KR910005624B1 - Method for producing of human garnulocyte colony stimulating factor - Google Patents

Abstract

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Description

Method for preparing glycoprotein having colony stimulating factor activity of human granulocytes

Figure 1 shows the sequence of three different probes, IWQ, A and LC.

2 shows the nucleotide sequence of the pHCS-1 insert.

Figure 3a shows the nucleotide sequence of the cDNA insert in pBRG4.

Figure 3b (I) shows the amino acid sequence of human G-CSF precursor inferred from pBRG4 cDNA.

Figure 3b (II) shows the amino acid sequence of human mature G-CSF inferred from pBRG4 cDNA.

Figure 4a shows the nucleotide sequence of a cDNA insert in pBRV2.

4b (I) shows the amino acid sequence of the human G-CSF precursor inferred from pBRV2 cDNA.

Figure 4b (II) shows the amino acid sequence of human mature G-CSF inferred from pBRV2 cDNA.

Figure 5 shows the restriction enzyme cleavage site of human G-CSF cDNA derived from pBRG4- or pBRV2-.

6 schematically shows the structure of pHGA410.

Figure 7 shows the preparation of the expression vector recombinant pTN-G4, pTN-G4VA α and pTN-G4VA β.

8A and 8B show a method of preparing pHGG4-dhfr.

Figure 8c shows the preparation method of pG4DR1 and pG4DR2.

9 schematically shows the structure of pHGV2.

Figure 10 shows a method for producing the expression vector recombinant pTN-V2, pTN-VA α and pTN-VA β.

11a and 11b show a method for producing a transgenic recombinant vector pHGV2-dhfr.

Figure 11c shows a method for producing pV2DR1 and pV2DR2.

The present invention relates to colony stimulating factors of human granulocytes. More specifically, the present invention relates to a method for producing a glycoprotein having the activity of a specific stimulator necessary for forming colonies of human granulocytes, that is, colony stimulator (hereinafter abbreviated as "CSF").

When bone marrow cells are used as target cells in the upper layer and kidney cells or fetal cells in the lower layer and cultured by two-layer soft agar culture, some of the cells in the upper layer proliferate and differentiate and are called neutrophil granulocytes (hereinafter, referred to as "granulocytes"). Or the fact that colonies of monocytes are formed, it is known that there are factors that promote colony formation in vivo [Pluznik and Sach; J. Cell, Comp. Physiol., 66, 319p (1965), and Bradley and Metcalf; Aust. J. Exp. Biol. Med. Sci., Vol. 44 287p (1966)].

Factors collectively called CSF are known to be produced in cells such as T-cells, monocytic macrophages, fibroblasts and endothelial cells, and these cells are normally widely distributed in vivo. CSF acts on the granulocytes or stem cells of monocytic macrophages to stimulate their proliferation and induce differentiation (in soft water) to form granulocytes or colonies of monocytic macrophages. Monocytic macrophage CSF (weak with GM-CSF); Monocytic macrophages CSF (weak M-CSF), which have the function of forming colonies of monocytes mainly; Pluripotent CSF (weakly multi-CSF) acting on more differentiated pluripotent stem cells; And subclasses such as granulocyte leukocytes CSF (weak to G-CSF) having an action of forming granular leukocyte colonies, as in the present invention, and target cells according to each subclass in recent years. The differentiation stage of is thought to be different. Asano; Metabolism and Disease, Volume 22, 249p (1985) and Yunis et al .; Growth and Maturation Factors, edited by Guroff, John Wiley & Sons, NY, Vol. 1, pp. 209p (1983).

Therefore, the purification of each CSF subclass to further elucidate their chemical and biological properties is very important in analyzing the pathological morphological aspects of various blood diseases and evaluating blood production mechanisms. The biological action of G-CSF, which has increased the interest of researchers, induces the differentiation of myeloid leukocytes and enhances the function of mature granulocytes, which has been shown to be of clinical importance for the treatment and prevention of leukemia. It is expected to be.

Conventionally, as a method for separating and purifying G-CSF, a cell culture method of culturing cells and separating G-CSF from its supernatant has been used. Since obtaining a -CSF is a complicated purification process, it has not been possible to obtain a uniform G-CSF in large quantities. Therefore, there is an urgent need for mass production of G-CSF using DNA recombination technology.

Under these circumstances, the inventors have isolated a gene encoding a polypeptide having human G-CSF activity and succeeded in expressing the gene in a host cell.

The present invention cultures mammalian cells transformed with a recombinant vector containing a cDNA fragment having a nucleotide sequence encoding the amino acid sequence shown in Fig. 3b (I) or 4b (I), and then produced a human body. It is to provide a method for producing a glycoprotein having human granulocyte leukocyte colony stimulator activity, characterized in that to separate the glycoprotein having granular leukocyte colony stimulator activity.

Hereinafter, the present invention will be described in detail.

The gene encoding a polypeptide having human G-CSF activity according to the present invention is a messenger RNA encoding a polypeptide having human G-CSF activity obtained in 15-17S fraction using sucrose density gradient centrifugation. complementary DNA) (cDNA).

The inventors obtained two such series of cDNAs.

One class of cDNAs has a gene or part thereof that encodes polypeptide I or II shown in FIG. 3B. More specifically, the sequence from the ATG at the 32-34 base position to the CCC at the 650-652 base position at the 5'-end of the nucleotide sequence shown in FIG. 3a, and at the 650-652 position from the ACC at 122-124 position To the CCC or the nucleotide sequence shown in FIG. 3a or part thereof. This series of cDNAs is hereinafter referred to as cDNA (+ VSE).

Another series of cDNAs has a gene encoding a polypeptide I or II shown in FIG. 4b or part thereof, and more specifically, an ATG at the 31-33 base position at the 5'-end of the nucleotide sequence shown in FIG. 4a. To the CCC at the 640-642 nucleotide position, the sequence from the ACC at the 121-123 position to the CCC at the 640-642 base position, or the sequence shown in FIG. 4A or part thereof. This series of cDNAs is hereinafter referred to as cDNA (-VSE).

The above-described genes can be obtained by the following method: First, mRNAs encoding G-CSFs are produced from mammalian cells or other host cells having the ability to produce polypeptides having G-CSF activity; This mRNA is then converted to double-stranded cDNA using any known technique; A group of recombinants containing the cDNA (hereinafter referred to as cDNA library) are continuously selected by known techniques.

A mammalian cell that can be used as an mRNA source is the cell line CHU-2 derived from human oral cancer (deposited with accession number I-483 at Collection Nationale de Cultures de Microrganismes (CNCM), France). However, instead of such tumor cell lines, cells that can be isolated from mammals or other suitably established cell lines can also be used. The preparation of mRNA may also use one of the proposed methods for cloning genes of several other bioactive proteins. For example, treatment with surfactants and phenols in the presence of ribonuclease inhibitors such as vanadil-ribonucleoside complexes [Berger and Birkenmeier; Biochemistry, Vol. 18, 5143p (1979)] or guanidine thiocyanate, followed by CsCl density gradient centrifugation [Chirgwin et al., Biochemistry, Vol. 18, 5294p (1979)] to obtain total RNA first, followed by the total RNA Poly (A ⁺ ) RNA is obtained by affinity column chromatography or batch adsorption using poly-U-sepharose and the like as a carrier using oligo (dT) -cellulose or Sepharose 2B.

In addition, the poly (A ⁺ ) RNA may be further fractionated by an appropriate method such as sucrose density gradient centrifugation. To confirm that the obtained mRNA can encode a polypeptide having G-CSF activity: transducing the mRNA into a protein and testing its physiological activity or identifying the protein using an anti-G-CSF antibody. And the like. In more detail, mRNA is injected into oocytes of Xenopus laevis (Gurdon et al., Nature, Vol. 233, 177p (1972)) or rabbit reticulocytes or wheat embryo system. Translation reactions are being performed (Schlief and Wensink, Practical Methods in Molecular Biology, Springer-Verlag, NY (1981)). Assay of G-CSF activity is carried out by applying soft agar culture method using bone marrow cells, and these methods are general. [Metcalf; Hemopoietic Colonies, Springer-Verlag, Berlin, Heidelberg, NY (1977).

Single-stranded cDNA was synthesized using the mRNA obtained in this manner as a template; Synthesizing a double-stranded cDNA from this single-stranded cDNA; Recombinant plasmids are prepared by insertion into appropriate vector DNA. This recombinant plasmid can be used to transform an appropriate host, for example Escherichia coli, to obtain a group of DNA (hereinafter referred to as cDNA library) in a transformant.

Double-stranded cDNA can be obtained from mRNA using one of two methods; For example, oligonucleotides (dT) complementary to the 3'-terminus poly A-chain of mRNA are treated with reverse transcriptase using initiators: or oligonucleotides corresponding to part of the amino acid sequence of the G-CSF protein are synthesized and CDNA complementary to mRNA is synthesized by treatment with reverse transcriptase, used as an initiator. Double-stranded cDNAs can also be prepared by the following methods: The single-stranded cDNA obtained after digestion and removal of mRNA by alkali treatment is first treated with reverse transcriptase or DNA polymerase I (e.g. Clenow fragment), Then treated with S1 nuclease; Alternatively, mRNA is treated directly with RNase H and DNA polymerase (eg E.coil polymerase I). For more information, see Maniatis et.al., Molecular Cloning, Cold Spring Harbor Laboratory (1982); And Gubler and Hoffman; Gene, 25, 263p (1983).

The double-stranded cDNA thus obtained is converted into suitable vectors, for example EK-type plasmid vectors represented by pSC101, pDF41, ColE1, pMB9, pBR322, pBR327, and pACYC1 or λgt, λc, λgt 10 and λgt WES and the like. E. coli strains (X1776, HB101, DH1 or C600) were transformed using these recombinant vectors by insertion into representative phage vectors to obtain cDNA libraries (see, eg, Molecular Cloning, ibid).

Double-stranded cDNAs can be linked to vectors by the following methods.

A chemically suitably synthesized DNA fragment is added to attach a connectable end to the end of the cDNA, and the vector DNA, previously cleaved using a restriction enzyme, and the cDNA are ligated by treatment with T4 phage DNA ligase in the presence of ATP. Alternatively, by adding dC, dG-chains (or dT, dA-chains) to vector DNA and double-stranded cDNAs previously cleaved using restriction enzymes, and then annealing the solution containing both DNAs. Can also be combined. (See Molecular Cloning ibid.)

Any known method can be used to transform host cells using the recombinant DAN thus obtained. Hanahan describes in detail when the host cell is E. coli [J. Mol. Biol., 166, 557p (1983)], i.e., by adding this recombinant DNA to competent cells prepared by coexisting CaCl ₂ , MgCl ₂ or RbCl.

Cells carrying the gene of interest can be searched by various methods described below: plus-minus method used for cloning of interferon cDNA [Taniguchi et al., Proc. Jpn. Acad., Vol. 55 Ser. B. , 464p (1979)], hybridization-translation assay method [Nagata et al., Nature, Vol. 284, 316p (1980)] and amino acid sequences of proteins with human G-CSF activity. Colony or plaque hybridization using chemically synthesized oligonucleotide probes (Wallace et al., Nucleic Acids Res., Vol. 9, 879p (1981) and Benton & Davis, Science, Vol. 196, 180p (1977)), etc. good.

In this way, a fragment having a gene encoding a polypeptide having cloned human G-CSF activity can be reinserted into an appropriate vector-DNA to transform host cells of other prokaryotes or eukaryotes. In addition, genes can be expressed in individual host cells by introducing sequences appropriate for promoters and expressions into their vectors.

Host cells derived from mammalian cells include COS cells, Chinese hamster ovary (CHO) cells, C-127 cells, and Hela cells. PSV2-gpt [Mulligan and Berg: Proc; Natl. Acad. Sci., USA. 78, 2072p (1981). These vectors include a replication origin, a selection marker, a promoter located in front of the gene to be expressed, an RNA junction, a polyadenylation code, and the like.

Examples of promoters that can be used for gene expression in mammalian cells include promoters such as retrovirus, polyomavirus, adenovirus, and simianvirus 40 (SV40). For example, when using the SV40 promoter, it is possible to easily perform the desired gene expression according to Mulligan et al. [Nature, Vol. 277 108p (1979)].

As the origin of replication, those derived from SV40, polyomavirus, adenovirus, bovine papillomavirus (BPV) and the like can be used. Selection markers include phosphate transferase APH (3 ′) II or I (neo) genes, thymidine kinase (TK) genes, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) genes, dehydro Folate reducing element (DHFR) gene and the like can be used.

The following methods can be used to obtain a polypeptide having human G-CSF activity using the above-described host-vector system: at an appropriate site in one of the vectors encoding the polypeptide having human G-CSF activity mentioned above. After inserting the gene to transform the host cell with recombinant DNA, the obtained transformant is cultured. Again it can be isolated and purified from the desired intracellular or culture using one of the known techniques.

Eukaryotic genes generally exhibit polymorphisms, known as human interferon genes, etc. (Nishi et al., J. Biochem., 97, 153 (1985)), whereby one or more amino acids are replaced by polymorphisms. Alternatively, even if there is a change in the nucleotide sequence, the amino acid sequence is not changed at all.

