CN107596361B - 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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- CN107596361B CN107596361B CN201710819084.5A CN201710819084A CN107596361B CN 107596361 B CN107596361 B CN 107596361B CN 201710819084 A CN201710819084 A CN 201710819084A CN 107596361 B CN107596361 B CN 107596361B
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Abstract
The invention provides a subunit vaccine of bovine clostridium perfringens type A, a preparation method and application thereof, wherein the subunit vaccine comprises a bovine clostridium perfringens type A alpha-toxin protein truncated body and a pharmaceutically acceptable adjuvant, and the alpha-toxin protein truncated body is an antigenic determinant amino acid sequence from amino acid 255 to amino acid 372 of alpha-toxin protein. The subunit vaccine has the following advantages: 1) the safety problem caused by incomplete inactivation does not exist; 2) the quality is controllable, and the difference between batches does not exist; 3) the production equipment and space requirements are low, the expression level is high, and the cost is low; 4) the risk of dispersing poison does not exist, and the safety of production operators is guaranteed.
Description
Technical Field
The invention relates to preparation and application of a bovine A-type clostridium perfringens subunit vaccine, belonging to the technical field of biological products for livestock.
Background
Clostridium perfringens type a (Clostridium perfringens) is an anaerobic, sporulating and thermophilic, opportunistic pathogen in nature and in the animal gut. The enterotoxemia and sudden death of cattle caused by the disease have the characteristics of acute morbidity, short course of disease, high mortality rate and the like. Therefore, the medicine often does not have good treatment effect.
Clostridium perfringens type a produces mainly alpha-toxin, which is the main immunogen and can stimulate the body to produce neutralizing antibodies. Thus, alpha-toxin is the main target antigen for subunit vaccine design. However, alpha-toxin has phospholipase C activity, can hydrolyze phosphatidylcholine in cell membranes, and the integrity of the cell membranes is damaged, finally, cells are broken and die, so that the alpha-toxin has the characteristics of cytotoxicity, hemolysis, lethality, skin necrosis and the like. Thus, inactivation of phospholipase C activity must be considered when designing for expression of the alpha toxin.
At present, alpha-toxin is subjected to truncation expression so as to obtain an alpha-toxin truncation body which does not contain toxicity and retains immunogenicity (see the invention patent with the publication number of CN 93107585); there is an inactivation treatment of the expressed alpha-toxin to obtain alpha-toxin with lost toxicity (see patent publication No. CN 200680021810). However, due to the limitations of the technical conditions at the time, the expression is truncated at a position which is either long or short, which may not only affect the immunogenicity of the target protein, but also reduce the yield of the target protein, resulting in higher production costs; in addition, toxicity caused by incomplete inactivation may exist when wild type alpha-toxin is inactivated, steps and time of a production process are increased, production cost is increased, and production and sale of vaccines are affected.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a bovine clostridium perfringens type A subunit vaccine capable of being industrially produced in a large scale and a preparation method thereof; secondly, the defect possibly caused by the shortening position or the length in the prior art is overcome; thirdly, the possible defects existing in the prior art when the alpha-toxin is inactivated are overcome.
According to one aspect of the invention, there is provided a bovine clostridium perfringens type a subunit vaccine comprising an alpha-toxin protein truncation of clostridium perfringens type a of a bovine, and a pharmaceutically acceptable adjuvant, wherein the alpha-toxin protein truncation is an epitope amino acid sequence of the alpha-toxin protein from amino acid 255 to amino acid 372
In the technical scheme of the invention, preferably, the amino acid sequence of the alpha-toxin protein truncation is shown in SEQ ID NO. 3.
In the technical scheme of the invention, preferably, the pharmaceutically acceptable adjuvant is ISA201VG adjuvant, and the weight ratio of the alpha-toxin protein truncation body to the adjuvant is 1: 1.
In the technical scheme of the invention, preferably, each head of the subunit vaccine contains 30-200 mu g of alpha-toxin protein truncation.
In the technical scheme of the invention, each head of the subunit vaccine preferably contains 100 mu g of alpha-toxin protein truncation.
