JP2006087427A - Method for producing heterooligomer protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stably producing a recombinant heterooligomer protein by preparing a transformed insect cell stably expressing the heterooligomer protein. <P>SOLUTION: This method for producing the oligomer protein composed of heterologous sub-units using the insect cell comprises the following steps. a step of transforming the insect cell with a plurality of vectors each expressing one of the heterologous sub-units, a step of culturing the transformed insect cell and a step of recovering the oligomer protein from the cultured product. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for stably producing a hetero-oligomeric protein using insect cells.

  Various methods for highly expressing proteins in microorganisms, animal cells and the like using genetic recombination techniques have been studied. Higher eukaryotic proteins undergo post-translational modifications (for example, addition of sugar chains, fatty acids, etc., phosphorylation) during expression, retain higher-order structures, and have biological activity (function). Therefore, when a higher eukaryotic protein is produced by a DNA recombination technique, it is preferable to use an animal cell that is a higher eukaryote as a host. However, since animal cells are difficult to handle and grow slowly, they are not well suited for substance production. Therefore, a protein expression system using an insect cell having post-translational modification activity as a host has been studied.

  Insect cell-baculovirus systems are generally used for protein production using insect cells as hosts. Baculovirus (nucleopolyhedrovirus) is a virus that produces a large amount of a protein called polyhedrin in infected insect cells. This insect cell-baculovirus system is a recombinant protein expression system that infects cultured insect cells with a recombinant baculovirus having a foreign gene inserted downstream of a polyhedrin promoter, and produces a foreign protein in the virus-infected cell. . However, since the cells are killed by virus infection, the target protein cannot be produced continuously.

  In addition to this insect cell-baculovirus system, various insect host-vector systems have been developed. For example, a human tissue plasminogen activator (tPA) gene is introduced into a vector having a baculovirus BmNPV-derived enhancer IE1 and a BmNPV-derived trans-acting factor HR3 upstream of the silkworm-derived actin promoter, and insect cells are transformed. There is an example in which tPA is expressed (Non-patent Document 1), but the protein is not necessarily stably produced.

Among proteins, when expressing oligomeric proteins composed of heterologous subunits (hereinafter referred to as hetero-oligomeric proteins), it is often necessary to use multiple vectors and to introduce a plasmid having a selection marker. It is difficult to obtain a transformant having the target gene. Moreover, it is difficult to maintain a vector in the obtained transformant, and it is difficult to stably produce a hetero-oligomer protein.
Farrell et al., Biotechnol. Bioeng., 64, 426-433 (1999)

  An object of the present invention is to provide a method for producing a recombinant heterooligomeric protein stably by producing a transformed insect cell capable of stably expressing a heterooligomeric protein.

  The present invention provides a method for producing an oligomeric protein composed of heterologous subunits using insect cells, wherein the method comprises a plurality of vectors capable of expressing each one of the heterologous subunits. Transforming the cells; culturing the transformed insect cells; and recovering the oligomeric protein from the culture.

  In a preferred embodiment, the oligomeric protein is an antibody.

  In a more preferred embodiment, each of the heterologous subunits is at least a partial peptide of the heavy chain constituting the antibody and at least a partial peptide of the light chain constituting the antibody.

  In another preferred embodiment, the plurality of vectors each have the same or different promoter.

  In still another preferred embodiment, at least one of the plurality of vectors has the insect cell selection marker.

  The present invention also provides a transformed insect cell that stably expresses an oligomeric protein composed of a heterologous subunit, wherein the insect cell has a plurality of vectors each capable of expressing one of the heterologous subunits. .

  In a preferred embodiment, at least one of the plurality of vectors has a selection marker for the insect cells.

  By transforming insect cells with a plurality of vectors capable of expressing each one of the heterologous subunits, a hetero-oligomeric protein is efficiently produced. Furthermore, since at least one of the plurality of vectors has an insect cell selection marker, transformation efficiency is improved, and the transformed cells can stably produce hetero-oligomeric proteins.

  First, various materials used in the present invention will be described.

(Host cell)
Any host cell can be used as long as it is an insect cell. Commercially available insect cell lines are also used. For example, BTI-TN5B1-4 (High Five) strain derived from Trichoplusia ni, Sf9 strain derived from Spodoptera frugiperda, etc. are preferably used. These strains are available from Invitrogen.

(Plasmid, vector)
In the present invention, any plasmid that can be expressed in insect cells is used without particular limitation. Preferably, a baculovirus-based plasmid is used. From the viewpoint of high expression efficiency, a plasmid having an enhancer IE1 derived from Baculovirus BmNPV and a trans-acting factor HR3 derived from BmNPV is more preferred upstream of the silkworm-derived actin promoter. Examples of such plasmids include pXINSECT-DEST38 and pXINSECT-DEST39. These plasmids are available from Invitrogen.

  A plasmid having an IE-2 promoter derived from Orgya pseudotsugata nuclear polyhedrosis virus is also preferably used. An example of such a plasmid is pIB / V5-His. This plasmid is available from Invitrogen.

  In addition, a plasmid having a selection marker is preferably used in that the selection efficiency of transformed cells and the stability of expression of the target gene can be improved. The aforementioned pIB / V5-His has a gene resistant to blasticidin and is preferable as a plasmid having a selection marker.

(Hetero-oligomer protein)
In the present invention, the hetero-oligomer protein refers to an oligomer protein composed of heterologous subunits. Examples of hetero-oligomeric proteins include antibodies, enzymes, receptors, ion channels, hemoglobin, blood clotting factors, and the like. Taking an antibody as an example, the antibody is a hetero-oligomeric protein composed of an H chain and an L chain. The H chain constituting the antibody may be full length, or may be a sequence of at least a part of the H chain, for example, a part of the H chain such as V _H domain, Fd fragment, Fc fragment. Further, it may be a fragment optionally containing these. The full length of the L chain constituting the antibody may be a _VL domain capable of interacting with the H chain. The H and L chains are selected so that the H and L chains can interact.

