CN106075416A

CN106075416A - The design of a kind of novel Echinococcus moltilocularis subunit vaccine, preparation method and application

Info

Publication number: CN106075416A
Application number: CN201610417912.8A
Authority: CN
Inventors: 樊海宁; 格日力; 阳旦才让; 汤锋; 周虎; 李润乐
Original assignee: Qinghai University
Current assignee: Qinghai University
Priority date: 2016-06-15
Filing date: 2016-06-15
Publication date: 2016-11-09

Abstract

The present invention provides a kind of novel Echinococcus moltilocularis subunit vaccine, and its active component is a polypeptide, and mainly surface antigen Emy162 and Mucosal Adjuvants b subunit of cholera toxin (CTB) by Echinococcus moltilocularis are constituted.The present invention mainly synthesizes Echinococcus moltilocularis surface antigen Emy162 gene order by gene synthesis technology, then by the gene order phase coupling of this gene with b subunit of cholera toxin, forms a fusion gene.Escherichia coli prokaryotic expression system is utilized to express this fusion gene, after protein purification, it is thus achieved that Echinococcus moltilocularis subunit vaccine.This Echinococcus moltilocularis subunit vaccine can induce body and produce for Echinococcus moltilocularis T cell and B cell immunne response and high titre specific antibody humoral immunoresponse(HI), can be used for preventing and to treat Echinococcus multilocularis diseases related.

Description

The design of a kind of novel Echinococcus moltilocularis subunit vaccine, preparation method and application

Technical field

The invention belongs to biomedicine field, be specifically related to the design system of a kind of novel Echinococcus moltilocularis subunit vaccine Preparation Method and application.

Background technology

Echinococcus moltilocularis (Alveolar Echinococcosis) causes alveolitoid echinococcosis, and Echinococcus moltilocularis is in centre Grows in the way of sprouting in host liver or infiltration type propagation, produces new vesicle, hepatic tissue of growing into, cyst wall exterior angle cortex the thinnest and The most imperfect, without obvious boundary between utricule and surrounding tissue, capsule liquid continues seepage and can contact with hepatic tissue, causes local hepatic tissue Pathological changes, hypertrophy, hepatic fibrosis, atrophy, degeneration and necrosis, late period can be transferred to the internal organs such as lung, brain, mammary gland like the transfer of hepatocarcinoma sample, Have the title of " worm cancer ", poor prognosis.In world wide, high altitude localities, the Northern Hemisphere is the region occurred frequently of alveolitoid echinococcosis.I State's Three River Sources areas is the rare hotspot of the world of echinococcosis multilocularis.It is wide, pernicious that echinococcosis multilocularis has morbidity scope The features such as degree height, early diagnosis difficulty, poor, the postoperative easy recurrence of outcome.There is no currently for echinococcosis multilocularis and make us full The therapeutic strategy of meaning.Just seeking medical advice during symptom occur in most of pastoral area patients, and the state of an illness is the most to late period, loses surgical engine meeting, Even if carrying out liver to divide or the excision of half leaf, Postoperative recurrent rate is the highest.The antibiotherapy such as mebendazole, albendazole has one Fixed therapeutic effect, but due to the high scarce limit of relapse rate after need to treating the appearance causing drug resistance for a long time continuously and treating.

