TWI396547B - Mannheimia haemolytica vaccine - Google Patents

Description

Hemolytic halhama vaccine

The present invention relates to a bacillus vaccine, and more particularly to a hemolytic H. haramella vaccine.

Pneumonic pasteurellosis is an acute fibrinous pleuropneumonia that causes animals that are infected with the disease to die rapidly, resulting in serious economic losses for the livestock producer. Hemolytic Manhattan Mia coli (Mannheimia haemolytica;. M haemolytica) belonging to Pasteurella Section (Pasteurellaceae), the hemolytic Manhattan Mia subtilis hereinafter abbreviated as M haemolytica, which M haemolytica based on a ubiquitous ruminant. The bacillus in the respiratory tract of the animal is the main pathogen causing inflammation of the ruminant infection with Pasteurella, and when the ruminant has sufficient health resistance, it will not cause disease, when the external environment changes or suffers from bacterial infection, etc. Due to the decreased resistance of ruminants, M. haemolytica can proliferate rapidly and enter the lungs through the respiratory action of ruminants, infecting alveolar epithelial cells, which leads to rapid inflammation of the ruminant disease. death.

At present, the prevention of ruminant infection in the market is caused by M. haemolytica , which is mainly caused by the use of M. haemolytica inactivated vaccine, but the use of M. haemolytica inactivated vaccine is not effective, and Can not cause cell type immune response, and M. haemolytica has many serotypes. Different serotypes of M. haemolytica have different specificity and toxicity to the host. The conventional M. haemolytica inactivated vaccine can only be used for single serotypes. The infection of M. haemolytica achieves an immunoprophylactic effect and only protects a few serotypes of M. haemolytica infection, so the use of conventional M. haemolytica inactivated vaccines does not achieve cross-protection of different M. haemolytica serotype infections, The use of M. haemolytica inactivated vaccines does not provide a complete preventive effect. Therefore, the development of a vaccine can prevent the serotypes of M. faecalis caused by M. haemolytica in different serotypes.

It is also known that the role of adjuvant in immunization is mainly to enhance the immune response of the animal's immune system to antigens or immunogens, including enhancing the intensity of the immune response and the persistence of the immune response, so that the adjuvant is combined with an antigen to form a vaccine preparation. To enhance the efficacy of the vaccine. Depending on the source, adjuvants are classified into chemical synthesis adjuvants and bio-classified adjuvants, and most adjuvants have certain defects in function. The mineral oil components in many adjuvants are mainly sustained-release, if The development of M. haemolytica itself as a natural adjuvant can be added as an adjuvant to the conventional M. haemolytica inactivated vaccine to enhance the inactivated vaccine for the treatment of Pasteurella pulmonary inflammation. The applicability in use, while improving the immune response of the animal immune system to M. haemolytica to achieve better immune prevention.

The invention provides a hemolytic H. haramii vaccine, which can prevent the ruminant animal from infecting the lung inflammation of the Pasteurella and rapidly die, thereby improving the purpose of immune prevention.

The second object of the present invention is to provide a hemolytic H. mobilis vaccine, which is suitable for the pulmonary inflammation of Pasteurella caused by different serotypes of hemolytic Haematococcus, which can effectively induce an immune response and can be achieved. Stimulates immune system cell proliferation and activation.

A further object of the present invention is to provide a hemolytic H. mobilis vaccine which produces a relatively complete and preferred cross-immunity protector.

In order to achieve the aforementioned object, the technical contents applied by the present invention are as follows:

A hemolytic H. haramella vaccine comprising a hemolytic H. halodii recombinant white blood cell toxin comprising an amino acid sequence as shown in SEQ ID NO: 2 as an adjuvant, and is inactivated with a hemolytic H. haramella The vaccine is co-prepared as a vaccine; another hemolytic H. haramella recombinant leukotoxin antigen line is prepared from the amino acid sequence of SEQ ID NO: 2 and a biphasic oil adjuvant as a hemolytic manami Both the sub-bacterial vaccine and the second vaccine have the effects of improving the immune response and activating immune cells.

The above and other objects, features and advantages of the present invention will become more <RTIgt;

M. haemolytica , hereinafter referred to as M. haemolytica , contains many virulence factors, such as: capsular, transferin binding protein, outer membrane Proteins; OMPs) and leukotoxin (LKT), etc., which are immunogenic for lung inflammation of Pasteurella; wherein the white blood toxin (LKT) is the main virulence factor of M. haemolytica , which is hereinafter referred to as LKT. The LKT is present in different M. haemolytica serotypes, so the LKT line is a common antigen of different serotypes M. haemolytica , which can utilize the common antigenic properties of LKT to resist infection of different serotypes M. haemolytica , thereby preventing Lung inflammation of the lungs.

