CN107502616A - A kind of soluble recombinant protein CTA CD154 and its preparation method and application - Google Patents
A kind of soluble recombinant protein CTA CD154 and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of soluble recombinant protein CTA CD154, its nucleotide sequence and amino acid sequence.The invention also discloses soluble recombinant protein CTA CD154 preparation method.The invention also discloses soluble recombinant protein CTA CD154 application.Soluble recombinant protein CTA CD154 can be as a kind of new immunopotentiator, and being capable of specific targeting DC cells, the immune response of body is significantly increased, and the Immune-enhancing effect CTA CD154 effects after fusion are significantly higher than the immunological enhancement of separate constituent.
Description
Technical field
The invention belongs to field of biological pharmacy, and in particular to a kind of soluble recombinant protein CTA-CD154 and its preparation side
Method and application.
Background technology
Mucosa-immune is the first line of defence of body resistance pathogenic microorganism invasion, and enhancing mucosa-immune effect is raising machine
One of effective means of body resistance.So increasing researcher explores how to improve body mucosal immunity effect, have
Scholar has found that cholera toxin (cholera toxin, CT) has preferable mucosal adjuvant activity, and it is identical by 1 A and 5
B subunits composition more subunit's macromoleculars.Confirm that Cholera Toxin A subunit (CTA) can make a small amount of resist through numerous studies
Original induction body produces early stage, efficient, lasting immune response；It can effectively strengthen body as mucosa-immune reinforcing agent
Mucosa-immune reacts, and it is horizontal can to significantly improve IgG antibody caused by related antigen stimulation.But CTA as immunopotentiator not
Function with selectively targeted DC cells, therefore, it plays immunological enhancement and is subject to certain restrictions.
It is in recent years delivery cell by professional antigen of concern that BMDC (Dendritic Cells, DC), which is,
(Antigen presentingCells, APC), antigen can be absorbed, processed and presented, start the immune response of T cell mediation.T
Lymphocyte played an important role in the activation of immune system, and its surface receptor CD40 and CD154 interacts
Promote cell-mediated be immunized by the activation of BMDC and the generation of natural killer cell.CD154 is otherwise known as
CD40L, is CD40 part, and a kind of II types transmembrane glycoprotein.As critical stimulus developed by molecule in CD4+T cell table
Face, also can be in cd8 t cell, B cell, macrophage, mast cell, surface of dendritic cells expression after activation.CD154 conducts
A kind of part can promote T, the differentiation of B cell, promotion macrophage activation after being combined with its acceptor, and with specifically
The function of DC cells is targetted, is had the function that in humoral and cellular immune response response important.Therefore, a kind of targeting DC of design is immunized
Reinforcing agent, new thinking is provided for follow-up test research.
The content of the invention
Technical problem：The technical problems to be solved by the invention there is provided one kind and be used for encoding soluble recombinant protein
CTA-CD154 DNA molecular.
The present invention also technical problems to be solved there is provided a kind of soluble recombinant protein CTA-CD154.
The present invention also technical problems to be solved there is provided containing described soluble recombinant protein CTA-CD154's
Recombinant expression carrier, transgenic cell line or the transgenosis recombinant bacterium of DNA molecular.
The present invention also technical problems to be solved there is provided a kind of immunopotentiator.
Last technical problems to be solved of the invention there is provided described immunopotentiator answering in terms of vaccine is prepared
With.
Technical scheme：In order to solve the above-mentioned technical problem, the invention discloses one kind to be used for encoding soluble recombinant protein
CTA-CD154 DNA molecular, its nucleotide sequence such as SEQ ID NO：Shown in 1.
Present invention also includes a kind of soluble recombinant protein CTA-CD154, its amino acid sequence such as SEQ ID NO：2
It is shown.
Present invention also includes the recombination expression of the DNA molecular containing described soluble recombinant protein CTA-CD154
Carrier, transgenic cell line or transgenosis recombinant bacterium.
Wherein, described recombinant expression carrier is that described DNA molecular is inserted into coli expression carrier to obtain
The recombinant expression carrier of DNA molecular containing soluble recombinant protein CTA-CD154.
Present invention also includes a kind of transgenosis recombinant bacterium, and the recombinant bacterium is by described recombinant expression carrier importing
In Escherichia coli, screening obtains transgenosis recombinant bacterium.
Present invention also includes soluble recombinant protein CTA-CD154 preparation method, comprises the following steps：
1) according to the inclined preferendum optimum synthesis CTA-CD154 gene orders of e. coli codon, and it is connected with carrier, builds
Recombinant plasmid, the CTA-CD154 gene orders such as SEQ ID NO：Shown in 1；
2) by recombinant plasmid transformed, induce and obtain soluble recombinant expression protein CTA-CD154 after purification.