Surface antigen is the basic function unit stimulating body to produce immunne response, and a lot of surface antigens can serve as vaccine Research and development.And subunit vaccine is prepared based on surface antigen, there is the vaccine design thinking of uniqueness, be to develop prevention and treatment The new direction of the vaccines such as infectious disease, malignant tumor and autoimmune disease.Subunit vaccine has the advantage that (1) Subunit vaccine reduce or eliminates conventional live vaccine or inactivated vaccine is difficult to remove pyrogen, allergen and other is harmful anti- Ying Yuan；And owing to subunit vaccine can not replicate in vivo, risk of not causing a disease host, is most safety and stability A kind of recombinant vaccine.(2) subunit vaccine comprises only one or more of pathogen and only produces protective immune response Necessary immunogenic ingredient, and do not contain other hereditary informations of pathogen, therefore do not contain infectious component, thus need not go out Live, also no pathogenicity, there is good stability.(3) subunit vaccine can use the prokaryotic expression such as escherichia coli, yeast system System is expressed.Therefore subunit vaccine yield is the highest, easy purification, it is simple to industrialized production.Lead at Echinococcus moltilocularis vaccine research Territory, subunit vaccine is compared with other vaccines, and same tool has great advantage, both can excite the more anti-Echinococcus moltilocularis of high specific Antibody, some autoimmune diseasees that can prevent again vaccine immunity from causing, there is higher immunity specific aim and safety.

Echinococcus moltilocularis antigen being found to one of focus of always current research, present stage echinococcosis research is main Concentrating on hydatidosis diagnosis antigen, the antigen research to prevention, suppression Echinococcus multilocularis is the most less；In addition Echinococcus moltilocularis The Immune escaping mechanism of infection host is complicated, and immunization therapy mode to Echinococcus moltilocularis is had not yet been formed at present.Research finds Emy162 has the strongest immunization, and is stably expressed in each stage (protoscolex, the spine ball of cultivation of echinococcus life cycle The larva of a tapeworm or the cercaria of a schistosome, immaturity adult and ripe adult), play an important role in the adhesion and athletic physiology of echinococcus.

This problem is planned the surface antigen Emy162 of Echinococcus moltilocularis and is carried out base with Mucosal Adjuvants b subunit of cholera toxin Because merging, obtain subunit vaccine CTB-Emy162, immune balb/c mice through prokaryotic system expression and purification, use spleen lymph thin The immunogenicity of the method confirmation subunit vaccines such as born of the same parents' propagation, specific ELISA and immunologic opsonin.

The thinking of the present invention is as follows: machine-processed to the immune protective of Echinococcus moltilocularis according to body, filters out and has well Immunogenic Echinococcus moltilocularis surface antigen Emy-162, utilizes Protocols in Molecular Biology and Mucosal Adjuvants cholera toxin B Subunit (CTB) phase coupling, designs scientific and reasonable, Echinococcus moltilocularis subunit vaccine CTB-Emy162 for novel structure. The panimmunity method such as mice spleen lymphocytes proliferation, ELISA is utilized to detect Echinococcus moltilocularis subunit vaccine CTB- The immunogenicity of Emy162 and immunologic opsonin.Research shows that Echinococcus moltilocularis subunit vaccine CTB-Emy162 can excite T cell, B cell immunne response and the high titre specificity that effectively suppression Echinococcus moltilocularis is active that BALB/c mouse produces resist Body, has good immunogenicity and immunologic opsonin.

Summary of the invention

The first aspect of the invention there is provided a kind of novel Echinococcus moltilocularis subunit vaccine.

The second aspect of the invention there is provided the preparation method of Echinococcus moltilocularis subunit vaccine.

The third aspect of the invention is the purposes disclosing Echinococcus moltilocularis subunit vaccine.

The first aspect of the invention there is provided a kind of for Echinococcus moltilocularis subunit vaccine CTB-Emy162.These are many Room echinococcus subunit vaccine is mainly made up of Emy162 and b subunit of cholera toxin, Echinococcus moltilocularis subunit vaccine CTB- The overall aminoacid sequence of Emy162 is as shown in (sequence 1), and the aminoacid sequence of surface antigen Emy162 is as shown in (sequence 3).

Novel Echinococcus moltilocularis subunit vaccine CTB-Emy162 of the present invention has the advantage that (1) Emy162 is many Room echinococcus surface antigen, the ideal candidates antigen being acknowledged as Echinococcus moltilocularis vaccine can obtain good protection work With.Echinococcus moltilocularis subunit vaccine CTB-Emy162 can cause T cell and B cell immunne response and specific antibody body Liquid immunne response.(3) molecular weight of Emy162 epitope peptide is the least, and immunogenicity is the lowest, and the Echinococcus moltilocularis of the present invention is sub- Subunit vaccine CTB-Emy162 takes the mentality of designing of " immunological adjuvant in coupling molecule " to strengthen exempting from of surface antigen Emy162 Epidemic focus, thus the specific antibody inducing high titre produces.