Polypeptides lacking or adding one or more amino acids of the amino acid sequences shown in FIGS. 3B or 4B, or polypeptides in which one or more amino acids are substituted with one or more other amino acids, may also have G-CSF activity. . For example, it has been known that a polypeptide obtained by converting each of the cysteine codons of the human interleukin-2 (IL-2) gene to serine codon retains the activity of interleukin-2 [Wang at el., Science, 224 Vol. 1431p (1984). ]. Therefore, genes encoding these polypeptides, recombinant vectors having these genes, and transformants obtained by the recombinant vectors, provided that they are naturally present or chemically synthesized, or that the polypeptides have human G-CSF activity. Or polypeptides or glycoproteins obtained by culturing a transformant are included in the scope of the present invention.

From now on, a gene encoding a polypeptide having human G-CSF activity, a recombinant vector having the gene and a transformant having the recombinant vector, and a polypeptide having human G-CSF activity expressed in the transformant Or a brief description of a method for producing a glycoprotein.

[(1) Probe Manufacturing]

The amino acid sequence is determined from the N terminus of the uniform human CSF protein obtained by purification from the culture supernatant of the tumor cell line CHU-2. It is digested with bromocyanate and treated with trypsin to obtain fragments, and amino acid sequences are also determined for these fragments (Examples 3 (i), (ii) and (iii)).

Of the determined amino acid sequences, three kinds of nucleotide probes a, (LC) and (IWQ) having the sequence shown in FIG. 1 are synthesized. (Example 4). Probe a is a mixed probe consisting of 14 consecutive nucleotides. Probe (IWQ) is a type of probe used for cloning the human clecistokinin gene [Takahashi et al., Proc. Natl. Acad. Sci., Vol. 82, 1931p (1985)] 30 contiguous nucleotides with deoxyinosine. The probe (LC) is a probe composed of 24 nucleotides synthesized based on the nucleotide sequence shown in FIG. 3, corresponding to the 32-39th portion from the N terminus of the amino acid sequence shown in Example 3 (i).

Chemical synthesis of nucleotides can be carried out by applying the improved phosphoester ester method to the solid phase method, which is described in the narrative of Narang (Narang, Tetrahedron, 39, 3 to 22 (1983)).

Probes prepared based on amino acid sequences at positions other than those mentioned above may also be used.

[(2) Construction of cDNA Library]

Guanidine thiocyanate solution was added to CHU-2 cells for homogenization, and CsCl density gradient centrifugation was performed to obtain total RNA.

Poly (A ⁺ ) RNAs are selected from this total RNA by oligo (dT) -cellulose column chromatography. Then, single-stranded cDNA is synthesized with reverse transcriptase, and RNase H and E. coli DNA polymerase I are added to obtain double-stranded cDNA. The dC chain is added to the double-stranded cDNA obtained above and conjugated with the pBR322 vector with the dG chain added to the Pst I cleavage site. The resulting recombinant DNA was used to transform E. coli strain X1776 and construct pBR322 based cDNA libraries (Examples 5 and 6).

In a similar manner, a double-stranded cDNA is linked to a λgt10 vector using an EcoRI linker to construct a λ-phage based cDNA library (Example 7).

[(3) screening]

Recombinant from pBR322 based cDNA library was immobilized on Whatman 541 filter paper and colonies hybridized using a 32p radiolabeled probe (IWQ) to select one clone. Southern blotting method: Southern, J. Mol. Biol., Vol. 98, 503p (1975)] showed that hybridization was also possible with probe a. The nucleotide sequence of this clone was determined by dideoxy method [Sanger, science, Vol. 214, 1205p (1981)].

The base sequence of the obtained cDNA insert is shown in FIG. As shown in Figure 2, this cDNA insert consists of 308 base pairs comprising a probe (IWQ) and a, and an open reading that encodes 83 amino acids comprising the amino acid sequence shown in Example 3 (iii). It can be seen that it has an open reading frame. The plasmid derived from pBR322 containing this 308 base pair is abbreviated as pHCS-1 below (Example 8).

DNA fragments containing 308 base pairs obtained from pHCS-1 were radiolabeled by the nick translation method (Molecular Cloning ibid), and used as probes to generate plaque hybridizations of λgt10-derived cDNA libraries [Benton and Davis, Science , 196, 180p (1977)] to obtain five clones. For clones that are thought to contain cDNA, their base sequences are determined in the same manner as described above (Figure 3a).

As shown in Figure 3a, it can be seen that this cDNA insert has one large open reading frame.

The amino acid sequence encoded by this cDNA can be inferred as shown in Figure 3a.

Compared with the N-terminal amino acid sequence of the G-CSF protein shown in Example 3 (i), the cDNA starts with an ATG sequence at 32-34 nucleotides at the 5 'end and ends with a GCC sequence at positions 119-121. A signal sequence encoded by 90 base pairs and a nucleotide sequence corresponding to a mature G-CSF polynucleotide encoded by 531 base pairs starting with an ACC sequence at positions 122-124 and ending with a CCC sequence at positions 650-652. It turned out to be doing. Therefore, the polypeptide of amino acid sequence I shown in FIG. 3b is composed of 207 amino acids and its molecular weight is calculated to be 22292.67 Daltons. The polypeptide of amino acid sequence II consists of 177 amino acids and has a molecular weight of 18986.74 Daltons (Example 9). However, the ATG of the 32-34 position or the 68-70 position can also be considered with respect to the starting site of a protein. Escherichia coli strain X1776, which maintains PBR322 with the cDNA (+ VSE) inserted into the EcoRI cleavage site, is located at the Ministry of Commerce, Industry and Technology, Microbial Technology Research Institute, the Agency of Industrial Science and Technology (Accession Number: FERM BP-954).

5 shows the restriction enzyme cleavage site of the obtained gene.

The plasmid in which the cDNA was bound to the EcoRI site of pBR327 (Soberon et al., G, Vol. 9, 287p (1980)) is hereinafter referred to as pBRG4.

PBRG4 thus obtained was treated with restriction enzyme EcoRI to obtain a DNA fragment containing cDNA of about 1500 base pairs. The fragments were radiolabelled by Molecular Cloning (ibid), and the λgt10-derived cDNA libraries were again selected by plaque hybridization (Benton and Davis, ibid) using this radiolabeled DNA fragment as a probe. At this time, two nitro cellulose filter papers on which λ-phage DNA was immobilized were prepared, one of which was used for the above-mentioned plaque hybridization, and the other one was subjected to plaque hybridization with the probe (LC) described above. Phage positive for both probes are selected. Clones considered to have full-length cDNA were selected and the base sequence of the cDNA insert was determined by dideoxy method as shown in FIG. 4a.

This cDNA has one large open reading frame, and the coding amino acid sequence is inferred as shown in Figure 4a.

Compared to the N-terminal amino acid sequence of the G-CSF protein shown in Example 3 (i), this cDNA is 90 starting from the ATG sequence at 31-33 nucleotides from the 5'-end and ending at the GCC sequence at 118-120 A signal sequence encoded with base pairs and a base sequence corresponding to a mature G-CSF polynucleotide encoded with 522 base pairs starting at the ACC sequence at positions 121-123 and ending at the CCC sequence at positions 640-642. Able to know. Therefore, the polypeptide of amino acid sequence I shown in FIG. 4B consists of 204 amino acids and its molecular weight was calculated as 21977.35 Daltons. The polypeptide of amino acid sequence II consisted of 174 amino acids and its molecular weight was calculated to be 18671.42 Daltons (Example 10).

However, it should be understood that in addition to the 31-33 'position, the ATG of the 58-60 position or the 67-69 position can be considered with respect to the protein start position.

Escherichia coli strain X1776, which retains pBR327 with the cDNA (-VSE) inserted into the EcoRI cleavage site, has been deposited with the Japan Institute of Commerce, Industry and Technology, Microbiological Technology Laboratory (Accession Number: FERM BP-). 955).

5 shows the restriction enzyme cleavage site of the gene.

The plasmid which bound this cDNA to the EcoRI site | part of pBR327 is called pBRV2 below.

[(4) Construction of Recombinant Vectors for Animal Cells]

Recombinant vectors (containing BPV) using C127 and NIH3T3 cells as host animal cells and recombinant vectors (containing dhfr) using CHO cells were prepared for + VSE and -VSE based cDNA. Representative examples are described below, and for more details, refer to related embodiments.

[Construction of a + VSE Recombinant Vector]

The cDNA (+ VSE) fragment obtained in (3) was inserted into the vector pdKCR to make plasmid pHGA410 (Examples 11 and 6). It is partially digested with EcoRI and treated with DNA polymerase I (cleno fragment) to make blunt ends. Hind III linker was attached to this DNA, followed by continuous treatment with Hind III and then T4 DNA ligase. The DNA is transformed into E. coli strain DH1 using rubidium chloride method (Molecular Cloning ibid.). The plasmid obtained at this time is called pHGA410 (H) (FIG. 7).

The pHGA410 (H) is treated with Sal I, then blunt ended with the ends and then again with Hind III to recover the Hind III-Sal I fragments. Meanwhile, the plasmid pdBPV-1 having the transformed fragment of bovine papilloma virus was treated with Hind III and Pvu II to separate large DNA fragments and conjugated with the Hind III-Sal I fragments previously separated. This is used to transform E. coli strain DH1 to obtain plasmid pTN-G4. This plasmid has CSF-cDNA from pHGG4 (Figure 7 and Example 12).

Further, pHGG4-dhfr, a CHO cell type recombinant vector (+ VSE), was prepared using pHGA410 or pHGA410 (H) plasmid and pAdD26SVpA plasmid (Examples 8a and 8b, Example 4).

The plasmid pAdD26SVpA was treated with EcoRI and BamHI to recover a 2-kb DNA fragment containing the dhfr gene and inserted into the Hind III site of pHGA410 (H) to prepare pG4DR1 and pG4DR2 (Figure 8c and Example 14). ).

[Construction of b-VSE Recombinant Vector]

The cDNA (-VSE) fragment obtained in (3) was inserted into the vector -pdKCR to make plasmid pHGV2 (Example 17), which was then partially digested with EcoRI and treated with DNA polymerase I (Cleno fragment). Make it a blunt end. After attaching Hind III linker to the DNA, followed by Hind III treatment and T4 DNA ligase treatment, E. coli strain DH1 was transformed using rubidium chloride method (Molecular Cloning, ibid.). The resulting plasmid is named pHGV2 (H) (FIG. 10).

The pHGV2 (H) is treated with Sal I and then the blunt end is terminated and then treated with Hind III to recover the Hind III-Sal I fragment. On the other hand, plasmid pdBPV-1 having a transformed fragment of bovine papilloma virus was treated with Hind III and Pvu II to separate large DNA fragments and to separate them with Hind III-Sal I fragments. This is used to transform E. coli strain DH1 to obtain plasmid pTN-V2. This plasmid has CSF-cDNA derived from pHGV2- (Figures 10 and 18).

In a similar manner to + VSE, pHGV2-dhfr, a recombinant vector for CHO cells (-VSE), was prepared using pHGV2 or pHGV2 (H) and pAdD26SVpA (Figures 11a and 11b, Example 20).

pAdD26SVpA was treated with EcoRI and BamHI to recover a DNA fragment of about 2 kb containing the dhfr gene, and then inserted into the Hind III site of pHGV2 (H) to prepare pV2DR1 and `` pV2DR2 (Figure 11c and Example). 20).

[(5) Expression in Animal Cells]

Representative examples are described below, and for more details, see the associated embodiments.

[Transduction in Mouse C127 Cells]

Plasmid pTN-G4 or pTN-V2 is treated with BamHI. This treated plasmid was transformed by C127TPVHFMF calcium phosphate method which was cultured. The transformed cells are cultured to select clones with high CSF production capacity. Glycoprotein containing transgenic G-CSF was recovered from the culture medium of the transformed cell and purified to confirm that it had G-CSF activity. Amino acid analysis and sugar content analysis of the sample were also carried out to confirm the presence of the desired glycoprotein.

To analyze the sugar content, the CSF sample used for amino acid analysis was quantified for amino sugars by the Elson-Margan method, neutral sugars by the orcinol sulfate method, or sialic acid by the thiobarbiturate method. Do each one. Each quantitative method is described in Biochemistry Experiment Lecture A Volume 4, Chapter 13, Chemistry of Carbohydrates (Bottom Volume) published by Tojin Kagaku. It can be seen that the sugar content of G-CSF obtained by converting each quantitative value into a weight% ranges from 1 to 20% by weight depending on the type of host cell, the expression vector, and the culture conditions.

EXAMPLE

Before the present invention is described in more detail with reference to examples, reference examples are first presented for the purpose of illustrating the method for analyzing CSF activity.

[Reference Example]

[Analysis of CSF Activity]

In the present invention, the following methods are used to measure CSF activity (hereinafter abbreviated as CSA).

