CN101914564B - Fusion of multiple enterotoxin genes of escherichia coli and application thereof - Google Patents
Fusion of multiple enterotoxin genes of escherichia coli and application thereof Download PDFInfo
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Abstract
The invention relates to an escherichia coli (E. coli) trivalent enterotoxin fusion gene and application thereof, and belongs to the field of genetic engineering subunit vaccines. The enterotoxigenic E. coli (ETEC) is a main pathogen causing baby-animal and infantile diarrhea. Aiming at the defects that the conventional TEC vaccine has narrow protection range and cannot induce high-level antitoxin, the invention designs a trivalent E. coli enterotoxin fusion with the fusion mode of 5'-STa-LT-STb-3' or 5'-STb-LT-STa-3'. After the multivalent enterotoxin gene or protein immunizes animals, high-level STa, STb and LT resistant antibodies can be induced to be generated. Therefore, the escherichia coli (E. coli) trivalent enterotoxin fusion gene can neutralize the toxicity of a key pathogenic factor enterotoxin, improve the immunity protection rate and widen the protection range without being limited by E. coli pilus types so as to effectively control ETEC diarrhea.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of genetic engineering subunit vaccine, particularly a kind of enterotoxigenic escherichia coli (ETEC) multivalence enterotoxin immunogen, be used for prevention and treatment by bacterial diarrhoea such as ETEC.
Background technology
Enterotoxigenic escherichia coli (ETEC) can cause young animal acute, the infectious diarrhea of (comprising piglet, calf, lamb etc.), the sound development of serious harm livestock breeding industry, and cause huge financial loss.ETEC or the infant of developing country and turista's main pathogens, the annual diarrhoea case that infects because of ETEC in the whole world is up to 6.5 hundred million person-times, cause approximately death of child below 380,000 5 years old (Sizemore etc.,, Expert Rev.Vaccines in 2004).
ETEC causes that the mechanism of action that diarrhoea occurs is: at first bacterium is colonizated on small intestinal epithelial cells by its surperficial adhesin such as pili, and then in enteron aisle amount reproduction and produce enterotoxin, enterotoxin as direct virulence factor makes the intestinal epithelial cells metabolism disorder, secretion large quantity of moisture and ionogen enter enteric cavity, finally cause the generation of suffering from diarrhoea.The pili of ETEC is of a great variety, the pili of animal-origin bacterial strain, and more important have F4 (K88), F5 (K99), F6 (987P), F18, a F41 etc.; The pili of people source ETEC is more, identified over 25 kinds.But the enterotoxin of ETEC secretion only has two kinds, and heat-labile toxin LT and heat-stable toxin ST, ST are divided into STa and two hypotypes of STb.
For the treatment of ETEC diarrhoea, main microbiotic and the chemicals used in production.But the key of controlling this disease is prevention, and the method for prevention is mainly used the vaccine immunity prevention except keeping health, sterile environment.Present commercial ETEC vaccine has full bacterium inactivated vaccine, pili subunit vaccine and toxoid vaccine.Below enumerate 4 vaccines that obtain China Ministry of Agriculture veterinary drug product authentication code, they are Typical Representatives of the bacillus coli vaccine for animals that uses in production practice:
(1) sheep colibacillosis inactivated vaccine, Heilongjiang Province's biological products one factories, veterinary drug new word (2006) 080074009.
(2) escherichia coli of piglets tervalence inactivated vaccine, Shandong animal health-care product health care company, veterinary drug new word (2006) 150251028.
(3) colibacillosis of piglet K88/K99 bivalent gene engineering inactivated vaccine, sea, Shanghai sharp biologics company limited, veterinary drug new word (2007) 090201027.
(4) colibacillosis of piglet K88, LTB bivalent gene engineering living vaccine, biological pharmaceutical factory, Zhongmu Industry Co.,Ltd Jiangxi, veterinary drug new word (2008) 140401025.
