KR101341061B1 - Polynucleotide for preparing human G-CSF and use thereof - Google Patents

Abstract

The present invention relates to a fusion polynucleotide in which a vesicle transfer signal sequence of a specific insect-derived secreted protein and a nucleotide sequence encoding a human-derived recombinant protein are fused, a vector comprising the same, a cell for transformation, and a method for producing a recombinant protein using the same. will be. When the human G-CSF recombinant protein is produced by the present invention, not only the burden of pathogens can be minimized, but also the expression can be increased, thereby producing a human G-CSF recombinant protein with high efficiency.

Description

Polynucleotide for preparing human G-CSF protein and use thereof

The present invention provides a fusion polynucleotide in which an vesicle transfer signal sequence of an insect-derived secreted protein PPAE (prophenoloxidase activating enzyme), PDI (protein disulfide isomerase), or BX (bombyxin) is fused with a nucleotide sequence encoding a target recombinant protein derived from humans, The present invention relates to a vector, a transformed cell, and a method for producing a recombinant protein using the same.

As molecular biology techniques have evolved, methods for mass production of useful metabolites, oral vaccines and medical proteins have been developed by genetic engineering methods. There are many ways to produce such a useful protein, but generally, it is intended to produce a protein of natural type with economical, stable, and high production efficiency of a biologically active protein. In addition, the choice of protein expression system that meets these needs should also consider the properties of the protein to be expressed.

Granulocyte colony stimulating factor (G-CSF) is a glycoprotein that stimulates the survival, proliferation, differentiation and function of neutrophil granulocyte progenitor cells and mature neutrophils. . Two forms of recombinant human G-CSF for clinical use are potent stimulators of neutrophil granulopoiesis and show efficacy in preventing infectious complications of some neutropenic conditions.

In addition, G-CSF lowers the mortality rate of cancer chemotherapy by reducing the incidence of febrile neutropenia, lowers the mortality rate of high-dose chemotherapy backed by bone marrow transplantation, and also severe chronic neutropenia. Reduces the incidence of infection in patients with In addition, G-CSF has been shown to have a therapeutic effect when administered after the onset of myocardial infarction.

Acute myelosuppression as a result of cytotoxic chemotherapy is widely known as a dose-limiting factor in the treatment of cancer. Other normal tissues may be adversely affected, but bone marrow is particularly sensitive to proliferation-specific treatments such as chemotherapy and radiation therapy. For some cancer patients, repeated or high dose cycles of chemotherapy may result in stem cell reduction of hematopoietic stem cells and their progeny. The prevention and protection from the side effects of such chemotherapy and radiation therapy can be of great benefit to cancer patients. G-CSF has been shown to be effective in alleviating such side effects by increasing the number of normal critical target cells, especially hematopoietic progenitor cells.

Because of its important use as a medicinal product, many studies are being conducted to synthesize human G-CSF. Various vectors and hosts are used to produce a large amount of recombinant protein, and generally, a method using E. coli, an animal cell line, or a yeast, a transgenic animal, or a transgenic plant is used.

The advantage of using prokaryotes such as Escherichia coli is that it has the advantage of ensuring high expression levels, but the addition of methionine (methionine), which is not present in human-derived natural proteins, to the amino terminus and endotoxins derived from prokaryotes. Pollution and often synthetic proteins require aggregation in prokaryotic cells to restore functional proteins. In addition, eukaryotic-derived proteins such as humans have many glycoproteins, but prokaryotes are incapable of synthesizing glycated proteins.

It is known that the use of yeast has the advantage of easy mass production, but the degree and pattern of glycosylation is very different from that of humans, resulting in many immunogenicity (Hermeling et al, Pharm. Res. 21 (6): 897-903 (2004). In the case of transgenic animals, it has not yet been commercialized due to problems related to the management of the animal itself and the possibility of contamination with pathogens. The method using animal cell lines is most widely used for the production of recombinant protein because the recombinant protein produced is very similar to human form and stable production and management is possible, but the production cost is high and the time and cost for cell line production are high. In the case of using mammalian cells such as CHO cells, there is a problem in that the stability and expression amount, such as contamination of a virus that can infect humans, are significantly low. Therefore, the development of a more efficient and safe recombinant protein production method is required.