According to another aspect of the present invention, there is also provided a method of preparing an alpha-toxin protein truncation, the method comprising the steps of: 1) carrying out codon optimization on the nucleotide sequence of the alpha-toxin protein to obtain a codon-optimized nucleotide sequence of the alpha-toxin protein, and amplifying the codon-optimized nucleotide sequence of the alpha-toxin protein by PCR to obtain a nucleotide sequence of an alpha-toxin protein truncation body; 2) inserting the nucleotide sequence of the alpha-toxin protein truncation into a pET28a vector to obtain a recombinant vector; 3) transferring the recombinant vector into E.coli BL21(DE3), inducing expression, and purifying to obtain the alpha-toxin protein truncated body.
In the technical scheme of the invention, preferably, the nucleotide sequence of the alpha-toxin protein truncation body amplified by PCR after codon optimization is shown as SEQ ID No. 1.
According to another aspect of the invention, the invention also provides a subunit vaccine for use in the treatment and prevention of clostridium perfringens type a in cattle.
According to another aspect of the invention, the invention also provides the use of an alpha-toxin protein truncation in the preparation of a bovine clostridium perfringens type a subunit vaccine.
Compared with the prior art, the subunit vaccine provided by the invention can well overcome the problem of incomplete inactivation probably existing in the prior art when the alpha-toxin is inactivated (the alpha-toxin protein in the subunit vaccine of the invention has no toxicity and does not need to be inactivated). Secondly, the alpha-toxin protein of the invention only cuts off the toxic structure domain in the alpha-toxin protein when cutting off, and retains all possible immunogenicity (in the comparative experiment of example 5, the immunogenicity of the alpha-toxin protein truncation of the invention is better than that of the alpha-toxin protein truncation of the prior art), therefore, the invention can well overcome the possible defects caused by the cutting off position or the length or the short length in the prior art. Thirdly, when the alpha-toxin protein truncation is prepared, the process is simple (the expressed protein can be obtained by purifying on a nickel column), the yield is high (about 800mg/L), the cost is low (escherichia coli is used for expression, the fermentation time is short, and the material consumption is low), and the method is suitable for large-scale industrial production. Finally, the α -toxin protein truncations of the invention were all normal within 7 days of the follow-up after injection of the mice, and thus, the α -toxin protein truncations of the invention were safe; and after the alpha-toxin protein truncation is used for preparing the vaccine, the rabbit or the mouse can be protected from being attacked by virulent viruses after the rabbit or the mouse is immunized, which shows that the vaccine can generate good immune response and is suitable for treating and preventing bovine A-type clostridium perfringens.
Drawings
FIG. 1 shows the results of the double restriction enzyme identification of the alpha-toxin protein truncation: m: marker, DL 10000; 1-4: the plasmid pET28a-PLC-C was double-digested with Nco I & Pvu I.
FIG. 2 results of purification of alpha-toxin protein truncations.
Figure 3 toxicity test results (weight gain tracking results) for α -toxin protein truncation mice: the experimental group is the weight gain result after the injection of the alpha-toxin protein truncation.
FIG. 4 shows the comparison of the nucleotide sequence of the alpha-toxin protein truncation before and after optimization, wherein OPTI-PLC-C is the nucleotide sequence after optimization, and PLC-C is the nucleotide sequence before optimization.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, which are only for illustrating the technical solutions of the present invention and are not to be construed as limiting the present invention.
The reagents are all domestic commercial products.
