JP2004242608A - Nontoxic variant pasteurella multocida toxin, gene thereof and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nontoxic variant Pasteurella multocida toxin (PTM) having entirely eliminated toxic activity and keeping the immunogenicity to swine to utilize the toxin as a main component of a medicinal composition such as a vaccine for preventing swine atrophic rhinitis; and to provide a method for producing the toxin. <P>SOLUTION: The entirely detoxified variant PTM is obtained by substituting serine present at 1,164th position and cysteine present at 1,165th position of a specific amino acid sequence of the PTM with other amino acids. The nontoxic variant PMT can be expressed in large amount by a genetic means, and can be purified by the provided production method. The nontoxic variant PMT can be utilized as a main component of a vaccine for preventing the swine atrophic rhinitis. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００６２】
表１に示した結果から明らかなとおり、モルモット皮膚壊死活性については、野生型ＰＭＴでは１６０倍希釈まで壊死斑の形成が認められたのに対し、変異型ＰＭＴでは原液においても壊死斑の形成は認められなかった。このように、変異型ＰＭＴにおいてモルモット皮膚壊死活性は消失しているものと考えられた。
【００６３】
マウス致死活性の検査には、４週齢のｄｄＹマウス５０匹を使用した。野生型ＰＭＴおよび変異型ＰＭＴをマウス１匹あたり１ｍｇ、１００μｇ、１０μｇ、１μｇおよび１００ ｎｇとなるよう調整し、各タンパク量当り５匹のマウスの腹腔内に接種した。接種後１週間観察し、生死を判定した。その結果を表２に示す。
【００６４】
【表２】

【００６５】
表２に示した結果から明らかなように、マウス致死活性については、野生型ＰＭＴでは、１ｍｇおよび１００μｇ注射群では５頭中全頭が、１０μｇ注射群では５頭中１頭が死亡した。一方、変異型ＰＭＴでは、最高量の１ｍｇ注射群においても死亡は認められなかった。本成績からＬＤ_５０（５０％致死量）は、野生型ＰＭＴで２０μｇタンパク量であったのに対して、変異型ＰＭＴでは３２００μｇタンパク量以上と計算された。このように、変異型ＰＭＴはマウス致死活性を示さなかった。
【００６６】
以上の成績から、変異型ＰＭＴではモルモットの皮膚壊死活性およびマウスの致死活性のいずれも検出されず、当該部位のアミノ酸置換により、毒素活性を完全に消失することが判明した。
【００６７】
（４）変異型ＰＭＴの免疫原性
作製された無毒変異型ＰＭＴが豚に対する免疫原性を保持しているかどうかを調べるため、無毒変異型ＰＭＴで免疫した豚を野生型ＰＭＴで攻撃することにより、その防御効果を調べた。
【００６８】
約５週齢のＢｂおよびｔＰｍフリー豚５頭を用いた。３頭は免疫群とし、１回あたり無毒変異型ＰＭＴの１６μｇタンパク量を水酸化アルミニウムゲルアジュバントとともに３週間隔で２回、筋肉内に注射した。残り２頭は非注射対照群とした。第２回注射後３週に免疫群の豚を、非注射対照群とともに体重１ｋｇあたり４μｇタンパク量の野生型ＰＭＴを筋肉内に注射することにより攻撃した。攻撃後２週に剖検して、鼻甲介に形成された病変の程度を肉眼的に観察した。また両群の豚について免疫前と攻撃時に採血を行い、血液中に産生されたＰＭＴに対する抗体をＥＬＩＳＡによって調べた。変異型ＰＭＴ免疫豚において、免疫時に発熱、沈うつ、食欲不振等の臨床的異常は認められず、また注射局所にも腫脹、発赤等の異常な反応はなく、変異型ＰＭＴの無毒化が完全であることが確認された。
【００６９】
抗体検査において、免疫前の血清では全例でＰＭＴに対する抗体は検出されなかった。免疫群では、第２回注射後２週の攻撃時には全例で抗体陽性となり、平均Ｅ値も０．２９まで上昇した（表３）。一方、対照群の２頭は陰性で経過した。
【００７０】
野生型ＰＭＴによる攻撃後３週に剖検して鼻甲介を肉眼的に調べた。その結果を図２に示す。免疫群の３頭にはいずれも萎縮性鼻炎を示す病変は認められなかった。一方で非免疫対照群の２頭ではそれぞれスコア３及び２の病変（鼻甲介の消失）が認められた（表３および図２）。
【００７１】
【表３】

【００７２】
このように、本発明において作製した無毒変異型ＰＭＴで免疫された豚は、ＰＭＴに対する抗体が誘導され、野生型ＰＭＴによる攻撃から防御されることが示された。
【００７３】
【発明の効果】
請求項１ないし３および請求項８に係る本発明は、毒素活性を発現せず豚萎縮性鼻炎に対する免疫活性を有するタンパク質であり、豚に免疫したときに野生型ＰＭＴによる攻撃から豚を防御でき、請求項１２に記載した豚萎縮性鼻炎に対するワクチンとして用いることができる。
【００７４】
また、請求項６または７に係る本発明によれば、この無毒変異型ＰＭＴをコードする遺伝子が得られ、請求項９および１０の遺伝学的手法により発現させることで、当該タンパク質を大量に得ることができる。さらに、請求項１１に記載した方法で精製することにより、ワクチンの成分として好適な不純物のない無毒変異型ＰＭＴを得ることができる。
【図面の簡単な説明】
【図１】野生型（Ｗｉｌｄ ｔｙｐｅ）および変異型（Ｍｕｔａｎｔ）ＰＭＴを陰イオン交換カラムクロマトグラフィーにより分画後、ピーク１、ピーク２および回収フラクションについてＳＤＳ−ポリアクリルアミドゲル電気泳動を行った時の電気泳動写真。
【図２】変異型ＰＭＴ免疫豚３頭および非注射対照豚２頭に対して野生型ＰＭＴで攻撃後、３週目に剖検して鼻甲介に形成された病変を示す写真。
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a nontoxic mutant toxin produced by genetically substituting some amino acids of Pasteurella multocida toxin, which is a toxin causing swine atrophic rhinitis. More specifically, the present invention relates to a nontoxic mutant toxin as an immunogen for providing a pharmaceutical composition such as a vaccine for preventing atrophic rhinitis in pigs upon immunization, and a method for producing the same.