Specifically, soluble recombinant protein CTA-CD154 preparation method comprises the following steps：
(1) according to the inclined preferendum optimum synthesis CTA-CD154 gene orders of e. coli codon, and with carrier pET32a phases
Even, construction recombination plasmid.
(2) by recombinant plasmid transformed BL21 competent cells, recombinant expression plasmid pET32a-CTA- is obtained after identification
CD154/BL21, recombinant expression protein is obtained after IPTG is induced, thalline carries out SDS-PAGE points after ultrasonication after expression
Analysis, it is found that recombinant protein mainly exists with soluble form, molecular weight of albumen is about 46kDa；Western-blot shows,
Go out the specific band for being now able to be combined with His monoclonal antibodies at 46kDa.
Present invention also includes described DNA molecular, described soluble recombinant protein CTA-CD154, described weight
The application of group expression vector, transgenic cell line or transgenosis recombinant bacterium in terms of immunopotentiator is prepared.
Present invention also includes a kind of immunopotentiator, and the immunopotentiator includes described soluble recombinant protein
CTA-CD154。
The immunopotentiator of the present invention, it is that a kind of immunopotentiator of targeting DC cells can target DC cells, improves DC
The activation efficiency of cell, the former immune response of enhancing body fight, and then improve vaccine immunity effect.
Present invention also includes the application described immunopotentiator in terms of vaccine is prepared.
The vaccine of the present invention includes but are not limited to inactivated foot-and-mouth disease vaccine, annulus vaccine or pseudo- rabies vaccine.The present invention
Vaccine can also include other inactivated vaccines, preferably pig inactivated vaccine.
Beneficial effect：The present invention possesses advantages below relative to prior art：The present invention carries out CTA and CD154 genes
After fusion, it is expressed using bacterium coli solubility expression system.Result of study confirms soluble recombinant protein CTA-
CD154 can as a kind of new immunopotentiator, and can specific targeting DC cells, significantly increase the immune of body
Responsing reaction, and the Immune-enhancing effect CTA-CD154 effects after fusion are significantly higher than the immunological enhancement of separate constituent.
Brief description of the drawings
Fig. 1, recombinant plasmid digestion qualification figure；1- molecular weight 10000bp standard Marker, 2-pET32a-CTA-
CD154 digestion products, 3-pET32a-CTA digestion products, 4-pET32a-CD154 digestion products；
Fig. 2, soluble recombinant protein SDS-PAGE analysis charts；1- protein standards Marker, 2-BL21 empty bacterium, 3-CTA-
The full bacterium of CD154, the inclusion body after 4-CTA-CD154 induced expressions, the supernatant after 5-CTA-CD154 induced expressions, 6- is after purification
CTA-CD154 albumen, the CTA albumen of 7- after purification, the CD154 albumen of 8- after purification；
Fig. 3, soluble recombinant protein Western-blot qualification figures；1- protein standards Marker, 2-CTA-CD154 is complete
Bacterium, the inclusion body after 3-CTA-CD154 induced expressions, the supernatant after 4-CTA-CD154 induced expressions, the CTA- of 5- after purification
CD154 albumen, the CTA albumen of 6- after purification, the CD154 albumen of 7- after purification；
LPB-ELISA antibody level testing result after Fig. 4, aftosa vaccine are immune；
CD11C accounts for the ratio chart of DC cells after Fig. 5, aftosa vaccine are immune；
CD11C+CTA-CD154 accounts for the ratio chart of DC cells after Fig. 6, aftosa vaccine are immune；
ELISA antibody test results after Fig. 7, annulus vaccine immunity；
CD11C accounts for the ratio chart of DC cells after Fig. 8, annulus vaccine immunity；
CD11C+CTA-CD154 accounts for the ratio chart of DC cells after Fig. 9, annulus vaccine immunity；
ELISA antibody test results after Figure 10, pseudo- rabies vaccine are immune；
CD11C accounts for the ratio chart of DC cells after Figure 11, pseudo- rabies vaccine are immune；
CD11C+CTA-CD154 accounts for the ratio chart of DC cells after Figure 12, pseudo- rabies vaccine are immune.
Embodiment
To further illustrate the details of the present invention, some embodiments are set forth below, but the present invention should not be limited thereto.