(Construction of vector)
In the present invention, in order to produce a hetero-oligomeric protein, transformation (co-transfection) is performed using a plurality of vectors each capable of expressing one of the heterologous subunits. Each of the plurality of vectors used in the present invention comprises a promoter and a DNA sequence encoding one different subunit constituting the hetero-oligomeric protein to be expressed. These vectors may further contain a signal sequence for secretion. For example, taking the case of a hetero-oligomeric protein composed of two subunits as an example, two vectors are used: a vector capable of expressing one subunit and a vector capable of expressing the other subunit. Each of the plurality of vectors is constructed so as to have a signal sequence for secretion and a DNA sequence encoding one subunit. These plurality of vectors may be those in which DNA sequences encoding different subunits are inserted into plasmids having the same structure, and DNA sequences encoding different subunits are inserted in plasmids having different structures. It may be what was done.

  When constructing a vector using plasmids having different structures, plasmids having different promoters may be used. Expression efficiency can be increased by expressing each subunit under the control of a different promoter. When expressing a hetero-oligomeric protein composed of two different subunits, in a preferred embodiment, either one of the two vectors has a silkworm-derived actin promoter and the other is an Orgya pseudotsugata nuclear polyhedrosis. It has a virus-derived IE-2 promoter.

  In addition, for the purpose of enhancing the selection efficiency of the transformant and the stability of expression of the target gene, it is preferable that at least one vector is constructed so as to contain a selection marker gene. The selection marker gene inherent in the plasmid may be used, or the selection marker gene may be inserted from the outside. Examples of selectable marker genes include neomycin resistance gene, blasticidin resistance gene, hygromycin resistance gene, puromycin resistance gene, bleomycin resistance gene and the like. For example, since the aforementioned pIB / V5-His has a resistance gene for the IE-2 promoter derived from Orgya pseudotsugata nuclear polyhedrosis virus and Blasticidin, the vector used in the present invention is constructed. It can be preferably used. Furthermore, the fact that the vector used in the present invention can be constructed to have an actin promoter, IE1 enhancer, and HR3 transactivator, and a selectable marker gene (eg, neomycin resistance gene, blasticidin resistance gene, etc.) This is preferable in that the strain selection efficiency and the stability of the vector can be improved.

  Preferably, each of the plurality of vectors used in the present invention contains a selectable marker gene. These selectable marker genes may be the same or different between vectors. It is more preferable that the plurality of vectors each contain a different selection marker gene from the viewpoint of enhancing the selection efficiency of the transformed strain.

(Insect cell transformation)
Insect cell transformation will be specifically described by taking the case of expressing an antibody as an example. In the following description, for example, in a plasmid of pXINSECT-DEST38 having an actin promoter derived from silkworm, a DNA sequence encoding an H chain (or part thereof) or a DNA sequence encoding an L chain (or part thereof) The inserted and constructed vectors are designated as vector H-1 and vector L-1. Alternatively, a pIB / V5-His plasmid having an IE-2 promoter derived from Orgya pseudotsugata nuclear polyhedrosis virus and a blasticidin resistance gene, a DNA sequence encoding an H chain (or part thereof), or an L chain (or Vectors constructed by inserting a DNA sequence encoding a part thereof are referred to as vector H-2 and vector L-2, respectively.

  For transformation, a combination of vector H-1 and vector L-1 derived from the same plasmid, or a combination of vector H-2 and vector L-2 can be used. In this case, the vector H-1 (or H-2) that expresses the H chain and the vector L-1 (or L-2) that expresses the L chain may have a mass ratio of 2: 1 or more. preferable. For transformation, a combination of vectors derived from different plasmids, a combination of vector H-1 and vector L-2, or a combination of vector H-2 and vector L-1 is used. Also good. When a combination of vectors derived from these different plasmids is used, the promoter contained in each vector is also different, and the expression efficiency of the antibody can be increased.

  Furthermore, the combination of vector H-2 and vector L-2 derived from pIB / V5-His is advantageous in that both vectors have a blasticidin resistance gene that is a selectable marker. For example, when obtaining an antibody-producing strain using a commercially available insect cell transformation kit, two different vectors (vectors H-1 and L-1) in which H chain and L chain are introduced into two pXINSECT-DEST38, respectively Then, a plasmid having a neomycin resistance gene is cotransfected to select cells that grow in the presence of neomycin. However, since all neomycin-resistant strains grow, including cells into which only the neomycin-resistant gene has been introduced, the efficiency of selection of cells into which the three genes have been introduced simultaneously becomes poor. However, when the vector H-2 or vector L-2 having a selectable marker (for example, blasticidin resistance gene) is used, the blasticidin resistant strain expresses at least the H chain or L chain, so that the selection efficiency of the transformed strain is improved. To do.

  A combination of a vector H-1 and a vector L-2, that is, a vector (vector H-1) in which a DNA sequence encoding an H chain (or a part thereof) is inserted into a plasmid having actin promoter derived from silkworm, Combination with a vector (vector L-2) in which a DNA sequence encoding an L chain (or part thereof) is inserted into a plasmid having an IE-2 promoter derived from Orgya pseudotsugata nuclear polyhedrosis virus and a blasticidin resistance gene Are preferably used. In this case, the ratio between the vector H-1 and the vector L-2 is preferably 10: 1 or more by weight, and more preferably 20: 1 or more. You may carry out by 50: 1 or more.