[Measuring method of CSA]

[a using human bone marrow cells:]

Single layer agar culture was performed according to the method of Bradly, TR, Metcalf, D. (Aust. J. Exp. Biol. Med. Sci., 44, 287-300p 1966). Specifically, 0.2ml bovine fetal serum, 0.1ml sample, 0.1ml (1-2 × 10 ⁵ nucleated cells) of non-adherent cell suspension of human bone marrow, modified McCoy's 5A containing 0.2ml of modified McCoy's 5A culture and 0.75% of agar. 0.4 ml of the culture solution is mixed, poured into a plastic plate for tissue cell culture (35 mm diameter), coagulated, and incubated at 37 ° C., 5% CO ₂ /95% air, and 100% humidity. After 10 days, the number of colonies formed (one colony is composed of 50 or more cells) is counted, and the CSA is measured using 1 unit of activity required to form one colony.

The modified McCoy's 5A culture and human bone marrow non-adherent cell suspension used in the above methods a and b are prepared as follows.

[Modified McCoy's 5A Medium (Twice Concentration)]

12 g of McCoy's 5A culture (Gibco), 2.55 g of MEM amino acid-vitamin medium (Nissui Seiyaku), 2.18 g sodium bicarbonate and 50,000 units of potassium penicillin G were dissolved twice in 500 ml of distilled water, followed by Millipore filter paper ( 0.22 μm) and sterilize by filtration.

[Human Bone Marrow Non-adherent Cell Suspension]

Bone marrow solution obtained by puncturing the sternum of a healthy person is diluted 5-fold with RPMI 1640 culture, placed on a picol-fake solution (Pharmacia Fine Chemicals), and centrifuged at 400 × g for 30 minutes at 25 ° C. The cell layer (specific gravity <1.077) at the interface is recovered. After washing these cells, adjust to a concentration of 5x10 ⁶ cells / ml using RPMI 1640 medium containing 20% fetal bovine serum. Pour this into a 25 cm 2 plastic flask for tissue culture, incubate for 30 min in a CO ₂ -cultivator, and then recover the non-adherent cells from the supernatant and place in a plastic flask (25 cm 2) again for 2 hours and 30 minutes. Non-adherent cells are recovered from the supernatant and used for analysis.

Example 1

[Establishment of "CHU-2"]

Tumors from patients with low oral cancer with a significant increase in the number of neutrophils are transplanted into nu / nu mice. About 10 days after transplantation, tumor weight and number of neutrophils increased significantly. After 12 days of transplantation, the tumors are aseptically extracted and cut into small pieces of 1-2 mm ³ cubes, which are then cultured by the following method.

10-15 pieces of the small tumors were placed in a 50 ml plastic centrifuge tube, 5 ml of trypsin solution (containing 0.25% trypsin and 0.02% EDTA), followed by shaking for 10 minutes in a warm bath at 37 ° C., followed by supernatant. Then, 5 ml of trypsin solution was added thereto, followed by decomposition at 37 ° C. for 15 minutes with stirring. The upper cell suspension is collected and 1 ml of bovine fetal serum is added to inactivate trypsin and preserve on ice.

The above operation is carried out again to recover the cell suspension, and combined with the one obtained above, the cell pellet is centrifuged at 15,000 rpm for 10 minutes to obtain a cell pellet. The cell pellet is washed twice with F-10 containing 10% fetal bovine serum, and then transplanted into a plastic culture flask (25 cm 2) with a cell concentration of 5 × 10 ⁶ / flask. Incubate overnight in a CO ₂ incubator (5% CO ₂ concentration, 100% humidity) using an F-10 culture containing 10% fetal bovine serum, remove supernatant with non-adherent cells, and add new culture Continue incubation. Six days after the start of the culture, the flasks are full of cells. Replace the culture with new ones. The next day, with 2 ml of anti-mouse erythrocyte antibody (Cappel) diluted 5 times with RPMI 1640, 2 ml of guinea pig complement (kyokuto seiyaku) diluted 2.5 times with RPMI 1640 was added, followed by 20 minutes at 37 ° C. Incubate. After the incubation was terminated, cardiac cells derived from nu / nu mice were removed by washing twice with F-10 containing 10% fetal bovine serum. Subsequently, F-10 culture solution containing 10% fetal bovine serum is added, and the cells are further incubated for 2 days, and some of the cells are collected and cloned by the limiting dilution method.

The 11 clones obtained were tested for CSF activity, resulting in clones (CHU-2) that exhibited about 10 times more activity than others.

Example 2

[Separation of "CSF"

The cells established as in Example 1 are grown in two culture flasks (150 cm 2) full. The cells are collected, suspended in 500 ml of F-10 culture solution containing 10% fetal bovine serum, transferred to a 1580 cm 2 rotary glass bottle (Belco), and cultured at a speed of 0.5.rpm.

When the cells have grown to the full wall of the vial, the cultures are exchanged for RPMI 1640 without serum. After incubation for 4 days, the culture supernatant is recovered and culture is continued by adding F-10 solution containing 10% fetal bovine serum. After incubation for 3 days, the culture medium is exchanged again with serum-free RPMI 1640 solution, and after 4 days, the culture supernatant is recovered. Repeat this procedure to obtain 500 ml of serum-free culture supernatant each week. Using this method, it was possible to recover the culture supernatant by maintaining the cells for a relatively long time.

5,000 ml of the culture supernatant obtained was added in one batch, and 0.01% Tween 20 was added thereto, followed by a single filtration using hollow fiber DC-4 and Amicon PM-10 (manufactured by Amicon). Concentrate to fold and then purify this concentrate in the following order.

: Concentrated culture using 0.01M Tris-HCI buffer (pH 7.4) containing 0.15M NaCl and 0.01Tween 20 (manufactured by Nakai Kagaku) by Ultrogel ACA 54 column (manufactured by LKB) with a length of 4.6 cm and 90 cm. 5 ml of the supernatant is gel filtered at a flow rate of approximately 50 ml / hour. The column was previously assayed with bovine serum albumin (Mw: 67,000), of albumin (Mw: 45,000) and chitochrome C (Mw: 12,400). After gel filtration turned off, 0.1 ml of each fraction was collected, diluted 10-fold, and the fractions showing activity were selected by the CSA assay method b. As a result, a fraction corresponding to Ve = 400-700 ml showed a macrophage-dominant CSA, whereas a fraction corresponding to Ve = 80-1200 ml showed a granular leukocyte-dominant CSA. Therefore, the latter fractions are collected and subjected to ultrafiltration using PM-10 (manufactured by Amicon) and concentrated to about 5 ml.

(ii) 0.1% trifluoroacetic acid solution containing 30% of n-propanol (for amino acid sequencing: manufactured by Tokyo Kasei KK) was added to the concentrated fraction, which was left on ice for 15 minutes and then centrifuged at 15,000 rpm for 10 minutes. Isolate to remove precipitate. Subsequently, the mixture was adsorbed onto a µ-Vondafac C18 column (semi-prepared 8 mm x 30 cm, manufactured by Waters) equilibrated with an aqueous solution containing n-propanol and trifluoroacetic acid, followed by a linear concentration gradient of 30 to 60%. Elution is successively with an aqueous 0.1% trifluoroacetic acid solution containing n-propanol. The apparatus for high pressure liquid chromatography measured the absorption at 220 nm and 280 nm simultaneously using the Hitachi model 685-50 and the detection using the Hitachi model 638-41 as a detector. After elution, 10 μιη of each fraction is aliquoted, diluted 100-fold, and the fractions showing activity are selected by the above-described "Measuring Method b of CAS". As a result, it was found that the peak eluting in 40% n-propanol had CSA activity, so that the peak was collected and chromatographed again under the same conditions, and the CSA was tested in the same manner. Activity was shown at the peak of and the peak was collected (4 fractions = 4 ml) and lyophilized.

(iii) The lyophilized powder was dissolved in 200μι of aqueous 0.1% trifluoroacetic acid solution containing 40% n-propanol, and high pressure using a TSK-G 3000SW column (orientated feeder: 7.5 mm x 60 cm) was applied to the solution. Perform liquid chromatography. Elution is carried out in the same aqueous solution at a flow rate of 0.4 ml / min, and taken up by 0.4 ml of CSA by a fractionation collector (type FRAC-100: Pharmacia Fine Chemicals). For each fraction fractionated, CSA was tested in the same manner as described above. As a result, activity was shown in a fraction having a retention time of 37 to 38 minutes (equivalent to about 20,000 MW). Purification with a Pak C18 column (4.6 mm x 30 cm) was followed by recovery of the main peak and freeze-drying. The obtained sample was assayed by the above-mentioned "Measurement method a of CSA", and found to have human G-CSF activity.

Example 3

[Determination of Amino Acid Sequences]

[(i) Determination of N-terminal amino acid sequence]

PTH amino acids obtained by Edman decomposition of samples with a meteorological analyzer (Applied BIOSYSTEMS) were subjected to conventional methods using a high pressure liquid chromatography apparatus (Beckman) and an ultrasphere-ODS column (Beckman). Analyze Equilibrate the column (5 μm: 4.6 mmΦ: × 250 mm ^L ) with the starting buffer solution (15 mM sodium acetate buffer solution (pH 4.5) and 40% acetonitrile) and then sample (dissolved with 20 μιη starting buffer solution). Inject and isolate using isocratic elution with starting buffer. The flow rate is 1.4 ml / min and the column temperature is maintained at 40 ° C. Detection of PTH amino acids uses ultraviolet absorption in the UV region of 269 nm and 320 nm. Each 2 nanomole of PTH amino acid standard sample (Sigma) is separated in situ to determine the holding time, and compared with the holding time of the sample. As a result, the sample was found to have the following 40 amino acid sequences from the N-terminus.

[(ii) Bromocyanide Decomposition]

The sample is dissolved in 70% formic acid. To the solution was added 200 equivalents of sublimated purified bromocyanate. The mixture is left to react at 37 ° C. overnight. The reaction was then lyophilized and separated by HPLC using a TSK G3000SW column (manufactured by Dongyang Procurement) to obtain four peaks. These peaks are named CN-1, CN-2, CN-3, and CN-4 from high molecular weight to amino acid sequence of CN-1, CN-2 with high yield using an automated meteorological analyzer (Applied Biosystems). Analyze under the same conditions as (i).

As a result, CN-1 was found to be a peptide from the N-terminus of the G-CSF protein, and CN-2 was shown to have the following amino acid sequence:

Pro-Ala-Phe-Ala-Ser-Ala-Phe-Gln-Arg-Arg-Ala-Gly-Gly-Val-Leu-Val-

Ala-Ser-His-Leu-Gln-

[(iii) trypsin digestion]

The sample was dissolved in 0.1M Tris-HCI buffer solution (pH7.4) containing 8M urea and mixed with 0.1M Tris-HCI Cheon buffer solution (pH7.4) containing 0.1% 2-mercapto ethanol. The final concentration is prepared to 2M. TPCK-treated trypsin (Sigma) was added to the sample to enzyme ratio of 50: 1 and reacted at 25 ° C. for 4 hours, followed by addition of trypsin treated with the same amount of TPCK, followed by further reaction at 25 ° C. for 16 hours. The reaction is then subjected to high speed reverse phase column chromatography using a C8 column (manufactured by Yamamura Kagaku K.K.). Elution is carried out with 0.1% TFA containing 5-60% linear density gradient n-propanol. Among the peaks obtained by measuring ultraviolet absorption at 280 nm, the amino acid sequence was analyzed under the same conditions as in (i) using an automatic meteorological analyzer. As a result, it was found that the main peak was a peptide having the following sequence comprising a portion of the CN-2 fragment shown in (ii):

Gln-Leu-Asp-Val-Ala-Asp-Phe-Ala-Thr-Thr-Ile-Trp-Gln-Gln-Met-Glu-

Glu-Leu-Gly-Met-Ala-Pro-Ala-Leu-Gln-Pro-Thr-Gln-Gly-Ala-Met-Pro-

Ala-Phe-Ala-Ser-

Example 4

Preparation of DNA Probes

[(i) Synthesis of Probe (IWQ)]

From the amino acid sequence obtained in Example 3 (iii), 30 contiguous nucleotides are obtained based on the 10 amino acid sequence represented by Ile-Trp-Gln-Gln-Met-Glu-Glu-Leu-Gly-Met (FIG. 1). ). The symbol of the nucleotide shown in FIG. 1 is briefly described. For example, the nucleotide at the 9-position from the 5'-end indicates that it is a mixture containing equimolar amounts of dA and dG. The nucleotide which is a raw material is mainly a dimer, but mononucleotide may be used as needed. A column equipped with a glass filter is filled with 20 mg of the starting material nucleotide resin Ap-d (G) (manufactured by Yamasa shoyu). After repeated washing with methylene chloride, the 4,4'-dimethoxytrityl group treated with methylene chloride solution containing 3% trichloroacetic acid is removed. The column is then washed several times with 1 ml of methylene chloride. The column was washed with anhydrous pyridine to replace the solvent, 20 mg of nucleotide dimer (DMTr) ApTp (NHR ₃ ) (from Nippon Zeon: NHR ₃ = triethylammonium: DMTr = dimethoxytrityl) and 0.2 ml of pyridine were added, Vacuum dry the inside of the column with a vacuum pump.

