KR102121817B1 - Vectors for expressing recombinant antigens using CRISPR and method for simultaneously multiplexing thereof - Google Patents

Abstract

The present invention provides a method of simultaneously inserting multiple vectors expressing a recombinant antigen into multiple positions of eukaryotic cell chromosomes through the CRISPR technology Homology directed recombination method. By inserting into two places at the same time, after selecting a human cell line capable of stably and overexpressing an immunogen, and using these cells to express a recombinant protein that can be used as an immunogen, it relates to various viruses that are socially problematic. It can be useful for the development of recombinant immunogens and vaccines.

Description

{Vectors for expressing recombinant antigens using CRISPR and method for simultaneously multiplexing thereof}

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.

In general, when proceeding with 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, glycosylation is induced differently from protein expression in eukaryotic cells such as human cells, and thus there is a limit in producing proteins requiring excessive glycosylation (Non-Patent Document 1). 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 (Non-Patent Document 2). 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 use a method of selecting and purifying a cell line capable of stably expressing a recombinant immunogen in human cells to obtain and continuously produce a recombinant immunogen. Homology-directed repair method of CRISPR gene editing technology is used to produce a cell line stably expressing an immunogen. In Non-Patent Document 3, 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, according to Non-Patent Document 4, 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 at the same time at two sites where expression is actively occurring in one cell.

Meanwhile, stably securing various recombinant antigens is a very important process in various studies using recombinant proteins. However, the technique of inducing stable overexpression of a recombinant antigen with the technique so far is very difficult, and no technique has been known to overcome this problem.

1.Nat Chem Biol (2012) 8(5): 434-436 2.Vaccine 32 (2014) 6170-6176 3.Nature Biotechnology 33 (2015) 543-550 4. Biotechnol. J 11 (2016) 1100-1109

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.

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.

In order to solve the above object,

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 technique, for inducing stable overexpression of a recombinant antigen in eukaryotic cells.

In addition, the present invention provides a method of simultaneously inserting multiple vectors expressing a recombinant antigen at multiple positions of eukaryotic chromosomes by a homology-directed repair method, which is a CRISPR gene editing technology, for increasing the stable overexpression efficiency of antigens in eukaryotic cells. .

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 to the Fc portion, or a signal peptide to induce glycosylation at the N-terminus.

In addition, the vector of the present invention includes sgRNAs targeting AAVS and SEMA6A positions, and preferably includes 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.

In addition, the present invention provides a vector capable of expressing the CRISPR-Cas system comprising the MERS virus antigen.

The vector includes sgRNA targeting AAVS and/or SEMA6A positions, and it is preferred that 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.

The present invention has the effect of producing a recombinant antigen in a eukaryotic cell stably and with high efficiency.

In addition, 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 into a specific position (chromosome 5 and 9) of a eukaryotic cell chromosome by a homology-directed repair method of CRISPR-cas9 editing technology. 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.
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 cells (Cell pellet) and outside (Supernatant) 2 It is a result of Western blotting showing an increase of more than doubled.
4 is a result of Western blotting demonstrating that the antigen expressed in Example 3 and released into the cell (Supernatant) is glycosylation.
5 is a result showing the high-purity purification of the antigen through silver staining (silver staining) after purification in large quantities of the antigen of Example 4 (mers virus receptor binding site, MERS-CoV RBD). The table below shows the high efficiency of the purified antigen.
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.
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.

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.

<Example 1> Recombinant antigen (mers virus receptor binding site) expression stable cell line selection

In order to produce a recombinant antigen (mers virus receptor binding site) stable cell line according to the present invention, experiments were conducted as follows.

To date, protein expression in eukaryotic cells has been achieved through transient expression. In the present invention, a cell line that stably expresses 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 a 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 simultaneously inserting the same recombinant antigen expression vector into two positions using a chromosome 5 SEMA6A position capable of overexpressing a protein similar to the AAVS position was developed (see FIG. 1 ). ).

By transfecting the Cas9 protein expression plasmid and the sgRNA expressing sgRNA targeting the AAVS and SEMA6A sites 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 are transfected. 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.