  As described above, a vector containing a selection marker gene can be constructed for the purpose of enhancing the selection efficiency of the transformant and the stability of expression of the target gene. Examples of such vectors are shown below. A DNA sequence encoding an H chain (or part thereof) or a DNA sequence encoding an L chain (or part thereof) in a vector having a silkworm-derived actin promoter and a neomycin resistance gene which is a selection marker gene The inserted and constructed vectors are designated as vector H-3 and vector L-3, respectively. On the other hand, a DNA sequence encoding an H chain (or a part thereof) or a DNA encoding an L chain (or a part thereof) in a vector having an actin promoter derived from silkworm and a blasticidin resistance gene which is a selection marker gene Vectors constructed by inserting sequences are referred to as vector H-4 and vector L-4, respectively.

  When using a plurality of vectors containing a silkworm-derived actin promoter and a selectable marker gene, transformation involves a combination in which each of different subunits is expressed and interacted (for example, H chain and L chain). Any combination can be used, if any. In the above case, vector H-3 and vector L-3, vector H-3 and vector L-4, vector H-4 and vector L-3, or vector H-4 and vector L-4 can be combined. In cotransfection, the ratio of the vector expressing the H chain and the vector expressing the L chain can be any weight ratio. The weight ratio is preferably 1: 1 or more, more preferably 2: 1 or more, further preferably 10: 1 or more, and still more preferably 20: 1 or more. You may carry out by 50: 1 or more.

  The means for transforming insect cells with these vectors is not particularly limited, and can be performed by means commonly used by those skilled in the art for insect cells. Specifically, it is carried out by the calcium phosphate method, lipofection (liposome method), electroporation (electroporation method) or the like. In this way, a transformed insect cell line capable of stably producing a hetero-oligomeric protein is obtained.

  Transformed insect cells can be selected using a selective medium. Selection using this selection medium may be performed using a selectable marker gene contained in a vector containing DNA encoding the subunit to be expressed, or by cotransfection with another vector having a selectable marker gene. . Antibiotics such as neomycin and blasticidin corresponding to the introduced selection marker gene can be added to the selection medium used for selecting transformed insect cells.

  In order to efficiently obtain stable transformed cells, it is necessary to culture the cells in the presence of an antibiotic concentration such that the cells into which the antibiotic resistance gene has not been introduced are completely killed. Usually, the antibiotic concentration for selecting stable transformed cells is optimally the concentration at which cells are completely killed in about one week. In view of this, the concentration at which the cells are given different antibiotics for selection of transformed cells can be determined as appropriate by one skilled in the art.

  For example, when at least one vector containing a blasticidin resistance gene as a selectable marker gene is used for cotransfection, the toxicity of blasticidin is strong. Therefore, if the ratio of the vector having the blasticidin resistance gene is reduced, the number of colonies formed decreases. This makes colony selection easier. The ratio of the vector having another antibiotic resistance gene (for example, neomycin resistance gene) and the vector having the blasticidin resistance gene during cotransfection can be any weight ratio, preferably 1: 1 or more, More preferably, it is 2: 1 or more, more preferably 10: 1 or more, still more preferably 20: 1 or more. You may carry out by 50: 1 or more.

  When vectors for expressing different subunits contain different selectable marker genes, cells having all of the genes to be expressed can be selected using antibiotics corresponding to the respective selectable marker genes. . For example, vector H-3 contains a neomycin resistance gene as a selectable marker gene, and vector L-4 contains a blasticidin resistance gene as a selectable marker gene. Cells introduced and resistant to blasticidin treatment have L-4 introduced. Cells that are resistant to both neomycin and blasticidin treatment have both H-3 and L-4, ie, both H and L chain genes introduced. In this way, selection of cells expressing all of the different subunits becomes more efficient. Of course, the same effect can be expected in the combination of the vector H-4 and the vector L-3.

  It can be confirmed that the obtained transformed cells produce a hetero-oligomeric protein using techniques well known to those skilled in the art, such as Western blotting and ELISA.

(Production of hetero-oligomeric protein)
Transformed insect cells that have been confirmed to produce hetero-oligomeric proteins are cultured under culture conditions commonly used by those skilled in the art. After the culture period, the produced hetero-oligomeric protein is recovered from the culture (culture supernatant) by an appropriate means according to the protein. The recovered protein can be appropriately purified as necessary.

  EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not restrict | limited to this Example.

  In the following examples, a Fab fragment of mouse-derived catalytic antibody 6D9 was used as a hetero-oligomeric protein. The Fab fragment of the catalytic antibody 6D9 is composed of an Fd fragment and an L chain, which are H chain portions thereof, and catalyzes the hydrolysis reaction of chloramphenicol monoester. The construction of the plasmid pARA7Fab containing the DNA sequence encoding this Fd fragment and L chain is described in Miyashita et al., J. Mol. Biol., 267, 1247-1257 (1997). This plasmid pARA7Fab was distributed by Professor Masao Fujii of the Institute of Advanced Science, Osaka Prefecture University.

Example 1
(Construction of plasmid expressing heavy chain)
The construction of plasmid pXIHA-6D9 Hc expressing the heavy chain is shown in FIG. First, a 6D9 Hc gene having a restriction enzyme AfeI recognition site and a Drosophila-derived BiP signal sequence in the upstream portion and a restriction enzyme XbaI recognition site in the downstream portion was prepared. This gene was amplified by PCR using plasmid pARA7Fab as a template and the primers of SEQ ID NOs: 1 and 2. The primer of SEQ ID NO: 1 has an AfeI recognition site and a BiP signal sequence, and the primer of SEQ ID NO: 2 has an XbaI recognition site. PCR was performed in the following cycle using Thermal cycler (BIO-RAD). First, it was treated at 94 ° C. for 2 minutes and at 72 ° C. for 2 minutes, and then 30 cycles of treatment at 94 ° C. for 30 seconds, 52.9 ° C. for 30 seconds, and 72 ° C. for 2 minutes, Finally, treatment at 72 ° C. for 5 minutes was performed. After confirming that the desired fragment was amplified by agarose gel electrophoresis, the 6D9 Hc gene was recovered by a method commonly used by those skilled in the art.

