WO2016013824A1

WO2016013824A1 - Bordetella pertussis strain for virus-neutralizing antigenic protein expression, and immunological composition using same

Info

Publication number: WO2016013824A1
Application number: PCT/KR2015/007499
Authority: WO
Inventors: 김태중; 서자영
Original assignee: 전남대학교산학협력단
Priority date: 2014-07-22
Filing date: 2015-07-20
Publication date: 2016-01-28
Also published as: KR20160011444A; KR101672719B1

Abstract

The present invention relates to a Bordetella pertussis strain for virus-neutralizing antigenic protein expression, and to an immunological composition using same. More specifically, the invention relates to: a Bordetella pertussis strain for expressing influenza virus hemagglutinin in the Fim2 gene; an immunological composition comprising same; and a method for producing the Bordetella pertussis strain. As a result of genetic modification by means of the Bordetella pertussis strain of the present invention, it is expected that there will be increased effects from use as a dead vaccine (killed whole cell vaccine) using a strain expressing the virus-neutralizing antigen as a dual antigenic protein, and it is expected that the invention can be used as a component vaccine (acellular vaccine) containing the dual antigenic protein via processing of a chemical drug of the Bordetella pertussis strain. When an immunological composition comprising the strain is administered into the human body, infection from Bordetella pertussis does not appear, and it is expected that said composition can be used as a virus-preventing vaccine or therapeutic strain via virally derived dual antigenic protein expression for inducing neutralizing antibodies, while maintaining the function whereby pertussis is prevented.

Description

Specification

NAME OF THE INVENTION: Bordetella pertussis strains for the expression of virus neutralizing antigen proteins and immunogenic compositions using them

[1] The present invention relates to Bordetella pertussis strains for the expression of virus neutralizing antigen proteins and to immunogenic compositions using the same, in particular Bordetella pertussis strains expressing influenza hamagglutinin in the Fim2 gene. It relates to an immunogenic composition and a method for producing the Bordetella pertussis strain.

Background

[2] Influenzavirus A is a mutation or

New antigenic strains are easy to emerge by genome shifts and cause a viral pandemic when humans have low antibody retention for the mutant antigens.The virus invades the mucosa and causes respiratory diseases. RNA virus belongs to the orsomyxoviride (Ortftomyjcovi dije).

The genome consists of eight segments, one of which is a protein per surface

Hemagglutinin (hemagglutinin or haemagglutinin (HA)) is a protein that attaches to the spherical surface of influenza and determines the subtype together with neuraminidase (NA). As a result, a new strain of influenza virus has developed, and up to now, the vaccine vaccines are selected each year and the vaccine vaccines have been introduced. The influenza vaccines currently sold are the first generation through virus culture and inactivation using fertilized eggs. The production of vaccines takes months to produce and prevents new virus outbreaks. It is also costly and expensive, which can be economical for a large number of people to be vaccinated.

[3] Pertussis is transmitted by Gram-negative hepatitis Bordete Ha pertussis (BPs) and is a highly contagious respiratory infection in infants and young children. Pertussis is characterized by a long-lasting severe convulsive cough, inability to expect passive passive immunization from the mother, and incidence in infants is severe and mortality is high, with approximately 80% of all deaths within 90% of infants within one year of birth. In Korea, it was designated as a disease that requires regular vaccination in 1954 by the infectious disease prevention method.

The use of diphtheria, tetanus, and pertussis combined vaccine (DTP) has begun, and three basic vaccinations have been carried out to date, including additional vaccinations. Systemic side effects such as back and severe side effects by the brain began to gradually become problematic. Several studies have been conducted to overcome this problem, and in 1979, diphtheria-acellular pertussis-tetanus (DaPT) vaccines containing component vaccines were developed in Japan and used throughout the country until now.

In Korea, no vaccine has been formed by influenza immunization with Bordetella pertussis vaccine.

Detailed description of the invention

Technical task

[4] In the present invention, the surface structure of Bordetella pertussis for solving the above problems

Another virus in the locus of the protein using the protein Fimbriae

By introducing and expressing the gene encoding the antigenic protein of the pathogen, Bordetella pertussis not only protects against the infection of the virus pathogens expressed by the antigenic protein, but also produces a multivalent live vaccine.

I wanted to develop.

Task solution

[5] The present invention relates to the Fim2 gene of Bordetella pertussis with the nucleotide sequence of SEQ ID NO: 1.

