JPH06181770A - Production of human lymphotoxin - Google Patents

Abstract

PURPOSE:To produce human lymphotoxin by gene recombination using a DNA sequence containing human lymphotoxin gene. CONSTITUTION:An animal cell transformed with a DNA having a DNA sequence wherein a human lymphotoxin chromosome DNA sequence is linked to a sequence of a promoter range to function with an animal cultured cell is cultured to form human lymphotoxin, which is collected. Human lymphotoxin having high safety can be mass produced.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a DNA sequence containing the human lymphotoxin gene and D having the DNA sequence.
Human using cultured cells transformed by NA
It relates to a method for producing lymphotoxin.

[0002] Lymphotoxin (LT) directly or indirectly attacks only cancer cells and causes necrosis (Ev.
ans CH et al. (1977) Cancer Research (Cance
r Res.), 37, 898), and is expected to be clinically applied as an anticancer agent.

[0003]

2. Description of the Related Art Lymphotoxin (LT) is a kind of lymphokine that is induced by stimulating lymphocyte cells of animals such as humans or mice with lectins such as phytohemagglutinin and concanavalin A or phorbol esters ( Devlin, JJ (1984) Lymphokines, vol. 9, p. 313). In humans, T cell or B cell line RDMI1788 (Agga selected as a representative producer cell by rosette formation with sheep red blood cells)
rwal, BB et al. (1984) The Journal of Biological Chemistry (J. Biol. Chem.), 259.
Vol., P. 686). Although LT is a glycoprotein (Toth, MK and Granger, GA (1979), Molcular Immunology (Mol. Immunol.), 16: 6.
(P. 71), can take various forms. Although the protein chemistry of LT has been studied by several groups, the smallest unit is a component having a molecular weight of about 20,000, and it is suggested that the unit component has an association or a complex with other components. (Aggarwal, BB et al. (1984) The Journal of Biological Chemistry, 259).
Vol., 686).

[0004] It is known that LT is produced by lymphocytes stimulated by phorbol ester, mitogen, etc. However, LT produced by such a production method is extremely small, and a large amount of fresh LT is produced. It requires large numbers of lymphocytes and is not suitable for mass production. In addition, when cells derived from established lymphocytes (cell lines) are stimulated with mitogen, LT
Is known to be produced inducibly, but the production capacity largely depends on the capacity of the cells used, and it cannot be said that the system is suitable for mass production. Recently, LT cDNA was cloned, and it became possible to produce LT-like protein in Escherichia coli (Gray, PW et al. (1984) Nature, 3).
12, p. 721). However, LT-like proteins produced by microorganisms often differ in the amino terminus of the produced protein from the natural ones because the protein synthesis mechanism between animal cells and microorganisms is somewhat different. Furthermore, in LT-like proteins produced by microorganisms, natural LT has sugar chains, whereas sugar chains are not bound. In this way, LT-like proteins produced by the protein synthesis system of microorganisms and natural LT are distinctly different from each other as substances, and there is a problem of antigen-antibody reaction when they are used as therapeutic agents for a long time or frequently. I'm worried.

[0005]

[Problems to be Solved by the Invention] In order to produce LT having the same amino terminus of protein as natural LT and having a sugar chain, application of a gene recombination technique using an animal cultured cell as a host is considered. To be In this case, it is presumed that the LT production cannot be observed simply by introducing the LT gene into the cultured animal cells. That is, LT is an inducible protein, and its expression is restricted at the gene level. Therefore, by the method of gene transfer, LT effective for cultured animal cells is obtained.
In order to impart productivity, it is necessary to improve the gene used.