  Next, pXINSECT-DEST38 (Invitrogen) was digested with restriction enzymes AfeI and XbaI, and DNA was purified using CONCERT Gel Extraction Systems (Invitrogen). This DNA and 6D9 Hc gene cleaved with restriction enzymes AfeI and XbaI are mixed and ligated, and transformed in E. coli K12 strain (NovaBlue, Merck Ltd.). By the method used, plasmid pXIHA-6D9 Hc was obtained. Although the recognition site AfeI does not exist in the catalog of pXINSECT-DEST38, since the Eco47III site of pXINSECT-DEST38 has the same recognition sequence as AfeI, this plasmid is cut with AfeI.

  On the other hand, the construction of another plasmid pIB-6D9 Hc expressing the H chain is shown in FIG. A plasmid pIB-6D9 Hc was obtained in the same manner except that pIB / V5-His (Invitrogen) was used instead of pXINSECT-DEST38.

(Preparation of plasmid expressing L chain)
The construction of plasmid pXIHA-6D9 Lc expressing the L chain is shown in FIG. First, a 6D9 Lc gene having a restriction enzyme BamHI recognition site and a Drosophila-derived BiP signal sequence in the upstream portion and a restriction enzyme XbaI recognition site in the downstream portion was prepared. This gene was amplified by PCR using plasmid pARA7Fab as a template and primers of SEQ ID NOs: 3 and 4. The primer of SEQ ID NO: 3 has a BamHI recognition site and a BiP signal sequence, and the primer of SEQ ID NO: 4 has an XbaI recognition site. PCR was performed in the following cycle using Thermal cycler (BIO-RAD). First, it was treated at 96 ° C. for 2 minutes and at 72 ° C. for 2 minutes, and then treated at 96 ° C. for 30 seconds, 57.4 ° C. for 30 seconds, and 72 ° C. for 2 minutes for 35 cycles, Finally, treatment at 72 ° C. for 5 minutes was performed. After confirming that the desired fragment was amplified by agarose gel electrophoresis, the 6D9 Lc gene was recovered by a method commonly used by those skilled in the art.

  Next, pXINSECT-DEST38 was digested with restriction enzymes BamHI and XbaI, and DNA was purified using CONCERT Gel Extraction Systems (Invitrogen). This DNA is mixed with 6D9 Lc gene cleaved with restriction enzymes BamHI and XbaI, ligated, transformed in E. coli K12 strain, and plasmid pXIHA-6D9 Lc is obtained by a method commonly used by those skilled in the art. It was.

  On the other hand, the construction of another plasmid pIB-6D9 Lc expressing the L chain is shown in FIG. A plasmid pIB-6D9 Lc was obtained in the same manner except that pIB / V5-His was used instead of pXINSECT-DEST38.

(Insect cell transformation)
BTI-TN5B1-4 strain (Invitrogen) derived from Trichoplusia ni (hereinafter referred to as High Five strain) and Sf9 strain (Invitrogen) derived from Spodoptera frugiperda were used as host cells. The Sf9 strain or High Five strain in the logarithmic growth phase is suspended in a fresh medium so that the viable cell density is 4 × 10 ⁵ cells / cm ^3, and 2 mL of the cell suspension medium is added to each of seven dishes having a diameter of 35 mm. Sowing. The cell density was measured using a hemocytometer and only live cells were counted by a dye exclusion method using trypan blue. For the Sf9 strain, serum-free medium EX-CELL 420 (JRH Biosciences) was used, and for the High Five strain, Express Five SFM (Invitrogen) was used.

  The constructed plasmids pXIHA-6D9 Hc and pXIHA-6D9 Lc were mixed at a mass ratio of 2: 1. 6 μl of FuGENE6 (Roche) consisting of positively charged lipid was added to 2 μg of this mixed plasmid DNA and incubated at room temperature for 45 minutes. After incubation, the medium was added to make a total volume of 100 μl, and each of the dishes containing the High Five strain and Sf9 strain was added for transformation. The supernatant was recovered by incubation at 27 ° C. for 72 hours, and antibody production was confirmed by Western blotting using alkaline phosphatase-labeled anti-mouse IgG (H + L).

  Next, 6 μl of FuGENE6 (Roche) consisting of positively charged lipid was added to 2 μg of 2: 1 mixed plasmid DNA of pIB-6D9 Hc and pIB-6D9 Lc, and incubated at room temperature for 45 minutes. After incubation, the medium was added to make a total volume of 100 μl, and each of the dishes containing the High Five strain and Sf9 strain was added for transformation. Antibody production was confirmed by Western blotting as described above. The results are shown in FIG. In FIG. 5, each lane indicates the following case. Lane 1: When using pXIHA-6D9 Hc and pXIHA-6D9 Lc and using High Five as a host; Lane 2: When using pXIHA-6D9 Hc and pXIHA-6D9 Lc and using Sf9 as a host; Lane 3: pIB- When 6D9 Hc and pIB-6D9 Lc are used as the host and the High Five strain is used as the host; Lane 4: When pIB-6D9 Hc and pIB-6D9 Lc are used and the Sf9 strain is used as the host.

  As shown in FIG. 5, antibody production was observed in the combination of lanes 1 to 3 by detection by Western blotting. In particular, when pXIHA-6D9 Hc and pXIHA-6D9 Lc were used and the High Five strain was used as the host (lane 1), significant antibody production was observed.

