KR101402272B1 - Novel G-CSF polynucleotide and use thereof - Google Patents

Abstract

The present invention relates to a synthetic G-CSF polypeptide comprising a polypeptide of SEQ ID NO: 1 capable of synthesizing a human G-CSF protein, and a polynucleotide encoding the same.
The present invention also relates to a method for producing human G-CSF from insects using an expression vector comprising the polynucleotide.

Description

Novel G-CSF polynucleotide and use thereof < RTI ID = 0.0 >

The present invention relates to novel synthetic G-CSF polynucleotides. More particularly, the present invention relates to polypeptides and polynucleotides of SEQ ID NOS: 1 and 2 capable of synthesizing human G-CSF, and can be used to produce human G-CSF from insect or insect cells.

As molecular biology techniques have evolved, methods for mass production of metabolites, oral vaccines, and medical proteins that are useful by genetic engineering methods have been developed. Although there are various methods for producing such a useful protein, in general, a biologically active protein is economical, stable, and produces a natural type protein with high production efficiency. Also, selection of a protein expression system that meets these needs should also consider the characteristics of the protein to be expressed.

Granulocyte colony stimulating factor (hereinafter referred to as 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 are effective in preventing some infectious complications of the neutropenic condition.

In addition, G-CSF reduces the incidence of febrile neutropenia (FN), thereby decreasing the mortality of cancer chemotherapy, reducing the mortality of high-dose chemotherapy supported by bone marrow transplantation, and reducing the severity of chronic neutropenia which reduces the incidence of infection in patients with neutropenia. In addition, G-CSF was shown to have a therapeutic effect when administered after the onset of myocardial infarction.

As a result of cytotoxic chemotherapy, acute myelosuppression 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 susceptible to proliferative-specific therapies such as chemotherapy and radiation therapy. For some cancer patients, repeated or high dose cycles of chemotherapy may result in stem cell loss of hematopoietic stem cells and their progeny. Prevention of these side effects of chemotherapy and radiation therapy and protection from side effects can be a 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, particularly hematopoietic progenitor cells.

Many studies have been conducted to synthesize human G-CSF because of its important use as such a drug. Various vectors and hosts have been used to produce recombinant proteins in large quantities. Generally, E. coli, animal cell lines, yeast, transformed animals, and transgenic plants are used.

The advantage of using prokaryotes such as Escherichia coli is that a high expression level can be secured, that methionine, which is not present in human-derived natural proteins, is added to the amino terminus, and that endotoxin contamination from prokaryotes And a process in which synthetic proteins often need to be coagulated in the prokaryotic cell and restored to a functional protein. Also, eukaryotic proteins such as human have many glycoproteins, but prokaryotes are not capable of synthesizing glycoproteins.

However, it is known that the degree and pattern of glycosylation is very different from that of humans, resulting in a lot of immunogenicity (Hermeling et al., Pharm. Res. 21 (6): 897-903 (2004)). In the case of transgenic animals, it has not yet reached commercialization due to the problem of contamination of the animal itself and the management of the pathogen. The method using an animal cell line is most widely used for the production of recombinant proteins because the produced recombinant protein is very similar to the human form and can be stably produced and managed. However, it has a high production cost, , And mammalian cells such as CHO cells are used, there is a problem that the stability and the expression amount such as contamination of viruses that can be infected to humans are remarkably low. Therefore, there is a need to develop a more efficient and safe recombinant protein production method.

In order to solve the problems of the prior art as described above, the present inventors have found that the present invention does not utilize Escherichia coli and animal cells, and that an unnecessary amino acid is not present at the N-terminus of the secretory protein, CSF and human endogenous G-CSF by minimizing the pollution burden of endotoxin or animal-derived foreign pathogens and enhancing the expression efficiency of natural type exocrine secretory proteins. Thereby completing the present invention.

It is an object of the present invention to provide novel synthetic G-CSF polypeptides and polynucleotides capable of synthesizing human G-CSF proteins in insect cells or insects.

In order to achieve the above object, the present invention provides a synthetic G-CSF polypeptide comprising the polypeptide of SEQ ID NO: 1 and a polynucleotide encoding the same.

The present invention also provides an expression vector comprising the polynucleotide and a cell transformed with the polynucleotide.

The present invention also relates to a method for producing an insect cell, Culturing the cells or raising the insects; And isolating human G-CSF from the cultured cell or the cultured insect.

According to the present invention, human G-CSF protein can be advantageously produced with high efficiency using insect cells.

Figures 1A and 1B show human G-CSF gene and amino acid sequence.
Figure 2 shows the synthesized novel G-CSF gene and amino acid sequence.
Fig. 3 shows the result of homology testing of the synthesized G-CSF gene using NCBI blast.
FIG. 4 shows the result of Western blotting that the synthesized G-CSF gene is introduced into silkworm moth cells to produce human G-CSF protein.

The present invention relates to a synthetic G-CSF polypeptide characterized by comprising the polypeptide of SEQ ID NO: 1.