Heat-labile toxin LT is comprised of two subunits, and A subunit (LTA) is the toxicity center, and B subunit (LTB) is a pentamer structure, there is no toxicity and good immunogenicity is arranged, and usually is used as the composition of vaccine.Heat-stable toxin STa and STb are the very little haptens of molecular weight, lack immunogenicity, if directly can't induce the body generation for the neutralizing antibody of STa and STb with the immunity of full bacterium inactivated vaccine.in order to address this problem, heat-stable toxin STa or STb can be coupled on macromolecular carrier proteins, make them possess immunogenicity (the Development of a vaccine of cross-linked heat-stable andheat-labile enterotoxins that protects against Escherichia coli producing eitherenterotoxin.Infect.Immun.1982 such as I.Klipstein, 37 (2): A recombinantEscherichia coli heat-stable enterotoxin b (STb) the fusion protein eliciting neutralizingantibodies such as 550-557.II.Dubreuil, FEMS Immunol.Med.Microbiol.1996, 13:317-323.).
Although ST is the important virulence factor that causes suffering from diarrhoea and occur, because natural STa and STb molecule do not have immunogenicity, the vaccine product of listing does not contain the ST composition now.In known prior art, in order to prepare effective STa or STb toxoid, the researchist has also built following fusion subunit vaccine:
1. the LTB-STa divalence merge enterotoxin (Zhang Zhaoshan etc., the fusion of heat-labile toxin and heat-stable toxin gene. Chinese microbiology and Journal of Immunology, 1994,14 (04): 219-222.)
2. the STa-LTB divalence merge enterotoxin (Xu Chongbo etc., the structure of enterotoxins of Escherichia coli ST1-LT-B fusion gene. Chinese animal doctor's journal, 1998,18 (05): 463-465.)
3. the K88ac-ST1-LTB trivalent merge enterotoxin and pilin (Xu Chongbo etc. express the immunogenicity research of intestinal bacteria K88ac-ST1-LTB fusion rotein engineering strain. Chinese Preventive Veterinary Medicine is reported, 2003,25 (01): 9-12.)
4. the LTB-STa-STb trivalent merge enterotoxin (Ge Yan etc., the construction and expression research of enterotoxigenic escherichia coli enterotoxin LTB, ST I and ST II fusion gene. Chinese Preventive Veterinary Medicine newspaper, 2002,24 (05): 27-29.)
5. the STa-STb-LTB trivalent merge enterotoxin (Wu Dongmei etc., the structure of enterotoxins of Escherichia coli STI, STII, LTB fusion gene plant expression vector. northwest agricultural journal, 2006,15 (05): 150-154.)
As previously mentioned; the pili numerous types of pathogenic bacterium ETEC, the clinical pathogenic strain that is separated to are usually expressed some still unidentified pili, thereby only use the ETEC of several frequently seen type to prepare the polyvalent inactivation whole-bacterial-vaccine; its protection domain is inadequate, often can run into protection ratio problem on the low side.Compare with diversified pili adhesin, enterotoxin only has LT and ST two classes, no matter ETEC produces the pili of what type, all finally to rely on these two kinds of enterotoxins to cause morbidity, therefore utilize enterotoxin to prepare vaccine and can realize purpose for all types ETEC bacterial strain, its advantage is: remove the trouble of preparation multiple (for different pili types) ETEC vaccine from, simplified immunization protocol, reduced cost; Can cause body and produce higher levels of LT antibody, the more important thing is the generation (inactivated vaccine is without this effect) that to induce ST antibody, thereby can improve the effect of immunoprophylaxis, and have certain therapeutic action.