  For each supernatant, the activity of the catalytic antibody 6D9 was measured by ELISA. ELISA was performed by the following method. A condensate of phosphonate and bovine serum albumin (hapten-BSA), which is a transition state analog of the hydrolysis reaction of chloramphenicol monoester, was added to each well of a 96-well plate (Corning) as an antigen. (50 μl) The mixture was allowed to stand at 4 ° C. for 16 hours and blocked by a conventional method. After each well was thoroughly washed, 50 μl of each supernatant was added and incubated at room temperature for 2 hours. After washing, 100 μl of goat anti-mouse IgG (κ chain specific) HRP conjugate (Exalpha Biologicals) diluted to 5 μg / mL with a washing solution was added and incubated at room temperature for 1 hour. The wells were washed, 100 μl each of the color developing solution was added, protected from light, and incubated at room temperature for 20 minutes, and then the absorbance at 405 nm of each well was measured using a microplate reader (Bio-Rad).

  By this method, it was confirmed that any of the supernatants in which no band was detected by Western blotting had antigen-binding activity, that is, the catalytic antibody 6D9 was produced (data not shown). It was also found that the antibody production efficiency was higher when the High Five strain was used as the host than when the Sf9 strain was used as the host. However, these transformants only transiently expressed antibodies.

(Example 2)
Using the vector obtained above, (1) a mixture of pIB-6D9 Hc and pXIHA-6D9 Lc, (2) a mixture of pXIHA-6D9 Hc and pIB-6D9 Lc, (3) pIB-6D9 Hc and Using the mixture of pIB-6D9 Lc and (4) the mixture of pXIHA-6D9 Hc and pXIHA-6D9 Lc (each 1: 1 mixture), the high five strain was transformed in the same manner as in Example 1. Converted.

  The result of the Western blotting about the expression of the antibody of the obtained transformant is shown in FIG. Lanes 1 to 4 show the results in the cases (1) to (4) above. As shown in FIG. 6, antibody production was confirmed by the combination of (1), (2), and (4). Among these, it was confirmed that antibodies can be expressed even with combinations of heterologous promoters as in (1) and (2). However, these transformants also only transiently expressed antibodies.

(Example 3)
In Example 2, (1) the combination of pIB-6D9 Hc and pXIHA-6D9 Lc and (2) the expression level of the antibody in the combination of pXIHA-6D9 Hc and pIB-6D9 Lc is shown in FIG. Thus, in the case of the combination (2), it was found that the expression level was large. Then, after transforming the High Five strain in the same manner as in Example 2 with the mixing ratio of pXIHA-6D9 Hc and pIB-6D9 Lc being 1: 1, 10: 1, 50: 1 and 100: 1, Incubated for 48 hours at 27 ° C. The cells were then transferred to a 12-well plate and incubated with medium containing 80 μg / ml blasticidin. The cells were incubated for 2 weeks while changing the medium with blasticidin-containing medium every 3 or 4 days. When the plasmid was mixed at a mass ratio of 1: 1 or 10: 1 and transformed, a large number of cells grew throughout the well and could not be isolated as colonies derived from a single cell. When the mass ratio of the plasmid was 100: 1, the growth of the transformed cells was not observed. In contrast, when the plasmid ratio was 50: 1, several colonies were obtained in each well. These colonies were sucked with a micropipette, transferred to a 12-well plate so that one colony was placed in each well, and subcultured in the presence of blasticidin. Western blotting was performed on the catalytic antibody 6D9 in the culture supernatant of each well of a 12-well plate 50 days after the transformation. The results are shown in FIG. As shown in FIG. 7, it can be seen that even after 50 days from the transformation, the transformed strain produced the antibody and produced the antibody stably for a long time. It can also be seen that when the plasmid was mixed at a mass ratio of 50: 1, a transformed strain was obtained efficiently.

Example 4
(Construction of plasmid having blasticidin resistance gene)
The construction of plasmid pIHA: bla having the blasticidin resistance gene is shown in FIG.

  First, a plasmid pIBΔpro / V5-His from which the OpIE-2 promoter of pIB / V5-His was deleted was constructed as follows. pIB / V5-His was digested with BspHI and HindIII and purified by agarose gel electrophoresis to obtain overhanging DNA fragments. Next, the blunt end DNA fragment was blunted using a DNA Blunting Kit (TaKaRa). Next, 10 μl of 2 × Ligation Mix (Ligation-Convenience Kit; Wako Pure Chemical Industries, Ltd.) was added to the solution containing blunt-end DNA, and incubated at 16 ° C. for 20 minutes. This was transformed into E. coli K12 strain, miniprep and alkali 1 + 2 + 3 method were performed to obtain pIBΔpro / V5-His.

  Next, in order to obtain a DNA fragment of IE-1 + HR3 + actin promoter as an insert, pXIHA-6D9 Lc obtained in Example 1 was digested with restriction enzymes KpnI and BamHI. As a vector, the above-mentioned pIBΔpro / V5-His was also digested with restriction enzymes KpnI and BamHI. A DNA fragment of about 3000 bp of vector-side pIBΔpro / V5-His and about 6600 bp of insert-side IE-1 + HR3 + actin promoter was separated by agarose electrophoresis and purified using Wizard SV Gel and PCR Clean-UP System (Promega). . Next, using 2 × Ligation Mix, ligation of the DNA fragment between the vector side and the insert side was performed. This ligation product was transformed into E. coli K12 strain, miniprep and alkaline 1 + 2 + 3 method were performed to obtain pIHA: bla.

(Construction of plasmid having blasticidin resistance gene and heavy chain gene)
Construction of the vector pIHA: bla-6D9 Hc for expressing the heavy chain was carried out as described below using the vector pIHA: bla. The construction of plasmid pIHA: bla-6D9 Hc having this blasticidin resistance gene and H chain gene is shown in FIG.