The operon of LKT consists of four genes, lktC , lktA , lktB and lktD . lktC translates the Lkt-C protein, which is responsible for acylation, and then activates the Lkt translated by lktA. -A structural proteins, however, transport and secretion of pulmonary disease caused by the main factor for the system was translated structural proteins Lkt-a, lktB and lktD Lkt-B and Lkt-D protein is responsible for the Lkt-a. Therefore, the present invention relates to a Lkt-A structural protein mainly causing pulmonary lesions of Pasteurella pulmonary inflammation, and a gene fragment of 1597 nucleotide to 3446 nucleotide of Lkt-A is selected by gene transfer technology. , transferred to recombinant leukotoxin and named rLkt-A2, which was prepared as recombinant leukotoxin rLkt-A2 subunit vaccine, and since rLkt-A2 is one of M. haemolytica components, rLkt-A2 itself is also The natural adjuvant can be combined with other M. haemolytica inactivated antigens to prepare a vaccine preparation, which can help to resist the infection of different serotypes of M. haemolytica , enhance the efficacy of the vaccine, and achieve cross protection of different serotypes M. haemolytica Lung lesions.

I. Preparation of recombinant white blood cell toxin protein rLkt-A2 of the hemolytic H. humilis M. haemolytica of the present invention in Escherichia coli

A. Screening of hemolytic H. mobilis M. haemolytica

The samples of cattle and sheep lesions and nasal cavity suspected of infection in livestock farms in various regions of Taiwan were collected and cultured on MacConkey medium, and the samples were confirmed by the hemolytic properties and fermentable lactose properties of the samples. The biochemical property of M. haemolytica can produce β-type hemolysis, which can be grown in MacConkey medium. The collected specimens containing many strains can be coated on MacConkey medium to preliminarily detect the lesions of cattle and sheep and the nasal cavity. The whole strain M. haemolytica was screened out, hereinafter referred to as M. haemolytica .

B. Gene colonization and expression vector construction of recombinant white blood cell toxin rLkt-A2

Leukocystoxin (LKT) is one of the major virulence factors of M. haemolytica , present in different M. haemolytica serotypes, which destroys neutrophils and other lymphocytes and increases lung lesions, and is attacked by LKT. Target cells are dose-dependent and can be divided into 3 categories. (1) When the concentration of white blood toxin is low, leukocyte toxin activates neutrophils, stimulates the release of macrophage hormones, reduces lymphocyte mitosis and stimulation. Mast cells release histamine, etc., (2) When the concentration of white blood toxin is increased, the target cells are stimulated to undergo apoptosis (apoptosis), and (3) when the concentration of white blood toxin is high, the cell membrane of the target cell will produce holes, thereby causing necrosis (necrosis). ). Therefore, the present invention adopts the common antigenic characteristics of leukotoxin to adopt the selection and sequencing of the leukotoxin. However, since the length of the leukotoxin is too long, it is difficult to express the full-length leukotoxin to Escherichia coli by gene transfer technology, and The Lkt-A structural protein is mainly caused by pulmonary inflammation of Pasteurella pulmonary inflammation, so the present invention selects the DNA of M. haemolytica by using the Blood & Tissue Genomic DNA Extraction Miniprep System, and extracts the extract. M. haemolytica DNA was used as a template for polymerase chain reaction (PCR) to design a set of primer pairs. The primer for Lkt-A full-length design was Lkt-A-Forward forward primer (Lkt-AF). With the Lkt-A-Reverse reverse primer (Lkt-AR) to increase the full-length Lkt-A gene, the primer sequence is as follows: Lkt-AF: 5'-TCAAGAAGAGCTGGCAAC-3' (SEQ ID NO: 3) Lkt-AR: 5'-AGTGAGGGCAACTAAACC-3' (SEQ ID NO: 4)