In order to attain the object of the present invention, functional analogues of synthetic G-CSF polypeptides may be included, and functional equivalents refer to sequences exhibiting activity equivalent to human G-CSF. And preferably has 90% or more sequence homology with the polypeptide sequence of SEQ ID NO: 1 of the present invention. The functional equivalent may be that some of the amino acid sequence of the synthetic G-CSF is produced as a result of addition, substitution or deletion, and the substitution of the amino acid is preferably a conservative substitution.

As a result, when the synthetic G-CSF protein is expressed in insect cells, an unnecessary amino acid is not present at the N-terminus of the secretory protein and secretion expression level is increased compared with the case of using Escherichia coli and animal cells, Or minimizing the pollution burden of the animal-derived foreign pathogens, thereby enhancing the expression efficiency of the natural type exocrine secretory proteins.

The present invention also provides an expression vector comprising the above-described polynucleotide.

The term " vector " in the present invention includes a plasmid vector or a baculovirus vector as a means for expressing a gene of a target recombinant protein in an insect cell that is a host cell according to the object of the present invention, Is a viral vector.

The vector according to the present invention may be used with various functional promoters known in the art, and the fusion polynucleotide is operably linked to a promoter. The term "promoter" refers to a DNA sequence that controls the expression of a nucleic acid sequence operatively linked to a particular host cell. The term "operably linked" means that a 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 transforming cell line resistance gene used as a selective marker for expression to induce high expression in the transforming cell line. In the present invention, a resistance gene for an insect cell used in the art can be used as a resistance gene.

In addition, the expression vector of the present invention may further include components of a general vector such as, for example, a replication origin and a multiple adenylation signal, and other transcription regulatory factors, and the like.

In a specific embodiment of the invention, the expression vector may be a pIZT / V5 His vector. By expressing the expression vector of the present invention, the desired recombinant protein can be expressed with high efficiency.

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

A preferred host cell for transformation usable in the present invention may be an insect cell. The insect cell base may include BM5 cell line, SF9 cell line, SF21 cell line, and the like. Preferably, the host cell is a BM5 cell line derived from a silkworm moth.

&Quot; Transformation " of an insect cell line in the invention includes any method of introducing a nucleic acid into a cell and can be carried out by selecting a suitable standard technique depending on the insect cell line used, as is known in the art. In a specific embodiment of the invention, transformation was carried out using a transfection reagent.

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

In another aspect, the present invention provides a method for producing an insect cell, comprising: introducing an expression vector of the present invention described in detail above into an insect cell or an insect; Culturing the cells or raising the insects; And separating human G-CSF from the cultured cell or the cultivated insect. The present invention also provides a method for producing human G-CSF.

The insect cells may include BM5 cells, SF9 cells, SF21 cells, and the like. Preferably, the host cells may be BM5 cells, which are silkworm-derived cell lines. The insect is preferably a silkworm moth. 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 a higher concentration of human G-CSF than can be found in naturally occurring insect cells have.

The step of introducing into an insect cell or an insect in the present invention includes any method of introducing an expression vector into an insect cell or an insect, and selecting a suitable standard technique according to an insect cell line or an insect to be used as is known in the art can do. In a specific embodiment of the present invention, BM5 cells, silk-derived cell lines, were transformed using transfection reagents.

In addition, the insect cell culture in the present invention may be performed according to a suitable culture medium and culture conditions known in the art. Such a culturing process can be easily adjusted according to the insect cell line selected by those skilled in the art. Suspension cultures and adherent cultures can be classified into batches, fed-batch and continuous-batch cultures according to the culture method. The medium used for culture should suitably meet the requirements of a particular cell line. In addition, for insect cell culture, the medium includes various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and 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. These carbon sources may be used singly or in combination. Examples of nitrogen sources that can be used include organic nitrogenous organic sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat and the like, such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate It contains inorganic minerals. These nitrogen sources may be used alone or in combination. In addition, amino acids, vitamins and suitable precursors and the like may be included.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  1. Human G- CSF  The new G- CSF  Sequence synthesis

Two types of human G-scf gene sequences are known as shown in Fig. The silkworm G-CSF polynucleotide synthesizes the same protein as the human G-CSF protein even if the gene sequence is different from human.

A new synthetic G-CSF polynucleotide was synthesized by suitably performing DNA modification so that the human G-CSF gene sequence can be expressed in silkworm moth. More specifically, based on the human DNA sequence, the codon usage of the insect gene is applied to transform the most frequent codon in the target organism, silkworm moth, and then the new DNA sequence is transferred through the DNA automatic synthesizer Were synthesized. Chemical DNA synthesis was accomplished by attaching the first base to which the DMP was attached to the support and sequencing the subsequent base to the oxidation reaction. This process was commissioned by Bioneer. The Sense strand (non-template strand, 5 '-> 3' strand) was chemically synthesized by attaching EcoRI restriction enzyme sequence to the 5 'end of the coding portion of the G-CSF gene and an AgeI restriction enzyme sequence at the 3' The double-stranded gene was synthesized by chemically synthesizing an anti-sense strand (3 '<- 5') and annealing these two strands.

