Song Nei Shi and Ipas specific IgA, IgG detects.ELISA result shows, the anti-Fu Shi of dysentery multivalent genetic engineering vaccine strain, anti-Song Nei Shi and IpaA, IpaB, IpaC, it is respectively 1280,1280 and 640,640,640,640 that IpaD serum IgA specific antibody is tired, and IgG is respectively 1280,1280 and 640,640,640,320; It is respectively 1280,1280 and 640,640,320,320 that ight soil sIgA specific antibody is tired, and IgG is respectively 1280,640 and 640,320,320,160; It is respectively 640,320 and 160,160,160,160 that saliva sIgA specific antibody is tired, and IgG is respectively 640,640 and 320,320,160,320.Simultaneously ELISA result shows, the anti-Fu Shi of dysentery bivalent gene engineering vaccine FSM2117, anti-Song Nei Shi serum IgA specific antibody is tired is respectively 640,320, and IgG is respectively 640,640; It is respectively 320,160 that ight soil sIgA specific antibody is tired, and IgG is respectively 640,320; It is respectively 320,160 that saliva sIgA specific antibody is tired, and IgG is respectively 160,80.The result shows that dysentery multivalent genetic engineering vaccine strain human oral special IgG of immunity and IgA antibody are higher than dysentery bivalent gene engineering vaccine FSM2117.The two valency specific IgA of the ight soil 4 multiplication high rates of tiring before than the clothes seedling are 68%-81%, and the saliva bivalent antibody 4 multiplication high rates of tiring are 75-91%, and invasin protein antibody is arranged.Prove that fully the dysentery multivalent genetic engineering vaccine strain has good multivalent immunogenic.Can effectively induce and produce special multivalence mucoantibody reaction.Further collect the case of suffering from diarrhoea half a year, by the laboratory bacteriological detection, make a definite diagnosis, add up, the result shows that dysentery multivalent genetic engineering vaccine strain oral immunity provides 91.8% to children, adult 86.5% protection ratio.Being 89.8% to Fu Shi 2a protection ratio wherein, to Song Nei Shi 84.6%, is 76.8% to the protection ratio of special-shaped dysentery.

C) immunity of dysentery multivalent genetic engineering vaccine strain collunarium has good immunogenicity and protectiveness: by at random, balanced, double blinding principle; successively 3000 routine volunteers are carried out collunarium immunity 0.01 hundred million CFU; immunity three times; immunity was respectively for 0,2,4 weeks pitch time; and successively 1400 routine volunteers are carried out collunarium immunity 1.0 hundred million CFU with dysentery bivalent gene engineering vaccine FSM2117; immunity three times, immunity was respectively for 0,2,4 weeks pitch time.The detection of 180d collection serum, ight soil and the anti-Fu Shi of saliva, anti-Song Nei Shi and Ipas specific IgA, IgG after both immunity are finished.ELISA result shows, the anti-Fu Shi of dysentery multivalent genetic engineering vaccine strain, anti-Song Nei Shi and IpaA, IpaB, IpaC, it is respectively (2560,1280 and 1280,1280,640,640) that IpaD serum IgA specific antibody is tired, and IgG is respectively (2560,2560 and 640,640,640,320); It is respectively (1280,1280 and 640,1280,640,640) that ight soil sIgA specific antibody is tired, and IgG is respectively (1280,1280 and 640,640,640,640); It is respectively (640,640 and 320,320,320,160) that saliva sIgA specific antibody is tired, and IgG is respectively (640,320 and 320,320,640,640).Simultaneously ELISA result shows, the anti-Fu Shi of dysentery bivalent gene engineering vaccine FSM2117, anti-Song Nei Shi serum IgA specific antibody is tired is respectively (640,320), and IgG is respectively (1280,640); It is respectively (640,320) that ight soil sIgA specific antibody is tired, and IgG is respectively (640,320); It is respectively (640,640) that saliva sIgA specific antibody is tired, and IgG is respectively (320,320).The result shows that dysentery multivalent genetic engineering vaccine strain people collunarium special IgG of immunity and IgA antibody are higher than dysentery bivalent gene engineering vaccine FSM2117.The two valency specific IgA of the ight soil 4 multiplication high rates of tiring before than collunarium seedling are 78%-88%, and the saliva bivalent antibody 4 multiplication high rates of tiring are 70-92%, and invasin protein antibody is arranged.Prove that fully the dysentery multivalent genetic engineering vaccine strain has good multivalent immunogenic.Can effectively induce and produce special multivalence mucoantibody reaction.Further collect the case of suffering from diarrhoea half a year, by the laboratory bacteriological detection, make a definite diagnosis, add up, the result shows that the immunity of dysentery multivalent genetic engineering vaccine strain collunarium provides 98.0% to children, adult 89.6% protection ratio.Being 93.4% to Fu Shi 2a protection ratio wherein, to Song Nei Shi 88.9%, is 89.1% to the protection ratio of special-shaped dysentery.

The production of embodiment 2, dysentery multivalent genetic engineering vaccine preparation

The production technique of dysentery multivalent genetic engineering vaccine oral capsule preparation is as follows: with the dysentery multivalent genetic engineering freeze-dried vaccine young plant (4,000,000,000 CFU/ prop up) in the seed pipe, (fresh beef 7500g adds 11250ml distilled water and boils 40min to insert the Hou Shi liquid medium, be cooled to 45 ℃ of fresh pig pancreas 1500mg that add homogenate, transfer pH8.0, add trichloromethane 200ml, digestion 7d with preceding 4 times of dilutions, and adds sodium-chlor 0.5%, pH7.2-7.4 is standby for accent) or LB substratum (peptone 10g, yeast leach liquor 5g, sodium-chlor 10g adds water to 1000ml, pH is 7.2-7.5) the 3.0L culturing bottle in, 37 ℃, 150-200rpm shaking culture 13-15h directly injects the 10L culturing bottle that 8500ml Hou Shi liquid medium or LB substratum are housed, 37 ℃, static cultivation 6-8h injects the 180L culture tank that 180 liters of Hou Shi liquid mediums or LB substratum are housed, 37 ℃, static cultivation 6-8h, regulate pH7.2, centrifugal, collect thalline.Vaccine and medical dressing composition mixture are made in single agent packaging container, are used to produce dysentery multivalent genetic engineering freeze-drying vaccine oral capsule, form of nose drops.