  As a vector, pIHA: bla was digested with AfeI and XbaI. In order to obtain an insert fragment, pXIHA-6D9 Hc obtained in Example 1 was also digested with AfeI and XbaI. A DNA fragment of about 9600 bp of vector side pIHA: bla and about 700 bp of insert side pXIHA-6D9 Hc was separated by agarose electrophoresis and purified using Wizard SV Gel and PCR Clean-UP System (Promega). Next, using 2 × Ligation Mix, ligation of the DNA fragment between the vector side and the insert side was performed. This ligated product was transformed into E. coli K12 strain, miniprep and alkali 1 + 2 + 3 method were performed to obtain pIHA: bla-6D9 Hc.

(Construction of plasmid having blasticidin resistance gene and L chain gene)
Construction of the vector pIHA: bla-6D9 Lc for expressing the L chain using the vector pIHA: bla was performed as described below. The construction of plasmid pIHA: bla-6D9 Lc having this blasticidin resistance gene and L chain gene is shown in FIG.

  As a vector, pIHA: bla was digested with BamHI and XbaI. In order to obtain an insert fragment, pXIHA-6D9 Lc obtained in Example 1 was also digested with BamHI and XbaI. A DNA fragment of about 9600 bp on the vector side pIHA: bla and about 700 bp on the insert side pXIHA-6D9 Lc was separated by agarose electrophoresis and purified using Wizard SV Gel and PCR Clean-UP System (Promega). Next, using 2 × Ligation Mix, ligation of the DNA fragment between the vector side and the insert side was performed. This ligated product was transformed into E. coli K12 strain, miniprep and alkali 1 + 2 + 3 method were performed to obtain pIHA: bla-6D9 Lc.

(Example 5)
(Construction of plasmid having neomycin resistance gene)
The blasticidin resistance gene of pIHA: bla obtained in Example 4 above was replaced with a neomycin resistance gene to construct pIHA: neo. The construction of plasmid pIHA: neo having the neomycin resistance gene is shown in FIG.

  The neomycin resistance gene was amplified by the PCR method using the plasmid pBmA: neo as a template and the primers shown in SEQ ID NOs: 5 and 6 in the sequence listing. These primers were designed from pBmA: neo so that the recognition base sequence of NcoI was located at one end of the neomycin resistance gene and SmaI was located at the other end. The primer of SEQ ID NO: 5 has an NcoI recognition site, and the primer of SEQ ID NO: 6 has a SmaI recognition site.

  PCR was performed by the following reaction cycle using Thermal cycler (BIO-RAD). First, treatment at 98 ° C. for 10 seconds, 98 ° C. for 2 minutes, followed by 30 cycles of treatment at 98 ° C. for 2 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute. Hold at 4 ° C. Thereafter, it was confirmed that the target fragment was amplified by agarose electrophoresis, and the neomycin resistance gene was recovered by a method commonly used by those skilled in the art.

Next, the prepared neomycin resistance gene was digested with restriction enzymes NcoI and SmaI. About 560 bp DNA fragment (neomycin ^r Nco-Nco) and about 260 bp DNA fragment (neomycin ^r Nco-Sma) of neomycin resistance gene were separated by agarose electrophoresis, and Wizard SV Gel and PCR Clean-UP System (Promega) The product was purified using Next, pIHA: bla constructed in Example 4 was digested with restriction enzymes NcoI and SmaI. A DNA fragment of about 9100 bp was separated by agarose electrophoresis and purified using Wizard SV Gel and PCR Clean-UP System (Promega). As a result, a DNA fragment pIHA: Δbla in which the blasticidin resistance gene was deleted from pIHA: bla was obtained.

Next, the above vector-side DNA fragment pIHA: Δbla and the insert-side DNA fragment neomycin ^r (Nco-Sma) were ligated using 2 × Ligation Mix. This ligated product was transformed into E. coli K12 strain and miniprep, alkali 1 + 2 + 3 method was performed to obtain pIHA: neo (Nco-Sma).

Next, the vector pIHA: neo (Nco-Sma) was digested with NcoI. An about 9,400 bp DNA fragment of the vector side pIHA: neo (Nco-Sma) was separated by agarose electrophoresis and purified using Wizard SV Gel and PCR Clean-UP System (Promega). Subsequently, using 2 × Ligation Mix, the obtained vector-side pIHA: neo (Nco-Sma) DNA fragment and the insert-side neomycin ^r (Nco-Nco) DNA fragment were ligated. This ligated product was transformed into E. coli K12 strain and miniprep, alkaline 1 + 2 + 3 method was performed to obtain pIHA: neo.

(Construction of plasmid having neomycin resistance gene and heavy chain gene)
As described below, a vector pIHA: neo-6D9 Hc for expressing an H chain was constructed using the above pIHA: neo. The construction of plasmid pIHA: neo-6D9 Hc having this neomycin resistance gene and heavy chain gene is shown in FIG.

  As a vector, pIHA: neo was digested with AfeI and XbaI. In order to obtain an insert fragment, pXIHA-6D9 Hc obtained in Example 1 was also digested with AfeI and XbaI. A DNA fragment of about 10,000 bp of vector side pIHA: neo and about 700 bp of insert side pXIHA-6D9 Hc was separated by agarose electrophoresis and purified using Wizard SV Gel and PCR Clean-UP System (Promega). Next, ligation of the DNA fragment between the vector side and the insert side was performed using 2 × Ligation Mix. This ligated product was transformed into E. coli K12 strain and miniprep, alkali 1 + 2 + 3 method was performed to obtain pIHA: neo-6D9 Hc.

(Construction of plasmid having neomycin resistance gene and L chain gene)
As described below, the pIHA: neo was used to construct a vector pIHA: neo-6D9 Lc for expressing the L chain. FIG. 13 shows the construction of plasmid pIHA: neo-6D9 Lc having this neomycin resistance gene and L chain gene.

