CN107596361A - Subunit vaccine of bovine A-type clostridium perfringens and preparation method and application thereof - Google Patents
Subunit vaccine of bovine A-type clostridium perfringens and preparation method and application thereof Download PDFInfo
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- CN107596361A CN107596361A CN201710819084.5A CN201710819084A CN107596361A CN 107596361 A CN107596361 A CN 107596361A CN 201710819084 A CN201710819084 A CN 201710819084A CN 107596361 A CN107596361 A CN 107596361A
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- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
the invention provides a subunit vaccine of a bovine clostridium perfringens type A, a preparation method and application thereof, wherein the subunit vaccine comprises a bovine clostridium perfringens type A α -toxin protein truncated body and a pharmaceutically acceptable adjuvant, and the α -toxin protein truncated body is an antigenic determinant amino acid sequence from an amino acid 255 position to an amino acid 372 position of α -toxin protein.
Description
Technical field
The present invention relates to the preparation and application of ox A type C.perfringens subunit vaccines, belong to veterinary biologicses skill
Art field.
Background technology
A types C.perfringens (Clostridium perfringens) is the anaerobism in nature and animal intestinal tract
Gemma and heat-resisting conditioned pathogen can be produced.It is anxious, sick that Roll toxaemia and ox Sudden Death Syndrome etc. caused by the disease are respectively provided with morbidity
The features such as journey is short, the death rate is high.So therapeutic effect that medicine has not often had.
A type C.perfringens mainly produces alpha-toxin, and the toxin is main immunogene, can stimulate in body generation
With property antibody.Therefore, alpha-toxin is the main target antigen of subunit vaccine design.But alpha-toxin has phospholipase C activity, can incite somebody to action
The hydrolysis of phosphatidlycholine of cell membrane, cell membrane integrity are destroyed, and ultimately result in cell rupture death, therefore it has carefully
Cellular toxicity, hemolytic, lethal and the characteristic such as cutaneous necrotizing.Therefore, must consider to live phospholipase C during design expression alpha-toxin
Property inactivation.
At present, have and reach alpha-toxin progress truncated form, to obtain without toxicity but retain the alpha-toxin truncation of immunogenicity
Body (referring specifically to Publication No. CN93107585 patent of invention)；Have and the alpha-toxin expressed is subjected to inactivation treatment, with
Obtain the alpha-toxin for losing toxicity (referring specifically to Publication No. CN200680021810 patent of invention).But due at that time
The limitation of technical conditions, position or length or short being truncated when truncated form reaches, this may not only influence the immunogenicity of destination protein,
The yield of destination protein may also can be reduced, causes production cost higher；The alpha-toxin of wild type is carried out into inactivation in addition may
In the presence of the not thorough and toxicity brought is inactivated, the step of also increasing production technology and time, the rise of production cost can be caused,
Influence the production and sales of vaccine.
The content of the invention
The technical problem to be solved in the present invention one be to provide it is a kind of can large-scale industrial production ox A type perfringens shuttles
Bacterium subunit vaccine and preparation method thereof；Position or length or short the defects of may bringing are truncated in the prior art second, overcoming；Three
It is to overcome the defects of there may be when being inactivated in the prior art to alpha-toxin.
According to an aspect of the present invention, it is described the invention provides a kind of ox A types C.perfringens subunit vaccine
The alpha-toxin protein truncation body of subunit vaccine including ox A type C.perfringens and pharmaceutically acceptable adjuvant, institute
Alpha-toxin protein truncation body is stated as antigenic determinant amino acid of the alpha-toxin albumen from 255, amino acid to 372, amino acid
Sequence
In technical scheme, it is preferable that the amino acid sequence of the alpha-toxin protein truncation body such as SEQ ID
Shown in NO.3.
In technical scheme, it is preferable that the pharmaceutically acceptable adjuvant is ISA 201VG adjuvants, institute
The weight ratio for stating alpha-toxin protein truncation body and adjuvant is 1:1.
In technical scheme, it is preferable that every part contains 30~200 μ g alpha-toxin eggs in the subunit vaccine
White truncate.
In technical scheme, it is preferable that every part is contained 100 μ g alpha-toxins albumen and cut in the subunit vaccine
Short body.
According to a further aspect of the present invention, present invention also offers a kind of method for preparing alpha-toxin protein truncation body, institute
The method of stating comprises the following steps：1) nucleotide sequence of alpha-toxin albumen is subjected to codon optimization, after obtaining codon optimization
Alpha toxin protein nucleotide sequence, the alpha toxin protein nucleotide sequence after codon optimization is amplified into alpha-toxin by PCR
The nucleotide sequence of protein truncation body；2) nucleotide sequence of alpha-toxin protein truncation body is inserted into pET28a carriers, obtained
To recombinant vector；3) recombinant vector is transferred to Transformed E .coli BL21 (DE3), induced expression, obtains alpha-toxin albumen after purification
Truncate.