Attenuated or poisoned Bordetella pertussis comprising a recombination gene having a structure in which a heterologous antigenic gene is inserted between the N-terminal 100 to 140 mer and the C-terminal 100 to 140 mer, preferably 120 mer, respectively.

pertussis).

[6] In the present invention, the heterologous antigenic gene may be, but not limited to, influenza virus-derived hemagglutinin (HA). In the present invention, the haemagglutinin may have a nucleotide sequence of SEQ ID NO: 2, although not limited thereto. .

[7] Influenza viruses are fragmented negative strand RNA viruses

A member of the Orthomyxoviridae family. Influenza A virus consists of nine proteins.

In addition to a single, unstructured NS 1 protein that is constructed and regulated

The non-structural NS1 protein is synthesized in large quantities during the regeneration cycle and concentrated in the cytosol and nucleus of infected cells. Due to the fragmented nature of the virus genome, the cells are mixed during infection by various viral strains. The mechanism of gene rearrangement (exchange of genomic fragments) can occur.

Influenza A viruses are classified into several subtypes, depending on the various proteins on their surface, such as magglutinin (HA) and neuroamidase (NA) virus proteins.

Influenza A virus subtypes are identified by two viral glycoproteins: haemagglutinin (HA or H) and neuroamidase (NA or N). Each influenza virus subtype is identified by a combination of H and N proteins. Eighteen HA subtypes and 11 NA subtypes are known. Influenza A viruses can infect humans, birds, pigs, horses and other animals, but wild birds are a natural host for these viruses. Influenza A subtype (ie, H1N1, Although H1N2, and H3N2) are spreading among people today, all combinations of 18 HA and 11 NA subtypes have been identified in birds, particularly wildlife and snipes.

In addition, evidence continues to confirm that H5 and H7 influenza viruses can cause human disease. HA of influenza A viruses includes two structurally specific regions, spherical head region and stem region. It contains receptor binding sites that induce viral adhesion to target cells and are responsible for the hemagglutination activity of HA. The stem region is essential for membrane fusion between the viral envelope and the endosomal membrane of the cell. Fusion proteins involved in activity. The present invention also relates to an immunogenic composition for the treatment or prevention of infection comprising the above Bordetella pertussis strain.

In the present invention, the infection may be, but is not limited to, a heterologous antigen pathogen infection to human or Bordetella pertussis infection. The present invention also relates to a heterologous strain between the N-terminal 100 to 140 mer and the C-terminal 100 to 140 mer, preferably 120 mer, of the Hm2 (fimbriae 2) gene of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: 1. A method for preparing a Bordetella pertussis strain comprising the steps of preparing an insert for homologous recombination by inserting an antigenic protein gene; and transforming the insert into Bordetella pertussis.

In the present invention, the heterologous antigenic gene may be, but not limited to, influenza virus derived hemagglutinin (HA).

In the present invention, the hemagglutinin is not limited, but the sequence of

It can have a sequence.

In the present invention, the insert is available, but not limited, linearized DNA cut with two restriction enzymes (BgHl + Kpnl) shown in 2.

Recombinant protein comprising influenza virus-derived magglutinin prepared through the embodiment of the present invention was modified by transforming Bordetella pertussis strain with 2x sample complete solution, followed by electrophoresis on 10% SDS-PAGE. After the transfer of the protein to the PVDF membrane, the electrophoresis resulted in rabbit-anti-Fim2 and rabbit anti-hamma-glutinin polyclonal antibodies produced by inoculating rabbits with recombinant Fim2 and recombinant hemagglutinin, which were separately expressed in E. coli. Was used as the primary antibody, followed by pure separation using a goat anti-rabbit IgG HRP conjugated antibody as a secondary antibody, thereby confirming that a recombinant protein having a size of about 37 kDa was formed and separated. The invention also relates to the N-terminal 100-140 mer and C-terminal 100-140 mer of the Bordetella pertussis Fim2 gene with a nucleotide sequence of SEQ ID NO: 1, preferably Preparing an insert for homologous recombination by inserting a heterologous antigenic gene between 120 mer and each; transforming the insert into Bordetella pertussis; culturing the transformed Bordetella pertussis to the heterologous antigen Expressing a protein; and making a vaccinated vaccine using the mutant strain; and determining whether the vaccinated vaccine is immunoreactive immunized into an animal.

It is about the method of immunoassay.

[19] In order to verify the immunity in the step of determining whether the immunization is performed, as shown in the embodiment of the present invention, the bacteriophage vaccine was prepared using the transformed mutant strain and administered to the experimental animal, and then blood was collected. Serum was isolated, serum was isolated, and ELISA method was used to compare IgG of Fim2 or hemagglutinin with negative control group to confirm antibody production and confirmed that antibody was successfully formed.