  As a vector, pIHA: neo was digested with BamHI-XbaI. In order to obtain an insert fragment, pXIHA-6D9 Lc obtained in Example 1 was also digested with BamHI-XbaI. A DNA fragment of about 10,000 bp of vector side pIHA: neo and about 700 bp of insert side pXIHA-6D9 Lc was separated by agarose electrophoresis, and purified using Wizard SV Gel and PCR Clean-UP System (Promega). Next, ligation of the DNA fragment between the vector side and the insert side was performed using 2 × Ligation Mix. This ligated product was transformed into E. coli K12 strain and miniprep, alkali 1 + 2 + 3 method was performed to obtain IHA: neo-6D9 Lc.

(Example 6)
(Confirmation of 6D9 Fab expression by transient expression using pIHA: bla and pIHA: neo)
Combinations of vectors constructed in Examples 4 and 5 above: (1) a mixture of pIHA: bla-6D9 Hc and pIHA: bla-6D9 Lc, (2) pIHA: bla-6D9 Hc and pIHA: neo-6D9 Lc (3) a mixture of pIHA: neo-6D9 Hc and pIHA: neo-6D9 Lc, (4) a mixture of pIHA: neo-6D9 Hc and pIHA: bla-6D9 Lc, and (5) pXIHA- Using the mixture of 6D9 Hc and pXIHA-6D9 Lc (each 1: 1 mixture), the High Five strain was transformed in the same manner as in Example 1 above. In the same manner as in Example 1, antibody production was confirmed by Western blotting.

  The result is shown in FIG. In the lanes 2 to 5 (showing the respective results of (1) to (4) above), Fab equal to or greater than that of pXIHA not having a selectable marker resistance gene ((5); lane 6 above) Was found to be secreted (lane 1 is a control in FIG. 14). This is considered to be due to the fact that the pIHA-derived vector (about 10500 bp) has a smaller vector size than pXIHA (about 11500 bp), so that the transfection efficiency to insect cells slightly increased.

(Example 7)
(Investigation of optimal antibiotic concentration for selection of stably transformed cells)
In this example, the optimum concentration for selection with two antibiotics (blasticidin and G418) was examined. In this example, stable transformed cells (pXIHA-6D9 Hc / pIB-6D9 Lc) established in a medium containing 80 μg / ml blasticidin in Example 3 were used. The cells were transferred to a 12-well plate, and the medium containing Express Five SFM supplemented with 80 μg / ml blasticidin and G418 at 0 μg / ml, 500 μg / ml, 1000 μg / ml, 1500 μg / ml, or 2000 μg / ml at 27 ° C. Incubated for 7 days. The appearance of cells before and 7 days after the addition of these antibiotics was observed using a differential interference microscope. The result is shown in FIG. The upper part of FIG. 15 shows the state of the cells before the addition of both antibiotics, and the lower part of FIG. 15 shows 80 μg / ml blasticidin and 0 μg / ml, 500 μg / ml, 1000 μg / ml, 1500 μg / ml. , Or the addition of 2000 μg / ml G418, shows the appearance of the cells 7 days later (the concentration in the figure represents the concentration of G418). As is clear from FIG. 15, the cells were completely killed in the medium supplemented with G418 of 1000 μg / ml or more. Therefore, in order to efficiently obtain stable transformed cells using two types of antibiotics, it is necessary to apply a selective pressure at a blasticidin concentration of 80 μg / ml and a G418 concentration of 1000 μg / ml in the medium. I found out.

(Example 8)
(Preparation of stably transformed cells using pIHA: neo-6D9 Hc and pIHA: bla-6D9 Lc)
PIHA: neo-6D9 Hc prepared in Example 5 and pIHA: bla-6D9 Lc prepared in Example 4 were mixed at a ratio of 50: 1, 10: 1, or 1: 1 and mixed into High Five. An attempt was made to produce stably transformed cells by transfection and applying selective pressure with two antibiotics (blasticidin and G418).

Cells suspended in 2 ml of medium so that the initial cell density was 2.0 × 10 ⁵ cells / cm ³ were seeded in a 35 mm dish and incubated at 27 ° C. for 24 hours. For cell density measurement, only a viable cell was counted by a dye exclusion method using trypan blue using a hemocytometer. 3 μl FuGENE 6 and 1 μg total DNA were added to the medium to a total volume of 100 μl and incubated at room temperature for 45 minutes. This reaction solution was added to the 35 mm dish and statically cultured at 27 ° C. until the cells became confluent. Thereafter, the cells were detached by pipetting, suspended in 10 ml of medium at a cell density of 1.0 × 10 ⁵ cells / cm ³ , and seeded in a 100 mm dish. 24 hours after seeding, blasticidin (final concentration 80 μg / ml) and G418 (final concentration 1000 μg / ml) were added. Thereafter, every four days, the medium was changed with Express Five SFM supplemented with 80 μg / ml blasticidin and 1000 μg / ml G418. When cell growth was slow, a medium in which conditioned medium and fresh medium were mixed in equal amounts was used. Approximately two weeks after the addition of antibiotics, colony formation was visually confirmed, the colonies were picked up with a pipette, dispensed into 96-well plates (one colony per well), and allowed to stand at 27 ° C. for 24 hours. Cultured. However, the culture medium was 100 μl per well without antibiotics. After 24 hours of culture, blasticidin (final concentration 80 μg / ml) and G418 (final concentration 1000 μg / ml) were added, and static culture was continued until the cells became confluent. In the same manner as in Example 1, the culture supernatant in each well was analyzed by Western blotting and ELISA, and several tens of wells having a large Fab expression level were selected. The cells in the selected wells were gently pipetted to suspend the cells, transferred to a 12-well plate, and then statically cultured at 27 ° C. for 24 hours. However, the medium was 1 ml per well without antibiotics. After 24 hours of culture, blasticidin (final concentration 80 μg / ml) and G418 (final concentration 1000 μg / ml) were added, and static culture was continued until the cells became confluent. The culture supernatant in each well was analyzed by Western blotting and ELISA as described above, and several wells having a large Fab expression level were selected. The cells in the selected wells were gently pipetted to suspend the cells, transferred to a 100 mm dish or 25 cm ² T flask, and then statically cultured at 27 ° C. for 24 hours. After 24 hours of culture, blasticidin (final concentration 20 μg / ml) and G418 (final concentration 200 μg / ml) were added, and static culture was continued until the cells became confluent. Thereafter, the passage was performed in the same manner.

