CN112891524A - Porcine mycoplasma pneumoniae subunit vaccine composition and preparation method and application thereof - Google Patents
Porcine mycoplasma pneumoniae subunit vaccine composition and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a swine mycoplasma pneumoniae subunit vaccine composition, which comprises PEA (delta III) -P42 recombinant protein, PEA (delta III) -P97 recombinant protein, PEA (delta III) -P95 recombinant protein, PEA (delta III) -P46 recombinant protein and PEA (delta III) -NrdF recombinant protein. The invention also discloses a preparation method and application of the swine mycoplasma pneumoniae subunit vaccine composition. The subunit vaccine for the mycoplasma hyopneumoniae of the invention has good immune effect and small immune dose, and can effectively prevent the mycoplasma hyopneumoniae related diseases and the related diseases caused by infection of the mycoplasma hyopneumoniae. The preparation method is simple, can prepare a large amount of antigen protein of the mycoplasma hyopneumoniae, has short time consumption and high expression level, greatly reduces the production cost, and is beneficial to large-scale production.
Description
Technical Field
The invention belongs to the field of biological products for livestock, and particularly relates to a swine mycoplasma hyopneumoniae subunit vaccine composition, and a preparation method and application thereof.
Background
Mycoplasma hyopneumoniae (MPS) is a chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae (Mhp) and mainly manifested by cough and asthma in pigs, and is characterized by chronic, contact, high infectivity, high morbidity and low mortality. The disease is widely distributed around the world, is often called local swine epidemic pneumonia abroad, and is often called swine enzootic pneumonia (asthma) in China. The growth rate of the pig is reduced by 12-16% due to mycoplasma hyopneumoniae infection, the feed conversion rate is reduced by 13-22%, the weight is relatively reduced by 10-25 kg, the marketing time of the commercial pig is delayed by 10-30 days, and meanwhile, the bronchus and bronchiole cilia of the pig are damaged, so that the physical barrier for preventing pathogenic microorganisms from invading is damaged, the huge salivary cells of the pig are damaged, the immune clearance capability of an organism is reduced, and the immunosuppression is caused. Therefore, mycoplasma hyopneumoniae is often mixed with pathogens such as porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus, actinobacillus pleuropneumoniae and pasteurella multocida clinically to infect, which increases the difficulty of diagnosis, prevention and control of the current swine disease. In addition, since Mycoplasma hyopneumoniae is a prokaryotic microorganism lacking a cell wall and is not sensitive to commonly used antibiotics acting on the cell wall (e.g., beta-lactam yeast antibiotics, etc.), the control and cleanup of swine herds once infected with Mycoplasma hyopneumoniae is difficult. According to preliminary statistics, the direct economic loss caused by mycoplasma hyopneumoniae infection worldwide is up to $ 2 hundred million each year. The 1999 statistics show that the cost of a vaccine for swine mycoplasmal pneumonia alone is as high as 1.1 billion pounds worldwide, which accounts for about one-fourth of the worldwide swine vaccine cost. It can be said that mycoplasma hyopneumoniae has become a major epidemic disease which seriously affects the economic benefit of the pig industry at present, and is likened to be a 'recessive killer' which affects the economic benefit of the pig industry.
At present, the control of the mycoplasma hyopneumoniae mainly depends on inactivated vaccines and attenuated vaccines, but the vaccines can only weaken the harm degree of diseases and cannot completely eradicate the mycoplasma hyopneumoniae. In addition, the culture conditions of mycoplasma hyopneumoniae are rigorous, the requirement on nutrition is high, the culture period is long, and the factors increase the production cost of the mycoplasma hyopneumoniae vaccine. Therefore, the development of a safe, efficient and low-cost novel vaccine is urgently needed.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a swine mycoplasma pneumoniae subunit vaccine composition, and a preparation method and application thereof. The vaccine composition has low production cost, high production efficiency and good immune effect.
In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:
the invention firstly provides a swine mycoplasma pneumoniae subunit vaccine composition, which comprises PEA (delta III) -P42 recombinant protein, PEA (delta III) -P97 recombinant protein, PEA (delta III) -P95 recombinant protein, PEA (delta III) -P46 recombinant protein and PEA (delta III) -NrdF recombinant protein; the PEA (delta III) -P42 recombinant protein is the fusion of a Pseudomonas aeruginosa exotoxin PEA (delta III) protein and a mycoplasma hyopneumoniae P42 protein fragment; the PEA (delta III) -P97 recombinant protein is the fusion of a Pseudomonas aeruginosa exotoxin PEA (delta III) protein and a mycoplasma hyopneumoniae P97 protein fragment; the PEA (delta III) -P95 recombinant protein is the fusion of a Pseudomonas aeruginosa exotoxin PEA (delta III) protein and a mycoplasma hyopneumoniae P95 protein fragment; the PEA (delta III) -P46 recombinant protein is the fusion of a PEA (delta III) protein of a pseudomonas aeruginosa exotoxin and a P46 protein fragment of mycoplasma hyopneumoniae, and the PEA (delta III) -NrdF recombinant protein is the fusion of the PEA (delta III) protein of the pseudomonas aeruginosa exotoxin and the NrdF protein of the mycoplasma hyopneumoniae.
Wherein the amino acid sequence of the Mycoplasma hyopneumoniae P42 protein fragment is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2.
Wherein the amino acid sequence of the Mycoplasma hyopneumoniae P97 protein fragment is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4.
Wherein the amino acid sequence of the Mycoplasma hyopneumoniae P95 protein fragment is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6.
Wherein the amino acid sequence of the Mycoplasma hyopneumoniae P46 protein fragment is shown as SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 8.
Wherein the amino acid sequence of the Mycoplasma hyopneumoniae NrdF protein is shown as SEQ ID NO.9, and the nucleotide sequence is shown as SEQ ID NO. 10.
The pseudomonas aeruginosa exotoxin PEA (delta III) protein is coded by a nucleotide sequence shown in SEQ ID NO.11 and is artificially synthesized.
Further, the vaccine composition also comprises an adjuvant.
Further, the adjuvant in the vaccine composition is SEPPIC Montanide IMS 1313VG NST adjuvant.
In the vaccine composition, the proportion of the PEA (delta III) -P42 recombinant protein, the PEA (delta III) -P97 recombinant protein, the PEA (delta III) -P95 recombinant protein, the PEA (delta III) -P46 recombinant protein and the PEA (delta III) -NrdF recombinant protein is 1:1:1:1: 1.
In the vaccine composition, the total content of PEA (delta III) -P42 recombinant protein, PEA (delta III) -P97 recombinant protein, PEA (delta III) -P95 recombinant protein, PEA (delta III) -P46 recombinant protein and PEA (delta III) -NrdF recombinant protein is 200-300 mu g/mL.
The invention also provides a preparation method of the swine mycoplasma hyopneumoniae subunit vaccine composition, which comprises the following steps:
(1) cloning and amplifying a P42 protein fragment gene, a P97 protein fragment gene, a P95 protein fragment gene, a P46 protein fragment gene and an NrdF protein gene, and respectively connecting the P42 protein fragment gene, the P97 protein fragment gene, the P95 protein fragment gene, the P46 protein fragment gene and the NrdF protein gene with a PEA (delta III) protein gene to construct a PEA (delta III) -P42 recombinant protein gene fragment, a PEA (delta III) -P97 recombinant protein gene fragment, a PEA (delta III) -P95 recombinant protein gene fragment, a PEA (delta III) -P46 recombinant protein gene fragment and a PEA (delta III) -NrdF recombinant protein gene fragment;
(2) constructing recombinant plasmids for expressing PEA (delta III) -P42 recombinant protein gene segments, PEA (delta III) -P97 recombinant protein gene segments, PEA (delta III) -P95 recombinant protein gene segments, PEA (delta III) -P46 recombinant protein gene segments and PEA (delta III) -NrdF recombinant protein gene segments;
(3) transforming the recombinant plasmid obtained in the step (2) into a DH10 Bac competent cell, and transposing to obtain a recombinant bacmid;
(4) transfecting the recombinant bacmid obtained in the step (3) into an insect cell to obtain a recombinant baculovirus;
(5) inoculating the obtained recombinant baculovirus into HF cells for mass culture, centrifuging and collecting culture solution supernatant to obtain PEA (delta III) -P42 recombinant protein, PEA (delta III) -P97 recombinant protein, PEA (delta III) -P95 recombinant protein, PEA (delta III) -P46 recombinant protein and PEA (delta III) -NrdF recombinant protein, purifying, adding an adjuvant, emulsifying and finally mixing uniformly.
The invention further provides application of the swine mycoplasma hyopneumoniae subunit vaccine composition in preparing a medicament for preventing and/or treating swine mycoplasma hyopneumoniae related diseases or related diseases infected by swine mycoplasma hyopneumoniae.
The invention has the beneficial effects that:
the subunit vaccine for the swine mycoplasma pneumoniae of the invention adopts PEA (delta III) -P42 recombinant protein, PEA (delta III) -P97 recombinant protein, PEA (delta III) -P95 recombinant protein, PEA (delta III) -P46 recombinant protein and PEA (delta III) -NrdF recombinant protein, has good immune effect and small immune dose, and can effectively prevent related diseases of the swine mycoplasma pneumoniae and related diseases infected by the swine mycoplasma pneumoniae.