The second aspect of the invention is to provide the preparation method of Echinococcus moltilocularis subunit vaccine, and its technology path describes in detail As follows:

(1) the structure design of novel Echinococcus moltilocularis subunit vaccine antigenic molecule

According to the body immune protective mechanism to Echinococcus moltilocularis, rationally select that there is good immunogenic Echinococcus moltilocularis Surface antigen Emy-162, and with Mucosal Adjuvants b subunit of cholera toxin (CTB) phase coupling, design one scientific and reasonable, Echinococcus moltilocularis subunit vaccine CTB-Emy162 of novel structure.

(2) recombinant expression plasmid pET28a-CTB-Emy162(contains fusion gene CTB-Emy162) structure

First a recombinant expression carrier pET-CTB containing b subunit of cholera toxin (CTB) gene is built；Then gene is passed through The nucleotide sequence of synthetic technology one Echinococcus moltilocularis surface antigen Emy162 of synthesis, and it is inserted into recombinant expression carrier In pET-CTB, build recombinant expression carrier pET28a-CTB-Emy162.

(3) prokaryotic expression of fusion protein CTB-Emy162 and purification

Recombinant expression carrier pET28a-CTB-Emy162 is transformed in e. coli bl21 (DE3), builds recombination engineering Bacterial strain BL21 (DE3)/pET28a-CTB-Emy162.Utilize IPTG abduction delivering, and by Ni-NTA nickel ion affinity chromatograph It is obtained in that high-purity fusion protein CTB-Emy162, is Echinococcus moltilocularis subunit vaccine.

The third aspect of the invention there is provided the purposes of Echinococcus moltilocularis subunit vaccine.Echinococcus moltilocularis subunit Vaccine CTB-Emy162 can be used for preventing and to treat Echinococcus multilocularis diseases related.

Accompanying drawing explanation

Fig. 1: the Molecular Design feature of novel Echinococcus moltilocularis subunit vaccine.

Fig. 2: Emy162 gene PCR result figure.

Swimming lane 1:DNA Marker；Swimming lane 2 is Emy162 DNA fragmentation result after PCR

The double digestion of Fig. 3: recombinant expression carrier pET28a-CTB-Emy162 is identified.

Swimming lane 1:DNA Marker；Swimming lane 2:pET28a-CTB-Emy162 plasmid is after Nco I and Xho I double digestion

Fig. 4: recombinant expression carrier pET28a-CTB-Emy162 plasmid map.

For the fusion gene CTB-Emy162 inserted between Nco I and Xho I

Fig. 5: the prokaryotic expression of Echinococcus moltilocularis fusion protein CTB-Emy162.

Swimming lane 1:BL21 (DE3)/pET-28a whole bacterial protein；Swimming lane 2: protein Marker；Swimming lane 3:BL21 (DE3)/ PET-28a-CTB-Emy162 whole bacterial protein；Swimming lane 4:BL21 (DE3)/pET-28a-CTB-Emy162 soluble protein；Swimming lane 5:BL21 (DE3)/CTB-Emy162 inclusion body protein.

Fig. 6: the Ni-NTA affinitive layer purification of Echinococcus moltilocularis fusion protein CTB-Emy162.

Swimming lane 1: CTB-Emy162 protein sample before purification；Swimming lane 2: through Ni-NTA CTB-Emy162 egg after purification White sample.

Fig. 7: Echinococcus moltilocularis subunit vaccine CTB-Emy162 induces anti-CTB-Emy162 and Emy162 epitope E_7-13、E_129-129The detection of antibody.