Above-mentioned document 1., 2., three classes in 3. merge enterotoxins and do not comprise STb, thereby its immune protective effect is not comprehensive; 4. and 5. comprised whole 3 kinds of important enterotoxins, but its integration program is not optimum.When three kinds of albumen merged, the albumen in the middle of generally being positioned at can be subject to larger impact than the albumen that is positioned at two ends, and its space structure can change because of the effect of both sides fusion rotein, thereby changes its immunogenicity.So for the fusion rotein LTB-STa-STb of document design 4., its deficiency is that the antigenic determinant of STa can be subject to the impact of both sides albumen and change, thereby can't present best immunogenicity.In like manner, the STa-STb-LTB that 5. document builds, defective is that STb can not present best immunogenicity.For their deficiency, the present invention has done effective improvement, and the immunogenicity of STa and STb all is improved.
Summary of the invention
The technical problem to be solved in the present invention is: overcome the deficiency that existing enterotoxigenic escherichia coli (ETEC) vaccine protection domain is narrower, can not induce generation high-caliber anti-heat-stable toxin (STa and STb) antibody; provide a kind of multivalence to merge the enterotoxin immunogen; can be used as vaccine and be used for controlling by bacterial diarrhoea such as ETEC, the effect that realize enlarging protection domain, improves protection ratio.
Technical scheme of the present invention is as follows:
Obtain three sections enterotoxin genes STa of enterotoxigenic escherichia coli, STb and LTB, the end to end multivalence that is joined together to form of three is merged enterotoxin genes, the order of its fusion is STa-LTB-STb or STb-LTB-STa from 5 ' end to 3 ' end, and namely LTB is positioned at centre, STa and STb and is positioned at both sides.In order to strengthen multivalence enterotoxin fusion gene as the effect of DNA vaccination immune hen, can connect at its upstream the signal peptide gene of the secreted protein of chicken.For the fusion gene that builds, LTB can be replaced by the fragment of LTB, and perhaps the fragment by CTB (b subunit of cholera toxin) or CTB replaces; STa and STb can be that individual gene or a plurality of gene are cascaded, or the series winding body of the fragment of STa and STb or a plurality of fragments.Described enterotoxin genes STa, STb, LTB, the fragment of CTB refers to the gene fragment that contains one or more antigenic determinants that intercepts from full gene, its length is 15 Nucleotide at least, can come prediction and choose these gene fragments by associated biomolecule software.The multivalent genetic that obtains is inserted suitable expression vector, then import host cell and express and to obtain multivalence enterotoxin fusion rotein.
Build the technical scheme of trivalent enterotoxin fusion gene STa-LTB-STb or STb-LTB-STa and use as follows:
(1) increase the respectively gene of STa, STb and LTB
With the STa (V00612.1), the STb (E00811.1) that announce in the GenBank database and the complete genome sequence of LTB (M17873.1), design has the primer of complementary end on two enterotoxin genes that needs connect, the ripe gene of conventional PCR reaction amplification STa, STb and LTB.
(2) use overlapping extension PCR method, first LTB is connected with STa (or STb), again the divalence fusion gene that obtains is connected with the 3rd gene STb (or STa), thereby obtains the fusion gene of 5 '-STa-LTB-STb-3 ' or 5 '-STb-LTB-STa-3 ' form.
(3) optional step: the signal peptide gene (called after L) of the secreted protein of amplification chicken, same with overlapping extension PCR method with its upstream that is fused to trivalent enterotoxin fusion gene, obtain the fusion gene as 5 '-L-STa-LTB-STb-3 ' or 5 '-L-STb-LTB-STa-3 ' form.Preferred signal peptide gene is the signal peptide of chicken insulin gene, and length is 72 bases, and its sequence is 5 '-ATGGCTCTCTGGATCCGATCACTGCCTCTTCTGGCTCTCCTTGTCTTTTCTGGCCC TGGAACCAGCTATGCA-3 '; Be perhaps the signal peptide of plasma urokinase-type plasminogen activator (upa) gene of chicken, length is 60 bases, and its sequence is 5 '-ATGAAGTTAATCATCTTTCTCACAGTAACTCTCTGCACACTTGTCACAGGACTTGA TTCT-3 '.