EXAMPLE 1 preparation of alpha-toxin protein truncations
1.1 selection of the truncation position of the alpha-toxin protein truncates
Alpha toxin is a main pathogenic factor of clostridium perfringens type a and can stimulate the body to produce neutralizing antibodies. It is itself a metalloenzyme that relies on the activity of zinc ion phospholipase c (plc), which hydrolyzes the phosphatidylcholine of the cell membrane, disrupting the integrity of the membrane, and ultimately leading to cell rupture and death. Analysis of the structure of the alpha-toxin protein (PDB:1GYG) divides the structure into two domains: the N-terminal structure comprises nine closely connected alpha-helices, the C-terminal structure consists of 8 antiparallel beta-sheets, and the N-terminal and the C-terminal are connected by a short, elastic linker. Wherein the phospholipase activity domain is completely N-terminal domain, and the C-terminal domain containing a plurality of antigenic determinants is free of phospholipase activity. To ensure immunogenicity (avoid truncations too short to lose part of immunogenicity; or truncations too long, resulting in low yields on subsequent preparations), the truncation position was chosen by analytical comparison to be the entire C-terminal domain, i.e.the sequence from amino acid 255 to amino acid 372 of the alpha-toxin.
1.2 codon optimization of alpha-toxin protein
According to GenBank (GenBank: AY823400) sequence number, the nucleic acid sequence of C57 is submitted to Nanjing Kingsley Biotechnology GmbH for optimized synthesis to obtain OPTI-PLC (codon optimized alpha toxin protein nucleotide sequence).
The nucleotide sequence comparison of α -toxin protein truncations before and after codon optimization is shown in fig. 4, where 77/354 ═ 21.75% of the nucleotides are different.
1.3 expression vector construction and validation
1.3.1PCR amplification of fragment of interest PLC-C (alpha toxin protein truncation)
1.3.1.1PCR reaction
(1) Primer design and Synthesis
Upstream primer 5'-CATGCCATGGGCAAGAACGTGAAAGAACTGGTT-3'
Downstream primer 5'-ccgCTCGAGTTTAATGTTGTAGGT-3'
(2) Sample loading system 50 μ L, as shown in the following table:
1.3.1.2PCR amplification procedure:
1.3.1.3PCR products for gel recovery
(1) Marking a sample collection EP tube, an adsorption column and a collection tube;
(2) weighing the weight of the marked empty EP pipe, and recording the numerical value;
(3) a single DNA band of interest was carefully excised from the agarose gel on a gel cutter with a scalpel and placed into a clean 1.5mL centrifuge tube;
(4) adding 600 mu L of PC buffer into the 1.5mL centrifuge tube in the step (3), placing in a water bath at 65 ℃ for about 5min, and turning the centrifuge tube up and down continuously and gently to ensure that the gel block is fully dissolved;
(5) column balancing: adding 500 μ L of balance liquid BL into adsorption column CB2 (the adsorption column is placed into the collection tube in advance), centrifuging at 12,000rpm/min for 1min, pouring off waste liquid in the collection tube, and placing the adsorption column back into the collection tube;
(6) adding the solution obtained in the step (5) into an adsorption column CB2, standing for 5min at 10,000rpm/min, centrifuging for 1min, pouring out waste liquid in a collecting pipe, and then putting the adsorption column CB2 into the collecting pipe;
(7) adding 700 μ L of rinsing liquid PW buffer into the adsorption column, standing for 5min, centrifuging at 10,000rpm/min for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CB2 into the collection tube;
(8) repeating the step (7);
(9) centrifuging with an empty adsorption column at 12,000rpm/min for 2min, removing rinsing liquid as much as possible, standing the adsorption column at room temperature for 10min, and completely air drying;
(10) placing adsorption column CB2 in a collecting tube, suspending and dropwise adding 50 μ L Elutionbuffer (preheated at 65 ℃) to the middle position of an adsorption film, standing for 3min, and centrifuging at 12,000rpm/min for 2 min;
(11) taking the centrifuge tube in the step (10) out of the centrifuge, discarding the middle adsorption column CB2, covering the centrifuge tube with a cover, and keeping the DNA sample in the centrifuge tube;
(12) and (3) storing the DNA sample in the step 11 at 4 ℃, and preparing an agarose gel electrophoresis identification gel to recover the DNA fragment.
1.3.2PCR product and vector double digestion reaction
(1) Marking a 200 mu L PCR tube which needs to be used, and loading and mixing the sample in the PCR tube according to the following table: 50 μ L reaction System
(2) And (3) placing the PCR tube in the step (1) in a corresponding enzyme constant-temperature water bath kettle with the optimal temperature, and carrying out water bath for 1-2 h.
