KR101672719B1 - Bordetella pertussis strain for expression of viral neutralizing antigen and immunogenic composition using the same - Google Patents

Abstract

The present invention relates to a Bordetella pertussis strain for the expression of a virus neutralizing antigen protein and an immunogenic composition using the Bordetella pertussis strain. More particularly, the present invention relates to a Bordetella pertussis strain expressing an influenza virus hemagglutinin in the Fim2 gene, And a method for producing the Bordetella pertussis strain.
As a result of transformation through the Bordetella pertussis strain of the present invention, a killed whole cell vaccine using a strain expressing the neutralizing antigen of the virus as a heterologous antigen protein is expected to be highly effective in utilization, and the Bordetella pertussis strain Of acellular vaccine containing a heterologous antigen protein through the treatment of the chemical agent of the present invention. The immunogenic composition comprising the strain does not show infection with Bordetella pertussis when administered in the human body and maintains the function of preventing pertussis, while preventing the virus from expressing a virus-derived heterologous antigen protein that induces neutralizing antibodies It is expected to be usable as a vaccine or a therapeutic strain.

Description

Bordetella pertussis strains for the expression of viral neutralizing antigen proteins and immunogenic compositions using the same -

The present invention relates to a Bordetella pertussis strain for the expression of a virus neutralizing antigen protein and an immunogenic composition using the Bordetella pertussis strain. More particularly, the present invention relates to a Bordetella pertussis strain expressing influenza hemagglutinin in the Fim2 gene, And a method for producing the Bordetella pertussis strain.

Influenza A virus type A is susceptible to emergence of new antigenic mutants due to drift or genome segment shift and causes a virus pandemic if the antibody level to the mutant antigen is low in the human population. The virus belongs to Orthomyxoviridae , which is a single strand RNA virus that invades the mucous membranes and causes respiratory diseases. One of the surface glycoproteins, hemagglutinin or haemagglutinin (HA), is attached to the spherical surface of influenza and forms a subtype with neuraminidase (NA) ). ≪ / RTI > In Korea, a new strain of influenza virus has been developed due to a mutation caused by a subtype derived from pigs. In the past, a vaccine vaccine which is popular every year has been selected and inoculated with a mixed vaccine. The current flu vaccine uses a first-generation vaccine production method through virus culture and inactivation using fertilized eggs, which takes several months to produce and does not prevent the occurrence of a new type of virus. Also, since the price is high, many people may have economic difficulties in vaccination.

Pertussis is spread by Gram for bacteria in Maribor between voice telra whooping cough (Bordetella pertussis, BPS), it is primarily a disease highly contagious respiratory tract infection in infants and young children. Pertussis is characterized by prolonged persistent coughing cough and is not expected to passively receive immunity from the mother. Infant illness is severe and mortality rate is high. About 80% of all deaths are infants within one year of age and 90 Gt;% < / RTI > Since 1958, the use of Diphtheria, Tetanus, and Pertussis combined vaccine (DTP) has been started in Korea since 1958, and the three basic inoculations . In the case of conventional commercial vaccines, systemic side effects such as fever, loss of appetite, severe irritability, convulsions and severe side effects due to cerebral infarction have gradually become problematic since the mid-1960s, and various studies have been conducted to overcome them. The acellular Pertussis-tetanus (DaPT) vaccine containing the component vaccine was developed in Japan and introduced into the country, and has been used nationwide until now. Currently, there is no case in Korea where a vaccine is made by combining influenza with Bordetella pertussis vaccine.

In order to solve the above problems, the present invention uses a surface structural protein of Bordetella pertussis, Fimbriae, to introduce a gene encoding an antigenic protein of another viral pathogen into the locus of the protein, And to develop a multivalent live vaccine that has a protective effect against the infection of the corresponding viral pathogen expressed as an antigenic protein.