It is known that many proteins of higher organisms are encoded in the nuclear DNA sequence by being divided into several parts. Mature messenger RNA (mRNA)
The DNA sequence encoding the sequence is exon,
Dividing sequences are intervening sequences or introns
is called. The biological significance and function of introns are currently unknown, but ovalbumin (Wickens, MP
Et al. (1980) Nature, 285, p. 628) and viral proteins (Lai, CJ. Et al. (1979) Proceedings of the National Academy of.
Science USA (Proc. Natl. Acad. Sci. U)
SA), Vol. 76, p. 71), the gene sequence not containing an intron is known to produce extremely less protein in the introduced animal cell than the sequence containing an intron. It was also known that stable mRNA accumulation occurs by adding the intron of the β-globin gene to the SV40 gene lacking the intron (Hamer, D.
H. et al. (1979) Cell, Volume 18, 1299.
page).

Splicing Removal of Intron Partial Sequence from Initial RNA Transcribed from Gene
However, splicing is presumed to be an event necessary for stable mRNA accumulation or mRNA translocation from the nucleus to the cytoplasm.

Although splicing at the correct position of the intron is essential for the expression of a normal and functional protein, the insulin gene and simian virus 40 (S
Aberrant splicing has been reported when the promoter region of V40) was bound and introduced into COS cells (Laub, O. et al. (1983) The Journal of Biological Chemistry, 258, 6043).
page). In addition, it is known that there is tissue-specific splicing in the expression of amylase, and salivary gland amylase and liver amylase are synthesized through two different splicing from the same gene (Young, RA
Et al. (1981) Cell, 23, 451). Also, S
V40 (Berk, AJ et al. (1978) Proceedings of the National Academy of Science USA, 75, 1274), adenovirus (Chow, LT (1977) cell, 12 volumes, 1
Page) and the like, a plurality of mRNAs and proteins are synthesized from the same gene through different splicing.
Therefore, in order to introduce the LT gene containing an intron into an animal cultured cell and produce LT, normal splicing needs to occur. However, the present inventors have found that a chromosomal DNA sequence coding for LT is introduced in the animal cultured cell. It was found that when a DNA sequence of a promoter region capable of functioning, that is, capable of initiating mRNA synthesis, is bound and introduced into various animal cultures, splicing normally occurs and LT is secreted in a large amount in the medium. It was

LT is a glycoprotein. Currently, there are many unclear points about the sugar chain structure of LT, and it is unknown whether there is a difference in the structure and antigenicity of the sugar chain of LT produced in cells other than human and the LT produced in human-derived cells. . However, LT produced by human-derived cells is considered to be very similar to natural LT, and L produced by non-human cells is considered.
It is considered to be safer than T. In addition, when LT is produced in cells other than humans, it is considered that constituents such as proteins and secretory components of organisms other than humans are mixed in LT products, and allergic reaction in long-term administration as a therapeutic agent, Although problems such as shock are expected, the products made using human-derived cells are essentially human ingredients,
That is, it is expected that the safety of the product will be improved because it does not contain substances other than those existing in human blood.

It is considered that the present invention enables a large amount of highly safe LT to be supplied. Hereinafter, the present invention will be described in more detail.

[0011]

[Means for Solving the Problems] The chromosomal gene region encoding LT has been clarified for the first time as a result of the cloning and analysis performed by the present inventors and has a restriction enzyme recognition site as shown in FIG. ing. The LT chromosomal DNA sequence is a DNA sequence containing a nucleotide sequence from the codon ATG of methionine, which is the amino acid for the initiation of protein synthesis of LT, to the termination codon TAG. For example, the plasmid pLTB shown in FIG.
BamHI 4.2Kb (kilobase) included in 4.2
Refers to the array of.

