CN108715607A - Recombinant cholera toxin b protein subunit, PEDV inactivated vaccines and preparation and application - Google Patents
Recombinant cholera toxin b protein subunit, PEDV inactivated vaccines and preparation and application Download PDFInfo
- Publication number
- CN108715607A CN108715607A CN201810325484.5A CN201810325484A CN108715607A CN 108715607 A CN108715607 A CN 108715607A CN 201810325484 A CN201810325484 A CN 201810325484A CN 108715607 A CN108715607 A CN 108715607A
- Authority
- CN
- China
- Prior art keywords
- cholera toxin
- protein
- recombinant cholera
- subunit
- pedv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55544—Bacterial toxins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Plant Pathology (AREA)
- Oncology (AREA)
- Gastroenterology & Hepatology (AREA)
- Communicable Diseases (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Physics & Mathematics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of recombinant proteins, and in particular to a kind of recombinant cholera toxin b protein subunit, PEDV inactivated vaccines and preparation and application.The present invention, through denaturation, purifying, renaturation and crosslinking, will obtain recombinant cholera toxin b protein subunit after the bacterial strain inducing expression for expressing recombinant cholera toxin b protein subunit.The chitosan solution that the recombinant protein is 0.4% with mass fraction, which is sufficiently mixed, is prepared into adjuvant, then is mixed with the PEDV after inactivation, and PEDV inactivated vaccines are made.The adjuvant can not only be obviously improved the local mucosa-immune effect of inactivation PEDV vaccines after oral immunity mouse, it can also reinforce its systemic immunity effect in serum, the vaccine is not pathogenic to experimental animal, and after immune animal, Porcine epidemic diarrhea virus antibody speed that serum and intestinal tissue generate is fast, the horizontal high and duration is long.
Description
Technical field
The present invention relates to a kind of recombinant proteins, and in particular to a kind of recombinant cholera toxin b protein subunit, PEDV inactivated vaccines
And preparation and application.
Background technology
Porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV) is pig epidemic diarrhea
The main pathogen of (Porcine Epidemic Diarrhea, PED), mainly causes chitling road contagious disease, it is characterized in that
Vomiting, diarrhea and dehydration etc..PEDV belongs to the more virales of Buddhist nun, coronaviridae, coronavirus genus, and being a kind of single-stranded positive has
Infective RNA virus, only there are one serotypes.1971 find on Britain's growing and fattening pigs pig farm for the first time, wean to 4~5 week old
Preceding piglet is affected.Then, Europe and Asia is multinational also begins to prevalence.North America starts to break out within 2013, and PEDV becomes the whole world
Popular virus and seriously compromise global aquaculture.
Prevention and control for PEDV, other than carrying out biological prevention and control, the inoculation of vaccine at present is the main of prevention and control PEDV infection
Means.The vaccine being commercialized at present is mainly based on totivirus inactivated vaccine and attenuated vaccine, but to the guarantor of currently a popular strain
It is less desirable to protect effect, tracing it to its cause may be as follows：Effective mucosa-immune, weak poison can not be generated after inactivated vaccine intramuscular injection
It is reduced in enteron aisle stability and proliferative capacity after vaccine cause is weak, the protection of generation is limited.Research for PEDV vaccine potencies
It was found that means best control PEDV are to generate a large amount of mucosal secretory immunoglobulin A in piglet gastrointestinal tract
(secretory immunoglobulin A, sIgA) antibody, but current inactivated vaccine is all made of the mode of injection, Wu Fayou
Activation mucosa-immune in effect ground goes to promote sIgA levels.In addition, some farms also carry out prevention and control by the anti-means raised, although
There is protecting effect in certain degree, but also exist simultaneously the risk of other virus infection, therefore this is mostly used it is not recommended that crossing
Method.For newborn piglet, do not have after birth and ripe gastrointestinal tract immune system also, therefore preferably immune at present
Mode is so that piglet is obtained protection from colostrum by the Immune transform in sow body by the way that prepartal sow is immunized.Though
Research hotspot so is still to the research and development of PEDV vaccines at present, but new generation vaccine is constantly subjected to the restriction of cost and batch production,
So the mucosa-immune effect for improving traditional vaccine is the key that current exploitation PEDV vaccines.
The cholera toxin (cholerae enterotoxin, CT) of comma bacillus is the mucous membrane for being currently research more hot topic
Adjuvant, while being also good immunostimulant, the B subunits (CTB) of wherein CT can recognize that Intestinal epithelial cells film by
Somatic nerves section glycosides fat GM1, and formation compound in combination, and itself does not have toxicity.Therefore using the characteristic of CTB,
There are many researchs to find that the crucial structural proteins amalgamation and expression of itself and virus uses, or mixes and make after expression with key protein
With the two has apparent enhancing immune effect to pass through drop currently, CTB has been widely used in the research of mucosal vaccine
Nose, subcutaneous and muscle injection mode, can be reacted with the mucosa-immune of enhancement antigen specificity.
Escherichia expression system is current most common exogenous protein expression system, have yield is high, the speed of growth is fast,
Advantage easy to operate, at low cost, but the foreign protein in the albumen of the expression of Escherichia coli is more, still needs to be further purified to obtain
Destination protein is obtained, purifying flow is more complex.The physics and chemistry and biology of the purifying Main Basiss related protein of protein at present
Matter difference distinguishes itself and foreign protein, and the soluble protein for no matter being generated in supernatant is in the inclusion body egg of precipitation
In vain, required destination protein can be purified by effective means.CTB albumen after purification using chitosan/TPP from
Sub- cross-linking method preparation processing, treated, and CTB albumen is used cooperatively effectively to promote PEDV traditional vaccines with various adjuvants
Immune effect.
Invention content
In order to overcome prior art PEDV tradition inactivated vaccine mucosa-immune effects poor, the shortcomings of immunization route is single with not
Foot, the primary purpose of the present invention is that providing a kind of preparation method of recombinant cholera toxin b protein subunit.
Another object of the present invention is to provide the recombinant cholera toxin b protein subunit that the above method is prepared, the eggs
The immune effect and improvement immunization route of inactivation PEDV can be effectively promoted in vain.
It is still another object of the present invention to provide the applications of above-mentioned recombinant cholera toxin b protein subunit.
Fourth object of the present invention is to provide a kind of PEDV inactivated vaccines.
The 5th application for being designed to provide above-mentioned PEDV inactivated vaccines of the present invention.
