CN106581667A - Design and preparation method and application of echinococcus multilocularis subunit vaccine LTB-Emy162 - Google Patents
Design and preparation method and application of echinococcus multilocularis subunit vaccine LTB-Emy162 Download PDFInfo
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Abstract
The invention relates to a design and preparation method and an application of an echinococcus multilocularis subunit vaccine LTB-Emy162. The active component of the echinococcus multilocularis subunit vaccine LTB-Emy162 is a polypeptide, which is mainly composed of an echinococcus multilocularis antigen protein Emy162 amino acid sequence and a mucosal immunoadjuvant E. coli heat-labile enterotoxin B subunit (LTB) amino acid sequence. In the invention, the gene sequence of the echinococcus multilocularis subunit vaccine LTB-Emy162 is synthesized through gene synthesis technology, the gene sequence is then linked to an expression vector through dual enzyme-cut, the expression vector then is converted into Arctic Express to perform expression of fusion protein, after purification of the protein, the echinococcus multilocularis subunit vaccine LTB-Emy162 is produced. The echinococcus multilocularis subunit vaccine can induce body to generate T-cell and B-cell immunologic response of the echinococcus multilocularis and humoral immune response of a high-titer specific antibody, and can be applied in prevention and therapy of echinococcus multilocularis infection related diseases.
Description
Technical field
The invention belongs to biomedicine technical field, and in particular to a kind of design of Echinococcus moltilocularis vaccine, preparation method
And related application.
Background technology
Also known as echinococcosis multilocularises, the disease is echinococcosis multilocularises-disease of kind of serious infecting both domestic animals and human, China's echinococcosis
Endemic Area occurred frequently is concentrated mainly on that the ground such as pastoral area and farming-pastoral region, Qinghai, Gansu and Northern Sichuan Province are more serious, and Qinghai is even more
The severely afflicated area that echinococcosis multilocularises are wreaked havoc.Echinococcus multilocularis early stage parasitize in intermediate host liver, cause local liver tissue lesions, hypertrophy,
Hepatic fibrosis, atrophy, degeneration and necrosis, and clinical symptoms are not obvious；Late period can be transferred to lung, brain, mammary gland like the transfer of hepatocarcinoma sample
In internal organs, even if carrying out liver point or the excision treatment of half leaf, Postoperative recurrent rate is also very high, therefore clinic has the title of " worm cancer ".According to system
Count 10 years case fatality rate of untreated AE patient and be up to 94%.AE is the specific type of echinococcosis, is Qinghai Province's Three River Sources areas
Characteristic endemic diseases, with morbidity scope it is wide, grade malignancy is high, outcome is poor the characteristics of, broad masses of the people endure many rooms to the fullest extent
The harm of echinococcuss, is listed in one of keypoint control parasitic disease.
Echinococcuss can parasitize any position of people, animal, because volume is big, vitality strong, not only be subject to surrounding tissue
High pressure and atrophy, are also also easy to produce secondary infection, if the rupture of larva of a tapeworm or the cercaria of a schistosome body, consequence is particularly acute.At present, AE is treated with surgery handss
Based on art, supplemented by Drug therapy, but and high recurrence rate big to injury of human of performing the operation, take Albendazole and mebendazole can be produced
Serious untoward reaction.Recently the subunit vaccine of invention have that safe, yield is high, purity is high relative to other vaccines,
The advantages of good stability, but immune effect is poor compared with traditional vaccine, need special structure design, special delivery system or
Adjuvant is improving its immunogenicity.Therefore, can be used for clinical prevention and treatment currently without the preferable medicine of effect and vaccine
Echinococcosis multilocularises.
The content of the invention
First purpose of the present invention is to provide a kind of subunit vaccine for Echinococcus moltilocularis.
Second object of the present invention is to provide a kind of preparation method of Echinococcus moltilocularis subunit vaccine.
Third object of the present invention is the purposes for providing Echinococcus moltilocularis subunit vaccine.
In order to realize the purpose of the present invention, the present invention is adopted the following technical scheme that：A kind of Echinococcus moltilocularis subunit vaccine
LTB-Emy162, the Echinococcus moltilocularis subunit vaccine is mainly by escherichia coli heat-labile（LTB）With Emy162 structures
Into, the overall aminoacid sequence of Echinococcus moltilocularis subunit vaccine LTB-Emy162 as shown in sequence 1, the aminoacid sequence of Emy162
Row are as shown in row 3；The nucleotide sequence of Echinococcus moltilocularis subunit vaccine LTB-Emy162 as shown in sequence 2, Echinococcus moltilocularis
Expression vector, transgenic cell line of the nucleotide sequence of antigen protein Emy162 as shown in sequence 4, also including nucleotide sequence
And Host Strains.HLT B subunits（LTB）And the intervening sequence between Echinococcus moltilocularis antigen protein Emy162
For DPRVPSS.
Second object of the present invention is to provide the preparation method of Echinococcus moltilocularis subunit vaccine, and its technology path is described in detail
It is as follows：
（1）The structure design of new Echinococcus moltilocularis subunit vaccine antigenic molecule：
Echinococcus moltilocularis antigen protein Emy162 and Mucosal Adjuvants escherichia coli heat-labile（LTB）Mutually it is coupled and sets
Count out Echinococcus moltilocularis subunit vaccine LTB-Emy162 that scientific and reasonable, structure is novel.