In technical scheme, it is preferable that the alpha-toxin protein truncation body that PCR is amplified after the codon optimization
Nucleotide sequence as shown in SEQ ID NO.1.
According to a further aspect of the present invention, present invention also offers a kind of subunit vaccine in ox A type C.perfringens
Application in treatment and prevention.
According to a further aspect of the present invention, present invention also offers a kind of alpha-toxin protein truncation body in ox A type aerogenesis pods
Application in the preparation of film clostridium subunit vaccine.
Compared with prior art, subunit vaccine provided by the invention can be good at overcoming first in the prior art to α-
(the alpha-toxin albumen in subunit vaccine of the invention does not have the halfway problem of inactivation that may be present when toxin is inactivated
It is toxic, it is not required that inactivation).Next, alpha-toxin albumen of the invention is when truncating only by the toxicity in alpha-toxin albumen
Domain is cut out, retain all possible immunogenicity (in the contrast experiment of embodiment 5, alpha-toxin protein truncation body of the invention
Immunogenicity alpha-toxin protein truncation body more of the prior art immunogenicity it is good), therefore, the present invention can be good at gram
Clothes truncate position or length or short the defects of may bringing in the prior art.Again, the present invention is preparing alpha-toxin protein truncation body
When, technique is simple (albumen expressed is i.e. available in ni-sepharose purification), and yield height (there are about 800mg/L), cost is low (to be used
Bacillus coli expression, fermentation time is short, and consumptive material is cheap), it is adapted to large-scale industrial production.Finally, alpha-toxin of the invention
In 7 days that protein truncation body tracks after mouse is injected, mouse be all it is normal, therefore, alpha-toxin protein truncation of the invention
Body is safe；And this alpha-toxin protein truncation body after immune rabbit and mouse, can protect rabbit after vaccine is prepared
Or mouse can produce good immune response, be suitable for the production of ox A types from the attack of strong poison, the vaccine of this explanation present invention
The treatment and prevention of gas capsular clostridium.
Brief description of the drawings
Fig. 1 alpha-toxin protein truncation body double digestion qualification results：M：Marker, DL10000；1-4：Plasmid pET28a-
The result of PLC-C Nco I＆Pvu I double digestions.
Fig. 2 alpha-toxin protein truncation body purification results.
Fig. 3 alpha-toxin protein truncation body mouse toxicity experimental results (weightening tracking result)：Experimental group is alpha-toxin albumen
Increase weight result after truncate injection.
The optimization of Fig. 4 alpha-toxin protein truncation bodies nucleotide sequence is front and rear relatively, and OPTI-PLC-C is the nucleotides after optimization
Sequence, PLC-C are the nucleotide sequence before optimization.
Embodiment
Below with reference to drawings and examples, the present invention will be further described, and embodiments of the invention are merely to illustrate this
The technical scheme of invention, and the non-limiting present invention.
Reagent is domestic commercially available prod.
The preparation of the alpha-toxin protein truncation body of embodiment 1
1.1 alpha-toxin protein truncation bodies truncate the selection of position
Alpha toxin is the main virulence factor of A type C.perfringens, and body can be stimulated to produce neutrality antibody.It is certainly
Body is a kind of metalloenzyme dependent on zinc ion phospholipase C (PLC) activity, the enzyme can by the hydrolysis of phosphatidlycholine of cell membrane,
Cell membrane integrity is destroyed, and ultimately results in cell rupture death.To the structure (PDB of alpha-toxin albumen:1GYG) divided
Analysis, its structure are divided into two domains：N-terminal structure includes nine close-connected alpha-helixs, and C-terminal structure is anti-flat by 8
Capable β-pleated sheet composition, N-terminal are connected with C-terminal by one section of short and flexible connexon.Wherein activity of phospholipase region is fully present
N-terminal domain, and the C-terminal region containing a large amount of antigenic determinants has no activity of phospholipase.In order to ensure immunogenicity (avoids cutting
Short too short mistake partial immunity originality；Or truncate oversize, and yield is not high when causing subsequently to prepare), by com-parison and analysis, selection
Truncate the sequence that position is whole C-terminal domain, i.e. 255 amino acids of alpha-toxin to 372 amino acids.
1.2 alpha-toxin albumen codon optimizations
According to GenBank (GenBank：AY823400) sequence number, C57 nucleotide sequence is committed to Nanjing Jin Siruisheng
Thing Science and Technology Ltd. optimum synthesis, obtain OPTI-PLC (the alpha toxin protein nucleotide sequence after codon optimization).