Thus, 20 mg of 2,4,6-chloromethylbenzenesulfonyl-3-nitrotriazolide (MSNT, manufactured by Wako Pure Chemical Industries) and 0.2 ml of anhydrous pyridine are added, and then the inside of the column is exchanged with nitrogen gas. The nucleotide resin is condensed with the dimer by reacting for 45 minutes with occasional shaking at room temperature. After completion of the reaction, the column is washed with pyridine and unreacted OH groups are acetonitriated with a pyridine solution containing excess acetic anhydride and 4-dimethyl aminopyridine, and then the column is washed with pyridine and then the next dimer or The monomers are condensed by repeating the above operations in the order recorded:

Equimolar mixtures of (DMTr) Ip (NHR ₃ ), (DMTr) GpGp (NHR ₃ ), (DMTr) Ip (NHR ₃ ), (DMTr) CpTp (NHR ₃ ) and (DMTr) TpTp (NHR ₃ ) Equimolar mixtures of DMTr) ApAp (NHR ₃ ) and (DMTr) ApGp (NHR ₃ ), equimolar mixtures of (DMTr) ApGp (NHR ₃ ) and (DMTr) GpGp (NHR ₃ ), (DMTr) GpAp (NHR ₃₎ ), (DMTr) TpGp (NHR 3), (DMTr) ApAp (NH R 3), and (DMTr) equimolar amounts mixture of _{GpAp (NHR 3), (DMTr} ) CpAp (NHR 3), (DMTr) Ap Ap ( Animal mass mixture of NHR ₃ ) and (DMTr) ApGp (NHR ₃ ), (DMTr) GpCp (NHR ₃ ), (DM Tr) TpGp (NHR ₃ ), (DMTr) Ip (NHR ₃ ) and (DMTr) ApTp ( NHR ₃ ). Of these, (DMTr) Ip (NHR ₃ ) is from Yamasa Shoyu and all others are from Nippon Zeon. At the end of the reaction up to the last step, the resin is washed and dried sequentially in the order of pyridine, methylene chloride and ether, without acetating. The dried resin was suspended in 1.7 ml of a mixed solution of 1 ml of dioxane containing 1 M tetramethylguanidine and 1 M α-picolin aloxime, 0.5 ml of pyridine, and 0.2 ml of water, and then allowed to stand overnight at room temperature and 100 to 200 µl under reduced pressure. Concentrate. A small amount (2-3 drops) of pyridine and 2-3 ml of concentrated ammonia water are added to the concentrate, and the mixture is heated to 55 ° C. for 6 hours. Subsequently, the aqueous layer obtained by extraction and extraction with ethyl acetate was concentrated under reduced pressure, and then the concentrate was dissolved in 50 mM triethyl ammonium acetate solution (pH 7.0) and added to chromatography using a C18 column (1.0 × 15 cm: Waters product). Elution was carried out with acetonitrile with a linear gradient of 10-30% in 50 mM triethyl ammonium acetate solution (pH 7.0) to concentrate the peak fraction eluted at a position near 25% acetonitrile concentration.

80% acetic acid was added to the concentrate, and the mixture was left at room temperature for 30 minutes, ethyl acetate was added thereto, followed by extraction and concentration. The aqueous layer was concentrated under reduced pressure. The resulting concentrate was purified by addition to high pressure liquid chromatography using a C18 column (Senhu Kagaku KK product: SSC-ODS-272: 6Φ x 200 mm ^L ). Eluate is carried out using acetonitrile with a linear gradient of 10-20% in 50 mM triethyl ammonium acetate solution (pH7.9) to obtain synthetic DNA in quantities of 10A ₂₆₀ or more.

[(ii) Synthesis of Probe a]

Of the amino acid sequences obtained in Example 3 (iii), 14 consecutive nucleotides were obtained based on the five amino acid sequences represented by Met-Pro-Ala-Phe-Ala (FIG. 1).

Synthesis condenses the following nucleotides with the nucleotide resin Ap-d (T) (manufactured by Yamasa Shoyu) in the same order as in the probe (IWQ):

(DMTr) CpAp (NHR ₃ ); (DMTr) GpGp (NHR ₃ ), (DMTr) CpAp (NHR ₃ ), (DM Tr) CpTp (NHR ₃ ); Equimolar mixtures of (DMTr) CpGp (NHR ₃ ) and (DMTr) CpCp (NHR ₃ ); Equimolar mixtures of (DMTr) ApGp (NHR ₃ ), (DMTr) TpGp (NHR ₃ ), (DMTr) GpGp (NHR ₃ ) and (DMTr) Cp Gp (NHR ₃ ); (DMTr) ApAp (NHR ₃ ); Equimolar mixtures of (DMTr) CpAp (NHR ₃ ) and (DMTr) CpGp (NHR ₃ ); And (DMTr) Gp (NHR ₃ ), all of which are from Nippon Zeon. Synthetic DNA of about 10A ₂₆₀ units was obtained. The nucleotide sequence of the obtained oligonucleotide was examined by Membrane-Gilbert sequencing, and found to have the nucleotide sequence shown in FIG.

[(iii) Synthesis of Probe (LC)]

DNA was synthesized automatically using Applied Biosystems DNA Synthesizer 380A. This method is based on the principles described by Carruter et al. (J. Am. Chem. Soc., 103 3185 (1981)) and is generally known as the phosphoamidite method.

DG-S (S: support) which deprotected the 5'-dimethoxytrityl group (DMTr), and the phosphoramidite body of (DMTr) -dT previously activated with tetrazole are condensed. Thereafter, unreacted hydroxyl groups are aceted and oxidized to iodine in the presence of water to form phosphate groups. 24 nucleotides having the sequence as shown in FIG. 1 were synthesized by deprotecting the DMTr group and repeating condensation in the same manner below. The obtained nucleotides are removed from the support and deprotected, followed by purification by reverse phase high pressure liquid chromatography using a C18 column (SSC-ODS-272 manufactured by Senshu Kagaku).

Example 5

Culture of CHU-2 Cells and Purification of mRNA

1) CHU-2 Cell Culture and Recovery

Established CHU-2 cells were completely grown in two culture flasks (150 cm 2), harvested and suspended in 500 ml of RPMI 1640 culture medium containing 10% fetal bovine serum. Belco) and incubated for 4 days at 0.5 rpm. When it was confirmed that the cells were completely proliferated on the inner wall of the rotating culture bottle, the culture medium was removed from the rotating culture bottle, and 100 ml of physiological saline containing 0.02% of EDTA, which had been previously heated to 37 ° C, was added and warmed at 37 ° C for 2 minutes. Detach the cells in the inner wall of the flask with a pipette. The obtained cell suspension is centrifuged at 1500 rpm for 10 minutes to obtain cell pellets.

The cells are resuspended in 5 ml of saline without EDTA and centrifuged at 1500 rpm for 10 minutes to obtain cell pellets (wet weight, about 0.8 g). Cells thus obtained are cryopreserved at −80 ° C. until RNA extraction is performed.

2) Purification of mRNA

MRNA is isolated from the CHU-2 cells obtained in 1) as described in Moleeular Cloning (Maniatis et al., Cold Spring Harbor, 196p 1982). Cryopreserved CHU-2 cells (3.8 g wet weight) were collected in 20 ml of 6 M guanidine solution [6 M guanidinium isothiocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M β-mercapto ethanol and 0.5% sodium sarco The actual sulphate], mixed well with a vortex mixer for 2-3 minutes, and then aspirated 10 times with a syringe of 18G (dose, 20 ml) is repeated. 6 ml of 5.7 M CsCl-0.1 M EDTA (pH 7.5) was first added to a polyaloma centrifuge tube fitted to a Beckman SW40 Ti rotor, and the cells were destroyed to fill the tube and loaded with about 6 ml of viscous guanidine solution. . The four centrifuge tubes thus prepared were centrifuged at 30,000 rpm for 15 hours at 20 ° C., and then the obtained pellet was washed three times with a small amount of 70% ethanol.

The pellets from each tube were combined and dissolved in 550μl of water to prepare a NaCl concentration of 0.2M, then treated with a mixture of phenol-chloroform (1: 1) and chloroform, and precipitated by adding 2.5-fold volume of ethanol. (Approximately 10.1 mg of total RNA is obtained from 3.8 g of wet cells.)

Purification of poly (A ⁺ ) -RNA from total RNA is carried out as follows. These methods are the following affinity chromatography method in which mRNA adds a polya chain to the 3 'end. Adsorption using oligo (dT) -cellulose (PL Biochemical Co., Ltd. Type 7) dissolves total RNA in adsorption buffer [containing 10 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1 mM EDTA and 0.1% SDS solution]. The mixture was heated at 65 ° C. for 5 minutes, and then passed through an oligo (dT) -cellulose column paralleled with the same solution, followed by poly (A ⁺ ) with TE solution [containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA). RNA is eluted. The non-adsorbed through solution is mixed with the first eluate by the same elution using the same column again. The result is 400 μg of poly (A ⁺ ) RNA.

The mRNA thus obtained was centrifuged by sucrose density gradient in the same manner as described in Schief and Wensink's Experimental Techniques [Practical Methods in Molecular Biology, Spinger-Verlag, New York, Heidelberg, Berlin (1981)]. Fractionation by size.

More specifically, Beckman makes a sucrose density gradient of 5-25% in a SW40 Ti centrifuge tube. Two sucrose solutions were RNase-free sucrose (Schwarz / Mann products) at 5% and 25%, respectively, in solutions containing 0.1M NaCl, 10mM Tris-HCl (pH 7.5), 1mM EDTA and 0.5% SDS. ) Is contained.

800 μg of mRNA [poly (A ⁺ ) -RNA] prepared by the method described above was dissolved in 200-500 μl of TE solution, the solution was heated at 65 ° C. for 5 minutes, quenched, and placed on top of a sucrose density gradient. Centrifuge for 20 hours at 30,000 rpm. Fractions are collected at 0.5 ml each to measure their absorbance at 260 nm. The size of the fractionated RNA was determined based on the position of the same standard RNA (28S, 18S, 5S ribosomal RNA), and the G-CSF activity of each fraction was determined by Xenopus laevis in the following manner. Investigate using oocyte system. First, the mRNA of each fraction is adjusted to an aqueous solution having a concentration of 1 μg / μl: oocytes collected from Xenopus (about 1 year old) are injected at a rate of 50 ng mRNA per one, followed by 96 pore microanalysis plates. 10 oocytes were inserted into one hole of 100 μl Barth medium [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO ₃ , 0.82 mM MgSO ₄ , 0.33 mM Ca (NO ₃ ) ₂ , and 0.41 mM CaCl, respectively. ₂ , 7.4 mM Tris-HCl (pH 7.6), penicillin 10 mg / L, and streptomycin sulfate 10 mg / L] at room temperature for 48 hours. : The culture supernatant is collected, concentrated and purified to measure G-CSF activity.

As a result, G-CSF activity was observed in 15-17S fraction.

Example 6

Synthesis of cDNA (Construction of pBP-based cDNA Library)

Land et al., Nucleic Acids Res., 9,2251 (1981), obtained in Example 5 according to a method modified by Gubler and Hoffman (Gubler and Hoffman, 25, 263 (1983)). Synthesize cDNA from poly (A ⁺ ) RNA.

1) Synthesis of single-stranded cDNA

Reagents are added to an Eppendorf tube (volume 1.5 ml) in the following order. : 80 μl reaction buffer (500 mM KCl, 50 mM MgCl ₂ , 250 mM Tris-HCl, pH 8.3), 20 μl of 200 mM dithiothreitol, 32 μl of 12.5 mM dNTP (containing 12.5 mM of dATP, dGTP, dCTP and dTTP, 10 μl) Α- ^32c P-dCTP (Amersham PB 10205), 32 μl oligo (dT0 _12-18 (PL Biochemicals: 500 μg / ml), 20 μl poly (A ⁺ ), RNA (2.1 μg / μl), distilled water 260 μl The total 400 μl of the reaction solution was heated at 65 ° C. for 5 minutes and then at 42 ° C. for 5 minutes.

120 units of reverse transcriptase (manufactured by Takara Shuzo) were added to the reaction solution, followed by further reaction at 42 ° C for 2 hours or more, 2 μl of RNase inhibitor (BRL product), 20 μl of TE solution, 16 μl of 100 mM sodium pyrophosphate, and 48 units of reverse transcriptase ( 4 μl) is added and reacted at 46 ° C. for 2 hours.

The reaction is stopped by addition of 0.5 M EDTA (8 μl) and 10% SDS (8 μl), then treated with phenol / chloroform and precipitated twice with ethanol to obtain single-stranded cDNA.

2) dC-chain Attachment to Single-Strand cDNA

After dissolving the single-stranded cDNA obtained in 1) in distilled water, it was added to 60 μl of dC-chained buffer [400 mM potassium cacodylate, 50 mM Tris-HCl (pH 6.9), 4 mM dithiothreitol, 1 mM CoCl ₂ , 1 mM DCTP] is added and the mixture is heated at 37 ° C. for 5 minutes. 3 μl of a terminal transferase (27 units / μl: PL Biochemicals) was added to the reaction solution, reacted at 37 ° C. for 2.5 minutes, treated once with phenol / chloroform, and precipitated with ethanol (twice). The cDNA to which this dC is linked is dissolved in 40 μl of TE solution containing 100 mM NaCl.

