CN114588256A

CN114588256A - Development and application of porcine infectious pleuropneumonia subunit vaccine

Info

Publication number: CN114588256A
Application number: CN202210242795.1A
Authority: CN
Inventors: 牛旻; 杨瑞华; 贾宾; 李婧
Original assignee: Muyuan Foods Co Ltd
Current assignee: Muyuan Foods Co Ltd
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2022-06-07

Abstract

The invention relates to the field of vaccine development, in particular to development and application of a subunit vaccine for porcine infectious pleuropneumonia. The invention realizes the efficient soluble expression of three toxins ApxI, ApxII and ApxIII and the OMP protein of the outer membrane protein through the optimization of the protein fragment. Further, ApxI, ApxII truncation proteins exhibit strong biological activity on VERO cells. Compared with the inclusion body purification mode, the truncated body protein screened by the invention has good solubility and simple purification mode, thereby having stronger biological activity. The purified protein is mixed with Gel/206 adjuvant to prepare vaccine, BALB/C mice of 6 weeks are immunized, and the APP7 type is used for attacking the virus, so that the vaccine also shows good attacking and protecting effects. The invention provides the soluble, easy-to-prepare and high-activity antigen fragment of the porcine infectious pleuropneumonia, so that the preparation of the subunit vaccine of the bacteria is simpler and more efficient.

Description

Development and application of porcine infectious pleuropneumonia subunit vaccine

Technical Field

The invention relates to the field of vaccine development, in particular to development and application of a porcine infectious pleuropneumonia subunit vaccine.

Background

Porcine contagious pleuropneumonia (Porcine infectious pleuropneumonia) is a highly contagious respiratory disease, also known as Porcine contagious pleuropneumonia. Acute forms are characterized by acute hemorrhagic, cellulosic and chronic, cellulosic necrotic pleuropneumonia, and exhibit high mortality. The porcine contagious pleuropneumonia is a worldwide disease, is widely distributed in all pig-raising countries of the world, such as the United kingdom, Germany, Switzerland, Denmark, Australia, Canada, Mexico, Argentina, Sweden, Poland, Japan, America, China, and the like, causes huge economic loss to the intensive pig-raising industry, and is internationally recognized as one of important epidemic diseases harming the modern pig-raising industry. The porcine infectious pleuropneumonia pathogen is caused by actinobacillus pleuropneumoniae (APP). APP is gram-negative coccobacillus or gracile bacilli. According to the reaction of bacterial capsular polysaccharide and bacterial lipopolysaccharide on serum, the bacteria are divided into APP 1-APP 15 types, wherein APP1 is divided into APP1a and APP1b subtypes, and APP5 is divided into APP5a and APP5b subtypes. APP has many virulence factors, which can cause swine diseases, including capsular polysaccharide, protease, Apx toxin and Outer Membrane Protein (OMP), etc., wherein the Apx toxin and the outer membrane protein OMP play the most important roles in the occurrence of pleuropneumonia and are also main immunogenic substances.

The Apx toxin is a secreted protease, the most prominent virulence factor of APP, with four distinct partial Apx toxins found in 15 serotypes of APP, including: ApxI with the molecular weight of 105-110 KD, ApxII with the molecular weight of 103-105 KD, ApxIII with the molecular weight of 120KD and ApxIV with the molecular weight of 220 KD. Apx iv is present in all serotypes of APP, and the other 3 toxins are synthesized and secreted only by certain serotypes of APP. The Apx toxin has cytotoxic effects on alveolar macrophages, neutrophils and erythrocytes, and destroys the immune system of the organism, thereby proliferating and causing diseases of APP in the organism. Apx is a virulence factor for APP and is also an important protective antigen. OMP is another important virulence factor of APP, and antibodies against OMP have opsonic activity, so OMP is also an important protective antigen of APP.

In order to prevent and treat porcine infectious pleuropneumonia, APP bacteria are used for preparing vaccines in the industry: the APP vaccine is prepared by carrying out scale-up culture on APP, then inactivating the APP by using an inactivating agent and adding an immunologic adjuvant. The vaccine has large total protein content and has the problem of immune stress, and the use of the vaccine can lead to the introduction of excessive ineffective protein, thereby causing poor immune effect.