Wherein, the nucleotide sequence comparison of the alpha-toxin protein truncation body before and after codon optimization as shown in Figure 4, there is 77/
354=21.75% nucleotides is different.
1.3 expression vector establishments and checking
1.3.1PCR purpose fragment PLC-C (alpha toxin protein truncation body) is expanded
1.3.1.1PCR reaction
(1) design of primers and synthesis
Sense primer:5’-CATGCCATGGGCAAGAACGTGAAAGAACTGGTT-3’
Anti-sense primer:5’-ccgCTCGAGTTTAATGTTGTAGGT-3’
(2) system 50 μ L are loaded, it is as shown in the table：
1.3.1.2PCR amplification program：
1.3.1.3PCR product carries out glue reclaim
(1) sample collection EP pipes, adsorption column and collecting pipe have been marked；
(2) the empty EP pipe weight marked is weighed, and records numerical value；
(3) single target DNA band is carefully cut with scalpel from Ago-Gel on bale cutting instrument be put into it is dry
In net 1.5mL centrifuge tubes；
(4) 600 μ L PC buffer are added in the 1.5mL centrifuge tubes into step (3), it is left that 5min is placed in 65 DEG C of water-baths
The right side, centrifuge tube is gently constantly spun upside down therebetween, to ensure that blob of viscose fully dissolves；
(5) column equilibration：Into adsorption column CB2, (adsorption column is placed in advance in collecting pipe) adds 500 μ L equilibrium liquid BL, centrifugation
12,000rpm/min, 1min, the waste liquid in collecting pipe is outwelled, adsorption column is placed back in collecting pipe；
(6) step (5) resulting solution is added in adsorption column CB2, stands 5min, 10,000rpm/min, centrifugation 1min,
The waste liquid in collecting pipe is outwelled, then adsorption column CB2 is put into collecting pipe；
(7) 700 μ L rinsing liquid PW buffer are added into adsorption column, stand 5min, centrifuge 10,000rpm/min,
1min, the waste liquid in collecting pipe is outwelled, adsorption column CB2 is put into collecting pipe；
(8) repeat step (7)；
(9) suction attached column centrifuges, and 12,000rpm/min, 2min, removes rinsing liquid as far as possible, adsorption column is placed in into room temperature puts
10min is put, is thoroughly dried；
(10) adsorption column CB2 is put into collecting pipe, 50 μ L Elution is vacantly added dropwise to adsorbed film centre position
Buffer (65 DEG C of preheatings), stands 3min, centrifuges 12,000rpm/min, 2min；
(11) centrifuge tube in step (10) is taken out from centrifuge, the adsorption column CB2 of centre is abandoned, covers centrifugation lid
Son, retain the DNA sample in centrifuge tube；
(12) DNA sample in step 11 is placed in 4 DEG C of preservations, prepares agarose gel electrophoresis identification glue reclaim DNA pieces
Section.
1.3.2PCR product and the reaction of carrier double digestion
(1) mark needs well the 200 μ L PCR pipes used, and sample-adding is carried out according to the following table in PCR pipe, mixes：50 μ L are anti-
Answer system
(2) PCR pipe in step (1) is placed in corresponding enzyme optimum temperature thermostat water bath, water-bath 1-2h.
Double digestion product glue reclaim：Above-mentioned double digestion system is taken out, enters row agarose gel electrophoresis to reclaim DNA therein
Fragment, method is the same as PCR primer glue reclaim in 1.3.1.3.
1.3.3 coupled reaction
(1) it is some to prepare 200 clean μ L PCR pipes, carries out mark, is placed in stand-by on EP pipe supports.
(2) sample-adding, mixing is carried out according to the following table in 200 μ L PCR pipes.
(3) after completing sample-adding according to form in step (2), each 10 μ l reaction systems are placed in 16 DEG C of reaction 2h of PCR instrument；
(4) the EP pipes in step (3) are taken out, are placed in 4 DEG C of preservations.
1.3.4 conversion reaction
(1) 10 μ L coupled reaction liquid are rapidly joined in 100 μ L competent cells, and blows and beats mixing, ice bath 30min；
(2) sample cell is taken out, is placed in 42 DEG C of water-bath 100s, immediately after ice bath 2min；
(3) sample cell is taken out, in superclean bench, 600 μ L LB liquid mediums are added into sample cell, then by sample
QC is placed in 37 DEG C of constant-temperature tables, 220rpm/min, cultivates 1h；
(4) coated plate：Sample cell in step (3) is taken out, room temperature centrifuges 8,000rpm/min, 2min, removes 600 μ L of supernatant liquid
The thalline of bottom of the tube is resuspended in body, remaining supernatant, and the bacterium solution of resuspension is put into corresponding conversion plate center, will be turned with bacteria stick is applied
The bacterium solution for changing plate center is uniformly spread out.