M. haemolytica DNA was used as a template, and the primers Lkt-AF and Lkt-AR were added to increase the Lkt-A full-length gene fragment by polymerase chain reaction. Since the full-length Lkt-A gene is too long, it is difficult to express E. coli in prokaryotic cells. The present invention selects the restriction fragment cleavage sites PstI and EcoRI on Lkt-A, and excises the 1597 nucleotide to 3446 nucleotide gene fragment on Lkt-A, and the amino acid size is 69 kDa. This gene fragment was named Lkt-A2. The Lkt-A2 gene fragment is further constructed in a expression vector, and the expression vector is preferably selected as a pGEX-4T-1 expression vector, and the competent cell is preferably selected as Escherichia coli BL21 (DE3), the pGEX The -4T-1 expression vector is a tagged protein with a glutathione S-transferase (GST) having a molecular weight of about 26 kDa, which helps to purify the protein expressed by the foreign gene; and the pGEX-4T-1 expression vector has lacZ ' Gene and ampicillin resistance gene, the lacZ' gene is located at the junction of the foreign gene. When the foreign gene is successfully constructed into the junction of the foreign gene, the lacZ' gene is destroyed and cannot express β-galactosidase. Competent cells with a vector for successful construction of a foreign gene are white incapable of expressing β-galactosidase; and the lacZ' gene possessed by the pGEX-4T-1 expression vector is selected to carry the successful construction of the Lkt-A2 gene. pGEX-4T-1 expresses the competent cells of the vector, and removes unnecessary bacteria by ampicillin drug resistance gene, and finally confirms the plastid Lkt-A2 sequence in sequence, and then carries out recombinant white blood toxin rLkt-A2 after correctness. Protein expression The following colonization transfected into E. coli for recombinant toxin Lkt-A2 called leukocytes rLkt-A2, which is the nucleic acid and amino acid sequence of the sequence listing as SEQ ID NO: 1 shown in FIG.

C. Recombinant white blood cell toxin rLkt-A2 expressed in Escherichia coli

The plastid with recombinant leukotoxin rLkt-A2 is transformed into a competent cell to express rLkt-A2, wherein the competent cell is preferably Escherichia coli BL21 (DE3), a preferred embodiment of the present invention For example, when culturing in LB medium at 37 ° C until the absorbance OD ₆₀₀ reaches 0.6, the concentration of 0.6 mM IPTG (isopropylthio-β-D galactoside) is added, but the concentration is not limited to the induction gene. Transcription to synthesize a large amount of rLkt-A2, and then select the optimal induction condition for the induction of the cells by IPTG for 4 hours but not limited to this time, the cells of the E. coli cells expressing rLkt-A2 were collected by centrifugation. Thus, the preparation of the recombinant leukocyte toxin rLkt-A2 of the present invention is completed.

After the preparation of the rLkt-A2 of the present invention, further use of the West is required. The correctness and antigenicity of the rLkt-A2 were confirmed by Western blotting. The Western blotting method used the immunized calf serum as a primary antibody and a goat anti-bovine IgG HRP-conjugated antibody (KPL, USA) as a secondary antibody. The results of the western blotting analysis of the rLkt-A2 are shown in Fig. 1, which shows that there is a binding reaction at the 95 kDa rLkt-A2, indicating that the rLkt-A2 can be recognized by the antibodies in the serum of the calf after immunization. This confirmed that the rLkt-A2 is antigenic. After Western blotting analysis, the rLkt-A2 protein is correct in size and can be identified by the serum of the calf after immunization. Therefore, the rLkt-A2 is antigenic and can stimulate the antigenicity of the immune reaction. The preparation of the rLkt-A2 is confirmed by the above test. . The rLkt-A2 is deposited with the Culture and Conservation Research Center (CCRC) of the Republic of China Food Industry Development Institute under the registration number BCRC940571.

From the results, it was found that rLkt-A2 can be identified as antigenic by the immunized calf serum. In the present invention, rLkt-A2 was selected to further prepare the first embodiment of the invention, rLkt-A2 adjuvant, with rLkt-A2 as an adjuvant. Adding a whole bacterium M. haemolytica non-activated antigen to prepare a hemolytic H. harpyella vaccine, and the second embodiment of the present invention, rLkt-A2 subunit vaccine, containing rLkt-A2 antigen and a biphasic oil Agent.