（2）Recombinant expression plasmid pCzn1-LTB-Emy162（Containing fusion gene LTB-Emy162）Structure：
Using the method synthetic gene LTB-Emy162 based on PAS (PCR-based Accurate Synthesis), by double
Enzyme action is connected between the Nde I and Xba I of pCzn1 carriers, the recombiant plasmid pCzn1-LTB-Emy162 of acquisition.
（3）The prokaryotic expression and purification of fusion protein LTB-Emy162：
Recombinant expression carrier pCzn1-LTB-Emy162 is transformed in escherichia coli Arctic Express, recombination is built
Engineered strain Arctic Express/pCzn1-LTB-Emy162.Using IPTG abduction deliverings, and by Ni-IDA nickel ions parent
The fusion protein LTB-Emy162 of electrophoresis purity, as Echinococcus moltilocularis subunit vaccine LTB-Emy162 are obtained with chromatography.
The 3rd purpose of the present invention is to provide the purposes of Echinococcus moltilocularis subunit vaccine LTB-Emy162.Echinococcus moltilocularis
It is diseases related that subunit vaccine LTB-Emy162 can be used for prevention and treatment Echinococcus multilocularis.
Echinococcus moltilocularis subunit vaccine LTB-Emy162 disclosed by the invention has advantages below：（1）Emy162 is many
Room echinococcuss surface antigen, the ideal candidates antigen for being acknowledged as Echinococcus moltilocularis vaccine can obtain good protection work
With.（2）Echinococcus moltilocularis subunit vaccine LTB-Emy162 will be Emy162 antigen proteins and Mucosal Adjuvants LTB scientific and reasonable
Combine, can excite for Echinococcus moltilocularis antigen Emy162 T cell immunne response and specific humoral immunity should
Answer.（3）Echinococcus moltilocularis subunit genetic engineering vaccine LTB-Emy162, safe, yield is high, purity is high, stability is strong.
（4）LTB-Emy162 has stronger immunogenicity, can induce for Echinococcus moltilocularis antigen protein Emy162 and total protein
The specific antibody of high titre is produced.
The present invention selects subunit vaccine, and subunit vaccine is relative to the safe of other vaccines, yield is high, purity
High, good stability, but immune effect is poor, needs special structure design, special delivery system or adjuvant and exempts from improving which
Epidemic focus, so employing LTB as Inner adjuvant to improve its immunogenicity.Research finds that Emy162 has very strong exempting from
Epidemic disease is acted on, and is stably expressed in each stage of echinococcuss life cycle（Procephalon knot, culture echinococcuss, immaturity adult with into
Maturation worm）, play an important role in the adhesion of echinococcuss and athletic physiology.
The present invention is by adopting molecule clone technology by the order of connection to epitope, intervening sequence and multiple copies
Several analysis design alveolitoid Echinococcus hydatid cyst subunit vaccines；And the expression of Jing prokaryotic systems purification acquisition recombiant protein LTB-Emy162；
Then by Jing Elisa, mice spleen lymphocytes proliferation experiment, GM1 specificitys after subunit vaccine LTB-Emy162 immune mouse
The immunogenicity and biological activity of technical research subunit vaccine LTB-Emy162 such as Elisa.
Description of the drawings
Fig. 1：The Molecular Design feature of Echinococcus moltilocularis subunit vaccine LTB-Emy162；
Fig. 2：The double digestion identification of recombinant expression carrier pCzn1-LTB-Emy162,
Swimming lane M：DNA Marker；Swimming lane 1：Plasmid before pCzn1-LTB-Emy162 enzyme action；Swimming lane 2：pCzn1-LTB-Emy162
/Nde I+Xba I；
Fig. 3：Recombinant expression carrier pCzn1-LTB-Emy162 vector construction collection of illustrative plates；
Fig. 4：The prokaryotic expression of Echinococcus moltilocularis multi-epitope peptide fusion protein LTB-Emy162,
Swimming lane M：Protein Marker；Swimming lane 1：Not plus IPTG induction bacterium liquid eggs is white；Swimming lane 2：Plus bacterium solution albumen after IPTG inductions；
Swimming lane 3：Supernatant after bacterium solution is centrifuged after adding 37 DEG C of inductions of 0.5mM IPTG；Swimming lane 4：Add 0.5mM 37 DEG C of induction bacteriums of IPTG
Liquid centrifuged deposit；
Fig. 5：The Ni-IDA affinitive layer purifications of Echinococcus moltilocularis multi-epitope peptide fusion protein LTB-Emy162,
Swimming lane M：Protein Marker；Swimming lane 1：The non-purifying proteins of LTB-Emy162；Swimming lane 2：The foreign protein of 20mM imidazoles eluting
With part destination protein；Swimming lane 3：LTB-Emy162 protein samples after purification；
Fig. 6：Echinococcus moltilocularis subunit vaccine LTB-Emy162 induces the detection of anti-Emy162 IgG antibodies, and Echinococcus moltilocularis are sub-
Subunit vaccine LTB-Emy162 can induce the IgG antibody for producing the anti-Emy162 of certain titre, and with higher antibody titer, and
RLTB can not cause the IgG antibody of anti-Emy162；
Fig. 7：Echinococcus moltilocularis subunit vaccine LTB-Emy162 induces the detection of anti-Echinococcus moltilocularis total protein IgG antibody；
Echinococcus moltilocularis subunit vaccine LTB-Emy162 can produce the IgG of the anti-Echinococcus moltilocularis total protein of certain titre and resist
Body；
Fig. 8：Echinococcus moltilocularis subunit vaccine LTB-Emy162 sensitized mice spleen lymphocyte is anti-to the propagation of antigenic stimulus
Should；
Fig. 9：The immunoblotting of Echinococcus moltilocularis subunit vaccine LTB-Emy162 immunologic opsonin（Western Blot）Identification；
Figure 10：Echinococcus moltilocularis subunit vaccine LTB-Emy162 is detected with the binding activity of Ganglioside GM1.