(4) utilize conventional Protocols in Molecular Biology, trivalent enterotoxin fusion gene is inserted carrier for expression of eukaryon obtain recombinant plasmid, namely can be used as trivalent enterotoxin DNA vaccination; Or trivalent enterotoxin fusion gene is inserted prokaryotic expression carrier, the recombinant plasmid that obtains obtains to merge enterotoxin albumen through expressing in intestinal bacteria, is the enterotoxin protein vaccine.
(5) application of trivalent enterotoxin vaccine: with the trivalent enterotoxin DNA vaccination that obtains or trivalent enterotoxin protein vaccine immunity pregnant female, can provide for the newborn young baby who sucks breast milk the passive immunization protection; This two classes vaccine also can be directly used in immune cub, makes cub produce active immunity, the generation of prevention diarrhoea; Two class vaccines can also be used for Immune Laying Hens, obtain containing the immune egg of anti-enterotoxin yolk antibody, and the antibody in the recycling immune egg is oral to cub, can make equally it obtain the passive immunization protection.
Beneficial effect of the present invention: advantage of the present invention is with 3 sections important enterotoxin STa, STb and LTB links together and carried out optimum combination, and the integration program of employing is " STa-LTB-STb " or " STb-LTB-STa ".That is to say that the present invention is with in the middle of carrier proteins LTB is placed in, and two haptens STa and STb separation two ends.In the middle of above 4. " background technology " Literature is not placed in LTB with 5. fusion enterotoxin, but be placed on an end, STa or STb are placed on the mid-way.Can not induce the deficiency of ST antibody in order to overcome inactivated vaccine and LT toxoid, the immunogenicity that strengthens STa and STb is the key that builds efficient enterotoxin vaccine, so STa and STb fusion can farthest be preserved its natural antigenic determinant at the two ends of carrier proteins, make them have maximum immunogenicity, thus enterotoxin amalgamation mode provided by the invention be better than document 4. with 5. integration program.Particularly, after document LTB-STa-STb fusion protein immunization animal 4., the active relative deficiency of the STa antibody of inducing; After document STa-STb-LTB fusion protein immunization animal 5., the active relative deficiency of the STb antibody of inducing.If use multivalence provided by the invention to merge enterotoxin " STa-LTB-STb " or " STb-LTB-STa " immune animal; than existing enterotoxin vaccine; it is advantageous that: can induce to produce higher levels of STa antibody and STb antibody; thereby obtain higher protection ratio; improve the protection effect of vaccine, reduce the incidence of ETEC diarrhoea.
Embodiment
Embodiment 1. preparation trivalent enterotoxin DNA vaccination and protein vaccines
(1) amplification enterotoxin genes STa
Do not use template, directly carry out conventional pcr amplification with following primer, condition: 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, 25 circulations, last 72 ℃ were extended 5 minutes.
STa-F1：5’-agggaattcaccatgaacacattctactgctgcgagctgtgctgcaat-3’
STa-R：5’-tagcaggtgggtagcagccagcggcggcgggattgcagcacagc-3’
(2) amplification enterotoxin genes LTB and STb
Template uses primer as follows with K88ac bacterial strain (C83902, be purchased from Chinese veterinary medicament supervisory institute) lysate, makes conventional pcr amplification, condition: 94 ℃ of denaturations 60 seconds, 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 40 seconds, 30 circulations, and last 72 ℃ were extended 5 minutes.
LTB-F：5’-gctacccacctgctagcccagctccccagactattacag-3’
LTB-R：5’-tggtgtggtgctggcgctgtttttcatactgattgcc-3’
STb-F：5’-gccagcaccacaccaccctctacacaatcaaataagaaagat-3’
STb-R：5’-cgctctagatcctcagcatccttttgctgcaaccat-3’
(3) connect LTB and STb to obtain LTB-STb
Template the PCR product LTB of previous step and the mixture of STb, the LTB-F that primer uses previous step to use, STb-R, PCR reaction conditions such as step (2).