1; the production of dysentery multivalent genetic engineering vaccine lyophilized oral capsule formulation: the above-mentioned wet thallus of collecting is suspended from contains 0.5% gelatin; 5% sucrose; 0.2% vitamins C; 0.1% thiaminogen; 0.1% skim-milk; in the phosphate buffered saline buffer of pH7.2; the concentration that makes bacterium liquid is 10,000,000,000 CFU/ml; be put in the freeze-drying dish;-30 ℃; 2-4h; vacuum reaches 85uHg, is warmed up to 30 ℃, constant temperature; dry 20h; obtain dysentery multivalent genetic engineering freeze-drying vaccine lyophilized powder, put 6-8 ℃, carry out pure bacterium test; the result shows survival rate＞40%; moisture＜3% is by the Congo red absorption of the biological phenotype of difference; contact hemolysis; Hela cell invasion identification protecting antigen monoclonal antibody (3G11; 2E8; 5F2; 5D3), with full bacterium WB; ELISA; screening of methods such as Immunoblot and evaluation invasion phenotype (inv ⁺) ability.Experimental result shows that the dysentery multivalent genetic engineering vaccine lyophilized powder can be expressed two valency LPS-O protective antigens and Ipas antigen.With dysentery multivalent genetic engineering vaccine lyophilized powder and sucrose, sodium starch glycolate, lactose, amylum pregelatinisatum, Magnesium Stearate mixing by a certain percentage, under oral capsule GMP condition, prepare capsule formulation.Make consisting of of every oral capsule: dysentery multivalent genetic engineering vaccine strain 1.0 hundred million CFU viable bacterias, sucrose 15mg, sodium starch glycolate 15mg, lactose 20mg, amylum pregelatinisatum 20mg, Magnesium Stearate 0.4mg.

2, the production of dysentery multivalent genetic engineering freeze-drying vaccine form of nose drops

The above-mentioned multivalent genetic engineering vaccine of collecting is suspended from contains 0.5% gelatin, 5% sucrose, 0.2% vitamins C, 0.1% thiaminogen, 0.1% skim-milk, in the phosphate buffered saline buffer of pH7.2, the viable count that makes bacterium liquid is 1,000,000,000 CFU/ml, in the packing freeze-drying dish,-30 ℃, 2-4h, vacuum reaches 85uHg, be warmed up to 30 ℃, constant temperature, dry 20h obtains dysentery multivalent genetic engineering freeze-drying vaccine pulvis.Put 6-8 ℃; carry out pure bacterium test; the result shows survival rate＞40%; moisture＜3%; by the Congo red absorption of the biological phenotype of difference, contact hemolysis, Hela cell invasion identification protecting antigen monoclonal antibody (3G11,2E8,5F2,5D3), with screening of methods such as full bacterium WB, ELISA, Immunoblot and evaluation invasion phenotype (inv ⁺) ability.Experimental result shows that dysentery multivalent genetic engineering vaccine bacterium lyophilized powder can be expressed two valency LPS-O protective antigens and Ipas antigen.Make consisting of of every bottle of form of nose drops: dysentery multivalent genetic engineering vaccine 0.1 hundred million/bottle CFU freeze-drying viable bacteria, it is standby to be uniform suspension with preceding and 0.2ml/ bottle physiological saline (including 1%BL-9 (9-lauryl ether), 2% whiteruss) dissolving.

3, two kinds of dysentery multivalent genetic engineering vaccine preparations that step 1 and step 2 are obtained carry out following evaluation:

1) purity test: after the goods dilution, (fresh beef 750g adds 1125ml distilled water and boils 40min to be inoculated in the big agar slant of Hou Shi, be cooled to 45 ℃ of fresh pig pancreas 150mg that add homogenate, transfer pH8.0, and adding trichloromethane 20ml, digestion 7d, with preceding 4 times of distilled water dilutings, and adding sodium-chlor 0.5%, transfer pH7.2-7.4, agar powder 1.5g) or LB agar slant (peptone 1.0g, yeast leach liquor 0.5g, sodium-chlor 1.0g, agar powder 1.5g adds water to 100ml), cultivate 48h for 37 ℃, two kinds of dysentery multivalent genetic engineering vaccine preparations all do not have varied bacteria growing.

2) antigen presentation: (fresh beef 7500g adds 11250ml distilled water and boils 40min the goods vaccine after will diluting in the big agar slant of Hou Shi, be cooled to 45 ℃ of fresh pig pancreas 1500mg that add homogenate, transfer pH8.0, and adding trichloromethane 200ml, digestion 7d, with preceding 4 times of distilled water dilutings, and adding sodium-chlor 0.5%, transfer pH7.2-7.4 standby,) or LB agar slant (peptone 1.0g, yeast is invaded fluid 0.5g, sodium-chlor 1.0g, agar powder 1.5g, add water to 100ml) line, cultivate 20-22h for 37 ℃, choose single bacterium colony and Fu Shi type II, group 3,4 and Song Nei Shi I phase factor, with invasin protein IpaA, IpaB, IpaC, IpaD serum is done slide agglutination test, and the above-mentioned two kinds of dysentery multivalent genetic engineering vaccine preparations of result all are the strong agglutination reaction of 4+ multivalence.

3) viable bacteria rate and water content detection: carry out live bacterial count with turbidimetry, survey moisture content with the Kai Shi method, three pull on state two kinds of dysentery multivalent genetic engineering vaccine preparation viable bacteria rates and the water content detection result as shown in table 1, three to pull on the biochemical reaction of stating two kinds of dysentery multivalent genetic engineering vaccine freeze-dried preparation as shown in table 2.

