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WO2020055163A1 - Vector expressing recombinant antigen by using crispr editing technology, and simultaneous multiple insertion method therefor - Google Patents

Vector expressing recombinant antigen by using crispr editing technology, and simultaneous multiple insertion method therefor Download PDF

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WO2020055163A1
WO2020055163A1 PCT/KR2019/011830 KR2019011830W WO2020055163A1 WO 2020055163 A1 WO2020055163 A1 WO 2020055163A1 KR 2019011830 W KR2019011830 W KR 2019011830W WO 2020055163 A1 WO2020055163 A1 WO 2020055163A1
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antigen
protein
vector
rbd
cells
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French (fr)
Korean (ko)
Inventor
김천생
김성준
안대균
김균도
구근본
김해수
신혜진
황인수
윤건영
장현주
응옥 유인 후인티
김미화
김범태
배상수
우재성
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Korea Research Institute of Chemical Technology KRICT
Korea Food Research Institute KFRI
Industry University Cooperation Foundation IUCF HYU
Korea University Research and Business Foundation
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Korea Research Institute of Chemical Technology KRICT
Korea Food Research Institute KFRI
Industry University Cooperation Foundation IUCF HYU
Korea University Research and Business Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20051—Methods of production or purification of viral material

Definitions

  • the present invention relates to a vector for expressing a recombinant antigen at a specific position of a human chromosome through a homology directed recombination method, which is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technique to induce stable overexpression of an antigen and a method for inserting the same.
  • a homology directed recombination method which is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technique to induce stable overexpression of an antigen and a method for inserting the same.
  • prokaryotic cells such as E.coli.
  • protein modification such as glycosylation may have a great influence on immunogenicity.
  • prokaryotic cells there is a limit in producing proteins that require excessive glycosylation due to induction of different glycosylation than expression of proteins in eukaryotic cells such as human cells (Nat Chem Biol (2012) 8 (5): 434-436). Therefore, when expressing a recombinant immunogen, many human cells such as 293 cells have been used.
  • the method mainly used to date is a method of purifying a protein that is transiently expressed after transfection of a recombinant immunogen-expressing plasmid into eukaryotic cells (Vaccine 32 (2014) 6170-6176).
  • This transient method is sufficient to test immunogenicity, but there is a limit to producing a large amount of recombinant immunogen.
  • Homology-directed repair method of CRISPR gene editing technology is used to produce a cell line stably expressing an immunogen.
  • Signal Peptide is a peptide that is located at the N-terminus of proteins and acts to be secreted out of the cell. They are characterized by being able to express them by fusing them to a desired protein and then releasing them out of cells.
  • the spike protein contains its signal peptide at the N-terminus.
  • Recombinant immunogen also has the advantage of obtaining the desired protein without lysis of the cell because it can be released outside the cell by fusion and expression of the signal peptide.
  • a desired expression vector can be additionally integrated, such as a method of integration into AAVS using a SEMA6A site.
  • the present invention was completed by discovering that the expression of a recombinant immunogen can be more than doubled by integrating the same expression vector simultaneously at two sites where expression is actively occurring in one cell.
  • An object of the present invention is to provide a stable cell line stably expressing an antigen by inserting a vector overexpressing the antigen at a specific position of a chromosome.
  • an object of the present invention is to provide a method of simultaneously inserting multiple vectors expressing a recombinant antigen at multiple sites of a chromosome in order to increase a stable overexpression rate of an antigen.
  • the present invention provides a vector for expressing an antigen at a specific position in a human chromosome by a homology-directed repair method, which is a CRISPR gene editing technology, for inducing stable overexpression of a recombinant antigen in eukaryotic cells.
  • the present invention provides a method of simultaneously inserting multiple vectors expressing a recombinant antigen at multiple positions of eukaryotic chromosomes using a homology-directed repair method, a CRISPR gene editing technique, to increase the stable overexpression efficiency of antigens in eukaryotic cells. .
  • the antigen is an antigen of the MERS virus
  • the MERS virus antigen is preferably a receptor binding domain (RBD) located in a spike protein.
  • RBD antigen is preferably a fusion of 6 histidine amino acid tags necessary for protein purification to the C-terminal, a fusion of the Fc portion, or a signal peptide to induce glycosylation at the N-terminus.
  • the vector of the present invention includes sgRNAs targeting AAVS and SEMA6A positions, and preferably contains Cas protein expression sequences and Cas protein is a Cas9 protein.
  • the method of the present invention may further include a Hygromycin resistant gene site or a Puromycin resistant gene site.
  • the eukaryotic cells also include 293 cells.
  • the present invention provides a vector capable of expressing the CRISPR-Cas system comprising the MERS virus antigen.
  • the vector comprises sgRNA targeting AAVS and / or SEMA6A positions, and the Cas is Cas9.
  • the MERS virus antigen is a receptor binding domain (RBD) located in a spike protein, and the six histidine amino acid tags necessary for protein purification are fused to the C-terminal or the Fc portion is fused behind the RBD protein.
  • RBD receptor binding domain
  • a signal peptide that induces glycosylation at the N-terminus may be fused, and may further include a Hygromycin resistant gene site or a Puromycin resistant gene site.
  • the present invention has the effect of producing a recombinant antigen in a eukaryotic cell stably and with high efficiency.
  • the present invention has the advantage of being able to secure a variety of recombinant antigens in large quantities.
  • FIG. 1 shows a stable cell line stably expressing an antigen by inserting an antigen expression vector at a specific position (chromosome 5 and 9) of a eukaryotic cell chromosome by a homology-directed repair method, which is a CRISPR-cas9 editing technique. It is a schematic diagram to produce.
  • Figure 2 is a schematic diagram showing the structure of the recombinant antigen (MERS virus receptor binding site, MERS-CoV RBD) over-expressed through the process of Example 1.
  • MERS virus receptor binding site MERS-CoV RBD
  • Figure 3 is after inserting the vector expressing the recombinant antigen of Example 2 at two chromosome specific positions (chromosome 5 and 9) simultaneously (integration), the amount of antigen expression in the cell (Cell pellet) and outside (Supernatant) 2 It is a result of Western blotting showing an increase of more than a factor.
  • Example 4 is a result of Western blotting demonstrating that the antigen expressed in Example 3 and released into the cell (Supernatant) is glycosylation.