[0002]
[Prior art]
[Non-patent document 1]
Petersen et. al, Infect. Immun. , 59, 1387-1393, 1991.
[Non-patent document 2]
Nielsen et. al. , Can. J. Vet. Res. , 55, 128-138, 1991.
[Non-Patent Document 3]
Ward et. al. , Infect. Immun. , 5636-5642, 1998.
[0003]
Atrophic rhinitis in pigs is a chronic disease characterized by atrophy or deformation of the turbinates and has caused enormous economic losses to the pig industry. The cause of this disease is a mixture of the gram-negative bacteria Bordetella bronchiseptica (Bb) and toxin-producing Pasteurella multitocida (tPm), or any of the infections, which causes abnormalities in the respiratory system, followed by delayed growth, feed efficiency. Often leads to significant economic losses, such as reduced infection and the induction of secondary infections. The prevalence of this disease in Japan is also high.
[0004]
Among them, Pasteurella multocida toxin (hereinafter abbreviated as "PMT") produced by tPm is an important cause of this disease. PMT is a protein consisting of 1285 amino acids with a molecular weight of 146 kDa (SEQ ID NO: 1), has strong toxicity, and has a strong cytopathic effect on various cultured cells. Injection into various animals such as mice and guinea pigs intravenously or intramuscularly kills them. In addition, when inoculated subcutaneously in the back of a guinea pig, necrotic spots are formed at the inoculated site within 2 to 3 days, and this reaction is often used for quantifying the amount of toxin. When the pigs are injected with the toxin, the pigs are killed at high concentrations and the nasal turbinate atrophy and nasal septum deformation are observed at low concentrations, similar to those seen in field cases. The formation of the turbinate lesion is considered to be caused by abnormal enhancement of bone resorption due to activation of osteoclasts by the action of toxin.
[0005]
In order to prevent atrophic rhinitis in swine caused mainly by PMT, it is effective to impart sufficient immunity to PMT to swine herds. In Japan, several types of vaccines containing components (toxoids) detoxified by chemically modifying PMT are commercially available and widely used for the prevention of this disease. However, the amount of PMT produced by this tPm is small, and it is extremely difficult to obtain a sufficient amount of pure PMT through a purification step. In addition, the operation of cultivating a large amount of bacteria retaining the toxin activity of PMT and preparing a large amount of the toxin through various operations is very dangerous.
[0006]
Therefore, researchers in various countries have searched for new molecules that are nontoxic and retain immunogenicity by changing the amino acid sequence of the toxin molecule itself by using genetic engineering techniques. Petersen et al. Used two restriction enzyme Bcl I cleavage sites present at the 5 'end of the structural gene present in PMT to delete the gap between them and deleted a 121 amino acid present in the N-terminal region. PMT was created (Non-Patent Document 1). This molecule was confirmed to have lost toxicity and to retain immunogenicity.