The chromosomal DNA sequence encoding LT is cloned from human DNA. For human DNA, for example, using human white blood cell culture cells or tissues, the method of Blin et al. (Blin, N. et al. (1976) Nucleic Acids Res., Vol. 3, 2303.
Page). Vectors used for cloning the LT gene include λ phage vector typified by Charon28, plasmid vector typified by pBR322, cosmid typified by pHC79, etc. A genetic engineering method using a λ phage as a vector capable of cloning the That is, human high molecular weight DNA is cleaved with an appropriate restriction enzyme and then inserted in place of the replaceable region of λ phage DNA to form a recombinant phage DNA. Next, the in vitro packaging method is used to prepare infectious phage particles. Next, it is plated with host E. coli to form plaques of recombinant phage (Enquist, L. et al. (1979) Methods in Enzymology, 68).
Volume, 281; Horn, B. (1979) Methods in Enzymology, 68, 299). To detect plaques of recombinant phages having a DNA fragment encoding LT, plaque hybridization method using cDNA or synthetic DNA (Woo, SLC (19
1979) Methods in Enzymology, Volume 68,
389; Szostak, JW et al. (1979) Methods.
In Enzymology, Vol. 68, p. 419) is available. In addition, a recombinant phage having the LT gene can be prepared in a large amount by recovering from the plaque selected by plaque hybridization and culturing with the host Escherichia coli. The DNA of recombinant phage can be prepared by the phenol method or the like (Maniatis, T. et al. (198
2 years) Molecular Cloning a Laboratory manual, Cold
Spring Harbor Laboratory).

[0013] As a method for introducing DNA into animal culture cells,
Although there is a difference in transfection efficiency, the calcium phosphate method (Wigler, M. et al. (1977) cell, 11 volumes, 2
23), microinjection method (Anderson, W.
F. et al. (1980) Proceedings of the National Academy of Science USA, 77, 5399), liposome method, DEAE-
Dextran method or cell fusion method (Schoffner.W. Et al. (1
980) Proceedings of the National Academy of Sciences USA, 77
Vol., P. 2163) and the like. As the DNA material used in the calcium phosphate method, microorganisms such as Escherichia coli and phages can be used in addition to the DNA solution. In the cell fusion method, a protoplast of a microorganism having a target DNA sequence as a plasmid is used.

LT is an inducing protein and is induced by stimulating human white blood cells with various mitogens. What kind of form is currently being used to stimulate mitogen LT
It is not known whether this gene acts on the gene to induce LT, but even if a gene containing the regulatory region of LT is introduced into cells that do not have such an induction mechanism, production of LT will be weak. The present inventors have determined that the sequences of promoter regions of other genes that function in cultured animal cells are 5'of the chromosomal DNA sequence of LT.
It was found that LT non-producing cells can be transformed into high producing cells by connecting to the side and introducing into cells. In this case, LT mRNA synthesis is under the control of the connected promoter region, for example, if the connected promoter region is the promoter region of a gene for a constitutive protein, LT mRNA is always synthesized in the cell, and The cell becomes a constitutive producer of LT. If the connecting promoter region is of an inducible protein, the transformed cell will produce an LT inducible protein.

The SV40 early gene promoter is known as a promoter that functions in animal cultured cells.
This promoter is Hind III-Pvu II of SV40 DNA.
The fragment is contained in an approximately 350 base pair (bp) DNA fragment. Also, this DNA fragment was reversed in SV
It has an activity as a promoter of 40 late genes. Transcriptional activity from the late promoter of SV40 is
Generally enhanced in the presence of SV40 T antigen. Therefore, the cells into which the LT gene connected to the late promoter of SV40 is introduced are preferably cells expressing T antigen. To produce cells expressing T antigen, a gene encoding T antigen may be introduced into cells. In addition, the DNA sequence encoding the T antigen of SV40 and SV40
When a cultured cell is transformed with a DNA sequence in which the LT gene sequence connected to the latter promoter sequence is present on the same sequence, a high-expressing strain of LT will be obtained in various cell lines.

Herpes simplex virus (H
SV) Type I thymidine kinase promoter is also SV
It is a constitutive promoter similar to the 40 early gene promoter, and the structure of the region is shown by Wagner et al. (Wagner, MJ et al. (1981) Proceedings of the National Academy of Science. (USA, 79, 1441). The functional promoter region includes the starting point of mRNA synthesis,
It refers to the sequence of the promoter region that does not include the codon for methionine, the first amino acid of proteins regulated by those promoters. Example 3 shows the preparation of a DNA sequence (LT expression vector) in which the sequence of the functional promoter region and the LT chromosomal DNA sequence were connected.