Another approach is to express and purify the important antigenic part of APP before use as an APP vaccine. The current research on subunit vaccines of porcine infectious pleuropneumonia is based on most of the major virulence factors of APP: apx toxin and OMP protein. The subunit vaccine avoids the problem of introducing excessive ineffective protein, but the APP antigen protein expressed by the tool bacteria mainly exists in the form of inclusion bodies, and the biological activity ratio of the protein purified and renatured by the inclusion bodies is poor, so that the subunit vaccine is difficult to use in production. In conclusion, the main problems in the development of the genetic engineering subunit vaccine are: the preparation and application of genetic engineering vaccines are limited due to the insolubility of the expression of target proteins, and the inclusion body proteins do not have biological activity and are difficult to exert immune effect, so that few vaccines are available in the market at present.

Therefore, in order to solve the problems of low activity of the inclusion body protein and complex preparation process, the solubility problem of the recombinant protein in the APP vaccine needs to be solved.

Disclosure of Invention

In view of this, the technical problem to be solved by the present invention is to provide a soluble antigenic protein of porcine infectious pleuropneumonia, and the present invention obtains a truncated protein with good solubility, easy preparation and high biological activity by optimizing fragments of three toxins, namely, ApxI, ApxII and ApxIII, and prepares a high-efficiency subunit vaccine of porcine infectious pleuropneumonia based on the truncated protein.

The invention provides an ApxI truncated protein which is characterized by having an amino acid sequence shown as SEQ ID NO. 1.

The invention also provides ApxII truncated protein which is characterized by having an amino acid sequence shown as SEQ ID NO. 2.

The invention provides nucleic acids encoding such truncation proteins.

The invention provides a vector, which is characterized by comprising a skeleton vector and a nucleic acid for encoding the truncated protein.

In some embodiments, the backbone vector is a pET-28s, pET32a, pETSUMO, phTO-BS, or PGE-6p-1 vector.

The invention also provides a recombinant host transformed with the vector.

In some embodiments, the host is escherichia coli BL 21.

The invention provides a preparation method of the truncated protein, which is characterized by comprising the following steps: culturing and inducing the recombinant host expression protein, wherein the induced expression temperature is 16-30 ℃.

In some embodiments, the culturing is to a bacterial OD₆₀₀And starting induction when the numerical value is 0.6-0.7, wherein the induction reagent is IPTG, the induction temperature is 25 ℃, and the induction time is 12-16 h.

The invention provides a porcine infectious pleuropneumonia vaccine, which comprises: ApxI truncation protein, ApxII truncation protein, and OMP protein.

Wherein the amino acid sequence of the ApxI truncated protein is shown as SEQ ID NO: 1.

Wherein the amino acid sequence of the ApxII truncated protein is shown in SEQ ID NO 2.

The amino acid sequence of the OMP protein is shown in SEQ ID NO. 4.

The vaccine also comprises ApxIII truncated protein, and the amino acid sequence of the ApxIII truncated protein is shown as SEQ ID NO. 3.

In the vaccine: the adjuvant is 206 adjuvant or Gel adjuvant, and the volume percentage of the adjuvant is 50%; the concentration of each protein was 25. mu.g/mL.

The invention also provides the truncated protein, a preparation method of the truncated protein, nucleic acid, a vector, a recombinant host and/or application of a vaccine in preparation of a medicament for preventing and treating porcine infectious pleuropneumonia.

The invention realizes the efficient soluble expression of three toxins ApxI, ApxII and ApxIII and the OMP protein of the outer membrane protein through the optimization of the protein fragment. Further, ApxI, ApxII truncation proteins exhibit strong biological activity on VERO cells. Compared with the inclusion body purification mode, the truncated body protein screened by the invention has good solubility and simple purification mode, thereby having stronger biological activity. The purified protein is mixed with Gel/206 adjuvant to prepare vaccine, BALB/C mice of 6 weeks are immunized, and the APP7 type is used for attacking the virus, so that the vaccine also shows good attacking and protecting effects. The invention provides the soluble, easy-to-prepare and high-activity antigen fragment of the porcine infectious pleuropneumonia, so that the preparation of the subunit vaccine of the bacteria is simpler and more efficient.

Drawings

FIG. 1 shows a schematic diagram of the pET-28s vector;

FIG. 2 shows a schematic diagram of the construction of pET-28s-APP-130 toxin fragments;

FIG. 3 shows a gel diagram of the expression of insoluble fragments ApxI, ApxII, ApxIII;

FIG. 4 shows purified gel maps of soluble fragments of ApxI, ApxII, ApxIII and OMP proteins;

FIG. 5 shows a diagram of a cell viability assay;

FIG. 6 shows an anatomical diagram of an experimental immune challenge mouse.