The present invention is characterized by the insertion of a heterologous antigen protein gene between 100 to 140 mer at the N-terminus of the Fim2 gene of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: 1 and 100 to 140 mer, preferably 120 mer each at the C-terminus ( Bordetella pertussis ) strain containing a recombinant gene having one structure.

In the present invention, the heterologous antigen protein gene may be hemagglutinin (HA) derived from influenza virus, although not limited thereto. In the present invention, the hemagglutinin is not limited, but it may have the nucleotide sequence of SEQ ID NO: 2.

Influenza viruses are RNA viruses of a fragmented negative strand and belong to the family Orthomyxoviridae. The influenza A virus further encodes one unstructured NS1 protein consisting of nine proteins with regulatory functions. The unstructured NS1 protein is synthesized in large quantities during the regeneration cycle and is concentrated in the cytosol and nucleus of infected cells. Due to the fragmented nature of the viral genome, mechanisms of gene rearrangement (exchange of genomic fragments) can occur during mixed infections of cells with different viral strains. Influenza A virus is further divided into several subtypes depending on the various hemagglutinin (HA) and neuroamininase (NA) viral proteins present on its surface. Influenza A virus subtypes are identified by two viral glycoproteins, hemagglutinin (HA or H) and neuroaminidase (NA or N). Each influenza virus subtype is identified by a combination of H and N proteins. 18 HA subtypes and 11 NA subtypes are known. Influenza A viruses can infect humans, birds, pigs, horses, and other animals, but wild birds are natural hosts for these viruses. All combinations of 18 HA and 11 NA subtypes have been identified in birds, particularly in wildlife and shorebirds, although only a subset of influenza A subtypes (i.e., H1N1, H1N2, and H3N2) are propagated among humans today. In addition, there is continuing evidence that H5 and H7 influenza viruses may cause human disease. The HA of influenza A virus contains two structurally specific regions, i.e., a globular head region and a stem region. The spherical head region contains a receptor binding site that results in viral attachment to the target cell and is involved in the hemagglutinating activity of HA. The stem region contains fusion proteins that are essential for membrane fusion between the viral envelope and the endosomal membrane of the cell and thus participate in fusion activity.

The present invention also relates to an immunogenic composition for the treatment or prevention of infectious diseases comprising the Bordetella pertussis strain.

In the present invention, the infectious disease is not limited, but may be a heterologous antigenic pathogen infection or Bordetella pertussis infectious disease to the human body.

The present invention also relates to a method for producing a heterozygous mutant of Bordetella pertussis Fim2 (fimbriae 2) gene having a nucleotide sequence of SEQ ID NO: 1, comprising 100 to 140 mers of the N-terminus and 100 to 140 mers of the C- Preparing an insert for homologous recombination by inserting an antigen protein gene; And transforming the insert into Bordetella pertussis; ( Bordetella pertussis ), which comprises a microorganism belonging to the genus Bordetella pertussis .

In the present invention, the heterologous antigen protein gene may be hemagglutinin (HA) derived from influenza virus, although not limited thereto.

In the present invention, the hemagglutinin is not limited, but it may have the nucleotide sequence of SEQ ID NO: 2.

In the present invention, the insert is not limited, but a linearized DNA fragment cut into two restriction enzymes ( Bgl II + Kpn I) shown in FIG. 2 can be used.

The transformed Bordetella pertussis strain transformed with influenza virus-derived hemagglutinin produced by the example of the present invention was denatured by adding 2x sample buffer and electrophoresed on 10% SDS-PAGE The resulting electrophoresis was transferred to a PVDF membrane, and then recombinant Fim2 expressed in E. coli was separately introduced and rabbit-anti-Fim2 produced by inoculating the rabbit with recombinant hemagglutinin and rabbit anti-hemagglutinin polyclonal antibody Was used as the primary antibody, and the purified anti-rabbit anti-rabbit IgG HRP-conjugated antibody was purified using a secondary antibody. As a result, it was confirmed that a recombinant protein having a size of about 37 kDa was formed and separated.