Echinococcus moltilocularis subunit vaccine CTB-Emy162 can induce higher titre for CTB-Emy162 and Emy162 epitope E_7-13、E_129-129Antibody.

Fig. 8: Echinococcus moltilocularis subunit vaccine CTB-Emy162 antibody titer detects.

Fig. 9: the Echinococcus moltilocularis subunit vaccine CTB-Emy162 sensitized mice spleen lymphocyte increasing to antigenic stimulus Grow reaction.

Figure 10: Echinococcus moltilocularis subunit vaccine CTB-Emy162 induces the detection of anti-Echinococcus moltilocularis antibody typing.

Detailed description of the invention

Material

1. IPTG solution: weigh 1.2g IPTG and be placed in 50ml centrifuge tube, adds 40ml sterilized water, after fully mixing is dissolved, It is settled to 50ml.With 0.22 μm filter filtration sterilization, aliquot subpackage ,-20 DEG C of preservations.

2. card that penicillin (Kana) reservoir (1g/mL): weigh that penicillin of 1g card (Kana) and be dissolved in 1mL sterilized water, Prepared concentration is the stock solution of 1g/mL, and by 0.22 m bacterial filter filtration sterilization, solution subpackage is stored in-20 DEG C of refrigerators In.

3. culture medium: (1) LB fluid medium: weigh 10g tryptone, 5g yeast extract and 5g NaCl, add steaming Distilled water, to 1000ml, adjusts pH to 7.4, autoclaving.(2) LB solid medium: 1.5g agar powder/100ml LB cultivates Liquid, after autoclaving, pour plate.

4. DNA electrophoretic buffer (50 x TAE): weigh 242g Tris, 37.2g Na₂EDTA·2H₂O and 57.1ml Glacial acetic acid, adds water to 1000ml, dilutes 50 times during use.

5. SDS-PAGE electrophoretic buffer (5X): weigh Tris powder 15.1g, glycine 94g, SDS 5.0g；Add The deionized water of about 800ml, stirring and dissolving；Add deionized water and be settled to 1L, room temperature preservation；Note: should allow when adding water water along Wall slowly flows down, to avoid owing to the reason of SDS produces a lot of foams.

6. coomassie brilliant blue R_250 dye liquor: 0.25g coomassie brilliant blue R_250 is dissolved in 100ml destaining solution.(3) fixing Liquid: 500ml ethanol, 100ml glacial acetic acid distilled water diluting to 1000ml.(4) destaining solution: 250ml ethanol, 80ml glacial acetic acid are used Distilled water diluting is to 1000ml.(5) preserve liquid: 25ml 87% glycerol to be dissolved in 225ml destaining solution.

9. laboratory animal: (2) BALB/c mouse: for SPF level, male, 6～7 week old, purchased from Beijing, credit number: SCXK (capital) 2012-0001.

12. lymphocyte proliferation assay main agents (1) mouse spleen lymphocyte separation liquid (Hao ocean, Tianjin biology skills Art company limited) (2) RPMI-1640 complete culture solution: add 10% hyclone, 100U/ at RPMI-1640 basic culture solution Ml penicillin, 100 μ g/ml streptomycins.

Example 1: the Molecular Design of Echinococcus moltilocularis subunit vaccine CTB-Emy162

On the basis of obtaining Emy162 aminoacid sequence, add intramolecular mucosal adjuvant CTB on this basis, design There is the Echinococcus moltilocularis fusion protein of scientific and reasonable structure, to reach to be effectively improved internal mucosal immunoreaction.

Result: the Molecular Design feature of Echinococcus moltilocularis subunit vaccine CTB-Emy162 and thinking such as (Fig. 1) institute Show.

