KR101908438B1 - A MANUFACTURING METHOD FOR SOLUBLE AND ACTIVE RECOMBINANT PROTEIN USING Aminoacyl tRNA synthetase N-terminal domain AS FUSION PARTNER AND A PRODUCT THEREOF - Google Patents

Abstract

The present invention relates to a method for efficiently producing a water-soluble recombinant protein in Escherichia coli using fusion protein expression technology, which is a protein folding induction technique, and more particularly to a method for efficiently producing an influenza antigen protein hemagglutinin (HA) HA-1 region that induces specificity and ARSNTD (Aminoacyl tRNA synthetase N-terminal domain), which is a fusion protein fusion protein-inducing fusion partner, are expressed to express the ARSNTD-HA fusion protein in a water-soluble state, The present invention relates to the development of a prophylactic vaccine that is developed as a preventive vaccine and also expresses L1 of human papilloma virus (HPV) as an ARSNTD-L1 fusion protein to form a water-soluble capsid and effectively protect against infection with HPV.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a water-soluble and active type recombinant protein using ARSNTD as a fusion partner,

The present invention relates to a method and a product for producing a water-soluble and active type recombinant protein using ARSNTD as a fusion partner.

In recombinant protein vaccines such as recombinant influenza vaccine and papilloma virus vaccine, a protein expression system using a fusion partner is required to make the vaccine soluble. In the case of existing fusion partners, the size of the fusion partner has a problem or has a problem in formation of a multimer. In particular, LysRS or LysN used in the existing patent [Korean Patent No. 10-0890579] may form a dimer to interfere with the formation of multimers. In the case of existing fusion partners, Escherichia coli is the origin of the vaccine. Therefore, And the like.

The high antigenic variability of seasonal influenza viruses and the frequent occurrence of pandemic species cause significant socioeconomic losses in human health, such as high medical burden and other infectious diseases such as Ebola and Mels [1, 42, 43, 44].

Hemagglutinin (HA) surface antigen of influenza A virus is an important component of innate immunity and plays an important role in cell adhesion such as receptor attachment or membrane fusion [5, 33]. Viral HA, a trimeric membrane penetration protein, consists of two units, HA1 and HA2, each with several disulfide bonds [14, 15]. The HA protein is known to be the major antigen in influenza vaccines leading to antibodies that protect against infection.

HA production using recombinant technology can solve many problems such as high production of HA in eggs, influenced by egg supply, and high cost of cell-based culture in traditional influenza virus antigen production [6 ]. Recombinant techniques enable the production of virus-like particle (VLP) vaccines in various cell culture systems [45]. In bacteria, recombinant antigens can be produced within a month, are less costly and are easier to scale up than traditional production methods [25]. However, these systems are faster and safer to produce recombinant HA in bacteria than traditional production systems, but there are some practical problems [9].

The challenge of assembling the HA protein and the difficulty in proper folding are the biggest obstacles to making vaccine antigens in E. coli [10]. In bacterial production, the expression level of recombinant HA is high but often fails to adequately fold trimers [27, 30, 36]. Previous studies using LysRS as a new fusion partner, as described in the existing patented technology (Patent Registration No. 10-0890579), inhibit the dimerization of LysRS from trimerization of HA. In addition, the antigenicity of LysRS was so high that antibody production to LysRS was more problematic than the desired target protein [11].

Recent studies suggest that the addition of the naturally trimerized bacteriophage T4 fibrin, known as the PODON, to the C-terminus of HA allows water-soluble expression of recombinant HA. It has also been demonstrated that recombinant HA bound to ferritin, an intracellular protein containing iron, expressed in mammalian cells, spontaneously assembles to produce nine trimeric viral spikes on the ferritin surface [8]. These studies show that other effective fusion tags are needed to help recombinant HA antigens fold properly to form trimers and to avoid production on unwanted antibodies produced against the fused protein [24, 27].

A recombinant protein vaccine using a virus-derived protein or a live vaccine using a virus having its pathogenicity weakened by adaptation to a low temperature has actually shown a good effect for the prevention of influenza virus. However, as described above, the recombinant protein vaccine has many difficulties in producing a functioning virus antigen. There is a problem that it takes much time and expense to use eukaryotic cells such as mammalian cells and there are problems that protein folding or neutralizing antibody can not be produced properly when prokaryotes such as Escherichia coli are used. Thus, in using recombinant viral antigens expressed in Escherichia coli as a vaccine, water-soluble expression of an influenza hemagglutinin glycoprotein membrane and formation of trimers important for the binding of HA monomer to cells and immunogenicity are required.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above problems, and it is an object of the present invention to provide a protein expression system using a fusion partner in order to render the recombinant protein soluble in water.

In order to accomplish the above object, the present invention provides a method for producing a protein using ARSNTD (Aminoacyl tRNA synthetase N-terminal domain) as a fusion partner.

In one embodiment of the present invention, the ARSNTD (Aminoacyl tRNA synthetase N-terminal domain) is preferably composed of the amino acid sequence set forth in SEQ ID NO: 1 or 2, but the mutation All variants which achieve the effect intended by the invention are also included in the scope of the present invention.

The present invention also provides a method for producing an influenza hemagglutinin (HA) antigen in the form of a water-soluble trimer using ARSNTD as a fusion partner.

In one embodiment of the present invention, the hemagglutinin (HA) antigen is preferably a HA1 site of hemagglutinin protein, and the hemagglutinin (HA) antigen is preferably H1 type, H3 type, H5 type , Or HA derived from influenza B virus, and the antigenic sequences are preferably composed of the amino acid sequences set forth in SEQ ID NOS: 3 to 6, respectively, but are not limited thereto.

In addition, the present invention provides an influenza preventive vaccine using an ARSNTD-HA trine fusion protein as a vaccine antigen.

In one embodiment of the present invention, it is preferred that the vaccine uses the N-terminal region of the ARSNTD as an ARSNTD fusion pair and the hemagglutinin HA1 region as an antigen, and the vaccine is H1 type, H3 type, H5 type , Or HA derived from influenza B virus is used as a vaccine antigen, but is not limited thereto.

The present invention also provides a method for producing a papilloma virus (HPV) protein in the form of a water soluble capsid using ARSNTD as a fusion partner.

In one embodiment of the present invention, the L1 region of the HPV protein is preferably used as an antigen, and the L1 region of the HPV protein is preferably HPV 16 L1 or HPV 18 L1 region, But are not limited to, those comprising the amino acid sequences shown in SEQ ID NOS: 7 to 8.

The present invention also provides a vaccine against papilloma virus (HPV) using ARSNTD-L1 capsid fusion protein as a vaccine antigen.

In one embodiment of the present invention, it is preferable that the vaccine uses the N-terminal region of the N-terminal domain of Aminoacyl tRNA synthetase as a fusion partner and the HPV L1 region as an antigen, and the vaccine uses HPV- But it is not limited thereto.

The present invention also provides a method for producing an ARSNTD vaccine comprising the steps of: a) producing a gene fused with ARSNTD, a gene of virus-like particle (VLP); And b) expressing the ARSNTD and the virus-like particle in a fusion state.

In one embodiment of the invention, the method further comprises the step of purifying the protein expressed in step b), wherein the method further comprises the step of polymerizing the purified protein over a monomer, Or more.

Hereinafter, the present invention will be described.

In the present invention, in order to improve the solubility of recombinant HA, we used the N-terminal domain of the RNA-interacting protein as a fusion partner of the viral antigen protein. This fusion resulted in a highly efficient production of influenza antigen protein hemagglutinin (trimer), which is water-soluble. This is a vaccine that provides excellent defense immunity to experimental animals. The efficacy was verified.

Hereinafter, the present invention will be described in detail.

In the present invention, the ARSNTD (aminoacyl tRNA synthetase N-terminal domain) of about 70 amino acids in size, the first of four domains of mammalian ARS, was used as a binding partner for the aqueous expression of viral proteins in bacteria. ARSNTD is absent in E. coli or yeast tRNA ligase and small in size, but has some of its functions to interact with RNA like LysRS. Because the interacting RNA acts like a chaperone, ARSNTD can improve the solubility of proteins that are prone to aggregation [12, 13]. The high solubility of ARSNTD can improve the solubility of the viral proteins bound in spite of its small size [28]. In addition, the small size of ARSNTD allows the production of proteins that must form multimers, and can reduce antibody-producing elements that cause unwanted immune responses. Therefore, ARSNTD is a convergence partner that surpasses existing limitations.