2. Preparation of the hemolytic H. hamatobacterium vaccine of the present invention

A. The hemolytic H. haramella vaccine according to the first embodiment of the present invention comprises a whole hemolytic Hamann bacillus inactivated antigen and an adjuvant, and the amino acid sequence of the adjuvant is SEQ ID NO: 2 is shown. The hemolytic H. hamatobacterium vaccine of the first embodiment of the present invention is prepared by the following method:

The first step of the hemolytic H. hamatobacterium vaccine of the first embodiment of the present invention is to prepare the recombinant leukotoxin rLkt-A2 antigen of the present invention, which is selected from a single colony sequenced by the gene of the present invention and cultured at 200 μg. In the LB medium of /mL ampicillin (MDBio, Taipei, Taiwan), the preferred embodiment of the present invention is selected to be cultured at 37 ° C until the absorbance OD _{600 nm} reaches 0.6, and the concentration of 1 mM IPTG (isopropyl sulphide) is added. Generation-β-D galactoside), but not limited to this concentration, induces transcription of the gene for 4 hours, causes Escherichia coli to express a large amount of rLkt-A2 protein, and collects the cells by centrifugation to prepare the recombinant white blood toxin rLkt- of the present invention. The A2 antigen, in this example, the recombinant white blood cell toxin rLkt-A2 antigen is used as an adjuvant, hereinafter referred to as rLkt-A2 adjuvant.

The second step of the hemolytic H. hamatobacterium vaccine of the first embodiment of the present invention is to prepare an inactivated vaccine having an M. haemolytica inactivated antigen, which is to culture a large amount of the whole bacteria collected by the present invention in a large amount. After the culture, M. haemolytica is inactivated, and the whole bacteria M. haemolytica is not activated, and the following is called whole bacteria M. haemolytica does not activate the antigen, and a dual-phase oily adjuvant (water-in-oil water-water) is added. In-oil-in-water; w/o/w), which was prepared by the ratio of inner layer antigen: oil layer: outer layer antigen: 1:1:2, and the M. haemolytica inactivated antigen system was selected as 1 × ¹⁰ 10 CFU / mL, hereinafter referred to as M. h inactivated vaccine.

A third step of the hemolytic H. haramella vaccine according to the first embodiment of the present invention is the addition of the rLkt-A2 adjuvant to the inactivated vaccine having the M. haemolytica inactivated antigen, completing the first embodiment of the present invention The preparation of the hemolytic H. mobilis vaccine, hereinafter referred to as the vaccine of the first embodiment of the present invention.

B. The hemolytic H. haramella vaccine according to the second embodiment of the present invention comprises a hemolytic H. halophilus leukotoxin antigen and a biphasic oily adjuvant, the hemolytic H. haemobacterium leukotoxin antigen It is composed of the amino acid sequence of SEQ ID NO: 2. The hemolytic H. haramella vaccine of the second embodiment of the present invention is prepared by the following method:

The first step of the hemolytic H. hamatobacterium vaccine of the second embodiment of the present invention: the antigen system used is the same as the first step of the first embodiment of the present invention, and the rLkt-A2 protein is expressed in a large amount by Escherichia coli. The cells were collected by centrifugation to obtain the recombinant white blood cell toxin protein rLkt-A2 antigen of the present invention, hereinafter referred to as rLkt-A2 antigen.

A second step of the hemolytic H. haramii vaccine according to the second embodiment of the present invention: the rLkt-A2 antigen of the present invention and a biphasic oily adjuvant are prepared as a primary unit vaccine, and the rLkt-A2 antigenic system is selected as 200 μ g / mL, the rLkt-A2 antigen line was selected for addition to the biphasic oil adjuvant (water-in-oil-in-water; w/o/w), from the inner layer of the antigen: oil layer The ratio of the outer layer antigen is 1:1:2 to prepare a unit vaccine, and the preparation of the hemolytic H. hamatobacterium vaccine of the second embodiment of the present invention is completed, which is hereinafter referred to as the vaccine of the second embodiment of the present invention.

So far, the vaccine of the first embodiment of the present invention and the preparation of the vaccine of the second embodiment can be completed, and the vaccine of the second embodiment is followed by the animal test and the immune reaction test. The following immunoassays are all calculated by Student's t test. In the figure, the p value is less than 0.05 in *, and the p value is less than 0.01 in ** to verify the efficacy of the vaccine of the second embodiment of the present invention.