The purpose of the invention is achieved by the following technical solution：
A kind of preparation method of recombinant cholera toxin b protein subunit, comprises the following steps：
(1) it expresses：The bacterial strain inducing expression that recombinant cholera toxin b protein subunit will be expressed divides then by bacterial cell disruption
From collecting broken thalline；
(2) it is denaturalized：Be added denaturing liquid after the broken thalline washing that step (1) is collected, ultrasound to clarifying, then 30~
40 DEG C of 1.5~2.5h of denaturation treatment；Centrifugation, collects supernatant, and filtering and impurity removing obtains clear liquid；
(3) it purifies：Clear liquid made from step (2) is used into ni-sepharose purification, obtains recombinant cholera toxin b subunit purifying egg
In vain；
(4) renaturation：By recombinant cholera toxin b subunit purifying protein obtained in step (3) successively in 8M, 6M, 4M, 2M and
It dialyses in 0M urea liquids, obtains the recombinant cholera toxin b subunit purifying protein after renaturation；
(5) it is crosslinked：Under condition of ice bath, by the recombination cholera poison after renaturation made from chitosan/acetic acid solution and step (4)
Plain B subunits purifying protein is uniformly mixed, and adjustment system pH is 5.3~5.8；Then 20~40 DEG C of 5~15min of stir process,
Sodium tripolyphosphate (TPP) solution is then slowly added dropwise while stirring, wherein the mass ratio of crosslinking agent sodium tripolyphosphate and chitosan
1:5；Continue stirring after being added dropwise to complete and carries out cross-linking reaction；Centrifugation, abandons supernatant, collects precipitation and lays equal stress on molten, obtains recombination cholera poison
Plain B protein subunits；
The amino acid sequence of recombinant cholera toxin b protein subunit described in step (1) such as SEQ ID NO:Shown in 1；
Recombinant cholera toxin b protein subunit described in step (1) is preferably by the b subunit of cholera toxin egg of comma bacillus
White gene carries out the recombinant protein that prokaryotic expression obtains；
The nucleotide sequence of the b subunit of cholera toxin protein gene such as SEQ ID NO:It is in the gene two shown in 2
5 '-BamH I and 3 '-Xho I restriction enzyme sites are added respectively and are obtained in end；
The carrier of the prokaryotic expression is preferably pET-32a carriers；
The bacterial strain of expression recombinant cholera toxin b protein subunit described in step (1) is preferably Escherichia coli；
The condition of induced expression described in step (1) is preferably：The isopropylthio galactolipin of final concentration of 1mmol/L
30 DEG C of induced expression 6h of glycosides (IPTG)；
The concrete operations of washing described in step (2) are preferably：
PBS is added in broken thalline to be resuspended, is then centrifuged for, abandons supernatant；Repeat aforesaid operations 2~4 times；Described
The condition of centrifugation is preferably 12000rpm centrifugations 10min；
Denaturing liquid described in step (2) includes the component of following final concentration：8mol/L urea, 0.1mol/L NaH2PO4,
0.01mol/L Tris, pH=8.0；
The condition of ultrasound described in step (2) is preferably power 200W, working time/intermittent time 5sec/5sec；
The condition of denaturation treatment described in step (2) is preferably 37 DEG C of denaturation treatment 2h；
The condition of centrifugation described in step (2) is preferably 12000rpm centrifugations 10min；
Filtering and impurity removing described in step (2) preferably uses 0.45 μm of membrane filtration impurity；
The concrete operations of ni-sepharose purification described in step (3) are preferably：
Nickel column on clear liquid after rinsing foreign protein using 20mmol/L imidazole elutions, reuses the elution of 500mmol/L imidazoles
Liquid rinses destination protein；
The condition of dialysis described in step (4) is 4 DEG C of 10~12h of dialysis；
The bag filter used during dialysis described in step (4) is preferably carried out activation process：Bag filter adds
Enter 1mM EDTA2Na and 2wt%NaHCO3In, it is put into micro-wave oven moderate heat and boils 15min, take out postcooling, use ddH2O cleanings three
It is secondary, 1mM EDTA are added after cooling2Na repeats to boil, cooling；
The concentration of chitosan is preferably 1mg/mL, the volume point of acetic acid in chitosan/acetic acid solution described in step (5)
Number is preferably 1%, pH=5；
The preparation method of chitosan/acetic acid solution described in step (5) is preferably：
0.1g chitosans are dissolved in the acetum that 100mL volume fractions are 1%, it is 5 to adjust pH value, is filtered to remove not
Molten impurity obtains the chitosan-acetic acid solution of 1mg/mL；
The recombinant cholera toxin b subunit purifying protein and three after chitosan/acetic acid solution, renaturation described in step (5)
The volume ratio of polyphosphate sodium solution is preferably 5:5:4, wherein recombinant cholera toxin b subunit purifying protein after renaturation it is initial
Concentration is preferably 0.5mg/mL, and the initial concentration of sodium tripolyphosphate is preferably 0.25mg/mL；
Uniformly mixed preferably ice bath oscillation mixing 30min described in step (5)；
System pH described in step (5) is preferably 5.5；
The condition of stir process described in step (5) is preferably 25 DEG C of 1200rpm stirrings 10min；
The time for continuing stirring described in step (5) is preferably 30min；
The condition of centrifugation described in step (5) is preferably 15000~20000rpm centrifugations 30min；
The molten concrete operations of weight described in step (5) are preferably：
Use ddH2Precipitation is resuspended O, then uses ultrasound that precipitation is made to dissolve；
The condition of the ultrasound is preferably 50W, working time/intermittent time 5sec/5sec；
A kind of recombinant cholera toxin b protein subunit, is prepared by above-mentioned preparation method；
Application of the recombinant cholera toxin b protein subunit in adjuvant and vaccine preparation field；
A kind of vaccine adjuvant, including above-mentioned recombinant cholera toxin b protein subunit and chitosan；
A kind of PEDV inactivated vaccines, including above-mentioned recombinant cholera toxin b protein subunit；
The preparation method of the PEDV inactivated vaccines, comprises the following steps：
(1) PEDV virus liquids are inactivated, obtains PEDV inactivation of viruses liquid；
(2) above-mentioned recombinant cholera toxin b protein subunit is added in chitosan solution, mixing obtains adjuvant；Then with
PEDV inactivation of viruses liquid mixings, obtain PEDV inactivated vaccines；
The titre of PEDV virus liquids described in step (1) is preferably 10-4TCID50；
The concrete operations of inactivation described in step (1) are preferably：Under aseptic condition, formaldehyde is added in PEDV virus liquids
Solution and mixing, 37 DEG C of inactivation 48h；After inactivation be added hypo solution in and formaldehyde；
The initial mass score of the formalin is preferably 40%；
End, mass fraction was preferably 0.3% after the formalin is added in PEDV virus liquids；
The dosage of the hypo solution is preferably PEDV virus liquids and the mixed volume of formalin；
The concentration of recombinant cholera toxin b protein subunit described in step (2) is preferably 0.5mg/mL；
The initial mass score of chitosan described in step (2) is preferably 0.4%；
The volume ratio of recombinant cholera toxin b protein subunit and chitosan solution described in step (2) is preferably 1:10；
The volume ratio of adjuvant and PEDV inactivation of viruses liquid described in step (2) is preferably 1:1；
The present invention has the following advantages and effects with respect to the prior art：
(1) present invention expresses recombinant cholera toxin b protein subunit using escherichia expression system, and the one side present invention carries
The purification process of confession prepares albumen, and low production cost is suitable for large-scale industrial production, and on the other hand, which has good
Immunostimulating effect, and can be suitably used for oral immunity.