ARSNTD was cloned from an animal (rats or rabbits) inoculated with the recombinant antigen to avoid the generation of unwanted antibodies to the fusion partner more thoroughly. Ultimately, human-derived ARSNTD can be used as a fusion partner to develop a vaccine against the human body.

These gene recombinant proteins were expressed, produced and purified. And proved a preventive function that excellently protects the purified antigen from attack immunization after immunization against rats. In other words, whether the water-soluble recombinant protein used in this study can be used as an effective antigen through the immunogenicity test in rats was determined by measuring the changes in mortality and weight by vaccinating the mice with the virus, And that it is effective. This experiment proved that a neutralizing antibody which inactivates the virus was produced through the neutralizing antibody test. These results are expected to be used as a way to develop pandemic, pandemic, and highly pathogenic avian influenza vaccines in a more inexpensive and rapid manner.

The preventive efficacy against the PR8 H1N1 virus shows that the vaccine antigen expressed in Escherichia coli is due to proper folding in its natural form. Therefore, there is strong evidence that suitable vaccine antigens for pathogenic influenza viruses and functional antimicrobials that may be expected in future vaccine studies on future antigen variants, seasonal influenza, and other species such as H3, H5, H7, and B virus , [26, 39, 40, 41].

The present invention is also applicable to the production of Human Papiloma Virus (HPV) vaccine in Escherichia coli.

Cervical cancer is the second leading cause of death in the world, with about 493,000 new diagnoses and 274,000 deaths each year. For the most part, about 85% of cases occur in developing countries, and cancer occurs in relatively young women, which is a reason for short life [46]. The eight major types (HPV 16, 18, 45, 31, 33, 52, 58 and 35) account for 85% of all cervical cancers globally, with HPV 16 and 18 being approximately 70% [47].

The papillomaviruses (PVs) are composed of quite diverse groups and can infect most mammals and birds. In humans, HPV types are divided into phylogenetically distinct groups, and the most common HPV types are alpha-papillomaviruses [48]. The genus includes HPV types 16 and 18, which can cause malignant neoplasms and cancer in the mucosa [49].

Human papillomavirus is a small, virus-free virus with two strands of DNA. It has an approximately 8 kb circular genome encoding six control proteins, E1, E2, E4, E5, E6 and E7, and capsid proteins, L1 and L2 [50]. The eight open reading frames (ORFs) are also divided into nonstructural and structural viral genes. The early (E) E1 and E2 proteins maintain viral DNA as an episome in the basal layer, and E6 and E7 are expressed at an early stage to stimulate cell cycle proliferation. Due to the regulation of cell cycle function, E6 and E7 are carcinogenic proteins due to the expressed high-risk HPV types. Although E4 and E5 are associated with improved proliferation, E5 is not necessarily involved in the progression of carcinogenesis by HPV. This is because the E5 coding sequence is lost during integration into the host cell DNA. L1 and L2 proteins in the late stage (L) are expressed in the upper layers of infected tissues. L1 is the major capsid protein, and the minor L2 protein will probably improve packaging and infectivity [51-54].

Human papillomaviruses consist of 72 pentameric capsomeres, which are the major capsid proteins L1 and are arranged in a regular dodecahedral lattice. The minor capsid protein, L2, is also present on the virus surface, but accounts for only about one-half of the L1 protein [55,56]. Virus particles are stabilized by intermolecular disulfide bonds. There are several cysteines in the L1 protein of HPV, but only two cysteines (175 and 428 amino acids in type 16) are involved in binding, and this cysteine is conserved among the other types. The reason why this cysteine is important for making human papillomavirus particles is that only cysteine 175 and 428 are exposed [57,58].

Virus-like particles (VLPs) are inherently self-assembled when only the L1 protein, except the others, or the L1 / L2 protein is expressed in various cell types. VLPs are very similar to infectious HPV viruses that occur naturally and structurally and immunologically, and can produce neutralizing antibodies after inoculation [59,60]. So researchers have focused on the L1 protein to develop the HPV vaccine. In general, reconstituted VLPs have been expressed in various cell types such as BSC-1, insect cells, yeast, and bacteria [61-64]. Currently, vaccines licensed for prevention include IgA and Saga vaccines. Cervarix ^TM (GlaxoSmith-Kline) is a divalent cervical cancer vaccine containing the L1 antigen of HPV types 16 and 18. Two types of HPV L1 protein were produced in insect cells and used as antigens. Gadasil (Merck and Co.) is a sagavirus vaccine against both HPV types 16 and 18, as well as HPV types 6 and 11. The vaccine also used L1 protein as an antigen, and the protein was produced in yeast (Saccharomyces cerevisiae). These vaccines were approved by the FDA in 2009 and 2006, respectively [65-67]. However, these vaccines have the disadvantage that they are relatively expensive due to low expression levels and high production costs. This problem is a major problem when it comes to developing countries that need the largest amount of HPV vaccine [68].

The present invention describes an efficient method for producing HPV types 16 and 18 in Escherichia coli. The recombinant HPV 16L1 and 18L1 were used as the previously described ARSNTD fusion partner to enhance their solubility. Through this, it was verified that VLPs of human papillomavirus were efficiently produced by E. coli in a water-soluble form, and that they have similar immunogenicity to VLP vaccine derived from insect cell which has been commercialized through animal experiments.

Specifically, ARSNTD (hARSNTD) was used as a fusion partner to prevent the generation of unwanted antibodies due to the small size of ARSNTD and ultimately to develop a vaccine against human body.

Using the bacterial system, these recombinant proteins produced high-risk HPV types 16 and 18 capsomeres and VLPs. The sequences of HPV 16L1 and 18L1 used were codon optimized to increase the expression level in E. coli and hARSNTD was fused to the N terminus of L1 to enhance the water solubility of L1 protein. With these trials, we obtained effectively soluble HPV 16L1 and 18L1 fusion proteins in E. coli, respectively.

Escherichia coli-derived purified antigens were analyzed by electron microscopy. The fusion protein was found to be homologous to 30-40 nm of caposomes, and 100 nm homologous VLPs were detected in the case of the TEV protease-cleaved protein, which is much larger than the size of the common HPVP VLPs of 55 nm. Since the size of E. coli-derived VLPs is cross-linked to a low level in the B cell receptors and antibodies against small size caposomes are induced weaker than antibodies to VLPs, our VLPs are higher than other VLPs Immunogenicity can be induced [75]. Similar results with Cervarix ^TM immunogenicity were obtained when mice were injected with VLPs from E. coli with various immunoadjuvants.

In conclusion, the present invention provides a method for replacing the vaccine production method which is being commercialized at a high cost. Currently, two commercialized vaccines, Gadasil and Cervarix ^TM, are not suitable for use in developing countries where these vaccines are most needed due to their high cost. These two vaccines are the most expensive vaccines ever produced [22]. Therefore, reducing production costs of preventive vaccines is an important step for developing countries. We produced HPV 16 and 18 VLPs and cap somae in E. coli. The E. coli-derived VLPs we have produced have been developed in a more inexpensive and effective alternative to commercially available vaccines and have shown potential to be used as vaccines for cervical cancer.

The present invention provides a method of producing milk-based vaccine and a method of replacing the cell-based vaccine production method, which takes 3-6 months in the previous production, and the existing long-term production method is a rapid pandemic There was a limit to using it as a preventive technique. This alias provides a stable pipeline for the production of influenza-preventive vaccines within a short period of time and can be applied not only to prevention of pandemics but also to annual seasonal influenza vaccines. HPV 16 and 18 VLPs, Respectively. The E. coli-derived VLPs produced by the present invention showed a potential to be used as a vaccine for cervical cancer by being developed in a more inexpensive and effective manner instead of a commercially available vaccine.