Third, the immunoprotective efficacy test of the hemolytic H. harveyii vaccine of the present invention in a mouse model

The protective efficacy test of the hemolytic H. haramii vaccine according to the second embodiment of the present invention selects ICR mice as experimental animals, and each of 5 mice as a group of animal experimental groups has 4 groups, which is small in the present invention. The white mouse immunization test was performed in three groups of the four groups of animal test groups, and the three groups were inoculated separately for the immunized group (A) M. h inactivated vaccine: prepared by M. haemolytica inactivated antigen, ( B) Vaccine according to the first embodiment of the present invention: M. haemolytica inactivated vaccine is added with the recombinant leukocyte toxin rLkt-A2 adjuvant of the present invention to prepare a cross-immunization vaccine and (C) the second embodiment of the present invention Vaccine: a subunit vaccine containing the recombinant white blood cell toxin rLkt-A2 antigen of the present invention and a biphasic oily adjuvant, and one group of animal test groups in which no preparation of any preparation was left in the group of four animal test groups ( D) Non-immune control group, respectively, immunoassay, antibody potency test, IgG subtype antibody analysis and cellular immunoreactivity test of mice were carried out to confirm the efficacy of the hemolytic H. mobilis vaccine of the second embodiment of the present invention. .

Mouse immune challenge test

Mouse immunization test system of the present invention take the immunized ICR mice challenged tests were first immunized subcutaneously three vaccines 0.2mL above (A) M. H inactivated vaccine, (B) a first embodiment of the present invention Vaccine and (C) vaccine of the second embodiment of the present invention, the dose is not limited to a dose of 0.2 mL, which stimulates the mouse to produce an immune response, and another (D) non-immunized control group does not apply any vaccine, the above four groups Two weeks after the immune reaction, the animal test group was selected to inoculate 2x MLD whole bacteria M. haemolytica , and the inoculated whole M. haemolytica line was selected as 1×10 ⁸ CFU/mL for abdominal cavity vaccination test, and then observed for two weeks. The results are shown in Table 1:

It is known from the table that the survival rate of the vaccine (A) M. h inactivated vaccine and (C) the vaccine of the second embodiment of the present invention is 80%, and the survival rate of the vaccine of the first embodiment of the present invention is inoculated (B). There was no survival of the mice at 60%, while (D) the non-immunized control group did not apply any of the preparations. It was found from the results that the vaccine was administered to the mice to produce immunity, so the survival rate was improved. For mice immune to challenge experiment, a second embodiment of the vaccine strain (C) of the present invention has (A) M. H inactivated vaccine survival rate of the same, but (A) M. H inactivated vaccine by It is limited to M. h serotype, while the vaccine of the second embodiment (C) of the present invention can break serotypes limited since rLkt-A2 is a common characteristic of the different serotypes of antigens from different serotypes resistant M. h of Infection, (C) The vaccine of the second embodiment of the present invention has the breadth and practicality of immunization application, and (B) the vaccine of the first embodiment of the present invention is a cross-immunization vaccine, which is greatly improved. The survival rate of mice, because the M. haemolytica inactivated vaccine has been able to achieve an immune effect against a serotype, in combination with the rLkt-A2 adjuvant of the present invention, the rLkt-A2 adjuvant can enhance the immunogen of the M. haemolytica inactivated vaccine. The sustainability of sexual and immune responses can also stimulate the immune response against the infection of different serotypes of M. haemolytica with the characteristics of its own common antigen to achieve cross-protection. The invention combines the common antigen leukotoxin Lkt-A2 of different serotypes M. haemolytica into recombinant white blood cell toxin rLkt-A2 by gene transfer technology, and prepares the rLkt-A2 as (B) according to the first embodiment of the present invention. The vaccine and (C) the vaccine of the second embodiment of the present invention can effectively achieve the efficacy of immunoprophylaxis.

Mouse antibody efficacy test

The safety test of the mouse of the present invention is tested by ICR mice, and each of the 6 mice is divided into four groups of animal test groups, and the three groups are the first in the immunization group. One muscle inoculation of 0.25 mL (A) M. h inactivated vaccine, (B) vaccine of the first embodiment of the invention: M. haemolytica inactivated vaccine, one of the rLkt-A2 adjuvants of the invention is added for cross-immunization efficacy vaccine, which rLkt-A2 selection of the concentration of 200 μ g / mL and (C) a second embodiment of the vaccine of the present invention: rLkt-A2 antigen of the present invention and a two-phase oily adjuvant of subunit vaccines, which rLkt selection -A2 concentration of 200 μ g / mL, the dose is not limited 0.25mL inoculum dose, and 4 groups of animals remain in the test group 1 group did not play any formulation applied to the test animal as a non-immunized control group, the above-described Four groups of animal test groups were vaccinated for two weeks after the first vaccination, and then vaccinated for the second time. The blood of the four groups of animals was collected at different times: before any preparation, two weeks after the first vaccination, and Two weeks after the second vaccination, the blood will be collected for three periods. ELISA was carried out together with the detection antibody titer, and analyzed for IgG by ELISA as blood test group of animals in each group, in the case IgG1 and IgG2a antibodies arising subtypes.