The present invention also relates to a method for producing a heterologous antigen protein gene, which comprises the step of selecting a heterologous antigen protein gene between 100 to 140 mer of the N-terminal of the Fim2 gene of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: 1 and 100 to 140 mer of the C- Thereby preparing a homologous recombination insert; Transforming the insert into Bordetella pertussis; Culturing the transformed Bordetella pertussis to express the heterologous antigen protein; And using the mutant to produce an inactivated vaccine; And determining whether or not the inoculum vaccine is immune to an animal; To a method for identifying an immunity.

In order to verify the immunity effect in the step of judging whether or not the immunity is present, a transformant vaccine is prepared using the transformed mutant as described in one embodiment of the present invention, and the vaccine is administered to an experimental animal, And IgG of Fim2 or hemagglutinin was compared with the negative control by ELISA to confirm the formation of the antibody. As a result, it was confirmed that the antibody was successfully formed.

In addition, the present invention provides a method for producing a heterologous antigen protein gene comprising the steps of: (a) providing a heterologous antigen protein gene between 100 to 140 mer of the N-terminal of the Fim2 gene of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: 1 and 100 to 140 mer of the C- Thereby preparing a homologous recombination insert; Transforming the insert into Bordetella pertussis; Culturing the transformed Bordetella pertussis to produce a dead mold vaccine; Immobilizing the prepared dead bacteria vaccine into an animal other than a human; And administering live influenza virus to said immunized animal and determining whether or not to defend; To a defense verification method.

In order to verify the defense effect against the influenza virus in the step of judging whether or not the virus is defected, an influenza virus is infected to an animal whose immunity has been confirmed as in the embodiment of the present invention, RNA is extracted from the animal tissue, The quantitative real-time polymerase chain reaction was performed, and compared with the negative control, the amount of viral RNA was similarly decreased and compared with that of the positive control administered with the inactivated influenza virus And the decrease was significantly higher than that of the control group.

As a result of transforming through the Bordetella pertussis strain of the present invention, a killed whole cell vaccine expressing a heterologous antigen protein is expected to be highly effective, and the treatment of the Bordetella pertussis strain with the chemicals and layer separation It is expected to be used as an acellular vccine.

The immunogenic compositions comprising the strains are expected to be useful as preventive vaccines or therapeutic strains through the expression of viral-derived heterologous antigenic proteins that induce neutralizing antibodies, without the infection of Bordetella pertussis when administered in the human body .

Fig. 1 shows the result of amplification of hemagglutinin in lane 1 as a result of PCR amplification, lane 2 shows the result of amplification of N-terminal and C-terminal region of Fim2 gene, and M is a 100 bp marker.
FIG. 2 is a schematic diagram showing that a gene coding for the hemagglutinin protein of the influenza virus is cloned and used as an insert for homologous recombination at the N-terminal 120mer and the C-terminal 120mer of the Fim2 gene .
FIG. 3 is a schematic diagram of a PCR for verifying the substitution of three loci.
Fig. 4 shows the result of PCR. As a result, lane 1 shows amplification result (0.95 kb) of N-terminal-hemagglutinin of Fim2 gene, lane 2 shows hemagglutinin amplification result (0.83 kb), lane 3 shows hemagglutinin The amplification result of the C-terminal of the nin-Fim2 gene (0.95 kb) and M is the 100 bp marker.
FIG. 5 shows Western blot results of the expression of hemagglutinin protein measured using a polyclonal antibody against Fim2. Lane 1 is a wild-type Bordetella pertussis (23 kDa), lane 2 is a mutant Bordetella pertussis (37 kDa) expressing hemagglutinin of the present invention, lane 3 is a positive control, and recombinant Fim2 expressed in E. coli Antigen (30 kDa).
FIG. 6 is a Western blot result of the expression of hemagglutinin protein measured using a polyclonal antibody against hemagglutinin. Lane 1 is a wild-type Bordetella pertussis (no band), lane 2 is a mutant Bordetella pertussis (37 kDa) expressing the hemagglutinin of the present invention, lane 3 is a positive control, It is a recombinant antigen of maggotinin (35 kDa).
FIG. 7 is a Western blot result obtained by using a polyclonal antibody against Fim3 to determine whether the structure of Fim3 is affected by Bordetella pertussis mutant. Lane 1 is a wild-type Bordetella pertussis (23 kDa), lane 2 is a mutant Bordetella pertussis (23 kDa) expressing the hemagglutinin of the present invention, lane 3 is a recombinant recombinant Fim3 expressed in Escherichia coli Antigen (35 kDa).
Figure 8 shows that the mutant Bordetella pertussis strain of the present invention was inoculated with the H1N1 virus inactivated with a positive control group of the immunoglobulin G (IgG) antibody of the group (middle) immunized with the formalin treatment and the vehicle mixed with the vehicle Figure 1 shows serum IgG concentration in one group (gray). As a negative control group, only PBS (black) inoculated group was used.
FIG. 9 shows the change in the threshold cycle (Ct) of the real-time PCR showing the protective effect when infected with influenza virus after immunization.
Fig. 10 quantitatively shows the relative amount of viruses calculated from the results of Fig.