[0007]
When Nielsen et al. Applied this molecule to a vaccine component, it was confirmed that the same efficacy as that of existing formalin-inactivated PMT was exhibited (Non-Patent Document 2). However, since the defective mutant PMT lacks amino acids, it is not able to obtain immunogenicity higher than that of the PMT toxoid, and its production is larger than that of the wild-type PMT, but it is not sufficient yet. Was.
[0008]
For this reason, an attempt was made to create a mutant PMT by replacing a specific amino acid without changing the size of the entire molecule. Ward et al. Reported that when cysteine at position 1165 was replaced with serine, no toxicity was observed in inoculation tests on cultured cells and pigs (Non-Patent Document 3). However, when the mutant PMT was intradermally injected into guinea pigs by the present inventors, formation of necrotic spots was observed at the injection site although weak, and detoxification was not complete. Also, the immunogenicity of this mutant PMT has not been investigated at present.
[0009]
[Problems to be solved by the invention]
From the above circumstances, it was necessary to create a completely detoxified mutant PMT which did not show any abnormality at the inoculated site even when inoculated in guinea pig skin. At that time, it is an essential condition that the immunogenicity is not impaired by this amino acid substitution in order to use it as a component of a vaccine for swine atrophic rhinitis.
[0010]
[Means for Solving the Problems]
In view of the above problems, the present inventors have aimed at providing an effective vaccine containing a mutant PMT which is completely nontoxic by amino acid substitution as a component and a method for producing the same. By substituting serine at position 1164 and cysteine at position 1165 with other amino acids, it was possible to produce a complete nontoxic mutant PMT which had no toxicity even by guinea pig subcutaneous injection. Furthermore, the present inventors have found that pigs injected with the purified nontoxic mutant PMT as a vaccine component can completely prevent the onset of the disease in response to wild-type PMT attack, thereby leading to the present invention.
[0011]
That is, the present invention has the following points.
[0012]
(1) A pig without expressing toxin activity, wherein the serine at position 1164 and the cysteine at position 1165 are substituted with other amino acids in the polypeptide of Pasteurella multocida toxin represented by SEQ ID NO: 1. A nontoxic mutant Pasteurella multocida toxin having immunological activity against atrophic rhinitis.
[0013]
(2) A nontoxic mutant Pasteurella multocida toxin which does not express toxin activity and has immunological activity against swine atrophic rhinitis, which is a polypeptide represented by SEQ ID NO: 4.
[0014]
(3) A Pasteurella multocida toxin characterized by comprising a region which does not express toxin activity in the polypeptide of the non-toxic mutant Pasteurella multocida toxin according to the above item 1 or 2 and has an immunological activity against swine atrophic rhinitis. Originating partial polypeptide.
[0015]
(4) a pig that does not exhibit toxin activity, wherein the serine at position 1164 and the cysteine at position 1165 are replaced with other amino acids in the polypeptide of Pasteurella multocida toxin represented by SEQ ID NO: 1. A method for producing a nontoxic mutant Pasteurella multocida toxin having immunological activity against atrophic rhinitis.
[0016]
(5) The production of the nontoxic mutant Pasteurella multocida toxin according to claim 4, wherein the amino acid that substitutes serine at position 1164 is alanine, and the amino acid that substitutes cysteine at position 1165 is serine. Method.
[0017]
(6) A gene encoding the following polypeptide (a), (b) or (c).
(A) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3
(B) a polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of (a), and which does not express toxin activity and has immunological activity against swine atrophic rhinitis
(C) a partial polypeptide derived from Pasteurella multocida toxin, which comprises a region having no immunological activity against swine atrophic rhinitis without expressing the toxin activity in the amino acid sequence of (a).
[0018]
(7) A gene comprising the following polynucleotide (a) or (b):
(A) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 2
(B) a polynucleotide that hybridizes under stringent conditions to the polynucleotide comprising the nucleotide sequence of (a) above
[0019]
(8) The following recombinant protein of (a) or (b).
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3
(B) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the above amino acid sequence (a), and which does not express toxin activity and has immunological activity against swine atrophic rhinitis
(C) a partial polypeptide derived from Pasteurella multocida toxin, which comprises a region having no immunological activity against swine atrophic rhinitis without expressing the toxin activity in the amino acid sequence of (a).
[0020]
(9) A recombinant expression vector comprising the gene described in the above item 6 or 7.
[0021]
(10) A transformant characterized by being transformed with the expression vector according to the above item 9 to produce the recombinant protein according to the above item 8.
[0022]
(11) A method for producing a nontoxic mutant Pasteurella multocida toxin, which comprises culturing the transformant according to the above item 10 and producing the recombinant protein according to the above item 8 in the transformant.
[0023]
(12) A vaccine for swine atrophic rhinitis, comprising the recombinant protein according to item 8 as an active ingredient.