  • Example 4 mers virus receptor binding site, MERS-CoV RBD
  • the table below shows the high efficiency of the purified antigen.
  • Example 6 is a result of analyzing the high purity of the purified antigen of Example 5 (mers virus receptor binding site, MERS-CoV RBD) through RP_HPLC.
  • Example 7 is a result of analyzing the high purity of the purified antigen (MERS virus receptor binding site, MERS-CoV RBD) of Example 5 through gel filtration.
  • Example 1 Recombinant antigen ( MERS virus Receptor binding site ) Expression stable cell line selection
  • a cell line stably expressing an immunogen is made using CRISPR-Cas9 technology, and a method capable of continuously expressing and refining the same recombinant antigen is used.
  • a method of integrating a recombinant antigen expression vector using a homology directed repair (HDR) method to the chromosome 19 AAVS site reported as the chromosomal site where protein expression is actively used was used (see FIG. 1).
  • HDR homology directed repair
  • the DNA at the desired position can be cut, and the left and right homology arms in the transfected recombinant antigen expression plasmid can be cut together.
  • HDR a system capable of inserting a desired gene into an accurate position has been developed. The left and right homology arms are shown in the recombinant antigen expression plasmid of FIG. 1.
  • a target protein was used as a receptor binding domain (RBD) located in the spike protein of MERS virus.
  • RBD receptor binding domain
  • six histidine amino acid tags necessary for protein purification were fused to the C-terminal (Fig. 2. Immunogen expression vector 1). Since the Fc portion of the human antibody has a function of stabilizing the recombinant antigen in the body, it was fusion followed by the RBD protein to produce it as immunogen expression vector 2 (Fig. 2 immunogen expression vector 2).
  • the Fc portion of the human antibody can be purified and purified purely through a Protein G column and used in the present invention. It is known that the natural MERS virus spike protein is glycosylated.
  • a signal peptide that induces glycosylation was fused to the two recombinant antigen N-terminus so that the glycosylation occurs while the recombinant MERS virus RBD antigen also undergoes the same discharge process.
  • Purifying the recombinant antigen discharged out of the cell has the advantage of efficiently and purely purifying the protein because it reduces the amount of other contaminating proteins that can be experienced while purifying the protein expressed in the cell.
  • an antigen-expressing plasmid was first inserted into the AAVS position of 293 cells.
  • Hygromycin resistant gene was inserted together, and only desired cells were selected by adding hygromycin to the cell culture medium.
  • cells expressing the immunogen efficiently were selected by Western method and expanded to use as immunogen expressing cells.
  • cells were selected in a similar manner using a SEMA6A position vector expressing the same immunogen in cells obtained from AAVS to make cells inserted at both positions simultaneously.
  • puromycin resistant gene was inserted together, and hygromycin and puromycin were added to the cell culture medium to select cells. Among the surviving cells, cells with high immunogen protein expression were selected and used for mass expression of the immunogen.
  • Recombinant antigen-expressing cells (AACY + SEMA6A) inserted simultaneously in the two positions of AAVS and SEMA6A prepared in Example 1 were compared with cells expressing only in one AAVS position (AACY) to confirm whether the expression of the recombinant antigen increased. Proceeded. This is a system designed to discharge the immunogen protein produced in the cell out of the cell, so the amount of the protein in the cell and the amount of the protein in the extracellular medium were compared. The results of experiments using the immunogen expression vector 2 of FIG. 2 are shown. In the case of this antigen (MERS-RBD-Fc), the Fc portion of the human antibody is fused, so that protein expression can be confirmed by using a secondary antibody against the human antibody by Western blotting.
  • Example 3 Recombinant antigen ( MERS virus Receptor binding site ) Glycosylation (glycosylation) check
  • Recombinant antigen secreted out of the cell by the signal peptide of MERS virus was tested using PNGase F, which can remove N-linked glycan to confirm that glycosylation occurred well.
  • the recombinant antigen used in the experiment is a MERS virus receptor binding site protein produced by the immunogen expression vector 1 of FIG. 2 (MERS-RBD).
  • PNGase F was treated with the MERS virus receptor binding site protein, and Western blotting was performed. Proteins were identified using anti-His tag antibodies capable of recognizing immunogens. As shown in FIG. 3, it was confirmed that glycosylation was well induced because the immunogen protein (MERS-RBD deglycosylated) treated with PNGase F was detected to be smaller in size than the untreated immunogen protein (MERS-RBD glycosylated). It may be used as an immunogen capable of inducing glycosylation similar to the surface protein of the virus and specifically helping to form an antibody capable of neutralizing the virus.
  • the cell lines selected in Example 1 were proliferated in large quantities to isolate the immunogen.
  • immunogen-expressing cell lines can be mass cultured using serum free media that can grow 293 cells without serum.
  • serum free media that can grow 293 cells without serum.
  • Sigma's Excell 293 serum free media or ThermoFisher Scientific's FreeStyle 293 expression media was used.
  • Sigma's CELLINE Classic bioreactor flask it is a flask that is frequently used for protein expression of antibodies or cells. The medium required for cell proliferation moves freely through the membrane inserted in the middle, but proteins released from cells or cells cannot.
  • the present invention has industrial applicability because it can be usefully used for the development of recombinant immunogens and vaccines against various viruses.

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Abstract

Provided in the present invention is a method for simultaneously inserting multiple vectors expressing recombinant antigens in multiple locations of eukaryotic chromosomes by using a homology directed recombination method, which is a CRISPR technology. The present invention relates to a method for expressing a recombinant protein which can be used as an immunogen, by selecting a human cell line that can stably over-express an immunogen, when simultaneous insertion into two sites is performed, and then using the cells, and which can be effectively used for the development of recombinant immunogens and vaccines against various socially problematic viruses.

Description

CRISPR 편집 기술을 이용한 재조합 항원을 발현시키는 벡터 및 이를 동시에 다중 삽입시키는 방법Vectors expressing recombinant antigens using CRISPR editing technology and multiplexing them simultaneously

본 발명은 항원의 안정적인 과 발현을 유도하기 위하여 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) 기술인 homology directed recombination 방법을 통하여 인간 크로모좀(chromosome) 특정 위치에 재조합 항원을 발현시키는 벡터 및 이를 삽입시키는 방법에 관한 것이다.The present invention relates to a vector for expressing a recombinant antigen at a specific position of a human chromosome through a homology directed recombination method, which is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technique to induce stable overexpression of an antigen and a method for inserting the same. will be.