Detailed Description

The invention provides a porcine infectious pleuropneumonia subunit vaccine and a preparation method thereof, and a person skilled in the art can use the contents for reference and appropriately improve process parameters to realize the vaccine. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 expression vector construction

And (3) constructing expression vectors of ApxI, ApxII, ApxIII and OMP proteins. ApxI, ApxII and Apx III full-length proteins are not soluble in expression, so that truncation and modification are required to be carried out on a whole protein sequence, and the solubility of the protein is improved. The interception of fragments is mainly based on three points: 1) in the codon bias, the GC content of the N-terminal codon is ensured to be about 40 percent as much as possible. 2) Ensuring that the hydrophilicity of the initial expression segment, namely the N-terminal amino acid, is high so as to ensure the soluble form of the subsequent protein. 3) Structurally, the truncated fragments have relatively independent structures, and obvious skeleton connection does not exist between the fragments.

130 different fragments are cut out on the whole protein sequences of ApxI, ApxII and ApxIII according to the principle, and are connected with pET28S, pET32a, pETSUMO, phTO-BS and PGE-6p-1 vectors to construct expression vectors of APX (taking X as 1-130). The specific operation steps are as follows:

1. after extracting the pig farm pathological material genome, identifying the positive APP toxin by using a specific primer PCR, and sequencing to obtain a sequence.

2. After obtaining the full sequence of the APP mycotoxin, the original sequence isolated was optimized to facilitate better translation and expression of the protein at the mRNA level, according to amino acid degeneracy preferences.

3. Truncating different fragments according to the complete sequences of the ApxI, ApxII and ApxIII proteins, and respectively amplifying primers to obtain 130 fragments in total.

4. The 130 fragments were digested with BamHI and XhoI restriction enzymes, respectively, and the matched fragments were separated by agarose gel electrophoresis.

5. After extraction and preparation of PET28S, pET32a, pETSUMO, phTO-BS and PGE-6p-1 plasmids, restriction enzymes were similarly used, i.e., BamHI and XhoI restriction enzymes were used, and the fragments were separated and recovered by agarose gel electrophoresis.

6. The 130 fragments were ligated to the vector pET-28S, respectively.

7. Then the ligation was performed with T4 ligase at 16 ℃ for 12-16 h.

8. The following day the ligation products were transformed into BL21 competent cells and plated on ampicillin resistant plates for selection.

9. And identifying by using a specific primer after 14-16 h.

10. The primers identify the correct monoclonal strain and send it to sequencing company for sequencing.

11. The correct plasmid will be identified, named and stored. The naming mode is as follows: the sequences were named pET28S-APPX, pET32a-APPX, pETSUMO-APPX, phTO-BS-APPX, and PGE-6p-1-APPX, respectively, according to the names of the ligation vector and the ligation fragment (wherein X is 1 to 130, corresponding to the fragment described in step 3 of example 1).

Example 2 Induction of expression

The expression vectors of the truncation proteins obtained in example 1 were transformed into E.coli expression systems, respectively, and then induced to express and examined whether the truncation proteins were expressed. The specific operation steps are as follows:

all plasmids obtained in example 1 were transformed into competent cells of the expression strain BL21(DE3), respectively. Firstly, taking out the preserved competent cells at the temperature of-80 ℃, adding the PET28S-APX plasmid when the competent cells are just thawed, gently and uniformly blowing the competent cells, and completely placing the competent cells in ice for 25-30 min. And (3) opening a metal incubator or a water bath kettle in advance, setting the temperature to be 42 ℃, immediately carrying out heat shock treatment at 42 ℃ for 90s after ice bath is finished, ensuring that the PET28S-APX plasmid enters cells, and then putting the cells into ice again to close the competent cell membranes for 3-5 min. Adding 600uL of non-resistant culture medium into a clean bench, culturing at 37 ℃ and 220rpm for 1 hour to ensure that the positive transformants grow into the first generation, then uniformly coating 100uL of non-resistant culture medium on a solid culture medium with corresponding resistance, and then staying overnight in a biochemical incubator at 37 ℃ for about 12-15 hours.