Hereinafter, the present invention will be described in detail with reference to examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited by the following examples.

[Example 1] Production of attenuated Bordetella pertussis strain expressing hemagglutinin (HA) of influenza virus

Disclosed strains and plasmids

Escherichia coli JM109 (Invitrogen; Carlsbad, CA, USA)

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

Red Helper Plasmid pKD46 (Datsenko and Wanner, 2000): A vector that produces Red and Gam proteins using the pBAD promoter

Bordetella pertussis standard strain Tohama I ATCC # BAA-589)

Influenza virus A / PR / 8/34 (H1N1) (VR1469): American Type Culture Collection (ATCC, Rockville, MD, USA).

Overlapping PCR

1) Primers for PCR of Fim2 and hemaglutinin

For amplification of N-terminal of Fim2 (GenBank # BX640414), hemagglutinin of influenza virus (GenBank # EF467821) and Fim2 C-terminal, forward and reverse primers were prepared and used as shown in Table 1 below. Total RNA was extracted from the virus using TRIzol ^® reagent (Invitrogen, Carlsbad, Calif., USA) to generate a template for cloning of influenza hemagglutinin, and a SuperScript III One-step RT-PCR system (Invitrogen, Carlsbad, Calif. , USA) and the primer Uni-12-F for cDNA synthesis (Table 1), and used as a template for PCR cloning.

[Table 1]

For each primer set, PCR was preliminary denaturation at 94 ° C for 5 minutes, 30 cycles at 94 ° C for 30 seconds, -57 ° C for 30 seconds and 1 minute at 72 ° C, and final extension at 72 ° C for 5 minutes. Each PCR product was loaded on 1.2% agarose gel and electrophoresed (FIG. 1).

In the PCR products for each of the amplified genes, overlapping PCR product 1 was prepared using Fim2 N-terminus and hemagglutinin PCR product. PCR was carried out at 94 ° C for 30 seconds at -57 ° C for 30 seconds at -72 ° C for 1 minute, followed by Fim2-NF primer and HA-R primer, pre-denaturing at 94 ° C for 5 minutes, 30 cycles were performed at 57 DEG C for 30 seconds and at 72 DEG C for 1 minute.

The overlapping PCR product 1 and the Fim2 C-terminal PCR product were used to generate the overlapping PCR product 2 by performing the PCR under the same conditions as in the production of the overlapping PCR product 1.

The PCR product was cloned into the pGEM-T Easy cloning vector, and digested with restriction enzymes Bgl II and Kpn I as shown in FIG. 2, and the prepared DNA of 1.07 kb was used as a linear DNA insert for homologous recombination.