Two kinds of dysentery multivalent genetic engineering vaccine freeze-dried preparation of table 1. viable bacteria rate and water content detection result

Lot number	Water content %	Viable bacteria rate (%)
					Before doing	After doing	After doing/before doing
		6010 6011 6012	1.81 2.54 2.13	53 38 32.6				28 21 23.6	53 55 72

Annotate: 6010 and 6011 is capsule, and 6012 is nasal drop

With the dysentery multivalent genetic engineering vaccine strain of incubated overnight, be inoculated in the biochemical reaction pipe (purchasing) of glucose, lactose, maltose, seminose and sucrose respectively in Nat'l Pharmaceutical ﹠ Biological Products Control Institute, cultivate 24h for 37 ℃, observe its reaction, reaction tubes becomes blue look positive (+) by green, and two kinds of dysentery multivalent genetic engineering vaccine preparation biochemical reactions the results are shown in Table 2

Two kinds of dysentery multivalent genetic engineering vaccine preparations of table 2. biochemical reaction result

Batch sugar	Glucose	Lactose	Maltose	Seminose	Sugarcane
						6010 6011 6012 FSM2117	+ + + +	- - - -	+ + + +	+ + + +	- - - -

Annotate: 6010 and 6011 is the dysentery multivalent genetic engineering vaccine oral capsule, and 6012 is the dysentery multivalent genetic engineering vaccine nasal drop; FSM2117 is a dysentery bivalent gene engineering vaccine oral preparations, "+" positive reaction, "-" negative reaction.

4) rabbit antibody produces test: get respectively to pull at every turn and state 1 in two kinds of preparations, be diluted to 6-20 hundred million/ml with sterile saline, ear vein approach immune health New Zealand white big ear rabbit, totally three times (0.25ml, 0.5ml, 0.5ml), each 7 days at interval, 3 rabbit of every batch of goods immunity, serum antibody titer is surveyed in blood sampling in 3-10 days after the last immunity.The main points that ELISA measures are, wrap respectively by elisa plate with Sh.Sonnei, Sh.flexneri, Ipas respectively, put 4 ℃ of refrigerator overnight, wash plate with PBS, the immunize rabbit serum that adds doubling dilution is put 37 ℃ of incubator 2.0h, washes plate with carbonic acid buffer, the enzyme mark goat anti-rabbit antibody that adds dilution is put 37 ℃ of incubator 1.0h, use 2N sulfuric acid stopped reaction subsequently, on the uv-spectrophotometric instrument, measure, and the conversion antibody titer.The result is as shown in table 3, shows that above-mentioned two kinds of dysentery multivalent genetic engineering vaccine freeze-dried preparation all have good immunogenicity.

The serum agglutinating antibody of two kinds of dysentery multivalent genetic engineering vaccine freeze-dried preparation of table 3. is tired

Batch	Lot number	Antibody titer
					Sh.Sonnei.	Sh.flexneri	Ipas
		6010	1 2 3	1280 1280 2560				5120 5120 10240	640 1280 1280
6011	1 2 3				1280 320 1280	10240 10240 10240	640 1280 2560
		6012	1 2 3	1280 1280 5120				5120 5120 5120	1280 1280 2560

Annotate: 6010 and 6011 is capsule, and 6012 is nasal drop.

5) virulence test: adopt the cavy corneal test, get lawn (the diameter 3mm after above-mentioned two kinds of dysentery multivalent genetic engineering vaccine preparations activate, about 5,000,000,000 viable bacterias), be coated with in the conjunctival sac of cavy angle, observed 7 days, three pull on and state two kinds of dysentery multivalent genetic engineering vaccine freeze-dried preparation and all do not cause any inflammatory reaction.

6) Ipas detects: (fresh beef 750g adds 1125ml distilled water and boils 40min with above-mentioned two kinds of dysentery multivalent genetic engineering vaccine freeze-dried preparation inoculation Hou Shi liquid medium, be cooled to 45 ℃ of fresh pig pancreas 150mg that add homogenate, transfer pH8.0, and adding trichloromethane 20ml, digestion 7d, with preceding 4 times of distilled water dilutings, and adding sodium-chlor 0.5%, pH7.2-7.4 is standby for accent) or LB substratum (peptone 10g, yeast leach liquor 5g, sodium-chlor 10g, add water to 1000ml, pH7.2-7.5), treat that bacteria concentration reaches 10 ¹⁰Cfu/ml (cultivating 20 hours for 37 ℃) uses the 10%SDS-PAGE electrophoresis detection, and the result shows that dysentery multivalent genetic engineering vaccine has all increased the antigen protein of invasin protein than the strain of dysentery bivalent gene engineering vaccine.

7) the invasion phenotypic evaluation of system: the oral capsule of the different batches that lasts 2 years or intranasal formulation are gathered ight soil to nearly 200 routine volunteers carry out biochemistry one by one, the system of genetics and antigen presentation detects, dysentery multivalent genetic engineering vaccine that proof ight soil is discharged and oral and collunarium dysentery multivalent genetic engineering vaccine are in the plasmid size, biochemical characteristic, aspect indifferences such as antigen presentation and cavy corneal test, with the Congo red absorption of a large amount of animals in laboratory, contact hemolysis, Hela cell invasion test-results unanimity proves that dysentery multivalent genetic engineering vaccine has good invasion phenotype.