일반적으로 단백질 연구를 진행할 때 발현은 E.coli와 같은 원핵세포를 이용하여 진행된다. 하지만 바이러스의 표면에 존재하는 단백질을 재조합 면역원으로 사용할 때는 glycosylation과 같은 단백질 수식이 면역원성에 큰 영향을 끼칠 수 있다. 원핵세포에서 발현을 수행하는 경우 인간세포와 같은 진핵세포에서 단백질을 발현하는 경우와 다른 glycosylation이 유도되어 과도한 glycosylation이 필요한 단백질을 생산하는데는 한계가 있다 (Nat Chem Biol (2012) 8(5): 434-436). 때문에 재조합 면역원을 발현하는 경우 293 세포와 같은 인간세포를 많이 이용해오고 있다. 현재까지 주로 사용되는 방법은 재조합 면역원 발현 플라스미드를 진핵세포에 transfection 한 후 transient하게 발현되는 단백질을 정제하는 방법이다 (Vaccine 32 (2014) 6170-6176). 이러한 transient한 방법으로 면역원성을 시험하기에는 충분하지만 대량의 재조합 면역원을 생산하기에는 한계가 존재한다. 이를 극복하기 위해서 인간세포에서 안정적으로 재조합 면역원을 발현할 수 있는 세포주를 선별하여 계속 생산되는 재조합 면역원을 얻어서 정제하는 방법이 사용 가능하다. 면역원을 안정적으로 발현하는 세포주를 제작하기 위해서 CRISPR gene editing 기술의 Homology-directed Repair 방법이 사용되고 있다. Nature Biotechnology 33 (2015) 543-550에서는 단백질 발현 벡터를 293 세포에서 비교적 transcription이 잘 일어나는 AAVS site에 삽입시키는 방법을 사용하였다. 이러한 방법은 세포 안에서 단백질 발현을 유도하는 방법이다. Signal Peptide는 단백질들의 N-말단에 위치하여 세포 밖으로 배출(secretion) 될 수 있도록 작용하는 펩타이드이다. 이들을 원하는 단백질에 융합시켜서 발현하면 세포 밖으로 배출시킬 수 있는 특징이 있다. 메르스 바이러스의 경우도 스파이크 단백질(S protein)의 경우 자신의 signal peptide를 N-말단에 포함하고 있다. 재조합 면역원 역시 signal peptide를 융합시켜서 발현시키면 세포 밖으로 배출이 가능해서 세포를 lysis 시키지 않고도 원하는 단백질을 얻을 수 있는 장점이 있다. AAVS site 이외에도 Biotechnol. J 11 (2016) 1100-1109에 따르면, gene의 transcription이 활발히 일어나는 hot spot이 보고되어 있다. 이러한 site 중에서 SEMA6A site를 사용하여 AAVS에 integration 시키는 방법처럼 추가로 원하는 발현 벡터를 integration 시킬 수 있다. 한 세포에 발현이 활발히 일어나는 두 site에 동시에 동일한 발현 벡터를 integration 시켜서 재조합 면역원 발현을 두 배 이상 향상시킬 수 있음을 알아내어 본 발명을 완성하였다.In general, when conducting protein research, expression is performed using prokaryotic cells such as E.coli. However, when a protein existing on the surface of a virus is used as a recombinant immunogen, protein modification such as glycosylation may have a great influence on immunogenicity. When expression is performed in prokaryotic cells, there is a limit in producing proteins that require excessive glycosylation due to induction of different glycosylation than expression of proteins in eukaryotic cells such as human cells (Nat Chem Biol (2012) 8 (5): 434-436). Therefore, when expressing a recombinant immunogen, many human cells such as 293 cells have been used. The method mainly used to date is a method of purifying a protein that is transiently expressed after transfection of a recombinant immunogen-expressing plasmid into eukaryotic cells (Vaccine 32 (2014) 6170-6176). This transient method is sufficient to test immunogenicity, but there is a limit to producing a large amount of recombinant immunogen. To overcome this, it is possible to select a cell line capable of stably expressing a recombinant immunogen in human cells and obtain and purify the recombinant immunogen that is continuously produced. Homology-directed repair method of CRISPR gene editing technology is used to produce a cell line stably expressing an immunogen. In Nature Biotechnology 33 (2015) 543-550, a method of inserting a protein expression vector into an AAVS site where transcription is relatively good in 293 cells was used. This method is a method for inducing protein expression in cells. Signal Peptide is a peptide that is located at the N-terminus of proteins and acts to be secreted out of the cell. They are characterized by being able to express them by fusing them to a desired protein and then releasing them out of cells. In the case of the MERS virus, the spike protein (S protein) contains its signal peptide at the N-terminus. Recombinant immunogen also has the advantage of obtaining the desired protein without lysis of the cell because it can be released outside the cell by fusion and expression of the signal peptide. In addition to the AAVS site, Biotechnol. According to J 11 (2016) 1100-1109, hot spots in which gene transcription is actively reported have been reported. Among these sites, a desired expression vector can be additionally integrated, such as a method of integration into AAVS using a SEMA6A site. The present invention was completed by discovering that the expression of a recombinant immunogen can be more than doubled by integrating the same expression vector simultaneously at two sites where expression is actively occurring in one cell.

한편, 다양한 재조합 항원을 안정적으로 확보하는 것은 재조합 단백질을 이용한 다양한 연구 진행에 있어서 매우 중요한 과정이다. 그러나, 현재까지의 기술로 재조합 항원의 안정적인 과 발현을 유도하는 기술은 매우 어렵고, 이러한 문제를 극복하는 기술이 공지된 바가 없다.Meanwhile, stably securing various recombinant antigens is a very important process in various studies using recombinant proteins. However, the technique to induce the stable overexpression of the recombinant antigen with the technique so far is very difficult, and no technique has been known to overcome this problem.

본 발명에서는 항원을 과 발현시키는 벡터를 크로모좀 특정 위치에 삽입시켜서 안정적으로 항원을 발현하는 안정 세포주를 제공하는데 목적이 있다.An object of the present invention is to provide a stable cell line stably expressing an antigen by inserting a vector overexpressing the antigen at a specific position of a chromosome.