Specific embodiment
Material：
1st, IPTG solution：Weigh 1.2g IPTG to be placed in 50ml centrifuge tubes, add 40ml sterilized water, after fully mixing dissolving,
50ml is settled to, with 0.22 μm of filter filtration sterilization, aliquot subpackage, -20 DEG C of preservations.
2nd, ampicillin（Amp）Reservoir（100 mg/mL）：Weigh 100mg ampicillin（Amp）It is dissolved in 1mL aseptic
Water, is obtained stock solution of the concentration for 100mg/mL, and by 0.22 m bacterial filter filtration sterilizations, solution subpackage is stored in -20 DEG C
In refrigerator.
3rd, culture medium：（1）LB fluid mediums：Weigh 10g tryptones, 5g yeast extracts and 5g NaCl, plus distillation
Water adjusts pH to 7.4, autoclaving to 1000ml.（2）LB solid mediums：1.5g agar powders/100ml LB cultures
Liquid, after autoclaving, pour plate.
4th, DNA electrophoretic buffers（50 x TAE）：Weigh 242g Tris, 37.2g Na2EDTA·2H2O and 57.1ml ice
Acetic acid, adds water to 1000ml, dilutes 50 times during use.
5th, SDS-PAGE electrophoretic buffers（5X）：Weigh Tris powder 15.1g, glycine 94g, SDS 5.0g；Add
The deionized water of about 800ml, stirring and dissolving；Plus deionized water is settled to 1L, room temperature preservation；Note：Should allow when adding water water along
Wall is slowly flowed down, to avoid many foams are produced due to due to SDS.
6th, Coomassie brilliant blue protein staining reagent：（1）Coomassie brilliant G-250 dye liquor（Quantification of protein is used）：Coomassie
Light blue G-250 100mg are dissolved in 95% ethanol of 50ml, are subsequently adding 86% phosphatase 11 00ml, with distilled water diluting extremely
1000ml.（2）Destaining solution：250ml ethanol, 80ml glacial acetic acid distilled water diluting to 1000ml.
8th, laboratory animal：BALB/c mouse：For SPF levels, male, 6～8 week old are dynamic purchased from the experiment of Beijing dimension tonneau China
Thing Technology Co., Ltd., credit number：SCXK（Capital）2012-0001.
9th, ELISA reagents：（1）Coating buffer：1.6g Na2CO3, 2.9g NaHCO3, 0.2g NaN3, plus distilled water is to 1L,
PH value is adjusted to 9.6.（2）Cleaning mixture：0.2g KH are weighed respectively2PO4, 2.9g Na2HPO4∙12H2O, 8.0g NaCl, 0.2g
KCl, 0.5ml Tween-20, adds ddH2O is settled to 1000 ml（PBST）.（3）Confining liquid：Weigh 3.0g BSA to be dissolved in
In 100ml lavation buffer solutions, 4 DEG C of preservations after filtration sterilization.（4）Substrate solution：Solubility one-component tmb substrate solution.（5）Eventually
Only liquid：Distilled water 178.3ml is measured, concentrated sulphuric acid 21.7ml is added dropwise over（1M H2SO4）.
10th, lymphocyte proliferation assay main agents（1）Mouse spleen lymphocyte separating liquid（It is purchased from CEDARLANE public
Department, CL5031）（2）RPMI-1640 complete culture solutions：10% hyclone, 100U/ are added in RPMI-1640 basic culture solutions
Ml penicillins, 100 μ g/ml streptomycins.（3）The incomplete culture fluid of RPMI-1640：Weigh 10.4g RPMI-1640 dry powder, 2.4g
HEPES、0.75g NaHCO3, plus deionized water is to 1000ml, pH 7.4, heat sterilization, subpackage.
Embodiment 1：The Molecular Design of Echinococcus moltilocularis subunit vaccine LTB-Emy162
In the design of Echinococcus moltilocularis subunit vaccine, the present invention selects Emy162 antigens, by escherichia coli intolerant to warmheartedness poison
Plain B subunits（LTB）As intramolecular immunological adjuvant, merge the N-terminal in Emy162, strengthen the immunogenicity of Emy162.