Used reagent in the present invention：DNA marker, restriction enzyme Nde I, Xho I are purchased from the precious biology in Dalian
Company；DH5 α, BL21 competent cells are purchased from Beijing Quanshijin Biotechnology Co., Ltd；Protein molecular weight Marker, BCA
Kit is Thermo Fisher Scientific Products；Pvdf membrane is Millipore Products；DAB nitrite ions,
HIS monoclonal antibodies are purchased from Nanjing Sheng Xing bio tech ltd.
The immunopotentiator CTA-CD154 of embodiment 1 preparation method
1st, the clone of CTA-CD154 target gene
According to GenBank log in CTA (accession number D30053.1), CD154 (accession number HQ110108.1) gene order,
Purpose of design gene order, CTA, CD154, CTA-CD154 are named as respectively, and according to the inclined preferendum of e. coli codon
After optimization, Nde I and Xho I restriction enzyme sites, commission Nanjing Genscript Biotechnology Co., Ltd. clone are added at sequence both ends
To pUC57 carriers, recombinant cloning vector pUC57-CTA, pUC57-CD154, pUC57-CTA-CD154 are formed.
2nd, the structure of recombinant plasmid
PUC57-CTA, pUC57-CD154, pUC57-CTA-CD154 and recombinant prokaryotic expression vector pET32a are distinguished
Double digestion is carried out with Nde I and Xho I and connection converts.Plasmid is extracted from transformed bacteria and does digestion identification, is coagulated through 1% agarose
Gel electrophoresis detect, and CTA purpose bands are about 774bp, and CD154 purpose bands are about 474bp, CTA-CD154 purpose bands
Size is about 1248bp.To identify correct recombinant expression plasmid be respectively designated as pET32a-CTA, pET32a-CD154,
pET32a-CTA-CD154.The digestion qualification figure is referring to Fig. 1.
3rd, the expression of destination protein
Recombinant plasmid pET32a-CTA, pET32a-CD154, pET32a-CTA-CD154 are converted into Escherichia coli respectively
BL21, the LB flat boards containing ampicillin are coated with, picking single bacterium colony, obtain recombinant bacterium pET32a-CTA/BL21, pET32a-
CD154/BL21、pET32a-CTA-CD154/BL21.By empty plasmid pET32a transformed competence colibacillus large intestine bars in the same manner
Bacterium BL21, obtain control strain pET32a/BL21.
Picking recombinant bacterium pET32a-CTA/BL21, pET32a-CD154/BL21, pET32a-CTA-CD154/BL21 with
PET32a/BL21 single bacterium colony, it is inoculated with respectively in 5mL LB fluid nutrient mediums (containing ampicillin, chloramphenicol), 37 DEG C of vibrations
Overnight incubation.The mother liquor is pressed 1 by next day:It is (mould containing ampicillin and chlorine that 100 ratios are transferred to new LB fluid nutrient mediums
Element), 37 DEG C, 220r/min, shaken cultivation 1.5-2h (OD600 reaches 0.5-1), final concentration of 0.4mmol/LIPTG is added, in 15
DEG C induction 24h, bacterium is harvested by centrifugation.Collect bacterial precipitation and it is resuspended with 1mL PBSs respectively, in ice-water bath
Middle carry out ultrasonic degradation.The supernatant after complete induction thalline and ultrasonic treatment, deposit sample is taken to carry out SDS- respectively
PAGE electrophoresis.Occur and target band of the same size at 28KDa, 18KDa, 46kDa respectively (referring to Fig. 2).
The above-mentioned sample handled well is detected with Western-blot, by separation gel after sample progress SDS-PAGE
Protein delivery to pvdf membrane.Take out the film to take a turn for the better to be put into the confining liquid of the TBST containing 5% skimmed milk power, 4 DEG C overnight.
Add 1:The His monoclonal antibodies of 2000 dilutions, after 37 DEG C are incubated 1h, TBST is washed 5 times, 5min/ times, adds 1:The HRP of 3000 dilutions
Sheep anti-mouse igg is marked, 37 DEG C are incubated 1h, and TBST is washed 5 times, and 5min/ times, DAB develops the color.Western-blot is confirmed, is existed respectively
Go out the specific band for being now able to be combined with His monoclonal antibodies at 46kDa, 28KDa, 18KDa (referring to Fig. 3).