또한, 본 발명은 항원의 안정적인 과 발현율 증대를 위하여 크로모좀의 다중 위치(multiple sites)에 재조합 항원을 발현시키는 벡터를 동시에 다중 삽입시키는 방법을 제공하는데 목적이 있다.In addition, an object of the present invention is to provide a method of simultaneously inserting multiple vectors expressing a recombinant antigen at multiple sites of a chromosome in order to increase a stable overexpression rate of an antigen.

상기 목적을 해결하기 위하여,In order to solve the above object,

본 발명은 진핵 세포에서 재조합 항원의 안정적인 과 발현 유도를 위해 CRISPR 유전자 편집기술인 homology-directed repair 방법으로 인간 크로모좀 특정 위치에 항원을 발현시키는 벡터를 제공한다.The present invention provides a vector for expressing an antigen at a specific position in a human chromosome by a homology-directed repair method, which is a CRISPR gene editing technology, for inducing stable overexpression of a recombinant antigen in eukaryotic cells.

또한, 본 발명은 진핵 세포에서 항원의 안정적인 과 발현 효율 증대를 위해 CRISPR 유전자 편집 기술인 homology-directed repair 방법으로 진핵 세포 크로모좀의 다중 위치에 재조합 항원을 발현시키는 벡터를 동시에 다중 삽입시키는 방법을 제공한다.In addition, the present invention provides a method of simultaneously inserting multiple vectors expressing a recombinant antigen at multiple positions of eukaryotic chromosomes using a homology-directed repair method, a CRISPR gene editing technique, to increase the stable overexpression efficiency of antigens in eukaryotic cells. .

본 발명에서 보다 구체적으로 항원은 메르스 바이러스의 항원이고, 상기 메르스 바이러스 항원은 스파이크 단백질 안에 위치한 수용체 결합부위(receptor binding domain; RBD)인 것이 바람직하다. 이 RBD 항원은 단백질 정제를 위해 필요한 6개의 histidine 아미노산 tag을 C-terminal에 fusion시킨 것, Fc 부분을 fusion시킨 것 또는 N-말단에 글리코실화를 유도하는 signal peptide가 융합된 것이 바람직하다.More specifically, in the present invention, the antigen is an antigen of the MERS virus, and the MERS virus antigen is preferably a receptor binding domain (RBD) located in a spike protein. The RBD antigen is preferably a fusion of 6 histidine amino acid tags necessary for protein purification to the C-terminal, a fusion of the Fc portion, or a signal peptide to induce glycosylation at the N-terminus.

또한 본 발명의 상기 벡터는 AAVS 및 SEMA6A 위치를 타겟팅하는 sgRNA를 포함하며, Cas 단백질 발현 서열을 포함하고 Cas 단백질은 Cas9 단백질인 것이 바람직하다.In addition, the vector of the present invention includes sgRNAs targeting AAVS and SEMA6A positions, and preferably contains Cas protein expression sequences and Cas protein is a Cas9 protein.

본 발명의 방법은 Hygromycin resistant gene 부위 또는 Puromycin resistant gene 부위를 더 포함할 수 있다. 또한 상기 진핵 세포는 293 세포를 포함한다.The method of the present invention may further include a Hygromycin resistant gene site or a Puromycin resistant gene site. The eukaryotic cells also include 293 cells.

또한 본 발명은 메르스 바이러스 항원을 포함하는 CRISPR-Cas 시스템을 발현할 수 있는 벡터를 제공한다.In addition, the present invention provides a vector capable of expressing the CRISPR-Cas system comprising the MERS virus antigen.

상기 벡터는 AAVS 및/또는 SEMA6A 위치를 타겟팅하는 sgRNA를 포함하고, 상기 Cas는 Cas9인 것이 바람직하다. 또한 상기 메르스 바이러스 항원은 스파이크 단백질 안에 위치한 수용체 결합부위(receptor binding domain; RBD)이고, 단백질 정제를 위해 필요한 6개의 histidine 아미노산 tag을 C-terminal에 fusion시키거나 RBD 단백질 뒤에 Fc 부분을 fusion시킨 것 또는 N-말단에 글리코실화를 유도하는 signal peptide가 융합된 것일 수 있고, Hygromycin resistant gene 부위 또는 Puromycin resistant gene 부위를 더 포함할 수 있다.Preferably, the vector comprises sgRNA targeting AAVS and / or SEMA6A positions, and the Cas is Cas9. In addition, the MERS virus antigen is a receptor binding domain (RBD) located in a spike protein, and the six histidine amino acid tags necessary for protein purification are fused to the C-terminal or the Fc portion is fused behind the RBD protein. Alternatively, a signal peptide that induces glycosylation at the N-terminus may be fused, and may further include a Hygromycin resistant gene site or a Puromycin resistant gene site.

본 발명은 진핵 세포에서 재조합 항원을 안정적이며 고효율로 생산할 수 있는 효과가 있다.The present invention has the effect of producing a recombinant antigen in a eukaryotic cell stably and with high efficiency.

또한 본 발명은 다양한 재조합 항원을 대량으로 확보할 수 있다는 장점이 있다.In addition, the present invention has the advantage of being able to secure a variety of recombinant antigens in large quantities.

도 1은 CRISPR-cas9 편집 기술인 homology-directed repair 방법으로 진핵 세포 크로모좀 특정 위치 (chromosome 5 및 9)에 항원 발현 벡터를 삽입(integration) 시켜서 항원을 안정적으로 발현하는 안정 세포주(stable cell line)를 제작하는 모식도이다.FIG. 1 shows a stable cell line stably expressing an antigen by inserting an antigen expression vector at a specific position (chromosome 5 and 9) of a eukaryotic cell chromosome by a homology-directed repair method, which is a CRISPR-cas9 editing technique. It is a schematic diagram to produce.

도 2는 실시예 1의 과정을 통해 과 발현되는 재조합 항원(메르스바이러스 수용체결합부위, MERS-CoV RBD) 구조를 보여주는 모식도이다.Figure 2 is a schematic diagram showing the structure of the recombinant antigen (MERS virus receptor binding site, MERS-CoV RBD) over-expressed through the process of Example 1.