(4) connect STa and LTB-STb to obtain STa-LTB-STb
Template uses primer as follows, PCR reaction conditions such as step (2) with step (1) and the PCR product S Ta of (3) and the mixture of LTB-STb.
STa-F2:5 '-acc
Accatgaacacattc-3 ' contains the EcoRI site
STbR1:5 '-acc
Tcctcagcatcc-3 ' contains the XbaI site
(5) build trivalent enterotoxin DNA vaccination pCI-STa-LTB-STb
Use conventional Protocols in Molecular Biology, utilize the restriction enzyme site at two ends to be cloned in carrier for expression of eukaryon pCI (U.S. Promega company) trivalent enterotoxin fusion gene STa-LTB-STb, and be transformed in bacillus coli DH 5 alpha.
(6) preparation of DNA vaccination pCI-STa-LTB-STb
A large amount of E.coli DH5 α that contain plasmid pCI-STa-LTB-STb that cultivate, use SDS alkaline lysis separation quality grain, polyoxyethylene glycol method plasmid purification (with reference to " molecular cloning experiment guide " third edition chapter 1 scheme 3 and scheme 8, the author is J.Sambrook and D.W.Russell), can obtain the trivalent enterotoxin DNA vaccination pCI-STa-LTB-STb for immune animal.
(7) build trivalent enterotoxin prokaryotic expression carrier pET30-STa-LTB-STb
Use following pair of primers, make template with the pCI-STa-LTB-STb plasmid that builds, amplification obtains trivalent enterotoxin fusion gene STa-LTB-STb, the restriction enzyme site that recycles its two ends is cloned in prokaryotic expression carrier pET30a (+) (U.S. Novagen company), obtains recombinant plasmid pET30-STa-LTB-STb and is transformed in e. coli bl21 (DE3).
SsF1:5 '-gcctaca
Aacacattctactg-3 ' contains the NdeI site
SsR1:5 '-acg
Gcatccttttgctgcaaccatta-3 ' contains the XhoI site
(8) express fusion enterotoxin STa-LTB-STb and separation and purification
Cultivate recombination bacillus coli BL21 (DE3) pET30-STa-LTB-STb, add IPTG and carry out abduction delivering, isolation of occlusion bodies (its main component is to merge enterotoxin Protein S Ta-LTB-STb), fully impurity is removed in washing, with the urea buffer solution dissolving inclusion body of high density, ultrafiltration process is removed urea and renaturation inclusion body, namely obtains the fusion enterotoxin STa-LTB-STb of solubility again, can be mixed and made into vaccine with suitable adjuvant, also can not add adjuvant and use separately.The STa-LTB-STb inclusion body can also be processed without dissolving, renaturation, only by directly using as vaccine after washing step, so also can excite the generation of anti-enterotoxin antibody, but than the immunogenicity of solubility STa-LTB-STb albumen slightly a little less than.
Embodiment 2. builds the trivalent enterotoxin DNA vaccination that contains chicken insulin signaling peptide
pCI-ins-STa-LTB-STb
(1) amplification chicken insulin signaling peptide gene ins
Do not use template, directly carry out conventional pcr amplification with following primer, condition: 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, 25 circulations, last 72 ℃ were extended 5 minutes again.
InsF:5 '-aac
Accatggctctctggatccgatcactgcctcttctggctctccttgt-3 ' contains the EcoRI site
insR：5’-gcagtagaatgtgtttgcatagctggttccagggccagaaaagacaaggagagccag-3’
(2) amplification heat-stable toxin gene STa
Do not use template, directly carry out conventional pcr amplification with following primer, the same step of reaction conditions (1).
STa-F：5’-aacacattctactgctgcgagctgtgctgcaatccc-3’
STa-R：5’-tagcaggtgggtagcagccagcggcggcgggattgcagcacagc-3’
(3) connect ins and STa to obtain ins-STa
Template is used insF and STa-R, PCR reaction conditions such as step (1) with the PCR product ins of first two steps and the mixture of STa, primer.