(5) step of converting (4) flat board is just being placed in biochemical constant incubator, after 37 DEG C of culture 1h, flat-plate inverted will be being converted
Put and carry out culture 15h；
(6) conversion results are observed.
1.3.5 plasmid extraction is identified with double digestion
1.3.5.1 plasmid extraction
(1) with 10 μ L liquid transfer gun heads from conversion flat board in picking monoclonal to 5mL that resistance containing card LB fluid nutrient mediums
In, 37 DEG C, 220rpm/min shakes bacterium and stayed overnight；
(2) bacterium solution is moved in 1.5mL EP pipes, room temperature centrifugation, 12,000rpm/min, 2min, abandons supernatant；
(3) 250 μ L plasmid extraction reagent P1buffer, thorough suspension thalline are added into the EP pipes of step (2)；
(4) 250 μ L P2buffer are added into step (3) solution, immediately gently reverse 5-10 mixing of centrifuge tube, room
Temperature stands 2-4min；
(5) 350 μ L P3buffer are added into step (4) solution, immediately gently reverse 5-10 mixing of centrifuge tube；
(6) by step (5) solution, room temperature centrifugation, 14,000rpm/min, 10min；
(7) supernatant solution in step (6) is moved into adsorption column center, room temperature centrifugation, 12,000rpm/min, 30s, outwelled
Liquid in collecting pipe；
(8) 500 μ L Buffer DW1 are added to adsorption column center, room temperature centrifugation, 12,000rpm/min, 30s, outwells receipts
Liquid in collector；
(9) 500 μ L wash solution are added to adsorption column center, room temperature centrifugation, 12,000rpm/min, 30s, fallen
Fall liquid in collecting pipe, be repeated once；
(10) suction attached column, room temperature centrifugation, 12,000rpm, 2min.
(11) adsorption column is put into a clean 1.5mL centrifuge tube, 50 μ L Elution is added to absorption center membrane
Buffer, it is stored at room temperature 5min, room temperature centrifugation, 12,000rpm, 2min.Preserve DNA solution in pipe.
1.3.5.2 double digestion is identified
(1) mark needs well the 200 μ L PCR pipes used, and sample-adding is carried out according to the following table：10 μ L reaction systems
(2) the μ L reaction systems of 200 μ L PCR pipes 10 in step (1) are placed in 37 DEG C of thermostat water baths, water-bath 1h.
(3) the double digestion system sample in step (2) is entered into row agarose gel electrophoresis, whether checks Insert Fragment size
Correctly；Experimental result is shown in Fig. 1：Purpose band is respectively 4124bp and 1467bp correct, and digestion identification structure is correct.
(4) selecting Insert Fragment, correctly clone send sequencing company to be sequenced.
1.4 alpha-toxin protein truncation body surfaces reach
1.4.1 e. coli bl21 is converted
Draw 1 μ l plasmids to add in 100 μ l BL21 competent cells, ice bath 30min；
42 DEG C of heat shock 90s；
Ice bath 2min；
The LB nutrient solutions of 500 μ l non-resistants are added in super-clean bench；
37 DEG C of 220rpm shake 1h；
100 μ l bacterium solutions card-coating that resistance LB flat boards are drawn, 37 DEG C are incubated overnight.