[0024]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, Pasteurella multocida toxin (hereinafter abbreviated as “nontoxic mutant PMT”) which does not express toxin activity and has immunological activity against swine atrophic rhinitis is a PMT having 1825 amino acids represented by SEQ ID NO: 1. It is a PMT mutant in which serine at position 1164 and cysteine at position 1165 in the peptide have been substituted with other amino acids.
[0025]
The amino acid to be substituted may be any one as long as the mutant PMT does not exhibit toxin activity and has immunological activity against swine atrophic rhinitis. For example, the serine at position 1164 and the cysteine at position 1165 of PMT are replaced with serine and alanine, respectively. Preferred variants are preferred.
[0026]
The amino acid sequence of a preferred nontoxic mutant PMT is shown in SEQ ID NO: 4.
[0027]
This non-toxic mutant PMT includes a mutant in which one or more other amino acids are substituted in addition to the two amino acids as long as it does not express toxin activity and has immunological activity against swine atrophic rhinitis, The PMT molecule including these mutations may be the entire molecule, or may be a partially added or deleted mutant.
[0028]
Specifically, the amino acids 1164 and 1165 are located in the active center of the present toxin, and mutation of these 2 amino acids is essential for detoxification and maintenance of immunogenicity of the present toxin. However, for amino acids located elsewhere in the toxin molecule, there are sites where substitutions, deletions or additions do not affect the structure or function of the toxin. Therefore, in addition to the above-mentioned specific two amino acids, a mutant in which substitution, addition or deletion of amino acids unrelated to the biological activity of the present toxin may be performed.
[0029]
Alternatively, a partial polypeptide of the mutant PMT may be used as long as it is a polypeptide that does not express toxin activity and has a region that has immunological activity against swine atrophic rhinitis.
[0030]
Specifically, in the present toxin, a region of several tens of amino acids around the amino acids at positions 1164 and 1165 is considered to be the active center. Therefore, it is expected that some of the partial polypeptides containing the present region also show immunogenicity, and even in such a partial polypeptide, a mutant in which the above-described specific two amino acids are substituted is included in the present patent. .
[0031]
The nontoxic mutant PMT and its partial polypeptide of the present invention may be translated from a gene encoding them, or may be produced by a chemical synthesis method or the like.
[0032]
The gene encoding the protein according to any one of claims 1 to 3 may have a mutation at another site, may have a partial addition or deletion, or may have a partial fragment containing these mutations. There may be. These base sequences may be obtained by any method such as a chemical synthesis method and a DNA replication method. As a specific embodiment of the gene encoding the nontoxic mutant PMT of claim 2, a gene having the nucleotide sequence shown in SEQ ID NO: 3 can be mentioned. The coding region (sequence shown in SEQ ID NO: 2) in this base sequence shows one combination example of codons indicating each amino acid residue of the amino acid sequence shown in SEQ ID NO: 1. The gene of the present invention is not limited to the specific gene, and may have a base sequence selected by combining any codon with each amino acid.
[0033]
The gene encoding the protein according to any one of claims 1 to 3, which hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions such as at 37 ° C in a hybridization solution containing 50% formaldehyde. Can be exemplified.
[0034]
The nontoxic mutant PMT or the partial polypeptide derived from the nontoxic mutant PMT of the present invention can produce a large amount of the protein by a gene recombination technique using the gene encoding the nontoxic mutant PMT.
[0035]
That is, a recombinant expression vector containing the gene according to claim 6 or 7 is prepared, and a transformant transformed to produce a nontoxic mutant PMT or a partial polypeptide thereof by the expression vector is cultured. Then, by producing a nontoxic mutant PMT or a partial polypeptide thereof in the transformant, a nontoxic mutant PMT can be produced.
[0036]
The above transformant can be produced, for example, as a cell that produces a nontoxic mutant PMT or a partial polypeptide thereof.
[0037]
That is, when the host cell is a eukaryotic cell such as a CHO cell, a BHK-1 cell and a Hela cell, for example, the amino acid sequence of the nontoxic mutant PMT described in claims 1 to 3 is encoded by the amino acid sequence of claim 6 or 7. The DNA fragment can be ligated to a promoter such as a virus and transformed into a eukaryotic cell to produce a nontoxic mutant PMT-producing cell. When a recombinant virus vector such as a baculovirus is used, a recombinant virus vector is produced based on, for example, the method of Smith et al. (Smith et al., Mol. Cel. Biol., 3, 2156-2165). be able to. That is, a DNA fragment encoding a nontoxic mutant PMT or a partial polypeptide thereof is introduced into insect cells together with baculovirus DNA, and a recombinant virus clone producing a nontoxic mutant PMT is selected from the obtained recombinant baculoviruses. Then, the recombinant clone can be infected to insect cells to produce nontoxic mutant PMT-producing cells. In addition, insects that produce the nontoxic mutant PMT can be produced by infecting insects with the recombinant clone.