4th, protein purification
Great expression recombinant protein pET32a-CTA/BL21, pET32a-CD154/BL21, pET32a- as stated above
CTA-CD154/BL21, bacterial sediment is collected by centrifugation.Soluble protein is obtained through multigelation and after being ultrasonically treated, and by albumen
Purified using Ni-Charged Resin affinity columns, concrete operation step by specification is carried out.Albumen passes through after purification
After ultrafiltration concentration pipe concentration desalination 3 times, verified with SDS-PAGE electrophoresis and Western-blot.With BCA kits to it
Concentration is measured, measure CTA-CD154 recombinant proteins concentration after purification for 1.2mg/mL (referring to the 6th swimming lane in Fig. 2,
The 5th swimming lane in Fig. 3), CTA protein concentrations after purification are 1.35mg/mL (referring to the in the 7th swimming lane, Fig. 3 in Fig. 2
6 swimming lanes), CD154 protein concentrations after purification (are swum for 1.25mg/mL referring to the 7th in the 8th swimming lane, Fig. 3 in Fig. 2
Road).The albumen can be used as immunopotentiator and directly use.
Influences of the immunopotentiator CTA-CD154 of embodiment 2 to inactivated foot-and-mouth disease vaccine immune efficacy
1st, experiment material
The O-shaped hoof-and-mouth disease venom (FMDV) of pig of inactivation, 146s contents are 6 μ g/mL, and Inner Mongol Jinyu Group give.
The ICR mouse of 4-5 week old are purchased from Yangzhou University's comparative medicine center, LPB-ELISA antibody titer≤1:4.
O-shaped aftosa LPB-ELISA antibody assay kit is purchased from Lanzhou veterinary institute.
GM-CSF, IL-4 are purchased from Gibco companies.
The anti-mouse CD11C monoclonal antibodies purchase spontaneous emerging Bioisystech Co., Ltd in Nanjing of FITC marks.
FITC-CTA-CD154 is prepared by this laboratory and marked.
CD154 albumen, CTA albumen are provided by this laboratory and (adjust concentration during seedling and immunopotentiator CTA-CD154 is dense
Degree is consistent).
Adjuvant ISA206 is bought matches BIC Corp in France.
2nd, vaccine formulation
Immunopotentiator CTA, CD154, CTA-CD154 are mixed by the dosage of 50 μ g/ head parts with the FMDV inactivated respectively,
Aqueous phase solution is obtained, then by aqueous phase solution and adjuvant ISA 206 according to volume ratio 1:1 ratio mixing, breast is carried out with emulsification instrument
CTA containing immunopotentiator, CD154, CTA-CD154 inactivated foot-and-mouth disease vaccine are obtained after change.
3rd, it is grouped, immune and immune rear coherent detection
3.1 experiment mouse are grouped and are immunized
Buy the ICR mouse 50 of 5 week old from Yangzhou University's comparative medicine center, random division is 5 groups, every group 10,
It is respectively labeled as G1, G2, G3, G4, G5 group.G1 groups are FMDV+CTA-CD154 groups；G2 groups are FMDV+CTA groups；G3 groups are FMDV
+ CD154 groups；G4 groups are FMDV control groups；G5 groups are PBS control group.The immunizing dose of every mouse is 0.25mL/, after 21d
Booster immunization once, respectively at 14d, 21d, 35d, 49d, 56d, 63d collection serum is used for ELISA antibody tests.Specifically it is shown in Table
1。
The mice group of table 1 and immune
3.2 immune rear liquid phase blocking antibody detections
14d, 21d, 35d, 49d, 56d, 63d after immune gather blood, separate serum, and it is anti-to carry out liquid phase blocking
Physical examination is surveyed.
Each immune group liquid phase blocking antibody testing result such as Fig. 4, the liquid phase blocking antibody of each immune group 35d after exempting from locate
Except the trend of rising, PBS blank control groups.Although the liquid phase blocking antibody of FMDV control groups has risen, exempt from rear 35d
There is obvious downward trend.FMDV+CTA groups improve with the antibody level of FMDV+CD154 groups compared with FMDV control groups
One titre, and the antibody level of whole duration declines slowly.The result illustrates that single CTA, CD154 can be improved
Mouse is tested to the immune response of antigen, there is immunological enhancement.The antibody level of FMDV+CTA-CD154 groups is significantly high
In FMDV control groups, 35 days two groups of antibody titer differences 2 after exempting from3.Compared with FMDV+CTA groups and FMDV+CD154 groups, resist
Body titre difference 22.The result illustrates that immunopotentiator CTA-CD154 can significantly improve the antibody level of experiment mouse, have
Preferable immunological enhancement, and its immunological enhancement is significantly higher than FMDV+CTA, FMDV+CD154 immune group.