The results of the antibody titer test are shown in Fig. 2, and the results show that after the first inoculation, the immunized group (A) M. h inactivated vaccine, (B) the vaccine of the first embodiment of the present invention, and (C) the present invention The vaccines of the second embodiment were significantly different from the (D) non-immunized control group. After the second vaccination, the antibody price difference between the three groups of immunized groups and (D) non-immunized control group was greater. This results in an increase in the antibody price of the immunized group once each vaccination is given, because after the first vaccination, the antibody first recognizes the antigen to be inoculated, and produces a certain amount of antibody to the antigen, but the second inoculation makes it possible The antibody recognizing the antigen is rapidly recognized and immediately produces a large amount of antibody against the antigen, amplifying the recognition of the antibody and enhancing immunity. From the results, it is known that the immunization group (A) M. h inactivated vaccine, (B) the vaccine of the first embodiment of the present invention, and (C) the vaccine of the second embodiment of the present invention all produce an immune response and increase the antibody in the animal. The content reaches the effect of immune prevention. Wherein (C) the vaccine of the second embodiment of the present invention has the highest antibody valence test amount, and (B) the vaccine of the first embodiment of the present invention, which represents the M. haemolytica common antigen rLkt-A2, which is prepared as a vaccine capable Effectively stimulate the immune system to produce antibodies, effectively fight against bacterial infections and achieve immune effects.

Two weeks after the second vaccination, the results of IgG, IgG1 and IgG2a antibody subtypes in the blood of each group of animals were measured by ELISA as shown in Fig. 3. The results showed that there was vaccination ( A) M. h inactivated vaccine, (B) Example of a first embodiment tuberculosis vaccines and (C according to the present invention) of the second embodiment of the vaccine of the present invention, the blood of the test animal immunized three groups are the high yield of the induced IgG In addition, IgG2a production is dominant, indicating that recombinant leukotoxin rLkt-A2 can be used as a secondary unit vaccine or adjuvant to effectively induce the immune system to produce a cellular immune response to T _H 1 to achieve memory. Cellular immune response, which in turn achieves long-lasting immune prevention.

Mouse cell immune response test

Three groups of immunized groups were inoculated with A) M. h inactivated vaccine, (B) vaccine of the first embodiment of the present invention, and (C) vaccine of the second embodiment of the present invention were administered 7 days after the second inoculation. (D) Non-immune control group: No preparation was applied, and the spleen cells of the spleen of the test group of the above four groups of animals were respectively taken out, and the antigen was not activated by M. haemolytica , and the rLkt-A2 adjuvant of the present invention was added with M. The haemolytica non-activated antigen, the rLkt-A2 antigen of the present invention and the phytohemagglutinin (PHA: an immune response capable of activating immune cells), and four different antigens are stimulated, and the results are shown in Fig. 4, and the immune group is subjected to The secondary vaccine has produced effective antibodies, so after stimulation with four different antigens, the stimulation index of the immunized group is effectively improved. Inoculation (B) The vaccine of the first embodiment of the present invention is stimulated by four different antigens, and the stimulation index of the group is effectively improved because of the characteristics of the common antigen of rLkt-A2 and the specificity of M. haemolytica inactivation of antigen. a serotype characteristic that substantially increases the immunogenicity of the antigen and the persistence of the immune response, which can direct the immune system to produce humoral or cellular immune responses to the antigen, and inoculate (C) the vaccine of the second embodiment of the present invention, After four different antigen stimuli, the stimulation index can be effectively improved. The results of the cellular immunoreactivity test showed that the rLkt-A2 preparation vaccine of the present invention can effectively elicit and assist in triggering a higher cellular immune response in experimental animals.

The above mouse test is completed, and the present invention is prepared as a vaccine by using rLkt-A2: (B) the vaccine of the first embodiment of the present invention and (C) the vaccine of the second embodiment of the present invention, which can improve the immune response and stimulate immunity. The system cells proliferate and activate and have a cross-immunization effect, and then test (C) how the vaccine of the second embodiment of the present invention is immune in the immune test of the sheep.