The amino acid sequence of LT can be deduced from the nucleotide sequence of the exon portion of the cloned LT gene. Nucleotide sequence is Maxam-Gilbert method (Maxam, A.
M. et al. (1980) Methods in Enzymology, Vol. 65, 499), or Sanger's dideoxy method (Sanger, F. (1981) Science),
214, p. 1205).

When the gene is introduced into a cell, the transgene may be stably integrated into the host chromosomal DNA.
The position on the chromosome where the gene is integrated is seemingly random, and the copy number of the integrated DNA is also irregular. When the LT gene is introduced into cells, the incorporated position and copy number differ from cell to cell, and the LT production amount of each cell differs. Therefore, cells having various production amounts can be obtained by cloning the cells. In order to introduce only the target gene and selectively propagate only cells that are stably expressed, a DNA sequence having a sequence in which a functional promoter sequence and the LT gene are connected and a selection marker gene on the same DNA sequence is suitable. . As a selectable marker gene in animal cells, Ecogpt (Mulligan, RC et al.
80) Science, 209, 1422), neo
(Southern, PJ et al. (1982) Journal of
Molecular and Applied Genetics
(J. Mol, Appl. Genet.), Vol. 1, p. 327), dhfr (Wig
ler, M. et al. (1980) Proceedings of
Genes such as The National Academy of Science USA, 77, 3567) are used. Further, in order to prepare such a DNA sequence in a large amount, it is desirable that such a DNA sequence is a plasmid or a phage which can be replicated in E. coli and can be prepared in a large amount. The LT expression vectors shown in Examples 3 to 5 are plasmids that serve the above purposes. That is, the origin of DNA replication (ori) that enables replication in E. coli, the selectable marker (ampicillin resistance gene), the selectable marker gene (Ecogpt) in animal culture cells, and the chromosomal DNA sequence of LT connected to a functional promoter are the same. It is a plasmid characterized by being present on a DNA sequence.

A cell introduced with the LT chromosomal DNA sequence connected to the promoter regions of other functional genes is L
In order to produce T, the DNA sequence must be compatible with the functions of the cell-specific RNA synthesis system, RNA maturation, protein synthesis system, protein maturation, secretion and the like. Although mRNA is synthesized from the introduced DNA, it is necessary to add a cap structure at the 5'end of the mRNA, splicing at the normal position and polyadenylation to the 3'end. In addition, for the expression of active LT, the synthesized L
It is necessary that the formation and maintenance of the normal higher-order structure of T-peptide, the cleavage of the signal peptide, and the secretion from the cell be performed accurately. The animal cultured cells used by the present inventors are American Type Culture Collection (A
The cells derived from hamsters, monkeys, and humans, which are available from TCC), but using the method for producing LT described herein, at least cultured cells derived from vertebrates, fused cells, normal and mutant cells, It is possible to produce active LT in a cell transformed with a virus or the like. In addition, using a cell line transformed from human cells with SV40 as a production cell improves the safety of the product by taking appropriate measures as compared with cells that have become cancerous or cell lines with unknown causes. There is expected. WI-26 VA4 is known as a transformed cell of SV40.

The LT chromosomal DNA sequence to which a functional promoter is connected is introduced into animal cultured cells by, for example, the calcium phosphate method, and cells which have started to produce LT can be obtained by using only the medium containing serum normally used for culturing cells. Furthermore, it was found that LT was produced even in a serum-free medium containing no serum. Using a serum-free medium for the production of LT not only makes recovery and purification of LT from the medium easier, but also prevents contamination of the product with serum components.

