KR100886358B1 - Avirulent attenuated strain of Pasteurella multocida and live vaccine containing the same - Google Patents

Abstract

The present invention relates to a P. multocida attenuated strain and a porcine pasteurella pneumonia containing the same, wherein the toxA gene of Pasteurella multocida is knocked out and the toxicity is removed. The present invention relates to a live vaccine for the prevention or treatment of P. multocida infection, which can be used for the immunological role of Pasteurella multocida toxin (PMT) as well as for the prevention or treatment of P. multocida infection. It can also be used as an expression vehicle system for expressing foreign heterologous proteins in multocida.

Pasteurella multocida, Pasteurella multocida, Homologous recombination, Knock-out, toxA, PMT, Attenuated, Live vaccine

Description

Non-pathogenic attenuated strain of Pasteurella multocida and vaccine containing the same

1 is a diagram showing the process of cloning the P. multocida toxA gene and kanamycin-resistant (kan ^R ) gene and inserting kan ^R into the toxA gene to prepare a DNA insert for homologous recombination;

Figure 2 is a photograph showing the results of PCR and restriction enzyme treatment carried out to prepare the insert for homologous recombination (lane 1: amplified toxA (3.9 kb), lane 2: pGEM-toxA treated with EcoRI (3.9 kb and 3 kb), lane 3: amplified kan ^R (0.8 kb), lane 4: pGEM-kan ^R treated with EcoRI (3 kb and 0.8 kb), lane 5: pGEM-toxA-kan ^R treated with EcoRI (4 kb and 0.8 kb), lane 6: pGEM-toxA-kan ^R treated with EcoRI (3 kb and 1.8 kb);

FIG. 3 is a diagram schematically illustrating PCR to confirm that kan ^R is inserted into the toxA locus (1: using toxA-F and kan-R primers, 2: using kan-F and toxA-R primers, and 3) : toxA-F and toxA-R primers);

4 is a photograph showing the results of investigating whether genomic DNA structure of P. multocida was converted due to kan ^R insertion by performing PCR as in FIG. 3;

5A shows the nucleotide sequence of a homologous recombination linear DNA insert (1.8 kb);

5B shows the structure of a pGEM®-T easy vector.

The present invention relates to a non-pathogenic attenuated strain of Pasteurella multocida and a vaccine containing the same. More specifically, the toxA gene of P. multocida is knocked out. A live vaccine for the prevention or treatment of P. multocida infections, including P. multocida attenuated strains, and porcine Pasteurella pneumonia containing the same, wherein the toxicity is eliminated.

Porcine Pasteurella pneumonia (Swine Pasteurellosis) is caused by P. multocida, causing delay in weight gain, vitality, and death. The main symptom is acute type of abdominal breathing, cough, small runny nose and fever of 40 ~ 42 ℃, open mouth breathing and breathing, and cyanosis on limb. Symptoms may last five to ten days and then die, or they may recover after three to five weeks. It usually occurs when the pigs or pigs are subjected to inadequate specification conditions, such as overcrowding or poor ventilation, or stresses such as coordination, weight measurement and transportation. For prevention, mixed vaccines of four diseases including swine Atrophic Rhinitis, Pasturela pneumonia, swine alone and E. coli diarrhea have been developed and used in Korea. The pattern of bacterial respiratory disease in Korean pigs, which has been examined through recent pathological evaluations in Korea, has been about 5.0 ~ 31.0%, although the proportion of total respiratory illnesses varies from year to year. The average rate is around 14.0%. However, other respiratory diseases, except pleural pneumonia, were reported to have higher rates of mortality in general, due to lower mortality rates and only a sustained reduction in the rate of referrals. As such, the respiratory disease of pigs related to P. multocida is dominant and the most serious and urgently needed prevention.

Pasteurella pneumonia is a pneumonia caused by the pathogen P. multocida. There are 4 types of bacterial antigens, A, B, D, and E. Types A and D are mainly associated with respiratory disease in pigs. Pneumonia is mainly caused by type A. The disease can be divided into acute and chronic with only acute pneumonia. Deep plastic death dies with symptoms of sudden high fever, shortness of breath, systemic bleeding, and lack of vigor. It shows a typical form of sepsis in which the color of the skin turns purple. In most cases, inflammatory exudates are sometimes observed in the nose with symptoms of acute, fever, difficulty breathing, loss of appetite, and lack of energy. If left untreated, death can occur after 5 to 10 days, but mortality is not high. The chronic form occurs when the farms that develop pneumonia fail to fully treat the acutely transmitted infection. Due to the constant growth of wet cough, weight loss, and systemic weakness, it is contracted and lost economic value, and it is a carrier and serves as an infectious agent. Preventive vaccines against the disease include P. multocida type A and D killed vaccines, combined with pleural pneumonia, atrophic rhinitis, and pasteurella pneumonia. However, P. multocida's toxigenicity is in the toxin of the bacterium released Pasteurella multocida toxin (PMT), so the vaccine will not produce PMT, so P. multocida is prevented by the vaccine. Is hard to expect. In addition, in order to study the toxicity of PMT itself and the immunological response caused by PMT, a situation in which PMT is removed is required.