3) Synthesis of Double-stranded cDNA

4 μl of oligo (dG) 12-18 (200 μg / ml: PL Biochemicals) was added to 40 μl of the DNA solution prepared in 2), and the mixture was heated at 65 ° C. for 5 minutes and then at 42 ° C. for 30 minutes. The solution was kept at 0 ° C. and 80 μl of buffer [100 mM-Tris-HCl (pH 7.5), 20 mM MgCl ₂ , 50 mM (NH ₄ ) ₂ SO ₄ , and 500 mM KCl], 4 μl of 4 mM dNTP (dATP, dCTP). , 4 mM of dGTP and dTTP, 60 μl of 1 mM β-NAD, 210 μl of distilled water, 20 μl of E. coli DNA plymerase I (from Takara Shuzo), 15 μl of E. coli DNA Liga The mixture was added (Trkara Shuzo and 15 μl of E. coli RNase H (Takara Shuzo)) and the mixture was reacted for 1 hour at 12 ° C. Again 4 μl of 4 mM dNTP was added and allowed to react for 1 hour at 25 ° C. Continuously phenol-chloroform Treatment and precipitation with ethanol (once) yielded about 8 μg of double-stranded cDNA, which was dissolved in TE solution and subjected to 1.2% agarose gel electrophoresis. . It is possible to recover the strand cDNA - is performed for about 560bp to the portion corresponding to the size of the 2kbp and only adsorbed to the DE81 Watts, double of about 0.2μg by carrying out the dissolution process.

4) Attach the dC-chain to the double-stranded cDNA

The double-stranded cDNA obtained in 3) was dissolved in 40 μl of TE solution, and 8 μl of the dC-chain attachment buffer described in 2) was added and heated at 37 ° C. for 2 minutes. After 1 μl of terminal transferase (27 units / μl) was added and reacted at 37 ° C. for 3 minutes, the reaction solution was immediately cooled to 0 ° C., 1 μl of 0.5 M EDTA was added to stop the reaction, followed by treatment with phenol-chloroform. The precipitate obtained by precipitation with ethanol is then suspended in 10 μl of TE solution.

5) Construction of pBR-based cDNA Library

4 μl of commercially available oligo (dG) -chain attached pBR 322 vector (10 ng / μl from BRL) and 2 μl of dC-chain attached double-stranded cDNA were annealed in a TE solution containing 75 μl of 0.1 M NaCl. Annealing consists of three steps: heating at 65 ° C. for 5 minutes, heating at 40 ° C. for 2 hours, and then cooling to room temperature.

Can be transformed from E. coli strain X1776 using the methods described in Maniatis et al. (Molecular Cloning, CSH, 249p (1982)). Cells are prepared and transformed with the annealed plasmid to obtain transformants.

Example 7

[Synthesis of cDNA (by λ phage library)]

1) Synthesis of single-stranded cDNA

3.8 g of cryopreserved CHU-2 cells were purified twice through an oligo (dT) -cellulose column according to the method of Example 5 to obtain 400 μg of poly (A ⁺ ) -RNA.

10 μl of the TE solution in which 12 μg of this poly (A ⁺ ) -RNA is dissolved is placed in a reaction tube containing 10 μg of actinomycin D (Sigma), and then reagents are added in the following procedure. 20 μl of reverse transcription buffer [250 mM Tris-HCl (pH 8.3): 40 mM MgCl ₂ : 250 mM KCl], 20 μl of 5 mM dNTP (containing 5 mM of dATP, dGTP, dCTP, dTTP, respectively), 20 μl of oligo (dT) _12-18 ( 0.2 μg / ml: RL Biochemicals), 1 μl 1M dithiothreitol, 2 μl RNasin (30 units / μl: Promega Biotech), 10 μl reverse transcriptase (10 units / μl: Seikagaku Kogyo), 1 μl α ^-32 P-dATP (10μCi: product of Amerscham) and 16μl of distilled water, total 100μl of reaction solution. The reaction solution was held at 42 ° C. for 2 hours, and then 0.5 M EDTA (5 μl) and 20% SDS (1 μl) were added to stop the reaction. Treatment with phenol / chloroform (100 μl) and precipitation with ethanol (twice) yield about 4 μg of single-stranded cDNA.

2) Synthesis of Double-stranded cDNA

The reaction solution was prepared by dissolving cDNA obtained in 1) in 29 μl of TE solution and adding reagents in the following order: 25 μl of polymerase buffer [400 mM Hepes (pH 7.6): 16 mM MgCl ₂ : 63 mM β-mercaptoethanol: And 270 mM KCl]: 10 μl 5 mM dNTP: 1.0 μl 15 mM β-NAD: 1.0 μl α- ³² P-dATP (10 μCi / μl): 0.2 μl E.coli DNA ligase (60 units / μl, manufactured by Takara Shuzo), 5.0μl of E. coli DNA polymerase I (New England Biolabs product: 10 units / μl): 0.1μl RNaseH (60 units / μl: Takara ShuZo) 28.7 μl of distilled water.

After incubating the reaction solution at 14 ° C. for 1 hour, the reaction solution is reduced to room temperature and reacted for another hour. 0.5 MEDTA (5 μl) and 20% SDS (1 μl) are then added to stop the reaction, treated with phenol / chloroform and precipitated with ethanol. The obtained DNA was dissolved in 20 μl of 0.5 mM EDTA, and 3 μl of Klenow buffer [500 mM Tris-HCl (pH 8.0) and 50 mM MgCl ₂ ], 3 μl of 5 mM dNTP, and 4 μl of distilled water were added to prepare a reaction solution. Add Lase (Clenow Fragment, manufactured by Takara Shuzo) and incubate at 30 ° C. for 15 minutes.

Dilute by adding 70 μl of TE solution to the culture reaction solution, and stop the reaction by adding 0.5 M EDTA (5 μl) and 20% SDS (1 μl). The reaction solution is treated with phenol / chloroform and precipitated with ethanol to yield about 8 μg double-stranded cDNA.

3) Methylation of Double-Stranded cDNA

40 μl of methylation buffer [500 mM Tris-HCl (pH 8.0), 50 mM EDTA], 20 μl of SAM solution [800 μM S-adenosyl-L-methylmethionine (SAM) in an aqueous solution of the double-stranded cDNA synthesized in 2) (30 μl) ): 50 mM β-mercapto ethanol] and 100 μl of water are added. 15 μl of EcoRI methylase (New England Biolabs: 20 units / μl) is added to the mixture to make 200 μl of the total reaction solution. The reaction is carried out at 37 ° C. for 2 hours, treated with phenol and ether, and then precipitated with ethanol to recover DNA.

4) Attachment of EcoRI Linker

1.5 μl of ligase buffer [250 mM TrisHCl (pH 7.5) and 100 mM MgCl ₂ ], 0.5 μl of prephosphorylated EcoRI linker (10-mer: Takara Shuzo), 1.5 μl in about 1.2 μg of the methylated double-stranded DNA 10 mM ATP, 1.5 μl of 100 mM dithiothreitol and ₂ μl of H ₂ O were added to make the reaction solution a total of 15 μl. 0.7 μl of T ₄ DNA ligase (3.4 units / μl, manufactured by Takara Shuzo) was added and allowed to react overnight at 4 ° C. The ligase is then deactivated by heating at 65 ° C. for 10 minutes. 100 mM Tris-HCl (pH 7.5), 5 mM MgCl ₂ , 50 mM NaCl, and 100 μg / ml gelatin were added to the reaction solution to make a total of 50 μl, followed by addition of EcoRI (3.5 μl: 10 units / μl), followed by 2 at 37 ° C. React time. 2.5 μl of 0.5 M EDTA and 0.5 μl of 20% SDS are then added, treated with phenol / chloroform and precipitated with ethanol to recover DNA. Subsequently, gel filtration or agarose gel electrophoresis of Ultrogel AcA34 (manufactured by LKB) is performed to remove unreacted EcoRI linker and recover about 0.5-0.7 μg of double-stranded cDNA with linker attached.

5) Combination of double-stranded cDNA with λgt10 vector

The above linker-attached double-stranded cDNA was mixed with 1.4 μl of λgt10 vector (product of Vector Cloning System) pretreated with 2.4 μg EcoRI, Ligase buffer solution (250 mM Tris-HCl and 100 mM MgCl ₂ ) and 6.5 μl of distilled water, and 42 ° C. After 15 minutes of heating at 1 μl of 10 mM ATP, 1 μl of 0.1 M dithiothreitol, and 0.5 μl of T ₄ DNA ligase, the total amount is made 15 μl and reacted overnight at 12 ° C.

6) in vitro packaging

About one third of the recombinant DNA obtained in 5) is packaged using an Invitro Packaging Kit (promegar Biotech) to obtain phage plaques.

Example 8

Screening colonies of pBR-based libraries using probes (IWQ) Place Whatman 541 filter paper on grown agar medium and leave at 37 ° C. for 2 hours. Taub and Thompson's method described below [Anal. Biochem., 126, 222 ( 1982).

That is, the colonies were transferred to 541 filter paper, and then transferred to agar medium containing coloramphenicol (250 μg / μl) and incubated overnight at 37 ° C.

Lift the 541 filter paper and leave it on another filter paper soaked in 0.5N NaOH solution for 3 minutes and repeat this twice. Hereinafter, the same operation was carried out twice for 3 minutes using 0.5 M Tris-HCl (pH 8) solution, and again 1.5 mg / ml lysozyme solution for 3 minutes with 0.05 M Tris-HCl (pH 8) solution at 4 ° C. [Contains 0.05M Tris-HCl (pH 8) and 25% sucrose] for 10 minutes in the same procedure: at 37 ° C. for 2 minutes with 1 × SSC solution (0.15M NaCl and 0.15M sodium citrate), After 30 minutes treatment with 1 × SSC solution containing 200 μg / ml proteinase K, and finally 2 minutes with 95% ethanol for 2 minutes with 1 × SSC solution at room temperature, 541 filter paper is dried. The obtained dry 541 filter paper was immersed in a solution of a 25: 24: 1 mixture of [phenol equilibrated with 100 mM Tris-HCl (pH 8.5), 100 mM NaCl, and 10 mM EDTA] for 30 minutes at room temperature in a phenol / chloroform / isoamyl alcohol.

The same operation is repeated three times with 5 × SSC solution for 3 minutes and twice with 95% ethanol solution for 3 minutes and then the filter paper is dried.

A probe (IWQ) was radiated at 32p in accordance with conventional methods (see Molecular cloning) to Wallace et al. Colony hybridization is performed according to Nucleic Acids Res., Vol. 9, 879 (1981). Hybridization buffer [6 × NET (0.9M NaCl: 0.09M Tris-HCl (pH 7.5): and 6 mM EDTA), 5 × Denhardt's solution, 0.1% SDS and 0.1 mg / ml denatured DNA (calf thymus) DNA)], followed by prehybridization at 65 ° C. for 4 hours, and then overnight at 56 ° C. using hybridization buffer (composition as above) containing 1 × 10 ⁶ cpm / ml of radiolabeled probe (IWQ). Hybridize. After the reaction, the 541 filter paper was washed twice with normal 6 × SSC solution (containing 0.1% SDS) for 30 minutes at room temperature, followed by 1.5 minutes at 56 ° C., and then the 541 filter paper was detected by radiograph.

Plasmids are isolated from positive clones and Southern blotting is performed using a probe (IWQ). Hybridization and radiographic autophotography were performed under the above conditions.

Similarly, Southern blotting is performed using probe a. The hybridization buffer having the above-mentioned composition is first hybridized at 49 ° C. for 1 hour. It is left to stand at 39 ° C and then hybridized at 39 ° C for 1 hour. After completion of the reaction, the nitro cellulose filter paper was washed with 6 × SSC containing 0.1% SDS twice for 30 minutes at room temperature and for 3 minutes at 39 ° C., followed by radiograph.

As a result, one clone was obtained positively, and nucleotide sequencing by the dideoxy method revealed that the clone was a clone having a DNA composed of 308 base pairs containing the probe (IWQ) and the probe a portion. The plasmid from pBR322 containing this insert was named pHCS-1.

Example 9

Screening of λ Phage Libraries Using a DNA Probe derived from pHCS-1

Plaque hybridization is performed according to the Benton and Davis method [Science, Vol. 196, 180p (1977)]. The pHCS-1 obtained in Example 8 was treated with Sau3A and EcoRI to obtain a DNA fragment of about 600 bp, which was radiolabeled by nick translation according to conventional methods. Nitrocellulose filter paper (S & S product) is placed on the agar medium on which phage plaque is formed, and the phage is transferred onto the filter paper. Phage DNA is denatured with 0.5 M NaOH and the filter paper is treated in the following order. 20 sec with 0.1 M NaOH and 1.5 M NaCl, followed by 20 sec with 0.5 M Tris-HCl (pH 7.5) and 1.5 M NaCl, finally 120 mM NaCl, 15 mM sodium citrate, 13 mM KH ₂ PO ₄ and 1 mM Treat with EDTA (pH7.2) for 20 seconds.