[0021]

EXAMPLES Examples will be shown below, but various experiments according to the present invention were carried out in accordance with "Recombinant DNA Experiment Guideline" established by the Prime Minister. In addition, phage, plasmid, D in the examples
Detailed operations for handling NA, various enzymes, Escherichia coli, etc. were referred to the following magazines and books.

1. Protein Nucleic Acid Enzyme, Vol. 26, No. 4 (1981) Extra number Gene manipulation (Kyoritsu Publishing) 2. Gene manipulation experiment method, edited by Yasutaka Takagi (1980)
Kodansha 3. Gene Operation Manual, edited by Yasutaka Takagi (1982)
Year) Kodansha 4. Molecular Cloning a laboratory manual, T. Mani
Edited by atis et al. (1982) Cold Spring Harbor Laborator
y 5. Methods in Enzymology, Volume 65, edited by L. Grossman et al. (1980) Academic Press 6. Methods in Enzymology, Volume 68, edited by R. Wu (1
997) AcademicPress

Example 1 Cloning of LT Gene Heparin blood was collected from a plurality of healthy adults, diluted 2-fold with a commercially available phosphate buffer (PBS) (manufactured by Flow Laboratories), and then applied as an upper layer on a Ficoll pack (manufactured by Pharmacia). Then, the white blood cell layer was separated by centrifugation at 2000 rpm for 30 minutes and further washed twice with PBS. 20m for 10 ⁸ cells
1 of 0.5 M EDTA-0.5% sarcosyl solution was added, 2 mg of protease K was added, and the mixture was incubated at 50 ° C. for 3 hours. Phenol extraction was performed twice, and the aqueous layer was dialyzed against 50 mM Tris-10 mM EDTA-10 mM sodium chloride (pH 8.0) overnight. RNaseA
Was added to 100 μg / ml, treated at 37 ° C. for 3 hours, then extracted twice with phenol, and the aqueous layer was dialyzed against 50 mM Tris-10 mM EDTA to give high molecular weight human DNA.
Got Human DNA was partially cleaved with the restriction enzyme Sau3AI and then subjected to sucrose density gradient centrifugation to prepare a Sau3AI DNA fragment having a size of about 15 to 20 kilobases. After cutting the lambda phage vector Charon28 DNA with BamHI, fractions containing the left end fragment and the right end fragment of Charon28 were collected by sucrose density gradient centrifugation and recovered by ethanol precipitation.

DNA fragments at both ends of Charon 28 and human 15
After binding the 20 kilobase Sau3AI fragment in T ₄ DNA ligase, Enkisuto and Sternberg methods (L. Enq
Uist and N. Sternberg (1979) Methods in Enzymology, 68, 281) were used for in vitro packaging to form recombinant phage plaques using E. coli LE392 as a host. Next, plaque hybridization methods (Benton, WD, Davi
s, RW (1977) Science, Volume 196, 180
Recombinant phage clones carrying the LT gene were selected according to page. As a probe, an oligonucleotide ATGACACCA having a sequence present in the LT gene is used.
CCTGAACGT, TCTACTCCCAGGTGGG
TC and ACTGTCTTCTTTGGAGCC (these sequences are LT amino acid sequences -34 to -29,75
To 80 and 163 to 168: Gray,
PW et al. (1984) Nature, Volume 312, 721
Phosphotriester method (Miyoshi. K. et al. (198)
0) Nucleic Assies Research, Volume 8, 550
7 ') and 5'-OH was labeled with [γ- 32 _P ] ATP and T4 polynucleotide kinase and used.