As a result：The Molecular Design feature of Echinococcus moltilocularis subunit vaccine LTB-Emy162 and thinking are as shown in Figure 1.
Embodiment 2：Recombinant expression carrier pCzn1-LTB-Emy162（Containing fusion gene LTB-Emy162）Structure
The aminoacid sequence of the multi-epitope peptide LTB-Emy162 that early stage is designed, turns according to e. coli codon preferences principle
Corresponding nucleotide sequence is melted into, using the method based on PAS (PCR-based Accurate Synthesis), design is complete
Long splicing primer, respectively devises protectiveness base synthetic gene LTB-Emy162 at the two ends of primer, by cloning site Nde I
Expression vector pCzn1 is connected into Xba I.
As a result：Using Nde I and Xba I double digestions recombiant plasmid pCzn1-LTB-Emy162 to be checked, 37 DEG C of reaction 2h, use
1% agarose gel electrophoresiies detection, it is found that the DNA fragmentation of double digestion is about 750bp, the reason with fusion gene LTB-Emy162
By in the same size, as shown in Figure 2.The vector construction collection of illustrative plates of recombinant expression carrier pCzn1-LTB-Emy162 is as shown in Figure 3.To obtain
Recombiant plasmid pCzn1-LTB-Emy162 proceed to TOP10 clone strains, the son sequencing of picking positive colony, sequencing result with it is pre-
Phase sequence is completely the same, and without frameshift mutation.
Embodiment 3：The prokaryotic expression of multi-epitope peptide fusion protein LTB-Emy162
To verify that correct recombinant expression plasmid pCzn1-LTB-Emy162 is transferred to escherichia coli Arctic Express bacterial strains
In.On the LB flat boards containing 50 μ g/mL Amp well prepared in advance, inoculating loop line engineering strain pCzn1-LTB-
Emy162/Arctic Express, are inverted in 37 DEG C of incubators, and after incubated overnight, picking single bacterium colony is inoculated in containing 50 g/
In the LB culture medium of mL Amp, 37 DEG C, 220rpm, overnight incubation.Recombinant bacterium is inoculated with respectively in containing 50 g/mL with 2% inoculum concentration
In Amp LB culture medium, 37 DEG C, 220rpm is cultivated to being 0.6-0.8 (about 2h) to thalline OD600, is added IPTG to make final concentration
Reach 1mmol/L, 37 DEG C, 220rpm abduction delivering 4h, with not plus IPTG inductions carrier bacterium pCzn1-LTB-Emy162/
Arctic Express are used as negative control.
As a result：Contrast with control strain, genetic engineering recombination strain pCzn1-LTB-Emy162/Arctic Express exist
Occurs destination protein band at about 29KD, be consistent Fig. 4 with the theoretical size of multi-epitope peptide fusion protein LTB-Emy162, multilist
Position peptide fusion protein LTB-Emy162 is present in inclusion body protein.
Embodiment 4：The purification of multi-epitope peptide fusion protein LTB-Emy162
（1）The refolding strategy of inclusion body protein
Bacterial sediment is resuspended in into 20 ml lysis buffer (20 mM Tris-HCl containing, 1 mM PMSF
And bacteria protease inhibitor cocktail, pH 8.0), ultrasonication (400 W of power, work 4
Sec, interval 8 sec, totally 20 min)；4 DEG C of 10000g of cell pyrolysis liquid of ultrasonication are centrifuged into 20 min, precipitation is collected；
Washed using inclusion body cleaning mixture (20mM Tris, 1mM EDTA, 2M carbamide, 1M NaCl, 1%Triton X-100, pH8.0)
Inclusion body 3 times；With dissolving buffer (20mM Tris, 5mM DTT, 8M carbamide pH8.0), inclusion body is dissolved by a certain percentage, 4
DEG C stand overnight；Room temperature, 15000rpm centrifugation 15min；By 20 mM Tris-HCL 5mM EDTA of above-mentioned solution Deca
In Buffer PH7.8 buffer, progressively gradient dilution is slowly stirred at double, and protein solution is loaded bag filter in PBS pH7.4
Dialysed overnight in solution.
（2）The purification of Ni-IDA nickel ion affinity chromatograph posts
Using low pressure chromatography system, protein solution is pre- flat to Ni-IDA Binding-Buffer with 0.5 ml/min flow velocitys loading
Ni-IDA-Sepharose CL-6B the affinity columns of weighing apparatus；With Ni-IDA Binding-Buffer with 0.5 ml/min flow velocitys
Rinse, baseline is reached to effluent OD280 values；With Ni-IDA Washing-Buffer (20 mM Tris-HCl, 20 mM miaows
Azoles, 0.15 M NaCl, pH8.0) rinsed with 1 ml/min flow velocitys, baseline is reached to effluent OD280 values；Use Ni-IDA
Elution-Buffer (20 mM Tris-HCl, 250 mM imidazoles, 0.15 M NaCl, pH8.0) is with 1 ml/min flow velocity eluting
Destination protein, collects effluent；During the protein solution of above-mentioned collection adds bag filter, using PBS（PH7.4）Carry out dialysing
Night.