IV. Vaccine protection efficacy test of the hemolytic H. haramii vaccine in the sheep model of the present invention

From the test of the aforementioned mouse, it is known that the rLkt-A2 of the present invention is prepared as a vaccine to achieve an effective immune response and has a cross-immunization effect. Therefore, the sheep immunoassay of the present invention is carried out by taking 6 healthy ruminants (reverse animals). Immunological test, randomly divided into two groups of animal test groups, three groups of three sheep, one of which is muscle inoculation (C) vaccine of the second embodiment of the present invention: containing the recombinant white blood toxin rLkt-A2 antigen of the present invention and a double phase preparation of oily adjuvant subunit vaccines, the antigen concentration rLkt-A2 selection of 200 μ g / mL, which is selected based vaccination dose 2 mL, 1 group was not immunized and control group: no any formulation administered to the animal playing In the test group, the antibody efficacy test, the cellular immunoreactivity test, and the inoculation of the sheep (C) the IFN-γ expression amount analysis of the vaccine of the second embodiment of the present invention, respectively, confirming the present invention (C) the second embodiment of the present invention The vaccine is vaccinated against the immune effect of larvae (ruminants).

Lamb antibody efficacy test

The above-mentioned (C) vaccine of the second embodiment of the present invention is a second embodiment of the present invention, and the vaccination is further vaccinated every two weeks after the first vaccination. The amount was selected to be 2 mL, and the inoculation was collected at different times. (C) The blood of the vaccine animal test group of the second embodiment of the present invention: blood before inoculation, two weeks and four weeks after immunization, and simultaneously collected at different times (D) The blood of the test group of the control group was not immunized; the collected blood was subjected to ELISA antibody titer detection.

The results are shown in Fig. 5. The results show that (C) the vaccine of the second embodiment of the present invention is significantly different from the (D) non-immunized control group two weeks after the first inoculation, after the second inoculation. (C) the difference in antibody valence between the hemolytic H. haramii vaccine of the second embodiment of the present invention and the (D) non-immunized control group is more than 6 times, because the reinforced vaccination enhances the antibody's memory of the antigen, Therefore, once the antigen is inoculated once, the recognition of the antigen by the antibody is enhanced once, and a larger amount of antibody is produced.

It is proved by the above test that (C) the vaccine of the second embodiment of the present invention can produce an immune reaction in ruminants (sheep), enhance the antibodies of ruminants, and enhance the immunity of ruminants to M. haemolytica . It can effectively prevent P. pastoris lung inflammation.

Cellular immune response test

7 days after the (C) vaccine of the second embodiment of the present invention and (D) the non-immune control group were vaccinated for the second time, the whole blood of the above two groups of animals was collected and the peripheral blood mononuclear cells were isolated. Stimulating the three different antigens of the M. haemolytica non-activated antigen, the rLkt-A2 antigen of the present invention, and ConA (Concanavalin A: activating cellular immune T cells to produce an immune response), the results are shown in Fig. 6, the ewes are inoculated After (C) the vaccine of the second embodiment of the present invention, the blood of the sheep has an antibody and an immune reaction for identifying the antigen, so that the blood of the sheep is collected and the M. haemolytica inactivated antigen, the rLkt-A2 antigen of the present invention is administered. After the ConA stimulation, the (C) stimulation index of the vaccine of the second embodiment of the present invention can be effectively stimulated to enhance the response.

Inoculation (C) In the vaccine animal experimental group of the second embodiment of the present invention, in addition to being stimulated by the rLkt-A2 antigen, the M. haemolytica non-activated antigen can also be effectively stimulated, confirming that the common antigen rLkt-A2 breaks through the M. haemolytica inactivated vaccine. Due to the limitation of the serotype, (C) the vaccine of the second embodiment of the present invention can be applied to the pulmonary inflammation of Pasteurella in different serotypes, and an effective and good immune response can be achieved.