(2) after the present invention is using chitosan/TPP ionic cross-linkings crosslinking recombinant cholera toxin b protein subunit, there is sustained release
Effect, can be used as adjuvant, with mass fraction be 0.4% chitosan adjuvant be used in mixed way, can as Mucosal Adjuvants
Preferably play the effect of adjuvant.
(3) inactivated vaccine produced by the present invention has good safety, after animal is immunized using oral immunization route,
It is suitble to farm's mass immunization, the PEDV antibody speed of generation is fast, level is high and the duration is long.
(4) recombinant cholera toxin b protein subunit produced by the present invention has good development prospect as adjuvant, not only may be used
To be used cooperatively with a kind of inactivation of viruses, moreover it is possible to and a variety of inactivation of viruses prepare into multi-joint seedling.
(5) present invention is addresses deficiency of traditional PEDV inactivated vaccines on mucosa-immune, by expressing mucous membrane assistant
Agent molecule is used in combination with conventional inactivated vaccine, makes up mutually, from the general immunity two in the local immunity and serum of mucous membrane
A aspect is started with, and control effect is immunized in the entirety for promoting existing PEDV vaccines.
Description of the drawings
Fig. 1 is the SDS-PAGE (coomassie brilliant blue staining) of the destination protein of Bacillus coli expression in the embodiment of the present invention 2
Electrophoretogram；Wherein, M：Albumen marker；1：The expression (full bacterium is unbroken) of pET-32a-CTB/BL-21 (DE3) pLysS bacterial strains；
2：The soluble expression (supernatant after bacterial cell disruption) of pET-32a-CTB/BL-21 (DE3) pLysS bacterial strains；3：pET-32a-
The non-solubility expression (precipitation after bacterial cell disruption) of CTB/BL-21 (DE3) pLysS bacterial strains；4：Empty carrier pET-32a/BL-21
(DE3) pLysS bacterial strains handle the control after induction with similarity condition；Left side of the digital is marker sizes, unit KDa, in figure
Arrow meaning is the destination protein of doubtful amalgamation and expression compared with the control.
Fig. 2 be in the embodiment of the present invention 2 before purification after destination protein SDS-PAGE (coomassie brilliant blue staining) electrophoresis
Figure；Wherein, M：Albumen marker；1：Albumen (precipitation of bacterial cell disruption, by the albumen of denaturation renaturation-after purification) after purification；
2：Unpurified albumen (precipitation after bacterial cell disruption is dissolved in denaturing liquid).
Fig. 3 is the Western blot electrophoretograms of the destination protein of Bacillus coli expression in the embodiment of the present invention 2；Wherein,
M：Albumen marker；1：The soluble expression of pET-32a-CTB/BL-21 (DE3) pLysS bacterial strains is (upper after bacterial cell disruption
Clearly)；2：The non-solubility expression (precipitation after bacterial cell disruption) of pET-32a-CTB/BL-21 (DE3) pLysS bacterial strains；3：pET-
The expression (full bacterium is unbroken) of 32a-CTB/BL-21 (DE3) pLysS bacterial strains；4：Empty carrier pET-32a/BL-21 (DE3) pLysS
Bacterial strain handles the control after induction with similarity condition.Left side of the digital is marker sizes, unit KDa, band and Fig. 1 knots in figure
Fruit is compared, and the destination protein of amalgamation and expression is determined that it is.
Fig. 4 is the interpretation of result figure of the destination protein slow release effect after being crosslinked in the embodiment of the present invention 2；Wherein, ordinate
The albumen concentration measured is represented, abscissa is the time of release.
Fig. 5 is that inactivated vaccine is special by PEDV in different time serum after oral immunity mouse in effect example of the present invention
The horizontal result analysis chart of specific IgG antibody；Wherein totally three groups：It is (real that respectively PEDV inactivates+0.4% chitosan+CTB crosslinking agents
Apply example 3) group, PEDV inactivations+0.4% chitosan of mass fraction (comparative example) group and PBS control group；Ordinate represent with
The sample absorbance of 450nm wavelength detectings；28d, 35d, 42d, 49d, 56d respectively represent one exempt from after 28 days, 35 days, 42 days, 49
It, 56 days.
Fig. 6 is that inactivated vaccine passes through in different time Intestinal Mucosal Tissues after oral immunity mouse in effect example of the present invention
The horizontal result analysis chart of PEDV Specific IgA antibodies；Wherein totally three groups：+ 0.4% chitosan+CTB crosslinkings of respectively PEDV inactivations
Agent (embodiment 3) group, PEDV inactivations+0.4% chitosan of mass fraction (comparative example) group and PBS control group；Ordinate generation
Table is with the sample absorbance of 450nm wavelength detectings.42d, 49d, 56d respectively represent one and exempt from 42 days, 49 days, 56 days afterwards.
Specific implementation mode
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
1mg/ml chitosan/acetic acid solution：It is 1% acetum (1ml to weigh a certain amount of chitosan to be dissolved in volume fraction
Glacial acetic acid is dissolved in 100ml ddH2O in), it is made into 1mg/ml, is put into magnetic agitation in conical flask (1200rpm), adjusting pH value is
5, successively with 0.45 μm and 0.22 μm of filter filtering, save backup；
The chitosan solution that mass fraction is 0.4%：It is that 1% acetic acid is molten to weigh a certain amount of chitosan to be dissolved in volume fraction
Liquid obtains the chitosan solution that mass fraction is 0.4%；
Sodium tripolyphosphate TPP solution：It weighs a certain amount of TPP to be dissolved in deionized water, is made into 0.25mg/ml, adjust pH
Value is 5；Successively with 0.45 μm and 0.22 μm of filter filtering, save backup；
Measure albumen concentration：Use BCA kit measurement albumen concentration；
PEDV GD-A strains, application No. is " 201310453489.3 ", entitled " the one plant of pig epidemics of application
It is disclosed in the patent of diarrhea virus and its cultural method and application "；
The structure of 1 soluble recombinant protein expression vector of embodiment
(1) acquisition of CTB protein gene
Based on the sequence of b subunit of cholera toxin albumen (CTB) albumen in ncbi database, after comparison, select
CTB protein amino acid sequences, amino acid is as follows, and commission Beijing six directions Hua Da Gene science company carries out gene chemical synthesis, base
Restriction enzyme site (BamH I and Xho I) is added in both ends when because of synthesis, and the nucleotide sequence being added after restriction enzyme site is as follows, closes
Unnamed gene after is CTB-gene.