To date, attempts have been made to develop attenuated strains or vaccines through mutation of genes in specific regions of P. multocida. For example, Korean Patent Publication No. 2005-16334 discloses a technique for preparing an attenuated strain of P. multocida by signature tagged transposon insertion or direct mutation. 2001-112937 discloses a technique for preparing an attenuated strain through mutation of a gene responsible for the toxicity of P. multocida and using it as an immunogenic composition or vaccine. However, the prior art did not use the toxA gene of P. multocida.

Furthermore, U.S. Patent Application Publication No. 2006-39923 discloses a technique for producing a vaccine against P. multocida mediated diseases using partial fragments of P. multocida toxin, and Japanese Patent Laid-Open No. 2004-242608 Discloses a technique for preparing a vaccine against P. multocida infection by substitution of the P. multocida toxin genes (Ser 1641 and Cys 1165), and International Publication No. 1989-9617 Techniques for making vaccines against P. multocida mediated diseases using mutations and fragments of the Toshida toxin gene are disclosed. However, these prior arts use polypeptide fragments which are not by direct mutation or post-transcriptional modification of genes or by attenuated strains as active ingredients.

The present inventors have developed a live vaccine that can effectively prevent and treat P. multocida infections, including porcine Pasteurella pneumonia, and remove PMT for use in studies such as toxicity of PMT itself and immunological reaction due to PMT. Ongoing studies have been conducted to prepare P. multocida strains. As a result, by introducing an antibiotic resistance gene by homologous recombination to the P. multocida toxA gene, an attenuated strain in which the toxA gene was knocked out was prepared and confirmed that the strain met the above purpose. The present invention has been completed.

It is therefore an object of the present invention to provide a polynucleotide in which an antibiotic resistance gene is introduced between specific N-terminal and C-terminal fragments of the P. multocida toxA gene.

Another object of the present invention is to provide a non-pathogenic, attenuated P. multocida strain, transformed with a polynucleotide of the antibiotic resistance gene and knocked out the P. multocida toxA gene by homologous recombination. will be.

Another object of the present invention is to provide a live vaccine for the prevention or treatment of P. multocida infection containing the strain as an active ingredient.

A first aspect of the invention relates to a polynucleotide consisting of an N-terminal 0.52 kb fragment of the P. multocida toxA gene, an antibiotic resistance gene and a C-terminal 0.44 kb fragment of the Pasteurella multocida toxA gene. The antibiotic resistance gene may be a kanamycin resistance gene.

Second, the present invention is a non-pathogenic, attenuated P. multocida strain, for example, Accession No. KCCM10824P, wherein the polynucleotide is transformed and knocked out the P. multocida toxA gene by homologous recombination. It is about.

Third, the present invention relates to a live vaccine for the prevention or treatment of P. multocida infections, such as porcine Pasteurella pneumonia, containing the P. multocida strain as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention uses homologous recombination technology, leaving only 0.52 kb of N-terminal fragment and 0.9 kb of C-terminal fragment in the toxA gene region of P. multocida, a pathogen of Pasteurella pneumonia, a swine respiratory disease. The attenuated non-pathogenic strain is prepared by knocking out the intermediate portion of the toxin (base sequence 522 to 2950 in the total of 3858 bases of the toxA gene) to be used as an attenuated live vaccine. In the present invention, the attenuated strain may be prepared through the following process.

A) Cloning the entire P. multosidosis toxin (PMT) gene, toxA.

B) Cloning the kanamycin resistance gene (kan ^R ) to be inserted between the EcoRV and HindIII positions of the PMT gene.

C) Two constructs are double digested with EcoRV and HindIII and ligated.

D) The construct is digested with EcoR I and the homologous recombination linear DNA insert is extracted by gel elution.

E. p.D46 plasmid is transformed into P. multocida dominant strain into an electrocompetent cell by electroporation.

F) L-arabinose is cultured in a medium prepared to a final concentration of 10 mM in order to induce λ Red recombinase in P. multocida transformed with pKD46 plasmid.