1.4.2 a large amount of induced expressions
Choose bacterium：Into 3ml cards that resistance LB nutrient solutions, 37 DEG C are incubated overnight picking monoclonal；
Switching：By 1:100 ratios transfer bacterium solution into 15ml cards that resistance LB nutrient solutions, and 37 DEG C of 220rpm cultivate 3.5-
4h；
Fermentation：By 1:150 ratios, which are seeded in 2L cards that resistance LB nutrient solutions, carries out shake flask fermentation, 37 DEG C of 220rpm cultures
3.5-4h to OD600It is worth 0.8-1.0；
Induction：1mL IPTG (1M) are added to the final concentration of 0.5mmol/L of IPTG, 37 DEG C of 220rpm Fiber differentiations 4h；
Microorganism collection：Bacterium solution 8,000rpm centrifugation 10min, collects thalline；With 40ml PBS thalline, 8,000rpm from
Heart 10min, thalline is collected, is placed in -20 DEG C of preservations；
1.5 alpha-toxin protein truncation bodies purify
(1) sample preparation：Lysate (10ml/g weight in wet bases) (50mM NaH are added in the thalline obtained in 1.42PO4,
500mM NaCl, 0.2%Triton X-114,0.05%Tween 20, pH 8.0；Membrane filtration using 0.8 μm), use
The piping and druming of 50mL syringes is uniformly to without graininess fungus block；Thalline sample is poured into cell homogeneous instrument sample cell, outlet is used
Beaker collects sample；Using 90% outflow outlet of sample as a circulation, after the completion of a circulation, outlet beaker is collected
Sample refund sample cell, continue repeat 5 circulation.Broken complete sample is dispensed into 250mL Beckman centrifuge tubes,
12,000rpm, 4 DEG C of centrifugation 30min, abandon supernatant, collect precipitation.Precipitation is resuspended with lysate to (can assisted milling), centrifugation
(12,000rpm, 4 DEG C of centrifugation 30min), repeats operation cleaning precipitation 3 times, finally by the precipitation cleaned up urea containing 8M
Lysate (1g/100mL) dissolve overnight, 12,000rpm, 4 DEG C of next day centrifugation 30min, collect supernatant and be used as sample, reserved 80
μ L samples detect for SDS-PAGE；
(2) column equilibration：With the ultrapure column volume of water balance 2~3 (CV), the ethanol for discharging 20% preserves liquid；Then with cracking
Liquid balances 2~3 column volumes (CV), 5mL/min.Control pressure is less than 0.5MPa.
(3) loading：2mL/min carries out loading, and collection flows through liquid (Flow through), takes 80 μ L to be examined for SDS-PAGE
Survey.
(4) 1 is rinsed：With (the 50mM NaH of cleaning buffer solution component 12PO4, 500mM NaCl, 8M urea, 0.2%Triton
X-114,0.05%Tween 20, pH 7.4, the membrane filtration using 0.8 μm) post is washed, 5mL/min, rinse 30 column volumes
(CV)。
(5) 2 are rinsed：With (the 50mM NaH of elution buffer component 1 of 10 times of column volumes (CV)2PO4, 500mM NaCl, 8M
Urea, 0.05%Tween 20, pH 7.4, the membrane filtration using 0.8 μm) post is washed, reduce Triton X-114 residual.
(6) wash miscellaneous：(the 50mM NaH of 4% elution buffer component 22PO4, 500mM NaCl, 20mM imidazoles, 8M urea,
0.05%Tween 20, pH 7.4, the membrane filtration using 0.8 μm) foreigh protein removing is removed, flat, speed 5ml/min is washed to baseline,
Collect, take 80 μ L to be analyzed for SDS-PAGE after mixing；
(7) elute：(the 50mM NaH of 50% elution buffer component 22PO4, 500mM NaCl, 250mM imidazoles, 8M urea,
0.05%Tween 20, pH 7.4, the membrane filtration using 0.8 μm) elution destination protein, flat, speed 5ml/ is washed to baseline
Min, collect, take 80 μ L to be analyzed for SDS-PAGE after mixing；The 100% de- component of buffer solution 2 cleaning pillar (50mM NaH2PO4,
500mM NaCl, 500mM imidazoles, pH 7.4, the membrane filtration using 0.8 μm), flat, 5mL/min is washed to baseline, is collected, mixing
After take 80 μ L for SDS-PAGE analyze.
(8) the outer liquid dialysis destination protein renaturation of 4M urea：The eluent of 50% elution buffer component 2 is collected, in aperture
Renaturation is carried out in 3KDa bag filter, dialyse more than 6h, and extracellular fluid dialysis are 20mM NaH2PO4, 200mM NaCl, 4M urea,
0.05%Tween 20, pH 7.4.
(9) the outer liquid dialysis destination protein renaturation of 2M urea：Bag filter is transferred to outside 2M urea renaturation is carried out in liquid, dialysed
More than 6h, extracellular fluid dialysis are 20mM NaH2PO4, 200mM NaCl, 2M urea, 0.05%Tween 20, pH 7.4.
(10) the outer liquid dialysis destination protein renaturation of 1M urea：Bag filter is transferred to outside 1M urea renaturation is carried out in liquid, thoroughly
More than 6h is analysed, extracellular fluid dialysis are 20mM NaH2PO4, 200mM NaCl, 1M urea, 0.05%Tween 20, pH 7.4.
(11) the outer liquid dialysis destination protein renaturation of 0.5M urea：Bag filter is transferred to outside 0.5M urea in liquid and answered
Property, dialyse more than 6h, and extracellular fluid dialysis are 20mM NaH2PO4, 200mM NaCl, 0.5M urea, 0.05%Tween 20,1% is sweet
Oil, pH 7.4.