Sequence table

<160>8

<210>1

<211>633

<212>PRT

＜213〉Shigella (Shigella)

<400>1

Met His Asn Val Asn Asn Thr Gln Ala Pro Thr Phe Leu Tyr Lys Ala

1 5 10 15

Thr Ser Pro Ser Ser Thr Glu Tyr Ser Glu Leu Lys Ser Lys Ile Ser

20 25 30

Asp Ile His Ser Ser Gln Thr Ser Leu Lys Thr Pro Ala Ser Val Ser

35 40 45

Glu Lys Glu Asn Phe Ala Thr Ser Phe Asn Gln Lys Cys Leu Asp Phe

50 55 60

Leu Phe Ser Ser Ser Gly Lys Glu Asp Val Leu Arg Ser Ile Tyr Ser

65 70 75 80

Asn Ser Met Asn Ala Tyr Ala Lys Ser Glu Ile Leu Glu Phe Ser Asn

85 90 95

Val Leu Tyr Ser Leu Val His Gln Asn Asp Leu Asn Phe Glu Asn Glu

100 105 110

Lys Gly Leu Gln Lys Ile Val Ala Gln Tyr Ser Glu Leu Ile Ile Lys

115 120 125

Asp Lys Leu Ser Gln Asp Ser Ala Phe Gly Pro Trp Ser Ala Lys Asn

130 135 140

Lys Lys Leu His Gln Leu Arg Gln Asn Ile Glu His Arg Leu Ala Leu

145 150 155 160

Leu Ala Gln Gln His Thr Ser Gly Glu Ala Leu Ser Leu Gly Gln Lys

165 170 175

Leu Leu Asn Thr Glu Val Ser Ser Phe Ile Lys Asn Asn Ile Leu Ala

180 185 190

Glu Leu Lys Leu Ser Asn Glu Thr Val Ser Ser Leu Lys Leu Asp Asp

195 200 205

Leu Val Asp Ala Gln Ala Lys Leu Ala Phe Asp Ser Leu Arg Asn Gln

210 215 220

Arg Lys Asn Thr Ile Asp Ser Lys Gly Phe Gly Ile Gly Lys Leu Ser

225 230 235 240

Arg Asp Leu Asn Thr Val Ala Val Phe Pro Glu Leu Leu Arg Lys Val

245 250 255

Leu Asn Asp Ile Leu Glu Asp Ile Lys Asp Ser His Pro Ile Gln Asp

260 265 270

Gly Leu Pro Thr Pro Pro Glu Asp Met Pro Asp Gly Gly Pro Thr Pro

275 280 285

Gly Ala Asn Glu Lys Thr Ser Gln Pro Val Ile His Tyr His Ile Asn

290 295 300

Asn Asp Asn Arg Thr Tyr Asp Asn Arg Val Phe Asp Asn Arg Val Tyr

305 310 315 320

Asp Asn Ser Tyr His Glu Asn Pro Glu Asn Asp Ala Gln Ser Pro Thr

325 330 335

Ser Gln Thr Asn Asp Leu Leu Ser Arg Asn Gly Asn Ser Leu Leu Asn

340 345 350

Pro Gln Arg Ala Leu Val Gln Lys Val Thr Ser Val Leu Pro His Ser

355 360 365

Ile Ser Asp Ala Val Gln Thr Phe Ala Asn Asn Ser Ala Leu Glu Lys

370 375 380

Val Phe Asn His Thr Pro Asp Asn Ser Asp Gly Ile Gly Ser Asp Leu

385 390 395 400

Leu Thr Thr Ser Ser Gln Glu Arg Ser Thr Asn Asn Ser Leu Ser Arg

405 410 415

Gly His Arg Pro Leu Asn Ile Gln Asn Ser Ser Thr Thr Pro Pro Leu

420 425 430

His Pro Glu Gly Val Thr Ser Ser Asn Asp Asn Ser Ser Asp Thr Thr

435 440 445

Lys Ser Ser Ala Ser Leu Ser His Arg Val Ala Ser Gln Ile Asn Lys

450 455 460

Phe Asn Ser Asn Thr Asp Ser Lys Val Leu Gln Thr Asp Phe Leu Ser

465 470 475 480

Arg Asn Gly Asp Thr Tyr Leu Thr Arg Glu Thr Ile Phe Glu Ala Ser

485 490 495

Lys Lys Val Thr Asn Ser Leu Ser Asn Leu Ile Ser Leu Ile Gly Thr

500 505 510

Lys Ser Gly Thr Gln Glu Arg Glu Leu Gln Glu Lys Ser Lys Asp Ile

515 520 525

Thr Lys Ser Thr Thr Glu His Arg Ile Asn Asn Lys Leu Lys Val Thr

530 535 540

Asp Ala Asn Thr Ile Asn Tyr Val Thr Glu Thr Asn Ala Asp Thr Ile

545 550 555 560

Asp Lys Asn His Ala Ile Tyr Glu Lys Ala Lys Glu Val Ser Ser Ala

565 570 575

Leu Ser Lys Val Leu Ser Lys Ile Asp Asp Thr Ser Ala Glu Leu Leu

580 585 590

Thr Asp Asp Ile Ser Asp Leu Lys Asn Asn Asn Asp Ile Thr Ala Glu

595 600 605

Asn Asn Asn Ile Tyr Lys Ala Ala Lys Asp Val Thr Thr Ser Leu Ser

610 615 620

Lys Val Leu Lys Asn Ile Asn Lys Asp

625 630

<210>2

<211>580

<212>PRT

＜213〉Shigella (Shigella)