As a result：After purification, collect each protein peak carries out SDS-PAGE analyses to Jing Ni-IDA nickel ion affinity chromatographs post,
It can be found that destination protein concentrates on the protein peak of 250mM imidazoles eluting generation.Analyzed in 250mM by gel imaging detector
The destination protein purity of imidazoles eluting is pure up to electrophoresis, as shown in Figure 5.
Embodiment 5：The immunogenicity of subunit vaccine LTB-Emy162 and immunologic opsonin research
（1）The immunity of BALB/c mouse
Experiment packet：SPF level BALB/c mouse is randomly divided into into 3 groups, respectively Multi-Epitope Fusion Protein LTB-Emy162 immunity
Group, recombinant cholera toxin b subunit（rLTB）Immune group and PBS immune group.Per group of 6 BALB/c mouse, are grouped totally in detail by 18
It is as shown below：
Table 1：SPF levels BALB/c mouse is grouped and immunization protocol
| Experimental group | Number | Immunization wayses | Immune time | Immunizing dose | Immunological adjuvant |
| LTB-Emy162 | 6 | Lumbar injection | 3 times | 50 μ g/ are only | Freund adjuvant |
| rLTB | 6 | Lumbar injection | 3 times | 50 μ g/ are only | Freund adjuvant |
| PBS | 6 | Lumbar injection | 3 times | 50 μ g/ are only | Freund adjuvant |
Immunization wayses：Sterilized with 75% chronic ethanol treated mice abdominal part, then abdominal part multiple spot subcutaneous injection epitope fusion protein LTB-
The blended emulsifier of Emy162 and Freund's complete adjuvant；Booster immunization once, adds Freund not exclusively to help for the 2nd, 3 weeks week about
Agent, the 4th week direct injection epitope fusion protein solution booster immunization.
Sero-fast collection：The 5th day after final immunization, eyeball of mouse blood sampling is plucked, collect blood, placement treat that serum is complete
Fully separating, 3000rpm is centrifuged 5 minutes subpackage serum, and -80 DEG C frozen standby.
（2）The ELISA detections of specific antibody in antiserum
By antigen（Emy162 and Echinococcus moltilocularis total protein）10 μ g/ml, 100 μ l/ holes coating are diluted to coating buffer respectively
Elisa plate, 4 DEG C overnight.Washed after 4 times with cleaning mixture, 300 μ l confining liquids, 37 DEG C of closing 2h are added per hole.4 times are washed with cleaning mixture
Afterwards, by antiserum（The anti-LTB-Emy162 antiserums of Mus and the anti-rLTB antiserums of Mus）Add with after mice negative serum doubling dilution
Elisa plate, 100 μ l/ holes, is incubated 60min at 37 DEG C.Washed after 4 times with cleaning mixture, add the sheep anti-mouse igg of HRP labellings（1:
10000）, 100 μ l/ holes are incubated 1h at 37 DEG C.Washed after 4 times with cleaning mixture, add 100 μ l/ holes tmb substrate nitrite ions, room temperature
Lucifuge reacts 10min, plus 50 μ l terminate liquid terminating reactions.Each hole OD450 values are determined with microplate reader.
As a result：The coating concentration of Emy162 is 10 μ g/ml, is detected by ELISA, Echinococcus moltilocularis subunit vaccine LTB-
Emy162 can produce the specific IgG antibodies for Emy162, and rLTB immune group can not produce anti-Emy162 antibody, such as scheme
6；The coating concentration of Echinococcus moltilocularis total protein is 10 μ g/ml, detects that subunit vaccine LTB-Emy162 can by ELISA
The antibody of anti-Echinococcus moltilocularis total protein is produced, and PBS immune group can not produce the specific IgG of anti-Echinococcus moltilocularis total protein
Antibody, such as Fig. 7.
（3）Mouse spleen lymphocyte proliferation experiment
The mice dislocation of Jing antigen immunes is put to death, after 75% ethanol 5min of bubble, superclean bench is moved into.Carefully cut off with shears
The abdominal part crust of mice, then the abdominal cavity of mice is cut off, mouse spleen is taken out with tweezers（Kermesinus）.2- is put in little plate
3ml Lympholyte-M lymphocyte separation mediums；Nylon wire is fixed with tweezers, is then lightly ground with syringe piston
Mouse spleen so that scattered unicellular transmission nylon wire is entered in lymphocyte separation medium；The outstanding separation for having spleen cell
Liquid is immediately transferred in centrifuge tube, covers 1640 culture medium of about 1ml before centrifugation again；1000g-1500g is centrifuged 20min.
Centrifugation terminates rear lymphocyte and can suspend in 1640 culture hypobasals.Prepared with the RPMI-1640 culture medium containing 10% calf serum
Into certain density cell suspension（5 x 105/ml）.In 96 hole flat-bottomed plates, 100 μ l cells are added per hole, while plus
Enter each 20 μ g/ml, Emy162 epitope peptides of LTB-Emy162 and Emy162（Emy16236-48And Emy1627-13）, TSP3 antigens
Epitope peptide（TSP333-42And TSP380-90）100 μ l each with normal saline, final volume are 200 μ l/ holes, and 3 parallel holes are done per group.