② selecting positive monoclone and expanding culture, preserving glycerol bacteria or further expanding induction expression on the next day. Real-time monitoring of OD during inducible expression₆₀₀When the value is 0.6-0.7, cooling immediately, adding IPTG with final concentration of 0.2mmol/L into the culture medium, and culturing at 25 deg.C and 180rp m respectivelyInducing for 12-16h under the condition, and setting a culture group without IPTG as a negative control.

(iii) when OD₆₀₀When the numerical value is 0.6-0.8, taking 1ml of negative control bacterial liquid as an uninduced sample, and taking 1ml of induced bacterial liquid as an induced sample. Centrifuging at 8000g for 3min, discarding the supernatant, and applying OD₆₀₀The value is multiplied by 100 times (volume, mL) of PBS to resuspend the thalli, 30 mu L of the resuspended thalli is sucked into a new centrifugal tube, equal volume of 2 Xprotein loading buffer (loading buffer) is added to mix evenly, after metal bath for 10min at 99 ℃, the mixture is centrifugated instantly, and then SDS-PAGE detection is carried out.

Example 3 cell treatment

The induced bacterial liquid of the truncated body protein obtained by screening in example 2 was further processed to determine whether the truncated body protein is expressed in inclusion bodies or soluble. The specific operation steps are as follows:

50ml of the bacterial solution is put into 8000g, centrifuged for 20min, the supernatant is discarded, the bacterial cells are washed three times by using a resuspension solution (2mmol/L Tris-HCl, 150mmol/L NaCl, pH 7.4), centrifuged for 20min at 8000g, the supernatant is discarded, the precipitate is resuspended by using 10ml of a lysis solution (20mmol/L Tris-HCl, 150mmol/L NaCl, 2% Triton X-100, 0.1mM PMSF, pH 7.4), and the suspension is subjected to ultrasonic disruption (lysozyme can be selectively added or freeze-thaw treatment can be repeatedly carried out before ultrasonic disruption).

② in order to prevent the protein destruction, the heavy suspension must be placed in the ice water mixture during the whole ultrasound process. Setting the parameters of the ultrasonic crusher to be on 5 s; off 7 s; total 5-10 min; the power was 35% x 100W.

③ after the crushing is finished, centrifuging for 30min at 12000g and 4 ℃. 30. mu.L of the supernatant was sampled by adding 30. mu.L of 2 × loading buffer (10 min at 99 ℃), and the pellet was resuspended in 10ml of lysis solution (here, 30. mu.L of the mixture was sampled by adding 30. mu.L of 2 × loading buffer and 10min at 99 ℃). SDS-PAGE detection was performed.

(iv) SDS-PAGE detection

The samples prepared four times above were spotted with 10. mu.L of each protein. In protein electrophoresis, the sample is first concentrated by electrophoresis in 5% concentration gel at 80V, and then protein separation is carried out in 10% separation gel at 120V. And after glue running, putting the gel into a Coomassie brilliant blue staining solution for staining for 2 hours, after staining is finished, recovering the Coomassie brilliant blue staining solution, then putting the protein gel into a destaining solution for destaining, and replacing the destaining solution in time until the background color is washed away. And (4) carrying out gray scanning on the bands by using Image J software, and calculating data such as specific gravity of the target protein and soluble expression level of the target protein.

The existing research results show that ApxI, ApxI and ApxIII proteins and truncation bodies thereof are often expressed in the form of inclusion bodies, and the soluble expression of the proteins and the truncation bodies is always a problem to be solved. The invention uses different vectors to express 130 truncates designed in the example 1, and the result shows that after codon optimization, most truncates can be expressed in an escherichia coli expression system, and a small part of truncates can not be expressed. In the expressed truncated body, more than 90% of the truncated body is expressed in the form of inclusion body, which is consistent with the existing research result.

The expression of partial truncates in different vectors is shown in Table 1, where the codon-unoptimized column represents the expression of the fragment when the codon is unoptimized and the other columns represent the expression of the codon-optimized fragment linked to the corresponding vector. It can also be seen from table 1 that truncated bodies of ApxI, ApxIII proteins are expressed mainly as inclusion bodies and a small portion in soluble form.