도 3은 실시예 2의 재조합 항원을 발현하는 벡터를 chromosome 특정 위치(chromosome 5 및 9) 2곳 동시에 삽입(integration) 시킨 후, 항원 발현양이 세포 안(Cell pellet)과 밖(Supernatant)에서 2배 이상 증가함을 보여주는 웨스턴 블로팅 결과이다.Figure 3 is after inserting the vector expressing the recombinant antigen of Example 2 at two chromosome specific positions (chromosome 5 and 9) simultaneously (integration), the amount of antigen expression in the cell (Cell pellet) and outside (Supernatant) 2 It is a result of Western blotting showing an increase of more than a factor.

도 4는 실시예 3에서 발현되어 세포 밖(Supernatant)으로 배출된 항원이 glycosylation이 됨을 입증하는 웨스턴 블로팅 결과이다.4 is a result of Western blotting demonstrating that the antigen expressed in Example 3 and released into the cell (Supernatant) is glycosylation.

도 5는 실시예 4의 항원(메르스바이러스 수용체결합부위, MERS-CoV RBD)을 대량으로 정제한 후, 은 염색법(silver staining)을 통하여 항원의 고순도 정제를 보여주는 결과이다. 아래 표는 정제된 항원의 고효율을 보여준다.5 is a result showing the high-purity purification of the antigen through silver staining (silver staining), after purifying the antigen of Example 4 (mers virus receptor binding site, MERS-CoV RBD) in large quantities. The table below shows the high efficiency of the purified antigen.

도 6은 실시예 5의 정제된 항원(메르스바이러스 수용체결합부위, MERS-CoV RBD)의 고순도를 RP_HPLC를 통해 분석한 결과이다.6 is a result of analyzing the high purity of the purified antigen of Example 5 (mers virus receptor binding site, MERS-CoV RBD) through RP_HPLC.

도 7은 실시예 5의 정제된 항원(메르스바이러스 수용체결합부위, MERS-CoV RBD)의 고순도를 gel filtration을 통해 분석한 결과이다.7 is a result of analyzing the high purity of the purified antigen (MERS virus receptor binding site, MERS-CoV RBD) of Example 5 through gel filtration.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are only for illustrating the present invention, it should not be construed that the scope of the present invention is limited to these examples.

<< 실시예Example 1> 재조합 항원( 1> Recombinant antigen ( 메르스바이러스MERS virus 수용체결합부위Receptor binding site ) 발현 안정 세포주 선별) Expression stable cell line selection

본 발명에 따른 재조합 항원(메르스바이러스 수용체결합부위) 안정 세포주를 제작하기 위해서 다음과 같이 실험하였다.To produce a recombinant antigen (mers virus receptor binding site) stable cell line according to the present invention was tested as follows.

현재까지, 진핵 세포에서의 단백질 발현은 일시적인 발현을 통하여 이루어져 왔다. 본 발명에서는 CRISPR-Cas9 기술을 이용하여 면역원을 안정하게 발현하는 세포주를 만들고, 이를 이용하여 계속해서 동일한 재조합 항원을 발현 정제할 수 있는 방법을 이용하였다. 단백질 발현이 활발히 이루어지는 크로모좀 위치로 보고된 크로모좀 19번 AAVS 위치에 homology directed repair (HDR) 방법을 이용하여 재조합 항원 발현 벡터를 삽입(integration) 시키는 방법을 이용하였다 (도 1 참조). 재조합 항원 발현율을 증가시키기 위하여 AAVS 위치와 유사하게 단백질을 과 발현시킬 수 있는 크로모좀 5번 SEMA6A 위치를 이용하여 동시에 두 위치에 같은 재조합 항원 발현 벡터를 삽입할 수 있는 시스템을 개발하였다(도 1 참조). To date, protein expression in eukaryotic cells has been achieved through transient expression. In the present invention, a cell line stably expressing an immunogen is made using CRISPR-Cas9 technology, and a method capable of continuously expressing and refining the same recombinant antigen is used. A method of integrating a recombinant antigen expression vector using a homology directed repair (HDR) method to the chromosome 19 AAVS site reported as the chromosomal site where protein expression is actively used was used (see FIG. 1). In order to increase the expression rate of the recombinant antigen, a system capable of inserting the same recombinant antigen expression vector into two positions at the same time using the chromosome 5 SEMA6A position capable of overexpressing the protein similar to the AAVS position was developed (see FIG. 1). ).

Cas9 단백질 발현 플라스미드와 AAVS 및 SEMA6A 위치를 타겟팅하는 sgRNA를 발현하는 플라스미드를 293 세포에 트랜스팩션 해주면 원하는 위치의 DNA를 모두 자를 수 있고, 같이 트랜스팩션한 재조합 항원 발현 플라스미드에 있는 왼쪽과 오른쪽 homology arm을 이용하여 HDR이 일어나서 원하는 유전자를 정확한 위치로 삽입할 수 있는 시스템을 개발하였다. 도 1의 재조합 항원 발현 플라스미드에 왼쪽과 오른쪽 homology arm을 표시하였다.By transfecting the Cas9 protein expression plasmid and the sgRNA expressing sgRNA targeting the AAVS and SEMA6A positions into 293 cells, the DNA at the desired position can be cut, and the left and right homology arms in the transfected recombinant antigen expression plasmid can be cut together. Using HDR, a system capable of inserting a desired gene into an accurate position has been developed. The left and right homology arms are shown in the recombinant antigen expression plasmid of FIG. 1.