Figure 1 is a schematic illustration of the pGE-ARSNTD vector used for the expression of fusion proteins in E. coli (A) ARSNTD and HAgd sequencing. (B) Comparison between each ARSNTD sequence derived from human, mouse and rabbit, E. coli has no ARSNTD sequence. (C) The spherical head domain consists of HA 63-286 amino acids at subunit HA1. (D) TEV protease can be used to obtain pure ARSNTD without six histidine tags, and the recombinant protein ARSNTD-HAgd can be divided into fusion tag MLNS and target protein HAgd. (E) Schematic illustration of the spherical head domain of trimer hemagglutinin.
FIG. 2 shows a comparison of soluble levels of ARSNTD in Escherichia coli (CE) of human, mouse and rabbit in E. coli (A) comparison of solubility of Escherichia coli in LysRS of mice and rabbits (B) Soluble expression of each ARSNTD-HAgd (F) soluble expression of each ARSNTD-eGFP.
Figure 3 is a purified (AB) purified ARSNTD-HAgd protein fraction of ARSNTD-fusion protein using nickel chromatography. (CD) purified ARSNTD fusion eGFP protein fraction.
FIG. 4 shows the determination of the subunit status of fusion antigens using gel filtration chromatography. (A) Molecular weight description determined using the partition coefficient (Kav) of mARSNTD-HAgd and rARSNTD-HAgd. (BC) Analysis of the ARSNTD-fused HAgd fraction by SDS PAGE (D) Molecular weight analysis determined using the partition coefficient (Kav) of the control mARSNTD-eGFP. (E) Fraction of each ARSNTD-fusion eGPF analyzed by SDS-PAGE. (F) A schematic illustration of the oligomeric state of ARSNTD-HAgd and ARSNTD-eGFP.
Figure 5 shows that ARSNTD-fused HAgd inoculation protects rats against the PR8 virus challenge. (A) Weight change of virus challenge mice inoculated with ARSNTD fusion HAgd antigen and control ARSNTD fusion eGFP. (B) Survival rate of inoculated virus-challenged rats. (C) Weight change in rats infected with virus without vaccination. (D) Prevention The survival rate of mice not infected with virus and infected.
Figure 6 shows that the anti-sera of rats against virus neutralization experiment A, C. mARSNTD-HAgd and rARSNTD-HAgd contain detectable HI and neutralizing antibodies. B, D. The rabbit antiserum to rARSNTD-HAgd contains more HI and neutralizing antibodies than the rat antiserum.
Figure 7 shows the aqueous expression and purification of the A / PR / 8 HAgd protein fused to E. coli LysRS. (A) Insert the HAgd gene into the E. coli vector pGE-LysRS (4) vector. (B) Soluble expression of LysRS-HAgd fusion protein in E. coli. Separation and purification of fusion proteins expressed in water soluble protein form.
(A) LysRS-HAgd protein vaccination was carried out twice weekly through the abdominal cavity of mice of three groups (Alum mixture of the target protein and the target protein, PBS alone) in the mouse model LysRS-HAgd fusion protein vaccination. . (B) boosting Two sera from mouse serum before inoculation and aggression inoculation and confirmation of viability using two weeks of influenza virus PR8.
Figure 9 shows LysRS-HAgd fusion protein specific antibody titer and virus neutralizing antibody (A) ELISA test to determine whether antibodies present in the mouse serum were inoculated with PBS, antibodies to LysRS and HAgd Experiment result. Data comparing the OD values obtained from plates coated with LysRS protein alone and plates coated with LysRS-HAgd protein. (B) Conducting hemagglutinin inhibition (HI) assay to test the virus neutralizing ability of vaccinated sera.
Figure 10 shows the protective ability of the LysRS-HAgd vaccine against the inoculation of the vaccine. When mice inoculated with three doses of the wild-type A / PR / 8 virus were vaccinated with three groups of mice, all mice including the LysRS-HAgd vaccinated group showed a rapid weight loss All died within 7 days of infection.
Figure 11: Expression and purification of HPV 16L1 and 18L1 fusion proteins. (A) Illustration of HPV 16L1 / 18L1 fusion protein (hARSNTD 16L1 / 18L1). (B) SDS-PAGE analysis of hARSNTD-16L1 and hARSNTD-18L1 expression in E. coli. Cultivation was carried out at 37 ° C and 30 ° C, and at 20 ° C with and without addition of IPTG. SM; size marker, T; total, S; soluble, P; pellet (C) Purification of hARSNTD-16L1 using nickel affinity chromatography. CE; crude extract, FT; flow through (unbound), W; washing. Number is the number of fractions eluted. (D) SDS-PAGE analysis of purified hARSNTD-16L1.
12 shows the cleavage of hARSNTD-16L1 using TEV protease. (A) Cleavage of the purified hARSNTD-16L1 protein. hARSNTD-16L1 and TEV protease were the control group. Protein was loaded at 2 ug and TEV protease was loaded at 2 U. (B) Identification of fusion proteins and fusion partners using western blot. The first antibody (1 ^st antibody) was anti-his (1: 1000) and the second antibody (2 ^nd antibody) was anti-mouse (1: 10000). (C) Water-solubility test after cutting. Each sample obtained by time was analyzed for supernatant after centrifugation. Samples are loaded at 1 ug each.
FIG. 13 is a structural characteristic analysis of a purified fusion protein and a protein cleaved by TEV protease. (A) The structure of the fusion protein diluted with PBS is confirmed by TEM. Photo enlarged 100,000 times. (B) TEM confirmation of lysate structure of proteins cleaved by TEV protease. The photo is enlarged to 50,000 times.
14 shows the immunoreactivity of Escherichia coli-derived VLPs with various immunoadjuvants. (A) Summary of groups for immunogenicity testing. (B) Vaccination of E. coli-derived VLP with various immunoadjuvants every 2 weeks. The serum of rats was used for the analysis of antibody production of VLP from Escherichia coli using ELISA. (C) Antibodies specific for E. coli -derived HPV 16L1 in serum were confirmed by ELISA coated with yeast-derived HPV 16L1. (D) Antibodies specific for ARSNTD in serum were analyzed by ELISA coated with purified ARSNTD protein. (E) Antibodies of B and C. (F) Use the antibody in the serum of group 1 for Western blotting. * For Cervarix ^TM , + for TEV protease. And not ARSNTD or TEV protease.
Fig. 15 confirms IFN-production specific to E. coli-derived HPV 16L1 VLP. (A) Stimulation of yeast-derived VLPs by isolating rat spleen cells inoculated with each group. Through ELISPOT, it was confirmed that cytokine was secreted from the cell culture medium.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

Example  1: gene Cloning

 The pGE-ARSNTD vector was used as an expression vector and the above vector was designed as the original pGE-LysRS (4) (12). First, the LysRS gene in pGE-LysRS (4) was removed using restriction enzymes NdeI and KpnI. Rats and rabbit ARSNTD were amplified by polymerase chain reaction (PCR) and then mARSNTD and rARSNTD were inserted into pGE-LysRS (4). The pGE-ARSNTD vector containing ARSNTD contains D6, ie, six aspartic acids linked to T7promoter (21), SG space linker, TEV proteinase protease recognition site (ENLYFQ /) and restriction (GTGSDIVDKL: KpnI / BamHI / EcoRV / SalI / HindIII) and six histidine tags (Fig. 1D). TEV protease can be used to obtain pure ARSNTD without 6 histidine tags, and the recombinant protein ARSNTD-HAgd is designed to separate into fusion tag ARSNTD and target protein HAgd. HAgd cDNA of A / Puerto Rico / 8/34 (H1N1) was amplified by RT-PCR and inserted between cleavage sites of KpnI and SalI of pGE-ARSNTD vector (Fig. 1A) . CDNAs of A / Fujian / 411/2002 (H3N2), A / Indonesia / 5/2005 (H5N1) and strain B / Victoria / 2/1987 (B) were obtained in the same manner and then KpnI of pGE-ARSNTD vector Lt; RTI ID = 0.0 > SalI. ≪ / RTI > Comparisons between ARSNTD sequences derived from humans, rats and rabbits were made, and E. coli has no ARSNTD sequence (Fig. 1B). The globular domain used in this experiment consisted of the HA 63-286 amino acid (in the case of H1N1) in the HA1 subunit, which corresponds to the part of the globular head domain of the naturally occurring trimeric hemagglutinin (Fig. 1E) present in the virus . H3N2, H5N1, and strainB were also designed for spherical hair domains corresponding to amino acids 81-288, 59-288, and 77-287 of HA, respectively.