Insertion into Fim2 locus through homologous recombination

Bordetella pertussis strain Bordet-Gengou blood agar the strain colonies cultured, and at 37 ℃ to (BBL ™, 297876, BD, USA) Stainer-Scholte (SS) broth (broth) was inoculated to 5 mL OD ₆₀₀ value Was incubated at 37 ° C until it became 0.5, cooled on ice for 20 minutes, and then centrifuged at 4000 xg for 15 minutes to collect the pellet of the bacteria. The cells were concentrated with 100 μL of chilled 10% glycerol (Sigma, USA) to prepare an electroporation competent cell. The pKD46 plasmid was transformed with the Gene Pulser Xcell electric perforation system (Bio-Rad, Hercules, Calif.) To express the λ Red recombinase in the cells, and then the ampicillin (ampicillin; Sigma, USA) and Stainer-Scholte (SS) broth supplemented with L-arabinose (Sigma, USA) and incubated at 30 ° C until OD ₆₀₀ value was 0.5. Then, the cells were concentrated with 100 μL of 10% glycerol (Sigma, USA) that was cooled again to prepare electrocompetent cells for transforming the previously prepared linear DNA for homologous recombination.

1 μL of insert DNA was transformed into 50 μL of electropneumatic competent cells by electroporation and cultured in Bordet-Gengou blood agar to obtain a mutant strain "BPS-Fim2-HA" expressing hemagglutinin as a heterologous antigen Respectively.

[Example 2] Hemagglutinin substitution confirmation

PCR assays were performed to verify the survival of BPS-Fim2-HA in the selection medium after transformation in Example 1 above. As shown in FIG. 3, three types of primer sets were used. PCR verification # 1 was Fim2-NF and HA-R primer, PCR verification # 2 was HA-F and HA-R primer, PCR verification # 3 was HA-F and Fim2-CR PCR amplification products of 0.95 kb, 0.83 kb and 0.95 kb, respectively, were obtained using primers.

As a result, it was confirmed from the result of FIG. 4 that a PCR product of the size expected for each PCR was generated.

[Example 3] Expression of a protein containing hemagglutinin (HA) as a heterologous antigen protein

1 ml of BPS-Fim2-HA culture broth was centrifuged at 4000 xg for 15 minutes to prepare bacterial pellets. The bacterial pellet was resuspended in 2x sample buffer (0.125M Tris (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol , 0.2% bromo-phenol-blue; Sigma, USA), followed by electrophoresis on 10% SDS-PAGE. The resultant electrophoresis was transferred to a PVDF membrane (Westeran ^® , Whatman (TM), GE Healthcare, UK), and the recombinant Fim2 expressed in Escherichia coli and the recombinant hemaglutin expressed in Escherichia coli were inoculated into rabbit- Fim2 and rabbit anti-hemagglutinin polyclonal antibody as the primary antibody and goat anti-rabbit IgG HRP conjugated antibody (Pierce, USA) as the secondary antibody to confirm the expression of the hemagglutinin protein Respectively.

As a result, the expression of the hemagglutinin-containing protein was confirmed by confirming the formation of a band for the 37 kDa protein from Fig. 5 and Fig. 6 (lane 2, respectively).

Fim3  ( fimbriae  3) Confirmation of structural mutation induction

Rabbit-anti-Fim3 polyclonal antibody produced by inoculating recombinant Fim3 expressed in Escherichia coli in the same conditions as the above experiment was used as a primary antibody, and then a goat anti-rabbit IgG HRP conjugated antibody (Pierce, USA) As a secondary antibody, normal expression of Fim3 was examined in order to examine whether normal Fim3 expression was affected by changes in Fim2 locus. As a result, it was found that Fim3 expression was not affected as shown in FIG.