G) Homologous recombination linear DNA is transformed into electrocompetent cells by electroporation and screened in medium containing kanamycin.

H) Confirm knock-out in genomic DNA of the transformed cells by PCR.

A manufacturing process of the attenuated strain according to the present invention is schematically illustrated in FIG. 1.

The homologous recombination linear DNA insert prepared from step a) to d) has the nucleotide sequence of SEQ ID NO: 1.

The attenuated P. multocida strain prepared from step a) to a) was deposited on December 14, 2006 at the Korea Microbiological Conservation Center, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea. No. KCCM10824P has been assigned.

The P. multocida attenuated strain may be used as a live vaccine for the prevention or treatment of diseases caused by P. multocida, for example, porcine Pasteurella pneumonia, by knocking out the toxA gene by homologous recombination. In addition, it can be usefully used to study the toxicity of PMT itself and the immunological response due to PMT.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended to aid the understanding of the present invention but are not intended to limit the scope of the present invention in any way.

Example 1 P. multocida toxA gene and kanamycin resistance gene (kan ^R Cloning of)

1) Cloning of the P. multocida toxA gene

5'-ATAT ATGAAAACAAAACATTTTTT-3 '(SEQ ID NO: 2) and reverse primer (toxA-) with forward primer (toxA-F) using known P. multocida toxA gene information (GenBank database: AF240778) R) was used to amplify the entire toxA gene site by PCR using 5'-TGTG TTATAGTGCTCTTGTTAAGC-3 '(SEQ ID NO: 3) as a template with P. multocida genomic DNA. P. multocida type D strains for toxA gene cloning were distributed from the National Veterinary Research and Quarantine Service. PCR conditions were 5 μl 10 × reaction buffer, 5 μl 25 mM MgCl ₂ , 5 μl 10 mM dNTP (thermal cycler (PTC-100 ™, MJ Research)) at a final volume of 50 μl, respectively. 2.5 mM) mixture and 5 μl (50 ng / μl) DNA and 1 μl of each primer (50 pM) in 1 μl 5 U Ex Taq DNA polymerase (TakaRa) PCR was incubated for 5 minutes at 94 ° C. 30 cycles of 1 minute at 94 ° C., 1 minute at 55 ° C. and 1 minute at 72 ° C., and 7 minutes at 72 ° C. The resulting amplification product (3.9 kb) was subjected to 0.8% (w / v) agarose gel. Electrophoresis followed by gel eluting harvested PCR products were cloned by inserting into pGEM®-T easy vector (see FIG. 5B) (Promega) (pGEM-toxA). E. coli JM109 (Invitrogen) was transformed, transformed colonies were screened again, subjected to enrichment and plasmid purification, and treated with EcoR I to insert the insert. The entire sequencing was performed only for the check and verify the production.

2) Cloning of the kanamycin resistance gene (kan ^R )

The kan ^R gene was inserted at the toxA gene position and used as a selection marker. That is, a 5'-primer (kan-F: 5'-GCGC GATATC ATGATTGAACAAGATGGATT-3 'designed to contain the EcoRV position using DNA of a pCR® Blunt vector (Invitrogen) as a template 4)) and the kan ^R gene were amplified by PCR using a 3'-primer (kan-R: 5'-CGCG AAGCTT TCAGAAGAACTCGTCAAGAA-3 '(SEQ ID NO: 5)) designed to contain the HindIII site. PCR conditions were the same as for the toxA gene, and PCR was performed with 94 cycles of 5 minutes incubation followed by 30 cycles of 94 ° C 30 seconds, 55 ° C 30 seconds and 72 ° C 1 minute, and final extension of 72 ° C 7 minutes. It was then cloned into pGEM®-T easy vector in the same manner as toxA gene cloning (pGEM-kan ^R ).

Example  2: Homologous  Recombination Linear DNA Insert  Produce

The two constructs prepared in Example 1, pGEM-toxA and pGEM-kan ^R, respectively, were cleaved using two restriction enzymes, EcoR V and HindIII, and the cleaved sites were ligated with each other so that the 2.4 kb portion of pGEM-toxA was 0.8 kb. was replaced with the kan ^R gene (pGEM-toxA-kan ^R ). Herein, to obtain a linear DNA fraction of about 1.8 kb (toxA-kan ^R -toxA: 1.8 kb), the enzyme was treated with restriction enzyme EcoR I and subjected to gel elution after electrophoresis to obtain a linear DNA insert for homologous recombination (SEQ ID NO: 1 and 5a) was prepared.