Thus, the present inventors do not use E. coli and animal cells to solve the above problems of the prior art, there is no unnecessary amino acid at the N-terminus of the secretory protein, the amount of secretion expression is increased, endotoxin As a result of diligent efforts to minimize the burden of contamination of endotoxins or foreign pathogens derived from animals and to increase the expression efficiency of naturally occurring foreign secreted proteins, vectors for insect cell expression have been developed. Specifically, the inventors fused insect DNA and human DNA by replacing a DNA sequence corresponding to a secreted signal amino acid sequence of a specific insect-derived secreted protein with a DNA sequence corresponding to the original secreted signal amino acid sequence possessed by hG-CSF. The new G-CSF gene was produced, and the production of the native G-CSF using this new gene confirmed that the expression of the secretory was significantly enhanced in insects, and the various secretions including the natural G-CSF were secreted in insects. The present invention has been completed for use in sex protein production.

It is an object of the present invention to provide a fusion polynucleotide in which a vesicle transfer signal sequence of an insect-derived secreted protein PPAE, PDI, or BX is fused with a base sequence encoding a human-derived recombinant protein.

It is another object of the present invention to provide a vector or a cell transformed with the fusion polynucleotide.

It is another object of the present invention to provide a method for mass production of human-derived recombinant proteins including human G-CSF using the vector or transformed cells.

In order to achieve the above object, the present invention encodes the vesicle transfer signal sequence of the insect-derived secreted protein PPAE (prophenoloxidase activating enzyme, PDI, protein disulfide isomerase (PDI), or BX (bombyxin) and the target recombinant protein derived from humans It provides a fusion polynucleotide fused to the base sequence.

Gene information of the insect-derived secretion protein may refer to NP_001036832.1 for PPAE, NP_001036997 for PDI, and NP_001121606.1 for BX.

As used herein, the term "endoplasmic reticulumn signal sequence" refers to a specific protein recognized by another protein that is required for the transfer of the protein from the cytoplasm to the endoplasmic reticulum when the protein is synthesized by ribosomes in the cell. Refers to an amino acid sequence.

In the present invention, the "human-derived recombinant protein" is generally a concept including a physiologically active polypeptide, a human-derived hormone, cytokine, interleukin, interleukin binding protein, enzyme, antibody, growth factor, transcription regulator And various proteins such as blood factors, vaccines, structural proteins, ligand proteins or receptors, cell surface antigens, receptor antagonists and derivatives and analogues thereof, specifically human growth hormone, interferon and interferon receptors , Colony stimulating factor, interleukin, erythropoietin, insulin, angiotensin, bone formation growth factor, B cell factor, T cell factor, nerve growth factor, cell surface antigen, monoclonal antibody and virus derived vaccine antigen It is not limited. Preferably, the human-derived recombinant protein may be human granulocyte colony stimulating factor (G-CSF).

In the present invention, the insect-derived secreted protein vesicle transfer signal sequence is preferably a vesicle transfer signal sequence of PPAE represented by SEQ ID NO: 1, a vesicle transfer signal sequence of PDI represented by SEQ ID NO: 2, and BX represented by SEQ ID NO: 3 It may be a nucleotide sequence corresponding to the amino acid sequence selected from the group consisting of vesicle transition signal sequence of. Preferably, the endoplasmic reticulum signal sequence may be from silkworm. In addition, the base sequence information of the endoplasmic reticulum signal sequence may refer to NM_001043367 (PPAE), NM_001043532.1 (PDI), and NM_001128134 (BX). Preferably, the vesicle transfer signal sequence of the PPAE is a nucleotide sequence represented by SEQ ID NO: 14, the vesicle transfer signal sequence of the PDI is a nucleotide sequence represented by SEQ ID NO: 15, the vesicle transfer signal sequence of the BX is represented by SEQ ID NO: 16 It may be a base sequence.

In addition, the base sequence encoding the human G-CSF may be any known sequence without limitation (NM_172220), but may preferably be a base sequence corresponding to the amino acid sequence represented by SEQ ID NO: 4, preferably the base sequence May be the nucleotide sequence represented by SEQ ID NO: 17.

The signal sequence or amino acid sequence of human G-CSF may include a functional equivalent, and the functional equivalent refers to a vesicle transfer signal sequence of the insect-derived secreted protein or a sequence exhibiting activity equivalent to that of human G-CSF. More preferably 90% or more of sequence homology. The functional equivalent may be the result of addition, substitution or deletion of part of the signal sequence or amino acid sequence of human G-CSF, respectively, and the substitution of the amino acid is preferably a conservative substitution.

The fusion polynucleotide is replaced with a vesicle transfer signal sequence of an insect secreting protein PPAE, PDI, or BX instead of a signal sequence of a human-derived recombinant protein, and encodes a human-derived recombinant protein from which the signal sequence and the signal sequence have been removed. The base sequences are fused to each other, and the fusion method may be performed by a method generally known in the art.