Recovering the double enzyme digestion product gel: taking out the double enzyme digestion system, and carrying out agarose gel electrophoresis to recover the DNA fragment in the double enzyme digestion system by the same method as that of the PCR product gel recovery in the 1.3.1.3.
1.3.3 ligation reactions
(1) A plurality of clean 200. mu.L PCR tubes are prepared, marked and placed on an EP tube rack for standby.
(2) Samples were loaded and mixed in a 200. mu.L PCR tube according to the following table.
(3) After sample adding is finished according to the table in the step (2), placing each 10 mu l reaction system in a PCR instrument for reaction for 2h at the temperature of 16 ℃;
(4) taking out the EP tube in the step (3), and storing at 4 ℃.
1.3.4 conversion reaction
(1) Quickly adding 10 μ L of the ligation reaction solution into 100 μ L of competent cells, uniformly mixing by blowing, and carrying out ice bath for 30 min;
(2) taking out the sample tube, placing in water bath at 42 ℃ for 100s, and immediately carrying out ice bath for 2 min;
(3) taking out the sample tube, adding 600 mu L of liquid LB culture medium into the sample tube in a super-clean workbench, then placing the sample tube in a constant temperature shaking table at 37 ℃, and culturing for 1h at 220 rpm/min;
(4) coating a plate: and (4) taking out the sample tube in the step (3), centrifuging at room temperature for 8,000rpm/min for 2min, removing 600 mu L of supernatant liquid, re-suspending the thalli at the bottom of the tube by the residual supernatant liquid, putting the re-suspended bacterial liquid into the center of a corresponding transformation plate, and uniformly spreading the bacterial liquid in the center of the transformation plate by using a bacteria coating rod.
(5) The flat plate in the transformation step (4) is placed in a biochemical constant-temperature incubator, and is cultured for 1h at 37 ℃, and then the transformation flat plate is inverted and cultured for 15 h;
(6) the transformation results were observed.
1.3.5 plasmid extraction and double restriction enzyme identification
1.3.5.1 plasmid extraction
(1) Picking the monoclonals from the transformation plate by using a 10-microliter pipette tip to 5mL of LB liquid culture medium containing kanamycin resistance, and shaking the monoclonals at 37 ℃ and 220rpm/min overnight;
(2) transferring the bacterial liquid into a 1.5mL EP tube, centrifuging at room temperature at 12,000rpm/min for 2min, and removing the supernatant;
(3) adding 250 mu L of plasmid extraction reagent P1buffer into the EP tube in the step (2) to completely suspend the thalli;
(4) adding 250 mu L P2buffer into the solution in the step (3), immediately and gently inverting the centrifuge tube for 5-10 times, uniformly mixing, and standing at room temperature for 2-4 min;
(5) adding 350 mu L P3buffer into the solution in the step (4), immediately and gently inverting the centrifuge tube for 5-10 times and uniformly mixing;
(6) centrifuging the solution in the step (5) at room temperature, and carrying out centrifugation at 14,000rpm/min for 10 min;
(7) transferring the supernatant solution in the step (6) to the center of an adsorption column, centrifuging at room temperature for 30s at 12,000rpm/min, and pouring out liquid in a collecting pipe;
(8) adding 500 μ L Buffer DW1 into the center of the adsorption column, centrifuging at room temperature at 12,000rpm/min for 30s, and pouring off the liquid in the collection tube;
(9) adding 500 μ L wash solution into the center of the adsorption column, centrifuging at room temperature, 12,000rpm/min for 30s, pouring off the liquid in the collection tube, and repeating once;
(10) the column was air-adsorbed, centrifuged at room temperature, 12,000rpm, 2 min.
(11) The column was placed in a clean 1.5mL centrifuge tube, 50. mu.L of Elutionbuffer was added to the center of the adsorption membrane, allowed to stand at room temperature for 5min, and centrifuged at room temperature at 12,000rpm for 2 min. The DNA solution in the tube was preserved.