재조합 항원을 생산하기 위해서 타겟 단백질은 메르스바이러스의 스파이크 단백질 안에 위치한 수용체결합부위(receptor binding domain, RBD)를 사용하였다. 여기에 단백질 정제를 위해 필요한 6개의 histidine 아미노산 tag을 C-terminal에 fusion 시켰다(도 2. 면역원 발현 벡터 1). 인간 항체의 Fc 부분은 재조합 항원을 체내에서 안정화시키는 기능을 가지고 있어서 RBD 단백질 뒤에 fusion 시켜서 면역원 발현 벡터 2로 제작하였다(도 2. 면역원 발현 벡터 2). 인간 항체의 Fc 부분은 프로테인 G 컬럼을 통하여 순수 분리 정제가 가능하고 이를 본 발명에 사용하였다. 자연상태의 메르스바이러스 스파이크 단백질은 글리코실화(glycosylation)가 된다고 알려져 있다. 따라서, 재조합 메르스바이러스 RBD 항원도 같은 배출 과정을 거치면서 글리코실화가 일어나도록 두 개의 재조합 항원 N-말단에는 글리코실화를 유도하는 signal peptide를 융합시켰다. 세포 밖으로 배출된 재조합 항원을 정제하면, 세포 안에서 발현되는 단백질을 정제하면서 겪을 수 있는 다른 오염 단백질들의 양을 줄일 수 있어 효율적이고 순수하게 단백질을 정제할 수 있는 장점이 있다.In order to produce a recombinant antigen, a target protein was used as a receptor binding domain (RBD) located in the spike protein of MERS virus. Here, six histidine amino acid tags necessary for protein purification were fused to the C-terminal (Fig. 2. Immunogen expression vector 1). Since the Fc portion of the human antibody has a function of stabilizing the recombinant antigen in the body, it was fusion followed by the RBD protein to produce it as immunogen expression vector 2 (Fig. 2 immunogen expression vector 2). The Fc portion of the human antibody can be purified and purified purely through a Protein G column and used in the present invention. It is known that the natural MERS virus spike protein is glycosylated. Therefore, a signal peptide that induces glycosylation was fused to the two recombinant antigen N-terminus so that the glycosylation occurs while the recombinant MERS virus RBD antigen also undergoes the same discharge process. Purifying the recombinant antigen discharged out of the cell has the advantage of efficiently and purely purifying the protein because it reduces the amount of other contaminating proteins that can be experienced while purifying the protein expressed in the cell.

실제로 재조합 항원 발현 안정 세포주를 만들기 위하여 먼저 293 세포의 AAVS 위치에 항원 발현 플라스미드를 삽입시켰다. Hygromycin resistant gene이 같이 삽입되어 원하는 세포만 세포 배양 배지에 hygromycin을 첨가하여 선별하였다. 이렇게 생성된 hygromycin resistant 콜로니 중에서 면역원을 효율적으로 발현하는 세포를 웨스턴 방법으로 골라내고 이를 확장하여 면역원 발현 세포로 사용하였다. 또한, 두 위치에 동시에 삽입된 세포를 만들기 위해서 AAVS에서 얻어진 세포에 동일한 면역원을 발현하는 SEMA6A 위치용 벡터를 이용하여 유사한 방법으로 세포를 선별하였다. SEMA6A 벡터는 puromycin resistant gene이 같이 삽입되어서 세포 배양 배지에 hygromycin과 puromycin을 같이 첨가하여 세포를 선별하였다. 살아남은 세포들 중에서 면역원 단백질 발현이 높은 세포를 골라서 면역원을 대량 발현하는데 사용하였다.In fact, in order to make a recombinant antigen-expressing stable cell line, an antigen-expressing plasmid was first inserted into the AAVS position of 293 cells. Hygromycin resistant gene was inserted together, and only desired cells were selected by adding hygromycin to the cell culture medium. Among the hygromycin resistant colonies thus produced, cells expressing the immunogen efficiently were selected by Western method and expanded to use as immunogen expressing cells. In addition, cells were selected in a similar manner using a SEMA6A position vector expressing the same immunogen in cells obtained from AAVS to make cells inserted at both positions simultaneously. In the SEMA6A vector, puromycin resistant gene was inserted together, and hygromycin and puromycin were added to the cell culture medium to select cells. Among the surviving cells, cells with high immunogen protein expression were selected and used for mass expression of the immunogen.

<< 실시예Example 2> 두 위치에 동시에 삽입한 재조합 항원 발현 세포에서 단백질 발현율 증가 확인 2> Confirmation of increase in protein expression rate in recombinant antigen-expressing cells inserted simultaneously in two positions

상기 실시예 1에서 제작된 AAVS와 SEMA6A 두 위치에 동시에 삽입된 재조합 항원 발현 세포(AACY + SEMA6A)가 AAVS 위치 1곳에만 발현하는 세포(AACY)보다 재조합 항원의 발현율이 증가하는지 알아보기 위하여 확인 실험을 진행하였다. 세포 안에서 만들어진 면역원 단백질이 세포 밖으로 배출되도록 제작한 시스템이어서 세포 안의 단백질의 양과 세포 밖 배지에 존재하는 단백질의 양을 비교하였다. 도 2의 면역원 발현 벡터 2번을 이용하여 실험한 결과를 보여주고 있다. 이 항원(MERS-RBD-Fc)의 경우 인간 항체의 Fc 부분이 fusion 되어 있어서, 웨스턴 블로팅 방법으로 인간 항체에 대한 2차 항체를 이용하면 단백질 발현을 확인할 수 있다.Recombinant antigen-expressing cells (AACY + SEMA6A) inserted simultaneously in the two positions of AAVS and SEMA6A prepared in Example 1 were compared with cells expressing only in one AAVS position (AACY) to confirm whether the expression of the recombinant antigen increased. Proceeded. This is a system designed to discharge the immunogen protein produced in the cell out of the cell, so the amount of the protein in the cell and the amount of the protein in the extracellular medium were compared. The results of experiments using the immunogen expression vector 2 of FIG. 2 are shown. In the case of this antigen (MERS-RBD-Fc), the Fc portion of the human antibody is fused, so that protein expression can be confirmed by using a secondary antibody against the human antibody by Western blotting.

도 3의 결과에서 나타난 바와 같이 AACY의 경우보다 AACY + SEMA6A의 경우가 세포 내에서의 발현(Cell pellet)과 세포 밖으로 secretion 된(supernatant) 항원 모두 발현이 2배 이상 증가하는 것을 확인하였다. 이러한 단백질 발현 증가는 항원 생산율의 증가로 이어져 실제 항원 생산에 있어서 경제성을 상승시킨다.As shown in the results of FIG. 3, it was confirmed that in the case of AACY + SEMA6A, the expression of both cell expression (Cell pellet) and the antigen secreted out of the cell (supernatant) increased by 2 times or more. This increase in protein expression leads to an increase in the antigen production rate, which increases the economic efficiency in actual antigen production.

<< 실시예Example 3> 재조합 항원( 3> Recombinant antigen ( 메르스바이러스MERS virus 수용체결합부위Receptor binding site ) ) 글리코실화Glycosylation (glycosylation) 확인 (glycosylation) check

메르스 바이러스의 signal peptide에 의해서 세포밖으로 secretion 된 재조합 항원이 glycosylation이 잘 일어났는지 확인하기 위하여 N-linked glycan을 제거할 수 있는 PNGase F를 이용하여 실험하였다.Recombinant antigen secreted out of the cell by the signal peptide of MERS virus was tested using PNGase F, which can remove N-linked glycan to confirm that glycosylation occurred well.