<400>2

Met His Asn Val Ser Thr Thr Thr Thr Gly Leu Pro Leu Ala Lys Ile

1 5 10 15

Leu Ala Ser Thr Glu Leu Gly Asp Asn Thr Ile Gln Ala Ala Asn Asp

20 25 30

Ala Ala Asn Lys Leu Phe Ser Leu Thr Ile Ala Asp Leu Ala Ala Asn

35 40 45

Lys Asn Ile Asn Thr Thr Asn Ala His Ser Thr Ser Asn Ile Leu Ile

50 55 60

Pro Glu Leu Lys Ala Pro Lys Ser Leu Asn Ala Ser Ser Gln Leu Thr

65 70 75 80

Leu Leu Ile Gly Asn Leu Ile Gln Ile Leu Gly Glu Lys Ser Leu Thr

85 90 95

Ala Leu Thr Asn Lys Ile Thr Ala Trp Lys Ser Gln Gln Gln Ala Arg

100 105 110

Gln Gln Lys Asn Leu Glu Phe Ser Asp Lys Ile Asn Thr Leu Leu Ser

115 120 125

Glu Thr Glu Gly Leu Thr Arg Asp Tyr Glu Lys Gln Ile Asn Lys Leu

130 135 140

Lys Asn Ala Asp Ser Lys Ile Lys Asp Leu Glu Asn Lys Ile Asn Gln

145 150 155 160

Ile Gln Thr Arg Leu Ser Glu Leu Ala Pro Asp Ser Pro Glu Asn Lys

165 170 175

Lys Leu Arg Arg Glu Glu Ile Gln Leu Thr Ile Lys Lys Asp Ala Ala

180 185 190

Val Lys Asp Arg Thr Leu Ile Glu Gln Lys Thr Leu Ser Ile His Ser

195 200 205

Lys Leu Thr Asp Lys Ser Met Gln Leu Glu Ser Leu Gln Glu Ser Arg

210 215 220

Lys Thr Glu Met Glu Arg Lys Ser Asp Glu Tyr Lys Glu Ile Asp Ser

225 230 235 240

Phe Ser Ala Phe Ser Asn Thr Ala Ser Ala Glu Gln Leu Ser Thr Gln

245 250 255

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

260 265 270

Phe Ile Gln Leu Val Gly Lys Asn Asn Glu Glu Ser Leu Lys Asn Asp

275 280 285

Leu Ala Leu Phe Gln Ala Ala Glu Val Arg Lys Ala Glu Glu Leu Asn

290 295 300

Arg Val Met Gly Cys Val Gly Lys Ile Leu Gly Ala Leu Leu Thr Ile

305 310 315 320

Val Ser Val Val Ala Ala Ala Phe Ser Gly Gly Ala Ser Leu Ala Leu

325 330 335

Ala Ala Val Gly Leu Ala Leu Met Val Thr Asp Ala Ile Val Gln Ala

340 345 350

Ala Thr Gly Asn Ser Phe Met Glu Gln Ala Leu Asn Pro Ile Met Lys

355 360 365

Ala Val Ile Glu Pro Leu Ile Lys Leu Leu Ser Asp Ala Phe Thr Lys

370 375 380

Met Leu Glu Gly Leu Gly Val Asp Ser Lys Lys Ala Lys Met Ile Gly

385 390 395 400

Ser Ile Leu Gly Ala Ile Ala Gly Ala Leu Val Leu Val Ala Ala Val

405 410 415

Val Leu Val Ala Thr Val Gly Lys Gln Ala Ala Ala Lys Leu Ala Glu

420 425 430

Asn Ile Gly Lys Ile Ile Gly Lys Thr Leu Thr Asp Leu Ile Pro Lys

435 440 445

Phe Leu Lys Asn Phe Ser Ser Gln Leu Asp Asp Leu Ile Thr Asn Ala

450 455 460

Val Ala Arg Leu Asn Lys Phe Leu Gly Ala Ala Gly Asp Glu Val Ile

465 470 475 480

Ser Lys Gln Ile Ile Ser Thr His Leu Asn Gln Ala Val Leu Leu Gly

485 490 495

Glu Ser Val Asn Ser Ala Thr Gln Ala Gly Gly Asn Val Ala Ser Ala

500 505 510

Val Phe Gln Asn Asn Ala Ser Lys Asn Leu Ala Asp Leu Thr Leu Ser

515 520 525

Lys Tyr Gln Val Glu Gln Leu Ser Lys Tyr Ile Gly Asp Ala Ile Glu

530 535 540

Lys Phe Gly Gln Leu Gln Glu Val Ile Ala Glu Leu Leu Ala Ser Met

545 550 555 560

Ser Asn Ser Gln Ala Asn Arg Thr Asp Val Ala Lys Ala Ile Leu Gln

565 570 575

Gln Thr Thr Ala

580

<210>3

<211>363

<212>PRT

＜213〉Shigella (Shigella)