Eleven phage clones were obtained from about 600,000 recombinant phage clones, which hybridized with all three synthetic DNA probes used. One of these phage clones 4-1 was cleaved with various restriction enzymes,
After performing agarose electrophoresis and transferring to a nitrocellulose filter, Southern hybridization (Southern. EM) using three kinds of synthetic DNA probes was performed.
(1975) Journal of Molecular Biology (J. Mol. Biol., 98, 503), BamHI 4.2 Kb, EcoRI 2.3.
The Kb and SmaI 2.7 Kb fragments hybridized with the three probes, and it was revealed that these fragments contained the DNA sequence encoding the amino acid sequence of LT. As a result of restriction enzyme analysis of DNA of several phage clones, the restriction enzyme recognition sites of the LT chromosomal DNA sequence and adjacent sequences were mapped as shown in FIG.

Example 2 Subcloning of LT Gene The DNA of phage clone 4-1 was digested with the restriction enzyme BamHI.
The resulting 4.2 Kb fragment was cut with the plasmid pUC.
9 (Vieira, J and Messing, J (1982) Gene (Gen
e), vol. 19, p. 259) and inserted into the BamHI site to obtain pL
TB4.2 was produced (Fig. 2).

Commercially available pUC9 primer CA
GGAAACAGCTATGAC, AGTCACGAC
Amino acid of GTTGTA (above Takara Shuzo) and LT-2
Oligomers corresponding to 3 to -28, 75 to 80, 163 to 168-ACCCTTGGGGAGGAAGA
G, TCTACTCCCAGGTGGTC, ACTGT
Nucleotide sequencing by the dideoxy method using CTTCTTTGGAGCC as a primer (Wallace, RB et al.
1981) Gene, 16:21, pLTB4.2. As a result, it was found that the 5 ′ side of LT had the sequence of SEQ ID NO: 1 and the 3 ′ side had the sequence of SEQ ID NO: 2.

Example 3 pSVeSmaILT, pSVpTKLT, pSV2L
Construction of LT and pSV3LLT pSVeSmaILT, which is a plasmid having a sequence in which the sequence of the early gene promoter region of SV40 and the LT chromosomal DNA sequence are connected, is pLTB4.2, pSV2g.
pt and pSV3gpt (Mulligan, RC and Berg, P.
(1980) Science, 209, 1422) was used as a starting material and was prepared by the procedure shown in FIGS.

That is, pSV3gpt was cleaved with HindIII, and the largest DNA fragment was circularized with T4 DNA ligase to prepare pHI. Next, the PvuII site of pHI was changed to a SalI site using a SalI linker,
II was produced. Furthermore, the HindIII site of pHII is
A SmaI site was introduced using a dIII-SmaI adapter to prepare pHSmaI. pHSmaI is Sal
I, EcoRI digestion and the BamHI site of pSV2gpt were digested with BamHI, followed by DNA polymerase I (Klenow)
The blunt end was blunt-ended and cyclized with T4 DNA ligase to prepare pSI, which was similarly digested with SalI and EcoRI, and ligated with a DNA fragment having an ampicillin resistance gene by T4 DNA ligase to prepare pSVeSmaI. Then pL
TB4.2 was digested with SmaI to obtain a DNA fragment having the LT gene sequence, which was digested with SmaI to obtain pSVeSm.
It was introduced into aI to prepare pSVeSmaILT.

The SalI linker and SmaI adapter used were d (pGGTGCGACC) and d (p
The one having the sequence of AGCTCCCGGG) was used.
As the DNA polymerase I, Klenow fragment was used.

Herpes simplex virus type 1
PSVpT, which is a plasmid having a sequence in which the promoter region of thymidine kinase of Escherichia coli is connected to the LT gene
KLT has pLTB4.2, pHSV106 (McKnigh
t, SL and Gabis, ER (1980) Nucleic
Assies Research, 8: 5931) and pSVe
It was prepared by the method shown in FIG. 5 using SmaI as a starting material. That is, LT gene B contained in pLTB4.2
The amHI-SmaI fragment was added to BglII-of pHSV106.
It was inserted into the SmaI site to prepare pHSVLT. Then p
LT gene B from HSVLT to TK promoter
The amHI-SmaI fragment was transformed into BamH of pSVeSmaI.
It was introduced into the I-SmaI site to prepare pSVpTKLT.