The detection of 3.3 targeting DC cells
Aseptic collection bone marrow cells in mice, stimulated through GM-CSF, IL-4, culture obtains enrichment mouse bone marrow cells after 7 days are come
The BMDC in source, adjustment cell density are 1 × 106Individual/mL.Add the anti-mouse of final concentration of 5 μ g/mLFITC marks
After CD11C monoclonal antibodies and FITC-CTA-CD154 are incubated three hours, PBS is washed 3 times, adds 300 μ L1% (w/v) paraformaldehydes,
It is transferred in streaming loading pipe, carries out the positive ratio that FACS detects each pipe DC cells.Testing result as shown in Figure 5, Figure 6,
The ratio of the CD11C DC cells of FMDV+CTA-CD154 immune groups is thin for the CD11C DC of 34.7%, FMDV+CD154 immune groups
The ratio of born of the same parents is 15.3%, but the ratio of the CD11C+CTA-CD154DC cells of FMDV+CTA-CD154 immune groups is
21.5%, and the ratio of the CD11C+CTA-CD154DC cells of FMDV+CD154 immune groups is 4%, two groups are compared, and difference is extremely
Significantly (P<0.05).The result shows that immunopotentiator CTA-CD154 can specifically target DC cells, and single
CD154 also has the function of targeting DC cells, but it is weaker to target ability.
In the present embodiment, the use volume of immunopotentiator can also adjust according to being actually needed, numerous to list herein.
Shadows of the immunopotentiator CTA-CD154 of embodiment 3 to commercialization porcine circovirus 2 type (PCV2) vaccine immunity effect
Ring 1, experiment material
Porcine circovirus 2 type inactivated vaccine (DBN-SX07 strains) is purchased from Chengdu day nation.
The ICR mouse of 4-5 week old are purchased from Yangzhou University's comparative medicine center.
Porcine circovirus 2 type ELISA antibody assay kits are purchased from Wuhan Ke Qian Biological Co., Ltd..
2nd, vaccine formulation
By immunopotentiator CTA, CD154, CTA-CD154 respectively by the dosage of 50 μ g/ head parts and the pig annulus of commercialization
Viral 2 type inactivated vaccines (DBN-SX07 strains) are mixed, and obtain CTA containing immunopotentiator, CD154, CTA-CD154 annulus
Vaccine.
3rd, it is grouped, immune and immune rear coherent detection
3.1 experiment mouse are grouped and are immunized
Buy the ICR mouse 50 of 5 week old from Yangzhou University's comparative medicine center, random division is 5 groups, every group 10,
It is respectively labeled as Z1, Z2, Z3, Z4, Z5 group.Z1 groups are PCV2+CTA-CD154 groups；Z2 groups are PCV2+CTA groups；Z3 groups are PCV2
+ CD154 groups；Z4 groups are PCV2 control groups；Z5 groups are PBS control group.The immunizing dose of every mouse is 0.2mL/, after 14d
Booster immunization once, respectively at 14d, 35d collection serum is used for ELISA antibody tests.Specifically it is shown in Table 2.
The mice group of table 2 and immune
3.2 immune rear annulus detection of specific antibody
14d, 35d after immune gather blood, separate serum, carry out PCV2 detection of specific antibody.Specific behaviour
Work is carried out according to kit specification.
After each 400 times of dilutions of immune group serum, antibody test result such as Fig. 7, it is special that head exempts from 14d collection Virus monitories PCV2
Property antibody, PCV2+CTA-CD154 immune groups antibody level is higher than other immune groups, but difference is not notable, small after booster immunization
Mouse PCV2 specific antibodies significantly raise, PCV2+CTA-CD154 immune groups antibody level apparently higher than PCV2+CTA immune groups,
PCV2+CD154 immune groups and PCV2 commercial seedling control groups, and significant difference (p<0.05).PBS negative controls PCV2 is special in experiment
Heterogenetic antibody detection is feminine gender.Experiment shows that immunopotentiator CTA+CD154 can significantly improve PCV2 inactivated vaccines
Immune efficacy, there is good immunological enhancement.
The detection of 3.3 targeting DC cells
Exempt from rear 21d aseptic collections bone marrow cells in mice, stimulated through GM-CSF, IL-4, culture obtains the mouse of enrichment after 7 days
The BMDC of derived from bone marrow, adjustment cell density are 1 × 106Individual/mL.Add the anti-of final concentration of 5 μ g/mLFITC marks
After mouse CD11C monoclonal antibodies and FITC-CTA-CD154 are incubated three hours, PBS is washed 3 times, adds 300 μ L1% paraformaldehydes,
It is transferred in streaming loading pipe, carries out the positive ratio that FACS detects each pipe DC cells.Testing result as shown in Figure 8, Figure 9,
The ratio of the CD11C DC cells of PCV2+CTA-CD154 immune groups is 18.5%, extremely notable with other immune group comparing differences
(P<0.05).The ratio of the CD11C+CTA-CD154DC cells of PCV2+CTA-CD154 immune groups is 10.98%, is exempted from other
Epidemic disease group is compared to difference extremely significantly (P<0.05).The result shows immunopotentiator CTA-CD154 and energy after annulus vaccine immunity
It is enough specifically to target DC cells.