The synthesized G-CSF sequence is shown in Fig.

The NCBI blast was performed to confirm that the synthesized G-CSF sequence was homologous to the existing sequence. As a result, it was confirmed that there was no nucleotide sequence having homology, and it was confirmed that the synthetic G-CSF sequence of the present invention was novel (FIG. 3).

Example  2. Human G- CSF  Expression

The synthesized polynucleotide was inserted into a pIZT / V5 His vector digested with the same restriction enzyme and cut with the same enzyme. At this time, to facilitate identification of the G-CSF protein to be produced, the G-CSF gene end codon was removed and inserted into the pIZT / V5 His vector so that the His tag was attached at the C-terminus.

The newly constructed vector pIZTbGCSF containing the G-CSF gene was transformed into a silkworm-derived cell line Bm5 with lipofectamine. An appropriate amount of each vector DNA was added to a 25 cm ² cell culture flask containing 4 x 10 ⁵ cells and transformed with lipofectamine (Lipofectamine ™ Invirogen, Life Technologies), a transfection reagent.

Two days after transformation, the medium was recovered and centrifuged to remove solids. To isolate human G-CSF present in 2 ml of each centrifugation supernatant, 60 μl of 50% nickel sepharose beads suspension was added and incubated at 4 ° C for one hour, and then nickel sepharose beads were washed three times. To the nickel beads thus obtained, 30 μl of 2 × PAGE gel loading buffer was added and heated, followed by PAGE (polyacrylamide electrophoresis). After completion of the electrophoresis, proteins present in the gel were transferred to a PROTRAN nitrocellulose membrane, and the presence of G-CSF was confirmed by Western blot analysis using an anti-his antibody according to a conventional method. Bm5 cell culture without transformation was used as a control.

As can be seen from FIG. 4, it was confirmed that the human G-CSF protein was expressed in insect cells into which the synthetic G-CSF gene was introduced. Thus, it was confirmed that the newly synthesized G-CSF polynucleotide sequence of the present invention can effectively synthesize human G-CSF protein in insect cells.

<110> Dong-eui University Industry-Academic Cooperation Foundation <120> Novel G-CSF polynucleotide and use thereof <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 195 <212> PRT <213> Artificial Sequence <220> <223> Novel G-CSF <400> 1 Met Phe Leu Ile Trp Thr Phe Ile Val Ala Val Leu Ala Ile Gln Thr   1 5 10 15 Lys Ser Val Val Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln              20 25 30 Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp          35 40 45 Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His      50 55 60 Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala  65 70 75 80 Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu                  85 90 95 Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala             100 105 110 Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln         115 120 125 Leu Asp Val Ala Asp Phe Ala Thr Ile Trp Gln Gln Met Glu Glu     130 135 140 Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala 145 150 155 160 Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser                 165 170 175 His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu             180 185 190 Ala Gln Pro         195 <210> 2 <211> 585 <212> DNA <213> Artificial Sequence <220> <223> Novel G-CSF <400> 2 atgttcctga tttggacttt cattgtggct gtacttgcaa ttcaaaccaa aagcgtggtg 60 gctacgccgt taggaccggc ttcatcactt cctcaatctt tcttgctcaa gtgtttagaa 120 caggttagaa aaatacaagg tgatggcgct gcgttacagg agaaattatg tgctacttac 180 aagttatgcc accctgagga actggtcctc ctgggtcata gcttaggaat accatgggcc 240 cccctgagtt cctgcccctc acaagcctta caactcgctg gatgcttgag tcagcttcat 300 agcggtcttt tcttatacca gggtctttta caggcactag agggcatctc gccagaatta 360 ggtcctacac tagacacatt gcaattggat gttgctgact ttgcgacaac catctggcaa 420 cagatggaag aattgggaat ggcacctgct ttacaaccaa cgcaaggtgc gatgcccgct 480 tttgcttctg cattccaaag aagagctggt ggcgtcctag ttgcgtccca cctacagtct 540 tttctggagg tatcgtatag agtcttgaga cacttagctc agccg 585

Claims (8)

A synthetic G-CSF polypeptide comprising the polypeptide of SEQ ID NO: 1.
A polynucleotide encoding a synthetic G-CSF, characterized in that it comprises a polynucleotide encoding the polypeptide of SEQ ID NO: 1.
3. The polynucleotide of claim 2, wherein said polynucleotide is SEQ ID NO: 2.
A polynucleotide encoding a synthetic G-CSF, wherein the polynucleotide of claim 2 or 3 is expressed in insect cells to synthesize human G-CSF.
An expression vector comprising the polynucleotide of claim 2 or 3.
An expression vector wherein the vector of claim 5 is a pIZT / V5 His vector.
An insect cell transformed with the expression vector of claim 5.
8. The transformed insect cell of claim 7, wherein the cell produces human G-CSF.

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