<400>3

Met Glu Ile Gln Asn Thr Lys Ser Ala Pro Ile Leu Tyr Thr Asp Ile

1 5 10 15

Ser Thr Lys Gln Thr Gln Ser Ser Ser Glu Thr Gln Lys Ser Gln Asn

20 25 30

Tyr Gln Gln Leu Ala Ala His Ile Pro Leu Asn Val Gly Lys Asn Pro

35 40 45

Val Leu Thr Thr Thr Leu Asn Asp Asp Gln Leu Leu Lys Leu Ser Glu

50 55 60

Gln Val Gln His Asp Ser Glu Ile Ile Ala Arg Leu Thr Asp Lys Lys

65 70 75 80

Met Lys Asp Leu Ser Glu Met Ser His Thr Ile Thr Pro Glu Asn Thr

85 90 95

Leu Asp Ile Ser Ser Leu Ser Ser Asn Ala ValSer Leu Ile Ile Ser

100 105 110

Val Ala Val Leu Leu Ser Ala Leu Arg Thr Ala Glu Thr Arg Leu Gly

115 120 125

Ser Gln Leu Ser Leu Ile Ala Phe Asp Ala Thr Lys Ser Ala Ala Glu

130 135 140

Asn Ile Val Arg Gln Gly Leu Ala Ala Leu Ser Ser Ser Ile Thr Gly

145 150 155 160

Ala Val Thr Gln Val Gly Ile Thr Gly Ile Gly Ala Lys Lys Thr His

165 170 175

Ser Gly Ile Ser Glu Gln Lys Gly Ala Leu Arg Lys Asn Leu Ala Thr

180 185 190

Ala Gln Ser Leu Glu Lys Glu Leu Ala Gly Ser Lys Leu Gly Leu Asn

195 200 205

Lys Gln Ile Asp Thr Asn Ile Thr Ser Pro Gln Thr Asn Ser Ser Thr

210 215 220

Lys Ile Leu Gly Lys Asn Lys Leu Ala Pro Asp Asn Ile Ser Leu Ser

225 230 235 240

Thr Glu His Lys Thr Ser Leu Ser Ser Pro Asp Ile Ser Leu Gln Asp

245 250 255

Lys Ile Asp Thr Gln Arg Arg Ala Tyr Glu Leu Asn Thr Leu Ser Ala

260 265 270

Gln Gln Lys Gln Asn Ile Gly Arg Ala Thr Met Glu Thr Ser Ala Val

275 280 285

Ala Gly Asn Ile Ser Thr Ser Gly Gly Arg Tyr Thr Ser Ala Leu Glu

290 295 300

Glu Glu Glu Gln Leu Ile Ser Gln Ala Ser Ser Lys Gln Ala Glu Glu

305 310 315 320

Ala Ser Gln Val Ser Lys Glu Ala Ser Gln Ala Thr Asn Gln Leu Ile

325 330 335

Gln Lys Leu Leu Asn Ile Ile Asp Asn Ile Asn Gln Ser Arg Asn Ser

340 345 350

Thr Ala Ser Gln Ile Ala Gly Asn Ile Arg Ala

355 360

<210>4

<211>332

<212>PRT

＜213〉Shigella (Shigella)

<400>4

Met Asn Ile Thr Thr Leu Thr Asn Ser Ile Ser Thr Ser Ser Phe Ser

1 5 10 15

Pro Asn Asn Thr Asn Gly Ser Ser Thr Glu Thr Val Asn Ser Asp Ile

20 25 30

Lys Thr Thr Thr Ser Ser His Pro Val Ser Ser Leu Thr Met Leu Asn

35 40 45

Asp Thr Leu His Asn Ile Arg Thr Thr Asn Gln Ala Leu Lys Lys Asp

50 55 60

Leu Ser Gln Lys Thr Leu Thr Lys Thr Ser Leu Glu Glu Ile Ala Leu

65 70 75 80

His Ser Ser Gln Ile Ser Met Asp Val Asn Lys Ser Ala Gln Leu Leu

85 90 95

Asp Ile Leu Ser Lys Lys Glu Tyr Pro Ile Asn Lys Asp Ala Arg Glu

100 105 110

Leu Leu His Ser Ala Pro Lys Glu Ala Glu Leu Asp Gly Tyr Glu Met

115 120 125

Ile Ser His Arg Glu Leu Trp Asp Lys Ile Ala Lys Ser Ile Asn Asn

130 135 140

Ile Asn Glu Gln Tyr Leu Lys Val Tyr Glu His Ala Val Ser Ser Tyr

145 150 155 160

Thr Gln Met Tyr Gln Asp Phe Ser Ala Val Leu Ser Ser Leu Ala Gly

165 170 175

Trp Ile Ser Pro Gly Gly Asn Asp Gly Asn Ser Val Lys Leu Gln Val

180 185 190

Lys Ser Leu Lys Asp Glu Leu Thr Lys Leu Lys Glu Lys Tyr Lys Asp

195 200 205

Lys Pro Leu Tyr Pro Ala Asn Asn Thr Val Ser Lys Glu Gln Ala Asn

210 215 220

Lys Trp Leu Thr Glu Leu Gly Gly Thr Ile Gly Lys Val Ser Glu Lys

225 230 235 240

Asn Gly Gly Tyr Val Val Asn Ile Asn Met Thr Pro Ile Asp Asn Met

245 250 255

Leu Lys Ser Leu Asp Asn Leu Gly Gly Asn Gly Glu Val Val Leu Asp

260 265 270

Asn Ala Lys Tyr Gln Ala Trp Asn Ala Gly Phe Ser Ala Glu Asp Glu

275 280 285

Thr Met Lys Asn Asn Leu Gln Thr Leu Val Gln Lys Tyr Ser Asn Ala

290 295 300

Asn Ser Ile Phe Asp Asn Leu Val Lys Val Leu Ser Ser Thr Ile Ser

305 310 315 320

Ser Cys Thr Asp Thr Asp Lys Leu Phe Leu His Phe

325 330

<210>5

<211>1902

<212>DNA

＜213〉Shigella (Shigella)