(4) connect ins-STa and LTB-STb to obtain ins-STa-LTB-STb
Template is used insF and STb-R (seeing embodiment 1), PCR reaction conditions such as step (1) with the mixture of PCR product ins-STa and LTB-STb (preparation method is with embodiment 1), primer.
(5) the trivalent enterotoxin DNA vaccination pCI-ins-STa-LTB-STb of construction expression chicken insulin signaling peptide
Use conventional Protocols in Molecular Biology, utilize the restriction enzyme site at two ends to be cloned in carrier for expression of eukaryon pCI ins-STa-LTB-STb, and be transformed in bacillus coli DH 5 alpha.
(6) preparation of DNA vaccination pCI-ins-STa-LTB-STb
The preparation method is with the step (6) of embodiment 1.
Embodiment 3. builds the trivalent enterotoxin DNA vaccination pCI-upa-STa-LTB-STb that contains chicken plasma urokinase-type plasminogen activator (upa) signal peptide
(1) the signal peptide gene upa of amplification chicken upa
Do not use template, directly carry out conventional pcr amplification with following primer, condition: 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, 25 circulations, last 72 ℃ were extended 5 minutes again.
UpaF:5 '-aac
Accatgaagttaatcatctttctcacagtaactctctgcac-3 ' contains EcoRI site upaR:5 '-gcagtagaatgtgttagaatcaagtcctgtgacaagtgtgcagagagttactg-3 '
(2) amplification enterotoxin genes STa
The preparation method is with the step (2) of embodiment 2.
(3) connect upa and STa to obtain upa-STa
Template is used upaF and STa-R (seeing embodiment 2), PCR reaction conditions such as step (1) with the PCR product upa of first two steps and the mixture of STa, primer.
(4) connect upa-STa and LTB-STb to obtain upa-STa-LTB-STb
Template is used upaF and STb-R (seeing embodiment 1), PCR reaction conditions such as step (1) with the mixture of PCR product upa-STa and LTB-STb (preparation method is with embodiment 1), primer.
(5) the trivalent enterotoxin DNA vaccination pCI-upa-STa-LTB-STb of construction expression chicken upa signal peptide
Use conventional Protocols in Molecular Biology, utilize the restriction enzyme site at two ends to be cloned in carrier for expression of eukaryon pCI upa-STa-LTB-STb, and be transformed in bacillus coli DH 5 alpha.
(6) preparation of DNA vaccination pCI-upa-STa-LTB-STb
The preparation method is with the step (6) of embodiment 1.
Embodiment 4. trivalent enterotoxin DNA vaccinations and protein vaccine Immune Laying Hens prepare toxinicide yolk antibody (IgY) and IgY to the protection effect of piglet
With 4 kinds of immunogens: trivalent enterotoxin DNA vaccination pCI-ins-STa-LTB-STb (pCI-ins-SLS), pCI-upa-STa-LTB-STb (pCI-upa-SLS), the blue brown hen of pCI-STa-LTB-STb (pCI-SLS) and trivalent enterotoxin protein vaccine STa-LTB-STb (SLS) immunity sea, every group of 5 hens, carry out first immunisation opening antenatal two weeks, after two weeks, two exempt from, then carry out three and exempt from after three weeks.2 of immunization ways: DNA vaccination---chest muscles add leg muscle 2 points; Protein vaccine---chest muscle 2 points, neck is more subcutaneous.Immunizing dose/chicken/time: 200 μ g plasmid DNA or 400 μ g albumen.Three exempt from beginning in rear 10 days collects egg, dilute with water method separating IgY continuously.The key step of water dilution method: separate yolk, add the distilled water of 6 times of volumes, it is 5.0,4 ℃ of hold over night that 0.1M dilute hydrochloric acid is regulated pH, and the centrifuging and taking supernatant is the water soluble ingredient that contains IgY, ammonium sulfate precipitation, ultrafiltration desalination.