In order to produce a recombinant antigen, the target protein used was 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 fused after the RBD protein to produce an 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 N-terminus of two recombinant antigens so that the glycosylation occurs while the recombinant MERS virus RBD antigen undergoes the same discharge process. Purifying the recombinant antigen discharged out of the cell has an advantage of efficiently and purely purifying the protein by reducing the amount of other contaminating proteins that may be experienced while purifying the protein expressed in the cell.

In order to make a recombinant antigen-expressing stable cell line, the 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 generated, cells expressing the immunogen efficiently were selected by Western method and expanded to be used 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.

<Example 2> Confirmation of increase in protein expression rate in recombinant antigen-expressing cells inserted simultaneously in two positions

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 rate of the recombinant antigen increased. Proceeded. Since the immunogen protein produced in the cell was designed to be discharged out of the cell, 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.

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 secreted (supernatant) antigen out of the cell increased more than 2 times than that of AACY. This increase in protein expression leads to an increase in the antigen production rate, which increases the economic efficiency in actual antigen production.

<Example 3> Recombinant antigen (mers virus receptor binding site) glycosylation confirmation (glycosylation)

To confirm that glycosylation of the recombinant antigen secreted out of the cell by the signal peptide of the MERS virus occurred well, experiments were performed using PNGase F, which can remove N-linked glycan.

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. to be. 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 is well induced because the immunogen protein (MERS-RBD deglycosylated) treated with PNGase F is detected in a smaller size than the untreated immunogen protein (MERS-RBD glycosylated). It may be used as an immunogen that can help to form antibodies capable of specifically neutralizing the virus by inducing glycosylation similar to the surface protein of the virus.

<Example 4> Mass culture of cells expressing antigen (MERS virus receptor binding site, MERS-CoV RBD) and protein purification and protein expression yield confirmation through this

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, many proteins are contained at high concentrations, which may interfere with the purification of antigenic proteins, and thus, mass production of immunogen-expressing cell lines 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. Sigma's CELLINE Classic bioreactor flask 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 the proteins released from the 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 and the cell culture medium using a centrifuge, the recombinant antigen in the cell culture medium was purified. MERS-RBD protein was purified using Ni-NTA column (QIAGEN), and 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 at both positions, it was confirmed that the yield (AACY + SEMA6A) increased more than 2 times.

<Experimental Example 5> Purified antigen (MERS virus receptor binding site, MERS-CoV RBD) purity analysis

In order to measure the purity of the purified recombinant antigen, it was analyzed by 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, 94% or more 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 further 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 highly purified.

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.

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.

Claims (18)

As a method of expressing a recombinant antigen in multiple positions of eukaryotic chromosomes by a homology-directed repair method, which is a CRISPR gene editing technology, to increase the stable overexpression efficiency of antigens in vitro eukaryotic cells,
A method of expressing a recombinant antigen by multiplexing simultaneously using a vector expressing a recombinant antigen, a vector expressing each sgRNA targeting AAVS and SEMA6A positions, and a Cas 9 protein expression vector The method of claim 1, wherein the antigen is a MERS virus antigen. The method of claim 2, wherein the MERS virus antigen is a receptor binding domain (RBD) located in a spike protein. The method according to claim 3, wherein the RBD antigen is a fusion of six histidine amino acid tags necessary for protein purification to the C-terminal. The method according to claim 3, wherein the RBD antigen is a fusion of an Fc portion. The method according to claim 3, wherein the RBD antigen is fused to an N-terminal signal peptide inducing glycosylation. delete delete delete The method of claim 1, further comprising a hygromycin resistant gene site in the vector expressing the recombinant antigen. The method of claim 1, further comprising a Puromycin resistant gene site in the vector expressing the recombinant antigen. The method of claim 1, wherein the eukaryotic cell is 293 cells.
CRISPR-Cas for inserting a MERS virus antigen at AAVS and SEMA6A positions, including a vector expressing the MERS virus antigen, a vector expressing each sgRNA targeting the AAVS and SEMA6A positions, and a Cas 9 protein expression vector 9 systems delete delete delete delete The system of claim 13, wherein the MERS virus antigen is a receptor binding domain (RBD) located in a spike protein.

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