The filter paper is then dried and heated at 80 ° C. for 2 hours to immobilize the DNA. Prehybridization overnight at 42 ° C. in prehybridization buffer containing 5 × SSC, 5 × Denhardt solution, 50 mM phosphate buffer, 50% formamide, 0.25 mg / ml denatured DNA (sperm DNA of salmon) and 0.1% SDS the embodiment, and nikkeu trans by negotiation radiation shown pHCS-1 probe 4 × 10 hybridization buffer containing ^{5 cpm / ml [5 × SSC} , 5 × Den Reinhardt solution, 20mM phosphate buffer (pH 6.0), 50% formamide , 0.1% SDS, 10% dextran sulfate and 0.1 mg / ml of denatured DNA (sperm DNA of salmon) is hybridized at 42 ° C. for 20 hours.

The hybridized nitrocellulose filter paper was washed with 0.1 × SSC containing 0.1% SDS at room temperature for 20 minutes, 0.1 × SSC containing 0.1% SDS at 44 ° C. for 30 minutes, and finally with 0.1 × SSC at room temperature for 10 minutes. It is detected by radiograph.

As a result, five positive clones (G1 to G5) were obtained. Among the clones obtained, the DNA nucleotide sequence of the clone containing the full-length cDNA was examined by the dideoxy method to confirm the nucleotide sequence as shown in FIG. 3A. The cDNA is cut out from the λgt10 vector and linked to the EcoRI site of pBR327 (Soberon et al., G, Vol. 9, 287p (1980)) to prepare a large amount of plasmid. This plasmid is called pBRG4.

Example 10

Screening of λ Phage Library Using pBRG4-derived DNA Probes and Probes (LC)

Plaque hybridization is performed according to the Benton and Davis method (Science, ibid) used in Example 9. The nitro cellulose filter paper (S & S product) is placed on the agar medium in which phage plaque is formed, and the phage is transferred onto the filter paper. After denaturing phage DNA with 0.5 M Na, the filter paper was treated with 0.1 M NaOH and 1.5 M NaCl for 20 seconds in the following order, followed by two treatments for 20 seconds with 0.5 M Tris-HCl (pH 7.5) and 1.5 M NaCl. Finally, treatment with 120 mM NaCl, 15 mM sodium citrate, 13 mM KH ₂ PO ₄ and 1 mM EDTA (pH 7.2) for 20 seconds. The filter paper is then dried and heated at 80 ° C. for 2 hours to immobilize DNA. Thus, two identical filter papers were prepared and screened using a DNA probe and a probe (LC) derived from pBRG4-.

When using a DNA probe derived from pBRG4, the following method is used. pBRG4 is treated with EcoRI to obtain a DNA fragment of about 1500 bp, and the DNA fragment is radiolabeled by nick translation according to a conventional method. One of the two nitrocellulose filter papers was prehybridized containing 5 × SSC, 5 × Denhardt's solution, 50 mM phosphate buffer, 50% formamide, 0.25 mg / ml denatured DNA (sperm DNA of salmon) and 0.1% SDS Pre-hybridization was carried out overnight at 42 ° C. in a buffer, and the hybridization buffer [5 × SSC, 5 × Denhardt's solution, 20 mM phosphate buffer containing a radioactive DNA probe of about 1500 bp (about 1 × 10 ⁶ cpm / ml). (pH 6.0), 50% formamide, 0.1% SDS, 10% dextran sulfate and 0.1 mg / ml of modified DNA (sperm DNA of salmon) for 20 hours at 42 ° C. The hybridized nitrocellulose filter paper was washed with 2 × SSC containing 0.1% SDS at room temperature for 20 minutes, at 0.1 × SSC containing 0.1% SDS at 44 ° C for 30 minutes, and finally with 0.1 × SSC at room temperature for 10 minutes. Detection by radiographs

When using a probe (LC), screening is carried out according to the following method. Another filter paper was previously treated for 2 hours at 65 ° C. in 3 × SSC containing 0.1% SDS, and then in a solution containing 6 × NET, 1 × Denhardt solution, and 100 μg / ml of denatured DNA (salmon sperm DNA). Prehybridization is carried out at 65 ° C. for 2 hours. Then at 63 ° C. in hybridization buffer [6 × NET, 1 × Denhardt's solution and 100 μg / ml of denatured DNA (salmon sperm DNA) containing radiolabeled probe (LC) (2 × 10 ⁶ cpm / ml) After overnight hybridization, the hybridized nitrocellulose filter paper was washed three times (6 minutes each) with 6 × SSC containing 0.1% SDS at room temperature and 6 minutes with 6 × SSC containing 0.1% SDS at 63 ° C.

The filter paper is dried and then detected by radioactive autophotography.

In the screening process described above, positive clones were selected for both probes, and the base sequence of the clone containing the full-length cDNA was examined by dideoxy method and found to have the nucleotide sequence shown in FIG. 4a. . Here, the cDNA was cut out of the λgt10 vector and linked to the EcoRI site of pBR327 to obtain plasmid pBRV2.

Example 11

Construction of pHGA410 Vector (For Animal Cells, + VSE System)

The EcoRI fragment having the cDNA shown in FIG. 3a obtained in Example 9 was treated with restriction enzyme DraI at 37 ° C. for 2 hours and then treated with cleno fragment of DNA polymerase I (manufactured by Takara Shuzo) to make blunt ends. . 1 μg of Bg1II linker (octamer, available from Takara Shuzo) is phosphorylated with ATP and separately bound to a mixture of about 1 μg of DNA fragment obtained. The bound fragment was subjected to agarose gel electrophoresis by treatment with restriction enzyme BglII, and only the largest DNA fragment was recovered.

This DNA fragment corresponds to about 710 base pairs containing the portion encoding the human G-CSF polypeptide (see Figure 5). Vector DNA obtained by treatment of vector pdKCR [Fukunaga et al. Proc. Natl. Acad. Sci., USA, Vol. 81, 5086p (1984)] with restriction enzyme BamHI and dephosphorylation with alkaline phosphatase (Takara Shuzo) T4DNA ligase (manufactured by Takara Shuzo) was added and linked with a 710 bp cDNA fragment to obtain pHGA410 (Figure 6). As shown in FIG. 6, this plasmid contains the promoter of the early SV40 gene, the replication initiation region of SV40, the rabbit β-globin gene, the replication initiation region of pBR322 and the β-lactamase gene (Amp ^r ) derived from pBR322. The human G-CSF gene is connected downstream of the promoter of the SV40 early gene.

Example 12

Construction of Recombinant Vector (+ VSE) for C127 Cell Transformation

1) Construction of pHGA410 (H)

PHGA410 Plasmid Obtained in Example 11 (Figure 6)

20 μg is dissolved in a reaction solution consisting of 5 mM Tris-HCl (pH 7.5), ₇ mM MgCl ₂ , 100 mM NaCl, 7 mM 2-mercapto ethanol and 0.01% bovine serum albumin (BSA), and the restriction enzyme EcoRI (10-15 units: Takara Shuzo) was added and reacted at 37 ° C. for about 30 minutes to partially cut into EcoRI. The DNA fragments are then treated twice with a 1: 1 mixture of phenol / chloroform and once with ether and precipitated with ethanol.

This DNA fragment was dissolved in 50 μl of a solution consisting of 50 mM Tris-HCl, 5 mM MgCl ₂ , 10 mM DTT, and 1 mM dATP, dCTP, dGTP and dTTP, and 5 μl of Cleno fragment of E. coli DNA polymerase (Takara Shuzo). Incubate at 14 ° C. for 2 hours to form a blunt end.

0.8% agarose gel electrophoresis was performed to recover 6 μg of a DNA fragment of about 5.8 kbp.

5 μg of the recovered DNA fragment was again dissolved in 50 μl of the reaction solution consisting of 50 mM Tris-HCl (pH 7.6), 10 mM MgCl ₂ , 10 mM DTT, and 1 mM ATP, ₂ μg HindIII linker (Takara Shuzo) and T4 DNA ligase (Takara Shuzo) ) 100 units are added and reacted overnight at 4 ° C.

Subsequently treated with phenol and ether: after precipitation with ol, the precipitate was dissolved in 30 μl of a solution consisting of 10 mM Tris-HCl (pH 7.5), ₇ mM MgCl ₂ and 60 mM NaCl, and incubated for 3 hours at 37 ° C. with 10 units of restriction enzyme HindIII. do. After treatment with T4 DNA ligase, the DNA was used to transform E. coli strain DH1 according to the rubidium chloride method (see Molecular Cloning, above) to obtain colonies of ampicillin-resistant transformants (Amp ^r ). Cells are selected in which the EcoRI site of the pHGA410 plasmid retains the plasmid substituted with HindIII. The plasmid thus obtained is named pHGA410 (H) (Fig. 7).

2) Construction of the expression vector recombinant PTN-G4

20 μg of pHGA410 (H) obtained in 1) above was dissolved in 50 μl of a reaction solution consisting of 10 mM Tris-HCl (pH 7.5), 7 mM MgCl ₂ , 175 mM NaCl, 0.2 mM EDTA, 7 mM 2-mercapto ethanol, and 0.01% bovine serum albumin. Add 20 units of restriction enzyme Sal I (manufactured by Takara Shuzo) and incubate at 37 ° C for 5 hours. Subsequently, after phenol treatment, precipitate with ethanol, add Cleno fragment of DNA polymerase (manufactured by Takara Shuzo) as in 1), and incubate at 14 DEG C for 2 hours to make a blunt end. This was subjected to agarose gel electrophoresis and the reaction solution was immediately precipitated with ethanol without DNA recovery. The resulting DNA fragment was treated with restriction enzyme HindIII and subjected to 1% agarose gel electrophoresis to recover 5 μg of HindIII-SalI fragment (about 2.7 kbp).

On the other hand, plasmid pdBPV-1 with bovine papillomavirus (BPV) [This plasmid was provided by Dr. Howley. Sarver, N., Sbyrne, J.C. & Howley, PM Proc. Natl. Aca d. Sci., USA, Vol. 79, 7147-7151p (1982), as described by Nagata et al. [Fukunaga, Sokawa and Nagata, Proc. Natl. Acad. Sci., USA] , 81, 5086-5090p (1984)], treated with HindIII and PvuII to obtain 8.4 kb DNA fragments. This 8.4 kb DNA fragment and the separately prepared HindIII-Sal I DNA fragment (2.7 kb) are ligated with T4DNA ligase according to a conventional method. This is used to transform E. coli strain DH1 according to the rubidium chloride method described in Molecular Cloning. E. coli colonies holding a plasmid with cDNA of G-CSF derived from pHGA410 are selected. This plasmid is named pTN-G4 (Figure 7).

Adenovirus type II (Tanpakushitsu, kakusan, koso (proteins, nucleic acids and enzymes), Vol. 27, Dec. 1982, Kyoritsu shuppam) was treated in the same manner, and contained Δ1 containing about 1700 bp SalI-HindIII fragments having VAI and VAII. The pVA plasmid is recovered and the fragment containing VAI and VAII is recovered from this plasmid. This fragment is inserted into the HindIII site of pTNG4 to obtain pTNG4VA α and pTNG4VA β (FIG. 7). The adenovirus VA gene allows the plasmid to enhance the expression efficiency of the transcriptional product of the early SV40 promoter.

Example 13

Transformation and Expression of C127 Cells (+ VSE)

PTN-G4 obtained in Example 12 was used to treat mouse C127 cells with restriction enzyme BamHI before transformation. 20 μg of pTN-G4 plasmid was dissolved in 100 μl of reaction solution [10 mM Tris-HCl (pH 8.0), ₇ mM MgCl ₂ , 100 mM NaCl, 2 mM 2-mercapto ethanol and 0.01% BSA] in 20 units of BamHI (Takara Shuzo) Treatment, treatment with phenol and ether, followed by precipitation with ethanol.

Mouse C127I cells are grown in minimal essential medium of Dolbecco containing 10% fetal bovine serum (Gibco). C127I cells grown in a culture dish (50 cm ^Φ ) are transformed using the DNA prepared separately by the calcium phosphate method at a rate of 10 μg per culture dish [Haynes, J. & Weissman, C., Nucleic Acids Res., 11, 687-706 (1983). The cells are treated with glycerol and then incubated at 37 ° C for 12 hours.

Transfer the cultured cells to three new culture dishes (5cm ^Φ ) and change the medium twice a week. On the 16th day, each of the foci-forming parts was transferred to a new culture plate, continuously cultured in a Dolbeco minimum essential medium containing 10% fetal bovine serum (Gibco), and high in G-CSF production capacity. Select clones. These clones produced about 1 mg / L of G-CSF. Subsequent cloning yields clones that produce 10 mg / L or more G-CSF. In addition to the C127I cells, NIH3T3 cells may also be used as host cells.