(12) albumen preserves buffer solution dialysis destination protein renaturation：Bag filter is transferred to the outer liquid of albumen preservation buffer solution
Middle carry out renaturation, dialyse more than 2h, and extracellular fluid dialysis are 20mM NaH2PO4, 200mM NaCl, 0.05%Tween 20,5% is sweet
Oil, 5% sucrose, pH 7.4 repeat step (12) 2-3 times.
(13) aseptic filtration：The protein solution of collection 12,000rpm centrifugation 15min, collects supernatant, moves to biology at 4 DEG C
Safety cabinet, the low syringe needle filter of 0.2 μm of protein binding rate is crossed, -80 DEG C of refrigerators are placed in after filtering and are preserved.
(14) purification result is as shown in Figure 2：The SDS-PAGE purity of alpha-toxin protein truncation body after purification can reach
More than 90%；By calculating, in the case of no optimization fermented and cultured, the expression quantity of alpha-toxin protein truncation body can reach
More than 800mg/L.
The alpha-toxin protein truncation body mouse toxicity of embodiment 2 is tested
10 18-20g BALB/C mices are bought, are randomly divided into 2 groups, every group 5.One of which as a control group, leg
The μ l PBS of intramuscular injection 200；One group of leg muscle injects 200 μ l (Tot Prot is 200 μ g) alpha-toxin protein truncation body.Note
Penetrate rear Continuous Observation 7 days, observe spiritual appetite and measurement body weight daily.
In 7 days of injection tracking, 2 groups of mouse spirit appetite are all fine, have no significant difference, and increase weight (see Fig. 3)
There is no significant difference.This explanation, the alpha-toxin protein truncation body that we prepare is safe, and the dosis tolerata of mouse is very high
(most low energy reaches 200 μ g), much meets the needs of preparing vaccine.
It is prepared by 3 Ns of A type C.perfringens subunit vaccines of embodiment
Appropriate alpha-toxin protein truncation body is added in ISA 201VG adjuvants to (antigen phase and adjuvant weight ratio are 1:1,
4 DEG C of preservations are placed in after emulsification, quality inspection are qualified；Specific vaccine information is as shown in the table：
The Immunization of embodiment 4 is tested
25 2kg of purchase or so NZw, 5 groups are randomly divided into, every group 5, embodiment is immunized in group 1- groups 3 respectively
The 4 vaccine 1- vaccines 3 prepared, the immune market seedling (inactivated vaccine) of group 4, group 5 are used as blank control group.Poison is attacked after immune within 21 days
(C57-1 toxin attacks 1 rabbit MLD), Continuous Observation 14 days.In 14 days of observation, control group is all dead, protective rate 0,4
For group immune group rabbit without death, and spiritual appetite is good, protective rate is 100%.
Next, the alpha-toxin protein truncation body that we prepare is prepared into vaccine (100 μ g/ head parts, specific preparation by us
Referring to embodiment 4), and truncation body protein of the same race is prepared referring to CN93107585 patent of invention, and it is prepared into vaccine (100 μ g/
Head part, specifically prepares referring to embodiments of the invention 4), by 2 kinds of vaccines while immune rabbit and attack poison.As a result show, we make
Vaccine prepared by standby alpha-toxin protein truncation body can protect rabbit from the attack of strong poison, 14 days observed after poison is attacked, rabbit
The spiritual appetite of son is normal, does not occur any adverse reaction, protective rate 100%, and referring to CN93107585 patent of invention
After vaccine immunity rabbit prepared by the alpha-toxin protein truncation body of preparation attacks poison, although death does not occur, 1/5 rabbit
The symptom that spiritedness appetite is deteriorated, therefore, protective rate 80%.
Prepared to further compare the patent of invention of the alpha-toxin protein truncation body of our preparations and CN93107585
The immunogenicity of alpha-toxin protein truncation body, we have carried out Immunization experiment using Bal b/c mouse.According to embodiment 4
Method prepare 2 kinds of vaccines, the concentration of alpha-toxin protein truncation body is 100 μ g/ head parts in vaccine.Buy 15 Bal b/c
Mouse, is randomly divided into 3 groups, every group 5, and 1 group of leg muscle injects prepared by 100 μ l our the alpha-toxin protein truncation bodies that prepare
Vaccine (is designated as experimental group 1), and alpha-toxin albumen prepared by the patent of invention that 1 group of leg muscle injects 100 μ l CN93107585 is cut
Vaccine (being designated as experimental group 2) prepared by short body, 1 group is used as blank control group, and leg muscle injects 100 μ l PBS.After immune
Attack within 21 days malicious (C57-1 toxin attacks 1 mouse MLD), Continuous Observation 14 days.In 14 days of observation, control group is all dead, protects
Shield rate is 0, and for experimental group 1 without death, and spiritual appetite is good, protective rate 100%, experimental group 2 has 1 dead mouse,
Protective rate is 80%.