1.3.5.2 double restriction enzyme identification
(1) The required 200. mu.L PCR tube was labeled and loaded as follows: 10 μ L reaction System
(2) And (3) putting the 200 mu L PCR tube 10 mu L reaction system in the step (1) into a constant-temperature water bath kettle at 37 ℃ and carrying out water bath for 1 h.
(3) Carrying out agarose gel electrophoresis on the double enzyme digestion system sample in the step (2), and checking whether the size of the inserted fragment is correct; the results are shown in FIG. 1: the target bands are 4124bp and 1467bp respectively, and the enzyme digestion identification construction is correct.
(4) Clones with correct inserts were selected for sequencing by the sequencing company.
1.4 alpha-toxin protein truncation expression
1.4.1 transformation of Escherichia coli BL21
Sucking 1 μ l plasmid, adding into 100 μ l BL21 competent cell, ice-cooling for 30 min;
heat shock at 42 ℃ for 90 s;
ice-bath for 2 min;
adding 500 mul of LB culture solution without resistance into a super clean bench;
shaking at 37 ℃ and 220rpm for 1 h;
100. mu.l of the inoculum was applied to kanamycin-resistant LB plates and incubated overnight at 37 ℃.
1.4.2 Induction of expression in large amounts
Selecting bacteria: selecting a monoclonal antibody to be added into 3ml of kanamycin-resistant LB culture solution, and carrying out overnight culture at 37 ℃;
transferring: transferring the bacterial liquid into 15ml of kanamycin-resistant LB culture solution according to the proportion of 1:100, and culturing at 37 ℃ and 220rpm for 3.5-4 h;
fermentation: inoculating into 2L Carna resistant LB culture solution at a ratio of 1:150Performing shake flask fermentation, culturing at 37 deg.C and 220rpm for 3.5-4h to OD600To a value of 0.8-1.0;
induction: adding 1mL of IPTG (1M) until the final concentration of the IPTG is 0.5mmol/L, and carrying out induced culture at 37 ℃ and 220rpm for 4 h;
and (3) collecting thalli: centrifuging the bacterial liquid at 8,000rpm for 10min, and collecting thalli; washing thallus with 40ml PBS, centrifuging at 8,000rpm for 10min, collecting thallus, and storing at-20 deg.C;
1.5 purification of alpha-toxin protein truncations
(1) Sample preparation: lysate (10ml/g wet weight) (50mM NaH) was added to the cells obtained in 1.42PO4500mM NaCl, 0.2% Triton X-114, 0.05% Tween 20, pH 8.0; filtering with 0.8 μm filter membrane), and blowing and beating with 50mL syringe until no granular bacteria block; pouring the thallus sample into a sample groove of a cell homogenizer, and collecting the sample by using a beaker at a sample outlet; and taking 90% of the sample flowing out of the sample outlet as a cycle, after the cycle is finished, pouring the sample collected by the sample outlet beaker back to the sample tank, and continuously repeating for 5 cycles. The completely crushed sample is divided into 250mL Beckman centrifuge tubes, centrifuged at 12,000rpm at 4 ℃ for 30min, the supernatant is discarded, and the precipitate is collected. Resuspending the precipitate with lysis solution (or grinding aid), centrifuging (12,000rpm, centrifuging at 4 deg.C for 30min), repeating the operation to wash the precipitate for 3 times, dissolving the washed precipitate with lysis solution containing 8M urea (1g/100mL) overnight, centrifuging at 4 deg.C for 30min at 12,000rpm the next day, collecting supernatant as sample, and reserving 80 μ L sample for SDS-PAGE detection;
(2) column balancing: balancing 2-3 Column Volumes (CV) by using ultrapure water, and discharging 20% ethanol preservation solution; then, the Column Volume (CV) is balanced by 2-3 columns with lysis solution, 5 mL/min. The pressure is controlled to be less than 0.5 MPa.
(3) Loading: the sample was loaded at 2mL/min, and the Flow-through (Flow through) was collected and 80. mu.L was taken for SDS-PAGE detection.