[Example 4] Production of vaccine for BPS-Fim2-HA bacteria

In order to test the immunological effect in an experimental animal mouse, a transformant vaccine was prepared using the mutant BPS-Fim2-HA prepared in the above example.

The strain prepared in the above example was cultured in a Stainer-Scholte (SS) broth, centrifuged at 4000 xg for 15 minutes, resuspended in PBS, formalin was added to a final concentration of 0.2% , And reacted for more than 48 hours to prepare killed-bacterial cells. Adjuvants for antigen recognition were mixed with Alum ^® (Imject Alum, Pierce, Rockford, IL, USA) at a ratio of 1: 1 to 1: 3. Total number of bacteria to be inoculated once was inoculated with cell number 1x10 ⁹ cells / mouse, which corresponds to the amount of recombinant H. mageul Ruti 50ug per mouse Nin can be inoculated.

[Example 5] Immunization test of mice using the prepared inoculum vaccine

5 days old ICR mice (DBL, Korea) were inoculated into the subcutaneously injected deadly bacillus. For this purpose, the experimental group was divided into three groups, and the influenza virus inoculated group prepared by inoculating the prepared inoculated vaccine group, the PBS inoculated group as the negative control group and the positive control group was constructed.

Antigen immunization was performed three times at intervals of 2 weeks. Blood was collected from the same vein in each mouse group and serum was isolated. Blood samples were collected three times at the interval of 7 days before and after the immunization. Diluted blood was diluted 1: 100 with distilled water and immunoglobulin G (IgG) was measured by enzyme immunoassay (ELISA).

As a result of comparison of IgG, antibody titers were induced on days 21 and 35 as compared with the negative control group (FIG. 8).

[Example 6] Verification of defense effect in mice using inoculum vaccine

To confirm the protective effect of the influenza virus infection after confirming the immune effect of Example 5, mice immunized with the immunoassay in Example 5 were administered sublethal dose (500 pfu / mouse, 40 LD ₅₀ , 10 ^4.1 EID ₅₀ ) Influenza virus A / PR / 8/34 (H1N1) was administered intranasally and infected. Five days after infection, the lung tissues were isolated from the mice, and total RNA was extracted using TRIzol ^® reagent (Invitrogen, Carlsbad, Calif., USA). SuperScript III One-step RT-PCR system (Invitrogen, Carlsbad, CA, USA) and the primer Uni-12-F (Table 2). Real-time quantitative PCR (Exicycler (TM) 96, Bioneer, Korea) was performed using the cDNA as a template and the primers shown in Table 2 were used. Since the RNA of a virus is interpreted as the equivalent amount of cDNA and the amount of cDNA will be proportional to the actual concentration of virus, the detection concentration in quantitative PCR can be interpreted as the amount of virus. For the detection of virus-derived RNA, cDNA, the hemagglutinin gene region, neuraminidase (NA) and M protein gene region were targets. Since the amount of total RNA is affected by the amount of tissue used in the sample, the amount of viral RNA detected by using the beta-actin gene RNA as a comparative group for indirect quantification was indirectly analyzed.

[Table 2] Quantitative real-time polymerase chain reaction primer design

As a result, as shown in FIG. 9, it was confirmed that the delta (Ct) value through the threshold cycle shows a difference of 10-13 cycles. One cycle difference in PCR is two times the concentration difference. Therefore, the difference in 10-13 cycles is interpreted as a difference in concentration of 2 ¹⁰ -2 ¹³ (1024 -8192 times). As shown in FIG. 10, the quantitative comparison of the amount of viral RNA with the amount of viral RNA revealed that the amount of viral RNA was significantly decreased as compared with that of the negative control, and a value similar to that of the positive control group of inactivated influenza virus Respectively.

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

In addition, from the results of immunity and defense effects confirmed by the inoculum vaccine containing the heterologous antigen protein expressed in the above-mentioned strain, the strain having the effect of preventing or treating influenza virus infection can be used as the heterologous antigen protein carrier And confirmed the present invention.