[20]

[21] The present invention also relates to Fim2 of Bordetella pertussis having the base sequence of SEQ ID NO: 1.

Preparing an insert for homologous recombination by inserting a heterologous antigenic gene between the N-terminal 100 to 140 mer and the C-terminal 100 to 140 mer of the gene, preferably 120 mer, respectively; the insert is a Bordetella pertussis E. the step of transforming; culturing the transformed Bordetella pertussis to prepare a bacteriophage vaccine; determining whether the immunized post-immune immunity in the animal except the human-generated bacterium vaccine; and the live influenza virus Determination of defense after administration into an immunized animal; relates to a method for identifying defense.

[22] In order to verify the protective effect against influenza virus in the step of determining whether to defend against the disease, as shown in one embodiment of the present invention, the influenza virus living in the immunized animal is infected and RNA is extracted from the animal tissue. CDNA was synthesized and subjected to quantitative real-time synthase chain reaction and compared with negative control group, and compared with the positive control group administered with influenza virus activated by the experimental group administered with the purely isolated protein. By measuring the decrease significantly compared to the negative control, the effective defense effect was confirmed.

Effects of the Invention

[23] As a result of the transformation through the Bordetella pertussis strain of the present invention, it is expected to be highly effective as a killed whole cell vaccine expressing the heterologous protein, and to treat and treat the chemicals of the Bordetella pertussis strain. It is expected to be available as acellular vccine using layer separation.

[24] The immunogenic composition containing the above strain does not show infection against Bordetella pertussis when administered in human body, and can be used as a vaccine or therapeutic strain through expression of a virus-derived antigenic protein that causes neutralizing antibodies. By It is expected.

Brief description of the drawings

Fig. 1 shows PCR amplification results, where lane 1 is the result of amplification of haemagglutinin, lane 2 is the N-terminal and C-terminal amplification results of Fim2 gene, and Μ is 100 bp marker.

2 shows the N-terminal 120 mer and the C-terminal 120 mer, influenza, of the Fim2 gene.

A schematic of cloning the gene encoding the virus's haemagglutinin protein and linking it to use as an insert for homologous recombination.

3 is a schematic diagram of PCR for verification of substitution of three genetic loci.

[28] Figure 4 is a PCR result, lane 1 is the amplification result of the N- terminal-hemagglutinin of the Fim2 gene

(0.95 kb), sewn 2 shows amplification of hemagglutinin (0.83 kb), and lane 3

Amplification of the C-terminus of the hemagglutinin-Fim2 gene (0.95 kb) and M is a 100 bp marker.

[29] Figure 5 shows the expression of hemagglutinin protein against Fim2.

Western Blot results were measured using lane 1 as wild Bordetella pertussis (23 kDa), lane 2 as mutant Bordetella pertussis (37 kDa) expressing hippocampus of the present invention, and lane 3 as a positive control. Is a recombinant antigen (30 kDa) of Fim2 expressed in E. coli.

6 shows the expression of hemagglutinin protein against hemagglutinin clone.

Western blot results measured using antibodies. Lane 1 is wild type Bordetella pertussis (no band), lane 2 is mutant Bordetella pertussis (37 kDa) expressing hemagglutinin of the present invention, and lane 3 Is a recombination antigen (35 kDa) of hemagglutinin expressed in E. coli as a positive control.

FIG. 7 shows western blot results using polyclonal antibodies against Fim3 to determine the effect of Fim3 on the Bordetella pertussis beach strain. Lane 1 is a wild type Bordetella pertussis (23 kDa), lane 2 Is a variant strain Bordetella pertussis (23 kDa) expressing hemagglutinin of the present invention, and Lane 3 is a recombinant control of Fim3 (35 kDa) expressed in Escherichia coli as a positive control.

8 shows H1N1 virus inactivated with the antibody titer and positive control of blood immunoglobulin G (IgG) of the group (middle) which immunized the mutant Bordetella pertussis strain with the formalin treatment and the vaccine mixed with excipients This is a diagram showing the IgG concentration of blood in the inoculated group (grey). A group inoculated with PBS (black) only as a negative control group was used.

9 is a result showing the change in the threshold cycle (Q) of the real-time PCR showing the protective effect when influenza virus infection after immunization.

10 quantitatively calculates the amount of relative virus calculated from the results of FIG.