In a specific embodiment of the present invention, the DNA sequence encoding the endoplasmic reticulum transition signal sequence inherent in human G-CSF from the base sequence encoding the human G-CSF protein, thereby enabling optimized secretion expression in insect cells By substituting the DNA portion of the vesicle transfer signal sequence of PPAE, PDI, or BX, a silkworm-derived secretion protein, a polynucleotide fused with an insect-derived signal sequence and the human G-CSF gene was synthesized and then used. As a result of transforming the derived cell line, it was confirmed that the expression level of human G-CSF was significantly increased (see FIGS. 3 and 4).

Through this, when human G-CSF protein is expressed in insect cells by using the signal sequence of the insect-derived specific protein as described above, unnecessary amino acids at the N-terminus of the secretory protein are compared with those of E. coli and animal cells. It does not exist, the amount of secretory expression is increased, it can be seen that there is an advantage to increase the expression efficiency of the natural type of foreign secretory proteins by minimizing the burden of contamination of foreign pathogens derived from endotoxin or animal.

In another aspect, the present invention provides an expression vector comprising the fusion polynucleotide.

As used herein, the term 'vector' includes a plasmid vector or a baculovirus vector as a means for expressing a gene of a recombinant protein of interest in an insect cell which is a host cell according to the object of the present invention, and preferably a baculo Rovirus vector.

The vector according to the present invention can be used a variety of functional promoters known in the art, the fusion polynucleotide is operably linked to the promoter. The term 'promoter' refers to a DNA sequence that regulates the expression of a nucleic acid sequence operably linked in a particular host cell. 'Operably linked' means that one nucleic acid fragment is combined with another nucleic acid fragment. Its function or expression is affected by other nucleic acid fragments. In addition, it may further comprise any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation.

The expression vector of the present invention may further comprise a resistance gene for transformation cell line used as a selection marker factor for expression in order to induce high expression in the transformation cell line. In the present invention, as a resistance gene, a resistance gene for insect cells commonly used in the art may be used.

In addition, the expression vector of the present invention may further include, but is not limited to, components of a general vector, such as origins of replication and multiple adenylation signals and other transcriptional regulators.

In a specific embodiment of the present invention, the expression vector may be a vector having a cleavage map disclosed in Fig. 8, 9, or 10, that is, pPPAE_GCSF, pPDI_GCSF, pBX_GCSF vector.

By expressing the expression vector of the present invention, a desired recombinant protein can be expressed with high efficiency.

In another aspect, the present invention provides a cell or cell line transformed with the expression vector.

Preferred transformation host cells usable in the present invention may be insect cells. The insect cell line may include a BM5 cell line, an SF9 cell line, an SF21 cell line, and the like, but is not limited thereto. Preferably, the host cell may be a BM5 cell which is a silkworm-derived cell line.

In the present invention, "transformation" into an insect cell line includes any method of introducing a nucleic acid into a cell, and may be performed by selecting a suitable standard technique according to the insect cell line used as known in the art. In a specific embodiment of the present invention it was transformed using a transformation reagent.

The host cell of the present invention may be a host cell producing only the desired human G-CSF, and the host cell genetically modified to produce a higher concentration of human G-CSF than can be seen in naturally occurring host cells. It includes.

In still another aspect, the present invention provides a method for preparing an insect cell, comprising the steps of: introducing into an insect cell or an insect the expression vector of the present invention described in detail above; Culturing the cells or breeding the insects; And separating human G-CSF from the cultured cells or reared insects.

The insect cells may include BM5 cells, SF9 cells, SF21 cells, and the like, but are not limited thereto. Preferably, the host cells may be BM5 cells which are silkworm-derived cell lines. The insect may preferably be a silkworm.

In addition, the insect cells of the present invention may be cells that produce only the desired human G-CSF, and may be genetically modified to produce higher concentrations of human G-CSF than those found in naturally occurring insect cells. have.

In the present invention, the step of introducing into an insect cell or insect may include any method of introducing an expression vector into an insect cell or insect, and is performed by selecting a suitable standard technique according to an insect cell line or an insect used as known in the art. can do. In a specific embodiment of the present invention, transforming reagents were used to transform BM5 cells, which are silkworm-derived cell lines.

In addition, the culture of the insect cells in the present invention can be made according to the appropriate medium and culture conditions known in the art. This culture process can be used by those skilled in the art can be easily adjusted according to the insect cell line selected. Depending on the cell growth method, suspension culture and adhesion culture are classified into batch, fed-batch and continuous culture according to the culture method. The medium used for culturing must adequately meet the requirements of the particular cell line. In addition, in the culture of insect cells, the medium includes various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources that can be used include glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, Alcohols such as glycerol and ethanol and organic acids such as acetic acid are included. These carbon sources may be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen organic nitrogen sources and urea such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate Inorganic nitrogen sources are included. These nitrogen sources may be used alone or in combination. In addition, amino acids, vitamins and suitable precursors may be included.