Amino acid sequence：
MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQ
HIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN.
Nucleotide sequence：
CGCGGATCCATGATTAAATTAAAATTTGGTGTTTTTTTTACAGTTTTACTATCTTCAGCATATGCACAT
GGAACACCTCAAAATATTACTGATTTGTGTGCAGAATACCACAACACACAAATACATACGCTAAATGATAAGATATT
TTCGTATACAGAATCTCTAGCTGGAAAAAGAGAGATGGCTATCATTACTTTTAAGAATGGTGCAACTTTTCAAGTAG
AAGTACCAGGTAGTCAACATATAGATTCACAAAAAAAAGCGATTGAAAGGATGAAGGATACCCTGAGGATTGCATAT
CTTACTGAAGCTAAAGTCGAAAAGTTATGTGTATGGAATAATAAAACGCCTCATGCGATTGCCGCAATTAGTATGGC
AAATTAACTCGAGCGG
(2) structure of recombinant expression carrier plasmid
CTB-gene and pET-32a is subjected to double digestion with two kinds of restriction enzymes of BamH I and Xho I simultaneously, then
Recovery purifying, 16 DEG C of connection 15h；Connection product is transformed into E.coli DH5 α competent cells, is coated with the LB containing ampicillin
Tablet, 37 DEG C are incubated overnight；Single bacterium colony on picking tablet, in the LB liquid medium culture 4h containing ampicillin, then
It carries out bacterium solution PCR identifications and Huada gene company is sent to be sequenced；Correct bacterium solution will be sequenced according to volume ratio 1:1000 are added to newly
In the fresh LB containing ampicillin, overnight incubation extracts plasmid and preserves for use later, and recombinant plasmid is named as pET-32a-
CTB。
(3) structure of recombinant strains
Recombinant plasmid pET-32a-CTB is converted into Escherichia coli BL-21 (DE3) pLysS, is applied to containing ampicillin
LB tablets, 37 DEG C are incubated overnight, and obtain recombinant strains pET-32a-CTB/BL-21 (DE3) pLysS bacterial strains.
The preparation of 2 recombinant cholera toxin b protein subunit of embodiment
(1) it expresses：PET-32a-CTB/BL-21 (DE3) pLysS bacterial strains made from embodiment 1 are taken out from -20 DEG C, with
Volume ratio 1:37 DEG C in 1000 LB liquid mediums of the access containing amicillin resistance, 220rpm shaker overnight cultures obtain
To seed liquor；Seed liquor is with volume ratio 1:100 LB liquid mediums of the access containing amicillin resistance, 37 DEG C of temperature,
4h or so, culture to cell concentration A are cultivated on 220rpm shaking tables600Reach 0.6；It is added final concentration of 1mM's in bacterium solution
IPTG carries out induction 6h with 150rpm rotating speeds, collects thalline, use PBS with volume ratio 1 at 30 DEG C of inducing temperature:10 are resuspended
Thalline after resuspension is crushed (condition by thalline using Ultrasonic Cell Disruptor：Power 200W, working time/intermittent time=5sec/
5sec), whether ultrasonic number is in limpid state according to broken rear bacterium solution；Then part bacteria liquid sample is directly taken to carry out SDS-PAGE
Analysis, the results are shown in Figure 1, it will be seen from figure 1 that empty carrier pET-32a/BL-21 (DE3) pLysS bacterial strain phases with swimming lane 4
Than, 1 and 3 swimming lanes obviously have more a protein band at about 30kDa, are consistent with expection, show CTB albumen energy successful expressions,
And major part is stored in 3 swimming lanes precipitation, is the inclusion body protein of insolubility；By remaining broken 4 DEG C of 12000rpm of bacterium solution
10min is centrifuged, supernatant precipitation is collected, carries out SDS-PAGE analyses respectively, it is final to determine that destination protein is sediment fraction (figure
1)；
(2) it is denaturalized：(1) precipitation obtained is resuspended in PBS, 12000rpm centrifuges 10min, abandons supernatant；PBS is used again
It is resuspended, repeated centrifugation abandons supernatant；It is washed 3 times with PBS in total；12000rpm centrifuges 10min after washing, precipitation is collected, with denaturation
Liquid (8mol/L urea, 0.1mol/L NaH2PO4, 0.01mol/L Tris, pH=8.0) precipitation is resuspended after, ultrasonic (power
200W, working time/intermittent time=5sec/5sec), until clarification (precipitation dissolving)；37 DEG C of shaking tables are then placed in, 150rpm becomes
Property processing 2h；Supernatant is collected by centrifugation in 12000rpm, and 0.45 μm of membrane filtration is used in combination, and standby sample carries out SDS-PAGE, remaining -20 DEG C guarantors
It deposits.
(3) it purifies：Albumen is purified using Ni-NTA affinity columns, is specifically comprised the following steps：
1. upper prop and pretreatment：Nickel column packing is filled in pillar, ddH is first used2O crosses column washing, then uses 500mmol/L
Imidazole elution crosses column, finally crosses column with 20mmol/L imidazole elutions；
2. loading：Supernatant made from step (2) is added in pillar, column 2 times are crossed；
3. rinsing foreign protein：Column, which is crossed, with 20mmol/L imidazole elutions rinses foreign protein；
4. eluting：Column is crossed with 500mmol/L imidazole elutions, obtains recombinant cholera toxin b subunit purifying protein；
(4) renaturation：The albumen of step (3) after purification carries out gradient dialysis renaturation, bag filter with different renaturation buffers
With final concentration 1mM EDTA2Na and final concentration 2wt%NaHCO3It impregnates, is put into microwave ingle 15min and boils, take out postcooling,
Use ddH2O is cleaned three times, and final concentration 1mM EDTA are added2Na repeats to boil, and the recombinant cholera toxin b of renaturation will be needed after cooling
Subunit purifying protein, which is transferred in bag filter, is put into large beaker, and the PBS solution of 8M urea is then added, is placed in 4 DEG C of refrigerator 11h, takes
Go out to discard the PBS solution of 8M urea；The PBS solution for adding 6M urea enters in beaker, repeats 4 DEG C of 11h, and taking-up discards 6M urine
The PBS solution of element；And so on, renaturation solution is replaced successively according to 8M, 6M, 4M, 2M, 0M urea PBS solution, finally takes out dialysis
Liquid in bag takes sample segment to carry out SDS-PAGE and western-blot detections.As a result as shown in Figures 2 and 3, by Fig. 2
It can be seen that, compare non-purifying protein (swimming lane 1), greatly improved by purity of protein after this research denaturation-purifying-refolding method
(swimming lane 2), while further confirming that CTB albumen can be with His tag monoclonal antibody specific recognitions by western-blot.