Further, pSV2LLT and pSV3LLT, which are plasmids having a sequence in which the LT gene is connected to the sequence of the late gene promoter region of SV40, are pLTB.
It was prepared by the method shown in FIGS. 6 and 7 using 4.2, pSV2gpt and pSV3gpt as starting materials. That is, the LT gene SmaI-S contained in pLTB4.2
The maI 2.5 Kb fragment was ligated into the PvuII site of pSV2gpt to create pSV2LLT. Then pSV2L
LT was partially cleaved with BamHI and pSV3gpt
PSV3 by ligation with BamHI fragment of T antigen gene
LLT was made.

Example 4 pSVeSmaILT, pSVpTKLT, pSV2L
Introduction of LT and pSV3LLT into cultured cells and production of LT Phenotypic expression vectors pSVeSmaILT, pSVpTK
L included in LT, pSV2LLT and pSV3LLT
To examine the expression of T gene, Wi
gler et al.'s method (Wigler et al. (1977) Cell, Volume 11,
The plasmid was introduced according to (Page 223). Cells (2 ×) in which the plasmid-calcium phosphate co-precipitate was previously grown in Eagle MEM medium containing 10% bovine neonatal serum.
10 ⁵ cells / 3 ml medium / 6 cm culture dish),
After 15 hours, the medium was renewed, and after 48 hours of culturing, LT contained in the medium was measured by a cell-killing effect using L929 cells as target cells (Ruff, MR and Gifford, GE).
(1981) Lynn Hokines, Vol. 2, p. 235). That is, 2 × 10 ⁴ cells / well in a 96-well multi-dish
After culturing for 1 day in 100 μl / 100 μl medium, the culture solution was removed, and samples diluted to various concentrations with Eagle MEM medium containing actinomycin D 1 μg / ml and 5% fetal bovine serum were prepared.
After adding 00 μl, the degenerative killing effect of the cells was measured 20 hours later. The LT1 unit was at a concentration that gave a lethality of 50%. As shown in Table 1, pSVeSmaILT, pSV
Expression of LT was observed in all cultured cells into which pTKLT, pSV2LLT or pSV3LLT had been introduced. Moreover, the expression of pSV3LLT was higher than that of pSV2LLT in all the cultured cells.

[0034]

[Table 1]

Example 5 Production of LT in normal medium and serum-free medium BHK-21 (C-1) into which pSVeSmaILT, pSVpTKLT or pSV3LLT was introduced in Example 4
MEM containing 10% fetal bovine serum, 25 μg / ml mycophenolic acid, 250 μg / ml xanthine as the medium 3)
The medium was replaced and the mycophenolic acid resistant strain was isolated. A mycophenolic acid resistant strain is grown on the entire bottom surface of a 24-well multi-dish, and ME containing 5% fetal calf serum (FCS) is added.
The cells were cultured in M medium and MEM medium containing no fetal calf serum for 24 hours, and the LT activity contained in the medium was measured. Table 2
As shown in, the isolated cell line produced LT with or without serum.

[0036]

[Table 2]

[0037]

[Brief description of drawings]

FIG. 1 is a schematic diagram showing recombinant phage DNA obtained by cloning a chromosomal DNA fragment containing the LT gene. E
Is the recognition site for the restriction enzyme EcoRI, B is the restriction enzyme Ba
The recognition site of mHI is shown.

FIG. 2 is a schematic diagram showing plasmid pLTB4.2.

FIG. 3 is a schematic diagram of construction of plasmid pSVeSmaI.

FIG. 4 is a schematic diagram of construction of plasmid pSVeSmaILT.

FIG. 5 is a schematic diagram of construction of plasmid pSVpTKLT.

FIG. 6 is a schematic diagram of construction of plasmid pSV2LLT.