Example 2: the structure of recombinant expression carrier (containing fusion gene CTB-Emy162)

(1) gene chemical synthesis of Echinococcus moltilocularis surface antigen Emy162 nucleotide sequence

By the aminoacid sequence of the Emy162 that early stage obtains, change into corresponding core according to e. coli codon preferences principle Nucleotide sequence, entrusts Nanjing Jin Sirui biotechnology company to carry out gene chemical synthesis.Emy162 after synthesis carries out PCR, reactant It is as follows:

Emy162(0.5mg/mL)	1 µL
		Emy162 primer	1 µL
Mix(0.1 mg/mL)	10µL
		ddH<sub>2</sub>O (without RNase)	8 µL
Total	20 µL

PCR reaction condition is: 95 DEG C of denaturations 5min, 95 DEG C of degeneration 30 sec, 72 DEG C of annealing 30 sec, and 72 DEG C extend 40sec, Totally 30 circulations；Last 72 DEG C extend 10 min.Then use 1% agarose gel electrophoresis, observe electrophoresis result (Fig. 2).Utilize DNA fragmentation gel purification test kit (Beijing Tian Gen Bioisystech Co., Ltd) reclaims the Emy162 base of purification PCR amplification Cause, in case subsequent experimental uses.

(2) recombinant expression carrier pET28a-CTB(contains cholera element element B subunit gene) structure

Extract recombiant plasmid pET-CTB-UE(by plasmid Mini Kit (sky root) to be given by professor Guo Le), useKpn I/XhoI carries out double digestion respectively, and double digestion reaction system is as follows:

pET-CTB-UE	24 µL
		0.1% BSA	4 µL
KpnI(15U/ L)	2µL
		XhoI(15U/ L)	2µL
K Buffer	4µL
		L Buffe	4µL
Total	40 µL

37 DEG C of endonuclease reaction 2 h, then use 1% agarose gel electrophoresis.It is utilized respectively agarose gel DNA and reclaims purification examination Agent box reclaims multi-epitope peptide UE gene and pET28a-CTB expression vector double digestion product, and pET28a-CTB is in case subsequent experimental makes With.

(3) Echinococcus moltilocularis surface antigen Emy162 gene and the connection of pET28a-CTB expression vector

The Emy162 gene reclaimed after the pET28a-CTB linearized plasmid vector reclaimed and PCR is passed through complementary viscosity end End, under the effect of T4 DNA ligase, (4 DEG C overnight) connects into the DNA molecular of closed hoop, i.e. forms recombinant expression plasmid pET28a-CTB-Emy162.T4 DNA ligase linked system is as follows:

PET28a-CTB linearized vector	2 µL
		Emy162 gene	14 µL
10 × T4 DNA ligase Buffer	2 µL
		T4-DNA ligase (3U/ L)	2 µL
Total	20 µL

Result: utilizeNco I/XhoI double digestion recombiant plasmid to be checked pET28a-CTB-Emy162,37 DEG C of reaction 2h, with 1% Agarose gel electrophoresis detection, find the DNA fragmentation about 790bp of double digestion, theoretical big with fusion gene CTB-Emy162 Little consistent (shown in Fig. 3).

PET28a-CTB-Emy162 is delivered Nanjing Jin Sirui biotechnology company, uses T7 promoter and terminator to lead to Gene sequencing is carried out, it was demonstrated that in pET28a-CTB-Emy162, the nucleotide sequence of fusion gene CTB-Emy162 is complete with primer Correctly, and without frameshift mutation.The plasmid map of recombinant expression carrier pET28a-CTB-Emy162 is as shown in (Fig. 4).

The prokaryotic expression of example 3 Echinococcus moltilocularis fusion protein CTB-Emy162

Successful for order-checking 100% recombinant expression plasmid pET28a-CTB-Emy162 is transformed into E.coli BL21 (DE3) bacterial strain In.On the LB flat board containing 50ug/mL Kana well prepared in advance, inoculating loop line engineering strain pET28a-CTB- Emy162/BL21 (DE3), is inverted in 37 DEG C of incubators, after incubated overnight 12～16h, and the single bacterium colony of picking, it is inoculated in containing 50 In the LB fluid medium of g/mL Kana, 37 DEG C, 190rpm, cultivates 6-8h.Recombinant bacterium is inoculated respectively in containing 50 with 1% inoculum concentration In g/mL Kana LB fluid medium, 37 DEG C, 180rpm shaken overnight, again this bacterium solution is inoculated with 1% inoculum concentration morning next day In the LB fluid medium containing 50 g/mL Kana, 37 DEG C, after 190rpm shaking flask 9h, add IPTG and make final concentration reach 1mmol/L, 28 DEG C, 190rpm abduction delivering, using empty carrier bacterium pET28a/BL21 (DE3) as negative control.