Example  2: Protein expression and purification

All recombinant proteins were overexpressed in E. coli host BL21 (DE3) pLysS and purified from soluble cell lysate fractions. The initial culture was incubated at 37 ° C in 3 ml LB containing 50 μg / ml of ampicillin and 30 μg / ml of chloramphenicol for one day. Large-scale cultures were performed at 37 ° C. until 500 μl of OD _{600 nm} reached 0.5-0.7 in 500 ml LB inoculated with 2.5 ml in the initial culture.

Cells were harvested at 3000 g. Cell lysates obtained by sonication were centrifuged at 12,000 g for 10 min and divided into aqueous and pellet fractions.

Protein expression was induced by addition of 1 mM IPTG at various temperatures (Figure 2). The soluble expression level of the ARSNTD-fusion protein and the LysN-fusion protein was tested. The solubility of all fusion proteins was slightly increased. There was no difference in the amount of soluble ARSNTD-fusion protein and LysN-fusion protein. An ARSNTD smaller than LysN was selected as the fusion partner. This is because the production of antibody to small size fusion partner is small. The ARSNTD fusion HAgd in the ARSNTD fusion protein was expressed 40% more water-soluble at 20 ° C (Fig. 2, D-E). mARSNTD-HAgd and rARSNTD-HAgd were selected as immunogens to be inoculated into rats and rabbits. ARSNTD-fusion eGFP was used as a control protein and its solubility was increased by 50% at 25 DEG C (Fig. 2F). As a result, the expression level of mouse-derived fusion partners (mARSNTD, mLysN, mLysRS) was higher than that of rabbit-derived fusion partners. Based on these results, the results of the application of mARSNTD fusion protein to several kinds of HA were as shown in the table below.

HA strain Expression rate (%) Water solubility (%) H1N1 20 50 H3N2 8 40 H5N1 10 30 B (victoria) 10 30

Table 1 shows the results of mARSNTD fusion experiments for various HA species.

 Proteins were purified by nickel affinity chromatography. Each water soluble fraction was equilibrated with A buffer [50 mM TrisHCl (pH 7.5), 300 mM sodium chloride, 10% glycerol, 2 mM 2-mercaptoethanol, Triton X-100 0.05%, and 10 mM imidazole] (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). After washing with buffer A, linear gradient imidazole protein is eluted in the range of 10-300 mM using B buffer. After SDS-PAGE, the fractions containing the protein of interest are pooled and dialyzed against C buffer [50 mM TrisHCl (pH 8.0), 100 mM NaCl, 0.1 mM EDTA, and 0.1% Tween 20]. The concentration of purified protein is determined using BSA (Amresco, Solon, OH, USA) with known concentration as a control.

Each ARSNTD fusion protein cultured at 500 ml was separated using single step nickel chromatography. Following cell lysis and fractionation, a water soluble fraction with most of the ARSNTD fusion protein was loaded onto the nickel column and then imidazoline linear changes in the range of 10-300 mM. The fusion proteins with the ARSNTD 6-histidine tag were effectively purified from the water-soluble fraction. In SDS-PAGE analysis of the purified protein, most of the proteins migrated to the expected molecular weight location and were successful in proper folding. The molecular weight of the ARSNTD fusion protein ranged from 36 kDa to 40 kDa (FIG. 3). The purified protein was collected and dialyzed for buffer exchange. Each purified fusion protein was quantitated by comparison with concentrations of known BSA. The final concentration of the fused protein was about 1 mg / ml.

Example  3: by gel filtration chromatography Trimer  analysis

  The oligomeric state of the purified recombinant protein is analyzed by gel filtration chromatography using a Superdex-200 analytical gel-filtration column at 4 ° C. T column is composed of a wide range of molecular weight markers, ferritin (440 kDa), aldolase (158), isoquinolones with buffer [50 mM Tris HCl (pH 7.5), 300 mM NaCl, 2 mM 2-mercaptoethanol Triton X- kDa), conalbumin (75 kDa), ovalbumin (44 kDa) and bulldextran 2000 (GE HealthCare).

mARSNTD-HAgd was eluted at 17.67 ml and the molecular weight was determined to be 140.54 kDa using the partition coefficient. 17.60 ml of rARSNTD-HAgd was eluted and its molecular weight was 143.87 kDa. Each molecular weight of the ARSNTD-fused HAgd showed that their biochemical state was mostly trimer (Fig. 4A). From the above results, it can be seen that the structure of newly formed HAgd originally has the structure of viral protein. The molecular weight of ARSNTD-HAgd was greater than 120 kDa (molecular weight of the expected trimer) because ARSNTD-HAgd forms a triangular shape. In addition, the flexible structure of ARSNTD can slightly shift the size of the fusion protein. Data from ARSNTD and ARSNTD fusion proteins analyzed by SDS-PAGE showed very similar results. The control protein mARSNTD-eGFP was subjected to gel filtration chromatography to elute 12.80 ml and 22.50 ml (Fig. 4B). The latter molecular weight was 34.2 kDa. The biochemical state of mARSNTD-eGFP was a mixture of oligomer (30%) and monomer (70%) (Fig. 4D-E). These results show that the fusion partner ARSNTD helps HAgd build the native trimer and help eGFP form monomers. The SDS-PAGE analysis of the separated proteins revealed that most of the proteins were purified corresponding to the expected molecular weights (FIGS. 4B, 4C and 4F).

Example  4: Immunization and attack inoculation

 The rabbit experiment was conducted at an animal facility located at Youngin Frontier (Gasan-dong, Korea). The rARSNTD fusion protein, which was emulsified with a complete Freund's adjuvant, was injected intradermally into various sites such as rabbits. Four weeks after the first inoculation, the protein and adjuvant mixture was boosted two additional times every two weeks. The rabbit serum was collected after one week from each booster and centrifuged and stored at 70 ° C. The above experimental plan was evaluated and approved by the Young Expert Frontier's Committee on Animal Experimentation (IACUC).

Rat experiments were performed on 6-week-old female BALB / c (12 mice, Orient-Bio). 4 μg of purified ARSNTD fused recombinant protein and 50 μg of a vaccine supplement (Thermo SCIENTIFIC) were added to each group of rats (3 per group) on day 0 (initial), 14 (first boost), 28 ) Were inoculated into the abdominal cavity. PBS-inoculated mice were used as controls. Serum samples were obtained via bleeding one week after each booster and some were stored at 70 ° C. The above experimental plan was evaluated and approved by YLARC (Permit number: IACUC-A-201503-202-02). After 21 days (after the second dose), all rats were challenged by inoculating intranasally with 10 ³ PFU of A / Puerto Rico / 8/34 (H1N1) in anesthetized with Avertin. Survival and weight changes of challenged rats were measured daily for two weeks. Rat experiments were conducted strictly according to the recommendations of the Korea Food and Drug Administration (KFDA) guidelines. The above experimental plan was evaluated and approved by YLARC (YLARC).

As a control for vaccination, all three rats infected with 10 ³ PFU of PR834 virus died and ^two rats infected with 10 ² PFU of PR8 virus showed up to 23% weight loss and recovered slowly (Figure 5C-D ).

 In the actual vaccination experiment, 21 days after the second inoculation day, all rats were challenged by intranasal inoculation of A / Puerto Rico / 8/34 (H1N1) virus of lethal dose (1LD50) 10 ^ 3 PFU in anesthetized with evertin . Survival and weight changes of challenged rats were measured daily for 14 days. The ARSNTD fusion HAgd (mARSNTD + HAgd: Marisu = 3, rARSNTD + HAgd: Marisu = 3) antigen was inoculated into 6 out of 12 animals. Although a few days later showed a 10% weight loss, all six immunized mice recovered and survived. ARSNTD fusion eGFP (mARSNTD + eGFP: 3 mice, rARSNTD + eGFP: 3 mice) were inoculated into 6 out of 12 mice and used as a control group. Six control rats increased weight loss and died one week later (Fig. 5A-B). From the above results, it was verified that the recombinant antigen protein developed in the present invention exhibits excellent vaccine efficacy.

Example  5: Virus neutralization experiment

    To test whether the ARSNTD-HAgd fusion protein can be used as a vaccine antigen and induce protective antibodies, microin- neutralization tests were performed with antiserum from rats and rabbits.