As described in detail above, human G-CSF using a fusion polynucleotide fused with a vesicle transfer signal sequence of an insect-derived secreted protein of the present invention and a human G-CSF sequence, a vector including the same or an insect cell transformed with the vector In the case of producing a recombinant protein, not only can minimize the burden of contamination of foreign pathogens, but also increase expression, thereby producing a high efficiency of human G-CSF recombinant protein.

1 shows a pGCSF vector comprising a human G-CSF gene.
Figure 2 shows that the endoplasmic reticulum transition signal sequence of the insect-derived secreted protein used as an embodiment of the present invention replaces the endoplasmic reticulum transition signal sequence of human G-CSF protein.
Figure 3 shows that the expression of hG-CSF significantly increased when the vesicle transfer signal sequence of the insect-derived secreted protein used in the embodiment of the present invention replaced the vesicle transfer signal sequence of the human G-CSF protein.
Figure 4 shows the expression of hG-CSF for each protein derived when the vesicle transfer signal sequence of human G-CSF protein is replaced with the vesicle transfer signal sequence of the silkworm-derived secretion proteins PPAE, PDI, and BX used in the embodiment of the present invention. The degree is shown.
Figure 5 shows the nucleotide sequence corresponding to the vesicle transfer signal sequence of silkworm-derived secretion proteins PPAE, PDI, and BX.
Figure 6 shows the nucleotide sequence of the mature G-CSF DNA linked to the vesicle transfer signal sequence of the insect-derived secretion proteins PPAE, PDI, and BX of the present invention.
Figure 7 shows the nucleotide sequence of precursor G-CSF DNA, the signal sequence portion is shown in shaded.
Figure 8 shows the pPPAE GCSF vector containing the vesicle transfer signal sequence and human G-CSF sequence of PPAE, an insect-derived secretion protein of the present invention.
Figure 9 shows the pPDI GCSF vector containing the vesicle transfer signal sequence and human G-CSF sequence of PDI, an insect-derived secretion protein of the present invention.
Figure 10 shows the pBX GCSF vector containing the vesicle transfer signal sequence and human G-CSF sequence of BX, an insect-derived secretion protein of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Human-derived G- with silkworm (insect) specific endoplasmic reticulum signal sequence CSF  DNA production

G-CSF cDNA clone was purchased from American type culture collection (ATCC), and the protein-coding DNA portion of the hG-CSF was encoded using JA6 and JB6. PCR products were digested with SacI and AgeI and inserted into the pIZTv5 / his vector to prepare new vectors containing hG-CSF. This new vector is used again as a template to the carboxyl terminus from the beginning of the mature protein after the hG-CSF has been removed from the endoplasmic reticulumn signal sequence (SEQ ID NO: 4). Corresponding regions were PCR using primers JA6F and JB6. A mature G-CSF DNA sequence linked to the signal sequence is shown in FIG. 6. The PCR product was again used as a template, and DNA portions of the vesicle transfer signal sequences of the insect secreting protein PPAE (prophenoloxidase activating enzyme), PDI (protein disulfide isomerase), and BX (bombyxin) (SEQ ID NO: 14, SEQ ID NO: 15, and PCR was performed using a primer comprising SEQ ID NO: 16), and the PCR product was digested with XbaI and BglII and inserted into the pIZTv5 / his vector, whereby each insect secretion protein signal sequence DNA was fused to hG-CSF, pPPAE_GCSF, pPDX_GCSF , pBX_GCSF vector was produced. The cleavage map of the vector is shown in FIGS. 8, 9, and 10, respectively. The primer sequences used in this example are shown in Table 1 below. In FIG. 2, the vesicle transfer signal sequences of the insect-derived secreted proteins PPAE, PDI, and BX replace the vesicle transfer signal sequences of the human G-CSF protein. Roughly Respectively.