(5) it is crosslinked：Under condition of ice bath, by the recombinant cholera toxin b subunit purifying protein (concentration after 5mL steps (4) renaturation
For 0.5mg/mL) 30min is mixed with the oscillation of the chitosan/acetic acid solution ice bath of 5mL 1mg/mL, adjustment system pH is 5.5；So
25 DEG C of 1200rpm stir process 10min of room temperature magnetic stirring apparatus are used afterwards；Then slowly dripped thereto with syringe while stirring
Add 4mL 0.25mg/mL TPP, wherein chitosan is 5 with TPP mass ratioes:1；Continue 25 DEG C of stirring 30min after being added dropwise to complete,
15000rpm, 4 DEG C of centrifugation 30min, abandon supernatant, collect precipitation, precipitate through ddH2O is washed three times, then uses ddH again2O will be precipitated
It is resuspended, sonic oscillation (50W, working time/intermittent time 5sec/5sec) makes precipitation dissolve, obtains recombinant cholera toxin b subunit
Albumen, 4 DEG C of preservations.
By the recombinant cholera toxin b protein subunit after crosslinking with volume ratio 1:1, which is added to the shell that mass fraction is 0.4%, gathers
In sugar juice, fully vibrate mixing, be placed in 37 DEG C of shaking tables 100rpm and vibrate, in different times (0h, 6h, 12h, 18h,
For 24 hours, 30h, 36h, 42h and 48h) it takes out, 30min is centrifuged with 15000rpm after taking-up, supernatant is taken to use spectrophotometric determination
Protein content.As a result see Fig. 4.It can be seen that preceding 18h slow releases, stablize in 24~48h and discharge, may persist to 48h.
The preparation of 3 recombinant cholera toxin b protein subunit of embodiment
(1) it expresses：PET-32a-CTB/BL-21 (DE3) pLysS bacterial strains made from embodiment 1 are taken out from -20 DEG C, with
Volume ratio 1:37 DEG C in 1000 LB liquid mediums of the access containing amicillin resistance, 220rpm shaker overnight cultures obtain
To seed liquor；Seed liquor is with volume ratio 1:100 LB liquid mediums of the access containing amicillin resistance, 37 DEG C of temperature,
4h or so, culture to cell concentration A are cultivated on 220rpm shaking tables600Reach 0.6；It is added final concentration of 1mM's in bacterium solution
IPTG carries out induction 6h with 150rpm rotating speeds, collects thalline, use PBS with volume ratio 1 at 30 DEG C of inducing temperature:10 are resuspended
Thalline after resuspension is crushed (condition by thalline using Ultrasonic Cell Disruptor：Power 200W, working time/intermittent time=5sec/
5sec), whether ultrasonic number is in limpid state according to broken rear bacterium solution；Then 4 DEG C of 12000rpm centrifugations of broken bacterium solution
10min collects precipitation；
(2) it is denaturalized：(1) precipitation obtained is resuspended in PBS, 12000rpm centrifuges 10min, abandons supernatant；PBS is used again
It is resuspended, repeated centrifugation abandons supernatant；It is washed 3 times with PBS in total；12000rpm centrifuges 10min after washing, precipitation is collected, with denaturation
Liquid (8mol/L urea, 0.1mol/L NaH2PO4, 0.01mol/L Tris, pH=8.0) precipitation is resuspended after, ultrasonic (power
200W, working time/intermittent time=5sec/5sec), until clarification (precipitation dissolving)；40 DEG C of shaking tables are then placed in, 150rpm becomes
Property processing 1.5h；Supernatant is collected by centrifugation in 12000rpm, and 0.45 μm of membrane filtration is used in combination；
(3) it purifies：Albumen is purified using Ni-NTA affinity columns, is specifically comprised the following steps：
1. upper prop and pretreatment：Nickel column packing is filled in pillar, ddH is first used2O crosses column washing, then uses 500mmol/L
Imidazole elution crosses column, finally crosses column with 20mmol/L imidazole elutions；
2. loading：Supernatant made from step (2) is added in pillar, column 2 times are crossed；
3. rinsing foreign protein：Column, which is crossed, with 20mmol/L imidazole elutions rinses foreign protein；
4. eluting：Column is crossed with 500mmol/L imidazole elutions, obtains recombinant cholera toxin b subunit purifying protein；
(4) renaturation：The albumen of step (3) after purification carries out gradient dialysis renaturation, bag filter with different renaturation buffers
With final concentration 1mM EDTA2Na and final concentration 2wt%NaHCO3It impregnates, is put into microwave ingle 15min and boils, take out postcooling,
Use ddH2O is cleaned three times, and final concentration 1mM EDTA are added2Na repeats to boil, and the recombinant cholera toxin b of renaturation will be needed after cooling
Subunit purifying protein, which is transferred in bag filter, is put into large beaker, and the PBS solution of 8M urea is then added, is placed in 4 DEG C of refrigerator 10h, takes
Go out to discard the PBS solution of 8M urea；The PBS solution for adding 6M urea enters in beaker, repeats 4 DEG C of 10h, and taking-up discards 6M urine
The PBS solution of element；And so on, renaturation solution is replaced successively according to 8M, 6M, 4M, 2M, 0M urea PBS solution, finally takes out dialysis
Liquid in bag；
(5) it is crosslinked：Under condition of ice bath, by the recombinant cholera toxin b subunit purifying protein (concentration after 5mL steps (4) renaturation
For 0.5mg/mL) 30min is mixed with the oscillation of the chitosan/acetic acid solution ice bath of 5mL 1mg/mL, adjustment system pH is 5.3；So
40 DEG C of 1200rpm stir process 5min of room temperature magnetic stirring apparatus are used afterwards；Then slowly dripped thereto with syringe while stirring
Add 4mL 0.25mg/mL TPP, wherein chitosan is 5 with TPP mass ratioes:1；Continue 25 DEG C of stirring 30min after being added dropwise to complete,
20000rpm, 4 DEG C of centrifugation 30min, abandon supernatant, collect precipitation, precipitate through ddH2O is washed three times, then uses ddH again2O will be precipitated
It is resuspended, sonic oscillation (50W, working time/intermittent time 5sec/5sec) makes precipitation dissolve, obtains recombinant cholera toxin b subunit
Albumen, 4 DEG C of preservations.
By the recombinant cholera toxin b protein subunit after crosslinking with volume ratio 1:1, which is added to the shell that mass fraction is 0.4%, gathers
In sugar juice, fully vibrate mixing, be placed in 37 DEG C of shaking tables 100rpm and vibrate, in different times (0h, 6h, 12h, 18h,
For 24 hours, 30h, 36h, 42h and 48h) it takes out, 30min is centrifuged with 15000rpm after taking-up, supernatant is taken to use spectrophotometric determination
Protein content.As a result with embodiment 2, preceding 18h slow releases are stablized in 24~48h and are discharged, may persist to 48h.