It can be seen from the results above that the immunogenicity for the alpha-toxin protein truncation body that we prepare is fine, can play
100% protective effect, alpha-toxin egg suitable with existing market seedling (inactivated vaccine) immune effect, and being prepared than prior art
The immunogenicity of white truncate is preferable.Because we use albumen to have as antigen than existing inactivated vaccine obvious excellent
Gesture：1) the not thorough caused safety issue of inactivation is not present；2) it is quality controllable, in the absence of difference between batch；3) production equipment
Relatively low with space requirement, expression quantity is high, and cost is low；4) risk for dissipating malicious (bacterium) is not present, the safety of production operation personnel has
Ensure.In addition, the alpha-toxin protein truncation body that we prepare can realize that industrialization production (does not protrude energy in prior art patent
No industrialization production, do not know specific yield can reach how many yet), Bacillus coli expression yield can reach 800mg/L,
If optimized to existing process, expression yield can also further increase, and can so significantly reduce production cost, favorably
In the promotion and application of vaccine.
The present invention is illustrated by above embodiment, it is understood, however, that the present invention is not limited to institute here
The particular example and embodiment of description.Purpose herein comprising these particular examples and embodiment is to help this area
In technical staff put into practice the present invention.Any those of skill in the art are easy to do not departing from spirit and scope of the invention
In the case of be further improved and perfect, therefore the present invention is only by the content of the claims in the present invention and limiting for scope
System, its intention cover the alternative in all spirit and scope of the invention for being included in and being limited by appendix claim and waited
Same scheme.
Sequence table
<110>Zhejiang oceanic rise bio tech ltd
<120>Ox A type C.perfringens subunit vaccines and its preparation method and application
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aagaacgtga aagaactggt tgcgtacatc agcaccagcg gcgagaagga tgcgggcacc 60
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gataacccgg gtaacgactt catgaccggc agcaaggata cctatacctt taagctgaaa 180
gacgagaacc tgaaaatcga cgatattcaa aacatgtgga tccgtaagcg taaatacacc 240
gcgttcccgg acgcgtataa gccggaaaac atcaaactga ttgcgaacgg caaggtggtt 300
gtggataaag acatcaacga gtggattagc ggcaacagca cctacaacat taaa 354
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aagaatgtaa aagaactagt agcttacata tcaactagtg gtgaaaaaga tgctggaaca 60
gatgactaca tgtattttgg aatcaaaaca aaggatggaa aaactcaaga atgggaaatg 120
gacaacccag gaaatgattt tatgactgga agtaaagaca cttatacttt caaattaaaa 180
gatgaaaatc taaaaattga tgatatacaa aatatgtgga ttagaaaaag aaaatataca 240
gcattcccag atgcttataa gccagaaaac ataaagttaa tagcaaatgg aaaagttgta 300
gtggacaagg atataaatga gtggatttca ggaaattcaa cttataatat aaaa 354
<210> 4
<211> 118
<212> PRT
<213>Alpha-toxin protein truncation body amino acid sequence (2 Ambystoma laterale x Ambystoma
jeffersonianum)
<400> 4
Lys Asn Val Lys Glu Leu Val Ala Tyr Ile Ser Thr Ser Gly Glu Lys
1 5 10 15
Asp Ala Gly Thr Asp Asp Tyr Met Tyr Phe Gly Ile Lys Thr Lys Asp
20 25 30
Gly Lys Thr Gln Glu Trp Glu Met Asp Asn Pro Gly Asn Asp Phe Met
35 40 45
Thr Gly Ser Lys Asp Thr Tyr Thr Phe Lys Leu Lys Asp Glu Asn Leu
50 55 60
Lys Ile Asp Asp Ile Gln Asn Met Trp Ile Arg Lys Arg Lys Tyr Thr
65 70 75 80
Ala Phe Pro Asp Ala Tyr Lys Pro Glu Asn Ile Lys Leu Ile Ala Asn
85 90 95
Gly Lys Val Val Val Asp Lys Asp Ile Asn Glu Trp Ile Ser Gly Asn
100 105 110
Ser Thr Tyr Asn Ile Lys
115
Claims (9)
1. a kind of subunit vaccine of ox A type C.perfringens, it is characterised in that the subunit vaccine produces including ox A types
The alpha-toxin protein truncation body of gas capsular clostridium and pharmaceutically acceptable adjuvant, the alpha-toxin protein truncation body be α-
The antigenic determinant amino acid sequence from 255, amino acid to 372, amino acid of toxin protein.
2. vaccine according to claim 1, it is characterised in that the amino acid sequence of the alpha-toxin protein truncation body is such as
Shown in SEQ ID NO.3.