Result: by genetic engineering recombination strain pET28a-CTB-Emy162/BL21 (DE3) pH value be 7, IPTG concentration It is to carry out abduction delivering in the case of 9h for 1mmol/L, inducing temperature 28 DEG C, induction time.Contrast with control strain, gene work Journey recombinant bacterial strain pET28a-CTB-Emy162/BL21 (DE3) occurs destination protein band at about 33KD, with Echinococcus moltilocularis The theoretical size of recombination fusion protein CTB-Emy162 is consistent (Fig. 5).Echinococcus moltilocularis recombination fusion protein CTB-Emy162 Inclusion body protein exists.

Example 4: the purification of Echinococcus moltilocularis multi-epitope peptide fusion protein CTB-EMY162

(1) purification of Ni-NTA nickel ion affinity chromatograph post

Adding after fully being mixed by Ni-NTA filler in chromatographic column, solution slowly can be flowed out by action of gravity, waits to be added completely into After, upper strata sieve plate is kept flat in post；In the post filled, add appropriate deionized water by after clean for alcohol flushing, add The Binding buffer balance of 8 times of column volumes, balance gets final product loading after terminating；After collecting thalline, every 100mg thalline adds 1- 5ml Binding Buffer, ice-bath ultrasonic cracking thalline；12000rpm is centrifuged, and abandons supernatant, precipitation is resuspended in Binding In Buffer, carry out ultrasonic Treatment, until inclusion body cleans up (milkiness in cleaner)；Precipitation is resuspended in In Binding Buffer containing 8M carbamide, ice bath 1h, make solubilization of inclusion bodies；Cellular lysate liquid is loaded upper prop, and flow velocity is 10 Times column volume/hour；The Wash buffer using 15 times of column volumes rinses pillar, collects and flows through peak；Use 5 times of column volumes Elution Buffer eluting, collects eluting peak；Use the Binding Buffer of 3 times of column volumes and 5 times of column volumes successively Deionized water wash after, with 3 times of column volumes 20% ethanol balance, 4 DEG C of preservations.

Result: through Ni-NTA nickel ion affinity chromatograph post after purification, each protein peak of collection carries out SDS-PAGE and divides Analysis, it appeared that destination protein concentrates on 50mM imidazoles and the protein peak of 250mM imidazoles eluting generation.Examined by gel imaging Survey instrument analysis 250mM imidazoles eluting destination protein purity all more than 85% (Fig. 6).

Embodiment 5: the immunogenicity of Echinococcus moltilocularis polyepitope vaccines CTB-EMY162 and immunologic opsonin research

(1) immunity of BALB/c mouse

Experiment packet: SPF level BALB/c mouse is randomly divided into 2 groups, the most novel Echinococcus moltilocularis Multi-Epitope Fusion Protein CTB-Emy162 immune group, PBS immune group.Often 12 BALB/c mouse of group, are grouped as follows shown in figure totally in detail by 24:

The packet of table 1:SPF level BALB/c mouse and immunization protocol

Experimental group	Number	Immunization ways	Immune time	Immunizing dose	Immunological adjuvant
						CTB-Emy162	12	Lumbar injection	4 times	100 μ g/ are only	Freund adjuvant
PBS	12	Lumbar injection	4 times	100 μ g/ are only	Freund adjuvant

Immunization ways: with 75% chronic ethanol treated mice abdominal part sterilization, then lumbar injection recombiant protein and Freund's complete adjuvant is mixed Co-emulsifier；Booster immunization is once week about, within the 2nd and 3 week, adds incomplete Freund's adjuvant, the 4th week direct injection recombiant protein Solution booster immunization.