The results of PCR and restriction enzyme treatment carried out to prepare the insert for homologous recombination are shown in FIG. 2. In Figure 2, lane 1 is amplified toxA (3.9 kb), lane 2 is treated with EcoR I pGEM-toxA (3.9 kb and 3 kb), lane 3 is amplified kan ^R (0.8 kb), lane 4 Is pGEM-kan ^R treated with EcoRI (3 kb and 0.8 kb), lane 5 is pGEM-toxA-kan ^R treated with EcoRI (4 kb and 0.8 kb), lane 6 is pGEM-toxA-kan ^As a result of treatment of ^R with EcoRI (3 kb and 1.8 kb), a portion corresponding to 1.8 kb was used as an insert for homologous recombination.

Example 3: Gene Breakdown by Homologous Recombination

5 ml of freshly prepared P. multocida culture was inoculated in 500 ml BHI broth (BBL ™, Becton, Dickinson and Company) and incubated until the OD value was 0.5. After cooling for 20 minutes on ice and centrifuged for 15 minutes at 4000 × g to recover the bacteria. It was concentrated to 2.5 ml of cooled 10% glycerol solution to prepare as competent cells for electroporation. Gene Pulser with a pKD46 plasmid (Datsenko and Wanner, 2000, PNAS 97 (12): 6640-5), Genbank AY048746 to express λ Red recombinase in this cell Transformation was performed using Xcell ™ electroporation system (Bio-Rad). The red helper plasmid pKD46 is a vector that produces red and gam proteins using the pBAD promoter. It acts as a homologous recombination when foreign DNA enters the cells. After transformation, in 250 ml SOB medium (per liter: 20 g of bacto-tryptone, 5 g of yeast extract, 0.584 g of NaCl, 0.186 g of KCl, pH 7.0) with ampicillin and L-arabinose (10 mM final) After selection, the cells were cultured at 30 ° C. until the OD value became 0.5. Concentrated again with 2.5 ml of cooled 10% glycerol solution to prepare electrocompetent cells to transform the homologous recombination linear DNA prepared earlier. 1 μl (100 ng) of insert DNA (toxA-kan ^R- toxA) was transformed into 40 μl of electrocompeted cells by electroporation and removed from LB medium with 50 μg / ml kanamycin added to remove toxA. Bacteria were selected.

The strain obtained as described above was deposited with the Korea Microorganism Conservation Center on December 14, 2006, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea, and was given accession number KCCM10824P.

Example 4: PCR Verification

Colonies surviving the kanamycin screening medium acted as a reporter to indicate that kan ^R was inserted, but once again PCR verification was performed to verify by molecular biological methods. That is, PCR was performed as follows to confirm that kan ^R was inserted into the toxA locus (see FIG. 3). That is, in performing PCR using three kinds of primer sets, the first toxA-F and kan-R primers, the second to kan-F and toxA-R primers, and the third toxA-F and toxA-R primers PCR products of 1.3 kb, 1.7 kb, and 2.2 kb, respectively, were expected.

Genomic DNA structure of P. multocida by PCR was performed by kan ^R Figure 4 shows the results of the conversion due to the insertion. As shown in Figure 4, it was confirmed that the PCR products of the expected size (1.3 kb (lane 1), 1.7 kb (lane 2) and 2.2 kb (lane 3) size was produced.

The P. multocida strain from which the toxA gene is removed according to the present invention can be used not only for probiotic vaccine against P. multocida infection but also for the immunological role of PMT and expressing foreign heterologous protein in P. multocida. It can also be used as an expression vehicle system.