The preparation of 4 recombinant cholera toxin b protein subunit of embodiment
(1) it expresses：PET-32a-CTB/BL-21 (DE3) pLysS bacterial strains made from embodiment 1 are taken out from -20 DEG C, with
Volume ratio 1:37 DEG C in 1000 LB liquid mediums of the access containing amicillin resistance, 220rpm shaker overnight cultures obtain
To seed liquor；Seed liquor is with volume ratio 1:100 LB liquid mediums of the access containing amicillin resistance, 37 DEG C of temperature,
4h or so, culture to cell concentration A are cultivated on 220rpm shaking tables600Reach 0.6；It is added final concentration of 1mM's in bacterium solution
IPTG carries out induction 6h with 150rpm rotating speeds, collects thalline, use PBS with volume ratio 1 at 30 DEG C of inducing temperature:10 are resuspended
Thalline after resuspension is crushed (condition by thalline using Ultrasonic Cell Disruptor：Power 200W, working time/intermittent time=5sec/
5sec), whether ultrasonic number is in limpid state according to broken rear bacterium solution；Then 4 DEG C of 12000rpm centrifugations of broken bacterium solution
10min collects precipitation；
(2) it is denaturalized：(1) precipitation obtained is resuspended in PBS, 12000rpm centrifuges 10min, abandons supernatant；PBS is used again
It is resuspended, repeated centrifugation abandons supernatant；It is washed 3 times with PBS in total；12000rpm centrifuges 10min after washing, precipitation is collected, with denaturation
Liquid (8mol/L urea, 0.1mol/L NaH2PO4, 0.01mol/L Tris, pH=8.0) precipitation is resuspended after, ultrasonic (power
200W, working time/intermittent time=5sec/5sec), until clarification (precipitation dissolving)；30 DEG C of shaking tables are then placed in, 150rpm becomes
Property processing 1.5h；Supernatant is collected by centrifugation in 12000rpm, and 0.45 μm of membrane filtration is used in combination；
(3) it purifies：Albumen is purified using Ni-NTA affinity columns, is specifically comprised the following steps：
1. upper prop and pretreatment：Nickel column packing is filled in pillar, ddH is first used2O crosses column washing, then uses 500mmol/L
Imidazole elution crosses column, finally crosses column with 20mmol/L imidazole elutions；
2. loading：Supernatant made from step (2) is added in pillar, column 2 times are crossed；
3. rinsing foreign protein：Column, which is crossed, with 20mmol/L imidazole elutions rinses foreign protein；
4. eluting：Column is crossed with 500mmol/L imidazole elutions, obtains recombinant cholera toxin b subunit purifying protein；
(4) renaturation：The albumen of step (3) after purification carries out gradient dialysis renaturation, bag filter with different renaturation buffers
With final concentration 1mM EDTA2Na and final concentration 2wt%NaHCO3It impregnates, is put into microwave ingle 15min and boils, take out postcooling,
Use ddH2O is cleaned three times, and final concentration 1mM EDTA are added2Na repeats to boil, and the recombinant cholera toxin b of renaturation will be needed after cooling
Subunit purifying protein, which is transferred in bag filter, is put into large beaker, and the PBS solution of 8M urea is then added, is placed in 4 DEG C of refrigerator 12h, takes
Go out to discard the PBS solution of 8M urea；The PBS solution for adding 6M urea enters in beaker, repeats 4 DEG C of 12h, and taking-up discards 6M urine
The PBS solution of element；And so on, renaturation solution is replaced successively according to 8M, 6M, 4M, 2M, 0M urea PBS solution, finally takes out dialysis
Liquid in bag；
(5) it is crosslinked：Under condition of ice bath, by the recombinant cholera toxin b subunit purifying protein (concentration after 5mL steps (4) renaturation
For 0.5mg/mL) 30min is mixed with the oscillation of the chitosan/acetic acid solution ice bath of 5mL 1mg/mL, adjustment system pH is 5.8；So
20 DEG C of 1200rpm stir process 15min of room temperature magnetic stirring apparatus are used afterwards；Then slowly dripped thereto with syringe while stirring
Add 4mL 0.25mg/mL TPP, wherein chitosan is 5 with TPP mass ratioes:1；Continue 25 DEG C of stirring 30min after being added dropwise to complete,
18000rpm, 4 DEG C of centrifugation 30min, abandon supernatant, collect precipitation, precipitate through ddH2O is washed three times, then uses ddH again2O will be precipitated
It is resuspended, sonic oscillation (50W, working time/intermittent time 5sec/5sec) makes precipitation dissolve, obtains recombinant cholera toxin b subunit
Albumen, 4 DEG C of preservations.
By the recombinant cholera toxin b protein subunit after crosslinking with volume ratio 1:1, which is added to the shell that mass fraction is 0.4%, gathers
In sugar juice, fully vibrate mixing, be placed in 37 DEG C of shaking tables 100rpm and vibrate, in different times (0h, 6h, 12h, 18h,
For 24 hours, 30h, 36h, 42h and 48h) it takes out, 30min is centrifuged with 15000rpm after taking-up, supernatant is taken to use spectrophotometric determination
Protein content.As a result with embodiment 2, preceding 18h slow releases are stablized in 24~48h and are discharged, may persist to 48h.
The preparation of 5 PEDV inactivated vaccines of embodiment
(1) inactivation of PEDV
By PEDV GD-A strains after cell culture expands, venom (titre 10 is normally withdrawn-4TCID50), sterile item
It is added in the formalin to PEDV virus liquids that initial mass score is 40% under part, until formaldehyde final volume score is 0.3%, shakes
It is even；It is then placed in 37 DEG C of incubators, inactivation 48h (shaking up at regular intervals)；Isometric sodium thiosulfate is added after inactivation
In solution and formaldehyde, cell experiment verify inactivation of viruses effect；
(2) PEDV oral vaccines are prepared
By the recombinant cholera toxin b protein subunit of a concentration of 0.5mg/mL made from embodiment 2 with volume ratio 1:1 is added to
In the chitosan adjuvant that mass fraction is 0.4%, mixing is fully vibrated, adjuvant is obtained；Then with PEDV inactivation of viruses liquid with body
Product ratio 1:1 mixing, obtains PEDV inactivated vaccines.
Comparative example
(1) inactivation of PEDV：With embodiment 3；
(2) vaccine is prepared:By mass fraction be 0.4% chitosan adjuvant and PEDV inactivation of viruses liquid with volume ratio 1:1
Mixing, obtains vaccine.