PNGase F를 이용하여 glycosylation 된 부분을 제거하면 단백질의 전체 무게가 감소하고, 이러한 glycosylation이 제거된 단백질이 SDS-PAGE gel 상에서 더 빨리 이동하게 되어 이러한 크기 차이를 비교하여 glycosylation이 되었는지 확인할 수 있는 실험이다. 실험에 사용한 재조합 항원은 도 2의 면역원 발현 벡터 1에 의해서 만들어진 메르스바이러스 수용체결합부위 단백질이다(MERS-RBD).When the glycosylation portion is removed using PNGase F, the total weight of the protein decreases, and the protein from which the glycosylation has been removed moves faster on the SDS-PAGE gel. . The recombinant antigen used in the experiment is a MERS virus receptor binding site protein produced by the immunogen expression vector 1 of FIG. 2 (MERS-RBD).

메르스바이러스 수용체결합부위 단백질에 PNGase F를 처리하고, 웨스턴 블로팅을 수행하였다. 면역원을 인지할 수 있는 anti-His tag 항체를 이용하여 단백질을 확인하였다. 도 3에서 확인한 바와 같이 PNGase F를 처리한 면역원 단백질(MERS-RBD deglycosylated)이 처리하지 않은 면역원 단백질(MERS-RBD glycosylated)보다 작은 크기로 검출되는 것으로 보아 glycosylation이 잘 유도됨을 확인하였다. 바이러스의 표면 단백질과 유사하게 glycosylation을 유도하여 특이적으로 바이러스를 중화시킬 수 있는 항체를 형성하는데 도움이 될 수 있는 면역원으로서 사용 가능할 것이다.PNGase F was treated with the MERS virus receptor binding site protein, and Western blotting was performed. Proteins were identified using anti-His tag antibodies capable of recognizing immunogens. As shown in FIG. 3, it was confirmed that glycosylation was well induced because the immunogen protein (MERS-RBD deglycosylated) treated with PNGase F was detected to be smaller in size than the untreated immunogen protein (MERS-RBD glycosylated). It may be used as an immunogen capable of inducing glycosylation similar to the surface protein of the virus and specifically helping to form an antibody capable of neutralizing the virus.

<< 실시예Example 4> 항원( 4> antigen ( 메르스바이러스MERS virus 수용체결합부위Receptor binding site , , MERSMERS -- CoVCoV RBDRBD ) 발현 세포 대량 배양 및 단백질 정제와 이를 통한 단백질 발현 수율 확인) Mass cell culture and protein purification and protein expression yield confirmation

실시예 1에서 선별된 세포주들을 대량으로 증식하여 면역원을 분리하였다. 세포를 키울 때 사용되는 소태아 혈청의 경우 여러 단백질들이 고농도로 포함되어 있어서 항원 단백질의 정제에 방해가 될 수 있어서, 혈청 없이 293 세포를 키울 수 있는 serum free media를 이용하여 면역원 발현 세포주들을 대량 배양하였다. 시그마 회사의 Excell 293 serum free media 혹은 ThermoFisher Scientific 회사의 FreeStyle 293 expression media를 사용하였다. Sigma 회사의 CELLINE Classic bioreactor flask의 경우 항체나 세포의 단백질 발현에 많이 사용되는 flask로 중간에 삽입된 membrane을 통해서 세포의 증식에 필요한 배지는 자유롭게 이동하지만 세포나 세포 밖으로 배출된 단백질들은 이동하지 못한다. 많은 수의 세포를 적은 볼륨 안에서 배양이 가능한 장점이 있고 고농도의 단백질을 얻을 수 있어서 정제 과정에서 훨씬 고순도의 단백질 정제에 도움이 된다. 원심분리기를 이용하여 세포와 세포배양액을 분리한 후 세포배양액 속에 있는 재조합 항원을 정제하였다. MERS-RBD 단백질은 Ni-NTA column (QIAGEN)을 이용하여 정제하였고, MERS-RBD-Fc 단백질은 protein G-column (GE healthcare)을 이용하여 정제하였다. 도 5에서 대량 정제된 재조합 항원을 은 염색법(silver staining)으로 염색하여 확인한 결과를 제시한다. 각 항원 단백질의 수율은 도 5의 테이블에 정리하였다. 두 위치에 삽입한 세포에서는 (AACY + SEMA6A) 수율이 2배 이상 증가하는 것을 확인할 수 있었다.The cell lines selected in Example 1 were proliferated in large quantities to isolate the immunogen. In the case of fetal bovine serum used to grow cells, a large number of proteins are contained, which may interfere with the purification of antigenic proteins, and thus, immunogen-expressing cell lines can be mass cultured using serum free media that can grow 293 cells without serum. Did. Sigma's Excell 293 serum free media or ThermoFisher Scientific's FreeStyle 293 expression media was used. In the case of Sigma's CELLINE Classic bioreactor flask, it is a flask that is frequently used for protein expression of antibodies or cells. The medium required for cell proliferation moves freely through the membrane inserted in the middle, but proteins released from cells or cells cannot. It has the advantage of culturing a large number of cells in a small volume and can obtain a high concentration of protein, which helps to purify a much higher purity protein in the purification process. After separating the cells from the cell culture medium using a centrifuge, the recombinant antigen in the cell culture medium was purified. The MERS-RBD protein was purified using Ni-NTA column (QIAGEN), and the MERS-RBD-Fc protein was purified using protein G-column (GE healthcare). In FIG. 5, the results obtained by staining the recombinant antigen purified in large quantities by silver staining are presented. The yield of each antigenic protein is summarized in the table in FIG. 5. In the cells inserted in both positions, it was confirmed that the yield (AACY + SEMA6A) increased more than 2 times.