[0038]
When the host cell is a prokaryote such as Escherichia coli or Bacillus subtilis, a DNA fragment encoding a nontoxic mutant PMT or a partial polypeptide thereof is ligated to an appropriate expression vector such as pBluescript II, pQE9, pET16 and pUB110. Then, prokaryotic cells such as Escherichia coli can be transformed to produce nontoxic mutant PMT-producing cells. At this time, a vector that can be expressed as a fusion protein with another protein may be selected.
[0039]
These genetic recombination operations may be performed by a known method, and are not particularly limited. By gene manipulation techniques and cell engineering techniques, expression efficiency can be controlled by, for example, addition of a signal sequence, suitable selection of a host-vector system, improvement of gene expression control sites, and the like. Such operations are described in Sambrook et al., 1989, Molecular cloning: a laboratory manual. 2nd edition, Cold Spring Harbor, New York; It can be done according to engineering technology.
[0040]
According to the invention of the nontoxic mutant PMT having the amino acid or a partial polypeptide thereof, a veterinary pharmaceutical composition such as a vaccine containing the same as an active ingredient can be provided, and the onset of swine atrophic rhinitis caused by infection with tPm can be provided. It can be used effectively as a vaccine for blocking.
[0041]
The nontoxic mutant PMT represented by SEQ ID NO: 3 of the present invention can be obtained by the following method.
[0042]
For example, a DNA primer (main chain and complementary strand) for mutation introduction of about 30 base pairs for mutating serine at position 1164 to alanine and cysteine at position 1165 to serine in wild-type PMT is synthesized. For example, using a DNA fragment of about 700 bp in the 3 'terminal region of the wild-type PMT gene containing amino acids 1164 and 1165 as a template, these primers and vector-derived primers such as M30-Universal and M30-Reversal primers are used, The 3′-side and 5′-side fragments sandwiching the are amplified by PCR. When the 3′-side and 5′-side fragments are further mixed and amplified again using, for example, M30-Universal and M30-Reversal primers, the 3′-terminal region of the PMT gene containing the sequence of the mutation-introducing primer is reduced. A 0.7 kbp DNA fragment can be obtained.
[0043]
When this fragment was replaced with the same fragment of about 0.7 kbp in the 3 'terminal region of the wild-type PMT gene by ordinary gene cloning using a restriction enzyme cleavage site, a primer for mutagenesis was obtained. Can be obtained. It can be confirmed by examining the entire nucleotide sequence of the gene encoding the mutated PMT that there is a mutation only in the relevant portion and no change in the nucleotide sequence of the other portion.
[0044]
The present gene can be inserted into a commercially available expression vector such as pBluescript II KS (+) and introduced into a derivative strain derived from Escherichia coli K12 to prepare a transformant for expressing a mutant PMT. . This transformant can be activated for transcription by a vector promoter, for example, by culturing it under 1 mM IPTG for 4 hours, to produce a large amount of mutant PMT protein or its partial polypeptide in E. coli cells. it can.
[0045]
The Escherichia coli can be disrupted by, for example, sonication or the like, and the mutant PMT protein can be extracted from the centrifuged supernatant. The extracted protein is dialyzed against, for example, a 0.02 M phosphate buffer, subjected to anion exchange column chromatography using DEAE sephacel or the like, and eluted with a continuous concentration gradient solution using 0.1 M to 0.6 M NaCl. The mutant PMT protein can be isolated and purified.
[0046]
Injection of 0.2 mL of the thus-purified mutant PMT protein subcutaneously on the back of guinea pigs did not show any deformation at the injection site, and did not kill the animals when injected intraperitoneally into mice. . Thus, it can be confirmed by an inoculation test to an animal that the produced mutant PMT is completely non-toxic.
[0047]
By immunizing a pig muscle twice, for example, with an amount of 16 micrograms of the mutant PMT protein together with an aluminum hydroxide gel adjuvant at three-week intervals, and intramuscularly injecting wild-type PMT two weeks after the final immunization. Make an attack. Control pigs are non-immunized and only challenge with toxin. Then, about 3 weeks after the challenge, the non-immune control pigs form remarkable atrophic rhinitis lesions on the turbinates, but the pigs immunized with the mutant PMT do not show such lesions. When an antibody test is performed on the serum collected at this time, it is found that the immunized pig has produced an antibody that reacts with wild-type PMT.
[0048]
As described above, the nontoxic mutant PMT or its partial polypeptide produced by the above method does not show any toxin activity and shows high immunogenicity to pigs. It can be used as a main component of a pharmaceutical composition such as a vaccine.