It is shown. Best Mode for Carrying Out the Invention

[35] Hereinafter, the present invention will be described in detail with reference to Examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited by the following Examples.

[36]

Example 1 Preparation of Attenuated Bordetella Pertussis Bacteria Expressing Hemagglutinin (HA) of Influenza Viruses

[38] Regulations Polismid

[39] Escherichia coli JM 109 (Invitrogen; Carlsbad, CA, USA)

[40] pGEM-T easy vector (Promega; Madison, WI, USA)

[41] Red helper plasmid pKD46 (Datsenko and Wanner, 2000): vector producing red and Gam proteins using pBAD promoter

[42] Bordetella pertussis standard strain Tohama I ATCC # B AA-589) [43] Influenza Virus A / PR / 8/34 (HlNl) (VR1469): American Type Culture

Collection (ATCC, Rockville, MD, USA).

[44]

[45] PCR (overlapping)

[46] 1) Primer for PCR of Fim2 and hemagglutinin

[47] N-terminus of Fim2 (GenBank # BX640414), Influenza Virus

Hippocampus article Ruti Nin (GenBank # EF467821) and C- to Fim2 for amplification of the terminal used by making the forward and reverse primers as shown in Table 1. H. influenza mageul Ruti TRIzol viruses to create a template for the cloning of non ^® Extract total RNA using reagents (Invitrogen, Carlsbad, CA, USA), Primer Uni-12-F for superscript ΠΙ One-step RT-PCR system (Invitrogen, Carlsbad, CA, USA) and cDNA synthesis (Table 1) CDNA was synthesized and used as a template for PCR cloning.

[48] [Table 1]

[50] For each primer set, PCR was carried out for 30 minutes at 94 ° C for 5 minutes, with 30 cycles at -94 ° C for 30 seconds -57 0C for 30 seconds -72 ° C for 1 minute, and the final extension was 72 ° C. Each PCR product was loaded on 1.2% agarose gel and subjected to electrophoresis (FIG. 1).

In the PCR product for each of the amplified genes, overlapping PCR product 1 was prepared using Fim2 N-terminal and hemagglutinin PCR products. PCR was performed for 15 minutes at 94 ° C for 30 minutes at -57 ° C for 30 minutes at -72 ° C for 1 minute, and then Fim2-NF primer and HA-R primer were added for 5 minutes at 94 ° C. 30 cycles were conducted at 30 seconds -57 ° C at 30 seconds -72 ° C for 1 minute at C.

The overlapping PCR product 2 was prepared by using the overlapping PCR product 1 and the Fim2 C-terminal PCR product prepared as described above under the same conditions as the overlapping PCR product 1 preparation.

The PCR product was cloned into a pGEM-T Easy cloning vector and cleaved using restriction enzymes g II and wl, as shown in FIG. 2, to prepare the prepared 1.07 kb of DNA.

It was used as a linear DNA insert for homologous recombination.

[54]

[55] Displacement of Fhn2 user account through association

[56] Bordet-Gengou blood agar (BBL ™, 297876, BD, Incubated at 37 ° C in USA), inoculated with 5 mL of Stainer-Scholte (SS) broth, incubated at 37 ° C until OD ₆₀₀ reached 0.5, and cooled on ice for 20 minutes. The pellets were then centrifuged at 4000 xg for 15 minutes to recover the pellets. The cells were concentrated with cold 10% glycerol (Sigma, USA) 100 to prepare competent cells for electroporation. To express Red recombinase in cells, pKD46 plasmids were generated using Gene Pulser Xcell electroporation system (Bio-Rad, Hercules, CA).

After transformation, ampicillin (Sigma, USA) with a final concentration of 10 mM

Selected from Stainer-Scholte (SS) broth with L-arabinose (Sigma, USA) and incubated at 30 ° C until the OD _6∞ value is 0.5. Sigma, USA) 100 to prepare an electrocompetent cell for transforming the linear DNA prepared for homologous recombination.

[57] A variant strain "BPS-Fim2- expressing hemagglutinin as a heterologous antigen by transforming 50 μί electrocompetent cells with l iL insert DNA by electroporation and culturing in Bordet-Gengou blood agar HA "was selected.

[58]

[Example 2] Hemagglutinin Substitution Confirmation

[60] BPS-Fim2-HA Survival in Selection Medium After Transforming in Example 1

PCR verification was performed for verification. Using three kinds of primer sets as shown in FIG. 3, PCR verification number 1 was Fim2-NF and HA-R primers, and PCR verification number 2 was HA-F and HA-R primers and PCR verification. Step 3 was used to verify that the amplification of PCR products of 0.95 kb, 0.83 kb and 0.95 kb was obtained using HA-F and Fim2-CR primers, respectively.