Example 14

Expression of G-CSF in CHO Cells (+ VSE)

1) Construction of pHGG4-dhfr

20 μg of the plasmid pHGA410 obtained in Example 11 was dissolved in 100 μl of the reaction solution containing 10 mM Tris-HCl (pH 7.5), 7 mM MgCl ₂ , 175 mM NaCl, 0.2 mM EDTA, 0.7 mM 2-mercapto ethanol and 0.01% BSA, 20 units of restriction enzyme Sal I (manufactured by Takara Shuzo) were added and reacted at 37 ° C. overnight, treated with phenol and ether and precipitated with ethanol.

This DNA precipitate was dissolved in 100 μl of a reaction solution consisting of 50 mM Tris-HCl, 5 mM MgCl ₂ , 10 mM DTT, and 1 mM dATP, dcTP, dGTP, and dTTP, and the Cleno fragment of E. coli DNA polymerase (10 μl: Takara Shuzo Product) and reacted at 14 ° C. for 2 hours, and treated with phenol and ether, followed by precipitation with ethanol.

EcoRI linker is added to this precipitated DNA. That is, the DNA was dissolved in 50 μl of a reaction solution consisting of 50 mM Tris-HCl (pH 7.4), 10 mM DTT, 0.5 mM spermidine, 2 mM ATP, 2 mM hexamine-cobalt chloride, and 20 μg / ml BSA, and EcoRI linker (Takara). Shuzo) and 200 units of T4 DNA ligase (Takara Shuzo) were added and allowed to react at 4 ° C for 12-16 hours. After treatment with phenol, followed by washing with ether, followed by precipitation with ethanol according to a conventional method, the precipitated DNA was digested with EcoRI and subjected to 1% agarose gel electrophoresis to recover 3 μg of DNA fragment of about 2.7 kbp.

On the other hand, plasmid pAdD26SVpA (Kaufman, RG & Sharp. PAMol. Cell Biol., Vol. 2, 1304 to 1319p (1982)) was treated with EcoRI, and dephosphorylation was performed using bacterial alkaline phosphatase (BAP). Namely, in more detail, 20 μg of pAdD26SVpA and 20 units of EcoRI were added to 100 μl of the reaction solution [50 mM Tris-HCl (pH 7.5), ₇ mM MgCl ₂ , 100 mM NaCl, 7 mM 2-mercapto ethanol and 0.01% BSA] at 37 ° C. After reacting for 10 hours, 5 units of BAP are added to the reaction solution, and the mixture is reacted at 68 ° C for 30 minutes. Subsequently, about 5 μg of the EcoRI fragment of pAdD26SVpA is recovered by treatment with phenol and electrophoresis. 0.5 μg of each of the above 2.7 kb fragment and pAdD26SVpA fragment is annealed. The resulting DNA was transformed into E. coli strain DH1 according to the rubidium chloride method, and colonies holding the plasmid of pHGG4-dhfr were selected. The resulting plasmid is named pHGG4-dhfr (Fig. 8a).

Another method is as follows: The pHGG4 plasmid is treated with SalI and partially cleaved with EcoRI without addition of the EcoRI linker. A DNA fragment of about 2.7 kbp is recovered and the DNA fragment treated with a Clenow fragment of E. coli DNA polymerase to make a blunt end at the end. On the other hand, the blunt-ended EcoRI fragment is prepared from pAdD26SVpA in the same manner as described above. The fragment (about 2.7 kb) prepared separately from the EcoRI fragment can also be treated with T4DNA ligase to prepare pHGG4-dhfr.

In addition, pHGA410 (H) prepared in Example 12) was treated with restriction enzymes hindIII and SalI as described in Example 12), and the HindIII-SalI fragment was linked to the blunt-ended EcoRI fragment of pAdD26SVpA. do. This method is also used to prepare pHGG4-dhfr (Figure 8b).

2) Construction of pG4DR1 and pG4DR2

10 μg of the plasmid pAdD26SVpA described in 1) was dissolved in 50 μl of the reaction solution containing 50 mM Tris-HCl (pH 7.5), ₇ mM MgCl ₂ , 100 mM NaCl, 7 mM 2-mercapto ethanol and 0.01% BSA, and the restriction enzymes EcoRI and BamHI. 10 units each was added and reacted at 37 ° C. for 10 hours. Successively treated with phenol and washed with ether. 1% low melting agarose gel electrophoresis is performed to recover a DNA fragment of about 2 kb. This recovered DNA fragment is treated with a cleno fragment of DNA polymerase according to a conventional method to make blunt ends. The blunt ended DNA fragments are treated with phenol, washed with ether and then precipitated with ethanol.

10 μg of the plasmid pHGA410 (H) obtained in Example 12) was dissolved in 50 μg of a reaction solution composed of 10 mM Tris-HCl (pH7.5), ₇ mM MgCl _2, and 60 mM NaCl, and 10 units of HindIII were added thereto at 37 ° C. for 6 hours. React. DNA fragments are recovered by conventional methods by 1% low melting agarose gel electrophoresis. The recovered DNA fragments are then treated with BAP and the ends are treated with Klenow fragments to make blunt ends. The DNA fragments were treated with phenol and washed with ether and then linked to the blunt end of the previously obtained 2 kb DNA fragment using T4 DNA ligase in the following manner: 1 μg of each DNA fragment was 66 mM Tris-HCl (pH 7.5). ), 6.6mM MgCl ₂ , 5mM DTT and 1mM ATP were dissolved in 30μl and the reaction was carried out at 6 ° C for 12 hours in the presence of 50 units of T4 DNA ligase. The ligated DNA is used to transform E. coli strain DH1. As a result, pG4DR1 and pG4DR2 shown in FIG. 8C are obtained.

3) Transformation and Expression

CHO cells (dhfr-strain: provided by Dr. L. Chasin of Columbia University) in a culture dish (9 cm ^Φ , Nunc) contain alpha minimum essential medium (α-MEM, adenosine, deoxyadenosine and thyme) containing 10% calf serum Cultured in the presence of Dean) and transformed by the calcium method (Wigler et al., Cell, 14, 725 (1978)) according to the following method.

Appropriate amount of carrier DNA (shipper thymus DNA) was added to 1 μg of pHGG4-dhfr plasmid prepared in 1), and the mixture was dissolved in 375 μl of TE solution, followed by addition of 125 μl of 1M CaCl ₂ . Cool on ice for 3-5 minutes and add 500 μl of 2 × HBS (50 mM Hepes, 280 mM NaCl and 1.5 mM phosphate buffer).

After cooling again on ice, 1 ml of the CHO cell culture solution was mixed, transferred to a culture dish, and incubated for 9 hours in a CO ₂ incubator. Remove the medium from the petri dishes, wash with TBS (tris-buffered saline), wash again with the addition of 20% glycerol-containing TBS and add non-selective medium (reinforced nucleotides to the α-MEM medium). After 2 days of incubation, the culture is diluted 10-fold and then transferred to selective medium (without enrichment of nucleotides). Continue culturing with fresh selective medium every 2 days, select colonies generated, transfer to new dish, grow cells in the presence of 0.02 μm methotrexate (MTX), continue to increase to 0.05 μm, later 0.1 μm Cloning is performed by propagation in the presence.

In addition, transformation of CHO cells can be performed by simultaneous transformation with pHGG4 and pAdD26SVpA (see Scahill et al. Proc. Natl. Acad. Sci., USA, 80, 4654-4658p (1983)).

CHO cells are also transformed by the following methods:

PG4DR1 or pG4DR2 prepared in 2) were previously treated with SalI and KpnI, respectively, to obtain DNA fragments and 10 μg of the fragments were used to transform CHO cells as described above: serially according to the method described for the transformed cells. Continue incubation in selective medium at: More than 100 positive colonies per culture plate appear after about 7 days. Transfer these colonies all at once to a new petri dish and continue incubating in a series of selective media in the presence of 0.01 μm MTX to repeat the above method by increasing the concentration of MTX to 0.02 μm, 0.05 μm and 0.1 μm. Selecting colonies to survive: Colony selection can be carried out in the same manner in the case of culturing while increasing the MTX concentration by selecting each of the obtained 10 colonies one by one.

Recombinant vectors with polycistronic genes can also be used for transformation of CHO cells. For example, pAdD26SVpA can be treated with PstI to recover two fragments and combine them with pBRG4-derived CSF cDNA fragments to obtain recombinant vectors inserted in the order of the polya sites of the adeno bicus promoter, CSF cDNA, DHFR and SV40. Manufacture. This recombinant vector is used to transform CHO cells.

Example 15

Transgene G-CSF Activity Assay (+ VSE)

Culture supernatants of C127 cells and CHO cells obtained in Examples 13 and 14 were adjusted to pH 4 with 1N acetic acid, respectively, and an equal volume of n-propanol was added and the resulting precipitate was removed by centrifugation. The supernatant is eluted with 50% n-propanol through an open column (1 ^Φ × 2 cm ^L ) filled with a C8 reversed phase carrier (manufactured by Yamamura Kagaku KK). The eluate is diluted twice with water and then eluted with n-propanol (30-60% primary density gradient) containing 0.1% TFA by reverse phase high pressure liquid chromatography using a YMC-C8 column (manufactured by Yamamura Kagaku KK). . Fractions eluting at a position near n-propanol concentration are collected and lyophilized to dissolve in 0.1 M glycine buffer (pH 9). Through this process, human G-CSF in C127 cells and CHO cells is enriched 20-fold.

To control, cells were transformed with plasmids containing no human G-CSF cDNA according to the method described above, and the culture supernatant was concentrated by the method described above. The human G-CSF activity of the sample is measured by the human G-CSA activity assay a described above. If the expression efficiency is high enough, the culture supernatant may be measured directly without concentration. The results are summarized in Table 1, which is data based on concentrated samples.

TABLE 1

Example 16

Amino Acid Analysis and Sugar Analysis (+ VSE)

The prepared CSF sample obtained in Example 15 was purified again according to the method described in Example 2 (i), and the purified CSF sample was hydrolyzed by a conventional method to automatically analyze the amino acid composition of the protein portion of the hydrolyzate. Analyze by special amino acid assay using a device (Hitachi). The results are shown in Table 2, and the hydrolysis conditions are as follows.

(Iii) 6N HCL, 110 ° C., 24 hours, in vacuum, (ii) 4N methanesulfonic acid + 0.2% 3- (2-aminoethyl) indole, 110 ° C., 24 hours, 48 hours, 72 hours, in vacuum.

The sample is dissolved in a solution containing 40% n-propanol and 0.1% trifluoro acetic acid (1.5 ml), each dried by 0.1 ml of dry nitrogen gas, and then the reagent of (i) or (ii) is added thereto. Vacuum seal and hydrolyze the contents.

Cys, Met, Val, Ile 및 Trp은 다음 방법으로 산출하였다. (＂Tampaku Kagaku(단백질화학) Ⅱ＂. 생화학 실험강좌, Tokyo Kagaku Dohjin 참조).The values shown in Table 2 are the average of four measurements obtained by hydrolysis of 24 hours (i) and 24, 48 and 72 hours of (ii). However, Thr, Ser,

Cys, Met, Val, Ile and Trp were calculated by the following method. (See Tampa Kagaku (Protein Chemistry II). Biochemistry Laboratory, Tokyo Kagaku Dohjin).

Cys 및 Met은 (ⅱ)의 24, 48, 72시간의 값의 시간경과에 따라 변화를 0시간에 대해 보외.-Thr, Ser,

Cys and Met are extrapolated to the change over time for values of 24, 48 and 72 hours in (ii) over 0 hours.

-For Val and Ile use the 72 hour value in (ii).

For Trp, use the average of the 24, 48 and 72 hour values of (ii).

TABLE 2

2) Sugar composition analysis

Internal standard (25 n moles of inositol) was added to 200 ng of purified CSF sample used in the amino acid composition analysis of 1), and methanol solution (500 μl) containing 1.5 N HCl was added thereto, and the tube was washed with N ₂ gas. It is made to react at 4 degreeC. Open the tube, add silver carbonate (Ag ₂ CO ₃ ) to neutralize the contents, add 50 μl of acetic anhydride, shake gently and leave overnight in a dark room at room temperature. The upper layer is placed in a sample tube and dried with nitrogen gas. 50 μl of a TMS reagent (5: 1 mixture of pyridine, hexamethyl disilazane and trimethyl chlorosilane) was added thereto, and the reaction was carried out at 40 ° C. for 20 minutes, and the product was stored in a room temperature cooler. Standard samples are prepared by combining 25 nanomoles of inositol with galactose (Gal), N-acetyl galactoseamine (Gal, NAc), sialic acid and other suitable reagents, each 50 nanomoles.

The sample thus prepared is gas chromatographed under the following conditions.

Analysis condition

Column: 2% OV-17 VIN port HP, 60-80 mesh, 3m, glass

Temperature: It rises from 110 degreeC to 250 degreeC at 4 degree-C / min.

Carrier Gas (N ₂ ) Pressure: First 1.2 ~ 1.6kg / cm ²

^{2 to} 2.5kg / cm2 at the end

Sensitivity: 10 ³ MΩ range, 0.1 ~ 0.4V

Pressure: H ₂ , 0.8kg / cm ²

Air, 0.8kg / cm ²

Sample volume: 2.5 to 3.0 μl

As a result, galactose, N-acetyl galactoseamine and sialic acid were identified in the CSF sample of the present invention.