<400>5

atgcataatg taaataatac tcaagcgcca acattcttat ataaggcaac ttcaccatca 60

tcaacagaat acagcgagtt aaaaagcaaa atatccgata tccatagttc gcaaacttct 120

ctaaaaacac cagcatcagt gtctgaaaaa gaaaactttg caacgtcttt taatcagaaa 180

tgtcttgatt ttttattttc ttcctcaggg aaagaagatg tgttaagaag catttattcc 240

aactcaatga atgcgtatgc caaaagcgag attctcgaat tttcaaatgt tttgtactcc 300

ttagtacatc aaaatgatct taattttgaa aacgaaaagg gacttcaaaa aattgtcgca 360

cagtattcgg aactaattat aaaagataaa ttatcccaag attctgcctt tggaccatgg 420

tcggcaaaga ataagaaact ccatcaatta cgacaaaaca ttgagcacag acttgcacta 480

ttagcacaac aacacacatc tggtgaagct ttatcattgg gacaaaaact cctcaatact 540

gaagtatcat catttatcaa gaataatatt cttgctgaat taaagttaag taatgaaact 600

gtttcatctc tcaaactaga tgatttagtt gacgcacagg caaaacttgc ctttgatagt 660

ttgcgcaatc aacgtaaaaa tactattgat agtaaaggat ttggtatagg taaactgtca 720

agagacttaa atacagtagc cgtgtttcct gagctgttga gaaaagtcct taatgatatt 780

ttagaagata taaaagattc gcatcctatc caagatggcc tccctacacc tcccgaagat 840

atgccagatg gcggaccaac ccccggagcc aatgagaaaa catcccaacc tgtaattcac 900

tatcatataa ataatgataa tagaacttac gataatagag tttttgacaa cagagtatat 960

gacaatagct atcacgagaa cccagaaaat gatgcacagt ctcctacttc tcagacaaac 1020

gatctattat cccgtaacgg aaactcatta ctaaatccac aaagagcact agttcaaaaa 1080

gtaacttccg ttctaccaca ctctatatca gatgctgtcc agacatttgc aaataattca 1140

gctttagaaa aggttttcaa ccatactcca gataattcgg atggaatagg ttcagacctg 1200

ttaactacga gtagtcaaga aagatctaca aataactctc tttctcgggg acacaggcct 1260

ctgaacatac agaactcttc aaccaccccc cctctccacc cggaaggagt gacaagcagt 1320

aatgataact catcagatac aactaaaagt agcgcttctc tttctcatag agtagcttcg 1380

caaatcaata aattcaactc aaacactgat tcaaaagtac ttcagactga ttttttatca 1440

agaaatggag acacatattt aacacgggaa acgatatttg aagcttcaaa aaaagtaaca 1500

aactccctaa gtaatcttat atctctcatt ggaactaaat caggaacaca agaacgagag 1560

ttacaggaaa aatcaaagga cattacaaaa tccacaacag aacacagaat aaacaacaaa 1620

ttaaaagtta cagatgcaaa tacaataaac tacgtaacag aaaccaacgc agatacaatt 1680

gataaaaatc atgcgatcta tgaaaaggca aaagaagtat ctagcgccct cagcaaggta 1740

ttgtcaaaaa ttgacgatac ctctgcagaa ttacttacag atgatatatc tgatttaaaa 1800

aataacaatg atattacagc tgaaaacaat aatatatata aagcagcaaa agatgtaacc 1860

acttccctat caaaagtatt aaagaatatc aataaggatt aa 1902

<210>6

<211>1743

<212>DNA

＜213〉Shigella (Shigella)

<400>6

atgcataatg taagcaccac aaccactggt ttgcctcttg ccaaaatatt ggcttccact 60

gagcttggag acaatactat ccaggctgca aatgatgcag ctaacaaatt attttctctt 120

acaattgctg atcttgctgc taacaaaaat attaatacaa ctaatgcaca ctcaacttcg 180

aatatattaa tccctgaact taaagcacca aagtcattaa atgcaagttc ccaactaacg 240

cttttaattg gaaaccttat tcaaatactt ggtgaaaaat ctttaactgc attaacaaat 300

aaaattactg cctggaagtc ccagcaacag gcaagacagc aaaaaaacct agaattctcc 360

gataaaatta atactcttct atctgaaact gaaggactaa ccagagacta tgaaaaacaa 420

attaataaac taaaaaacgc agattcaaaa ataaaagacc tagaaaataa aattaaccaa 480

attcaaacaa gattatccga actcgcccca gattcaccag aaaataaaaa attaagacgg 540

gaagaaatac aactcactat caaaaaagac gcagcagtta aagacaggac attgattgag 600

cagaaaactc tgtcaattca tagcaaactt acagataaat caatgcaact cgaaaaagaa 660

atagactctt tttctgcatt ttcaaacaca gcatctgctg aacagctatc aacccagcag 720

aaatcattaa ccggacttgc cagtgttact caattgatgg caacctttat tcaactagtt 780

ggaaaaaata atgaagaatc tttaaaaaat gatctggctc tattccagtc tctccaagaa 840

tcaagaaaaa ctgaaatgga gagaaaatct gatgagtatg ctgctgaagt acgtaaagca 900

gaagaactca acagagtaat gggttgtgtt gggaaaatac ttggggcact tttaactatc 960

gttagtgttg ttgcagcagc tttttctgga ggagcatctc tagcactggc agctgttggt 1020

ttagctctta tggttacgga tgctatagta caagcagcga ccggcaattc cttcatggaa 1080

caagccctga atccgatcat gaaagcagtc attgaaccct taatcaaact cctttcagat 1140

gcatttacaa aaatgctcga aggcttgggc gtcgactcga aaaaagccaa aatgattggc 1200

tctattctgg gggcaatcgc aggcgctctt gtcttggttg cagcagtcgt tctcgtagcc 1260

actgttggta aacaggcagc agcaaaactt gcagagaata ttggcaaaat aataggtaaa 1320

accctcacag accttatacc aaagtttctc aagaattttt cttctcaact ggacgattta 1380

atcactaatg ctgttgccag attaaataaa tttcttggtg cagcgggtga tgaagtaata 1440

tccaaacaaa ttatttccac ccatttaaac caagcagttt tattaggaga aagtgttaac 1500

tctgccacac aagcgggagg aaatgtcgct tctgctgttt tccaaaacaa cgcgtcgaaa 1560

aatctagcag acctgacatt atcgaaatat caagttgaac aactgtcaaa atacatcggt 1620

gacgcaatag aaaaattcgg ccaattgcag gaagtaattg cagagctgtt agcctcaatg 1680

tccaactctc aggctaatag aactgatgtt gcaaaagcaa ttttgcaaca aactactgct 1740

tga 1743

<210>7

<211>1092

<212>DNA

＜213〉Shigella (Shigella)