[0049]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0050]
(1) Introduction of site-specific mutation into PMT gene
In order to substitute an amino acid at a predetermined site of the PMT gene, the following primers were designed.
[0051]
The sequence of 30 bases from base Nos. 3480 to 3509 in the wild-type PMT gene is g, gaa, gct, ggc, tct, tgt, gat, tca, gta, agc, cc and is translated from this sequence. The amino acid sequence is Glu-Ala-Gly-Ser-Cys-Asp-Ser-Val-Ser-Pro (1161 to 1170). In order to mutate the 1164th Ser (serine) into Ala (alanine) and the 1165th Cys (cysteine) Ser (serine) among them, primers for mutagenesis (SEQ ID NO: 4) g, gaa, gct, ggc , Gct, AgC, gat, tca, gta, agc, cc and (PMT-MF) and its complementary sequence (SEQ ID NO: 5) gg, gct, tac, tga, atc, GcT, agC, gcc, agc, ttc , C (PMT-MR) were prepared. The amino acid sequence encoded by these mutated primers is Glu-Ala-Gly-Ala-Ser-Asp-Ser-Val-Ser-Pro. In addition, the substitution of these bases creates a new restriction enzyme cleavage site in the mutant primer that is cleaved with the restriction enzyme NheI.
[0052]
To introduce a mutation into the actual PMT gene using these primers, a DNA fragment of about 0.7 kbp from the EcoRI restriction enzyme cleavage site at base number 3391, which is the 3 ′ terminal region of the gene to the 3 ′ end, was introduced. Was inserted into the EcoRI-SacI restriction enzyme cleavage site in the multicloning site of the vector pBluescript II KS (+) (Stratagene).
[0053]
Using the 3 ′ terminal portion of the PMT gene as a template, a PCR reaction using a vector-derived M30-20 Reverse primer and a PMT gene mutation primer PMT-MR Reverse primer, and a PMT gene mutation primer PMT-MF Two PCR reactions using the Forward primer and the M30-20 Universal primer were performed according to a conventional method. Next, 2 μL of each of the reaction solutions obtained by these two PCR reactions was mixed, and a heat-resistant DNA polymerase, dNTP and a reaction buffer were added again, and further, M30-20 Revalsal primer and M30-20 Universal primer were added. Was added to perform a secondary PCR reaction. As a result, a DNA fragment having a base length of about 0.7 kbp, which was the same size as the DNA fragment before the mutation, was amplified. The presence of a restriction enzyme NheI cleavage site, which was not present in the original fragment, in this fragment indicated that the same sequence as the primer used for the mutation was introduced into this 0.7 kbp fragment. Was.
[0054]
Therefore, in order to replace this DNA fragment with the same part of the original PMT gene, the original PMT gene was digested with EcoRI and SacI restriction enzymes, and a DNA fragment of about 0.7 kbp digested with the same restriction enzymes was digested according to a conventional method. Inserted. In this manner, a mutant PMT gene (SEQ ID NO: 2) was prepared in which the 3 ′ terminal site of the original PMT gene was replaced with the mutant DNA fragment.
[0055]
In order to confirm whether or not the same sequence as the mutation-introducing primer used initially exists in the mutant PMT gene thus prepared, the present gene was analyzed using a PERKIN-ELMAR 310-type automatic base sequencer. Was determined. As a result, the sequence from base number 3480 to base 3509 of the mutant PMT gene is certainly g, gaa, gct, ggc, Gct, AgC, ga, tca, gta, agc, cc, and No mutation was found. In this way, a gene encoding a mutant PMT (SEQ ID NO: 3) in which Ser (serine) at position 1164 is replaced with Ala (alanine) and Cys (cysteine) at position 1165 is replaced with Ser (serine). Was completed.
[0056]
(2) Purification of mutant PMT
In order to confirm properties such as toxicity and immunogenicity of the mutant PMT, respective proteins were purified from original (wild-type) and recombinant E. coli expressing the mutant PMT.
[0057]
The wild-type or mutant PMT gene was incorporated into a pBluescript II KS (+) vector, and this was introduced into XL1-Blue (manufactured by STRATAGENE), which is a derivative of Escherichia coli K12. Recombinant Escherichia coli containing each gene was expressed in an LB medium containing 1 mM IPTG by shaking culture at 37 ° C. for 4 hours. After the expression culture, Escherichia coli was centrifuged and collected, and treated with an ultrasonic crusher (Sonic Power manufactured by Branson) for about 5 minutes to crush the cells. This was centrifuged at 10,000 × g for 20 minutes, and ammonium sulfate was added to the collected supernatant so as to be 50% saturated, and the protein was salted out. This was centrifuged at 10,000 × g for 20 minutes to collect a precipitate, which was then dissolved in a 0.02 M phosphate buffer (pH 6.6). This solution was dialyzed overnight against a 0.02 M phosphate buffer (pH 6.6), and then subjected to column chromatography.