Additionally, expression purified SDS-PAGE protein gel images of several representative truncations are shown in FIG. 3, where the English letters are the exemplary truncation numbers and the Arabic numerals are the lane numbers. The 'whole bacteria' is a strain sample after induction expression, the 'supernatant' is a supernatant sample after centrifugation of broken bacteria, the 'precipitate' is a precipitate sample after centrifugation of broken bacteria, and the 'elution' is a sample eluted after NTA adsorption of the supernatant. As can be seen from FIG. 3, the truncated bodies a, b, c, e, h, i, j, k, l, p and q were substantially fully expressed in the inclusion bodies, a large number of bands of interest were present in the pellet, and substantially no bands of interest were present in the supernatant or eluted samples. Other non-displayed truncations expressed in inclusion bodies were expressed similarly to these. Both the supernatants and pellets of truncations m and r had comparable bands of interest, these truncations were expressed partially in the supernatant and partially in inclusion bodies, and the other undelayed truncations "partially expressed in inclusion bodies" had a similar proteoglial pattern to these. While the pellet of truncations d, n and o has substantially only a very small number of bands of interest, mostly expressed in the supernatant, these can be used as alternative truncations for the subsequent steps, and the other non-displayed protein gel maps of truncations "expressed in the supernatant, soluble" are similar to these.

In summary, only a very small portion of the 130 truncations designed in example 1 was expressed in soluble form. In the invention, among the fragments capable of being expressed, a truncated ApxI with higher expression quantity and containing more antigen segments (predicted by software) is selected_691-1031、ApxII_22-907、ApxIII_438-737As an alternative antigen, further separation, purification and activity test can be carried out.

TABLE 1 truncated protein Induction expression

EXAMPLE 4 protein purification

Further purification of OMP proteins and ApxI from the screening of example 3_691-1031、ApxII_22-907、ApxIII_438-737Soluble truncation proteins. The method comprises the following specific steps:

protein purification is carried out by adopting an affinity chromatography (Ni-NTA gravity column), and the purification steps (whole low-temperature treatment) are as follows:

firstly, liquid preparation:

balance liquid: 20mmol/L Tris-Hcl, 150mmol/L Nacl, pH 7.4;

washing the impurities: 20mmol/L Tris-Hcl, 150mmol/L Nacl, 20mM imidazole, pH 7.4;

eluent: 20mmol/L Tris-HCl, 150mmol/L NaCl, 500mM imidazole, pH 7.4.

Secondly, washing the column by using 5-10 CV pure water;

thirdly, balancing the column by using 5-10 CV balancing liquid;

adding a protein sample (no more than 10CV), and collecting the flow-through liquid;

5-10 CV of balancing liquid is used for balancing the column;

sixthly, washing the column by using 5-10 CV impurity washing liquid (the impurity washing concentration is required to be groped);

seventhly, washing the column by using 0.5CV eluent, and collecting the eluent;

and (8) washing the Ni column with 5-10 CV eluent after the Ni column is used, washing the column with 5-10 CV pure water, finally sealing with 20% ethanol, and storing at 4 ℃.

In the process, the sample is analyzed by SDS-PAGE to analyze the purification effect. As shown in fig. 1: the protein can be obtained by purifying Ni-NAT, and the recovery efficiency can be further improved after condition optimization.

Ninthly, nickel removal and regeneration of the nickel column are carried out according to the using condition:

a. and washing the column with 5-10 CV pure water.

b. Washing with 5-10 CV 0.02M Tris-HCl, 0.1M EDTA, pH 7.4, and washing with 5-10 CV pure water.

c. Washing with 5-10 CV 1.0M NaOH, standing for 10 hours, and washing with purified water to neutrality.

d. And (3) washing with 5-10 CV 0.1M NiSO4, standing for 0.5 hour, and washing with purified water until the flowing liquid does not contain nickel sulfate (green liquid).

e. And washing the mixture with 5-10 CV of 20% ethanol and then storing the mixture.

Purification of ApxI_691-1031、ApxII_22-907、ApxIII_438-737And OMP proteins as shown in figure 4. The left-most panel is purified ApxI_691-1031Four samples of protein, which had good solubility and higher purity. The middle graph is ApxII_22-907And ApxIII_438-737The lane from left is: ApxII_22-907Post-induction samples, disrupted supernatant, purified protein samples; ApxIII_438-737Post-induction samples, disrupted supernatant, purified protein samples. Thus, after NTA adsorption, ApxII with higher purity can be obtained by purification_22-907And ApxIII_438-737A protein. The right-most panel is the protein gel diagram of the OMP protein purification, from left to right, the uninduced sample, the induced expression sample, the disrupted supernatant, the disrupted precipitate, the NTA adsorption flow-through sample, and the eluted protein sample. It was observed that high purity soluble OMP protein was obtained after elution.