Primer sequence Name nucleotide sequence Region JA6
(SEQ ID NO: 5) ATA CTG AGC TCT CTA GAA TGT CTC CTG AGC CCG CTC TG 5'of G-CSF precursor JA4-F
(SEQ ID NO: 6) CCG AGC TCT CTA GAA TGT TTT TAA TTT GGA CAT TCA TCG TGG CTG TTC TGG CGA TCC AGA CCA AAA GT 5'of PPAE G-CSF JA6-F
(SEQ ID NO: 7) CTG GCG ATC CAG ACC AAA AGT GTT GTT GCA ACC CCC CTG GGC CCT GCC 5'of PPAE G-CSF JB6
(SEQ ID NO: 8) ATC TGA CCG GTA GAT CTC CCG ACC CGT TCC ACC GCG GGC TGG GCA AGG TGG CG 3'of G-CSF JA9
(SEQ ID NO: 9) TAT TAG AAT TCG GAT CCG AAA TGT TCG GAT CAC TAA AGT TTG TTC TTT TAT TGG GCA TA 5'of PDI JA10
(SEQ ID NO: 10) ATT GGG CAT AAT TTA TTT ATG TAA AGC GAC CCC CCT GGG CCC TGC C 5'of PDI and G-CSF JA11
(SEQ ID NO: 11) TAT TAG AAT TCG GAT CCG AAA TGA TGA AGA CTG CAG TAA TGT TCA TAT TAG TAG TCG TGA TCA G 5'of BX JA12
(SEQ ID NO: 12) GTC GTG ATC AGT TTG ACG TAT TCA ACC CCC CTG GGC CCT GCC 5'of BX and G-CSF JB8
(SEQ ID NO: 13) ATA TAC CGG TCT CGA GGG GCT GGG CAA GGT GGC G 3'of GCSF

Example  2: G- using human-derived G-CSF DNA with silkworm (insect) specific vesicle transfer signal sequences CSF  Protein production

Each of the vectors prepared above was transformed into BM5 cells, silkworm-derived cell lines. That is, an appropriate amount of each vector DNA was put into a 25 cm ² cell culture flask containing 4 x 10 ⁵ cell numbers and transformed with FuGENE (Roche applied science, FuGeneHD transfection Reagent), a transformation reagent. The transformed BM5 cell culture solution was recovered after 4 days and centrifuged to remove impurities solids. In order to separate hG-CSF present in 2 ml of each centrifugal supernatant, 60 μl of 50% nickel sepharose beads suspension was added and bound for 1 hour at 4 ° C., and the nickel sepharose beads were washed three times. 30 μl 2x PAGE gel loading buffer was added to the nickel beads thus obtained and heated, followed by PAGE (polyacrylamide electrophoresis). After electrophoresis, the proteins in the gel were transferred to the PROTRAN nitrocellulose membrane, and then analyzed by western blot using an anti-his antibody. At this time, the presence and relative concentration of hG-CSF recombinant protein was detected by chemoluminescent method, and the relative concentration was calculated using Alpha Innotech FluorChem FC2 MultiImage II device.

As a result, as shown in Figure 3, when the vesicle transfer signal sequence of the human G-CSF protein is replaced with the vesicle transfer signal sequence of PPAE, an insect-derived secretion protein, the expression level of human G-CSF is significantly higher than that of the natural type before the replacement. It was confirmed that the increase. In addition, as shown in Figure 4, when the vesicle transfer signal sequence of the insect-derived secretion proteins PPAE, PDI and BX of the insect-derived protein used in the present invention was excellently expressed hG-CSF, but in particular replaced by PPAE-derived vesicle transfer signal sequence It can be seen that it shows a relatively high efficiency.

Attach an electronic file to a sequence list

Claims (13)

A fusion polynucleotide in which a vesicle transfer signal sequence encoding an amino acid sequence of SEQ ID NO: 1 of an insect-derived secreted protein PPAE (prophenoloxidase activating enzyme) is fused with a base sequence encoding human G-CSF.
delete The fusion polynucleotide according to claim 1, wherein the human G-CSF is the amino acid sequence of SEQ ID NO. The fusion polynucleotide according to claim 1, wherein the vesicle transfer signal sequence of the PPAE is a nucleotide sequence represented by SEQ ID NO: 14. The fusion polynucleotide according to claim 1, wherein the nucleotide sequence encoding human G-CSF is a nucleotide sequence represented by SEQ ID NO: 17. An expression vector comprising the fusion polynucleotide of any one of claims 1 to 3. The expression vector of claim 6, wherein the vector is a pPPAE GCSF vector having a cleavage map disclosed in FIG. 8.
8

A cell transformed with the expression vector of claim 6. A transgenic insect cell wherein the vector of claim 6 is introduced to produce human G-CSF. The insect cell of claim 9, wherein the insect cell is a BM5 cell, SF9 cell, or SF21. Introducing the expression vector of claim 6 into an insect cell or insect;
Culturing the cells or breeding the insects; And
Human G-CSF production method comprising the step of isolating human G-CSF from the cultured cells or reared insects. The production method according to claim 11, wherein the expression vector has a cleavage map as described in FIG.
8

The method of claim 11, wherein the insect cells are BM5 cells, SF9 cells, or SF21.

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