(4) Washing 1: with Wash buffer fraction 1(50mM NaH)2PO4500mM NaCl, 8M urea, 0.2% TritonX-114, 0.05% Tween 20, pH 7.4, filtered using a 0.8 μ M filter), the column was washed, 5mL/min, and 30 Column Volumes (CV) were washed.
(5) And (3) washing 2: by using 10 times of columnVolume (CV) of elution buffer component 1(50mM NaH)2PO4500mM NaCl, 8M urea, 0.05% Tween 20, pH 7.4, filtered using a 0.8 μ M filter) to reduce Triton X-114 residue.
(6) Impurity washing: 4% elution buffer fraction 2(50mM NaH)2PO4500mM NaCl, 20mM imidazole, 8M urea, 0.05% Tween 20, pH 7.4, filtered using a 0.8 μ M filter) to remove contaminating proteins until the baseline is washed flat at a rate of 5ml/min, collected, mixed and 80 μ L used for SDS-PAGE analysis;
(7) and (3) elution: 50% elution buffer fraction 2(50mM NaH)2PO4500mM NaCl, 250mM imidazole, 8M urea, 0.05% Tween 20, pH 7.4, filtered using a 0.8 μ M filter membrane) until the baseline is flush, at a rate of 5ml/min, collected, mixed and 80 μ L used for SDS-PAGE analysis; 100% Decuffer 2 fraction column wash (50mM NaH)2PO4500mM NaCl, 500mM imidazole, pH 7.4, filtered using a 0.8 μm filter), to baseline flush, 5mL/min, collected, mixed and 80 μ L used for SDS-PAGE analysis.
(8) Renaturation of target protein by 4M urea external fluid dialysis: collecting 50% eluate of elution buffer component 2, renaturing in dialysis bag with pore diameter of 3KDa, dialyzing for more than 6 hr, and collecting dialysate of 20mM NaH2PO4200mM NaCl, 4M urea, 0.05% Tween 20, pH 7.4.
(9) Renaturation of target protein by 2M urea external fluid dialysis: transferring the dialysis bag into 2M urea external solution for renaturation, dialyzing for more than 6h, wherein the dialysis external solution is 20mM NaH2PO4200mM NaCl, 2M urea, 0.05% Tween 20, pH 7.4.
(10) Renaturation of target protein by 1M urea external fluid dialysis: transferring the dialysis bag into 1M urea external solution for renaturation, dialyzing for more than 6h, wherein the dialysis external solution is 20mM NaH2PO4200mM NaCl, 1M urea, 0.05% Tween 20, pH 7.4.
(11) Renaturation of target protein by 0.5M urea external fluid dialysis: transferring the dialysis bag into 0.5M urea external solution for renaturation, dialyzing for more than 6h, wherein the dialysis external solution is 20mM NaH2PO4200mM NaCl, 0.5M urea,0.05% Tween 20, 1% glycerol, pH 7.4.
(12) Protein storage buffer dialysis renaturation of the protein of interest: transferring the dialysis bag into external solution of protein storage buffer solution for renaturation, dialyzing for more than 2h, wherein the dialysis external solution is 20mM NaH2PO4200mM NaCl, 0.05% Tween 20, 5% glycerol, 5% sucrose, pH 7.4, and repeating step (12)2-3 times.
(13) And (3) degerming and filtering: the collected protein solution was centrifuged at 12,000rpm for 15min at 4 ℃ and the supernatant was collected and transferred to a biosafety cabinet, passed through a 0.2 μm syringe filter with low protein binding rate, filtered and stored in a refrigerator at-80 ℃.
(14) The purification results are shown in FIG. 2: the purity of SDS-PAGE of the purified alpha-toxin protein truncation can reach more than 90 percent; through calculation, the expression quantity of the alpha-toxin protein truncation body can reach more than 800mg/L under the condition of not optimizing fermentation culture.