The preparation method is simple, can prepare a large amount of antigen protein of the mycoplasma hyopneumoniae, has short time consumption and high expression level, greatly reduces the production cost, and is beneficial to large-scale production.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a swine mycoplasma pneumoniae subunit vaccine composition, which comprises an immunizing amount of PEA (delta III) -P42 recombinant protein, an immunizing amount of PEA (delta III) -P97 recombinant protein, an immunizing amount of PEA (delta III) -P95 recombinant protein, an immunizing amount of PEA (delta III) -P46 recombinant protein and an immunizing amount of PEA (delta III) -NrdF recombinant protein; the PEA (delta III) -P42 recombinant protein is the fusion of a Pseudomonas aeruginosa exotoxin PEA (delta III) protein and a mycoplasma hyopneumoniae P42 protein fragment; the PEA (delta III) -P97 recombinant protein is the fusion of a Pseudomonas aeruginosa exotoxin PEA (delta III) protein and a mycoplasma hyopneumoniae P97 protein fragment; the PEA (delta III) -P95 recombinant protein is the fusion of a Pseudomonas aeruginosa exotoxin PEA (delta III) protein and a mycoplasma hyopneumoniae P95 protein fragment; the PEA (delta III) -P46 recombinant protein is the fusion of a PEA (delta III) protein of a pseudomonas aeruginosa exotoxin and a P46 protein fragment of mycoplasma hyopneumoniae, and the PEA (delta III) -NrdF recombinant protein is the fusion of the PEA (delta III) protein of the pseudomonas aeruginosa exotoxin and the NrdF protein of the mycoplasma hyopneumoniae.
Wherein the amino acid sequence of the mycoplasma hyopneumoniae P42 protein, the amino acid sequence of the mycoplasma hyopneumoniae P97 protein, the amino acid sequence of the mycoplasma hyopneumoniae P95 protein, the amino acid sequence of the mycoplasma hyopneumoniae P46 protein and the amino acid sequence of the mycoplasma hyopneumoniae NrdF protein are all the amino acid sequences of corresponding proteins of the mycoplasma hyopneumoniae virus 7448 strain (the NCBI accession number is AE 017244.1).
The amino acid sequence of the mycoplasma hyopneumoniae P42 protein fragment is shown as SEQ ID NO.1, and is the 434 th to 600 th amino acid sequence based on the P42 protein amino acid sequence of mycoplasma hyopneumoniae 7448 strain shown as SEQ ID NO. 12; the nucleotide sequence of the P42 protein fragment is shown in SEQ ID NO. 2.
The amino acid sequence of the mycoplasma hyopneumoniae P97 protein fragment is shown as SEQ ID NO.3, and is the 788 th to 915 th amino acid sequence based on the P97 protein amino acid sequence of mycoplasma hyopneumoniae 7448 strain shown as SEQ ID NO. 13; the nucleotide sequence of the P97 protein fragment is shown in SEQ ID NO. 4.
The amino acid sequence of the mycoplasma hyopneumoniae P95 protein fragment is shown as SEQ ID NO.5, and the amino acid sequence of the mycoplasma hyopneumoniae P7448 strain P95 protein shown as SEQ ID NO.14 is the amino acid sequence from the 604 th position to the 750 th position of the benchmark; the nucleotide sequence of the P95 protein fragment is shown in SEQ ID NO. 6.
The amino acid sequence of the mycoplasma hyopneumoniae P46 protein fragment is shown as SEQ ID NO.7, and is the 323 rd-419 th amino acid sequence based on the P46 protein amino acid sequence of mycoplasma hyopneumoniae 7448 strain shown as SEQ ID NO. 15; the nucleotide sequence of the P46 protein fragment is shown in SEQ ID NO. 8.
The amino acid sequence of the mycoplasma hyopneumoniae NrdF protein is shown as SEQ ID NO.9, and the nucleotide sequence is shown as SEQ ID NO. 10.
The pseudomonas aeruginosa exotoxin PEA (delta III) protein is coded by a nucleotide sequence shown in SEQ ID NO.11 and is artificially synthesized.
The vaccine composition further comprises an adjuvant; the adjuvant is SEPPIC Montanide IMS 1313VG NST adjuvant.
In the vaccine composition, the proportion of PEA (delta III) -P42 recombinant protein, PEA (delta III) -P97 recombinant protein, PEA (delta III) -P95 recombinant protein, PEA (delta III) -P46 recombinant protein and PEA (delta III) -NrdF recombinant protein is 1:1:1:1: 1.
In the vaccine composition, the total content of PEA (delta III) -P42 recombinant protein, PEA (delta III) -P97 recombinant protein, PEA (delta III) -P95 recombinant protein, PEA (delta III) -P46 recombinant protein and PEA (delta III) -NrdF recombinant protein is 200-300 mu g/mL.
Examples
mu.L of Mycoplasma hyopneumoniae culture was taken and Mycoplasma hyopneumoniae DNA was extracted according to Kit instructions of Mini BEST Universal Genomic DNA Extraction Kit Ver.5.0 of TaKaRa.
1. Amplification of fragment gene of Mycoplasma hyopneumoniae P42 protein
The gene encoding the 434 nd to 600 th amino acid fragments of the P42 protein was amplified using the extracted DNA as a template and primers P42F and P42R. The primers are shown in Table 1, the PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.
TABLE 1P 42 protein fragment Gene amplification primers
TABLE 2P 42 protein fragment Gene PCR System
TABLE 3 PCR reaction conditions for P42 protein fragment genes
2. Amplification of Mycoplasma hyopneumoniae NrdF protein Gene
And (3) amplifying the NrdF protein gene by using the extracted DNA as a template and by using primers NrdF-F and NrdF-R according to the PCR reaction system and conditions in the step 1. The primers are shown in Table 4.
TABLE 4 NrdF protein Gene amplification primers
3. Amplification of fragment gene of Mycoplasma hyopneumoniae P97 protein
And (3) amplifying the gene of 788-915 amino acid fragments of the encoded P97 protein by using the extracted DNA as a template and primers P97-F and P97-R according to the PCR reaction system and conditions in the step 1. The primers are shown in Table 5.
TABLE 5P 97 protein fragment Gene amplification primers
4. Amplification of fragment gene of Mycoplasma hyopneumoniae P95 protein
And (3) amplifying the gene of the 604 th-750 th amino acid fragment of the P95 protein by using the extracted DNA as a template and using primers P95-F and P95-R according to the PCR reaction system and conditions in the step 1. The primers are shown in Table 6.
TABLE 6P 95 protein fragment Gene amplification primers
5. Amplification of fragment gene of Mycoplasma hyopneumoniae P46 protein
And (3) amplifying a gene encoding 323-419 amino acid fragment of the P46 protein by using the extracted DNA as a template and using primers P46-F and P46-R according to the PCR reaction system and conditions in the step 1. The primers are shown in Table 7.
TABLE 7P 46 protein fragment Gene amplification primers
6. Connection of recombinant protein gene and construction of recombinant protein expression vector
And (2) performing double enzyme digestion on the target gene obtained by amplification in the steps 1-5 by using Kpn I and Not I respectively, performing double enzyme digestion on the artificially synthesized PEA (delta III) gene by using BamH I and Kpn I, and performing double enzyme digestion on the pFastBacHT A vector by using BamH I and Not I. Respectively connecting 5 segments of target gene products with PEA (delta III) gene enzyme digestion products and pFastBacHT A carrier enzyme digestion products, transforming the connecting products into escherichia coli DH 5 alpha competent cells after connection, and respectively naming the obtained positive recombinant plasmids as: pFastBac-PEA (. DELTA.III) -P42, pFastBac-PEA (. DELTA.III) -NrdF, pFastBac-PEA (. DELTA.III) -P97, pFastBac-PEA (. DELTA.III) -P95, pFastBac-PEA (. DELTA.III) -P46.
7. Acquisition and passage of recombinant baculovirus
Recombinant rBacmid-PEA (. DELTA.III) -P42, rBacmid-PEA (. DELTA.III) -NrdF, rBacmid-PEA (. DELTA.III) -P97, rBacmid-PEA (. DELTA.III) -P95, rBacmid-PEA (. DELTA.III) -P46 were transfected into insect cells sf9, respectively, specifically with reference to CellfectinTM II Reagent(InvitrogenTM) The instructions are transfected, and after the cells are cultured until obvious lesions of visible cells, culture supernatants are harvested, namely recombinant baculovirus F1 generation rBac-PEA (delta III) -P42, rBac-PEA (delta III) -NrdF, rBac-PEA (delta III) -P97, rBac-PEA (delta III) -P95 and rBac-PEA (delta III) -P46.
The recombinant baculovirus of F1 generation harvested above was inoculated into sf9 cells (2.0X 10) at the log phase of growth at a virus inoculation amount of 1%6cfu/mL), culturing at 27 ℃ and 110rpm until the cytopathic effect is more than 80%, and harvesting the culture supernatant, wherein the supernatant is the F2 generation recombinant baculovirus. The operations are repeated, and F3\ F4 generation recombinant baculovirus is harvested.
8. Protein expression
The recombinant baculovirus of F4 generation obtained in step 7 was inoculated into HF cells in an inoculation amount of MOI 5 to 10, the cells were harvested about 7 days after inoculation, and the expression of the target protein was confirmed by SDS-PAGE of the supernatant obtained by centrifugation. The target protein was purified by nickel column, and protein quantification was performed with reference to Shanghai-Production BCA kit, and the results showed that the target protein concentrations after purification were 1.21mg/mL, 0.85mg/mL, 1.66mg/mL, 1.02mg/mL, and 1.53mg/mL, respectively.
Preparation of subunit vaccine composition for mycoplasma hyopneumoniae
Adding adjuvants into the obtained purified PEA (delta III) -P42 recombinant protein, PEA (delta III) -NrdF recombinant protein, PEA (delta III) -P97 recombinant protein, PEA (delta III) -P95 recombinant protein and PEA (delta III) -P46 recombinant protein, emulsifying, mixing, and storing at 4 deg.C. The specific ratio of the vaccine is shown in Table 8.
TABLE 8 Mycoplasma hyopneumoniae virus subunit vaccine component ratios
The amount of adjuvant suitable for use in the compositions of the present invention is preferably an effective amount. By "effective amount" is meant the amount of adjuvant necessary or sufficient to exert their immunological effect in a host when administered in combination with the antigen of the invention without causing undue side effects. The precise amount of adjuvant to be administered will vary depending on factors such as the ingredients used and the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.
The amount of an ingredient or component of the composition of the present invention is preferably a therapeutically effective amount. The therapeutically effective amount refers to the amount necessary to exert their immunological effects in the host to which the composition is administered without causing undue side effects. The precise amounts of the ingredients used and the composition to be administered will vary depending on factors such as the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.
Immunogenicity test of subunit vaccine for Mycoplasma hyopneumoniae
Selecting 35 weaned piglets, randomly dividing into 7 groups, and 5 weaned piglets in each group, wherein 6 groups correspond to 6 vaccines prepared in the immunization table 8 respectively, the immunization mode is neck intramuscular injection, and the immunization dose is 2 ml/head; group 7 served as a control group, and the neck was injected intramuscularly with the same dose of sterile PBS; 28 days after immunization, the immunized group and the control group were challenged with virulent strain YN14 strain, clinical isolate, Mycoplasma hyopneumoniae efficacy test, 0.2g of diseased lung tissue (100MID) was injected into each pig trachea, and each group of pigs were kept separately without antibiotics in the feed. And (5) killing the lung by dissecting 25-28 days after the virus attack, and calculating the reduction rate of the lung pathological changes. The reduction rate of the lung lesion of the immune group reaches 60 percent and is qualified by vaccine inspection. The results of the specific tests are shown in Table 9. According to the judgment of the virus attacking result, the vaccines 4, 5 and 6 have better immune protection effect and meet the requirements.
TABLE 9 protective results against challenge following immunization of piglets with Mycoplasma hyopneumoniae subunit vaccine
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents made by the contents of the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
Sequence listing
<110> Youbang, Yangzhou biopharmaceutical Co Ltd
<120> mycoplasma hyopneumoniae subunit vaccine composition, and preparation method and application thereof
<130> 2020
<141> 2020-12-09
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 167
<212> PRT
<213> Amino acid sequence of P42 protein fragment (Amino acid sequence of P42 protein fragment)
<400> 1
Ile Glu Ala Ala Pro Arg Gly Leu Pro Gln Ile Glu Val Ser Phe Ser
1 5 10 15
Ile Asp Val Asn Gly Ile Thr Thr Val Ser Ala Lys Asp Lys Lys Thr
20 25 30
Gly Lys Glu Gln Thr Ile Thr Ile Lys Asn Thr Ser Thr Leu Ser Glu
35 40 45
Glu Glu Ile Asn Lys Met Ile Gln Glu Ala Glu Glu Asn Arg Glu Ala
50 55 60
Asp Ala Leu Lys Lys Asp Lys Ile Glu Thr Thr Val Arg Ala Glu Gly
65 70 75 80
Leu Ile Asn Gln Leu Glu Lys Ser Ile Thr Asp Gln Gly Glu Lys Ile
85 90 95
Asp Pro Lys Gln Lys Glu Leu Leu Glu Lys Gln Ile Gln Glu Leu Lys
100 105 110
Asp Leu Leu Lys Glu Glu Lys Thr Asp Glu Leu Lys Leu Lys Leu Asp
115 120 125
Gln Ile Glu Ala Ala Ala Gln Ser Phe Ala Gln Ala Thr Ala Gln Gln
130 135 140
Ala Asn Thr Ser Glu Ser Asp Pro Lys Ala Asp Asp Ser Asn Thr Ile
145 150 155 160
Asp Ala Glu Ile Lys Gln Asp
165
<210> 2
<211> 501
<212> DNA
<213> P42 protein fragment nucleotide sequence (P42 protein fragment nucleotide sequence)
<400> 2
attgaagctg ctccacgagg tcttccccag attgaagtta gtttttcaat tgatgtcaac 60
gggattacaa cggtttcagc aaaagataaa aaaaccggca aagaacaaac aattacaatt 120
aaaaatactt caactttatc agaagaagaa attaataaga tgattcagga agccgaagaa 180
aatcgtgaag ctgatgctct taaaaaagac aaaatcgaga caacagttcg tgccgaaggg 240
cttattaatc aacttgagaa atcaataact gatcaaggtg aaaaaattga tccaaaacaa 300
aaagaattac ttgaaaaaca aattcaagaa ttaaaagatc ttctaaaaga agaaaaaact 360
gacgaattaa aattaaaatt agaccaaatt gaagcagctg cccaatcttt tgcgcaggca 420
accgcgcagc aagcaaatac atctgaatct gatccaaaag ctgatgattc aaacacaatt 480
gatgctgaaa tcaagcagga t 501
<210> 3
<211> 128
<212> PRT
<213> Amino acid sequence of P97 protein fragment (Amino acid sequence of P97 protein fragment)
<400> 3
Glu Gly Lys Arg Glu Glu Val Asp Lys Lys Val Lys Glu Leu Asp Asn
1 5 10 15
Lys Ile Lys Gly Ile Leu Pro Gln Pro Pro Ala Ala Lys Pro Glu Ala
20 25 30
Ala Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Val Ala Ala
35 40 45
Lys Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys
50 55 60
Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Glu Ala Ala Lys Pro
65 70 75 80
Val Ala Thr Asn Thr Asn Thr Asn Thr Gly Phe Ser Leu Thr Asn Lys
85 90 95
Pro Lys Glu Asp Tyr Phe Pro Met Ala Phe Ser Tyr Lys Leu Glu Tyr
100 105 110
Thr Asp Glu Asn Lys Leu Ser Leu Lys Thr Pro Glu Ile Asn Val Phe
115 120 125
<210> 4
<211> 384
<212> DNA
<213> P97 protein fragment nucleotide sequence (P97 protein fragment nucleotide sequence)
<400> 4
gaaggtaaaa gagaagaagt agataaaaaa gttaaagaat tagataataa aataaaaggt 60
atattacctc agcccccagc agctaaacct gaagcagcaa aaccagtagc agctaaacct 120
gaagcagcaa aaccagtagc agctaaacct gaagcagcaa aaccagtagc agctaaacct 180
gaagcagcaa aaccagtagc ggctaaacct gaagcagcta aacctgaagc agccaaacca 240
gttgctacta atactaatac taatactggc ttttcactta caaataaacc aaaagaagac 300
tatttcccaa tggcttttag ttataaatta gaatatactg acgaaaataa attaagccta 360
aaaacaccgg aaattaatgt attt 384
<210> 5
<211> 147
<212> PRT
<213> Amino acid sequence of P95 protein fragment (Amino acid sequence of P95 protein fragment)
<400> 5
Glu Ile Ser Glu Lys Glu Ser Lys Lys Leu Glu Lys Glu Pro Thr Asn
1 5 10 15
Leu Asn Gln Lys Thr Glu Lys Asn Ser Ser Thr Thr Ala Ile Ser Ser
20 25 30
Glu Lys Asn Glu Lys Pro Glu Lys Ser Met Ser Glu Thr Pro Pro Glu
35 40 45
Leu Val Lys Val Gln Gln Asn Gln Pro Lys Thr Glu Glu Glu Pro Lys
50 55 60
Gln Glu Thr Asp Lys Gln Glu Lys Pro Asn Asn Gln Lys Asn Thr Glu
65 70 75 80
Gln Ala Lys Glu Ser Lys Glu Lys Glu Lys Asn Gly Asn Ser Ser Leu
85 90 95
Lys Ser Pro Asn Leu Pro Val Glu Lys Ser Ser Asp Ser Gln Glu Glu
100 105 110
Gly Gln Lys Thr Asp Arg Leu Gln Gln Glu Asn Glu Gln Ala Ala Phe
115 120 125
Asn Asp Lys Ile Val Ser Ile Leu Gln Ser Gln Tyr Glu Ile Ser Leu
130 135 140
Ile Lys Asn
145
<210> 6
<211> 441
<212> DNA
<213> P95 protein fragment nucleotide sequence (P95 protein fragment nucleotide sequence)
<400> 6
gagatttccg aaaaagagtc aaaaaaacta gaaaaagaac caacaaattt aaaccaaaag 60
acagagaaaa atagttctac aaccgcaatt tcttctgaaa aaaatgaaaa accagaaaaa 120
tcaatgtctg aaacaccgcc cgagttagta aaggttcagc aaaatcagcc aaaaaccgaa 180
gaagaaccaa agcaagaaac tgataaacaa gaaaaaccca ataaccaaaa aaatacagaa 240
caagctaaag agagcaagga aaaggaaaaa aacggtaatt cttctttaaa aagtccgaat 300
ttacctgtgg aaaaatcctc tgattcccag gaagaaggtc aaaaaactga tagattacag 360
caagaaaatg aacaagccgc ttttaatgat aaaatagttt ctattctgca aagtcagtat 420
gaaatttcat taattaaaaa t 441
<210> 7
<211> 188
<212> PRT
<213> Amino acid sequence of P46 protein fragment (Amino acid sequence of P46 protein fragment)
<400> 7
Lys Asn Ser Gly Asn Lys Ile Ile Asp Leu Ser Pro Glu Gly Glu Asn
1 5 10 15
Ala Val Tyr Val Pro Gly Trp Asn Tyr Gly Thr Ala Gly Gln Arg Ile
20 25 30
Gln Ser Phe Leu Thr Ile Asn Lys Asp Pro Ala Gly Gly Asn Lys Ile
35 40 45
Lys Ala Ile Gly Ser Lys Pro Ala Ser Ile Phe Lys Gly Phe Leu Ala
50 55 60
Pro Asn Asp Gly Met Ala Glu Gln Ala Ile Thr Lys Leu Lys Leu Glu
65 70 75 80
Gly Phe Asp Thr Gln Lys Ile Phe Val Thr Gly Gln Asp Tyr Asn Asp
85 90 95
Lys Ala Lys Thr Phe Ile Lys Asp Gly Asp Gln Asn Met Thr Ile Tyr
100 105 110
Lys Pro Asp Lys Val Leu Gly Lys Val Ala Val Glu Val Leu Arg Val
115 120 125
Leu Ile Ala Lys Lys Asn Lys Ala Ser Arg Ser Glu Val Glu Asn Glu
130 135 140
Leu Lys Ala Lys Leu Pro Asn Ile Ser Phe Lys Tyr Asp Asn Gln Thr
145 150 155 160
Tyr Lys Val Gln Gly Lys Asn Ile Asn Thr Ile Leu Val Ser Pro Val
165 170 175
Ile Val Thr Lys Ala Asn Val Asp Asn Pro Asp Ala
180 185
<210> 8
<211> 564
<212> DNA
<213> P46 protein fragment nucleotide sequence (P46 protein fragment nucleotide sequence)
<400> 8
aaaaattcgg gaaataaaat aattgattta tctcctgaag gcgaaaatgc tgtttatgtc 60
ccaggatgga attatggaac tgccggtcaa agaatccaat cttttctaac aattaacaaa 120
gatccagcag gtggtaataa aatcaaagct attggttcaa aaccagcttc tattttcaaa 180
ggatttcttg caccaaatga tggaatggcc gaacaagcaa tcaccaaatt aaaacttgaa 240
ggatttgata cccaaaaaat ctttgtaact ggtcaagatt ataatgataa agccaaaact 300
tttatcaaag acggcgatca aaatatgaca atttataaac ctgataaagt tttaggaaaa 360
gttgctgttg aagttcttcg ggttttaatt gcaaagaaaa ataaagcatc tagatcagaa 420
gttgaaaacg aactaaaagc aaaactacca aatatttcat ttaaatatga taatcaaaca 480
tataaagtgc aaggtaaaaa tattaataca attttagtaa gtccagtaat tgttacaaaa 540
gctaatgttg ataatcctga tgcc 564
<210> 9
<211> 353
<212> PRT
<213> amino acid sequence of NrdF protein (NrdF protein amino acid sequence)
<400> 9
Met Ile Asn Ser Lys Leu Asn Ser Asn Ser Asn Asn Asn Lys Leu Lys
1 5 10 15
Ile Lys Asn Asp Tyr Tyr His Gln Ser Val Ser Pro Leu Glu Tyr Ala
20 25 30
Leu Asn Asn Phe Ser Gly Lys Met Arg Ser Ile Asn Trp Asn Ile Ile
35 40 45
Asn Asp Pro Lys Asp Leu Glu Val Trp Thr Arg Ile Val Gln Asn Phe
50 55 60
Trp Ile Pro Glu Lys Ile Pro Leu Ser Asn Asp Leu Glu Ser Trp Arg
65 70 75 80
Thr Leu Ser Pro Thr Trp Lys Lys Val Val Thr Arg Thr Phe Thr Gly
85 90 95
Leu Thr Leu Leu Asp Thr Ile Gln Ala Thr Ile Gly Asp Val Ala Gln
100 105 110
Ile Lys His Ser Leu Thr Asp His Glu Gln Val Ile Tyr Thr Asn Phe
115 120 125
Ala Phe Met Val Gly Val His Ala Arg Ser Tyr Gly Ser Ile Phe Ser
130 135 140
Thr Leu Cys Ser Ser Glu Glu Ile Glu Glu Ala His Asn Trp Val Ile
145 150 155 160
Asn Asn Glu Lys Leu Gln Lys Arg Ala Arg Ile Leu Ile Pro Phe Tyr
165 170 175
Val Gly Glu Asp Pro Leu Lys Ser Lys Val Ala Ala Ala Leu Met Pro
180 185 190
Gly Phe Leu Leu Tyr Gly Gly Phe Tyr Leu Pro Phe Tyr Leu Ser Ala
195 200 205
Arg Ser Lys Leu Pro Asn Thr Ser Asp Ile Ile Arg Leu Ile Leu Arg
210 215 220
Asp Lys Val Ile His Asn Tyr Tyr Ser Gly Tyr Lys Phe Gln Lys Lys
225 230 235 240
Val Glu Lys Leu Ser Pro Glu Lys Gln Asp Glu Ile Lys Lys Phe Thr
245 250 255
Phe Asp Leu Leu Tyr Lys Leu Ile Glu Leu Glu Lys Asp Tyr Leu Tyr
260 265 270
Asp Leu Tyr Ser Glu Val Gly Leu Ala Glu Ser Ala Ile Lys Phe Ser
275 280 285
Ile Tyr Asn Ala Gly Lys Phe Leu Gln Asn Leu Gly Tyr Asp Ser Pro
290 295 300
Phe Ser Lys Glu Glu Thr Glu Ile Glu Pro Glu Ile Phe Ser Gln Leu
305 310 315 320
Ser Ala Arg Ala Asp Glu Asn His Asp Phe Phe Ser Gly Asn Gly Ser
325 330 335
Ser Tyr Val Met Ala Leu Ala Glu Glu Thr Glu Asp Glu Asp Trp Glu
340 345 350
Phe
<210> 10
<211> 942
<212> DNA
<213> nucleotide sequence of NrdF protein (NrdF protein nucleotide sequence)
<400> 10
atgcgatcaa tcaactggaa tatcataaat gatccaaaag atcttgaagt ttggacccga 60
atagtgcaaa atttctggat tccagaaaaa attccgcttt caaatgatct tgaatcctgg 120
agaactttat cacctacctg gaaaaaagta gttactcgca cttttaccgg acttacttta 180
cttgatacaa ttcaggcaac aatcggtgat gtagcccaaa ttaaacactc tttaaccgat 240
catgaacagg taatttatac aaatttcgct tttatggttg gggttcatgc tcgttcttat 300
ggttcaattt tttctaccct ttgctcaagt gaagaaatag aagaggcgca taactgggta 360
atcaataacg aaaaattgca aaaaagggcc agaattctta taccttttta tgtaggtgaa 420
gatcctttaa aatcaaaagt tgctgctgct ttaatgccag gttttctctt atatggtggt 480
ttttatcttc ctttttatct ttcagcgcgt tcaaaactgc caaatacatc cgatattatt 540
cgactgattt tacgcgataa agtgattcat aattattatt caggttataa atttcaaaaa 600
aaagtggaaa aattaagccc ggaaaaacaa gacgaaatta aaaaatttac ctttgatcta 660
ttatataaac taattgaatt agaaaaagat tatctctatg atttatattc tgaagttgga 720
cttgctgaat cagcaataaa atttagcatt tataatgccg ggaaattctt gcaaaatcta 780
ggatatgatt cacctttttc aaaagaggaa accgaaattg aacctgaaat ttttagtcaa 840
ttatcggctc gagctgatga aaatcatgac tttttttcag gaaatggctc ttcttatgta 900
atggcgcttg ctgaagaaac tgaagatgaa gattgggagt tt 942
<210> 11
<211> 1236
<212> DNA
<213> Pseudomonas aeruginosa exotoxin PEA (DeltaIII protein nucleotide sequence)
<400> 11
atgcacctga cccctcactg gattcctctg gtggcctccc tgggactgct ggccggtggc 60
tcattcgctt ccgctgccga ggaggctttc gacctgtgga acgagtgcgc caaggcttgc 120
gtgctggacc tgaaggacgg agtcaggtcc agcagaatgt cagtggaccc tgccatcgct 180
gacactaacg gccagggagt gctgcactac tccatggtcc tggaaggagg taacgacgct 240
ctgaagctgg ccatcgacaa cgctctgagc atcacctctg acggtctgac tatccgcctg 300
gagggcggag tggaaccaaa caagcctgtc cgttactctt acactaggca ggctaggggc 360
agctggtctc tgaactggct ggtcccaatc ggacacgaga agccttctaa catcaaggtg 420
ttcatccacg aactgaacgc tggtaaccag ctgtcacaca tgtcccctat ctacactatc 480
gagatgggcg acgaactgct ggccaagctg gctagggacg ccaccttctt cgtcagagct 540
cacgagagca acgaaatgca gcctactctg gccatcagcc acgctggcgt gtctgtggtc 600
atggctcaag ctcagccaag gagggagaag aggtggtcag aatgggcttc tggcaaggtc 660
ctgtgcctgc tggaccccct ggacggcgtg tacaactacc tggctcagca gaggtgcaac 720
ctggacgaca cctgggaggg caagatctac agagtcctgg ccggcaaccc tgctaagcac 780
gacctggaca tcaagcccac cgtgatctca cacaggctgc acttccctga gggtggctcc 840
ctggctgctc tgactgctca ccaggcctgc cacctgccac tggaaacctt cactaggcac 900
agacagcctc gtggatggga gcagctggaa cagtgcggtt acccagtgca gaggctggtc 960
gccctgtacc tggctgccag actgagctgg aaccaggtgg accaggtcat caggaacgct 1020
ctggcttctc ctggctctgg aggtgacctg ggagaggcta tccgcgagca gccagaacaa 1080
gctaggctcg ctctgaccct ggctgctgct gagtctgaaa ggttcgtcag acagggtact 1140
ggcaacgacg aagccggagc cgcttcagct gacgtggtct ccctgacttg ccctgtggct 1200
gctggagagt gcgctggtcc tgctgactca ggtgac 1236
<210> 12
<211> 599
<212> PRT
<213> Mycoplasma hyopneumoniae 7448 strain P42 protein Amino acid sequence (Amino acid sequence of P42 protein of Mycoplasma hyopneumoniae 7448 strain)
<400> 12
Met Ala Lys Glu Ile Ile Leu Gly Ile Asp Leu Gly Thr Thr Asn Ser
1 5 10 15
Val Val Ala Ile Ile Glu Asn Gln Lys Pro Val Val Leu Glu Asn Pro
20 25 30
Asn Gly Lys Arg Thr Thr Pro Ser Val Val Ala Phe Lys Asn Asn Glu
35 40 45
Glu Ile Val Gly Asp Ala Ala Lys Arg Gln Leu Glu Thr Asn Pro Glu
50 55 60
Ala Ile Ala Ser Ile Lys Arg Leu Met Gly Thr Asp Lys Thr Val Arg
65 70 75 80
Ala Asn Glu Arg Asp Tyr Lys Pro Glu Glu Ile Ser Ala Lys Ile Leu
85 90 95
Ala Tyr Leu Lys Glu Tyr Ala Glu Lys Lys Ile Gly His Lys Val Thr
100 105 110
Lys Ala Val Ile Thr Val Pro Ala Tyr Phe Asp Asn Ala Gln Arg Glu
115 120 125
Ala Thr Lys Asn Ala Gly Lys Ile Ala Gly Leu Gln Val Glu Arg Ile
130 135 140
Ile Asn Glu Pro Thr Ala Ala Ala Leu Ala Phe Gly Leu Asp Lys Thr
145 150 155 160
Glu Lys Glu Met Lys Val Leu Val Tyr Asp Leu Gly Gly Gly Thr Phe
165 170 175
Asp Val Ser Val Leu Glu Leu Ser Gly Gly Thr Phe Glu Val Leu Ser
180 185 190
Thr Ser Gly Asp Asn His Leu Gly Gly Asp Asp Trp Asp Asn Glu Ile
195 200 205
Val Asn Leu Val Lys Lys Ile Lys Glu Glu Tyr Asp Phe Asp Pro Lys
210 215 220
Ser Asp Lys Met Ala Leu Thr Arg Leu Lys Glu Glu Ala Glu Lys Thr
225 230 235 240
Lys Ile Asn Leu Ser Asn Gln Ser Val Ser Thr Val Ser Leu Pro Phe
245 250 255
Leu Gly Met Gly Lys Asn Gly Pro Ile Asn Val Glu Leu Glu Leu Lys
260 265 270
Arg Ser Glu Phe Glu Lys Met Thr Ala His Leu Ile Asp Arg Thr Arg
275 280 285
Lys Pro Ile Val Asp Ala Leu Lys Gln Ala Lys Ile Glu Ala Ser Asp
290 295 300
Leu Asp Glu Val Leu Leu Val Gly Gly Ser Thr Arg Met Pro Ala Val
305 310 315 320
Gln Ser Met Ile Glu His Thr Leu Asn Lys Lys Pro Asn Arg Ser Ile
325 330 335
Asn Pro Asp Glu Val Val Ala Ile Gly Ala Ala Ile Gln Gly Gly Val
340 345 350
Leu Ala Gly Glu Ile Ser Asp Val Leu Leu Leu Asp Val Thr Pro Leu
355 360 365
Thr Leu Gly Ile Glu Thr Leu Gly Gly Ile Ala Thr Pro Leu Ile Pro
370 375 380
Arg Asn Thr Thr Ile Pro Val Thr Lys Ser Gln Ile Phe Ser Thr Ala
385 390 395 400
Glu Asp Asn Gln Thr Glu Val Thr Ile Ser Val Val Gln Gly Glu Arg
405 410 415
Gln Leu Ala Ala Asp Asn Lys Met Leu Gly Arg Phe Asn Leu Ser Gly
420 425 430
Ile Glu Ala Ala Pro Arg Gly Leu Pro Gln Ile Glu Val Ser Phe Ser
435 440 445
Ile Asp Val Asn Gly Ile Thr Thr Val Ser Ala Lys Asp Lys Lys Thr
450 455 460
Gly Lys Glu Gln Thr Ile Thr Ile Lys Asn Thr Ser Thr Leu Ser Glu
465 470 475 480
Glu Glu Ile Asn Lys Met Ile Gln Glu Ala Glu Glu Asn Arg Glu Ala
485 490 495
Asp Ala Leu Lys Lys Asp Lys Ile Glu Thr Thr Val Arg Ala Glu Gly
500 505 510
Leu Ile Asn Gln Leu Glu Lys Ser Ile Thr Asp Gln Gly Glu Lys Ile
515 520 525
Asp Pro Lys Gln Lys Glu Leu Leu Glu Lys Gln Ile Gln Glu Leu Lys
530 535 540
Asp Leu Leu Lys Glu Glu Lys Thr Asp Glu Leu Lys Leu Lys Leu Asp
545 550 555 560
Gln Ile Glu Ala Ala Ala Gln Ser Phe Ala Gln Ala Thr Ala Gln Gln
565 570 575
Ala Asn Thr Ser Glu Ser Asp Pro Lys Ala Asp Asp Ser Asn Thr Ile
580 585 590
Asp Ala Glu Ile Lys Gln Asp
595
<210> 13
<211> 1082
<212> PRT
<213> Mycoplasma hyopneumoniae 7448 strain P97 protein Amino acid sequence (Amino acid sequence of P97 protein of Mycoplasma hyopneumoniae 7448 strain)
<400> 13
Met Ser Lys Lys Ser Lys Thr Phe Lys Ile Gly Leu Thr Ala Gly Ile
1 5 10 15
Val Gly Leu Gly Val Phe Gly Leu Thr Val Gly Leu Ser Ser Leu Ala
20 25 30
Lys Tyr Arg Ser Glu Ser Pro Arg Lys Ile Ala Asn Asp Phe Ala Ala
35 40 45
Lys Val Ser Thr Leu Ala Phe Ser Pro Tyr Ala Phe Glu Thr Asp Ser
50 55 60
Asp Tyr Lys Ile Val Lys Arg Met Leu Val Asp Ser Asn Asn Asn Ile
65 70 75 80
Arg Asn Lys Glu Lys Val Ile Asp Ser Phe Ser Phe Phe Thr Lys Asn
85 90 95
Gly Asp Gln Leu Glu Lys Ile Asn Phe Gln Asp Pro Glu Tyr Thr Lys
100 105 110
Ala Lys Ile Thr Phe Glu Ile Leu Glu Ile Ile Pro Asp Asp Val Asn
115 120 125
Gln Asn Phe Lys Val Lys Phe Gln Ala Leu Gln Lys Leu His Asn Gly
130 135 140
Asp Ile Ala Lys Ser Asp Ile Tyr Glu Gln Thr Val Ala Phe Ala Lys
145 150 155 160
Gln Ser Asn Leu Leu Val Ala Glu Phe Asn Phe Ser Leu Lys Lys Ile
165 170 175
Thr Glu Lys Leu Asn Gln Gln Ile Glu Asn Leu Ser Thr Lys Ile Thr
180 185 190
Asn Phe Ala Asp Glu Lys Thr Ser Ser Gln Lys Asp Pro Ser Thr Leu
195 200 205
Arg Ala Ile Asp Phe Gln Tyr Asp Leu Asn Thr Ala Arg Asn Pro Glu
210 215 220
Asp Leu Asp Ile Lys Leu Ala Asn Tyr Phe Pro Val Leu Lys Asn Leu
225 230 235 240
Ile Asn Arg Leu Asn Asn Ala Pro Glu Asn Lys Leu Pro Asn Asn Leu
245 250 255
Gly Asn Ile Phe Glu Phe Ser Phe Ala Lys Asp Ser Ser Thr Asn Gln
260 265 270
Tyr Val Ser Ile Gln Asn Gln Ile Pro Ser Leu Phe Leu Lys Ala Asp
275 280 285
Leu Ser Gln Ser Ala Arg Glu Ile Leu Ala Ser Pro Asp Glu Val Gln
290 295 300
Pro Val Ile Asn Ile Leu Arg Leu Met Lys Lys Asp Asn Ser Ser Tyr
305 310 315 320
Phe Leu Asn Phe Glu Asp Phe Val Asn Asn Leu Thr Leu Lys Asn Met
325 330 335
Gln Lys Glu Asp Leu Asn Ala Lys Gly Gln Asn Leu Ser Ala Tyr Glu
340 345 350
Phe Leu Ala Asp Ile Lys Ser Gly Phe Phe Pro Gly Asp Lys Arg Ser
355 360 365
Ser His Thr Lys Ala Glu Ile Ser Asn Leu Leu Asn Lys Lys Glu Asn
370 375 380
Ile Tyr Asp Phe Gly Lys Tyr Asn Gly Lys Phe Asn Asp Arg Leu Asn
385 390 395 400
Ser Pro Asn Leu Glu Tyr Ser Leu Asp Ala Ala Ser Ala Ser Leu Asp
405 410 415
Lys Lys Asp Lys Ser Ile Ile Leu Ile Pro Tyr Arg Leu Glu Ile Lys
420 425 430
Asp Lys Phe Phe Ala Asp Asp Leu Tyr Pro Asp Thr Lys Asp Asn Ile
435 440 445
Leu Val Lys Glu Gly Ile Leu Lys Leu Thr Gly Phe Lys Lys Gly Pro
450 455 460
Lys Ile Asp Leu Pro Asn Ile Asn Gln Gln Ile Phe Lys Thr Glu Tyr
465 470 475 480
Leu Pro Phe Phe Glu Lys Gly Lys Glu Glu Gln Ala Lys Leu Asp Tyr
485 490 495
Gly Asn Ile Leu Asn Pro Tyr Asn Thr Gln Leu Ala Lys Val Glu Val
500 505 510
Glu Ala Leu Phe Lys Gly Asn Lys Asn Gln Glu Ile Tyr Gln Ala Leu
515 520 525
Asp Gly Asn Tyr Ala Tyr Glu Phe Gly Ala Phe Lys Ser Val Leu Asn
530 535 540
Ser Met Thr Gly Lys Ile Gln His Pro Glu Lys Ala Asp Ile Gln Arg
545 550 555 560
Phe Thr Arg His Leu Glu Gln Val Lys Ile Ser Ser Asn Ser Val Leu
565 570 575
Asn Gln Pro Gln Thr Thr Lys Glu Gln Val Ile Ser Ser Leu Lys Ser
580 585 590
Asn Asn Phe Phe Lys Asn Gly His Gln Val Ala Ser Tyr Phe Gln Asp
595 600 605
Leu Leu Thr Lys Asp Lys Leu Thr Val Leu Glu Thr Leu Tyr Asp Leu
610 615 620
Ala Lys Lys Trp Gly Leu Glu Thr Asn Ser Ala Gln Phe Pro Lys Gly
625 630 635 640
Ala Phe Gln Tyr Thr Lys Asp Ile Phe Ala Glu Ala Asp Lys Leu Glu
645 650 655
Phe Leu Glu Trp Lys Lys Lys Asp Pro Tyr Asn Gln Ile Lys Glu Ile
660 665 670
His Gln Leu Ser Phe Asn Ile Leu Ala Arg Asn Asp Val Ile Lys Ser
675 680 685
Asp Gly Phe Tyr Gly Val Leu Leu Leu Pro Gln Ser Val Lys Thr Glu
690 695 700
Leu Glu Gly Lys Asn Glu Ala Gln Ile Phe Glu Ala Leu Lys Lys Tyr
705 710 715 720
Ser Leu Ile Glu Asn Ser Ala Phe Lys Thr Thr Ile Leu Asp Lys Asn
725 730 735
Leu Leu Glu Gly Thr Asp Phe Lys Thr Phe Gly Asp Phe Leu Lys Ala
740 745 750
Phe Phe Leu Lys Ala Ala Gln Phe Asn Asn Phe Ala Pro Met Ala Lys
755 760 765
Leu Asp Asp Asn Leu Gln Tyr Ser Phe Glu Ala Ile Lys Lys Gly Glu
770 775 780
Thr Thr Lys Glu Gly Lys Arg Glu Glu Val Asp Lys Lys Val Lys Glu
785 790 795 800
Leu Asp Asn Lys Ile Lys Gly Ile Leu Pro Gln Pro Pro Ala Ala Lys
805 810 815
Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro
820 825 830
Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Val Ala Ala Lys Pro Glu
835 840 845
Ala Ala Lys Pro Val Ala Ala Lys Pro Glu Ala Ala Lys Pro Glu Ala
850 855 860
Ala Lys Pro Val Ala Thr Asn Thr Asn Thr Asn Thr Gly Phe Ser Leu
865 870 875 880
Thr Asn Lys Pro Lys Glu Asp Tyr Phe Pro Met Ala Phe Ser Tyr Lys
885 890 895
Leu Glu Tyr Thr Asp Glu Asn Lys Leu Ser Leu Lys Thr Pro Glu Ile
900 905 910
Asn Val Phe Leu Glu Leu Val His Gln Ser Glu Tyr Glu Glu Gln Lys
915 920 925
Ile Ile Lys Glu Leu Asp Lys Thr Val Leu Asn Leu Gln Tyr Gln Phe
930 935 940
Gln Glu Val Lys Val Ala Ser Asp Gln Tyr Gln Lys Leu Ser His Pro
945 950 955 960
Met Met Thr Glu Gly Ser Ser Asn Gln Gly Lys Lys Gly Glu Gly Thr
965 970 975
Pro Asn Gln Gly Lys Lys Ala Glu Gly Ala Pro Ser Gln Gly Lys Lys
980 985 990
Ala Glu Gly Thr Ser Asn Gln Gln Asn Thr Thr Thr Glu Leu Thr Asn
995 1000 1005
Tyr Leu Pro Glu Leu Gly Lys Lys Ile Asp Glu Ile Ile Lys Lys Gln
1010 1015 1020
Gly Lys Asn Trp Lys Thr Glu Val Glu Leu Ile Glu Asp Asn Ile Ala
1025 1030 1035 1040
Gly Asp Ala Lys Leu Leu Tyr Phe Val Leu Arg Asp Asp Ser Lys Ser
1045 1050 1055
Gly Asp Pro Lys Lys Ser Ser Leu Lys Val Lys Ile Thr Val Lys Gln
1060 1065 1070
Ser Asn Asn Asn Gln Glu Leu Lys Ser Lys
1075 1080
<210> 14
<211> 1147
<212> PRT
<213> Mycoplasma hyopneumoniae 7448 strain P95 protein Amino acid sequence (Amino acid sequence of P95 protein of Mycoplasma hyopneumoniae 7448 strain)
<400> 14
Met Lys Ile Val Lys Lys Ile Lys Lys Thr Lys Tyr Leu Thr Arg Lys
1 5 10 15
Ser Lys Ile Leu Leu Gly Leu Gly Leu Ser Ser Ser Ile Leu Gly Ile
20 25 30
Leu Gly Ile Ser Val Ala Val Ser Tyr Gly Leu Ala Val Ile Lys Lys
35 40 45
Asn Ser Tyr Asn Thr Thr Ile Ala Asp Leu Asn Arg Leu Ala Gln Lys
50 55 60
Ile Asn Gly Leu Ser Phe Asn Ala Gln Lys Ile Ser Pro Phe Ser Thr
65 70 75 80
Tyr Ala Ser Leu Lys Lys Glu Trp Lys Asn Leu Glu Ser Ser Glu Lys
85 90 95
Ser Gly Glu Phe Phe Asp Phe Tyr Thr Leu Asn Tyr Lys Arg Leu Gln
100 105 110
Pro Tyr Lys Leu Pro Asn Gly Ile Trp Ala Glu Phe Leu Lys Val Glu
115 120 125
Pro Asp Asp Ala Asn Gln Gln Phe Asn Val Glu Phe Val Leu Lys Ser
130 135 140
Phe Asn Gly Ser Arg Val Ile Lys Ser Asp Ile Lys Ser Glu Lys Val
145 150 155 160
Ala Ile Ser Pro Asn Ser Thr Phe Phe Leu Glu Asn Phe Tyr Gln Ala
165 170 175
Leu Glu Ile Asp Leu Lys Asn Ile Ser Pro Tyr Ser Asn Gly Asp Lys
180 185 190
Gly Lys Lys Asn Pro Lys Asn Trp Leu Ala Ser Asp Phe Leu Thr Glu
195 200 205
Ile Asn Ser Arg Glu Thr Ala Gln Asp Ala Ile Lys Arg Ile Arg Asp
210 215 220
Phe Phe Asn Phe Asp Phe Asp Ser Val Leu Lys Asn Lys Asn Phe Ala
225 230 235 240
Ile Lys Tyr Lys Asp Asn Leu Ile Phe Pro Tyr Lys Ile Glu Ile Leu
245 250 255
Lys Asn Asp Asp Asp Ser Trp Ile Lys Pro Ser Gln Leu Asn Pro Asp
260 265 270
Phe Leu Glu Ile Gln Gly Arg Val Ser Phe Thr Asp Gln Ala Lys Lys
275 280 285
Leu Phe Pro Lys Ser Phe Asn Thr Asn Ile Thr Lys Asn Phe Ser Phe
290 295 300
Leu Leu Phe Asp Leu Ala Leu Asn Lys Ser Ala Phe Ala Asp Pro Lys
305 310 315 320
Val Phe Ile Arg Ile Pro Lys Leu Val Glu Pro Lys Ile Asp Glu Phe
325 330 335
Ser Ser Glu Asn Pro Gln Glu Lys Ile Asp Leu Ser Gln Lys Ser Ile
340 345 350
Phe Trp Ile Tyr Asn Phe Leu Lys Tyr Lys Glu Asp Asn Ile Ser Leu
355 360 365
Lys Thr Pro Glu Glu Ala Lys Asn Ala Leu Asn Ser Leu Leu Asn Asn
370 375 380
Asp Leu Glu Leu Asp Phe Ser Gln Asn Asn Asn Leu Glu Pro Asn Ile
385 390 395 400
Lys Glu Lys Phe Glu Tyr Gln Leu Leu Val Asp Lys Ile Asn Phe Asn
405 410 415
Ser Asp Gln Asn Ser Ser Phe Ile Asn Ile Pro Phe Glu Ile Ser Tyr
420 425 430
Pro Leu Asp Ser Glu Lys Lys Asn Lys Leu Lys Thr Glu Thr Gln Val
435 440 445
Leu Leu Arg Lys Phe Lys Asn Ser Ser Ser Leu Asp Ser Thr Val Phe
450 455 460
Asp Pro Lys Asn Phe Ser Ser Leu Pro Ile Val Asn Leu Lys Phe Ile
465 470 475 480
Ser Glu Lys Asn Arg Asp Lys Asp Asp Ile Phe Glu Ala Phe Glu Thr
485 490 495
Val Ser Lys Phe Glu Leu Glu Arg Leu Leu Asn Leu Asn Gln Gln Gln
500 505 510
Gln Ile Tyr Gln Ile Leu Thr Asp Pro Thr Lys Phe Asn Leu Thr Phe
515 520 525
Pro Glu Lys Glu Ile Leu Asp Ala Trp Ile Ser Ser Tyr Asn Phe Pro
530 535 540
Ser Val Gln Glu Phe Ser Arg Arg Thr Leu Val Ala Lys Lys Leu Glu
545 550 555 560
Asp Gly Ser Gln Ser Arg Pro Phe Phe Glu Asn Ser Arg Glu Phe Ile
565 570 575
Ala Phe Thr Lys Lys Ile Leu Ser Leu Glu Lys Ala Glu Ala Lys Lys
580 585 590
Tyr Ile Glu Pro Phe Phe Glu Ala Leu Lys Thr Glu Ile Ser Glu Lys
595 600 605
Glu Ser Lys Lys Leu Glu Lys Glu Pro Thr Asn Leu Asn Gln Lys Thr
610 615 620
Glu Lys Asn Ser Ser Thr Thr Ala Ile Ser Ser Glu Lys Asn Glu Lys
625 630 635 640
Pro Glu Lys Ser Met Ser Glu Thr Pro Pro Glu Leu Val Lys Val Gln
645 650 655
Gln Asn Gln Pro Lys Thr Glu Glu Glu Pro Lys Gln Glu Thr Asp Lys
660 665 670
Gln Glu Lys Pro Asn Asn Gln Lys Asn Thr Glu Gln Ala Lys Glu Ser
675 680 685
Lys Glu Lys Glu Lys Asn Gly Asn Ser Ser Leu Lys Ser Pro Asn Leu
690 695 700
Pro Val Glu Lys Ser Ser Asp Ser Gln Glu Glu Gly Gln Lys Thr Asp
705 710 715 720
Arg Leu Gln Gln Glu Asn Glu Gln Ala Ala Phe Asn Asp Lys Ile Val
725 730 735
Ser Ile Leu Gln Ser Gln Tyr Glu Ile Ser Leu Ile Lys Asn Leu Thr
740 745 750
Asn Gln Ile Glu Ala Leu Lys Asn Ser Pro Glu Lys Gln Lys Ala Thr
755 760 765
Leu Asp Ala Ala Ser Phe Thr Asp Leu Phe Ile Glu Thr Tyr Ile Asn
770 775 780
Asn Asp Leu Val Ser Gln Phe Asp Ser Phe Ala Ala Gly Leu Asn Tyr
785 790 795 800
Lys Ile Val Phe Val Ala Asn Gln Asp Asp Glu Ile Ile Asp Gln Val
805 810 815
Gln Leu Pro Glu Pro Glu Lys Ser Lys Lys Thr Glu Lys Thr Pro Asp
820 825 830
Phe Lys Glu Glu Ser Glu Lys Lys Glu Leu Glu Asn Lys Glu Asn Ser
835 840 845
Pro Ala Val Asp Gln Lys Ser Asp Gln Lys Ile Ser Leu Ser Phe Phe
850 855 860
Gln Glu Glu Lys Asn Asn Thr Gln Asn Gln Gly Gln Glu Glu Ser Asp
865 870 875 880
Lys Lys Gln Gln Thr Pro Lys Thr Ala Gln Glu Asn Ser Gln Asn Leu
885 890 895
Val Gln Gln Pro Lys Pro Glu Gln Glu Thr Glu Ile Phe Lys Leu Gly
900 905 910
Tyr Tyr Tyr Ile Phe Thr Ala Pro Asn Ser Glu Lys Ile Val Phe Arg
915 920 925
Thr Pro Ile Lys Ser Leu Lys Leu Lys Val Phe Leu Ala Glu Lys Gly
930 935 940
Gly Ile Thr Leu Glu Lys Leu Ser Tyr Asn Met Leu Asn Phe Pro Gln
945 950 955 960
Ser Leu Leu Lys Leu Glu Leu Ala Asp Ser Asn Phe Thr Ser Ala Glu
965 970 975
Ala Leu Lys Lys Gln Pro Glu Asp Ile Leu Lys Ala Glu Phe Ser Ser
980 985 990
Gln Asp Lys Asp Phe Lys Ala Thr Met Thr Ser Phe Lys Lys Leu Phe
995 1000 1005
Gly Asn Lys Thr Phe Met Lys Ile Tyr Pro Leu Leu Ser Gly Asn Gly
1010 1015 1020
Leu Ile Tyr Lys Ala Asn Ser Val Phe Lys Asp Lys Phe Gly Asn Leu
1025 1030 1035 1040
Lys Ile Arg Phe Ala Val Lys Asp Leu Asp Ala Ser Glu Lys Lys Gln
1045 1050 1055
Ile Val Phe Pro Asn Ile Leu Glu Pro Glu Leu Glu Lys Lys Glu Gln
1060 1065 1070
Asp Val Arg Pro Lys Ala Thr Glu Ala Glu Ser Lys Gln Glu Lys Gln
1075 1080 1085
Glu Lys Gln Glu Lys Ser Gln Leu Asn Ser Glu Ser Ser Gln Thr Ser
1090 1095 1100
Thr Thr Ser Ser Gln Asn Gln Glu Asn Asn Gln Lys Asn Gln Leu Glu
1105 1110 1115 1120
Ile Phe Lys Pro Ala Glu Lys Glu Ala Lys Tyr Pro Leu Val Phe Thr
1125 1130 1135
Val Ile Lys Pro Asn Arg Gln Phe Arg Arg Asn
1140 1145
<210> 15
<211> 419
<212> PRT
<213> Mycoplasma hyopneumoniae 7448 strain P46 protein Amino acid sequence (Amino acid sequence of P46 protein of Mycoplasma hyopneumoniae 7448 strain)
<400> 15
Met Lys Lys Met Leu Arg Lys Lys Phe Leu Tyr Ser Ser Ala Ile Tyr
1 5 10 15
Ala Thr Ser Leu Ala Ser Ile Ile Ala Phe Val Ala Ala Gly Cys Gly
20 25 30
Gln Thr Glu Ser Gly Ser Thr Ser Asp Ser Lys Pro Gln Ala Glu Thr
35 40 45
Leu Lys His Lys Val Ser Asn Asp Ser Ile Arg Ile Ala Leu Thr Asp
50 55 60
Pro Asp Asn Pro Arg Met Ile Ser Ala Gln Lys Asp Ile Ile Ser Tyr
65 70 75 80
Val Asp Glu Thr Glu Ala Ala Thr Ser Thr Ile Thr Lys Asn Gln Asp
85 90 95
Ala Gln Asn Asn Met Leu Thr Gln Gln Ala Asn Leu Ser Pro Ala Pro
100 105 110
Lys Gly Phe Ile Ile Ala Pro Glu Asn Gly Ser Gly Val Gly Thr Ala
115 120 125
Val Asn Thr Ile Ala Asp Lys Gly Ile Pro Ile Val Ala Tyr Asp Arg
130 135 140
Leu Ile Thr Gly Ser Asp Lys Tyr Asp Trp Tyr Val Ser Phe Asp Asn
145 150 155 160
Glu Lys Val Gly Glu Leu Gln Gly Leu Ser Leu Ala Ala Gly Leu Leu
165 170 175
Gly Lys Glu Asp Gly Ala Phe Asp Ser Ile Asp Gln Met Asn Glu Tyr
180 185 190
Leu Lys Ser His Met Pro Gln Glu Thr Ile Ser Phe Tyr Thr Ile Ala
195 200 205
Gly Ser Gln Asp Asp Asn Asn Ser Gln Tyr Phe Tyr Asn Gly Ala Met
210 215 220
Lys Val Leu Lys Glu Leu Met Lys Asn Ser Gly Asn Lys Ile Ile Asp
225 230 235 240
Leu Ser Pro Glu Gly Glu Asn Ala Val Tyr Val Pro Gly Met Asn Tyr
245 250 255
Gly Thr Ala Gly Gln Arg Ile Gln Ser Phe Leu Thr Ile Asn Lys Asp
260 265 270
Pro Ala Gly Gly Asn Lys Ile Lys Ala Ile Gly Ser Lys Pro Ala Ser
275 280 285
Ile Phe Lys Gly Phe Leu Ala Pro Asn Asp Gly Met Ala Glu Gln Ala
290 295 300
Ile Thr Lys Leu Lys Leu Glu Gly Phe Asp Thr Gln Lys Ile Phe Val
305 310 315 320
Thr Gly Gln Asp Tyr Asn Asp Lys Ala Lys Thr Phe Ile Lys Asp Gly
325 330 335
Asp Gln Asn Met Thr Ile Tyr Lys Pro Asp Lys Val Leu Gly Lys Val
340 345 350
Ala Val Glu Val Leu Arg Val Leu Ile Ala Lys Lys Asn Lys Ala Ser
355 360 365
Arg Ser Glu Val Glu Asn Glu Leu Lys Ala Lys Leu Pro Asn Ile Ser
370 375 380
Phe Lys Tyr Asp Asn Gln Thr Tyr Lys Val Gln Gly Lys Asn Ile Asn
385 390 395 400
Thr Ile Leu Val Ser Pro Val Ile Val Thr Lys Ala Asn Val Asp Asn
405 410 415
Pro Asp Ala
Claims (10)
1. A porcine mycoplasma pneumoniae subunit vaccine composition is characterized by comprising PEA (delta III) -P42 recombinant protein, PEA (delta III) -P97 recombinant protein, PEA (delta III) -P95 recombinant protein, PEA (delta III) -P46 recombinant protein and PEA (delta III) -NrdF recombinant protein; the PEA (delta III) -P42 recombinant protein is the fusion of a Pseudomonas aeruginosa exotoxin PEA (delta III) protein and a mycoplasma hyopneumoniae P42 protein fragment; the PEA (delta III) -P97 recombinant protein is the fusion of a Pseudomonas aeruginosa exotoxin PEA (delta III) protein and a mycoplasma hyopneumoniae P97 protein fragment; the PEA (delta III) -P95 recombinant protein is the fusion of Pseudomonas aeruginosa exotoxin PE A (delta III) protein and a mycoplasma hyopneumoniae P95 protein fragment; the PEA (delta III) -P46 recombinant protein is the fusion of a PEA (delta III) protein of a pseudomonas aeruginosa exotoxin and a P46 protein fragment of mycoplasma hyopneumoniae, and the PEA (delta III) -NrdF recombinant protein is the fusion of the PEA (delta III) protein of the pseudomonas aeruginosa exotoxin and the NrdF protein of the mycoplasma hyopneumoniae.
2. The mycoplasma hyopneumoniae subunit vaccine composition of claim 1, wherein the mycoplasma hyopneumoniae P42 protein fragment has an amino acid sequence shown as SEQ ID No.1 and a nucleotide sequence shown as SEQ ID No. 2.
3. The mycoplasma hyopneumoniae subunit vaccine composition of claim 1, wherein the mycoplasma hyopneumoniae P97 protein fragment has an amino acid sequence shown as SEQ ID No.3 and a nucleotide sequence shown as SEQ ID No. 4.
4. The mycoplasma hyopneumoniae subunit vaccine composition of claim 1, wherein the mycoplasma hyopneumoniae P95 protein fragment has an amino acid sequence shown as SEQ ID No.5 and a nucleotide sequence shown as SEQ ID No. 6.
5. The mycoplasma hyopneumoniae subunit vaccine composition of claim 1, wherein the mycoplasma hyopneumoniae P46 protein fragment has an amino acid sequence shown as SEQ ID No.7 and a nucleotide sequence shown as SEQ ID No. 8.
6. The mycoplasma hyopneumoniae subunit vaccine composition of claim 1, wherein the mycoplasma hyopneumoniae NrdF protein has an amino acid sequence shown in SEQ ID No.9 and a nucleotide sequence shown in SEQ ID No. 10.
7. The swine mycoplasma pneumoniae subunit vaccine composition of claim 1, wherein the pseudomonas aeruginosa exotoxin PEA (Δ iii) protein is encoded by a nucleotide sequence shown in SEQ ID No. 11.
8. The swine mycoplasma pneumoniae subunit vaccine composition according to any one of claims 1 to 7, wherein the vaccine composition further comprises an adjuvant.
9. A method of preparing the mycoplasma hyopneumoniae subunit vaccine composition of any one of claims 1-8, comprising the steps of:
(1) cloning and amplifying a P42 protein fragment gene, a P97 protein fragment gene, a P95 protein fragment gene, a P46 protein fragment gene and an NrdF protein gene, and respectively connecting the P42 protein fragment gene, the P97 protein fragment gene, the P95 protein fragment gene, the P46 protein fragment gene and the NrdF protein gene with a PEA (delta III) protein gene to construct a PEA (delta III) -P42 recombinant protein gene fragment, a PEA (delta III) -P97 recombinant protein gene fragment, a PEA (delta III) -P95 recombinant protein gene fragment, a PEA (delta III) -P46 recombinant protein gene fragment and a PEA (delta III) -NrdF recombinant protein gene fragment;
(2) constructing recombinant plasmids for expressing PEA (delta III) -P42 recombinant protein gene segments, PEA (delta III) -P97 recombinant protein gene segments, PEA (delta III) -P95 recombinant protein gene segments, PEA (delta III) -P46 recombinant protein gene segments and PEA (delta III) -NrdF recombinant protein gene segments;
(3) transforming the recombinant plasmid obtained in the step (2) into a DH10 Bac competent cell, and transposing to obtain a recombinant bacmid;
(4) transfecting the recombinant bacmid obtained in the step (3) into an insect cell to obtain a recombinant baculovirus;
(5) inoculating the obtained recombinant baculovirus into HF cells for mass culture, centrifuging and collecting culture solution supernatant to obtain PEA (delta III) -P42 recombinant protein, PEA (delta III) -P97 recombinant protein, PEA (delta III) -P95 recombinant protein, PEA (delta III) -P46 recombinant protein and PEA (delta III) -NrdF recombinant protein, purifying, adding an adjuvant, emulsifying and finally mixing uniformly.
10. Use of a mycoplasma hyopneumoniae subunit vaccine composition according to any one of claims 1-8 in the preparation of a medicament for preventing and/or treating a mycoplasma hyopneumoniae-associated disease or a disease associated with a mycoplasma hyopneumoniae infection.
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