Inoculation (C) Analysis of the IFN-γ expression of the vaccine of the second embodiment of the present invention

The blood of the vaccination of the vaccine of the second embodiment of the present invention is collected twice (C), and the blood of the non-immunized control group is collected and the peripheral blood mononuclear cells are separated, and the blood mononuclear sphere is separated. The RNA of the cell is reverse transcribed into cDNA, and the IFN-γ primer pair is used for RT-PCR to detect the inoculation (C) whether the vaccine of the second embodiment of the present invention produces a cellular immune response to achieve antiviral activity. Immunity and immune response; another set of β-actin primer pairs, as internal control, because β-actin is a protein expressed by a housekeeping gene, which is expressed in tissues and cells. Constant, so when detecting changes in protein, β-actin is usually used as a standard control group. As a result, as shown in Fig. 7, only the inoculation (C) of the vaccine of the second embodiment of the present invention detected IFN-γ in the blood of the sheep, and (D) the non-immunized control group did not detect the IFN-γ. It is known that vaccination (C) of the vaccine of the second embodiment of the present invention stimulates entry into a cellular immune response, triggers an immune response to promote activation of T cells, and enhances immunity of the animal.

The immune efficacy of the vaccinated sheep of the hemolytic H. haramii vaccine of the present invention was examined by gene level, and it was confirmed that the vaccinated sheep can effectively produce cellular immunity and induce a good immune response.

The hemolytic H. hamatobacterium vaccine of the present invention selects mice and larvae (ruminants) as experimental animals, and after a series of immunoassays, double confirms the hemolytic H. hamatobacterium vaccine of the present invention, and is effective It induces a cellular immune response, prevents the ruminant from infecting the lung inflammation of Pasteurella, and rapidly dies, and has the effect of preventing immunity.

The hemolytic H. hamatobacterium vaccine of the present invention utilizes the characteristic of the common antigen leukotoxin of Hemolyzed Mania bacillus, and is genetically transfected into rLkt-A2 and prepared as a hemolytic Hm. Breaking through the limitations of the hemolytic H. haramella inactivated vaccine, it is suitable for the inflammation of Pasteurella caused by different serotypes of Haemolyticus, which can effectively induce immune response and stimulate immunity. The efficacy of system cell proliferation and activation.

The hemolytic H. hamatobacterium vaccine of the present invention is prepared by using the common antigen recombinant leukotoxin rLkt-A2 of Hemolyzed Mania bacillus as an adjuvant, together with other conventional hemolytic H. haemobacterium inactivated vaccines. For a cross-immunization vaccine, the additive effect of the two produces a more complete and better cross-immunoprotection than the conventional hemolytic H. mobilis non-activated vaccine, and has the utility and utility of immunological application.

While the present invention has been disclosed in its preferred embodiments, the invention is not intended to limit the invention, and the invention may be variously modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

Figure 1: Results of Western blotting analysis of rLkt-A2 recombinant protein.

Figure 2: Results of the ELISA antibody titer test for each mouse test animal group.

Fig. 3 is a graph showing the results of IgG, IgG1 and IgG2a antibody subtypes in the blood of each mouse test animal group.

Figure 4: Results of the cellular immune response test in each mouse test animal group.

Figure 5: Results of ELISA antibody titer test for each sheep test animal group.

Figure 6: Results of the cellular immune response test in each sheep test group.

Figure 7: Results of the amount of IFN-γ in the blood of each sheep test group.

<110> National Pingtung University of Science and Technology

<120> Hemolytic Mania Bacillus Vaccine

<160> 4

<210> 1

<211>

<212> DNA

<213> Hemolytic Mania bacillus (M. haemolytica)

<220>

<221> CDS

<222> (1)...(1725)

<400> 1

<210> 2

<211>

<212> PRT

<213> Hemolytic Mania bacillus (M. haemolytica)

<400> 2

<210> 3

<211>

<212> DNA

<213> Artificial sequence

<223> Lkt-A-F

<400> 3

<210> 4

<211>

<212> DNA

<213> Artificial sequence

<223> Lkt-A-R

<400> 4

Claims (5)

A hemolytic H. haramella vaccine comprising: an adjuvant, the amino acid sequence of the adjuvant is as shown in SEQ ID NO: 2; and a whole hemolytic Hamannia bacterium does not activate the antigen. According to the vaccine preparation described in Item 1 of the patent application, the method of inoculation is by injection. A hemolytic H. haramella vaccine comprising a hemolytic H. halodae leukotoxin antigen line consisting of the amino acid sequence of SEQ ID NO: 2, and a biphasic oily adjuvant. According to the third aspect of the patent application, the hemolytic H. haramii vaccine is inoculated by an injection method. The hemolytic H. hamatobacterium vaccine according to item 3 of the patent application scope is a sub-unit vaccine.