Example 2 toxicity test of alpha-toxin protein truncation mice
10 BALB/C mice, 18-20g, were purchased and randomly divided into 2 groups of 5 mice each. One group served as a control group and was injected intramuscularly in the legs with 200. mu.l PBS; one leg was intramuscularly injected with 200. mu.l (total protein 200. mu.g) of a-toxin protein truncation. After injection, the observation was continued for 7 days, and appetite was observed and body weight was measured every day.
Within 7 days of the injection follow-up, 2 mice were well appetizing, no significant difference was seen, and no significant difference was seen in weight gain (see fig. 3). This indicates that the alpha-toxin protein truncation prepared by the method is safe, and the tolerance of the mouse is very high (the minimum can reach 200 mu g), so that the method is far enough for preparing the vaccine.
Example 3 bovine clostridium perfringens type a subunit vaccine preparation
Adding a proper amount of alpha-toxin protein truncation into ISA201VG adjuvant (the weight ratio of an antigen phase to the adjuvant is 1:1, and after emulsification and quality inspection are qualified, storing at 4 ℃, wherein the specific vaccine information is shown in the following table:
example 4 immune challenge experiment
About 25 New Zealand white rabbits (2 kg) were purchased and randomly divided into 5 groups of 5, each group consisting of 5, and the vaccine 1-vaccine 3 prepared in example 4, the vaccine market vaccine (inactivated vaccine) in group 4, and group 5 were immunized as a blank control group, respectively, in groups 1-3. The virus was removed 21 days after immunization (C57-1 toxin removed 1 rabbit MLD) and observed for 14 consecutive days. Within 14 days of observation, all the control groups died, the protection rate was 0, and all the rabbits in the 4 immune groups did not die, and the animals had good appetite and the protection rate was 100%.
Next, we prepared the α -toxin protein truncates we prepared into vaccines (100 μ g/head, see example 4 for specific preparation), and the patent of invention CN93107585 prepared the homologous truncates proteins and prepared into vaccines (100 μ g/head, see example 4 for specific preparation), and 2 vaccines were used to immunize rabbits and challenge simultaneously. The results show that the vaccine prepared by the alpha-toxin protein truncation prepared by the inventor can protect rabbits from the attack of strong toxin, the rabbit appetite is normal and no adverse reaction occurs 14 days after the attack, the protection rate is 100 percent, and the rabbit with 1/5 has the symptom of appetite deterioration although death does not occur after the rabbit is immunized by the vaccine prepared by the alpha-toxin protein truncation prepared by the patent of CN93107585, so the protection rate is 80 percent.
To further compare the immunogenicity of the α -toxin protein truncations we prepared with the α -toxin protein truncations prepared in the patent of the invention of CN93107585, we performed immune challenge experiments using Bal b/c mice. 2 vaccines were prepared according to the method of example 4, each vaccine having an alpha toxin protein truncation concentration of 100. mu.g/head. 15 Bal b/c mice were purchased and randomly divided into 3 groups of 5, 1 group of which was injected intramuscularly in the legs with 100. mu.l of the vaccine prepared from the alpha-toxin protein truncate we prepared (designated as experimental group 1), 1 group of which was injected intramuscularly in the legs with 100. mu.l of the vaccine prepared from the alpha-toxin protein truncate of the invention patent CN93107585 (designated as experimental group 2),1 group served as a blank control group, and 100. mu.l of PBS was injected intramuscularly in the legs. The virus challenge was carried out 21 days after immunization (C57-1 toxin challenge 1 mouse MLD) and 14 consecutive days were observed. Within 14 days of observation, the control group died completely, the protection rate was 0, none of the experimental group 1 died, and the appetite was good, the protection rate was 100%, 1 mouse died in the experimental group 2, and the protection rate was 80%.