<400>7

atggaaattc aaaacacaaa atcagccccg attttatata cagatatatc tacaaaacaa 60

actcaaagtt cttccgaaac acaaaaatca caaaattatc agcagttagc agcacatatt 120

ccacttaatg tcggtaaaaa tcccgtatta acaactacat taaatgatga tcaactttta 180

aagttatcag agcaggttca gcatgattca gaaatcattg ctcgccttac tgacaaaaag 240

atgaaagatc tttcagaaat gagtcacaca attaccccag agaacaccct ggatatttcc 300

agtctttctt ctaatgctgt ttctttaatt attagtgtag ccgttctact ttctgctctc 360

cgcactgcag aaactagatt gggctctcaa ttgtcattga ttgcgttcga tgctacaaaa 420

tcagctgcag agaacattgt tcggcaaggc ctagcagccc tatcatcaag cattactgga 480

gcggtcacac aagtaggtat aacgggtatc ggtgccaaaa aaacgcattc agggattagc 540

gaacaaaaag gagccttaag aaagaacctt gccactgctc aatctcttga aaaagagctt 600

gcaggttcta aattagggtt aaataaacaa atagatacaa atatcacctc accacaaact 660

aactctagca caaaaatttt aggtaaaaat aaactggcac cagataatat atccctgtca 720

actgaacata aaacttctct tagttctccc gatatttctt tgcaggataa aattgacacc 780

cagagaagag cttacgagct caataccctt tctgctcagc aaaaacaaaa cattggccgt 840

gcaacaatgg agacatcagc cgttgctggt aatatatcca catcaggagg gcgttataca 900

tctgctcttg aagaagaaga acaactaatc agtcaggcca gcagtaaaca agcagaggaa 960

gcatcccaag tatctaaaga agcatcccaa gcgacaaatc aattaataca aaaattattg 1020

aatataattg acaacatcaa ccaatcaagg aattctacag ccagtcagat tgctggtaac 1080

attcgagctt aa 1092

<210>8

<211>998

<212>DNA

＜213〉Shigella (Shigella)

<400>8

atgaatataa caactctgac taatagtatt tccacctcat cattcagccc aaacaatacc 60

aacggttcat caaccgaaac agttaattct gatataaaaa caacgaccag ttctcatcct 120

gtaagttccc ttactatgct caacgacacc cttcataata tcagaacaac aaatcaggca 180

ttaaagaaag atctctcaca aaaaacgttg actaaaacat cgctagaaga aatagcatta 240

cattcatctc agattagcat ggatgtaaac aaatccgctc aactattgga tattctttcc 300

aagaaagaat atccaattaa taaagacgca agagaattat tacattcagc cccgaaagaa 360

gccgagcttg atggatatga aatgatatct cacagagaac tgtgggataa aattgcaaag 420

tcaatcaata atattaatga acagtatctg aaagtatatg aacatgccgt tagttcatat 480

actcaaatgt atcaagattt tagcgctgtt ctttccagtc ttgccggctg gatctctccc 540

ggaggtaacg acggaaactc cgtgaaatta caagtcaaat cgcttaaaga cgaattgaca 600

aagctcaagg aaaaatataa gataaaccgc tatatccagc aaataatact gttagtaagg 660

aacaagcaaa taaatggctt acagaattag gtggaacaat cggcaaggta tctgaaaaaa 720

acgggggata tgttgtcaat ataaacatga ccccaataga caatatgtta aaaagcttag 780

ataatctagg tggaaatggc gaggttgtgc tagataatgc aaaatatcag gcatggaatg 840

ccggattctc tgccgaagat gaaacaatga aaaataatct tcaaacttta gttcaaaaat 900

acagtaatgc caatagtatt tttgataatt tagtaaaggt tttgagtagt acaataagct 960

catgtacaga tacagataaa ctttttctcc atttctga 998

Claims (10)

1, a kind of dysentery multivalent genetic engineering vaccine, its activeconstituents are both to have expressed invasin protein antigen I paA, IpaB, and IpaC and IpaD express the dysentery multivalent genetic engineering vaccine strain of the two valency LPS-0 polysaccharide antigens of Fu Shi and Song Nei Shi again.

2, vaccine according to claim 1 is characterized in that: the production substratum of described dysentery multivalent genetic engineering vaccine is that pH is Hou Shi substratum or the LB substratum of 6.5-7.6.

3, vaccine according to claim 2 is characterized in that: the pH of described production substratum is 7.2-7.5.

4, according to claim 1 or 2 or 3 described vaccines, it is characterized in that: the formulation of described dysentery multivalent genetic engineering vaccine is capsule oral formulation or form of nose drops.

5, vaccine according to claim 4 is characterized in that: the dysentery multivalent genetic engineering vaccine of described formulation adopts lyophilization to be prepared.

6, vaccine according to claim 5, it is characterized in that: the lyophilized vaccine that is adopted in the dysentery multivalent genetic engineering vaccine process of the described formulation of preparation is 0.5% gelatin that contains of pH7.0-7.6, the phosphoric acid buffer of 5% sucrose, 0.2% vitamins C, 0.1% thiaminogen, 0.1% skim-milk; Described percentage composition is a quality quality percentage composition.

7, a kind of method for preparing the described dysentery multivalent genetic engineering vaccine strain of claim 1 may further comprise the steps:

1) will contain invasin protein IpaA, IpaB, the I of IpaC and IpaD gene plasmid mutually transfer among the shigella flexneri 2a type II, obtain containing the reorganization shigella flexneri 2a type II of described I phase plasmid;

2) will contain aroD genetically deficient among the reorganization shigella flexneri 2a type II of described I phase plasmid, both expressed invasin protein antigen I paA, IpaB, IpaC and IpaD, expressed the dysentery multivalent genetic engineering vaccine strain of the two valency LPS-O polysaccharide antigens of Fu Shi and Song Nei Shi again.

8, method according to claim 7 is characterized in that: the described invasin protein IpaA that contains, IpaB, the I of IpaC and IpaD gene plasmid mutually come from Song Nei Shi dysentery bacillus strain 48025-11 or Song Nei Shi dysentery bacterial strain S63.

9, method according to claim 7 is characterized in that: the described invasin protein IpaA that contains, IpaB, the I of IpaC and IpaD gene plasmid mutually transfer among the shigella flexneri 2a type II by luring kinoplaszm grain R386; The described kinoplaszm grain R386 that lures comes from E.coli.

10, according to claim 7 or 8 or 9 described methods, it is characterized in that: described shigella flexneri 2a type II is a shigella flexneri 2a type II T32 bacterial strain.

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