Toxin 1, toxin 2, toxin 3 and outer membrane protein OMP of actinobacillus pleuritis can be expressed in colibacillus in the form of inclusion body, and after denaturation and renaturation treatment, the protein with certain activity can be obtained by NTA purification. According to the invention, soluble expression ApxI, ApxII and ApxIII fragments are obtained by screening 130 protein fragments, and soluble protein with higher purity is obtained after further purification. These high purity soluble proteins can be used for further activity verification and animal experimental verification.

Example 5 verification of protein Activity

OMP protein is mycoprotein and has no cytotoxic function. ApxI, ApxII and ApxIII are bacterial toxins, the immunogenicity of the bacterial toxins comes from the toxin injection of animals, and high antibodies generated after the bacterial toxins are tolerated and aimed at the toxins protect animal bodies; therefore, the protein function verification is carried out preliminarily to judge whether the protein has biological activity, and if the protein has corresponding functions, the purified protein can be preliminarily judged to have normal activity.

Separately taking purified ApxI_691-1031、ApxII_22-907、ApxIII_438-737And (3) quantifying protein, filtering and sterilizing through a 0.22um filter membrane, adding the protein into VERO cells with good growth state (cultured in a 6-well plate and 2mL of cell culture medium until the confluence degree is 60% -70%) according to the volumes of 1uL, 5uL, 10uL, 20uL, 50uL and 100uL shown in figure 5, uniformly mixing, culturing in a 37-degree incubator, observing once every 6h, and recording the cell state.

The result shows that ApxIII_438-737The protein has a very weak cytotoxic effect and no apparent macroscopic results are obtained at the cellular level. And toxin fragment ApxI_691-1031、ApxII_22-907The two protein fragments have higher biological activity, and can be presumed to have more complete protein functions (as shown in figure 5), have the potential to have better immunogenicity.

Example 6 animal immune challenge protection experiment

As shown in table 2, different components of vaccine A, B, C, D were prepared by mixing proteins and adjuvants, and used to perform challenge protection experiments on mice in order to evaluate the challenge protection effect of homemade APP vaccine on mice. The specific operation steps are as follows:

ApxI obtained by purification in example 4_691-1031、ApxII_22-907、ApxIII_438-737And OMP protein, respectively adding 50% (V/V) of 206 adjuvant or 15% (V/V) of Gel adjuvant, and emulsifying for 30 min. After the emulsion is ensured to be uniform and not to be layered, BALB/C mice of 6 weeks are immunized by self-made seedlings A, B, C, D respectively, and 2 times of boosting immunization is carried out 3 weeks after the first immunization. 15 days after immunization, challenge was performed using APP7 type strain to verify the protection rate, all mice were subjected to a dissection 10 days after challenge, visceral lesions of the mice were observed, and the results are shown in fig. 6 and are counted in table 2.

As shown in table 2, the challenge protection experiment results show that: the protection rates of the A \ B \ C groups are all 100 percent, and the protection rate of the D group is 66.67 percent; the commercial shoot control had only a 33.33% poor protection, followed by a blank control. The homemade APP vaccine shows a good immune protection effect after APP7 type toxicity challenge.

TABLE 2 APP vaccine immunization/challenge protection Experimental Table

The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention.

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Ser Ser Gly Lys Ala Tyr Val Asp Tyr Phe Gln Glu Gly Lys Leu Leu