[61] As a result, from the results in Fig. 4, PCR products of a size expected for each PCR verification were obtained.

It was confirmed that it was generated.

[62]

Example 3 Confirmation of Expression of Heteroglutinin (HA) Protein Containing Heterologous Antibody

[64] 1 mL of the mutant strain BPS-Fim2-HA culture was centrifuged at 4000 xg for 15 minutes to prepare bacterial pellets, and 2x sample buffer (0.125M Tris (pH 6.8), 4% SDS, 20% glycerol 10% 2). After denatured by adding -mercaptoethanol, 0.2% bromo-phenol-blue; Sigma, US A), electrophoresis was performed on 10% SDS-PAGE. The electrophoresis result was PVDF membrane (Westeran ^® , Whatman ™, GE Healthcare). Rabbit-anti-Fim2 and rabbit-anti-hemagglutinin-clonal antibodies produced by inoculating rabbits with recombinant Fim2 expressed in Escherichia coli and recombinant hemagglutin expressed in Escherichia coli The expression of hemagglutinin protein was confirmed using a goat anti-rabbit IgG HRP conjugated antibody (Pierce, USA) as a secondary antibody.

As a result, the expression of the protein containing hemagglutinin was confirmed by confirming that bands for the protein of 37 kDa were formed from FIGS. 5 and 6 (lane 2, respectively). [67] Investigation of structural variation in Fim3 (fimbriae 3)

[68] Recombinant Fim3 expressed in Escherichia coli under the same conditions as above experiment

Inoculation of rabbit-anti-Hm3 polyclonal antibody produced as a primary antibody, followed by the use of goat anti-rabbit IgG HRP conjugated antibody (Pierce, USA) as a secondary antibody resulted in normal Fim3 development due to changes in the Fim2 locus. As a result of checking whether Fim3 is normally expressed to see if the current is affected, it was found that the expression of Fim3 was not affected.

[69]

Example 4 Preparation of BPS-Fim2-HA Bacterial Vaccine

[71] In order to verify the immune effects in mice, experimental animals,

The vaccinated vaccine was prepared using the prepared strain BPS-Fim2-HA.

[72] The strain prepared according to the above example was transferred to Stainer-Scholte (SS) broth.

After incubation, centrifugation at 4000 X g for 15 minutes, resuspended in PBS, and after the reaction was repeated at 37 ° C for more than 48 hours, including formalin to reach a final concentration of 0.2% prepared for killed-bacterial cells. Adjuvant was mixed with Alum ^® (Imject Alum, Pierce, Rockford, IL, USA) at a ratio of 1: 1-1: 3. The total number of cells inoculated once was inoculated with lxlO ⁹ cells / mouse, which was quantified with recombinant hemagglutinin so that 50 ug per mouse could be inoculated.

[73]

Example 5 Validation of Immune Effect in Mice Using Prepared Bacillus Vaccine

[75] A five-week-old ICR mouse (DBL, Korea) was prepared by subcutaneous subcutaneous examples.

For this purpose, the experimental group was divided into three groups, and the influenza virus inoculated group, which was inactivated by the PBS vaccine group and the positive control group, was prepared as the normal control vaccine vaccine group and the negative control group.

[76] Antigen immunization was performed three times at two-week intervals.

Blood was drawn from the vein to separate the serum.

The test was carried out three times for 7 days. After dilution to 1: 100 with distilled water

Enzyme immunoassay (ELISA) was used to compare immunoglobulin G (IgG) in blood.

[77] The antibody value was increased at 21 and 35 days in comparison with the IgG control result and negative control group.

Induction was confirmed (FIG. 8).