Blood samples from rats were collected via iblinding and allowed to condense at room temperature. The coagulants were removed and the samples were centrifuged at 5500 rpm for 10 min at 4 ° C in a microcentrifuge. The purified serum was transferred to a sterile microcentrifuge container and heat inactivated at 56 ° C for 30 minutes. Heat-inactivated serum was diluted with MEM and diluted in 96-well plates in 1/2 serial dilution. To analyze the activity of neutralizing antibodies against influenza virus, the same amount (50 μl) of influenza virus (100 PFU) was added to diluted serum and mixed. 96-well plates containing virus and serum were incubated at 37 ° C for 2 hours. The above mixture was absorbed into a 24-well plate containing fused MDCK cells. Plates covered with agar were incubated at 32 ° C in a 5% CO ₂ incubator. A single layer of cells was stained with crystal violet for 4 days after infection. In each analysis, sera were diluted and analyzed in duplicate, and each analysis was performed at least twice. The neutralization area was calculated as a diluted volume corresponding to the reduced amount of 50% plaque compared to the control.

HI for homologous PR8 virus or antiserum against ARSNTD-eGFP protein without neutralizing antibody was used as negative control (Fig. 6A-D). On the other hand, detectable levels of HI and neutralizing antibodies were found in antisera against mARSNTD-HAgd and rARSNTD-HAgd in rats (Figs. 6A and C). In addition, more HI and neutralizing antibodies were produced in rabbit antiserum against rARSNTD-HAgd than in rat antisera (Fig. 6B and D). The above results show that the vaccine efficacy verified by the animal experiment in FIG. 5 is in agreement with the neutralization function which actually inactivates the virus.

Example  6: Escherichia coli LysRS  Through fusion Flu virus  A / PR / 8 HAgd  Protein expression and purification

The HA-GD gene was inserted into the E. coli vector pGE-LysRS (4) vector (Fig. 7A). As a result of expressing LysRS-HAgd fusion protein in E. coli, most of them were expressed as a water-soluble protein form as shown in Fig. 7B. The expressed fusion protein was isolated and purified to obtain high purity protein. The concentration of the produced fusion protein was about 2.31 mg / ml.

Example  7: Mouse model LysRS - HAgd Fusion protein  vaccination

Balb / c mice were divided into three groups as shown in Fig. 8A, and LysRS-GD protein was administered twice per week through the abdominal cavity. Alum was used as an immunity enhancer and the PBS inoculation group was used as a negative control. To determine the HA GD-specific antibody titers in the serum, two blood samples of the mouse were obtained before boosting and before inoculation, and serum was isolated (Fig. 8B).

Example  8: LysRS - HAgd Of the fusion protein Specific antibody titer  And virus Neutralizing antibody  Measure

Specific antibody titers to HA GD in mouse serum isolated after Boosting inoculation were measured. In order to perform the ELISA, the LysRS protein and the LysRS-HA GD protein were coated on different plates, and then the mouse serum dilution was incubated and the OD value was measured (Fig. 9A) , Whereas the LysRS-HA GD fusion protein showed an OD value of more than 1 in inoculated sera. However, it is expected that most of the antibodies present in the serum are specific antibodies to the LysRS protein, considering that the plate coated with the LysRS protein shows a similar OD value. The hemagglutinin inhibition (HI) assay was performed to test the virus neutralizing ability of the vaccinated sera, showing no virus neutralizing ability despite the vaccination (FIG. 9B). These results show that most of the antibodies are produced to the fusion protein in the mouse even when the fusion protein derived from Escherichia coli is a highly soluble protein. This can be a serious problem in the production of a specific antibody to a target protein and its efficacy as a vaccine.

Example  9: LysRS -H GD Fusion protein Specific antibody titer  And virus Neutralizing antibody  Measure

When each vaccinated group mouse was inoculated with a lethal dose of wild-type A / PR / 8 virus, all mice, including the LysRS-HA GD inoculated group, showed rapid weight loss and died within 7 days of infection (FIG. 10). This means that the neutralizing antibody of Example 8, as well as the experimental results, did not provide any LysRS-HA GD inoculation against the wild-type virus at all.

Example  10: In E. coli Fusion protein  Expression and purification

For the water-soluble expression of HPV 16L1 and 18L1 proteins in E. coli cytoplasm, HPV 116L1 and 18L1 were fused at the C-terminus of hARSNTD to form hARSNTD-L1. In addition, a site recognized by the protein cleavage enzyme TEV protease and six histidine tags were introduced into the hARSNTD and HPV in the form of a linking peptide. The site recognized by TEV protease is ENLYFQG, which has seven amino acids. Since the glycine is left in the target protein after being cleaved by TEV protease, we designed the recognition site of TEV protease with six amino acids except glycine (Fig. 11A) . These linked peptides were introduced for purification of HPV L1 from hARSNTD by single purification using nickel affinity chromatography and subsequent cleavage of the proteolytic enzyme TEV protease.

hARSNTD-16L1 and hARSNTD-18L1 were overexpressed by 1 mM IPTG in E. coli host BL21 (DE3) pLysS. A small amount of culture to confirm the degree of water solubility of the protein was incubated in 3 ml LB containing 50 ug / ml of ampicillin and 30 ug / ml of chloramphenicol for one day at 37 ° C, 0.5 ml of cultured cells were transferred to 20 ml LB and cultured at 37 ° C until OD reached 0.5 at 600 nm. Protein expression was induced by addition of 1 mM IPTG at various temperatures. After adding IPTG, proteins were expressed at 37 ° C for 3 hours, at 30 ° C for 4 hours and at 20 ° C for 5 hours, and the cultured cells were harvested at 3000 rpm. Cell lysates obtained by sonication were centrifuged at 12,000 rpm for 10 minutes and divided into water soluble and insoluble fractions. This fusion protein was expressed insoluble at 37, while having a water solubility of greater than 90% at 20 < 0 > C. The degree of water solubility of the expressed hARSNTD-16L1 and hARSNTD-18L1 proteins was analyzed by SDS-PAGE (FIG. 11B). On the first day, large-scale cultures were incubated in 3 ml LB containing 50 μg / ml of ampicillin and 30 μg / ml of chloramphenicol for 8 hours at 37 ° C. and then cultured in 50 ml LB containing the same concentrations of ampicillin and chloramphenicol And then cultured overnight. The next day, 20 ml of 50 ml of cells cultured the previous day in 500 ml LB containing the same concentration of antibiotics were transferred and cultured at 37 ° C until the OD reached 0.5-0.7 at 600 nm. Protein expression was induced by adding 1 mM IPTG and incubating at 20 ° C for 5 hours.

Proteins were purified by nickel affinity chromatography. To this end, the A buffer used for chromatography was sonicated, and the cell lysate obtained by centrifugation was bound to Ni-NTA column (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) pre-equilibrated with buffer A. The composition of the buffer A used for the purification was 50 mM TrisHCl (pH 8.2), 300 mM sodium chloride, 10% glycerol, 2 mM beta-mercaptoethanol, 0.1% Tween-20 and 10 mM imidazole. A buffer, the protein was eluted at a gradually imidazole concentration (10-300 mM) using B buffer containing 300 mM imidazole. Fractions containing the protein of interest were pooled in a buffer (50 mM Tris HCl, pH 8.2, 500 mM NaCl, 5 mM EDTA, and 0.01% Tween-80), analyzed by SDS-PAGE , And concentrated (Fig. 11D). The molecular weight of the fusion protein is 66.2 kDa. The concentration of purified protein was determined using BSA (Amresco, Solon, OH, USA) with known concentration as a control. The final concentration of fusion protein was about 0.8 mg / ml, and the overall separation purification efficiency was about 1.5 mg / 0.5 L.

Example  11: TEV  protease Of the fusion protein  cut

The fusion protein hARSNTD-16L1 is cleaved with AcTEV protease (Invitrogen) in TEV buffer [50 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 1 mM DTT]. In order to optimize the cutting conditions, several reaction temperatures and conditions were tested. As a result, it was confirmed that when the fusion protein was reacted at 25 ° C for 5 hours, it was gradually cleaved into hARSNTD-6xhis-tev site (Gx) and HPV 16L1 by TEV protease (FIG. 12A). We also confirmed the fusion protein and the cleaved fusion partner by western blot using a histidine tag antibody (Fig. 12B). The truncated fusion partner hARSNTD can be detected because of the histidine tag at the C-terminus of hARSNTD. The AcTEV protease used in this study was also detected because it has a histidine tag.