Sero-fast collection: after final immunization the 5th day, plucks eyeball of mouse blood sampling, collects blood, place and treat that serum is complete Fully separating, 3000rpm is centrifuged 5 minutes subpackage serum, and-80 DEG C frozen standby.

(2) the ELISA detection of specific antibody in antiserum

By antigen Emy162, Emy162 specific antigen epitope little peptide (E_7-13、E_129-139), PBS be diluted to 5 μ g/ with being coated liquid Ml, 100 μ l/ holes are coated elisa plate, and 4 DEG C overnight.After washing 4 times with cleaning mixture, every hole adds 300 μ l confining liquids, 37 DEG C of closings 2h.After washing 4 times with cleaning mixture, by antiserum (mouse-anti CTB-Emy162 antiserum and mouse-anti CTB antiserum) and mice feminine gender blood Add elisa plate, 100 μ l/ holes after clear doubling dilution, at 37 DEG C, hatch 60min.After washing 4 times with cleaning mixture, add HRP mark The sheep anti-mouse igg (1:2000) of note, 100 μ l/ holes, at 37 DEG C, hatch 1h.After washing 4 times with cleaning mixture, add tmb substrate colour developing Liquid, room temperature lucifuge reaction 15min, add 50 μ l stop buffers and terminate reaction.Each hole OD is measured by microplate reader₄₅₀Value.By OD value more than setting The dilution factor that the hole of 2.1 times of fixed negative control OD value is corresponding, it is determined as the titer of this sample.

The concentration that is coated of result: Emy162 is 5 μ g/ml, is detected by ELISA, and CTB-EMY162 subunit vaccine can Produce the IgG antibody for Emy162 antigen, and PBS immune group can not produce the IgG antibody for Emy162 antigen；E_7-13And E_129-139Being coated concentration is 5 μ g/ml, is detected by ELISA, and CTB-EMY162 subunit vaccine can produce special for Emy162 Property epitope peptide E_7-13And E_129-139IgG antibody, and PBS can not produce for Emy162 specific antigen epitope peptide E_7-13 And E_129-139IgG antibody (Fig. 7,8).The above results explanation subunit vaccine CTB-Emy162 can stimulate body produce for The high titre specific antibody of Emy162, has good immunogenicity.

(3) mouse spleen lymphocyte proliferation experiment

Mice through antigen immune is dislocated and puts to death, after steeping 75% ethanol 5min, move into superclean bench.Carefully cut off with shears The abdominal part crust of mice, then cut off the abdominal cavity of mice, take out mouse spleen (kermesinus) with tweezers.Aseptic little burning at 20 ml 4 ~ 5 ml EZ-Sep Mouse 1X lymphocyte separation mediums are put in Bei；Fix nylon wire with tweezers, then use syringe Piston is lightly ground mouse spleen so that scattered unicellular enter in lymphocyte separation medium through nylon wire；Have outstanding The separation liquid of spleen cell is immediately transferred in centrifuge tube, covers 1640 culture medium of about 200 μ l before being centrifuged again；800 g Centrifugal 30 min.After centrifugal end, lymphocyte can floating come up, and assembles below 1640 cover layers.With containing 10% calf serum RPMI-1640 culture medium be prepared as certain density cell suspension (5 x 10⁴/ ml).In 96 hole flat-bottomed plates, every hole Add 100 μ l cells, be simultaneously introduced CTB-Emy162, E_7-13、E_129-139, PBS(20 μ g/ml), final volume is 200 μ l/ holes, often Group does 3 parallel holes.The 96 hole flat-bottomed plates added are placed 37 DEG C of 5% CO₂After incubator cultivates 48h, in each hole Adding MTT solution (5mg/ml) 30 μ l, after continuing to cultivate 4h, sucking-off supernatant gently, every hole adds DMSO 100 μ l vibration mixing OD is read afterwards by microplate reader₅₇₀Value.