FIG. 7 is a schematic diagram of construction of plasmid pSV3LLT. 2 to 7, SmaI, AvaI, BamHI,
AccI, ScaI, EcoRI, SacI, Hind
III, PvuII, SalI and BglII represent the recognition sites for the respective restriction enzymes. HuLT is human LT gene, pU
C9 is a region derived from the vector plasmid pUC9, Amp
r is an ampicillin resistance gene, Ecogpt is an E. coli guanine phosphoribosyl transferase gene,
SVe is the SV40 viral early gene promoter region, T-ag is the SV40 T-antigen gene, pTK is the thymidine kinase promoter region, TK is the thymidine kinase gene, and (PvuII / SmaI) is the restriction enzyme P.
The figure shows a combination of the vuII cleavage site and the SmaI cleavage site.

[Sequence list]

SEQ ID NO: 1 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: Genomic DNA sequence GGATCCCCGG CCTGCCTGGG CCTGGGCCTT G GTGGGT ... Intron ... GTTCTCCCCC ATG ACA CCA CCT rProG Tet ... Sequence number: 2 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA sequence GCC TAC TCT CCC AAG GCC ACC TCC TCC Ala Tyr Ser Pro Lys Ala Thr Ser Ser CCA 90 CTC TAC CTG GCC CAT GAG GTC CAG CTC Leu Tyr Leu Ala His Glu Val Gln Leu TTC 100 Phe TCC TCC C G TAC CCC TTC CAT GTG CCT Ser Ser Gln Tyr Pro Phe His Val Pro CTC 110 Leu CTC AGC TCC CAG AAG ATG GTG TAT CCA Leu Ser Ser Gln Lys Met Val Tyr Pro GGG Gly CTG CAG GAA CCC TGG CTG CAC TCG ATG Leu Gln Glu Pro Trp Leu His Ser Met TAC 130 Tyr CAC GGG GCT GCG TTC CAG CTC ACC CAG His Gly Ala Ala Phe Gln Leu Thr Gln GGA 140 Gly GAC CAG CTA TCC ACC CAC ACA GAT GGC Asp Gln Leu Ser Thr His Thr Asp Gly ATC 1 0 lle CCC CAC CTA GTC CTC AGC CCT AGT ACT Pro His Leu Val Leu Ser Pro Ser Thr GTC 160 Val TTC TTT GGA GCC TTC GCT CTG Phe Phe Gly Ala Phe Ala Leu TAG AACTTGG 170 STOP AAAAATCCAG AAAGAAAAAA TAATTGATTT CAAGACCTTC TCCCCATTCT GCCTCCATTC TGACCATTTC AGGGGTCGTC ACCACCTCTC CTTTGGCCAT TCCAACAGCT CAAGTCTTCC CTGATCAAGT CACCGGAGCT TTCAAAGAGA GAATTCTAGG CATCCCAGGGG GACCCACACT CCCTGAA CCA TCCCTGATGT CTGTCTGGCT GAGGATTTCA AGCCTGCCTA GGAATTCCCA G

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (72) Inventor Hajime Kawarada 2183-4 New House, Hiraoka-cho, Kakogawa-shi, Hyogo Prefecture (72) Inventor Kiyoshi Watanabe 5-15 41, Matsugaoka, Akashi-shi, Hyogo Prefecture

Claims (3)

[Claims] 1. A human lymphotoxin chromosomal DNA sequence characterized by the restriction enzyme cleavage map of FIG. 1 having the sequence of SEQ ID NO: 1 on the 5 ′ side and the sequence of SEQ ID NO: 2 on the 3 ′ side and an animal. Human lymphotoxin is produced by culturing an animal cell transformed with a DNA having a DNA sequence connected to a sequence of a promoter region which functions in the cultured cell to produce human lymphotoxin, which is collected. Production method. 2. The method according to claim 1, wherein the cultured animal cells are cultured in a culture medium, and human lymphotoxin is recovered from the culture medium. 3. The method according to claim 2, wherein the culture solution is a serum-free culture solution.