The mouse immuning test of effect example PEDV inactivated vaccines
By random point of the BALB/c mouse (being purchased from Guangdong Medical Lab Animal Center) of 27 6 week old PEDV negative antibodies
It is 3 groups, every group 9.Every group is all made of oral mode and is immunized, and is immunized altogether twice, every minor tick one week.First group：It is every small
PEDV inactivated vaccines made from 100 μ L embodiments 3 are immunized in mouse；Second group：Made from every 100 μ L comparative example of mouse immune
Vaccine；Third group：Every mouse immune sterile PBS of 100 μ L (control group).
The 5th week after second week (experiment 4th week, the 28th day) is immunized to second after immune for the second time (experiment the 8th week,
56th day), it selects 3 mouse at random weekly and carries out orbital vein blood sampling, the serum of precipitation uses PEDV prepared by this laboratory
ELISA detects IgG kits and carries out antibody test.Testing result is shown in Fig. 5.
The 6th week (the 42nd day) is tested to experiment the 8th week (the 56th day), weekly after having acquired blood, selects 3 at random
Mouse is euthanized, and acquires intestinal tissue, and antibody test is carried out using PEDV ELISA detection IgA kits after grinding.Detection
As a result see Fig. 6.
As a result it shows：PEDV inactivated vaccines made from embodiment 3 can be quick by oral immunity approach after mouse is immunized
Generate the antibody (being respectively IgG antibody and IgA antibody) for PEDV in serum and intestinal tissue, and with made from comparative example
Vaccine more rapid, higher level compared to the antibody generated.
Therefore PEDV inactivated vaccines produced by the present invention fully have activated mouse vivo immuning system, from the serum of acquisition
From the point of view of, blood-serum P EDV specific IgG antibodies are obviously improved, in experiment periods the 35th day with mass fraction is used alone
0.4% chitosan solution has apparent difference as the inactivation PEDV groups of adjuvant, then peaks within the 49th day in experiment periods,
This shows that the vaccine effectively has activated systemic immunity.For IgA antibody in intestinal tissue, equally reach within the 49th day in experiment periods
Peak, and 0.4% chitosan solution is used alone and is only carried on a small quantity with respect to PBS control group as the inactivation PEDV groups of adjuvant
It rises, therefore the immunoenhancement result of recombinant cholera toxin b protein subunit produced by the present invention is obviously.Finally, this hair
Bright PEDV inactivated vaccines obtained can promote IgG antibody and IgA antibody respectively by oral immunity mouse in serum and enteron aisle
Level, effectively activate systemic immunity and enteron aisle part mucosa-immune, play stronger protecting effect and disease-resistant toxic effect
Fruit.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>Recombinant cholera toxin b protein subunit, PEDV inactivated vaccines and preparation and application
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 124
<212> PRT
<213> Artificial Sequence
<220>
<223>B subunit of cholera toxin protein amino acid sequence
<400> 1
Met Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser Ser
1 5 10 15
Ala Tyr Ala His Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu
20 25 30
Tyr His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr
35 40 45
Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys
50 55 60
Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp
65 70 75 80
Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala
85 90 95
Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys
100 105 110
Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn
115 120
<210> 2
<211> 393
<212> DNA
<213> Artificial Sequence
<220>
<223>B subunit of cholera toxin protein gene nucleotide sequence
<400> 2
cgcggatcca tgattaaatt aaaatttggt gtttttttta cagttttact atcttcagca 60
tatgcacatg gaacacctca aaatattact gatttgtgtg cagaatacca caacacacaa 120
atacatacgc taaatgataa gatattttcg tatacagaat ctctagctgg aaaaagagag 180
atggctatca ttacttttaa gaatggtgca acttttcaag tagaagtacc aggtagtcaa 240
catatagatt cacaaaaaaa agcgattgaa aggatgaagg ataccctgag gattgcatat 300
cttactgaag ctaaagtcga aaagttatgt gtatggaata ataaaacgcc tcatgcgatt 360
gccgcaatta gtatggcaaa ttaactcgag cgg 393
Claims (10)
1. a kind of preparation method of recombinant cholera toxin b protein subunit, it is characterised in that comprise the following steps：
(1) it expresses：The bacterial strain inducing expression for expressing recombinant cholera toxin b protein subunit is detached then by bacterial cell disruption, is received
Collect broken thalline；
(2) it is denaturalized：Denaturing liquid is added after the broken thalline washing that step (1) is collected, ultrasound is extremely clarified, then 30~40 DEG C
1.5~2.5h of denaturation treatment；Centrifugation, collects supernatant, and filtering and impurity removing obtains clear liquid；
(3) it purifies：Clear liquid made from step (2) is used into ni-sepharose purification, obtains recombinant cholera toxin b subunit purifying protein；
(4) renaturation：Recombinant cholera toxin b subunit purifying protein obtained in step (3) is urinated in 8M, 6M, 4M, 2M and 0M successively
It dialyses in plain solution, obtains the recombinant cholera toxin b subunit purifying protein after renaturation；
(5) it is crosslinked：Under condition of ice bath, by the recombinant cholera toxin b after renaturation made from chitosan/acetic acid solution and step (4)
Subunit purifying protein is uniformly mixed, and adjustment system pH is 5.3~5.8；Then 20~40 DEG C of 5~15min of stir process, then
Sodium tripolyphosphate solution is slowly added dropwise while stirring, wherein the mass ratio 1 of crosslinking agent sodium tripolyphosphate and chitosan:5；It is added dropwise
Continue stirring after the completion and carries out cross-linking reaction；Centrifugation, abandons supernatant, collects precipitation and lays equal stress on molten, obtains recombinant cholera toxin b subunit egg
In vain.
2. the preparation method of recombinant cholera toxin b protein subunit according to claim 1, it is characterised in that：
The amino acid sequence of recombinant cholera toxin b protein subunit described in step (1) such as SEQ ID NO:Shown in 1.
3. the preparation method of recombinant cholera toxin b protein subunit according to claim 1, it is characterised in that：
Denaturing liquid described in step (2) includes the component of following final concentration：8mol/L urea, 0.1mol/L NaH2PO4,
0.01mol/L Tris, pH=8.0.
4. the preparation method of recombinant cholera toxin b protein subunit according to claim 1, it is characterised in that：
The condition of denaturation treatment described in step (2) is 37 DEG C of denaturation treatment 2h.
5. the preparation method of recombinant cholera toxin b protein subunit according to claim 1, it is characterised in that：
The concrete operations of ni-sepharose purification described in step (3) are：
Nickel column on clear liquid 2 reuses 500mmol/L imidazole elutions after rinsing foreign protein using 20mmol/L imidazole elutions
Rinse destination protein.
6. the preparation method of recombinant cholera toxin b protein subunit according to claim 1, it is characterised in that：
Chitosan/acetic acid solution, recombinant cholera toxin b subunit purifying protein described in step (5) and sodium tripolyphosphate solution
Volume ratio be 5:5:4, wherein the initial concentration of recombinant cholera toxin b subunit purifying protein is 0.5mg/mL, sodium tripolyphosphate
Initial concentration be 0.25mg/mL.