Result: the mouse spleen lymphocyte that Echinococcus moltilocularis subunit vaccine CTB-Emy162 sensitization is crossed, through CTB- Emy162、E_7-13、E_129-139Stimulate and obvious lymphproliferation response the most all can occur, and the mouse spleen of PBS immune group Lymphocyte accepts, during above-mentioned antigenic stimulus, lymphproliferation response does not all occur, and Echinococcus moltilocularis subunit vaccine is described CTB-Emy162 maintains its immunological characteristic, it is possible to stimulate body to produce specificity cellular immunity response (Fig. 9).

(4) the ELISA detection of specific antibody typing in antiserum

With being coated liquid, antigen Emy162, PBS are diluted to 5 μ g/ml, and 100 μ l/ holes are coated elisa plate, and 4 DEG C overnight.Use cleaning mixture After washing 4 times, every hole adds 300 μ l confining liquids, closes 2h for 37 DEG C.After washing 4 times with cleaning mixture, by antiserum and mice negative serum Add elisa plate, 100 μ l/ holes after doubling dilution, at 37 DEG C, hatch 60min.After washing 4 times with cleaning mixture, add HRP labelling Sheep anti mouse IgA, IgG1, IgG2a, antibody diluent dilute, dilution ratio (1:2000), 100 μ l/ holes, hatch at 37 DEG C 1h.After washing 4 times with cleaning mixture, add tmb substrate nitrite ion, room temperature lucifuge reaction 15min, add 50 μ l stop buffers and terminate reaction. Each hole OD is measured by microplate reader₄₅₀Value.

Result: as shown in (Figure 10), PBS immunized controls group compares, Echinococcus moltilocularis subunit vaccine CTB-EMY162 energy Enough produce IgG1, IgGA and IgG2a antibody of anti-Emy162.

Claims

1. an Echinococcus moltilocularis subunit vaccine, its active component is a polypeptide, mainly by b subunit of cholera toxin (CTB) Constituting with echinococcus surface antigen Emy162, its aminoacid sequence is as shown in sequence 1.

2. as claimed in claim 1, a kind of Echinococcus moltilocularis subunit vaccine CTB-Emy162, its nucleotide sequence such as sequence 2 Shown in.

3. the aminoacid sequence of an Echinococcus moltilocularis surface antigen Emy162 is as shown in sequence 3.

4. as claimed in claim 3, the nucleotide sequence of Echinococcus moltilocularis surface antigen Emy162 is as shown in sequence 4.

5. comprise the expression vector of the nucleotide sequence described in claim 2 and 4, transgenic cell line and Host Strains.

6. as claimed in claim 1, the interval between b subunit of cholera toxin (CTB) and Echinococcus moltilocularis surface antigen Emy162 Sequence is DPRVPSS.

7. a preparation method for Echinococcus moltilocularis subunit vaccine, is resisted by gene synthesis technology synthesis Echinococcus moltilocularis surface The nucleotide sequence of former Emy162, is merged the downstream at b subunit of cholera toxin (CTB) gene, builds containing fusion gene The recombinant expression carrier pET28a-CTB-Emy162 of CTB-Emy162 and recombination engineering bacteria BL-21 (DE3) thereof,

After the fermentation of recombination engineered strain, through Ni-NTA nickel ion displacement chromatography purification, it is thus achieved that the fusion protein of this vaccine CTB-Emy162。

8. according to the Echinococcus moltilocularis subunit vaccine described in claim 1 and 6, it is characterised in that pin can be produced by excitating organism To Echinococcus moltilocularis T cell immunne response and effective high titre specific antibody suppressing Echinococcus moltilocularis.

9., according to the Echinococcus moltilocularis subunit vaccine described in claim 1 and 7, it is characterized in that can serve as prevention and treatment is many The drug regimen that room echinococcus infects.