3. subunit vaccine according to claim 1, it is characterised in that the pharmaceutically acceptable adjuvant is
ISA201VG adjuvants, the weight ratio of the alpha-toxin protein truncation body and adjuvant is 1:1.
4. subunit vaccine according to claim 1, it is characterised in that in the subunit vaccine every part contain 30~
Alpha-toxin protein truncation body described in 200 μ g.
5. subunit vaccine according to claim 4, it is characterised in that every part contains 100 μ in the subunit vaccine
Alpha-toxin protein truncation body described in g.
A kind of 6. method for preparing the alpha-toxin protein truncation body as described in claim 1-5 is any, it is characterised in that methods described
Comprise the following steps：
1) nucleotide sequence of alpha-toxin albumen is subjected to codon optimization and obtains the alpha toxin protein nucleotides after codon optimization
Sequence, the alpha toxin protein nucleotide sequence after codon optimization is amplified to the nucleotides of alpha-toxin protein truncation body by PCR
Sequence；
2) nucleotide sequence of the alpha-toxin protein truncation body is inserted into pET28a carriers, obtains recombinant vector；And
3) recombinant vector is transferred to Transformed E .coli BL21 (DE3), induced expression, obtains alpha-toxin protein truncation body after purification.
7. according to the method for claim 6, it is characterised in that the alpha-toxin egg that PCR is amplified after the codon optimization
The nucleotide sequence of white truncate is as shown in SEQ ID NO.1.
8. a kind of subunit vaccine as described in Claims 1 to 5 any claim is in the treatment of ox A types C.perfringens and in advance
Application in anti-.
9. one kind alpha-toxin protein truncation body as described in claim 6~7 any claim is sub- in ox A types C.perfringens
Application in the preparation of subunit vaccine.
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| CN201710819084.5A CN107596361B (en) | 2017-09-12 | 2017-09-12 | Subunit vaccine of bovine A-type clostridium perfringens and preparation method and application thereof |
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| CN201710819084.5A CN107596361B (en) | 2017-09-12 | 2017-09-12 | Subunit vaccine of bovine A-type clostridium perfringens and preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108578686A (en) * | 2018-04-23 | 2018-09-28 | 武汉中拓康明生物科技有限公司 | A method of preparing clostridiosis of sheep genetic engineering subunit vaccine |
| CN111983240A (en) * | 2020-08-27 | 2020-11-24 | 天津大学 | Visual detection method for clostridium perfringens alpha toxin |
| CN114621963A (en) * | 2022-03-11 | 2022-06-14 | 牧原食品股份有限公司 | A-type subunit vaccine of clostridium welchii and preparation thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1057533C (en) * | 1992-05-20 | 2000-10-18 | 大不列颠及北爱尔兰联合王国国防大臣 | Method for producing clostridium perfringens vaccines |
| CN101198352A (en) * | 2005-04-18 | 2008-06-11 | 谢尔英·普劳有限公司 | C. perfringens alpha toxoid vaccine |
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2017
- 2017-09-12 CN CN201710819084.5A patent/CN107596361B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1057533C (en) * | 1992-05-20 | 2000-10-18 | 大不列颠及北爱尔兰联合王国国防大臣 | Method for producing clostridium perfringens vaccines |
| CN101198352A (en) * | 2005-04-18 | 2008-06-11 | 谢尔英·普劳有限公司 | C. perfringens alpha toxoid vaccine |
Non-Patent Citations (1)
| Title |
|---|
| S. G. VACHIERI ET AL.: "Comparison of a nontoxic variant of Clostridium perfringens a-toxin with the toxic wild-type strain", 《ACTA CRYST.》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108578686A (en) * | 2018-04-23 | 2018-09-28 | 武汉中拓康明生物科技有限公司 | A method of preparing clostridiosis of sheep genetic engineering subunit vaccine |
| CN108578686B (en) * | 2018-04-23 | 2022-07-01 | 杨凌凯瑞生物科技有限公司 | Method for preparing genetic engineering subunit vaccine for clostridium aegypti |
| CN111983240A (en) * | 2020-08-27 | 2020-11-24 | 天津大学 | Visual detection method for clostridium perfringens alpha toxin |
| CN111983240B (en) * | 2020-08-27 | 2023-11-21 | 天津大学 | Visual detection method for clostridium perfringens alpha toxin |
| CN114621963A (en) * | 2022-03-11 | 2022-06-14 | 牧原食品股份有限公司 | A-type subunit vaccine of clostridium welchii and preparation thereof |
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| Publication number | Publication date |
|---|---|
| CN107596361B (en) | 2020-08-14 |
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