From the results, the prepared alpha-toxin protein truncation body has good immunogenicity, can play a 100% protection role, has equivalent immune effect with the existing market vaccine (inactivated vaccine), and has better immunogenicity than the alpha-toxin protein truncation body prepared by the prior art. Because we use protein as antigen, the vaccine has obvious advantages compared with the existing inactivated vaccine: 1) the safety problem caused by incomplete inactivation does not exist; 2) the quality is controllable, and the difference between batches does not exist; 3) the production equipment and space requirements are low, the expression level is high, and the cost is low; 4) the risk of toxin (bacteria) dispersion does not exist, and the safety of production operators is guaranteed. In addition, the prepared alpha-toxin protein truncation can realize industrial production (whether industrial production can be highlighted or not is not highlighted in the prior art patent, and the specific yield can be reached is not clear), the expression yield of escherichia coli can reach 800mg/L, if the prior art is optimized, the expression yield can be further increased, so that the production cost can be remarkably reduced, and the popularization and the application of the vaccine are facilitated.
The invention is illustrated by the above examples, but it should be understood that the invention is not limited to the particular examples and embodiments described herein. These specific examples and embodiments are included to assist those skilled in the art in practicing the present invention. Further modifications and improvements will readily occur to those skilled in the art without departing from the spirit and scope of the invention and, accordingly, it is intended that the invention be limited only by the terms of the appended claims, along with the full scope of equivalents to which such terms are entitled.
Sequence listing
<110> Zhejiang Hilon Biotechnology Ltd
<120> bovine A-type clostridium perfringens subunit vaccine, and preparation method and application thereof
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<213> codon-optimized nucleotide sequence of truncated alpha-toxin protein (2 Ambystoma lateralex Ambystoma jeffersonanum)
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aagaacgtga aagaactggt tgcgtacatc agcaccagcg gcgagaagga tgcgggcacc 60
gacgattaca tgtacttcgg tatcaagacc aaggacggca aaacccagga gtgggaaatg 120
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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<211>354
<212>DNA
<213> codon-optimized nucleotide sequence of truncated alpha-toxin protein (2 Ambystoma lateralex Ambystoma jeffersonia)
<400>4
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> amino acid sequence of truncated alpha-toxin protein (2 Ambystoma laterale x Ambystomajeffersonanium)
<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
6570 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 (8)
1. A subunit vaccine of a bovine clostridium perfringens type A, which comprises an alpha-toxin protein truncation of the bovine clostridium perfringens type A and a pharmaceutically acceptable adjuvant, wherein the alpha-toxin protein truncation is an epitope amino acid sequence from amino acid 255 to amino acid 372 of an alpha-toxin protein, and the amino acid sequence of the alpha-toxin protein truncation is shown as SEQ ID NO. 3.
2. The subunit vaccine of claim 1, wherein the pharmaceutically acceptable adjuvant is ISA201VG adjuvant, and the weight ratio of the alpha-toxin protein truncation to the adjuvant is 1: 1.
3. The subunit vaccine of claim 1, wherein each head of the subunit vaccine contains 30-200 μ g of the alpha-toxin protein truncation.
4. The subunit vaccine of claim 3, wherein each serving of the subunit vaccine contains 100 μ g of the alpha-toxin protein truncation.
5. A method of preparing a truncation of the α -toxin protein of any one of claims 1 to 4, comprising the steps of:
1) carrying out codon optimization on the nucleotide sequence of the alpha-toxin protein to obtain a codon-optimized nucleotide sequence of the alpha-toxin protein, and amplifying the codon-optimized nucleotide sequence of the alpha-toxin protein by PCR to obtain a nucleotide sequence of an alpha-toxin protein truncation body;
2) inserting the nucleotide sequence of the alpha-toxin protein truncation body into a pET28a vector to obtain a recombinant vector; and
3) transferring the recombinant vector into E.coliBL21(DE3), inducing expression, and purifying to obtain alpha-toxin protein truncated body.
6. The method according to claim 5, wherein the nucleotide sequence of the alpha-toxin protein truncation amplified by PCR after codon optimization is shown as SEQ ID No. 1.
7. Use of a subunit vaccine according to any one of claims 1 to 4 in the manufacture of a vaccine for the treatment and prevention of clostridium perfringens type a in cattle.
8. Use of an alpha-toxin protein truncation as defined in any one of claims 1 to 4 in the preparation of a bovine clostridium perfringens type a subunit vaccine.
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