JPH0834725B2 - Lymphotoxin - Google Patents

Description

TECHNICAL FIELD The present invention uses a DNA sequence containing a human lymphotoxin gene, a cultured cell transformed with a DNA having the DNA sequence, and a transformed cell thereof. The present invention relates to a method for producing human lymphotkin.

Lymphotoxin (LT) directly or indirectly attacks only cancer cells and causes necrosis (Evans CH et al. (1977) Cancer Res., 37
Vol., Page 898), and is expected to be clinically applied as an anticancer drug.

(Prior Art) Lymphotoxin (LT) is a kind of lymphokine induced by stimulating lymphocyte cells of animals such as humans or mice with lectins such as phytohemagglutinin and concanavalin A or phorbol esters ( De
vlin, JJ (1984) Lymphokines, 9
Vol., Pp. 313). As a typical producer cell, in humans, T cell or B cell line selected by rosette formation with sheep erythrocyte RPMI 1788 (Aggarwal, BB et al. (1984) The Journal of Biological Chemistry (J. Bio
l. Chem.), 259, 686) are known. LT is a glycoprotein (Toth, MK and Granger, GA (1979), but Morikiurler Immunol., 16: 671.
Page), and can take various forms. Although the protein chemistry of LT has been studied by several groups, it has a molecular weight of about 2
It is said that 0,000 components are the minimum units, and that the unit components are associated or complex with other components (Aggarwal, BB et al. (1984) The Journal of
Biological Chemistry, 259, 686).

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,
Moreover, a large amount of fresh lymphocytes are required, which is not suitable for mass production. Moreover, it is known that LT is induced by inducing lymphocyte-derived cells (cell lines) that have been established by mitogen, etc., but the production capacity largely depends on the capacity of the cells used. However, it cannot be said that the system is suitable for mass production. Recently, the LT cDNA was cloned and transformed into E. coli.
-Like protein production became possible (Gray, PW et al. (1984)
Nature, 312, 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, the LT-like protein produced by a microorganism has a sugar chain, whereas a natural LT has a sugar chain. In this way, LT-like protein produced by the protein synthesis system of microorganisms and natural LT are distinctly different from each other as substances, and when used as a therapeutic drug for a long period of time or when used frequently, the antigen-antibody reaction I'm worried about the problem.

(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 method using an animal cultured cell as a host is considered. To be In this case, simply LT
It is presumed that the production of LT cannot be observed even when the gene is introduced into cultured animal cells. That is, LT is an inducible protein, and its expression is restricted at the gene level. Therefore, in order to impart an effective LT-producing ability to animal culture cells by the gene introduction method, 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. The DNA sequence encoding the mature form of the mRNA (mRNA) sequence is called exon, and the split sequence is called an intervening sequence or intron.
The biological significance and function of introns are currently unknown, but ovalbumin (Wickens, MP et al. (1980) Nature, 285, 628) and viral proteins (Lai, C-J. Et al. (197).
9 years) Proceedings of the National Academy of Sciences USA (Proc.N
atl.Acad.Sci.USA), 76, p. 71) is known to produce extremely less protein in the introduced animal cells than the intron-containing gene sequence. . In addition, by adding the intron of the β-globin gene to the SV40 gene lacking the intron,
It was known that stable mRNA accumulation occurs (Hamer, DHR
(1979) Cell, 18: 1299).

The removal of the sequence of the intron portion from the initial RNA transcribed from the gene is called splicing.
Splicing is presumed to be a necessary event for stable mRNA accumulation or mRNA translocation from the nucleus to the cytoplasm.

Splicing at the correct position of the intron is essential for the expression of a normal and functional protein, but it is abnormal when the insulin gene and the simian virus 40 (SV40) promoter region are linked and introduced into COS cells. Splicing has been reported (Laub, O. et al. (1983
Year) The Journal of Biological Chemistry, 258, 6043). Further, in the expression of amylase, there is tissue-specific splicing, and it is known that salivary amylase and pancreatic amylase are synthesized from the same gene through two different splicings (Young, RA ( 1981) Cell, 23, 451). In addition, SV40 (Berk, AJ et al. (1978) Proceedings of the National Academy of Science USA, Vol. 75, p. 1274), Adenovirus (Ch
In ow, LT (1977) cell, vol. 12, p. 1), 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 animal culture cells and produce LT, normal splicing needs to occur.
When the LT-encoding chromosomal DNA sequence functions in animal cultured cells, that is, a DNA sequence of a promoter region capable of initiating mRNA synthesis is bound and introduced into various animal cultured cells, splicing is normally performed. Occurring, it was found that LT was secreted into the medium in a significant amount.