Detect the tiring of specific antibody of three kinds of enterotoxin STa, STb and LTB with indirect elisa method.The antigen that is used for coated 96 orifice plates in the ELISA experiment is the fusion enterotoxin G ST (Thiadiazolidine isomerase) of use escherichia coli expression-STa, Trx (Trx)-STb and LTB.Two anti-be the anti-chicken IgG of rabbit of HRP mark, chromogenic substrate TMB, the OD value under detection 450nm.Tiring of antibody is defined as OD
450nmThe maximum dilution multiple of the IgY of value 〉=0.2.
Three immunity of table 1. are tiring of anti-three kinds of enterotoxin special yolk antibodies afterwards
Every group of 8 newborn piglets of piglet protest test, artificial feeding's milk and breast nursing not, experimental group oral 10ml yolk every day (coming from the egg that the hen of accepting immunity produces), the oral common yolk 10ml of control group took 3 days.The 4th day oral ETEC K88 bacterial strain 1 * 10
12The CFU/ head is attacked poison.After this observed 10 days, record morbidity and death condition, each organizes the ordinary milk of feeding during this period.
Piglet survival condition after 10 days, the yolk antibody that obtains with trivalent enterotoxin DNA vaccination and trivalent enterotoxin protein vaccine Immune Laying Hens can both play effective provide protection, the diarrhoea that prevention ETEC causes.
Table 2. piglet first takes anti-enterotoxin 1 gY, the survival condition after ETEC attacks poison again
The passive protection effect that embodiment 5. trivalent enterotoxin protein vaccine immunity pregnant sows produce piglet
Use following three kinds of vaccines:
(1) commercialization piglet colibacillosis tervalence inactivated vaccine (by K88, K99, three kinds of strain bacterium compositions of 987P)
(2) trivalent enterotoxin Protein S Ta-LTB-STb (SLS)
(3) mass mixing (SLS+K88 pili) such as trivalent enterotoxin Protein S LS and K88 pili
Heat extraction prepares the K88 pili: in the LB liquid nutrient medium, 36h is cultivated in 37 ℃ of concussions with the K88 inoculation.Centrifugal collection thalline, PBS is resuspended, puts 60 ℃ of water-bath 30min, vibration 10min, the centrifuging and taking supernatant, with diameter 0.22 μ m membrane filtration, filtrate is K88 pili crude extract.Crude extract is transferred pH to 4.0 with 2.5% citric acid, 4 ℃ of standing 2h, 4 ℃ of centrifugal 30min of 11000r/min abandon supernatant, and precipitation is with the PBS dissolving, and this process triplicate finally obtains the K88 pilin of purifying.
12 sows are used in experiment altogether, are divided into 4 groups---3 vaccine immunity groups and 1 control group, 3 every group.Pregnant sow is front 40 days of farrowing and each intramuscular injection on the 15th 1 time.Immunizing dose: the tervalence inactivated vaccine by specification uses, and the SLS protein groups is the 10mg/ head, and SLS+K88 pili group is the 20mg/ head.Get the milk of respectively organizing sow after farrowing and mix, indirect elisa method detects tiring of anti-K88 pili in milk, STa, STb, LTB specific antibody, and concrete grammar is with embodiment 4.Challenge test: oral ETEC K88 or F41 bacterial strain 1 * 10 when each organizes piglet 3 age in days
12The CFU/ head is attacked poison.After this observed 10 days, each is organized piglet and continues to take breast milk, record morbidity and death condition.