To investigate the water solubility of HPV 16L1 cut after TEV protease cleavage, protease-treated whole lysates were separated by centrifugation into soluble and insoluble fractions and confirmed by SDS-PAGE (FIG. 12C). Most fusion proteins, cleaved fusion partners, and cleaved HPV 16L1 retained their water-solubility, which allows the fusion protein to function properly at a recognition site where a particular protease can recognize it, Indicating that the target protein can be derived.

Example  12: Structural characterization of purified proteins

Transmission electron microscopy was used to analyze the structure of the purified fusion protein and the cleaved protein. To this end, the lysates treated with fusion protein and TEV protease were diluted to 50 ng / ul with PBS [2.67 mM KCl, 1.47 mM KH ₂ PO ₄ , 137.93 mM NaCl, 8.1 mM Na ₂ HPO ₄ ] 3 ul of the sample was placed on a carbon-coated grid and stained with negative staining. As a result, the fusion proteins formed capsaicin of 30-40 nm size, and even cleaved HPV 16L1 formed virus-like particles (VLPs) of 100 nm or more larger than 50 nm, the size of the original HPV virus (Figures 13A and 13B). This indicates that E. coli-derived proteins can self-assemble into self-assembled VLPs and that fusion proteins can also form homogenous capsomeres.

Example  13: Various immune supplements ( adjuvants ) ≪ / RTI >

To confirm the immunogenicity of the cleaved proteins, we injected three Balb / c mice (three per group) at various intervals of two weeks with various adjuvants. The immunoadhesin used was L-pampo (CHA vaccine institute), CIA06 (EYEGENE Inc.) and alum. The control group consisted of yeast-derived HPV 16L1 antigen in EYEGENE, Cervarix ^TM , a commonly used yeast-derived HPV VLP, and hARSNTD, Respectively. The reason for using the fusion partner as a control is that the protein used in the immunogenicity test contains a cleaved fusion partner. Rats used as negative controls were injected with PBS only. Eight groups for immunogenicity testing are summarized in Figure 14A. All the antigens were injected into the muscle of the rats at the same concentration of 4 ug.

One week after the third inoculation, serum obtained from eye-bleeding was used to identify specific IgG antibodies to HPV 16L1 using ELISA. The highest immunoreactivity was observed in the Cervarix ^TM -infected rats when coated with E. coli-derived VLPs and yeast-derived VLPs used in the inoculation, and antibody values obtained from the mice inoculated with the alum-derived VLPs were similar to those of Cervarix ^TM Respectively. Even HPV 16L1 specific immune responses were evident in mice inoculated with E. coli-derived VLPs inoculated without the adjuvant (FIGS. 14B, C and E). However, as expected, the fusion partner hARSNTD did not show any immune response (Fig. 14D).

To confirm the above results, we performed western blotting using serum from group 1 as the first antibody (1 ^st antibody). Antibodies to the fusion partner hARSNTD were not induced in mice inoculated with E. coli-derived VLPs, and Cervarix ^™ bound antibodies obtained from mice inoculated with E. coli-derived VLPs (FIG. 14F and G). These results demonstrate that certain antibodies against HPV have been produced when inoculated with purified VLPs in E. coli.

Example  14: cytokine through immune response of cleaved protein ( cytokine ) produce

In order to evaluate the level of Th1-type from splenocytes of mice inoculated with various immunoadjuvants, the production of IFN- was measured by ELISPOT (Enzyme-Linked ImmunoSpot). Spindle cells were isolated from the mice that had been inoculated until the third inoculation, which was stimulated by yeast-derived VLPs. The IFN-level in the Cervarix ^TM -injected rats was highest, but the IFN-level in the 5th group inoculated with our cut proteins with alum was also significantly higher than the other groups (Fig. 15A). From the above results, we confirmed that VLP produced in Escherichia coli produces a cytokine specific for HPV similar to Cervarix ^™ .

[references]

1.World Health Organization Influenza fact sheet. 2014. Available at www.who.int/mediacentre/factsheets/fs211/en. March 2014. Retrieved 25 November 2014.

2 . Hurtado JC, Mosquera MM, de Lazzari E, MartE, Torner N, Isanta R, et al. BMC Infect Dis. 2015; 15: 44. doi: 10.1186 / s12879-015-0775-5.

3 . Scott A. Harper, et al., Clin Infect Dis. 2009; 48 (8): 10031032.

4 . Jing Qi Feng, et al., Vaccine. 2009; 27, pp. 63586362.

5. Wiley, D. C., I. A. Wilson, and J. J. Skehel. 1981. Nature 289: 373-378.

6 . Wong SS, Webby RJ. Clin Microbiol Rev. 2013; 26: 476492. doi: 10.1128 / CMR.00097-12.

7. Wei, C. J., et al. 2008. J. Virol. 82: 6200-6208.

8 . Kanekiyo, M. et al. Nature 499, 102106 (2013).

9. Alymova, I. V., et al. 1998. J. Virol. 724472-4477.

10. Choi et al., 2011. Chaperoning roles of macromolecules interacting with proteins in vivo. Int. J. Mol. Sci., 12, 19791990.

11. Jang et al., 2014. J. Virol. 196, 56-64.

12. Choi et al., 2008. PLoS ONE 3, e2677

13. Choi et al., 2009. RNA Biol. 6, 2124

14. Chen, J., et al., Cell 95: 409-417.

15. Garten, W., F. X. Bosch, D. Linder, R. Rott, and H. D. Klenk. 1981. Virology 115: 361-374.

16. Stevens, J., et al. 2004. Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus. Science 303: 1866-1870.

17. Wiley, D. C., I. A. Wilson, and J. J. Skehel. 1981. Nature 289: 373-378.

18. Aguilar-Yanez et al., 2010. PLoS ONE, 5, p. e11694.

19 . DuBois et al., 2011. J. Virol. 85, pp. 865872.

20. Frankel and Smith, 1998. Cell, 92, pp. 149151.

21. Kim et al., 2007. Protein Sci. 16, 635-643.

22. Jang et al., 2013. PLoS ONE, 8, e75043

23. Gerhard W. Curr Top Microbiol Immunol. 2001; 260: 171190.

24. Esposito and Chatterjee, 2006. Curr. Opin. Biotechnol. 17, pp. 353358.

25. Baneyx, F., 1999. Curr. Opin. Biotechnol., 10, 411421.

26. Pica N, Palese P. Annu Rev Med. 2013; 64: 189202

27. Khurana et al., 2010. PLoS ONE, 5, p. e11548.

28. Choi et al., 2012. Int. J. Mol. Sci., 13, 1036810386

29 . Meier et al., 2004. J. Mol. Biol., 344, pp. 10511069.

30 . Khurana et al., 2011. J. Virol. 85, pp. 12461256.

31. Ofir-Birin Y., et al., Mol. Cell. 2013; 49: 3042.

32. Skehel, J. J., and D. C. Wiley. 2000. Annu. Rev. Biochem. 69: 531-569.

33. Davis et al., 1999. Biotechnol. Bioeng. 65, pp. 328382.

34. Seong et al., 2010. Method for increasing solubility of target protein using RNA-binding protein as fusion partner. U.S. Patent 11 / 482,401.

35. Stevens J, et al. 2006. Science, 312 (5772), 404410.

36. Bizebard, T., et al. 1995. Nature 376: 92-94.

37. Wiley, D. C., I. A. Wilson, and J. J. Skehel. 1981. Nature 289: 373-378.

38. Weldon, W. C., et al. 2010. PLoS One 5: e12466.

39. Copeland CS, et al., 1986. J Cell Biol, 103 (4), 11791191.

40. Chiu, F. F., et al., Biochem. Biophys. Res. Commun. 383: 27-31.

41. Shien, J. H., et al. Microbiol. Immunol. Infect. 41: 369-376.

42. World Health Organization Ebola response roadmap situation report 3 June 2015. Geneva: WHO, 2015. Available online at http://apps.who.int/ebola/en/current-situation/ebola-situation-report-3-june -2015 [Accessed 24 July 2015].