The 96 hole flat-bottomed plates for having added are placed into 37 DEG C of 5% CO2After 60h is cultivated in incubator, 40 μ of MTS solution is added in each hole
L, reads OD570 values with microplate reader after continuing culture 2h.Stimulation index（SI）When >=2, it is judged as the positive；Stimulation index is calculated
Formula is as follows：
As a result：Mouse spleen lymphocyte Jing LTB-Emy162 that subunit vaccine LTB-Emy162 sensitization is crossed, Emy162,
Emy162 epitope peptides（Emy16236-48And Emy1627-13）Stimulation can occur obvious lymphproliferation response, and
The mouse spleen lymphocyte of PBS immune group receives that lymphproliferation response does not occur during above-mentioned antigenic stimulus, illustrates many
In room echinococcuss subunit vaccine LTB-Emy162, Emy162 epitope peptides maintain its immunological characteristic, can stimulate machine
Body produces the cellullar immunologic response for respective Th epitopes, such as Fig. 8.
（4）Protein immunoblot（Western blot）
Taking protein sample LTB-Emy162 carries out 15% SDS-PAGE electrophoresis.With half dry type electrotransfer method by the protein on gel
It is transferred on pvdf membrane, constant current 1mA/cm2, 2h；With 10 % skim milks confining liquid, 28 DEG C of shaking table closings 2 after pvdf membrane is cleaned
h；PBST washes film 3 times, each 10min.It is separately added into by 1:The good antiserum of 2500 multiple dilutions（The anti-LTB-Emy162 blood of Mus
Clearly）, 4 DEG C overnight；Film 3 times, each 10min are washed with PBST within second day.Add by 1:10000 multiple dilutions it is good two resist（HRP
Enzyme mark sheep anti-mouse igg）, 28 DEG C of shaking tables incubation 1h；Film 3 times, each 10min are washed with PBST.In darkroom (permission red light source),
Pvdf membrane albumen is faced up, is placed on preservative film, absorbent paper sops up unnecessary PBST solution, uniform Deca is mixed in advance on film
The ECL luminescent solutions for getting togather, react 1min；PDVF films are wrapped in preservative film, one piece of big X-ray film of grade is cut（Film
Make shear angle to process to distinguish band order）, placing clamp film magazine 0.5 ~ 30min of exposure；Take out X-ray film, float in a developer to
Band occurs, and is put into and stops shadow liquid（1.5% glacial acetic acid）In stop shadow 1min, with flowing water rinse 1min after float to background in fixative
Partially transparent, last flowing water rinses 20min stabilized images.
As a result：The anti-LTB-Emy162 serum of Mus can occur association reaction with the LTB-Emy162 albumen of purification, illustrate many
Room echinococcuss subunit vaccine LTB-Emy162, can excitating organism produce for LTB-Emy162 antibody with high specificity, such as
Fig. 9.
Embodiment 5：The molecular immune adjuvanticity of LTB components in GM1-ELISA detection subunit vaccines LTB-Emy162
By Ganglioside GM1 or bovine serum albumin（BSA）10 μ g/ml, 100 μ l/ holes, coating are diluted to coating buffer
Elisa plate, 4 DEG C overnight.4 times are washed with PBST；300 μ l confining liquids, 37 DEG C of closing 2h are added per hole；4 are washed with cleaning mixture PBST
After secondary, 4 DEG C of preservations.By multi-epitope peptide fusion protein LTB-Emy162 and recombinant cholera toxin b subunit（rLTB）According to a definite proportion
Example dilution（The 100 μ g/ml of μ g/ml to 0.78）, add 100 μ l/ holes, 37 DEG C of placement 60min.Washed 4 times with cleaning mixture PBST, added
The anti-LTB polyclonal antibodies of Mus（1:1000）, 100 μ l/ holes, 37 DEG C of incubation 60min.Washing 4 times；Add HRP labelling sheep anti-mouse iggs
（1:10000）, 100 μ l/ holes, 37 DEG C of placement 30min.After washing 4 times, 100 μ l/ holes tmb substrate nitrite ions, 37 DEG C of incubations are added
10min, 50 μ l terminate liquid terminating reactions.Each hole OD450 values are detected with microplate reader.
As a result：Detect whether multi-epitope peptide fusion protein LTB-Emy162 and rLTB have by GM1-ELISA and form five
The activity that aggressiveness and Ganglioside GM1 are combined.The OD450 of multi-epitope peptide fusion protein LTB-Emy162 and rLTB is significantly high
In negative control group（Coating BSA）, it was demonstrated that multi-epitope peptide fusion protein LTB-Emy162 and rLTB be respectively provided with to be formed pentamer and
The activity that Ganglioside GM1 is combined, also illustrates that novel subunit vaccine LTB-Emy162 and rLTB component has and preferably divides
Sub- immunolgical adjuvant activity, such as Figure 10.