Example 17

Construction of pHGV 2 Vector (VSE System for Animal Cells)

The EcoRI fragment of cDNA shown in FIG. 4a obtained in Example 10 was treated with a restriction enzyme Dral at 37 ° C. for 2 hours, and then treated with cleno fragment of DNA polymerase I (Takara Shuzo) to make a blunt end. . 1 μl of a Bal II linker (8-mer, Takara Shuvo) is phosphorylated with ATP and then combined with about 1 μg of the DNA fragment mixture obtained above. The bound fragment was treated with restriction enzyme BglII and subjected to agarose gel electrophoresis to recover only the largest DNA fragment.

This DNA fragment corresponds to about 700 bp containing the portion encoding the human G-CSF polypeptide (see Figure 5). Vector pdKCR [Fukunaga et al., Proc. Natl. Acad. Sci., USA, Vol. 81, 5086 (1984)] is treated with restriction enzyme BamHI and then dephosphorylated using alkaline phosphatase (from Takara Suzo). The resulting vector DNA is combined with about 700 bp cDNA fragment in the presence of T ₄ DNA ligase (Takara Shuvo) to prepare pHGV2 (Figure 9). As shown in FIG. 9, the plasmid is composed of a promoter of the SV 40 early gene, an SV40 replication initiation region, a part of the rabbit β-globin gene, a replication initiation region of pBR-322 and a pBR-322-derived β-lactamase gene (Amp ^r ), and the human G-CSF gene is connected downstream of the promoter of the SV40 early gene.

Example 18

Recombinant vector (-VSE) construction for transformation of C127 cells

1) Preparation of pHGV2 (H)

20 µl of the plasmid pHGV2 (Fig. 9) obtained in Example 17 was treated by the method described in 1) of Example 12 to obtain a plasmid named pHGV2 (H) (Fig. 10).

2) Preparation of the expression vector recombinant pTN-V2, pRNVA α and pTNVA β.

E. coli holding a plasmid having cDNA of pH-GV-2-derived G-CSF by the method described in 2) of Example 12 using 20 μg of pHGV2 (H) obtained in 1) above was prepared. Select. This plasmid is named pTN-V2 (Figure 10).

Adenovirus type II [Tampakushisu, Kakusan Koso (proteins, nucleic acids and enzymes), Vol. 27, Dec., 1982, Kyoritsu Shuppan] was treated in the same manner, and ΔpVA containing about 1700 bp Sall-HindIII fragment with VAI and VAII. A plasmid is obtained and a fragment with VAI and VAII is recovered from this plasmid. This fragment was inserted into the HindIII site of pTN-2 to obtain pTNVA α and pTNVA β (FIG. 10). Due to the VA gene of adenovirus, this plasmid can enhance the expression of transcription products from the SV40 early promoter.

Example 19

Transformation of C127 Cells and Expression of G-CSF (-VSE)

PTN-V2 obtained in Example 18 is treated with restriction enzyme BamHI before use for transformation of mouse C127 cells. Subsequently, mouse C127I cells are transformed with the DNA prepared above to express G-CSF (see Example 13), and clones with high G-CSF production capacity are selected. These clones produce G-CSF at the level of about 1 mg / L.

Cloning is continued to select clones capable of producing G-CSF at a level of 10 mg / L. In the same manner, C127 cells were transformed with pTNVA α and pTNVA β obtained in Example 19 and transformants were selected with clones with high G-CSF production capacity. From pTNVA α, clones producing 20 mg / L or more of G-CSF were obtained, but low-productivity (several mg / L) clones were obtained in the transformation using pTNVA.

As host cells, NIH3T3 cells can be used in addition to the C127I cells described above.

Example 20

Expression of G-CSF in CHO Cells (-VSE)

1) Construction of pHGV2-dhfr

From 20 μg of the pHGV2 plasmid (Example 17), the DNA fragment (0.5 μg) and the EcoRI fragment of pAdD26SVpA (0.5 μg) obtained according to the method described in Example 1 1) are annealed. Using the resulting plasmid, E. coli strain DH1 was transformed according to the rubidium chloride method, and colonies holding the plasmid pHGV2-dhfr were selected. The plasmid thus obtained is named pHGV2-dhfr (Fig. 11a).

Another method is as follows: plasmid pHGV2 is treated with SalI and partially digested with EcoRI without addition of the EcoRI linker to recover a DNA fragment of about 2.7 kb and treated with this DNA fragment with a Klenow fragment of E. coli DNA polymerase. To make blunt ends.

Meanwhile, pHGV2-dhfr was prepared by conjugating a separately prepared fragment (about 2.7 kb) and T4 DNA ligase by preparing a blunt-ended EcoRI fragment from pAdD26SVpA in the same manner as described above.

PHGV2 (H) prepared in 1) of Example 18 was treated with restriction enzymes HindIII and SalI as in 2) of Example 12, and then the HindIII-SalI fragment was blunt-ended of pAdD26SVpA as described above. Combine with EcoRI fragments. This method also applies to the preparation of pHGV2-dhfr (Fig. 11b).

2) Construction of pV2DR1 and pV2DR2

10 μg of the plasmid pAdD26SVpA described in 1) was dissolved in 50 μg of the reaction solution containing 50 mM Tris-HCl (pH 7.5), ₇ mM, MgCl ₂ , 100 mM NaCl, 7 mM 2-mercapto ethanol and 0.01% BSA, and the restriction enzyme EcoRI and Each BamHI was added 10 units each, and reacted at 37 DEG C for 10 hours, followed by phenol treatment in a conventional manner, followed by washing with ether. A 1% low melting point agarose gel electrophoresis is performed to recover a DNA fragment of about 2 kb and treated with a Clenow fragment of DNA polymerase to make a blunt end in a conventional manner. The blunt ended DNA fragments are treated with phenol, washed with ether and then precipitated with ethanol.

10 μg of the plasmid pHGV2 (H) obtained in Example 19) was dissolved in 50 μl of a reaction solution containing 10 mM Tris-HCl (pH 7.5), ₇ mM MgCl _2, and 60 mM NaCl, and 10 units of HindIII were added to react at 37 ° C. for 6 hours. Let's do it. DNA fragments are recovered by conventional 1% low melting agarose gel electrophoresis. The recovered DNA fragments are treated with BAP and treated with Klenow fragments to make blunt ends. This DNA fragment is treated with phenol and washed with ether and then treated with T4 DNA ligase in the following manner to bind the blunt end with the approximately 2 kb DNA fragment obtained earlier. 1 μg of each DNA fragment is dissolved in 30 μl of a reaction solution containing 66 mM Tris-HCl (pH 7.5), 6.6 mM MgCl ₂ , 5 mM DTT, and 1 mM ATP, and 50 units of T4 DNA ligase are added and reacted at 6 ° C. for 12 hours. This ligation product is used to transform E. coli strain DH 1. As a result, pV2DR1 and pV2DR2 shown in FIG. 11C are obtained.

3) Transformation and Expression

According to the method described in 3) of Example 14 using the pHGV2-dhfr plasmid prepared in 1), CHO cells are transformed to express G-CSF.

Transformation of CHO cells may be performed by co-transfection with pHGV2 and pAdD26SVpA.

CHO cells are also transformed by the following methods: pV2DR1 or pV2DR2 prepared in 2) were pretreated with SalI and KpnI, respectively, to obtain DNA fragments, and 10 μg of this fragment was used to transform CHO cells as above: Inverted cells are continuously cultured in a series of selective media in the manner described above: after 7 days, more than 100 positive colonies appear per culture dish: These colonies are transferred all at once to a new culture dish and a series of selective in the presence of 0.01 μM MTX. Continue incubation in the medium: Here over 10 colonies appear: Repeat the above procedure to select surviving colonies by continuously increasing the MTX concentration to 0.02 μM, 0.05 μM and 0.1 μM. The same method can be used to select colonies individually and culture with increasing MTX concentration. It can be carried out.

CHO cells can also be transformed by using a recombinant vector having a so-called polycistronic gene. For example, pAdD26SVpA is treated with PstI and the two fragments are recovered to combine them with the pBRV2-derived CSF cDNA fragments to produce recombinant vectors in which the poly a sites of the adenovirus promoter, CSF cDNA, DHFR and SV40 are inserted in sequence. . This recombinant vector is used to transform CHO cells.

Example 21

Assay of Transduced G-CSF Activity (-VSE)

Human G-CSF is prepared from the culture supernatant of C127 cells and CHO cells obtained in Examples 19 and 20 according to the method described in Example 15, and the liquid G-CSF activity of the sample is assayed. The results are as shown in Table 3.

TABLE 3

Example 22

Amino Acid Analysis and Sugar Analysis (-VSE)

1) Analysis of Amino Acid Composition

The crude CSF sample obtained in Example 21 is purified again by the method described in Example 2 (iii). The purified CSF sample is analyzed for amino acid composition according to the method described in 1) of Example 16. The results are as shown in Table 4.

TABLE 4

2) Sugar composition analysis

The purified CSF sample used for the amino acid composition analysis of 1) was subjected to the glycosylation analysis by the same method and the same analysis conditions as described in 2) of Example 16. As a result, it was confirmed that galactose, N-acetyl galactoseamine and sialic acid were present in the CSF sample of the present invention.

Example 23

Infection Defense Effect of Human G-CSF

[Test Methods]

1. Protective effect against Pseudomonas aeruginosa infection

Intraperitoneal administration of endoxan (Endoxan: Shionogi brand name) 200 mg / kg to ICR mice (male) of 8 to 9 weeks of age (weight 35.3 ± 1.38 g) was divided into three groups and human G-CSF to two groups. (25,000 units / mouse or 50,000 units / mouse) of solvent [physiological saline of 1% propanol and 0.5% (W / V) mouse serum albumin], and the remaining group 1 each with only 0.1 ml of solvent every 24 hours. Four subcutaneous injections were made. Three hours after the fourth dose, each group of mice was infected subcutaneously with Pseudomonas aeruginosa GNB-139 (3.9 × 10 ⁵ CFU / mouse). 21 hours after infection, the first two groups were once again subcutaneously injected with a solvent containing human G-CSF (25,000 units / mouse or 50,000 units / mouse) and the other group was injected only with solvent.

The infection defense effect of human G-CSF is examined by counting the number of live mice on day 10 after infection.

Preparation of Cell Suspension

Pseudomonas aeruginosa GNB-139 is incubated overnight at 37 ° C. in a Heart-infusion liquid medium (trade name of Difco). The culture is suspended in physiological saline.

[result]

i) The test is performed on purified human G-CSF samples (+ VSE) derived from CHO cells as used in the amino acid composition analysis of Example 16.

The results are shown in Table 7.

TABLE 5

When the infection protection effect of the said test was investigated using the purified human G-CSF sample derived from the same cell used for the amino acid composition analysis of Example 16, the same result is obtained.

ii) Test 1 is performed on purified human G-CSF samples (-VSE) derived from the same CHO cells as those used for the amino acid composition analysis of Example 22, and the results are shown in Table 8.

TABLE 6

The same results are obtained when the infection protection effect of the test was investigated using purified human G-CSF samples derived from the same C127 cells as those used for the amino acid composition analysis of Example 22.

Claims (3)

Culturing mammalian cells transformed with a recombinant vector containing a cDNA fragment having a nucleotide sequence encoding the following amino acid sequence, and then separating the produced glycoprotein having human granular leukocyte colony stimulator activity A method for producing a glycoprotein having human granulocyte leukocyte colony stimulating factor activity. Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu (Val Ser Glu) _m Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Leu Val Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Gln Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (Wherein m represents 0 or 1). The production method according to claim 1, wherein the base sequence is the following base sequence. ATG GCT GGA CCT GCC ACC CAG AGC CCC ATG AAG CTG ATG GCC CTG CAG CTG CTG CTG TGG CAC AGT GCA CTC TGG ACA GTG CAG GAA GCC ACC CCC CTG GGC CCT GCC AGC TCC CTG CCC CAG AGC TTC CTG CTC AAG TGC TTA GAG CAA GTG AGG AAG ATC CAG GGC GAT GGC GCA GCG CTC CAG GAG AAG CTG (GTG AGT GAG) _m TGT GCC ACC TAC AAG CTG TGC CAC CCC GAG CTG GTG GTG CTG CTC GGA CAG TCT CTG GGC ATC CCC TGG GCT CCC CTG AGC AGC TGC CCC AGC CAG GCC CTG CAG CTG GCA GGC TGC TTG AGC CAA CTC CAT AGC GGC CTT TTC CTC TAC CAG GGG CTC CTG CAG GCC CTG GAA GGG ATC TCC CCC GAG TTG GGT CCC ACC TTG GAC ACA CTG CAG CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GAA GAA CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT GCC ATG CCG GCC TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG GTC CTG GTT GCC TCC TAT CTG CAG AGC TTC CTG GAC GAG TCG TAC CGC GTT CTA CGC CAC CTT GCC CAG CCC (Wherein m represents 0 or 1). The production method according to claim 1, wherein the base sequence is the base sequence shown in FIGS. 3A or 4A.

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