In the present embodiment, the use volume of immunopotentiator can also adjust according to being actually needed, numerous to list herein.
Influences of the immunopotentiator CTA-CD154 of embodiment 4 to pseudorabies (PRV) vaccine immunity effect
1st, experiment material
Pseudo- mad net vaccine (pseudo- mad dog gene-deleted vaccine) is purchased from great Bei agricultures group.
The ICR mouse of 4-5 week old are purchased from Yangzhou University's comparative medicine center.
PRV gB ELISA kits are purchased from Dutch Biochek.
2nd, vaccine formulation
By immunopotentiator CTA, CD154, CTA-CD154 respectively by 50 μ g/ head parts dosage and commercialization it is pseudo- it is mad only
Vaccine is mixed, and obtains CTA containing immunopotentiator, CD154, CTA-CD154 pseudo- rabies vaccine.
3rd, it is grouped, immune and immune rear coherent detection
3.1 experiment mouse are grouped and are immunized
Buy the ICR mouse 50 of 5 week old from Yangzhou University's comparative medicine center, random division is 5 groups, every group 10,
It is respectively labeled as P1, P2, P3, P4, P5 group.P1 groups are PRV+CTA-CD154 groups；P2 groups are PRV+CTA groups；P3 groups are PRV+
CD154 groups；P4 groups are PRV control groups；P5 groups are PBS control group.The immunizing dose of every mouse is 0.2mL/, is adopted in 21d
Collection serum is used for ELISA antibody tests.Specifically it is shown in Table 3.
The mice group of table 3 and immune
3.2 immune rear pseudo- mad dog detection of specific antibody
21d collection blood after immune, separates serum, carries out PRV detection of specific antibody.Concrete operations are according to kit
Specification is carried out.
Each immune group serum presses 1：Detected after 10 times of dilutions, antibody test result such as Figure 10,3 weeks collection blood after being immunized
Clear to carry out PRV detection of specific antibody, PRV+CTA immune groups and PRV+CD154 immune groups antibody level are higher than PRV commercial seedlings pair
It is not notable according to group, difference.Other three immune groups and PRV+CTA-CD154 immune groups compare, antibody level is considerably higher, poor
It is different notable, and antibody test homogeneity is more preferable.PBS control group testing result is feminine gender in experiment.Experiment shows, immunopotentiator
CTA-CD154 can significantly improve the immune efficacy of PRV attenuated vaccines, have good immunological enhancement.
The detection of 3.3 targeting DC cells
Exempt from rear 21d aseptic collections bone marrow cells in mice, stimulated through GM-CSF, IL-4, culture obtains the mouse of enrichment after 7 days
The BMDC of derived from bone marrow, adjustment cell density are 1 × 106Individual/mL.Add the anti-of final concentration of 5 μ g/mLFITC marks
After mouse CD11C monoclonal antibodies and FITC-CTA-CD154 are incubated three hours, PBS is washed 3 times, adds 300 μ L1% paraformaldehydes,
It is transferred in streaming loading pipe, carries out the positive ratio that FACS detects each pipe DC cells.Testing result as shown in Figure 11, Figure 12,
The ratio of the CD11C DC cells of PRV+CTA-CD154 immune groups is thin for the CD11C DC of 13.54%, PRV+CD154 immune groups
The ratio of born of the same parents is 4.05%, but the ratio of the CD11C+CTA-CD154DC cells of PRV+CTA-CD154 immune groups is
The ratio of the CD11C+CTA-CD154DC cells of 8.54%, PRV+CD154 immune group is 2.05%, compared with other immune groups
Significant difference.The result shows to add that can specifically to target DC after immunopotentiator CTA-CD154 thin in pseudo- rabies vaccine
Born of the same parents.
In the present embodiment, the use volume of immunopotentiator can also adjust according to being actually needed, numerous to list herein.