Sequence table
Sequence 1
<110>Qinghai University
<120>A kind of design of Echinococcus moltilocularis subunit LTB-Emy162, preparation method and application
<130> 1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 250
<212> PRT
<213>Artificial sequence
<400> 1
Met Thr Ala Pro Gln Thr Ile Thr Glu Leu Cys Ser Glu Tyr Arg Asn
1 5 10 15
Thr Gln Ile Tyr Thr Ile Asn Asp Lys Ile Leu Ser Tyr Thr Glu Ser
20 25 30
Met Ala Gly Lys Arg Glu Met Val Ile Ile Thr Phe Lys Ser Gly Glu
35 40 45
Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys
50 55 60
Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Thr Tyr Leu Thr
65 70 75 80
Glu Thr Lys Ile Asp Lys Leu Cys Val Trp Asn Asn Lys Thr Pro Asn
85 90 95
Ser Ile Ala Ala Ile Ser Met Glu Asn Asp Pro Arg Val Pro Ser Ser
100 105 110
Met Glu Glu Val Gly Val Asp Pro Glu Leu Ile Ala Lys Leu Thr Lys
115 120 125
Lys Leu Gln Thr Thr Leu Pro Glu His Phe Arg Trp Ile His Val Gly
130 135 140
Ser Arg Ser Leu Glu Leu Gly Trp Asn Ala Thr Gly Leu Ala Asn Leu
145 150 155 160
His Ala Asp His Ile Lys Leu Thr Ala Asn Leu Tyr Thr Thr Tyr Val
165 170 175
Ser Phe Arg Tyr Arg Asn Val Pro Ile Glu Arg Gln Lys Leu Thr Leu
180 185 190
Glu Gly Leu Lys Pro Ser Thr Phe Tyr Glu Val Val Val Gln Ala Leu
195 200 205
Lys Gly Asp Ser Glu Val Tyr Lys Tyr Thr Gly Phe Ile Arg Thr Leu
210 215 220
Ala Pro Gly Glu Asp Gly Ala Asp Arg Ala Gly Gly Phe Ala Leu Ile
225 230 235 240
Phe Ala Met Ala Gly Leu Leu Leu Leu Thr
245 250
Sequence 2
<210> 2
<211> 750
<212> DNA
<213>Artificial sequence
<400> 2
atgacagcac ctcagacgat taccgaattg tgtagcgaat atcgcaatac ccagatctat 60
actatcaatg acaaaattct cagctacaca gaatccatgg ccggtaaacg cgagatggta 120
atcattactt ttaagtcggg cgaaaccttt caagtggaag tccccggcag ccaacatatt 180
gactcgcaga aaaaagcgat tgaacgcatg aaagataccc tccgtatcac ctatctgacc 240
gagactaaga tcgataagct gtgtgtatgg aacaacaaga caccgaacag tattgcggcg 300
atcagcatgg aaaacgatcc tcgtgttccc agtagtatgg aagaggttgg tgtagacccg 360
gaactgattg cgaaactgac gaagaaatta cagacgactc tcccggaaca ttttcgctgg 420
attcatgtag gctcccgtag cctggaactg ggctggaatg cgacaggtct ggccaacctc 480
catgcggatc atattaaact gaccgcgaat ctgtacacca catacgtgag ctttcgctat 540
cgcaacgtgc cgattgaacg ccaaaaactc accctggagg gcttgaaacc atcaaccttc 600
tatgaggtcg tggttcaagc gctgaaaggg gactcggagg tttataaata cacgggtttc 660
atccgcacgc tcgcgcctgg cgaagatggt gcagatcgcg ccggcggttt cgccttgatt 720
tttgcaatgg ctggccttct gctgctgacg 750
Sequence 3
<210> 3
<211> 138
<212> PRT
<213>Artificial sequence
<400> 3
Met Glu Glu Val Gly Val Asp Pro Glu Leu Ile Ala Lys Leu Thr Lys
1 5 10 15
Lys Leu Gln Thr Thr Leu Pro Glu His Phe Arg Trp Ile His Val Gly
20 25 30
Ser Arg Ser Leu Glu Leu Gly Trp Asn Ala Thr Gly Leu Ala Asn Leu
35 40 45
His Ala Asp His Ile Lys Leu Thr Ala Asn Leu Tyr Thr Thr Tyr Val
50 55 60
Ser Phe Arg Tyr Arg Asn Val Pro Ile Glu Arg Gln Lys Leu Thr Leu
65 70 75 80
Glu Gly Leu Lys Pro Ser Thr Phe Tyr Glu Val Val Val Gln Ala Leu
85 90 95
Lys Gly Asp Ser Glu Val Tyr Lys Tyr Thr Gly Phe Ile Arg Thr Leu
100 105 110
Ala Pro Gly Glu Asp Gly Ala Asp Arg Ala Gly Gly Phe Ala Leu Ile
115 120 125
Phe Ala Met Ala Gly Leu Leu Leu Leu Thr
130 135
Sequence 4
<210> 4
<211> 414
<212> DNA
<213>Artificial sequence
<400> 4
atggaagagg ttggtgtaga cccggaactg attgcgaaac tgacgaagaa attacagacg 60
actctcccgg aacattttcg ctggattcat gtaggctccc gtagcctgga actgggctgg 120
aatgcgacag gtctggccaa cctccatgcg gatcatatta aactgaccgc gaatctgtac 180
accacatacg tgagctttcg ctatcgcaac gtgccgattg aacgccaaaa actcaccctg 240
gagggcttga aaccatcaac cttctatgag gtcgtggttc aagcgctgaa aggggactcg 300
gaggtttata aatacacggg tttcatccgc acgctcgcgc ctggcgaaga tggtgcagat 360
cgcgccggcg gtttcgcctt gatttttgca atggctggcc ttctgctgct gacg 414
Claims (9)
1. a kind of Echinococcus moltilocularis subunit vaccine LTB-Emy162, its active component is a polypeptide, mainly by escherichia coli
Heat-labile toxin B subunits（LTB）Constitute with Echinococcus moltilocularis antigen protein Emy162, its aminoacid sequence is as shown in sequence 1.