<110> Industry Foundation of Chonnam National University <120> Avirulent attenuated strain of Pasteurella multocida and live          vaccine containing the same <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 1766 <212> DNA <213> Artificial Sequence <220> <223> 0.52 kb N-terminal fragment of toxA-0.8 kb of kanR-0.44 kb          C-terminal fragment of toxA <400> 1 atgaaaacaa aacatttttt taactcagat tttactgtaa aaggaaaaag tgccgatgaa 60 atttttagaa gattgtgtac tgatcatcct gacaagcaat taaacaatgt aaaatggaaa 120 gaagttttta ttaatcgttt tggtcagatg atgctagata ctcctaatcc gagaaagatt 180 gtagaaaaaa ttattaatga agggcttgaa aaacaaggcc tgaaaaatat agatcctgaa 240 actacatatt tcaacatttt ttcatcttct gacagctccg atgggaacgt ttttcattat 300 aactctttat cagaatccta tcgagttact gatgcctgcc taatgaatat ttttgtggag 360 cgttattttg atgattggga cttgctaaat agcttagcca gtaatggaat atattcagta 420 ggaaaagaag gagcttatta tcctgatcat gattatggtc cagaatataa ccctgtttgg 480 ggaccaaacg aacaaattta ccattctaga gtgattgcag atatcatgat tgaacaagat 540 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 600 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 660 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg 720 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 780 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 840 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 900 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 960 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 1020 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc 1080 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 1140 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 1200 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 1260 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 1320 aagcttctgc aatcagcaaa agataataat ataaaattta gagctatagg gcattcagat 1380 aattctgttc cgccatttaa taacccttat aagtctttat attataaagg aaatataata 1440 gctgaagcaa ttgaaaaact agatcgagaa ggtcaaaaat ttgttgtatt tgctgatagt 1500 tctctgctca acagcacgcc tgggacaggt cgtcctatgc caggactagt tcaatattta 1560 aaaataccag caactgtagt agatagcgat ggtgcatggc aatttcttcc agatgtagct 1620 tcaagcagag ttcctattga agttacagag ttagaaaatt ggcaagtctt aactcctcca 1680 caaggtaaga ttcttggatt aaagcaattt aagttaacgg caggttttcc aacagaacaa 1740 agtcgcttac ctcttttaga gaattc 1766 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer toxA-F <400> 2 atatatgaaa acaaaacatt tttt 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer toxA-R <400> 3 tgtgttatag tgctcttgtt aagc 24 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer kan-F <400> 4 gcgcgatatc atgattgaac aagatggatt 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer kan-R <400> 5 cgcgaagctt tcagaagaac tcgtcaagaa 30 <210> 6 <211> 525 <212> DNA <213> Artificial Sequence <220> <223> 0.52 kb N-terminal fragment of toxA <400> 6 atgaaaacaa aacatttttt taactcagat tttactgtaa aaggaaaaag tgccgatgaa 60 atttttagaa gattgtgtac tgatcatcct gacaagcaat taaacaatgt aaaatggaaa 120 gaagttttta ttaatcgttt tggtcagatg atgctagata ctcctaatcc gagaaagatt 180 gtagaaaaaa ttattaatga agggcttgaa aaacaaggcc tgaaaaatat agatcctgaa 240 actacatatt tcaacatttt ttcatcttct gacagctccg atgggaacgt ttttcattat 300 aactctttat cagaatccta tcgagttact gatgcctgcc taatgaatat ttttgtggag 360 cgttattttg atgattggga cttgctaaat agcttagcca gtaatggaat atattcagta 420 ggaaaagaag gagcttatta tcctgatcat gattatggtc cagaatataa ccctgtttgg 480 ggaccaaacg aacaaattta ccattctaga gtgattgcag atatc 525 <210> 7 <211> 446 <212> DNA <213> Artificial Sequence <220> <223> 0.44 kb C-terminal fragment of toxA <400> 7 aagcttctgc aatcagcaaa agataataat ataaaattta gagctatagg gcattcagat 60 aattctgttc cgccatttaa taacccttat aagtctttat attataaagg aaatataata 120 gctgaagcaa ttgaaaaact agatcgagaa ggtcaaaaat ttgttgtatt tgctgatagt 180 tctctgctca acagcacgcc tgggacaggt cgtcctatgc caggactagt tcaatattta 240 aaaataccag caactgtagt agatagcgat ggtgcatggc aatttcttcc agatgtagct 300 tcaagcagag ttcctattga agttacagag ttagaaaatt ggcaagtctt aactcctcca 360 caaggtaaga ttcttggatt aaagcaattt aagttaacgg caggttttcc aacagaacaa 420 agtcgcttac ctcttttaga gaattc 446  

Claims (7)

A 0.52 kb fragment from the N-terminus of the Pasteurella multocida toxA gene of SEQ ID NO: 6 to 0.52 kb; Antibiotic resistance genes; And a 0.44 kb fragment from 0.46 kb to 0.9 kb from the C-terminus of the Pasteurella multocida toxA gene of SEQ ID NO: 7. The polynucleotide of claim 1 wherein the antibiotic resistance gene is a kanamycin resistance gene. The polynucleotide of claim 1, wherein the entire nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 1. A non-pathogenic, attenuated Pasteurella mulch transformed with a polynucleotide according to any one of claims 1 to 3 and knocked out the Pasteurella multocida toxA gene by homologous recombination. Toshida strain. The Pasteurella multocida strain of claim 4, wherein Accession No. KCCM10824P is used. Live vaccine for the prevention or treatment of Pasteurella multocida infection containing the Pasteurella multocida strain according to claim 4. 7. The live vaccine of claim 6, wherein the Pasteurella multocida infection is porcine Pasteurella pneumonia.

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