[0058]
DEAE sephacel (manufactured by Pharmacia), which is an anion exchanger, was used as a carrier for chromatography, and packed in a glass column having a diameter of 40 mm and a height of 50 cm. The sample was added, the column was washed, the unadsorbed fraction was discharged, and elution was performed with a continuous concentration gradient solution of 0.1 M to 0.6 M NaCl. The final peak portion of the protein in the fractionated eluate was collected, and concentrated using polyethylene glycol 8000 (manufactured by Wako Pure Chemical Industries, Ltd.) to a volume of about 30 mL.
[0059]
The fractions (peak 1, peak 2 and collected fraction) collected by the above-mentioned chromatography for the wild-type and mutant PMT were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (FIG. 1). As a result, the mutant PMT prepared this time was eluted in the same fraction as the wild-type PMT. Each fraction also contained a single protein with a molecular weight of about 146 kDa. This indicates that the mutant PMT can be purified by the present method, similarly to the wild-type PMT.
[0060]
(3) Toxin activity of mutant PMT
Regarding the toxin activity of the mutant PMT, the skin necrosis toxin activity by intradermal inoculation in guinea pigs and the lethal activity by intraperitoneal inoculation of mice were examined.
For the measurement of guinea pig skin necrosis activity, a Hartley guinea pig weighing about 350 g was used. Wild-type PMT (80 μg protein amount / mL) and mutant PMT (34 μg protein amount / mL) were serially diluted with phosphate buffered saline, and 0.2 mL was injected into the back skin of guinea pigs. Two days after the injection, necrotic spots formed at the injection site were observed. Table 1 shows the results.
[0061]
[Table 1]

[0062]
As is clear from the results shown in Table 1, the formation of necrotic spots was observed in the guinea pig skin necrosis activity up to 160-fold dilution in the wild-type PMT, whereas the necrotic spot formation was not observed in the undiluted solution in the mutant PMT. I was not able to admit. Thus, it was considered that the guinea pig skin necrosis activity had disappeared in the mutant PMT.
[0063]
For the test of the mouse lethal activity, 50 4-ddY ddY mice were used. Wild-type PMT and mutant-type PMT were adjusted to 1 mg, 100 μg, 10 μg, 1 μg, and 100 ng per mouse, and inoculated intraperitoneally into 5 mice per protein amount. One week after the inoculation, the cells were observed to determine whether they were alive or dead. Table 2 shows the results.
[0064]
[Table 2]

[0065]
As is evident from the results shown in Table 2, with respect to the lethal activity of the mice, all of the 5 animals in the 1 mg and 100 μg injection groups and 1 in 5 animals in the 10 μg injection group died in the wild-type PMT. On the other hand, with the mutant PMT, no death was observed even in the highest dose of 1 mg injection group. LD from the result ₅₀ The (50% lethal dose) was calculated to be 20 μg protein in wild-type PMT, but 3200 μg protein in mutant PMT. Thus, the mutant PMT did not show mouse lethal activity.
[0066]
From the above results, in the mutant PMT, neither the skin necrosis activity of guinea pig nor the lethal activity of mouse was detected, and it was found that the toxin activity was completely eliminated by amino acid substitution at the site.
[0067]
(4) Immunogenicity of mutant PMT
In order to examine whether the produced nontoxic mutant PMT retains immunogenicity to pigs, the protective effect was examined by attacking pigs immunized with nontoxic mutant PMT with wild-type PMT.
[0068]
Five Bb and tPm-free pigs of about 5 weeks of age were used. Three animals were immunized, and 16 μg of a nontoxic mutant PMT protein was injected intramuscularly twice at three-week intervals with aluminum hydroxide gel adjuvant at one time. The other two animals were non-injected control groups. Three weeks after the second injection, the pigs in the immunized group were challenged with the non-injected control group by intramuscular injection of 4 μg protein / kg body weight of wild-type PMT. Necropsy was performed 2 weeks after the challenge, and the extent of the lesion formed in the turbinate was visually observed. In addition, blood was collected from the pigs of both groups before immunization and at the time of challenge, and antibodies to PMT produced in the blood were examined by ELISA. In the mutant PMT-immunized pigs, there were no clinical abnormalities such as fever, depression, and anorexia during immunization, and there were no abnormal reactions such as swelling or redness at the injection site, and the detoxification of the mutant PMT was complete. It was confirmed that there was.