43. Prasannavenkatesh Durai, et al., Exp Mol Med. 2015 Aug; 47 (8): e181.

44. Tackling emerging infections: clinical and public health lessons from the West African Ebola virus disease outbreak, 20142015

45. Chia-Ying Wu, et al., PLoS One. 2012; 7 (8): E42363.

46. D. Maxwell Parkin, Freddie Bray. Vaccine 24 (2006) S11-S25.

47. Gary Clifford, et al., Vaccine 24 (2006) S26-S34.

48. Anna Rosa Garbuglia. Cancer 6 (2014) 1705-1726.

49. Ethel-Michele de Villiers, et al., Virology 324 (2004) 17-27.

50. Tao Bian, et al., Mol Cancer Ther 7 (2008) 1329-1335

51. M.J. Conway, C. Meyers. J Dent Res 88 (2009) 307-317.

52. Harald zur Hausen. Nature 2 (2002) 342-350

53. John Doorbar. Journal of Clinical Virology 32S (2005) S7-S15

54. Kamal Morshed, et al., Otolaryngologia Polska 68 (2014) 213-219.

55. Xiaojiang S. et al., Molecular Cell 5 (2000) 557-567.

56. Katharina Slupetzky, et al., Vaccine 25 (2007) 2001-2010.

57. Martin Sapp, et al., J Virol 72 (1998) 6186-6189.

58. Yorgo Modis, Benes L. Trus, Stephen C. Harrison. The EMBO Journal 21 (2002) 4754-4762.

59. R. Kirnbauer, F. Booy, N. Cheng, D. R. Lowy, J. T. Schiller. Proc. Natl. Acad. Sci. 89 (1992) 12180-12184.

60. Brooke Bishop, et al., The Journal of Biological Chemistry 282 (2007) 31803-31811.

61. Michael E. Hagensee, Nobuo Yaegashi, Denise A. Galloway. Journal of Virology 67 (1993) 315-322

62. Robert C. Rose, et al., Journal of Virology 67 (1993) 1936-1944.

63. Wei Zhang, et al., Virology 243 (1998) 423-431.

64. Toshiyuki Sasagawa et al., Virology 206 (1995) 126-135.

65. Hyoung Jin Kim, Su Jeung Lim, Hye-Lim Kwag, Hong-Jin Kim. PLoS one 7 (2012) e35893

66. Sabitha Ramakrishnan, Steena Partricia, Ganeshan Mathan. Biomedicine & Pharmacotherapy 70 (2015) 103-110.

67. Vicente Madrid-Marina, et al., Archives of Medical Research 40 (2009) 471-477.

68. Jan M. Agosti, M.D., Seu J. Goldie, M.D., M.P.H. Introducing HPV vaccine in developing countries-key challenges and issues. N ENGL J MED 356 (2007) 1908-1910.

69. Francois Baneyx, Mirna Mujacic. Nat Biotechnol 22 (2004) 1399-1408.

70. Lysann Schadlich, et al., Journal of Virology 83 (2009) 7690-7705.

72. Dominic Esposito, Deb K Chatterjee. Current Opinion in Biotechnology 17 (2006) 353-358.

73. Choi SI et al., PLoS ONE 3 (2008) e2677.

74. Choi SI, Ryu K, Seong BL. RNA Biol 6 (2009) 21-24.

75. Choi SI, Son A, Lim KH, Jeong H, Seong BL. Int J Mol Sci 13 (2012) 10368-10386.

76. Nadja Thones, Anna Herreiner, Lysann Schadlich, Konrad Piuko, Martin Muller. J Virol. 82 (2008) 5472-5485.

Abbreviation:

 LysRS: Lysyl tRNA Synthetase.

 HAgd: Hemagglutinin globular domain

 eGFP: Enhanced Green Fluorescent Protein

 TEV protease: Tobacco Etch Virus nuclear inclusion a endopeptidase

 PR8 (H1N1): A / Puerto Rico / 8/34 (H1N1)