<110> University Industry Liaison Office of Chonnam National University <120> Bordetella pertussis strain for expression of viral neutralizing          antigen and immunogenic composition using the same <130> P14061130752 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 633 <212> DNA <213> Bordetella pertussis Fim2 <400> 1 ctaggggtag accacggaaa aacccacata agtggtgatg gcgctggctt cgacgtcgcc 60 gttctttttg acgtaggagg ccaggtagcg catcgtgacg gttctgctgg tcccaccggt 120 ctgcacttcg gggtcgaagc ctgccgcctg ctgggtcgcc tcgttcgcac ccatggtgat 180 cttggagtca ttgagattgg aaatccgtac ctgcacgccc tgggcctcgg ttgcggcggt 240 gatgttgctc agctgggttt gcggattggt ggcatagacc atcttgtatg cccgcaagtc 300 gccggtgctg tagtcagtgg tcgggcccgg ttcgaaatag gccttgacgc cgttgcccag 360 gctggacggg caatccttca gcttgatgat gaaaggggta cggccggcct ggtcgccgtt 420 ggccttcaat gcgttcttgg agattttggg tagctgtacg accttggtgt gattcgggcc 480 tgaggggtcc tcgatgacgc aggtggtgtc ggtgatggtg ccggtgatga cgatggtgcc 540 gtcgtcggcg tgcgccgcgg acgcaatggc cgccagagcg gcccgcaagc acaggcgcag 600 ggcgcgttgg aaagggattt gcatgggtaa cac 633 <210> 2 <211> 1698 <212> DNA <213> Influenzae hemagglutinin <400> 2 atgaaggcaa acctactggt cctgttatgt gcacttgcag ctgcagatgc agacacaata 60 tgtataggct accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat 120 gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa actatgtaga 180 ttaaaaggaa tagccccact acaattgggg aaatgtaaca tcgccggatg gctcttggga 240 aacccagaat gcgacccact gcttccagtg agatcatggt cctacattgt agaaacacca 300 aactctgaga atggaatatg ttatccagga gatttcatcg actatgagga gctgagggag 360 caattgagct cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg 420 cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa aagcagtttt 480 tacagaaatt tgctatggct gacggagaag gagggctcat acccaaagct gaaaaattct 540 tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg gtattcatca cccgcctaac 600 agtaaggaac aacagaatct ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660 aattataaca ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct 720 gggaggatga actattactg gaccttgcta aaacccggag acacaataat atttgaggca 780 aatggaaatc taatagcacc aatgtatgct ttcgcactga gtagaggctt tgggtccggc 840 atcatcacct caaacgcatc aatgcatgag tgtaacacga agtgtcaaac acccctggga 900 gctataaaca gcagtctccc ttaccagaat atacacccag tcacaatagg agagtgccca 960 aaatacgtca ggagtgccaa attgaggatg gttacaggac taaggaacaa tccgtccatt 1020 caatccagag gtctatttgg agccattgcc ggttttattg aagggggatg gactggaatg 1080 atagatggat ggtatggtta tcatcatcag aatgaacagg gatcaggcta tgcagcggat 1140 caaaaaagca cacaaaatgc cattaacggg attacaaaca aggtgaacac tgttatcgag 1200 aaaatgaaca ttcaattcac agctgtgggt aaagaattca acaaattaga aaaaaggatg 1260 gaaaatttaa ataaaaaagt tgatgatgga tttctggaca tttggacata taatgcagaa 1320 ttgttagttc tactggaaaa tgaaaggact ctggatttcc atgactcaaa tgtgaagaat 1380 ctgtatgaga aagtaaaaag ccaattaaag aataatgcca aagaaatcgg aaatggatgt 1440 tttgagttct accacaagtg tgacaatgaa tgcatggaaa gtgtaagaaa tgggacttat 1500 gattatccca aatattcaga agagtcaaag ttgaacaggg aaaaggtaga tggagtgaaa 1560 ttggaatcaa tggggatcta tcagattctg gcgatctact caactgtcgc cagttcactg 1620 gtgcttttgg tctccctggg ggcaatcagt ttctggatgt gttctaatgg atctttgcag 1680 tgcagaatat gcatctga 1698 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Fim2-N-F <400> 3 ggcagatcta tggtgttacc catgcaaatc cct 33 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Fim2-N-R <400> 4 tagcaaggtc cagtaatagt tggatccgat ggtgccggtg atgac 45 <210> 5 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> HA-F <400> 5 gtcatcaccg gcaccatcgg atccaactat tactggacct tgcta 45 <210> 6 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> HA-R <400> 6 gctggtccca ccggtctgaa gctttttcac tccatctacc ttttccct 48 <210> 7 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Fim2-C-F <400> 7 agggaaaagg tagatggagt gaaaaagctt cagaccggtg ggaccagc 48 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Fim2-C-R <400> 8 aatggtaccg gggtagacca cggaaaaacc cacata 36 <210> 9 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Uni-12-F <400> 9 agcaaaagca gg 12 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HA-real-878-F <400> 10 cgaagtgtca aacacccctg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HA-real-1170-R <400> 11 cccgttaatg gcattttgtg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-real-187-F <400> 12 tcaagatttg aatcggtcgc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NA-real-460-R <400> 13 ccttcccctt ttcgatcttg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> M-217-F <400> 14 ggactgcagc gtagacgctt 20 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> M-405-R <400> 15 catcctgttg tatatgaggc ccat 24