LT is a glycoprotein. Currently, there are many unclear points about the structure of LT sugar chains, and it is not clear whether there is a difference in the structure and antigenicity of LT sugar chains formed in cells other than human and LT derived from human cells. . But made with human-derived cells
LT is believed to be very similar to natural LT,
It is considered to be safer than LT made of non-human cells. 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 reactions in long-term administration as therapeutic agents, Although problems such as shocks are expected, the products made from human-derived cells contain essentially no human components, that is, substances that are present in human blood, improving the safety of the product. There is expected.

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.

(Means for Solving 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. are doing. LT
The 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, BamHI4 contained in plasmid pLTB4.2 shown in FIG. An array of 2 Kb (kilobase).

The chromosomal DNA sequence encoding LT is cloned from human DNA. For human DNA, for example, human white blood cell culture cells or tissues are used, and the method of Blin et al.
976) Nucleic Aids Research (Nucleic A
cids Res.), 3, 2303). Vectors used for cloning the LT gene include a λ-fage vector typified by Charon28, a plasmid vector typified by pBR322, and a cosmid typified by pHC79, etc. A genetic engineering method is used in which a λ phage capable of cloning a fragment is used as a vector. 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-fuged DNA to form a recombinant-forged DNA.
Next, an infectious forage particle is prepared by using an in vitro packaging method. Then, it is plated with host E. coli to form plaques of recombinant phage (Enquist, L. et al. (1979) Methods in Enzymology, 68, 281; Hor.
n, B. (1979) Methods in Enzymology, 68
Vol., P. 299). For detection of plaques in recombinant forages containing LT-encoding DNA fragments, plaque-high redidation using cDNA or synthetic DNA as a probe (Woo, S.
LC (1979) Methods in Enzymology, 68
Volume, 389; Szostak, JW et al. (1979) Methods in.
Enzymology, Vol. 68, p. 419) is available. Also LT
Recombinant phage having the gene can be prepared in a large amount by recovering from the plaque selected by plaque hybridization and culturing with the host Escherichia coli. In addition, the DNA of recombinant phage can be prepared by the phenol method or the like (Maniatis, T. et al. (1982) Molecular Clo
ning a Laboratory manual, Cold Spring Harbor Labora
tory).

As a method of introducing DNA into animal culture cells, although there is a difference in the transfer efficiency, the calcium phosphate method (Wigl
er, M. et al. (1977) Cell, Vol. 11, p. 223), Micro-injection method (Anderson, WF et al. (1980) Proceedings of the National Academy of
Science USA, Vol. 77, p. 5399), liposome method, DEAE-dextran method or cell fusion method (Schoffne
r, W. et al. (1980) Proceedings of the National Academy of Sciences USA, 77, 2163). As the DNA material used in the calcium phosphate method, microorganisms such as Escherichia coli, and phage can be used in addition to the DNA solution. In the cell fusion method, microbial brotoplasts having a target DNA sequence as a plasmid are used.

LT is an inducible protein and is induced by stimulating human white blood cells with various mitogens and the like. At present, it is unclear how the stimulation of mitogens acts on the LT gene and induces LT, but even if a gene containing the LT regulatory site is introduced into cells without such induction mechanism,
LT production will be weak. The present inventors connected a sequence of the promoter region of another gene that functions in an animal cultured cell to the 5 ′ side of the chromosomal DNA sequence of LT and introduced it into the cell to transform a non-LT producing cell into a high producing cell. It has been found that can be transformed into. 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 constantly synthesized in the cell and The cell then becomes a constitutive producer of LT. If the promoter region to be connected is of the inducible protein, the transformed cell produces the LT inducible protein.

The SV40 early gene promoter is known as a promoter that functions in animal cultured cells. This promoter is contained in the Hind III-Pvu II fragment of SV40 DNA, an approximately 350 base pair (bp) DNA fragment. Also this DNA
The fragment reversely has the activity as a promoter of the late gene of SV40. Transcriptional activity from the SV40 late promoter 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 prepare cells expressing T antigen, T
A gene encoding an antigen may be introduced into cells.
When a cultured cell is transformed with a DNA sequence in which the LT gene sequence connecting the SV40 T antigen-encoding DNA sequence and the SV40 late promoter sequence is present on the same sequence,
A variety of cell lines will yield high LT expressing strains.