[78]

Example 6 Investigation of Protective Effect in Mice Using Bacillus Vaccine

In order to confirm the protective effect upon infection with influenza virus after confirming the immune effect of Example 5, a sublethal dose (500 pfu / mouse, 40 LD ₅₀ , 10 ^{4 1} EID ₅₀ ) of influenza virus

The infection was administered intranasally with A / PR / 8/34 (HlNl). Five days after infection,

Lung tissue was isolated from the mouse, and TRIzol ^® reagent (Invitrogen, Carlsbad, Total RNA was extracted using CA, US A) and cDNA was synthesized using Superscript III One-step RT-PCR system (Invitrogen, Carlsbad, CA, USA) and primer Uni-1 ² -F (Table ¾). Real-time quantitative PCR using the cDNA as a template and using the primers shown in Table 2;

Exicycler ™ 96, Bioneer, Korea). The RNA concentration of the virus will be interpreted as the equivalent cDNA amount and the amount of cDNA will be proportional to the actual concentration of the virus, so the detection concentration in quantitative PCR can be interpreted as the amount of virus. Virus-derived RNA, ie cDNA, was detected by targeting the hemagglutinin gene region, neuraminidase (NA), and M protein gene region. In this study, we analyzed the amount of viral RNA detected using the beta-actin gene RNA as a comparative group to confirm the relative quantification.

[81]

[82] [Table 2]

[83]

As can be seen from Fig. 9, the delta (Ct) value through the threshold cycle was shown to be the difference of 10-13 cycles. Since the difference of 1 cycle in PCR is twice the concentration difference, the difference of 10-13 cycles is interpreted as the concentration difference of 2 ^t0 -2 ¹³ (1024 times -8192 times). As a result, as shown in FIG. 10, it was confirmed that the control group was significantly reduced compared to the negative control group, and that the positive control group showed similar values to the group immunized with inactivated influenza virus.

[85]

From the above results, it was confirmed that the modified Bordetella pertussis strain of the present invention was capable of expressing the hemagglutinin (HA) protein of influenza virus, a heterologous antigen, while retaining the original pathogen.

[87] Also, prevention of influenza virus infection from the results of immunization and defense effect confirmed by the bacterium vaccine containing the heterologous antigen protein expressed in the above strain. Alternatively, the present invention was completed by confirming that the strain of the present invention can be used as a heterologous antigenic protein carrier.

[88]

Sequence List Free Text

[89] The sequence listing was attached as a sequence listing file.

Claims

Claim

Borde containing a recombination gene having a structure in which the heterologous antigen gene is inserted between the N-terminal 100 and 140 mer and the C-terminal 100 to 140 mer of the Bordetella pertussis with SEQ ID NO: 1 and the base sequence. Tela pertussis (fl ifefe / to pertussis) strain. According to claim 1,

Bordetella pertussis strain, characterized in that the heterologous protein gene is influenza virus-derived hemagglutinin (HA).

According to claim 2,

The seahorse glutenin is Bordetella pertussis strain, characterized in that having the base sequence of SEQ ID NO: 2.

An infectious immunogenic composition comprising the Bordetella pertussis strain of any one of claims 1 to 3.

In paragraph 4,

The infectious disease is a heterologous antigen pathogen infection into human or

A composition characterized by being Bordetella pertussis.

Preparing an insert for homologous recombination by inserting a heterologous antigenic protein gene between the N-terminal 100 to 140 mer and the C-terminal 100 to 140 mer of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: 1; and

Transforming the insert to Bordetella pertussis;

Bordetella pertussis (Bi fe // fl pertussis) strain comprising a.

In paragraph 6,

The heterologous antigen protein gene is a production method characterized in that the influenza virus-derived hemagglutinin (HA).

According to claim 7,

The hemagglutinin has a nucleotide sequence of SEQ ID NO: 2, characterized in that the manufacturing method.

In paragraph 6,

The insert is a manufacturing method characterized by using linearized DNA cut with two restriction enzymes (BglU + Kpnl) shown in (but not limited to) 2.

For homologous recombination by inserting a heterologous antigenic protein gene between the N-terminal 100 to 140 mer and the C-terminal 100 to 140 mer of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: 1 Manufacturing an insert;

Transforming the insert to Bordetella pertussis;

Expressing the heterologous antigen protein by culturing the transformed Bordetella pertussis;

Preparing the bacteriophage vaccine using the transformed Bordetella pertussis strain; and

Determining whether the immunized immunity is immunized into an animal excluding the germ vaccine;

Immune confirmation method comprising a.

11. The Fim2 of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: 1

Inserting a heterologous antigenic protein gene between the N-terminal 100 to 140 mer and the C-terminal 100 to 140 mer of the gene to prepare an insert for homologous recombination;

Transforming the insert to Bordetella pertussis; culturing the transgenic Bordetella pertussis to express the heterologous antigen protein;

Preparing the bacteriophage vaccine using the transformed Bordetella pertussis strain;

Determining whether the immunized immune vaccine is immunized into an animal except human; and

Administering an influenza virus into the immunized animal to determine defense;

Defense check method comprising a.