Claims (11)

A hemagglutinin having a nucleotide sequence of SEQ ID NO: 2 derived from influenza virus between 100 to 140 mer of N-terminal and 100 to 140 mer of C-terminal of Fim2 gene of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: Bordetella pertussis strain comprising a recombinant gene having a structure in which HA is inserted. delete delete An immunogenic composition for treating or preventing an infectious disease comprising the Bordetella pertussis strain of claim 1. 5. The method of claim 4,
Wherein the infectious disease is an influenza viral infection or a Bordetella pertussis infectious disease to the human body. A hemagglutinin having a nucleotide sequence of SEQ ID NO: 2 derived from influenza virus between 100 to 140 mer of N-terminal and 100 to 140 mer of C-terminal of Fim2 gene of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: (HA) to produce a homologous recombination insert; And
Transforming the insert into Bordetella pertussis;
( Bordetella pertussis ) strain. delete delete The method according to claim 6,
The insert is not limited but is a linearized DNA fragment cut into two restriction enzymes ( Bgl II + Kpn I) shown in FIG. A hemagglutinin having a nucleotide sequence of SEQ ID NO: 2 derived from influenza virus between 100 to 140 mer of N-terminal and 100 to 140 mer of C-terminal of Fim2 gene of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: (HA) to produce a homologous recombination insert;
Transforming the insert into Bordetella pertussis;
Culturing the transformed Bordetella pertussis to express the hemagglutinin;
Producing a dead mold vaccine using the transformed Bordetella pertussis strain; And
Determining whether or not the inactivated vaccine is immune to an animal other than a human;
&Lt; / RTI &gt; A hemagglutinin having a nucleotide sequence of SEQ ID NO: 2 derived from influenza virus between 100 to 140 mer of N-terminal and 100 to 140 mer of C-terminal of Fim2 gene of Bordetella pertussis having the nucleotide sequence of SEQ ID NO: (HA) to produce a homologous recombination insert;
Transforming the insert into Bordetella pertussis;
Culturing the transformed Bordetella pertussis to express the hemagglutinin;
Producing a dead mold vaccine using the transformed Bordetella pertussis strain;
Determining whether or not the inactivated vaccine is immune to an animal other than a human; And
Administering the influenza virus to the immunized animal and determining whether the virus is defensible;
The method comprising:

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