  FIG. 16 shows the result of Western blotting analysis of the culture supernatant after transfection with an expression vector and culturing for 16 days in the presence of antibiotics. Regardless of the co-transfection ratio of pIHA: neo-6D9 Hc and pIHA: bla-6D9 Lc, it was confirmed that Fab was secreted and expressed stably for a relatively long period of time (lanes 2 to 4; pIHA, respectively). : neo-6D9 Hc / pIHA: bla-6D9 Lc is 50/1, 10/1, 1/1; where lane 1 is a control). In addition, the expression of the L chain or the dimer of the L chain was decreased compared to the results of the expression confirmation by transient expression in Example 6. This suggests that only cells into which both Hc and Lc genes have been introduced are efficiently selected.

Example 9
Insect cells were co-transfected and stable transformed cells were selected in the same manner as in Example 8 except that culture was performed in the presence of two types of antibiotics for 30 days.

  FIG. 17 shows the results of analyzing the culture supernatant after transfection with an expression vector and culturing for 30 days in the presence of antibiotics (lanes 1 to 3, pIHA: neo-6D9 Hc / pIHA). : bla-6D9 Lc 10/1; Lanes 4-5, pIHA: neo-6D9 Hc / pIHA: bla-6D9 Lc 50/1; Lane 6, pIHA: neo-6D9 Hc / pIHA: bla-6D9 Lc 1/1, in these, the total DNA amount is constant). As can be seen from FIG. 17, even after 30 days from transfection, the Fab fragment is secreted and expressed in large quantities. In addition, when the ratio of the vector having the neomycin resistance gene is increased (in other words, the ratio of the vector having the blasticidin resistance gene is decreased) by observation under a microscope, the number of colonies formed decreases, and the colonies are selected. I found it easier. In lane 5, the expression amount of the Fab fragment is large, and it can be seen that a high expression strain can be obtained efficiently.

  Since the method of the present invention can efficiently and stably secrete and produce hetero-oligomeric proteins in insect cells, it can be widely used in the field of recombinant protein production. In particular, it may be useful for the production of antibodies and enzymes derived from higher eukaryotes.

It is a schematic diagram showing the construction of the vector pXIHA-6D9 Hc. FIG. 3 is a schematic diagram showing the construction of vector pIB-6D9 Hc. FIG. 2 is a schematic diagram showing the construction of vector pXIHA-6D9 Lc. FIG. 3 is a schematic diagram showing the construction of vector pIB-6D9 Lc. It is a photograph which shows the result of the western blotting about the production of the antibody in a transformed insect cell. It is a photograph which shows the result of the western blotting about the production of the antibody in the insect cell transformed with the combination of various vectors. It is a photograph which shows the result of the western blotting about the antibody in a culture supernatant at the time of culture | cultivating the insect cell transformed by vector pXIHA-6D9 Hc and pIB-6D9 Lc for 50 days. It is a schematic diagram showing the construction of the vector pIHA: bla. FIG. 3 is a schematic diagram showing the construction of vector pIHA: bla-6D9 Hc. FIG. 3 is a schematic diagram showing the construction of vector pIHA: bla-6D9 Lc. It is a schematic diagram showing the construction of the vector pIHA: neo. It is a schematic diagram showing the construction of the vector pIHA: neo-6D9 Hc. It is a schematic diagram showing the construction of the vector pIHA: neo-6D9 Lc. It is a photograph which shows the result of the western blotting about the production of the antibody in the insect cell transformed by the combination of various vectors using pIHA: bla and pIHA: neo. It is a differential interference micrograph showing the state of insect cells transformed with pXIHA-6D9 Hc and pIB-6D9 Lc when cultured in a selective medium containing 80 μg / ml blasticidin and various concentrations of G418. It is a photograph which shows the result of having analyzed the culture supernatant after transfecting expression vectors pIHA: neo-6D9 Hc and pIHA: bla-6D9 Lc and culturing for 16 days in the presence of antibiotics by Western blotting. It is a photograph which shows the result of having analyzed the culture supernatant after transfecting expression vectors pIHA: neo-6D9 Hc and pIHA: bla-6D9 Lc and culturing for 30 days in the presence of antibiotics by Western blotting.

Claims (7)

A method for producing an oligomeric protein composed of heterologous subunits using insect cells,
Transforming the insect cell with a plurality of vectors capable of expressing each one of the heterologous subunits;
Culturing the transformed insect cell; and recovering the oligomeric protein from the culture.   The method of claim 1, wherein the oligomeric protein is an antibody.   The method according to claim 2, wherein each of the heterologous subunits is at least a part of a peptide of an H chain constituting an antibody and at least a part of a peptide of an L chain constituting the antibody.   The method according to any one of claims 1 to 3, wherein each of the plurality of vectors has the same or different promoter.   The method according to any one of claims 1 to 4, wherein at least one of the plurality of vectors has a selection marker for the insect cells.   A transformed insect cell that stably expresses an oligomeric protein composed of heterologous subunits, the transformed insect cell having a plurality of vectors each capable of expressing one of the heterologous subunits.   The transformed insect cell according to claim 6, wherein at least one of the plurality of vectors has a selection marker for the insect cell.

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