Sequence table
<110>Jiangsu Province Agriculture Science Institute
<120>A kind of soluble recombinant protein CTA-CD154 and its preparation method and application
<130> 17NJ1V0310082
<141> 2017-10-18
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1185
<212> DNA
<213>Recombinant protein c TA-CD154 (artificial sequence)
<220>
<221> misc_feature
<222> (1)..(1185)
<400> 1
aacgacgata agctgtaccg tgcggacagc cgtccgccgg atgagatcaa acaaagcggt 60
ggcctgatgc cgcgtggtca gagcgaatac tttgaccgtg gcacccaaat gaacattaac 120
ctgtatgatc acgcgcgtgg tacccagacc ggtttcgtgc gtcacgacga tggttacgtt 180
agcaccagca tcagcctgcg tagcgcgcac ctggtgggtc aaaccattct gagcggccac 240
agcacctact atctgtatgt tctggcgacc gcgccgaaca tgtttaacgt gaacgacgtt 300
ctgggtgcgt acagcccgca cccggatgag caggaagtga gcgcgctggg tggcatcccg 360
tatagccaaa tttacggttg gtatcgtgtg cactttggcg ttctggacga gcagctgcac 420
cgtaaccgtg gttaccgtga tcgttactat agcaacctgg acatcgcgcc ggcggcggat 480
ggttatggtc tggcgggttt cccgccggaa caccgtgcgt ggcgtgagga accgtggatt 540
caccacgctc cgccgggttg cggtaacgcg ccgcgtagca gcatgagcaa cacctgcgac 600
gagaagaccc agagcctggg tgtgaaattt ctggatgaat accagagcaa ggttaaacgt 660
caaatcttca gcggctatca gagcgacatc gatacccaca accgtattaa ggacgagctg 720
ggatccaagg gtgaccagga cccgcagatc gcggcgcacg ttattagcga ggcgagcagc 780
aagaccgcga gcgtgctgca gtgggcgccg aaaggctact ataccctgag caccaacctg 840
gttaccctgg aaaacggtcg tcagctggcg gtgaaacgtc aaggcatcta ctatatttac 900
gcgcaggtta ccttctgcag caaccgtgac gcggcgggtc aagcgccgtt tatcgcgagc 960
ctgtgcctgc gtagcccgag cggcagcgag cgtattctgc tgcgtgcggc gaacacccac 1020
agcagcagca agccgtgcgg tcagcaaagc atccacctgg gtggcgtttt cgaactgcag 1080
ccgggcgcga gcgtgtttgt taacgtgacc gatccgagcc aagtgagcca cggtaccggc 1140
ttcaccagct ttggtctgct gaaactgcac caccaccacc accac 1185
<210> 2
<211> 395
<212> PRT
<213>Recombinant protein c TA-CD154 (artificial sequence)
<400> 2
Asn Asp Asp Lys Leu Tyr Arg Ala Asp Ser Arg Pro Pro Asp Glu Ile
1 5 10 15
Lys Gln Ser Gly Gly Leu Met Pro Arg Gly Gln Ser Glu Tyr Phe Asp
20 25 30
Arg Gly Thr Gln Met Asn Ile Asn Leu Tyr Asp His Ala Arg Gly Thr
35 40 45
Gln Thr Gly Phe Val Arg His Asp Asp Gly Tyr Val Ser Thr Ser Ile
50 55 60
Ser Leu Arg Ser Ala His Leu Val Gly Gln Thr Ile Leu Ser Gly His
65 70 75 80
Ser Thr Tyr Tyr Leu Tyr Val Leu Ala Thr Ala Pro Asn Met Phe Asn
85 90 95
Val Asn Asp Val Leu Gly Ala Tyr Ser Pro His Pro Asp Glu Gln Glu
100 105 110
Val Ser Ala Leu Gly Gly Ile Pro Tyr Ser Gln Ile Tyr Gly Trp Tyr
115 120 125
Arg Val His Phe Gly Val Leu Asp Glu Gln Leu His Arg Asn Arg Gly
130 135 140
Tyr Arg Asp Arg Tyr Tyr Ser Asn Leu Asp Ile Ala Pro Ala Ala Asp
145 150 155 160
Gly Tyr Gly Leu Ala Gly Phe Pro Pro Glu His Arg Ala Trp Arg Glu
165 170 175
Glu Pro Trp Ile His His Ala Pro Pro Gly Cys Gly Asn Ala Pro Arg
180 185 190
Ser Ser Met Ser Asn Thr Cys Asp Glu Lys Thr Gln Ser Leu Gly Val
195 200 205
Lys Phe Leu Asp Glu Tyr Gln Ser Lys Val Lys Arg Gln Ile Phe Ser
210 215 220
Gly Tyr Gln Ser Asp Ile Asp Thr His Asn Arg Ile Lys Asp Glu Leu
225 230 235 240
Gly Ser Lys Gly Asp Gln Asp Pro Gln Ile Ala Ala His Val Ile Ser
245 250 255
Glu Ala Ser Ser Lys Thr Ala Ser Val Leu Gln Trp Ala Pro Lys Gly
260 265 270
Tyr Tyr Thr Leu Ser Thr Asn Leu Val Thr Leu Glu Asn Gly Arg Gln
275 280 285
Leu Ala Val Lys Arg Gln Gly Ile Tyr Tyr Ile Tyr Ala Gln Val Thr
290 295 300
Phe Cys Ser Asn Arg Asp Ala Ala Gly Gln Ala Pro Phe Ile Ala Ser
305 310 315 320
Leu Cys Leu Arg Ser Pro Ser Gly Ser Glu Arg Ile Leu Leu Arg Ala
325 330 335
Ala Asn Thr His Ser Ser Ser Lys Pro Cys Gly Gln Gln Ser Ile His
340 345 350
Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
355 360 365
Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
370 375 380
Gly Leu Leu Lys Leu His His His His His His
385 390 395
Claims (10)
1. a kind of DNA molecular for encoding soluble recombinant protein c TA-CD154, its nucleotide sequence such as SEQ ID NO：1
It is shown.