2. Echinococcus moltilocularis subunit vaccine LTB-Emy162 as claimed in claim 1, its nucleotide sequence such as 2 institute of sequence
Show.
3. Echinococcus moltilocularis subunit vaccine LTB-Emy162 as claimed in claim 1, the Echinococcus moltilocularis antigen protein
The aminoacid sequence of Emy162 is as shown in sequence 3.
4. Echinococcus moltilocularis subunit vaccine LTB-Emy162 as claimed in claim 3, the Echinococcus moltilocularis antigen protein
The nucleotide sequence of Emy162 is as shown in sequence 4.
5. Echinococcus moltilocularis subunit vaccine LTB-Emy162 as described in claim 2 or 4, comprising the nucleotide sequence
Expression vector, transgenic cell line and Host Strains.
6. Echinococcus moltilocularis subunit vaccine LTB-Emy162 as claimed in claim 1, HLT B subunits
（LTB）And the intervening sequence between Echinococcus moltilocularis antigen protein Emy162 is DPRVPSS.
7. a kind of preparation method of Echinococcus moltilocularis subunit vaccine LTB-Emy162, synthesizes many room spine by gene synthesis technology
The nucleotide sequence of ball larva of a tapeworm or the cercaria of a schistosome multi-epitope peptide LTB-Emy162, builds the recombinant expression carrier containing fusion gene LTB-Emy162
PCzn1-LTB- Emy162 and its recombination engineering bacteria Arctic Express；After the fermentation of recombination engineered strain, Jing
Ni-IDA nickel ion displacement chromatography purification, obtains the fusion protein LTB- Emy162 of the vaccine.
8. Echinococcus moltilocularis subunit vaccine LTB-Emy162 described in any one of claim 1-6, it is characterised in that can swash
Send out body and produce the immunne response for Echinococcus moltilocularis B cell and T cell.
9. Echinococcus moltilocularis subunit vaccine LTB-Emy162 described in any one of claim 1-7 is preventing and treating many room spine
Application in the infection of the ball larva of a tapeworm or the cercaria of a schistosome.
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| CN112979780A (en) * | 2021-02-25 | 2021-06-18 | 青海大学 | Echinococcus multilocularis glucose transporter polypeptide vaccine GLEP, and preparation method and application thereof |
| CN113181349A (en) * | 2021-04-25 | 2021-07-30 | 新疆医科大学 | M cell-targeted multi-epitope oral vaccine and application thereof in hydatid vaccine |
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| CN102675466A (en) * | 2011-03-09 | 2012-09-19 | 冯强 | Epitope delivery system with Escherichia coli heat labile enterotoxin B subunits serving as carriers |
| JP5055522B2 (en) * | 2007-01-31 | 2012-10-24 | 国立大学法人北海道大学 | A novel protein derived from polychaete |
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| JP5055522B2 (en) * | 2007-01-31 | 2012-10-24 | 国立大学法人北海道大学 | A novel protein derived from polychaete |
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| RUNLE LI等: "Immunological features and efficacy of the recombinant subunit vaccine LTB-EMY162 against Echinococcus multilocularis metacestode", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| 周虎: "新型多房棘球蚴亚单位疫苗CTB-Emy162的研究", 《中国优秀硕士学位论文全文数据库》 * |
| 庞明泉: "多房棘球蚴抗原蛋白Emy162及TSP3的抗原表位的预测及鉴定", 《中国优秀硕士学位论文全文数据库》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112979780A (en) * | 2021-02-25 | 2021-06-18 | 青海大学 | Echinococcus multilocularis glucose transporter polypeptide vaccine GLEP, and preparation method and application thereof |
| CN112979780B (en) * | 2021-02-25 | 2022-02-08 | 青海大学 | Echinococcus multilocularis glucose transporter polypeptide vaccine GLEP, and preparation method and application thereof |
| CN113181349A (en) * | 2021-04-25 | 2021-07-30 | 新疆医科大学 | M cell-targeted multi-epitope oral vaccine and application thereof in hydatid vaccine |
| CN113181349B (en) * | 2021-04-25 | 2023-02-28 | 新疆医科大学 | M cell-targeted multi-epitope oral vaccine and application thereof in hydatid vaccine |
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