65 70 75 80

Glu Lys Lys Pro Asp Asp Phe Ser Lys Val Val Phe Asp Pro Thr Lys

85 90 95

Gly Glu Ile Asp Ile Ser Asn Ser Gln Thr Ser Thr Leu Leu Lys Phe

100 105 110

Val Thr Pro Leu Leu Thr Pro Gly Thr Glu Ser Arg Glu Arg Thr Gln

115 120 125

Thr Gly Lys Tyr Glu Tyr Ile Thr Lys Leu Val Val Lys Gly Lys Asp

130 135 140

Lys Trp Val Val Asn Gly Val Lys Asp Lys Gly Ala Val Tyr Asp Tyr

145 150 155 160

Thr Asn Leu Ile Gln His Ala His Ile Ser Ser Ser Val Ala Arg Gly

165 170 175

Glu Glu Tyr Arg Glu Val Arg Leu Val Ser His Leu Gly Asn Gly Asn

180 185 190

Asp Lys Val Phe Leu Ala Ala Gly Ser Ala Glu Ile His Ala Gly Glu

195 200 205

Gly His Asp Val Val Tyr Tyr Asp Lys Thr Asp Thr Gly Leu Leu Val

210 215 220

Ile Asp Gly Thr Lys Ala Thr Glu Gln Gly Arg Tyr Ser Val Thr Arg

225 230 235 240

Glu Leu Ser Gly Ala Thr Lys Ile Leu Arg Glu Val Ile Lys Asn Gln

245 250 255

Lys Ser Ala Val Gly Lys Arg Glu Glu Thr Leu Glu Tyr Arg Asp Tyr

260 265 270

Glu Leu Thr Gln Ser Gly Asn Ser Asn Leu Lys Ala His Asp Glu Leu

275 280 285

His Ser Val Glu Glu Ile Ile Gly Ser Asn Gln Arg Asp Glu Phe Lys

290 295 300

Gly Ser Lys Phe Arg Asp Ile Phe His Gly Ala Asp Gly Asp Asp Leu

305 310 315 320

Leu Asn Gly Asn Asp Gly Asp Asp Ile Leu Tyr Gly Asp Lys Gly Asn

325 330 335

Asp Glu Leu Arg Gly Asp Asn Gly Asn Asp Gln Leu Tyr Gly Gly Glu

340 345 350

Gly Asp Asp Lys Leu Leu Gly Gly Asn Gly Asn Asn Tyr Leu Ser Gly

355 360 365

Gly Asp Gly Asn Asp Glu Leu Gln Val Leu Gly Asn Gly Phe Asn Val

370 375 380

Leu Arg Gly Gly Lys Gly Asp Asp Lys Leu Tyr Gly Ser Ser Gly Ser

385 390 395 400

Asp Leu Leu Asp Gly Gly Glu Gly Asn Asp Tyr Leu Glu Gly Gly Asp

405 410 415

Gly Ser Asp Phe Tyr Val Tyr Arg Ser Thr Ser Gly Asn His Thr Ile

420 425 430

Tyr Asp Gln Gly Lys Ala Ser Asp Ser Asp Lys Leu Tyr Leu Ser Asp

435 440 445

Leu Ser Phe Asp Asn Ile Leu Val Lys Arg Val Asn Asp Asn Leu Glu

450 455 460

Phe Arg Ser Asn Asn Asn Ser Asn Ser Gly Val Leu Thr Ile Lys Asp

465 470 475 480

Trp Phe Lys Gly Gly Asn Ser Tyr Asn His Lys Ile Glu Gln Ile Val

485 490 495

Asp Lys Asn Gly Arg Lys Leu Thr Ala Gly Asn Leu Gly Asn Asn Phe

500 505 510

His Asp Thr Gln Gln Ala Ser Ser Leu Leu Lys Asn Val Thr Gln Glu

515 520 525

Gln Asn Glu Ser Asn Leu Ser Ser Leu Lys Thr Glu Leu Gly Lys Ile

530 535 540

Ile Thr Asn Ala Gly Asn Phe Gly Val Ala Lys Gln Gly Asn Thr Gly

545 550 555 560

Ile Asn Thr Ala Ala Leu Asn Asn Glu Val Asn Lys Ile Ile Ser Ser

565 570 575

Ala Asn Thr Phe Ala Thr Ser Gln Leu Gly Gly Ser Gly Met Gly Thr

580 585 590

Leu Pro Ser Thr Asn Val Asn Ser Met Met Leu Gly Asn Leu Ala Arg

595 600 605

Ala Ala

610

<210> 4

<211> 392

<212> PRT

<213> Susscrofa domestica

<400> 4

Met Ser Ser Gly Leu Val Pro Arg Gly Ser His Met Ala Ser Met Thr

1 5 10 15

Gly Gly Gln Gln Met Gly Arg Gly Ser Glu Phe Met Asn Ile Ala Thr

20 25 30

Lys Leu Ile Ala Gly Leu Val Ala Gly Leu Val Leu Thr Ala Cys Ser

35 40 45

Gly Gly Gly Ser Ser Gly Ser Ser Pro Lys Pro Asn Ser Glu Ser Thr