[0069]
In the antibody test, no antibody against PMT was detected in the serum before immunization in all cases. In the immunized group, at the time of challenge 2 weeks after the second injection, all cases became antibody-positive, and the average E value also increased to 0.29 (Table 3). On the other hand, two animals in the control group were negative.
[0070]
Three weeks after challenge with wild-type PMT, autopsy was performed and the turbinates were examined visually. The result is shown in FIG. No lesion showing atrophic rhinitis was observed in any of the three immunized animals. On the other hand, lesions with a score of 3 and 2 (loss of the turbinate) were observed in two non-immune control groups, respectively (Table 3 and FIG. 2).
[0071]
[Table 3]

[0072]
Thus, it was shown that pigs immunized with the nontoxic mutant PMT produced in the present invention were induced with antibodies against PMT and were protected from challenge with wild-type PMT.
[0073]
【The invention's effect】
The present invention according to claims 1 to 3 and claim 8 is a protein that does not express toxin activity and has immunological activity against swine atrophic rhinitis, and can protect swine from attack by wild-type PMT when immunizing swine. , Can be used as a vaccine against swine atrophic rhinitis.
[0074]
Further, according to the present invention according to claim 6 or 7, a gene encoding the nontoxic mutant PMT is obtained, and the protein is obtained in a large amount by being expressed by the genetic technique of claims 9 and 10. be able to. Further, by purifying by the method described in claim 11, a nontoxic nontoxic mutant PMT suitable as a vaccine component without impurities can be obtained.
[Brief description of the drawings]
FIG. 1 shows the results of fractionation of wild type (Wild type) and mutant type (Mutant) PMT by anion exchange column chromatography, followed by SDS-polyacrylamide gel electrophoresis of peak 1, peak 2 and the recovered fraction. Electrophoresis photograph.
FIG. 2 is a photograph showing lesions formed in the turbinate after autopsy at 3 weeks after challenge with three mutant PMT-immunized pigs and two non-injected control pigs with wild-type PMT.
[Sequence list]

Claims (12)

Swine atrophic rhinitis which does not express toxin activity, wherein the serine at position 1164 and the cysteine at position 1165 are substituted with other amino acids in the polypeptide of Pasteurella multocida toxin represented by SEQ ID NO: 1. Nontoxic mutant Pasteurella multocida toxin having immunological activity against A non-toxic mutant Pasteurella multocida toxin which does not express toxin activity and has immunological activity against swine atrophic rhinitis, which is a polypeptide represented by SEQ ID NO: 4. A partial polysaccharide derived from Pasteurella multocida toxin, which comprises a region having no immunological activity against swine atrophic rhinitis without expressing toxin activity in the polypeptide of the nontoxic mutant Pasteurella multocida toxin according to claim 1 or 2. peptide. Swine atrophic rhinitis which does not express toxin activity, wherein the serine at position 1164 and the cysteine at position 1165 are replaced with other amino acids in the polypeptide of Pasteurella multocida toxin represented by SEQ ID NO: 1. A method for producing a non-toxic mutant Pasteurella multocida toxin having an immunological activity against A. The method for producing a nontoxic mutant Pasteurella multocida toxin according to claim 4, wherein the amino acid that substitutes serine at position 1164 is alanine and the amino acid that substitutes for cysteine at position 1165 is serine. A gene encoding the following polypeptide (a), (b) or (c).
(A) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 3 (b) consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the above amino acid sequence (a), and having a toxin activity A polypeptide which does not express and has immunological activity against swine atrophic rhinitis (c) a pasteurella characterized by comprising a region which does not express the toxin activity in the amino acid sequence of (a) and has immunological activity against swine atrophic rhinitis・ Multocida toxin-derived partial polypeptide A gene comprising the following polynucleotide (a) or (b):
(A) a polynucleotide consisting of the base sequence represented by SEQ ID NO: 2 (b) a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the base sequence of (a) above The following recombinant protein (a) or (b):
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3; (b) a protein consisting of the amino acid sequence of the above (a) in which one or more amino acids are deleted, substituted or added, and expresses toxin activity (C) a protein having immunological activity against swine atrophic rhinitis which does not express the toxin activity in the amino acid sequence of the above (a) and comprising a region having immunological activity against swine atrophic rhinitis; Toxin-derived partial polypeptide A recombinant expression vector comprising the gene according to claim 6 or 7. A transformant transformed by the expression vector according to claim 9 to produce the recombinant protein according to claim 8. A method for producing a nontoxic mutant Pasteurella multocida toxin, comprising culturing the transformant according to claim 10 and producing the recombinant protein according to claim 8 in the transformant. A vaccine for swine atrophic rhinitis, comprising the recombinant protein according to claim 8 as an active ingredient.

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