By experimental result as seen from Table 3; after trivalent enterotoxin Protein S Ta-LTB-STb immunity pregnant sow provided by the invention; three kinds of toxinicides of high-titer can be detected in its milk; after piglet is taken in such milk; make it can obtain effective protection when suffering ETEC virulent strain artificial challenge; survival rate reaches 70-80%, takes in the control group piglet survival rate of common breast milk not as good as 10%.Commercial tervalence inactivated vaccine has better protecting rate (70%) for the infection of homologous strain (being K88 in this example), but the protection ratio lower (40%) that infects for allos bacterial strain F41.On the contrary; SLS restructuring enterotoxin is all very high to the protection ratio of the bacterial strain (being K88 and F41 in this example) of various pili types; prove absolutely that trivalent enterotoxin SLS provided by the invention compares with traditional inactivated vaccine, be not subjected to the restriction of ETEC pili type, wider protection domain is arranged.Trivalent enterotoxin SLS is mixed with pilin; the protection better effects if of the polyvalent vaccine of preparation to homologous strain; the survival rate of piglet reaches 90%, thereby trivalent enterotoxin SLS provided by the invention can be used as a component of other multivalence or multiple vaccines and is used.
In table 3. pregnant sow milk, specific antibody tires and the survival condition of piglet after attacking poison
Claims (8)
1. the syzygy of a plurality of enterotoxin genes of an enterotoxigenic escherichia coli, it is characterized in that containing 3 kinds of not ripe genes of band signal peptide sequence: heat-labile toxin LT, heat-stable toxin STa and STb, the order of its fusion is that LT is placed in the middle, STa and STb are in both sides, and connected by the catenation sequence of at least 15 length of nucleotides between any two fusion genes of STa, LT and STb, this multivalence fusion gene is " 5 '-STa-LT-STb-3 ' " or " 5 '-STb-LT-STa-3 ' ".
2. the syzygy of a plurality of enterotoxin genes according to claim 1, it is characterized in that: heat-labile toxin LT refers to the B subunit LTB of heat-labile toxin LT or the analogue b subunit of cholera toxin of LTB.
3. the syzygy of a plurality of enterotoxin genes according to claim 1 is characterized in that: 5 ' end in the syzygy of a plurality of enterotoxin genes adds signal peptide sequence.
4. the syzygy of a plurality of enterotoxin genes according to claim 3, it is characterized in that: described signal peptide sequence refers to the signal peptide of the Regular Insulin of chicken, its sequence length is 72 bases, and its sequence is atggctctctggatccgatcactgcctcttctggctctccttgtcttttctggccc tggaaccagctatgca.
5. the syzygy of a plurality of enterotoxin genes according to claim 3, it is characterized in that: described signal peptide sequence refers to the signal peptide of the plasma urokinase-type plasminogen activator of chicken, its sequence length is 60 bases, and its sequence is atgaagttaatcatctttctcacagtaactctctgcacacttgtcacaggacttga ttct.
6. the nucleic acid vaccine of an anti-Experimental Enterotoxigenic Escherichia coli Infection is characterized in that it is that syzygy by claim 1 or 3 described a plurality of enterotoxin genes is cloned into suitable carrier for expression of eukaryon and prepares and gets.
7. the fusion rotein of a plurality of enterotoxin genes, its syzygy by a plurality of enterotoxin genes claimed in claim 1 produces at suitable host cell inner expression.
8. the fusion rotein of a plurality of enterotoxin genes according to claim 7, is characterized in that: use as vaccine separately, perhaps be added in other multivalence or multiple vaccines as one of component.
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| CN109608541B (en) * | 2018-08-24 | 2020-04-07 | 湖北神地农业科贸有限公司 | Yolk antibody for resisting swine enterotoxigenic escherichia coli and preparation method thereof |
| CN109467606A (en) * | 2018-11-15 | 2019-03-15 | 大连理工大学 | A kind of escherichia coli enterotoxin STa-LTB-STb fusion protein and its encoding gene and application |
| CN115725002B (en) * | 2022-12-08 | 2024-02-13 | 中国动物卫生与流行病学中心 | Coli specific antigen fusion protein and recombinant lactococcus lactis thereof |
| CN115850405B (en) * | 2022-12-12 | 2024-02-02 | 中国动物卫生与流行病学中心 | Antigen fusion protein and application thereof in preparation of vaccine |
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