&Lt; 110 > PnP Biopharm Co., Ltd. <120> A MANUFACTURING METHOD FOR SOLUBLE AND ACTIVE RECOMBINANT PROTEIN          USING Aminoacyl tRNA synthetase N-terminal domain AS FUSION          PARTNER AND A PRODUCT THEREOF <130> P17-0016HS <150> 10-2016-0015124 <151> 2016-02-05 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 72 <212> PRT <213> mouse <400> 1 Met Ser Glu Gln Ala Thr Leu Gln Glu Ser Glu Val Lys Val Asp Gly   1 5 10 15 Glu Gln Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala Glu              20 25 30 Lys Lys Leu Ala Glu Lys Glu Ala Lys Gln Lys Glu Leu Ser Glu Lys          35 40 45 Gln Leu Asn Gln Thr Ala Ser Ala Pro Asn His Thr Ala Asp Asn Gly      50 55 60 Val Gly Ala Glu Glu Glu Thr Leu  65 70 <210> 2 <211> 77 <212> PRT <213> Homo sapiens <400> 2 Met Ser Glu Gln His Ala Gln Ala Ala Val Gln Ala Ala Glu Val Lys   1 5 10 15 Val Asp Gly Ser Glu Pro Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg              20 25 30 Leu Lys Ala Glu Lys Lys Val Ala Glu Lys Glu Ala Lys Gln Lys Glu          35 40 45 Leu Ser Glu Lys Gln Leu Ser Gln Ala Thr Ala Ala Ala Thr Asn His      50 55 60 Thr Asp Asn Gly Val Gly Pro Glu Glu Glu Ser Val  65 70 75 <210> 3 <211> 344 <212> PRT <213> H1N1 <400> 3 Met Lys Ala Lys Leu Leu Le Le Leu Cys Ala Leu Ala Ala Ala Asp   1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr              20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn          35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile      50 55 60 Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly  65 70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile                  85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe             100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe         115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn     130 135 140 Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys                 165 170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu             180 185 190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Leu Tyr         195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg     210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile                 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala             260 265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser Met         275 280 285 His Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser     290 295 300 Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn                 325 330 335 Ile Pro Ser Ile Gln Ser Arg Gly             340 <210> 4 <211> 566 <212> PRT <213> H3N2 <400> 4 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala   1 5 10 15 Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly              20 25 30 His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp          35 40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr      50 55 60 Gly Gly Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly Glu Asn Cys  65 70 75 80 Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys Asp Gly Phe Gln                  85 90 95 Asn Lys Lys Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn             100 105 110 Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val         115 120 125 Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser Phe Asn Trp Thr     130 135 140 Gly Val Thr Gln Asn Gly Thr Ser Ser Ala Cys Lys Arg Arg Ser Asn 145 150 155 160 Lys Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr His Leu Lys Tyr Lys                 165 170 175 Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp Lys             180 185 190 Leu Tyr Ile Trp Gly Val His His Pro Gly Thr Asp Ser Asp Gln Ile         195 200 205 Ser Leu Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val Ser Thr Lys Arg     210 215 220 Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Ser Arg Pro Arg Ile Arg 225 230 235 240 Asp Val Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly                 245 250 255 Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly             260 265 270 Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala         275 280 285 Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile     290 295 300 Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala 305 310 315 320 Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met                 325 330 335 Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala             340 345 350 Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly         355 360 365 Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys     370 375 380 Ser Thr Gln Ala Ile Asn Gln Ile Asn Gly Lys Leu Asn Arg Leu 385 390 395 400 Ile Gly Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser                 405 410 415 Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr             420 425 430 Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu         435 440 445 Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe     450 455 460 Glu Arg Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 465 470 475 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser                 485 490 495 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu             500 505 510 Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys         515 520 525 Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys     530 535 540 Val Ala Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile 545 550 555 560 Arg Cys Asn Ile Cys Ile                 565 <210> 5 <211> 569 <212> PRT <213> H5N1 <400> 5 Met Glu Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser   1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val              20 25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile          35 40 45 Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys      50 55 60 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn  65 70 75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val                  85 90 95 Glu Lys Ala Asn Pro Thr Asn Asp Leu Cys Tyr Pro Gly Ser Phe Asn             100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu         115 120 125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser     130 135 140 Ser Gly Val Ser Ser Ala Cys Pro Tyr Leu Gly Ser Pro Ser Phe Phe 145 150 155 160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile                 165 170 175 Lys Lys Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp             180 185 190 Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln         195 200 205 Asn Pro Thr Thr Tyr Ile Ser Ile Gly Thr Ser Thr Leu Asn Gln Arg     210 215 220 Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225 230 235 240 Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn                 245 250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile             260 265 270 Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Leu Glu Tyr Gly         275 280 285 Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser     290 295 300 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310 315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser                 325 330 335 Pro Gln Arg Glu Ser Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile             340 345 350 Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr         355 360 365 Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys     370 375 380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser 385 390 395 400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe                 405 410 415 Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp             420 425 430 Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met         435 440 445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu     450 455 460 Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465 470 475 480 Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu                 485 490 495 Ser Ile Arg Asn Gly Thr Tyr Asn Tyr Pro Gln Tyr Ser Glu Glu Ala             500 505 510 Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly         515 520 525 Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala     530 535 540 Leu Ala Ile Met Met Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550 555 560 Ser Leu Gln Cys Arg Ile Cys Ile Lys                 565 <210> 6 <211> 527 <212> PRT <213> strain B / Victoria / 2/1987 <400> 6 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp   1 5 10 15 Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys              20 25 30 Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr          35 40 45 Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr      50 55 60 Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp Val  65 70 75 80 Ala Leu Ala Arg Pro Lys Cys Met Gly Thr Ile Pro Ser Ala Lys Ala                  85 90 95 Ser Ile Leu His Glu Val Lys Pro Val Thr Ser Gly Cys Phe Pro Ile             100 105 110 Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly         115 120 125 Tyr Glu His Ile Arg Leu Ser Thr His Asn Val Ile Asn Ala Glu Thr     130 135 140 Ala Pro Gly Gly Pro Tyr Lys Val Gly Thr Ser Gly Ser Cys Pro Asn 145 150 155 160 Val Thr Asn Gly Asn Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro                 165 170 175 Lys Asn Asp Asn Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val             180 185 190 Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe         195 200 205 His Ser Asp Ser Glu Thr Gln Met Val Lys Leu Tyr Gly Asp Ser Lys     210 215 220 Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val 225 230 235 240 Ser Gln Ile Gly Gly Phe Pro Asn Gln Ala Glu Asp Gly Gly Leu Pro                 245 250 255 Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Ser Gly Lys             260 265 270 Thr Gly Thr Ile Thr Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val         275 280 285 Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu     290 295 300 Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys 305 310 315 320 Ser Lys Pro Tyr Tyr Thr Gly Gly His Ala Lys Ala Ile Gly Asn Cys                 325 330 335 Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr             340 345 350 Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile         355 360 365 Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His     370 375 380 Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu 385 390 395 400 Lys Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser                 405 410 415 Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met             420 425 430 Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp         435 440 445 Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu     450 455 460 Ser Asn Glu Gly Ile Asle Ser Glu Asp Glu His Leu Leu Ala Leu 465 470 475 480 Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly                 485 490 495 Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp             500 505 510 Arg Ile Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro         515 520 525 <210> 7 <211> 505 <212> PRT <213> HPV 16 <400> 7 Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val   1 5 10 15 Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn              20 25 30 Ile Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro          35 40 45 Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys      50 55 60 Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro  65 70 75 80 Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln                  85 90 95 Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro             100 105 110 Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp         115 120 125 Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg     130 135 140 Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly 145 150 155 160 Cys Lys Pro Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr                 165 170 175 Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn             180 185 190 Thr Val Ile Gln Asp Gly Asp Met Val His Thr Gly Phe Gly Ala Met         195 200 205 Asp Phe Thr Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile     210 215 220 Cys Thr Ser Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu 225 230 235 240 Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe                 245 250 255 Val Arg His Leu Phe Asn Arg Ala Gly Thr Val Gly Glu Asn Val Pro             260 265 270 Asp Asp Leu Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser         275 280 285 Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala     290 295 300 Gln Ile Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn 305 310 315 320 Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr                 325 330 335 Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu             340 345 350 Thr Thr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu         355 360 365 Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr     370 375 380 Ala Asp Val Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu 385 390 395 400 Asp Trp Asn Phe Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp                 405 410 415 Thr Tyr Arg Phe Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr             420 425 430 Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu         435 440 445 Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu     450 455 460 Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe 465 470 475 480 Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr                 485 490 495 Thr Ala Lys Arg Lys Lys Arg Lys Leu             500 505 <210> 8 <211> 472 <212> PRT <213> HPV 18 <400> 8 Glu Ala Leu Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro   1 5 10 15 Phe Val Ala Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr Ser              20 25 30 Ile Phe Tyr His Ala Gly Ser Phe Arg Leu Leu Thr Val Gly Asn Pro          35 40 45 Tyr Phe Arg Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro Lys      50 55 60 Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro  65 70 75 80 Asn Lys Phe Gly Leu Pro Asp Thr Ser Ile Tyr Asn Pro Glu Thr Gln                  85 90 95 Arg Leu Val Trp Ala Cys Ala Gly Val Glu Ile Gly Arg Gly Gln Pro             100 105 110 Leu Gly Val Gly Leu Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp         115 120 125 Thr Glu Ser Ser Ala Ala Thr Ser Ser Val Ser Glu Asp Val Arg     130 135 140 Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly 145 150 155 160 Cys Ala Pro Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys                 165 170 175 Ser Arg Pro Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu Leu Lys Asn             180 185 190 Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met         195 200 205 Asp Phe Ser Thr Leu Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile     210 215 220 Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp 225 230 235 240 Pro Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe                 245 250 255 Ala Arg His Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro             260 265 270 Gln Ser Leu Tyr Ile Lys Gly Thr Gly Met Arg Ala Ser Pro Gly Ser         275 280 285 Cys Val Tyr Ser Pro Ser Ser Ser Ser Val Ser Ser Ser Val Ser     290 295 300 Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn 305 310 315 320 Asn Gly Val Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr                 325 330 335 Thr Pro Ser Thr Asn Leu Thr Ile Cys Ala Ser Thr Gln Ser Ser Val             340 345 350 Pro Gly Gln Tyr Asp Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val         355 360 365 Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu     370 375 380 Thr Ala Asp Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu 385 390 395 400 Glu Asp Trp Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser Leu Val                 405 410 415 Asp Thr Tyr Arg Phe Val Gln Ser Val Ala Ile Thr Cys Gln Lys Asp             420 425 430 Ala Ala Pro Ala Glu Asn Lys Asp Pro Tyr Asp Lys Leu Lys Phe Trp         435 440 445 Asn Val Asp Leu Lys Glu Lys Phe Ser Leu Asp Leu Asp Gln Tyr Pro     450 455 460 Leu Gly Arg Lys Phe Leu Val Gln 465 470

Claims (18)

delete delete A method for producing an influenza hemagglutinin (HA) antigen in a water soluble form using a domain consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2 as a fusion partner. The production method according to claim 3, wherein the hemagglutinin (HA) antigen is HA derived from H1, H3, H5 or B influenza virus. The production method according to claim 3 or 4, wherein the hemagglutinin (HA) antigen is H1N1 type. A vaccine for the prevention of influenza by using a protein consisting of a fusion of an influenza hemagglutinin (HA) antigen and a domain comprising the amino acid sequence of SEQ ID NO: 1 or 2 as a vaccine antigen. The influenza vaccine according to claim 6, wherein the hemagglutinin (HA) antigen is HA derived from H1, H3, H5 or B influenza virus. The influenza vaccine according to claim 6 or 7, wherein the hemagglutinin (HA) antigen is H1N1 type. delete  A method for producing papilloma virus (HPV) 16L1 protein or HPV 18L1 protein in a water soluble form using a domain consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2 as a fusion partner. [Claim 11] The method according to claim 10, wherein the method comprises injecting a vector encoding a domain consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2 and a vector comprising a gene encoding papilloma virus (HPV) 16L1 protein or HPV18L1 protein into a host Followed by culturing at 20 占 폚 to express the protein. A vaccine for the prevention of papilloma virus (HPV) using as a vaccine antigen a protein consisting of a domain consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2 and a protein consisting of a papilloma virus (HPV) 16L1 protein or a fusion of HPV18L1 protein. delete delete delete delete delete delete

Images