<< 실험예Experimental example 5> 정제된 항원( 5> purified antigen ( 메르스바이러스MERS virus 수용체결합부위Receptor binding site , , MERSMERS -- CoVCoV RBDRBD ) 순도 분석) Purity Analysis

정제된 재조합 항원들의 순도를 측정하기 위하여 RP-HPLC, Gel filtration 방법을 통하여 분석하였다. 도 6에서는 MERS-RBD 항원과 MERS-RBD-Fc 항원의 순도를 RP-HPLC 방법으로 분석한 그래프이다. 단 1회의 column 정제만을 이용하여서도 94% 이상의 고순도의 항원들이 정제된 것을 확인할 수 있었다. 정제된 각 항원들의 순도를 그래프 밑의 표로 제시하였다. Gel filtration 방법을 통하여 추가적으로 MERS-RBD-Fc 항원의 순도를 분석하였다(도 7). MERS-RBD-Fc 면역원의 경우 대부분 single peak를 나타내는 것으로 확인되어 순도가 높은 항원임을 확인하였다.In order to measure the purity of the purified recombinant antigen, it was analyzed through RP-HPLC, Gel filtration method. 6 is a graph analyzing the purity of MERS-RBD antigen and MERS-RBD-Fc antigen by RP-HPLC method. It was confirmed that even with only one column purification, more than 94% of high-purity antigens were purified. The purity of each purified antigen is presented in a table below the graph. The purity of the MERS-RBD-Fc antigen was additionally analyzed through a gel filtration method (FIG. 7). In the case of MERS-RBD-Fc immunogen, it was confirmed that most of them showed a single peak, and thus it was confirmed that the antigen was high in purity.

이상의 설명은 본 발명을 예시적으로 설명한 것에 불과한 것으로, 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자라면 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 다양한 변형이 가능할 것이다. 따라서, 본 명세서에 개시된 실시 예들은 본 발명을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시 예에 의하여 본 발명의 사상과 범위가 한정되는 것은 아니다.The above description is merely illustrative of the present invention, and those skilled in the art to which the present invention pertains will be capable of various modifications without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed herein are not intended to limit the present invention, but to explain the present invention, and the spirit and scope of the present invention are not limited by these embodiments.

본 발명의 보호범위는 아래의 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술은 본 발명의 권리범위에 포함하는 것으로 해석되어야 할 것이다.The scope of protection of the present invention should be interpreted by the following claims, and all technologies within the equivalent range should be interpreted as being included in the scope of the present invention.

본 발명은 다양한 바이러스에 대한 재조합 면역원, 백신 개발에 유용하게 사용될 수 있어 산업상 이용가능성이 있다.The present invention has industrial applicability because it can be usefully used for the development of recombinant immunogens and vaccines against various viruses.

Claims (18)

진핵 세포에서 항원의 안정적인 과 발현 효율 증대를 위해 CRISPR 유전자 편집 기술인 homology-directed repair 방법으로 진핵 세포 크로모좀의 다중 위치에 재조합 항원을 발현시키는 벡터를 동시에 다중 삽입시키는 방법In order to increase the stable overexpression efficiency of antigens in eukaryotic cells, a method of simultaneously inserting a vector expressing a recombinant antigen into multiple positions of eukaryotic cell chromosomes using the CRISPR gene editing technology homology-directed repair method 제1항에 있어서, 상기 항원은 메르스 바이러스의 항원인 방법The method of claim 1, wherein the antigen is an antigen of the MERS virus. 제2항에 있어서, 상기 메르스 바이러스 항원은 스파이크 단백질 안에 위치한 수용체 결합부위(receptor binding domain; RBD)인 방법The method of claim 2, wherein the MERS virus antigen is a receptor binding domain (RBD) located in a spike protein. 제3항에 있어서, 상기 RBD 항원은 단백질 정제를 위해 필요한 6개의 histidine 아미노산 tag을 C-terminal에 fusion시킨 것인 방법The method according to claim 3, wherein the RBD antigen is obtained by fusion of six histidine amino acid tags necessary for protein purification to the C-terminal. 제3항에 있어서, 상기 RBD 항원은 Fc 부분을 fusion시킨 것인 방법The method according to claim 3, wherein the RBD antigen is obtained by fusion of an Fc portion. 제3항에 있어서, 상기 RBD 항원은 N-말단에 글리코실화를 유도하는 signal peptide가 융합된 것인 방법The method according to claim 3, wherein the RBD antigen is fused with a signal peptide that induces glycosylation at the N-terminus. 제1항에 있어서, 벡터는 AAVS 및 SEMA6A 위치를 타겟팅하는 sgRNA를 포함하는 방법The method of claim 1, wherein the vector comprises sgRNA targeting AAVS and SEMA6A positions. 제1항에 있어서, 벡터는 Cas 단백질 발현 서열을 포함하는 방법The method of claim 1, wherein the vector comprises a Cas protein expression sequence. 제8항에 있어서, 상기 Cas 단백질은 Cas9 단백질인 방법The method of claim 8, wherein the Cas protein is a Cas9 protein. 제1항에 있어서, Hygromycin resistant gene 부위를 더 포함하는 방법 The method of claim 1, further comprising a Hygromycin resistant gene site. 제1항에 있어서, Puromycin resistant gene 부위를 더 포함하는 방법The method of claim 1, further comprising a Puromycin resistant gene site. 제1항에 있어서, 상기 진핵 세포는 293 세포인 방법The method of claim 1, wherein the eukaryotic cell is 293 cells. 메르스 바이러스 항원을 포함하는 CRISPR-Cas 시스템을 발현할 수 있는 벡터A vector capable of expressing the CRISPR-Cas system containing the MERS virus antigen 제13항에 있어서, AAVS 위치를 타겟팅하는 sgRNA를 포함하는 벡터14. The vector of claim 13, comprising a sgRNA targeting the AAVS position. 제13항에 있어서, SEMA6A 위치를 타겟팅하는 sgRNA를 포함하는 벡터14. The vector of claim 13, comprising a sgRNA targeting the SEMA6A position. 제13항에 있어서, AAVS 및 SEMA6A 위치를 타겟팅하는 sgRNA를 포함하는 벡터14. The vector of claim 13 comprising sgRNA targeting AAVS and SEMA6A positions. 제13항에 있어서, 상기 Cas는 Cas9인 벡터The vector according to claim 13, wherein the Cas is Cas9. 제13항에 있어서, 상기 메르스 바이러스 항원은 스파이크 단백질 안에 위치한 수용체 결합부위(receptor binding domain; RBD)인 벡터The vector of claim 13, wherein the MERS virus antigen is a receptor binding domain (RBD) located in a spike protein.
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