2. a kind of soluble recombinant protein CTA-CD154, its amino acid sequence such as SEQ ID NO：Shown in 2.
3. the recombinant expression carrier of the DNA molecular containing the soluble recombinant protein CTA-CD154 described in claim 1, turn base
Because of cell line or transgenosis recombinant bacterium.
4. recombinant expression carrier according to claim 3, the DNA molecular described in claim 1 is inserted into Escherichia coli
The recombinant expression carrier of the DNA molecular containing soluble recombinant protein CTA-CD154 is obtained in expression vector.
5. a kind of transgenosis recombinant bacterium, the recombinant bacterium is that the recombinant expression carrier described in claim 3 or 4 is imported into large intestine bar
In bacterium, screening obtains transgenosis recombinant bacterium.
6. the preparation method of the soluble recombinant protein CTA-CD154 described in claim 2, it is characterised in that including following step
Suddenly：
1）According to the inclined preferendum optimum synthesis CTA-CD154 gene orders of e. coli codon, and it is connected with carrier, structure restructuring
Plasmid, the CTA-CD154 gene orders SEQ ID NO as claimed in claim 1：Shown in 1；
2）By recombinant plasmid transformed, induce and obtain soluble recombinant expression protein CTA-CD154 after purification.
7. the soluble recombinant protein CTA-CD154 described in DNA molecular, claim 2 described in claim 1, claim 3
Application of the described recombinant expression carrier, transgenic cell line or transgenosis recombinant bacterium in terms of immunopotentiator is prepared.
8. a kind of immunopotentiator, it is characterised in that the immunopotentiator includes the soluble restructuring egg described in claim 2
White CTA-CD154.
9. application of the immunopotentiator described in claim 8 in terms of vaccine is prepared.
10. application according to claim 9, it is characterised in that the vaccine be inactivated foot-and-mouth disease vaccine, annulus vaccine or
Pseudo- rabies vaccine.
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| CN116019899A (en) * | 2022-12-06 | 2023-04-28 | 江苏省农业科学院 | Mucosal immunopotentiator for improving targeting intestinal DC, and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001074383A1 (en) * | 2000-04-03 | 2001-10-11 | Uab Research Foundation | Chimeric antigen-enterotoxin mucosal immunogens |
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2017
- 2017-10-18 CN CN201710969969.3A patent/CN107502616B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001074383A1 (en) * | 2000-04-03 | 2001-10-11 | Uab Research Foundation | Chimeric antigen-enterotoxin mucosal immunogens |
Non-Patent Citations (4)
| Title |
|---|
| ANNIE GEORGE-CHANDY等: "Cholera Toxin B Subunit as a Carrier Molecule Promotes Antigen Presentation and Increases CD40 and CD86 Expression on Antigen-Presenting Cells", 《INFECTION AND IMMUNITY》 * |
| MARIA CRISTINA GAGLIARDI等: "Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming", 《EUR. J. IMMUNOL.》 * |
| 解晓燕: "霍乱毒素A、B亚单位对HIV-1调节/辅助基因DNA疫苗的佐剂效应研究", 《苏州大学硕士学位论文》 * |
| 郝海霞等: "霍乱毒素作为黏膜佐剂的研究进展", 《中国病原生物学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116019899A (en) * | 2022-12-06 | 2023-04-28 | 江苏省农业科学院 | Mucosal immunopotentiator for improving targeting intestinal DC, and preparation method and application thereof |
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