7. a kind of recombinant cholera toxin b protein subunit, it is characterised in that pass through claim 1~6 any one of them preparation side
Method is prepared.
8. application of the recombinant cholera toxin b protein subunit in adjuvant and vaccine preparation field described in claim 7.
9. a kind of PEDV inactivated vaccines, it is characterised in that include recombinant cholera toxin b protein subunit according to any one of claims 8.
10. the preparation method of the PEDV inactivated vaccines described in claim 9, it is characterised in that comprise the following steps：
(1) PEDV virus liquids are inactivated, obtains PEDV inactivation of viruses liquid；
(2) above-mentioned recombinant cholera toxin b protein subunit is added in chitosan solution, mixing obtains adjuvant；Then with PEDV
Inactivation of viruses liquid mixing, obtains PEDV inactivated vaccines.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810325484.5A CN108715607B (en) | 2018-04-12 | 2018-04-12 | Recombinant cholera toxin B subunit protein, PEDV inactivated vaccine, preparation and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810325484.5A CN108715607B (en) | 2018-04-12 | 2018-04-12 | Recombinant cholera toxin B subunit protein, PEDV inactivated vaccine, preparation and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108715607A true CN108715607A (en) | 2018-10-30 |
| CN108715607B CN108715607B (en) | 2021-12-10 |
Family
ID=63898952
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810325484.5A Active CN108715607B (en) | 2018-04-12 | 2018-04-12 | Recombinant cholera toxin B subunit protein, PEDV inactivated vaccine, preparation and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108715607B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115044524A (en) * | 2022-06-13 | 2022-09-13 | 山东大学 | Genetic engineering recombinant lactic acid bacteria and application thereof in aspect of resisting vibrio mimicus infection |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997020576A1 (en) * | 1995-12-07 | 1997-06-12 | Danbiosyst Uk Limited | Vaccine compositions for intranasal administration comprising chitosan and use thereof |
| CN106928373A (en) * | 2017-03-17 | 2017-07-07 | 青岛明勤生物科技有限公司 | A kind of porcine mycoplasmal pneumonia multi-epitope mucosal vaccine |
| CN106946984A (en) * | 2017-03-17 | 2017-07-14 | 中国科学院武汉物理与数学研究所 | A kind of preparation method for the b subunit of cholera toxin albumen for having bioactivity |
-
2018
- 2018-04-12 CN CN201810325484.5A patent/CN108715607B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997020576A1 (en) * | 1995-12-07 | 1997-06-12 | Danbiosyst Uk Limited | Vaccine compositions for intranasal administration comprising chitosan and use thereof |
| CN106928373A (en) * | 2017-03-17 | 2017-07-07 | 青岛明勤生物科技有限公司 | A kind of porcine mycoplasmal pneumonia multi-epitope mucosal vaccine |
| CN106946984A (en) * | 2017-03-17 | 2017-07-14 | 中国科学院武汉物理与数学研究所 | A kind of preparation method for the b subunit of cholera toxin albumen for having bioactivity |
Non-Patent Citations (4)
| Title |
|---|
| S A MOSCHOS,ET AL.: "Adjuvant synergy: The effects of nasal coadministration of adjuvants", 《IMMUNOLOGY AND CELL BIOLOGY》 * |
| 傅婷等: "壳聚糖季铵盐纳米粒的制备及其应用研究进展", 《国际流行病传染病学杂志》 * |
| 刘晨光等: "壳聚糖作为药物缓释材料的研究进展", 《高技术通讯》 * |
| 磨健悦等: "壳聚糖包被的口服猪流行性腹泻病毒亚单位疫苗的免疫效果", 《中国畜牧兽医学会2014年学术年会论文集》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115044524A (en) * | 2022-06-13 | 2022-09-13 | 山东大学 | Genetic engineering recombinant lactic acid bacteria and application thereof in aspect of resisting vibrio mimicus infection |
| CN115044524B (en) * | 2022-06-13 | 2023-11-21 | 山东大学 | Genetically engineered recombinant lactobacillus and application thereof in resisting vibrio mimicus infection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108715607B (en) | 2021-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106220716B (en) | Infectious coryza of chicken subunit vaccine and preparation method thereof | |
| CN108486067B (en) | Porcine epidemic diarrhea virus variant strain, inactivated vaccine prepared from same and application of inactivated vaccine | |
| CN107337718A (en) | A kind of gene for encoding carrying Cap gene of porcine circovirus type 2 and its application | |
| CN108619503A (en) | A kind of pig circular ring virus genetic engineering subunit vaccine and the preparation method and application thereof | |
| CN105949287B (en) | A kind of A type pair chicken poultry bacillus immune protective antigen and its application | |
| CN107899008B (en) | Sick three subunit vaccines of a kind of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus | |
| CN107267466A (en) | A kind of method for mass producing swine pseudorabies vaccine | |
| CN111393531A (en) | Subunit fusion protein CD2V-Fc and preparation method and application thereof | |
| CN107098974A (en) | A kind of fusion protein and its application | |
| CN108823218A (en) | Chicken infectivity bursa of Fabricius virus VP 2 gene, its expression product, its subunit vaccine and application | |
| CN104630151A (en) | Monoclonal antibody for detecting porcine C-reactive protein (CRP) | |
| CN102816246B (en) | Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof | |
| CN111471701A (en) | Method for efficiently expressing ORF2 gene of goose star virus soluble capsid protein and application thereof | |
| CN107510841A (en) | Source blocks the vaccine that echinococcosis cause of disease Echinococcus granulosus is propagated | |
| CN111978411B (en) | Porcine reproductive and respiratory syndrome subunit vaccine and preparation method and application thereof | |
| CN109021115A (en) | A kind of pig circular ring virus trivalent subunit vaccine | |
| CN108715607A (en) | Recombinant cholera toxin b protein subunit, PEDV inactivated vaccines and preparation and application | |
| CN106754981A (en) | A kind of method that utilization E. coli system prepares Goose Parvovirus sample particle | |
| CN111440815A (en) | Novel duck reovirus composite vaccine and preparation method of yolk antibody | |
| CN106008678A (en) | Fusion protein for inhibiting clostridium perfringens infection and related biological material and application thereof | |
| CN102604993A (en) | Immunologic adjuvant-Helicobacter pylori antigen fused protein oral vaccine and preparation method thereof | |
| CN105267988A (en) | Novel echinococosis granulosis vaccine with CPG DNA (deoxyribonucleic acid) immune adjuvants | |
| CN105267989A (en) | Novel water-soluble echinococosis granulosis vaccine with immune adjuvants | |
| CN102183658B (en) | Recombinant UL55 protein-based duck plague virus antibody detection method | |
| CN109053896A (en) | A kind of pig circular ring virus bivalent genetic engineering vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |