TW201221139A - Recombinant outer membrane lipoprotein E of Pasteurella haemolytica and vaccine composition containing the same - Google Patents

Abstract

A vaccine composition is disclosed, which includes a recombinant outer membrane lipoprotein E (rPlpE) of Pasteurellaceae bacilli and a double oil emulsion adjuvant. The rPlpE largely expressed by a prokaryotic expression system serves as an antigen. The vaccine composition with one serotype antigen can be subjected to immunize at least one immunized animal to have immunity against Pasteurellaceae bacilli with different serotypes.

Description

201221139 VI. Description of the Invention: [Technical Field] The present invention relates to a bacterial recombinant protein and its use in a vaccine composition, in particular to a Pasteurella recombinant outer membrane lipoprotein E and a vaccine composition thereof Application of things. [Prior Art] The bacterium of the genus J. bacillus is also called hemolysis. The bacterium of H. sphaeroides belongs to the genus Pasiewre/ZAfeeae in Gram-negative bacteria, and is a ruminant (such as cattle, sheep, etc.). One of the common bacteria on the respiratory tract. However, when the animal's resistance declines, the hemolytic H. haramella has the opportunity to multiply, which causes acute fibrinous pleuropneumonia in animals, leading to rapid death of infected animals, resulting in serious economic losses in the livestock industry. For Pasteurella-induced pneumonia in animals, conventional techniques vaccinate animals by using an inactivated vaccine of Pasteurella, thereby providing animal immunity. Inactivated vaccines, also known as killed bacterial vaccines or bacterins, use physical or chemical methods to treat bacteria and cause them to lose their vitality. Although inactivated vaccines are easy to manufacture' however, inactivated vaccines only provide short-term immunity. The reason is that the non-activated vaccine provides ex-genous antigens and the immune cells elicited by the exogenous antigen in the animal are mainly CD4+ helper T cells. After helping the tau cells to recognize the antigen, it is necessary to undergo a series of reactions and a series of reactions to produce cytokines and activate the immune cells, so that the immune effect is slower and the effect of the immune memory is less. Usually, the vaccine does not activate the vaccine for a short period of time. Therefore, after a few weeks of the first dose of vaccination, it is often necessary to boost the second dose of the vaccine, and even multiple immunizations are required to obtain a better immune effect. Secondly, the serotype of the hemolytic Pasteurella is large, and the use of a single serotype of inactivated vaccine has difficulty achieving cross-protection. Furthermore, regarding the current epidemic situation of Pasteurella hemolyticum, there is no domestic commercial vaccine introduced in the country, and no localized vaccine has been developed. _ In summary, the conventional inactivated vaccine of Pasteurella has its limitations in application and does not provide perfect preventive effects. In view of this, there is an urgent need to provide effective common antigens to help immunized animals to resist infection with different serovars. SUMMARY OF THE INVENTION Accordingly, it is an aspect of the present invention to provide a vaccine composition which utilizes a prokaryotic expression system to express a large amount of Pasteurella recombinant outer membrane lipoprotein E • (rPlpE) as an antigen and binds to a biphasic oily substance. The agent is formulated into a vaccine to effectively provide immunity of at least one immunized animal against Pasteurella of different serotypes. According to the above aspect of the invention, a vaccine composition is proposed. In one embodiment, the vaccine comprises a Pasteurella recombinant outer membrane lipoprotein E and a biphasic oily adjuvant, wherein the Pasteurella recombinant outer membrane lipoprotein E comprises an amino acid sequence as identified by the identification number 1, and The two-phase oily adjuvant is a water-in-oil-in-water (w/o/w) dual-phase oil emulsion. The vaccine induces the production of 201221139 ^ antibody in at least one immunized animal, and the antibody is specific against the hemolytic H. haramii registry number BCRC 13948, BCRC ' 13946) and the hemolytic H. haramii B2 . According to an embodiment of the invention, the at least one immunized animal is, for example, a cow, a sheep or a mouse. Application of the Pasteurella recombinant outer membrane lipoprotein E (rPlpE) of the present invention and a vaccine composition thereof, which utilizes a prokaryotic expression system to express a large amount of Pasteurella recombinant outer membrane lipoprotein E (rPlpE) in combination with a biphasic The oleaginous adjuvant produces φ to obtain a vaccine composition, thereby effectively providing immunity to at least one immunized animal having Pasteurella against different serotypes. [Embodiment] As described above, the present invention provides a vaccine composition which utilizes a prokaryotic expression system to express a large amount of a full-length Pasteurella recombinant outer membrane lipoprotein E (rPlpE) as an antigen, and binds to a biphasic oily adjuvant. The vaccine composition is prepared to induce the production of antibodies in at least one of the immunized animals, and the anti-? body may have a cross-protective effect. Vaccine Composition The "P. Pasteurian recombinant outer membrane lipoprotein E (fpE)" as referred to herein is mainly referred to as the Pasteurella outer membrane lipoprotein E(tetra)pE) which comprises an amino acid such as a sequence recognition sequence (IV). In one embodiment, the Pasteurellae outer membrane lipoprotein E(tetra)pE) of f has about one amino acid derived from, for example, a haemolytic Mania bacillus m, wherein the hemolytic H. haramii strain It belongs to the i-type blood 5 201221139 Qing type 'purchased from Taiwan Hsinchu Food and Welfare Research and issued number BCRC 13948. The nine "storage", referred to herein as "biphasic oil adjuvant" (water-ln_oil good water; w / 〇 / w) type two-phase * f milk-packed oil water letter 18 ^), which is Oil (oil phase) and surfactant (water phase II, 〇U three-layer structure. Applicant 'this w/〇/w type dual-phase oil emulsion system antigen (aqueous phase), oil layer, outer layer immunogen (Aqueous phase) The structure of the inner layer = the inner layer of the oil layer and the outer layer of the antigen is 丨: 丨: 2, in a 曰 antigen, the aqueous phase portion can be, for example, a water-soluble = intermediate 4-phase portion containing an immunogen and a physiological saline solution, for example It may be a surfactant, such as Tween 8 〇. Oil, the term "cross-protection effect" as used herein refers to a vaccine prepared by using the recombinant outer membrane lipoprotein (4) of Pasteurella as a common antigen and prepared with a biphasic adjuvant. 'Effectively inducing at least an antibody produced in the body of an immunized animal, and the antibody is specifically specific against Pasteurella different from the antigen serotype. In the embodiment, the above-mentioned immunized animal may be a cow or a sheep. Or one of the mice. The antibody produced by immunization against the hemolytic Haemophilia (Mcmnheimia haemofyt Ica; the accession number is BCRC 13948, the accession number is BCRC 13946) and the hemolytic H. haramii B2. The above-mentioned hemolytic H. haramella (BCRC 13948) belongs to serotype type 1, hemolytic manhamiya Bacillus (BCRC 13946) belongs to serotype type 5, which is obtained from the lungs of infected lambs, and the serotype of hemolytic H. hamidobacter B2 (native type) is unknown. In one embodiment, the vaccine composition described above can mix Pasteurella recombinant 201221139 outer membrane lipoprotein E (rPlpE) with a biphasic oily adjuvant to prepare a vaccine, wherein rPlpE can be, for example, the content of the vaccine. It is about 200 pg/mL. In other embodiments, the content of the Pasteurella recombinant outer lipoprotein E in this vaccine composition is adjusted according to different immunized animals, and may be more or less than 200 pg/mL. For example, from 100 pg/mL to 400 pg/mL. Method for producing Pasteurella recombinant outer membrane lipoprotein E (rPlpE) In one embodiment, the above-mentioned Pasteurella recombinant outer membrane lipoprotein E (rPlpE) φ can be further utilized. Prokaryotic expression system The nucleotide sequence shown in SEQ ID NO: 2 is identified to obtain an amino acid sequence as shown in SEQ ID NO: 1. In this embodiment, the nucleotide sequence shown in SEQ ID NO: 2 contains the outer membrane of Pasteurella. The open reading frame (ORF) sequence of the start codon (GTG) to stop codon (ΤΑA) of lipoprotein E is about 1071 base pairs in length. In an example, a nucleotide sequence represented by SEQ ID NO: 2 can be obtained by performing a polymerase chain reaction (PCR) amplification using a specific primer pair. Suitable primer pairs described above may be based on, for example, the GenBank of the National Center for Biotechnology Information (NCBI) website, Pasteurella K. et al., 1998, Pasteurella Outer Membrane Protein E (PlpE) gene sequence (number: AF059036, a total of 1310 bases), using commercially available sequence analysis software or other functionally equivalent software, such as EditSeq 5.0 DNASTAR professional sequence analysis software (Expert Sequence Analysis Software; DNASTAR Inc.) To design a sequence of specific primer pairs. The above primer 201221139 includes an upstream primer and a downstream primer, wherein the 5th end of the upstream primer is designed with an alkali restriction enzyme and is complementary to the ith base to the 18th of the PlpE gene sequence, as shown in sequence identification number 3; At the 5th end of the downstream primer, the sequence restriction enzyme cleavage site is set, and the gene 4 of the outer membrane lipoprotein E of Pasteurella is shown. 1 〇 54 bases to 1071 bases are complementary, as in the sequence identification number 鲦 for example b, the first restriction enzyme may be, for example, five (3) still, and the second restriction 13940^ is especially 1. Using the above primers, the sequence indicated by No. 2 can be identified from the bacillus bacillus (BCRC segment, such as =, the nucleus DNA is increased by about 1071 bp rpipE). The above cited examples are discussed. Table 1 is shown. Sequence 1 of Table 1 cgcgaattc|g tg|aaattcaa taaaaaa -:

EcoRl start codon ATACTCGAG|T TAjTTTTTTCT CGCTAAC -3 __Xhol stop codon__ it's use of the first restriction enzyme and the second restriction enzyme to cleave the ρ1ρΕ^ & w nucleic acid fragment and a commercially available vector, respectively, using a ligase a recombinant expression plastid comprising ρ"' to bind the nucleic acid fragment of PlpE described above and a commercially available vector to form a nucleic acid fragment of p. This recombinant expression is then transformed into a prokaryotic expression system in the gamma prokaryotic expression system Performance (or called transgenic recombinant outer membrane lipoprotein E. When translating the above-mentioned PlpE nucleic acid fragment, the transgenic protein will also be translated together with the tagged protein, which is translated from the Pasteurella 201221139 bacillus Membrane lipoprotein E (PlpE) can be directly linked to this marker protein. Therefore, in other embodiments, the above mentioned Pasteurella recombinant outer membrane lipoprotein E (rPlpE) can also be referred to as Pasteurella outer membrane lipoprotein. E (plpE) and its directly linked marker protein. In one example, the above marker proteins may include, but are not limited to, histidine tag (histidine tag; his-tag) protein, S antigen expression base. Marker (s-epitope tag; s-tag) protein and thioredoxin tag; trx-tag protein 'only the invention is not limited thereto. In other embodiments, other commercially available Vectors are available, and such vectors may also carry other marker proteins or other types of proteins. The recombinant outer membrane lipoprotein E of the Pasteurella expressed by the prokaryotic expression system is about 63 kDa, wherein the portion of PlpE is about 45 kDa, and the portion of the labeled protein is about 18 kDa. Method for producing dual-phase oily adjuvant In other embodiments, the above-mentioned dual-phase oily adjuvant can be further prepared by referring to a conventional method' or by reference to Miyakawa T. et al. In the March 1993 issue of the Journal of Biological and Pharmaceutical Bulletin, Vol. 16 No. 3, pp. 268-272, entitled "Intravenous Stabilized Water-in-Oil-in-Water Composite Emulsion in Rats" Release of water-soluble agents in vivo (/« Fzvo Release of Water-Soluble Drugs from Stabilized Water-in-Oil-in-Water (W/O/W) Type Multiple Emulsions Following Intravenous Administrations Using Rats) The preparation method disclosed in the article is listed here as a reference. Briefly, when the above-mentioned two-phase oily adjuvant is a w/o/w type dual-phase oil emulsion, the w/o/w type dual-phase oil emulsion can be formed by, for example, a two-stage emulsification method 201221139. An equal weight ratio of the aqueous phase to the oil phase is used to form a water-in-oil (w/〇) type oily emulsion using, for example, a homomixer or other functionally equivalent equipment. Then, this water-in-oil (w/0) type oil emulsion is equal to the water phase in an equal weight ratio, and the above-mentioned homogenizer or other functional equivalent equipment is used to form a w/o/w type dual phase oil emulsion. In the following, several embodiments are used to illustrate the application of the present invention, and it is not intended to limit the present invention to those skilled in the art, and Φ can be modified in various ways without departing from the spirit and scope of the invention. Reconstruction of the full-length gene of the recombinant outer membrane lipoprotein E of Pasteurella and a large number of cultures 1. Synthesis of nucleic acid fragments of the full-length gene of Pasteurella outer membrane lipoprotein E by polymerase chain reaction In the examples, the recombinant plastids, transformed strains and large-scale culture of the recombinant outer membrane lipoprotein E (rPlpE) group of Pasteurella were constructed. The nucleic acid fragment of the full-length gene of the recombinant outer membrane lipoprotein E (rPlpE) contained in the Pasteurella is extracted from the DNA of the hemolytic H. haemobacterium (like (10); the accession number is BCRC 13948). , obtained by increasing the polymerase chain reaction. It is stated that the DNA of the hemolytic H. haramella can be extracted by a commercially available kit, and the primer pair shown in Table 1 and the DNA are added to a commercially available PCR reaction reagent (TaKaRa, Shiga, Japan). In the above, the polymerization cycle control reaction of the 201221139 was carried out using a temperature cycle controller (Thermocycler; TaKaRa, Shiga, Japan) to obtain a nucleic acid fragment. The reagent for the above PCR reaction is as shown in Table 2: _ 2nd table ____ Reagent ___ volume (μ! 〇 upstream primer (10 mM) 0.5 downstream primer (10 mM) 0.5 1 Ox PCR buffer (TaKaRa, Shiga , Japan) 5 dNTPs (2.5 mM; TaKaRa, Shiga, Japan) 4

Taq DNA polymerase (5 U/pL, TaKaRa, Shiga, Japan) 0.25 hemolytic H. cerevisiae chromosomal DNA 1 secondary deionized water ___38.75 Total 50 Polymerase chaining with DNA using primer pairs In the reaction, the reaction conditions may be, for example but not limited to, exemplified by about 1 minute at about 94 ° C, about 30 seconds at 94 ° C, about 30 seconds at 52 ° c, and about 1 minute at 72 ° C. A total of 30 cycles of the reaction were repeated to obtain a nucleic acid fragment of about 1071 base pairs PlpE. 2. Constructing a recombinant plastid containing the recombinant outer membrane lipoprotein E of P. striata and preparing a transformed strain by the above polymerase-locking reaction, and using the primer to modulate the rplpE full-length gene obtained by the primer, for example [ (10) and for ! (both TaKaRa, Shiga, Japan), after cutting and purifying the nuclear 201221139 acid fragment of the rpipE full-length gene, 'can be constructed and ligated into a vector treated and purified by the same restriction enzymes', for example, mammalian cell expression Vector pET-32a(+) (Novagen, USA) vector' to form a recombinant plasmid to obtain a recombinant plasmid containing a nucleic acid fragment of the full-length gene of pplE or (pET_32a/13948-PlpE). The resulting recombinant plasmid was also confirmed by PCR and DNA sequencing. However, the restriction enzyme action, the construction of recombinant plastids, and the ligation reaction relating to recombinant plastids are well known to those of ordinary skill in the art, and therefore will not be further described herein. The recombinant plastid can be further transformed into a suitable host, such as a competent cell of the E. coli (5. cW) BL21 (DE3) strain, as a host for the transfer and preservation of the recombinant plastid. . Thereafter, blue-white screening was performed using isopropyl-beta-D-thiogalactopyranoside ( IPTG;

Amresco, USA) induces the recombination of the plastid lactose operator (/ac operon) and utilizes 5·bromo-4-chloro-3-吲哚-bD-galactose bud (5-bromo-4-chloro-3- Indolyl-bD-galactopyranoside ; X-gal) allows the colonization of successful colonies to develop color, and by PCR and DNA sequencing to determine the sequence of the correct bacteria behind, a large number of cultures can be carried out. The above DNA sequencing sequence was aligned with the gene sequence of the Pasteurella exoprotein E (PlpE) of the GenBank (number: AF059036), and the sequence was confirmed to be correct. However, there are common knowledge in the technical field for constructing recombinant plastids, transforming to competent cells, mass culture, extracting plastid DNA, measuring optical density (OD) values, establishing standard curves and DNA sequencing. They are well known, so they are not described here. [S] 12 201221139 Example 2: Preparation and characterization of recombinant outer membrane lipoprotein E of Pasteurella

(I) Preparation of Pasteurella recombinant outer membrane lipoprotein E In this example, first, a transformant strain containing the above recombinant expression vector (pET-32a/13948-PlpE) was cultured in 200 mL of Luria-Bertani culture solution (containing In Ampicillin), when cultured at 37 ° C until the OD_nm value was about 0.6, the recombinant protein was induced by 1 mM isopropyl-pD-thiogalactopyranoside (IPTG) and induced for 4 hours. (II) Evaluation of qualitative and biological activity of recombinant outer membrane lipoprotein E of Pasteurella 1. Sodium-sulphate sodium-polyacrylamide gel electrophoresis (SDS-PAGE) Completed transformed strains (containing Pasteurella recombinant outer membrane lipoprotein E) and 2-fold sample buffer (2X sample buffer; eg Φ contains 100 mM Tris-HCl pH 6.8, 200 mM dithiothreitol, 40/〇sodium dodecyl Sulfate, 0.2% bromophenol blue, 20% glycerol) mixed in equal volume 'at 100 ° C, boiled for 1 minute and cooled, then placed on ice and prepared for 8% SDS-PAGE analysis, about 1〇〇 Volt's electrophoresis analysis for approximately 90 minutes' was followed by standard procedure staining and destaining. Since SDS-PAGE can be carried out using commercially available products, it is not mentioned here. Please refer to FIG. 1 , which is a SDS-PAGE electrophoresis analysis diagram of recombinant outer membrane lipoprotein E according to an embodiment of the present invention, wherein the centromere indicates that the standard protein molecular weight marker indicates '' 26 kDa, kDa, 201221139

43 kDa, 55 kDa, 72 kDa, 95 kDa" protein of equal molecular weight. Lanes 1 to 4 indicate concentrations of looo pg/mL, 500 pg/mL·, 250 pg/mL, 125 pg/mL, respectively. The relative position of bovine serum albumin (B SA), and lane 5 indicates that the Pasteurella recombinant outer membrane lipoprotein E (rPlpE) expressed after 4 hours of induction of the transformant of Example 1 by IPTG From the results of Fig. 1, it was revealed that after 4 hours of inducing the transformed strain of Example 1 by IPTG, the Pasteurella recombinant outer membrane lipoprotein E (rPlpE) was obtained, and its molecular weight was about 63 kDa as compared with the standard protein molecular weight marker. Western blotting assay The above SDS_PAGE electrophoresis gel (containing Pasteurella recombinant outer membrane lipoprotein E) is then used in Western gelset kits (GE Healthcare, WI, USA) to protein in the electrophoresis gel. Transfer (using about 300 mA) to the transfer film (Amersham Biosciences, Buckinghamshire, UK). Since the transfer step can be carried out using a commercially available product, it will not be described here. After that, the transferred PVDF film is utilized 1X. BSA (KPL, Inc., MD, USA) or 5% The milk powder was dissolved in Phosphate buffer saline (PBS) and applied at 37 Torr for about 1 hour. Then 'washed twice with PBST (lx phosphate buffer saline, 0.5% Tween 20) for 10 minutes each time. The goat immunized serum of type 1 hemolytic H. haramella (BCRC 13948 non-activated vaccine) was added as a first antibody (l: 15,000x), and it was perceived as overnight at 4eC. PBST was washed three times for 10 minutes each time to wash away non-specific primary antibody binding. 201221139 Subsequently 'added to rabbit anti-goat IgG (rabbit anti-goat IgG) diluted 10,000 times (1: 10,000) in combination with wasabi peroxidase -HRP) (KPL, Inc" MD, USA) as a secondary antibody, after feeling about 1 hour at about 37 ° C, was washed three times with PBST for 10 minutes each time. Then, after adding light-lighting agent (for example, Enhanced ChemiLuminescence (ECL) Plus Western blotting detection reagent; GE Healthcare, Newcastle, UK), the mixture is uniformly mixed in a ratio of 40:1, and then subjected to a color reaction on the PVDF film. Whether the above-described performance of the Pasteurella recombinant outer membrane lipoprotein E (rPlpE)' can be detected by a luminescence fluorescence image analysis system (for example, G:Box luminescence fluorescence image analysis system; Syngene, Frederick, MD) The goat immune serum was identified, and the results are shown in Fig. 2.第 Refer to Fig. 2, which shows the results of western knockdown analysis of the recombinant outer membrane lipoprotein E (rPlpE) of Pasteurella not according to an embodiment of the present invention, wherein the first lane is the transformation of the first embodiment by IPTG induction. When the strain is small, the second to third lanes are 4 hours, and the obtained rPlpE has a size of about 63 kDa. From the results of Fig. 2, it can be seen that after 4 hours of transformation of the transformed strain of Example 1 by iptg, the recombinant outer membrane lipoprotein e (rPlpE) of Pasteurella was obtained. Example 3: Evaluation of recombinant outer membrane lipoprotein of Pasteurella Immunization effect in animal model and cell model 1. Preparation of animal vaccine containing Pasteurella recombinant outer membrane lipoprotein E. Pasteurella recombinant outer membrane lipoprotein E (rPlpE) obtained in Example 2 and biphasic oil adjuvant Kunhe, a vaccine, in which rpipE is about 200 pg/mL in this vaccine group 201221139. In addition, a vaccine of a positive control group (hereinafter referred to as a non-activated vaccine) was prepared by mixing an inactivated vaccine of hemolytic type Hm. mannii (BCRC 13948) of serotype type 1 with a biphasic oily adjuvant. The inactivated vaccine is present at a level of about 2 x 109 CFU/mL. 2. Evaluation of safety efficacy test (I)--The mice were randomly divided into experimental group, positive control group and negative control group Lu, each of which was a 3-week-old imprinting control area (Imprinting Control Region; There were 9 ICR mice. The experimental group and the positive control group were subcutaneously injected with 0.25 mL of rPlpE vaccine and non-activated vaccine for the first immunization, and the negative control group was not immunized. The experimental group and the positive control group were subjected to immunization. Two weeks after the first immunization, 0.25 mL of rPlpE vaccine and non-activated vaccine were injected subcutaneously as a booster, and blood was collected before the first immunization, 2 weeks after the first immunization, and 2 weeks after the booster immunization. Indirect enzyme-linked immunosorbent assay (indirect φ ELIS A) method for measuring antibody titers in serum. The method for indirect ELISA is as follows: First, in a 96-well V-type microplate, each The wells were coated with a hemolytic H. haemobacterium (BCRC 13948; 1.25 /zg/mL) in a well using a fixing solution (containing 15 mM Na2C03, 35 mM NaHC03, 3 mM NaN3, pH 9.6) at about 4 °C. The next action was overnight. Next, after washing with PBST three times for about 5 minutes each time, about 150 μί of an embedding solution (KPL, Inc., MD, USA) was added to each well for about 1 hour at 37 ° C. Then Wash with PBST three times for about 5 minutes each time. After 201221139, add the serum to be tested at different dilution rates, and apply for about 1-2 hours or about 90 minutes at 37 ° C. After washing three times with PBST, add dilution 2500. Double (1:2,500), HRP-conjugated goat anti-mouse IgG-HRP (CALTAG, CA, USA) as a secondary antibody, after feeling at 37 ° C for about 60 minutes, Wash three times with PBST for about 5 minutes each time. Then, add an equal volume of luminescent toner, such as TMB perosidase substrate (KPL, Inc., MD, USA), at room temperature in the dark. After 5 minutes, an equal amount of stop solution (KPL, Inc., MD, USA) was added to stop the color reaction. Subsequently, a cold light fluorescence analyzer such as an Anthos 2020 ELISA reader (Anthos 2020 ELISA reader; Anthos 2020, Cambridge, UK) 'Detected in waves The 450 nm absorbance (〇D450nm) to analyze the antibody titers in mouse serum, the results as shown in Figure 3. Please refer to FIG. 3, which is a diagram showing the ELISA antibody valency diagram of a vaccine composition of a Pasteurella recombinant outer membrane lipoprotein E (rpipE vaccine) according to an embodiment of the present invention, wherein the horizontal axis indicates different blood collection. Time (〇, 2, 4 weeks), the vertical axis represents the s/p ratio (s/Prati〇) calculated according to formula (1), the blank column represents the negative control group, and the diagonal column represents the test group inoculated with the rPlpE vaccine. Figure number * indicates a statistically significant difference (p < 0.05). In addition, when the s/p ratio was calculated, the serum of the ICR mice immunized with the non-activated vaccine was used as a positive control group. S/P ratio = - group 〇 D450nm - negative control group 〇 D4snnm positive control group OD450nm - negative control group OD45 〇 nm (1) From the results of Fig. 3, the 201221139 test group inoculated with the rplpE vaccine of Example 3 was reinforced. After 2 weeks of immunization, the antibody titer was significantly higher than that of the group. The rPlpE vaccine was effective in inducing the body of the mouse. 3. Evaluation of safety efficacy test (2) ~ Goat The experimental goats were randomly divided into a test group and a negative control site, each of which was 3 healthy goats. The goats in the experimental group were intramuscularly injected; the E vaccine (first immunization), and the goat in the negative control group were not paralyzed. The goats in the experimental group were supplemented with rPlpE vaccine (reinforcing immunity) at a dose of 2 mg of muscle after 2 weeks of the first immunization. Then, after all the observations of 2-4 weeks in the mountain behavior period, blood samples were taken before the first immunization, the first week, and 2 and 4 weeks after the booster immunization, and the antibody titer in the goat serum was analyzed by ELISA. . The method for indirect ELISA can be considered as above. It is not worthy of praise here. Each well is added with a dilution of 300 times : _). The blood price of the blood to be tested is analyzed, and the results are shown in Fig. 4. Please refer to FIG. 4, which is a diagram showing the vaccine composition of the bacillus recombinant outer membrane lipoprotein E according to an embodiment of the present invention (the rplpE epidemic EUSA antibody price map, wherein the horizontal axis indicates different blood sampling times (〇, 2 sheep*

6 weeks), the vertical axis represents the S/P ratio calculated according to the above formula (I) (S/P represents the negative control group, the slanted column represents the experimental group inoculated with the lynx vaccine, and the figure * indicates There is a significant difference (Ρ<〇.05). The results of the map can be seen as 'inoculation of the rPlpE vaccine of the third example, after 2 weeks of immunization, 2 weeks after boosting immunity, The antibody sputum was significant in the negative control group, indicating that the rPlpE vaccine could indeed produce antibodies in goats induced by 201221139. 4. Evaluation of mouse challenge test (1) The experimental mice were randomly divided into experimental group and positive control. Group and negative control group '5 of ICR mice each of which was 3 weeks old. The mice in the experimental group and the positive control group were subcutaneously injected with 0 25 mL of rPlpE vaccine (containing rpipE, dose). Mice in the negative control group were not immunized with approximately 2〇〇pg/mL) and inactivated vaccine (containing B. meliloti BCRC 13948 at a dose of 2xl09 • CFU/mL). All mice were After 2 weeks of the first immunization, each mouse was intraperitoneally injected with 0.2 mL of hemolytic activity. The live bacteria of B. cerevisiae (BCRC 13948) (dose was 2 X 1〇9 CFU/mL), and after 2 weeks of observation, the survival rate of the mice was calculated, and the results are shown in Table 3. Table 3 survived. Number of deaths (%) 4 1 80 4 1 80 1 4 20

5 test group positive control group according to the group of three ιί, see Table 3, which shows a vaccine against the recombinant outer membrane lipoprotein of Pasteurella sinensis according to an embodiment of the present invention. Immune protection. As can be seen from the results of Table 3, the use of the test-off heart, the mice in the group after the challenge of 'the survival rate of the two is much higher than the yin immunity, which means the rplpE vaccine and the inactivated vaccine for the mice Μ 201221139 5. Evaluation of mouse challenge test (2) The experimental mice were randomly divided into two groups: the experimental group, the positive control group and the negative control group, each of which was 3 weeks old. 5 ICR mice. The mice in the 2 groups were injected subcutaneously with 0.25 mL of rPlpE vaccine (first immunization; containing rPlpE 'dose of about 200 | ng/mL), and the mice in the positive control group were injected subcutaneously. 0.25 mL of non-activated vaccine (first immunization; containing hemolytic H. haramii BCRC 13948 at a dose of 2xl09 CFU/mL), and mice in the 2 φ group negative control group were not immunized. After 2 weeks, all mice were challenged. One group and the negative group The mice were each injected intraperitoneally with a live bacterial culture of mL. 2 mL of hemolytic H. haramella (BCRC 13948) (dose 2 χ 109 CFU/mL·; Table 4 referred to as BCRC 13948); another group of experiments The mice in the group and the negative control group were intraperitoneally injected with a live culture of 0.2 mL of hemolytic H. mobilis B2 (dose of 2×10 CFU/mL; Table 4 is abbreviated as B2). A viable culture (at a dose of 2xl09 CFU/mL) of 0.2 mL of hemolytic H. glutinosa (BCRC 13948) was injected intraperitoneally. The above hemolytic H. haramella (BCRC 13948) belongs to serotype 1 The type 'is isolated from infected bovines. The PlpE gene sequence of the hemolytic H. hamidobacter (BCRC 13948) and the PlpE gene sequence of the hemolytic H. haramella (BCRC 13946) are known. The same. The hemolytic H. haramella (BCRC 13946) belongs to the serotype type 5, which is obtained from the lungs of infected lambs. In addition, the hemolytic H. haramella B2 (native type) ) is isolated from cattle infected with Taiwan [si 20 201221139, but its serotype After 2 weeks of inoculation of live bacteria, the survival rate of the mice was calculated, and the results are shown in Table 4. Table 4 Survival rate of survival number of immune challenge (%) ΓΡφΕ vaccine BCRC 13948 4 1 80 rPlpE Vaccine B2 5 0 100 Not activated vaccine BCRC 13948 4 1 80 Not immunized BCRC 13948 1 4 20 Not immunized B2 2 3 40 Group

Test group Test group Positive control group Negative control group Negative control group Please refer to Table 4, which shows a vaccine containing the Pasteurella recombinant outer membrane lipoprotein E according to another embodiment of the present invention. Vaccine) is also immune to the protection of mice. From the results of the fourth |, it was found that the mice of the test group and the positive control group were challenged with the serotype of Pasteurella, and the survival rates of the two were in the control group. Secondly, the test results of the different types of clearing pasteurization (four), the survival rate is higher than the pasteurization of the Pasteurella, the main group, which means that the vaccine can provide mouse confrontation Pasteurella with different antigen β1. 6. Evaluation of the stimulation index of mouse pancreatic cell proliferation 64 Isolation and culture of spleen lymphocytes from mice [S1 21 201221139 Three-week-old ICR mice were randomly divided into a test group and a negative control group, each of which was 9 Only ICR mice. The ICR mice of the experimental group were subcutaneously injected with 0.25 mL of rPlpE vaccine (first immunization). The ICR mice of the negative control group were not immunized. The ICR mice of the experimental group were injected subcutaneously with a 25 mL rplpE vaccine (reinforcing immunity) 2 weeks after the first immunization. Then, all ICR mice were sacrificed for 2 weeks, and ICR mice were sacrificed before the first immunization, 2 weeks after the first immunization, and 2 weeks after the booster immunization. Then, the spleen lymphocytes of 3 weeks old ICR mice were obtained by aseptic technique, washed with Hank's balanced salt solution, and then gently ground on a 1 μm mesh to prepare a single cell. The suspension was washed repeatedly with Hank's balanced salt solution and centrifuged 3 times (centrifugation at 900 xg for 1 minute). Thereafter, the cell concentration was adjusted to 1 χ 1 〇 6 cells/mL with RPMI-1640 complete medium and dispensed into a 96-well plate, wherein each well was 100 细胞 of cell fluid. 6.2 Evaluation of the stimulation index of cell proliferation 6.1·1 rPlpE stimulation Next, 10 //g of the rPlpE of Example 2 was added to the spleen lymphocytes (Spien0Cytes) of the isolated and cultured ICR mice per well, and placed in Incubate in 7 7 C, 5% C02 incubator for 72 hours. Then, it was added to each well and placed at 5% CO 2 and 37. (: Incubate for 72 hours in an incubator. As for the positive control group, 5 //g of mitogen was added per well, such as Concanavalin A (Con A A A Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con Con In the negative control group, rPlpE and T cell lysin were not added. There was a significant difference between the above groups (Ρ<〇.〇5). 6.2.2 ΜΤΤ test In each of the above cells, 20 "L was added per well. MTS reagent, for example [s] 22 201221139 such as 3-(4,5-dimercaptozol-2)-5-(3-carboxymethoxyphenyl)-2-(4) in the CellTiter 96® kit reagent - sulfophenyl)-2H-tetrazolidine[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2 -(4-sulfophenyl)-2H-tetrazolium ; MTS tetrazolium ; 5 mg / mL ; Promega, WI, USA] After mixing evenly, avoiding light at 37 ° C, 5% CO 2 reaction for 4 hours ' using an ELISA reader (Anthos 2020, Cambridge, Austria), detected at a wavelength of 492 nm Absorbance value (〇D492nm) 〇6·2·3 Cell proliferation index (SI) calculation The calculation of the cell proliferation index (SI) can be calculated according to the following formula (II): Cell proliferation index ( SI) = _ test OD492nm - background value 〇 D492nm negative control group OD492nm - background value 〇 D492nm In the formula (π), the above background value is the value of 0 〇 492 (10) detected after the cell is only reacted with RPMI_164 〇 medium and 20 μί of MTS. As shown in Figure 5.

Referring to Figure 5, there is shown a graph showing the results of a cellular immunoreactivity test of an rpipE vaccine according to an embodiment of the present invention in a mouse, wherein the horizontal axis represents the vertical axis of the antigen representing the cell proliferation index (SI)' blank column. Representing the negative control group, the slanted column represents the rPlpE vaccine test group, and the figure number* indicates that there is a 'statistically significant difference (p&lt;5). From the results of Fig. 5, it can be seen that the test group has a cell proliferation effect after the mouse is immunized with the kiss saplings. The sprint needs to be supplemented, although the present invention is illustrated by a specific plastid, expression system, reaction conditions, immunized animal, analytical method, material condition or specific instrument 23 201221139, illustrating the Pasteurella recombinant outer membrane lipid of the present invention. Protein E (PlpE) can be applied to a vaccine composition. It is to be understood by those skilled in the art that the present invention is not limited thereto, and the present invention is not limited thereto, and the present invention does not depart from the spirit and scope of the present invention. Recombinant outer membrane lipoprotein E (PlpE) can be performed using other plastids, expression systems, reaction conditions, immunized animals, analytical methods, induction conditions, or instrumentation. For example, a large number of prokaryotic expression systems are used to express rPlpE as an antigen, in addition to effectively inducing antibodies in sheep and mice to counter serotype type 1 hemolytic H. haramii (BCRC 13948) and hemolysis In addition to H. sphaeroides B2, antibodies can also be produced in sheep and mice to counteract serotype type 5 hemolytic H. hamidobacteria (BCRC 13946). Secondly, the rPlpE of the present invention is isolated from the serotype type 1 hemolytic mania bacillus (BCRC 13948), and can effectively induce the production of antibodies in sheep, and can also effectively induce bovine bodies. The antibody is produced in end against the hemolytic H. hamidobacterium (BCRC 13948), the hemolytic H. haramella (BCRC 13946), and the hemolytic H. haramii B2. According to the above invention, the Pasteurella recombinant outer membrane lipoprotein E (rPlpE) vaccine has the advantage of utilizing a prokaryotic expression system to express a large amount of Pasteurella recombinant outer membrane lipoprotein E (rPlpE) as an antigen, thereby facilitating mass production. Vaccination. Secondly, the dual-phase oil adjuvant used in this vaccine composition is a water-in-oil-in-water dual-phase oil emulsion, which can effectively prolong the release time of the antigen in the animal and also help to improve the antibody of the vaccinated animal. Reaction and cellular immune response. Furthermore, it is more effective to provide at least one immune animal with the efficacy against different serotypes. While the present invention has been described above in terms of several embodiments, it is not intended to limit the invention, and it is to be understood by those skilled in the art without departing from the spirit and scope of the invention. Various modifications and refinements are made, and the scope of the present invention is defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; SDS-PAGE of the sodium dodecyl sulfate-polyacrylamide colloidal electrophoresis analysis of the recombinant outer membrane lipoprotein E of Bacillus. Fig. 2 is a view showing the results of Western collapse analysis of the Pasteurella recombinant outer membrane lipoprotein E (rPlpE) according to an embodiment of the present invention. Fig. 3 is a graph showing the ELISA antibody titer of a vaccine of the Pasteurella recombinant outer membrane lipoprotein E (rPlpE vaccine) in mice according to an embodiment of the present invention. Fig. 4 is a graph showing the ELISA antibody titer of a vaccine of the Pasteurella recombinant outer membrane lipoprotein E (rPlpE vaccine) in goats according to an embodiment of the present invention. Fig. 5 is a graph showing the results of a cellular immunoreactivity test of a vaccine of the Pasteurella recombinant outer membrane lipoprotein E (rPlpE vaccine) in mice according to an embodiment of the present invention. [Main component symbol description] (none) [S] 25 201221139 Sequence table &lt;110&gt; National Pingtung University of Science and Technology

&lt;120&gt; Pasteurella recombinant outer membrane lipoprotein E and vaccine composition containing the same &lt;130&gt; None &lt;160&gt; 4 &lt;210&gt; 1 &lt;211&gt; 356 &lt;212&gt; PRT

&lt;213&gt; Hemolytic Mangiferella outer membrane lipoprotein E (PlpE) &lt;400&gt;

Met Lys Phe Asn Lys Lys Leu lie Leu Thr Phe Ala Ala Thr Leu Val Leu Ser Ala Cys 15 10 15 20

Gly Gly Ser Gly Ser Gly Gly Ser Ser Ser Ser Pro Asn His Pro Lys Pro Val Leu Val 21 25 30 35 40

Pro Lys Thr Gin Asn Asn Leu Gin Ala Gin Asn Val Pro Gin Ala Gin Asn Ala Ser Gin 41 45 50 55 60

Ala Gin Asn Ala Pro Gin Ala Gin Asn Ala Pro Gin Ala Gin Asn Ala Pro Gin Val Glu 61 65 70 75 80

Asn Ala Pro Gin Ala Gin Asn Ala Pro Gin Val Glu Asn Ala Pro Gin Ala Glu Val Thr 81 85 90 95 100

Pro Pro Val Pro Gin Pro Gin Ser Gin Lys lie Asp Gly Ser Phe Asp Lys lie Gly Ser 101 105 110 115 120

Val Lys Leu Asn Lys Glu Ala Gin Thr Leu Glu Leu Ser Arg Phe Thr Leu Val Asp Lys 121 125 130 135 140

Leu Gly Thr Pro Pro Lys Phe Asp Lys Val Ser Gly Lys Lys lie lie Glu Glu Lys Asp 141 145 150 155 160

Phe Leu Val Leu Asn Leu Ser Asp lie Asn Ala Glu Gin Leu Ser Gly Asp Phe Leu lie 161 165 170 175 180

Arg Arg Ser Asp Asp Leu Phe Tyr Gly Tyr Tyr His Asp Thr Asn Gly Lys Asn Leu Val 181 185 190 195 200

Asp Ala Ala Asp Lys Phe Ser Gin Tyr Phe Val Val Tyr Asp Glu Lys Arg Val Asn Asp 201 205 210 215 220

Asn lie Ser Asp Lys Leu Thr Ala Thr Tyr Arg Lys Lys Glu Gly Phe Val Tyr Gly Ser 221 225 230 235 240

Asn Pro His Thr Lys Glu Phe Ala Ala Arg lie Ser Lys Leu Gly Asp Val Glu lie Lys 241 245 250 255 260 201221139

Phe Glu Asn Gly Gin Ala Gin Gly Ser He Lys Asp Glu Lys Asp Gly Asn Ala Glu He 261 265 270 275 280

Phe Thr lie Lys Gly Asp Thr Lys Gin Leu Glu lie Thr Pro Thr Glu Ser Asn Arg lie 281 285 290 295 300 , lie lie Ala lie Leu Asp Gin Asn Gin Lys Ser Tyr Thr Pro Gly Met Glu Lys Ala He 301 305 310 315 320

Met Glu Thr Lys Phe lie Asp Ser Lys Ala Gly Asn Ser Asp Gin Lys Tyr Leu lie Gly 321 325 330 335 340

Glu Ala Lys Ser Asp Asn Trp Gin Ala lie Met Val Ser Glu Lys Lys 341 345 350 355

&lt;210&gt; 2 &lt;211&gt; 1071 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;400&gt; 2

gtgaaattca ataaaaaatt aattttaaca tttgctgcaa ccttagtttt aagtgcttgc 60 ggaggaagcg gtagcggagg ttcgtcttca acaccgaatc accccaaacc agtactagta 120 ccaaaaacac aaaataatct tcaagcacaa aatgttcctc aggcacaaaa tgcctctcag 180 gcacaaaatg cccctcaggc acaaaatgct cctcaggcac aaaatgctcc tcaggtggaa 240 aatgctcctc aggcacaaaa tgctcctcag gtagaaaatg ctcctcaagc agaggttact 300 ccgcctgtac cacagccaca atcacaaaaa attgacggtt cttttgataa aattggttca 360 gtaaaactca ataaagaggc tcaaactctt gagcttagta gattcacttt ggtggataaa 420 ttaggcacac caccgaagtt tgataaagta agcggtaaaa aaattattga agaaaaagat 480 tttctcgtat taaatttgtc tgatattaat gctgaacaac tctctggcga ttttcttatt 540 cgccgtagcg atgatctatt ctatggctac tatcacgata caaatggcaa aaatcttgtc 600 gatgctgccg ataaattcag tcaatatttt gtcgtgtatg atgagaaacg ggtaaatgat 660 aatatctctg ataaattaac agcaacttac cgtaaaaaag aaggctttgt atatggttca 720 aatccacata ctaaagaatt tgccgcacgg atcagcaaat tgggggatgt agaaattaaa 780 tttgaaaatg gtcaagctca aggaagtata aaagacgaaa aagatggaaa tgctgagatc 840 tttacta tta aaggtgatac aaaacagtta gagattaccc caacggaaag taaccgaatc 900 m 2 201221139 attatagcaa ttttagacca aaatcaaaaa agctatactc caggaatgga aaaagcaatt 960 atggaaacta agtttattga ttcaaaggct ggtaattccg accaaaaata cttaatcggt 1020 gaagcaaaaa gcgataactg gcaagcaata atggttagcg agaaaaaata a 1071 &lt; 210 &gt; 3 &lt; 211 &gt; 27 &lt; 212 &gt; DNA &lt; 213> Artificial sequence

&lt;400&gt; 3 cgcgaattcg tgaaattcaa taaaaaa &lt;210&gt; 4 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;400&gt; 4 atactcgagt tattttttct cgctaac

Claims (1)

201221139 VII. Patent application scope: 1. A vaccine composition comprising: Pasteurella recombinant outer membrane lipoprotein E, wherein the Pasteurella recombinant outer membrane lipoprotein E comprises an amino acid sequence as shown in SEQ ID NO: And a two-phase oily adjuvant, wherein the dual-phase oily adjuvant is a water-in-oil-in-water; w/o/w type dual-phase oil emulsion (double oil emulsion), and wherein the vaccine composition induces production of antibodies in at least one animal, and the anti-system specificity against hemolysis of Mannheimia; registration numbers BCRC 13948 and BCRC 13946) and hemolytic Hamiabacterium B2). 2. The vaccine composition of claim 1, wherein the at least immunized animal is a cow, a sheep or a mouse. 3. The vaccine composition according to claim 1, wherein the Pasteurella recombinant outer membrane lipoprotein E has a concentration of 200 pg/mL in the vaccine composition. 4. The vaccine composition of claim 3, wherein the Pasteurella recombinant outer membrane lipoprotein E is derived from a recombinant expression plastid expressed in a prokaryotic expression system. 5. The vaccine composition according to claim 4, wherein the recombinant expression plastid comprises, for example, a sequence identification number 2 plant and a second column. 6. The vaccine composition according to claim 4, wherein the prokaryotic expression system is Escherichia coli μ&quot;). 7. The vaccine composition according to claim 4, wherein the Pasteurella recombinant outer membrane lipoprotein E is more linked to a marker protein, and the marker protein comprises a histidine tag; his-tag a protein, an S-epitope tag (s-tag) protein, and a thioredoxin tag; trx-tag protein.