50 55 60

Pro Lys Val Asp Met Ser Ala Pro Lys Ala Glu Gln Pro Lys Lys Glu

65 70 75 80

Glu Ala Pro Gln Ala Asp Ser Pro Lys Ala Glu Lys Pro Lys Ser Ile

85 90 95

Ala Pro Leu Met Met Glu Asn Pro Lys Val Glu Lys Gln Lys Glu Asn

100 105 110

Asn Leu Gln Glu Lys Ser Pro Lys Ala Asp Glu Pro Gln Val Met Asp

115 120 125

Pro Lys Leu Gly Ala Pro Gln Lys Asp Asp Gln Lys Leu Glu Glu Pro

130 135 140

Lys Asn Lys Ser Asn Ala Glu Ile Leu Lys Glu Leu Gly Ile Lys Asp

145 150 155 160

Ile Lys Thr Gly Ile Ile Thr Arg Ser Asp Val Val Leu Asn Leu Thr

165 170 175

Leu Asp Glu Gln Glu Asn Ile Gln Ile Arg Leu Ser Glu Ser Asp Ile

180 185 190

Val Arg Asn Asp Leu Lys Ile Thr Asn Thr Ile Pro Asn Gln Asp Ile

195 200 205

Arg Thr Leu Lys Asp Ser Thr Gly Arg Leu Leu Gly Tyr Tyr Gly Tyr

210 215 220

Met Gln Leu Asn Gln Val Arg Glu Gly Glu Arg Tyr Gly Ile Asn Asn

225 230 235 240

Val Asp Leu Val Gly His Tyr Leu Leu Ser Met Asp Glu Ser Thr Lys

245 250 255

Thr Ala Pro Asn Lys Ser Ile Glu Tyr Arg Gly Lys Met Leu Tyr Gly

260 265 270

Tyr Lys Asn Val Asp Asn Arg Asn Leu Val Ala Asp Val Gln Ala Ser

275 280 285

Tyr Asn His Ser Asp Lys Lys Leu Ser Met Glu Ile Phe Gly Asp His

290 295 300

Gly Asp Tyr Trp Lys Leu Gly Ala Ile Gly Asn Asn Arg Leu Pro Lys

305 310 315 320

Asp Met Val Thr Gly Val Val Val Asp Lys Asp Gly Thr Ile Ser Asn

325 330 335

Ala Gly Leu Tyr Ser Lys Ile Asp Asn Thr Pro Gly Lys Leu Thr Pro

340 345 350

Asp Ala Thr Phe Ser Gly Gly Ile Phe Gly Lys Asn Gly Asp Val Leu

355 360 365

Ala Gly Ser Ala Asp Gly Lys Asn Trp Gln Gly Val Ile Gly Ala Thr

370 375 380

Ala Thr Thr Lys Glu Asp Lys Lys

385 390

Claims

1. A porcine infectious pleuropneumonia vaccine, comprising: ApxI truncated protein, ApxII truncated protein and OMP protein;

wherein the amino acid sequence of the ApxI truncated protein is shown as SEQ ID NO:1,

the amino acid sequence of the ApxII truncated protein is shown in SEQ ID NO 2.

2. The vaccine of claim 1, wherein the amino acid sequence of the OMP protein is set forth in SEQ ID NO 4.

3. The vaccine of claim 1 or 2, further comprising an ApxIII truncation protein having the amino acid sequence set forth in SEQ ID No. 3.

4. The vaccine of claim 1, comprising:

the adjuvant in the vaccine is 206 adjuvant or Gel adjuvant;

the concentration of each protein in the vaccine was 25. mu.g/mL.

ApxI truncation protein having an amino acid sequence shown in SEQ ID NO. 1.

The ApxII truncated protein is characterized by having an amino acid sequence shown as SEQ ID NO. 2.

7. A nucleic acid encoding the truncation protein of claim 5 or 6.

8. A vector comprising a backbone vector and the nucleic acid of claim 7.

9. A recombinant host transformed with the vector of claim 8.

10. A method for producing the truncated protein of claim 6 or 7, comprising: culturing and inducing the recombinant host expression protein of claim 9, wherein the induced expression temperature is 16-30 ℃.