The herpes simplex virus (HSV) type I thymidine kinase promoter is also a constitutive promoter similar to the SV40 early gene promoter, and the structure of the region has been shown by Wagner et al. (Wagner, MJ
Et al. (1981) Proceedings of the National Academy of Sciences USA, 79
Vol., 1441). A functional promoter region refers to a sequence of a promoter region that includes the initiation sites for mRNA synthesis but does not include the codon for methionine, which is the first amino acid of the proteins regulated by those promoters. DNA in which the sequence of a functional promoter region and the LT chromosomal DNA sequence are connected
The construction of the sequence (LT expression vector) is shown in Example 3.

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 Maxa Moog Gilbert method (Maxam, AM et al. (1980) Metz Tunes in Enzymology, 65, 499 pages) or Sanger's dideoxy method (Sanger, F. (1981) Science (Science) , 214, p. 1205), etc.

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 selectively propagate only the stably expressing cells by introducing the target gene, a DNA sequence having a sequence in which the functional promoter sequence and the LT gene are connected and a selectable marker gene on the same DNA sequence is suitable. . Ecogpt as a selectable marker gene in animal cells
(Mulligan, RC et al. (1980) Science, Volume 209, 1422
P.), Neo (Southern, PJ et al. (1982) Journal of Morel-Killer and Applied Geneetics (J.MOl.Appl.Genet.), Vol. 1, 327), dhfr (Wigle
r, M. et al. (1980) Proceedings of the National Academy of Sciences USA, 77, p. 3567). Moreover, in order to prepare such a DNA sequence in large quantities, such D
It is desirable that the NA sequence is a plasmid or phage that can be replicated in E. coli and can be prepared in large quantities. The LT expression vectors shown in Examples 3 to 5 are plasmids that serve the above purposes. That is, DNA that enables replication in E. coli
The replication origin (ori), 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 present on the same DNA sequence. It is a plasmid characterized by that.

LT connecting promoter regions of other functional genes
To produce LT in cells into which the chromosomal DNA sequence of is introduced, the DNA sequence is compatible with the cell-specific RNA synthesis system, RNA maturation, protein synthesis system, protein maturation, secretion, etc. Need to be MRNA is synthesized from the introduced DNA, but less than 5'of mRNA requires the addition of a cap structure, splicing at the normal position, and polyadenylation to the 3'end. In addition, for the expression of active LT,
The formation and maintenance of the normal higher-order structure of the synthesized LT peptide, the cleavage of the signal peptide, and the secretion from the cell must be accurately performed. The animal cultured cells used by the present inventors are hamster, monkey, and human-derived cells available from American Type Culture Collection (ATCC). If used, at least vertebrate-derived cultured cells,
It is possible to produce active LT in fused cells, normal and mutant cells, virus transformed cells and the like. In addition, using cell lines transformed from human cells with SV40 as production cells improves the safety of the product by taking appropriate measures compared to 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.

LT chromosomal DNA sequence connected to a functional promoter, for example, introduced into animal culture cells by the calcium phosphate method, cells that are now capable of producing LT, not only the medium containing serum that is usually used for cell culture, 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.

(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. Further, detailed operations for handling the phages, plasmids, DNA, various enzymes, Escherichia coli, etc. in the examples are referred to the following magazines and books.

1. Protein Nucleic Acid Enzymes, Vol. 26, No. 4, (1981) Extra edition Gene manipulation (Kyoritsu Shuppan) 2. Gene manipulation experiment method, edited by Yasutaka Takagi (1980) Kodansha 3. Manipulated by gene manipulation, Yasutaka Takagi (1982) Year) Kodansha 4.Molecular Cloning a laboratory manual, T.Maniatis
Et al. (1982) Cold Spring Harbor Laboratory 5.Methods in Enzymology, Volume 65, L. Grossman et al. (1980)
Year) Academic Press 6.Methods in Enzymology, Volume 68, edited by R. Wu (1979) Academic
mic Press 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 solution (PBS) (manufactured by Flow Laboratories), and then an upper layer was added to FICOAL PAK (manufactured by Pharmacia). Centrifuge at 2000 rpm for 30 minutes to separate the white blood cell layer, and then add PBS.
It was washed twice with. 20 ml of 0.5 M EDT for 10 ⁸ cells
A-0.5% sarcosyl solution was added, and 2 mg of protease K was added.
, And incubated at 50 ° C. for 3 hours. The phenol layer was extracted twice, and the aqueous layer was 50 mM Tris-10 mM EDTA-10 mM.
It was dialyzed against sodium chloride (pH 8.0) overnight. R Nase A to 1
It was added to 00 μg / ml and treated at 37 ° C for 3 hours, followed by phenol extraction twice, and the aqueous layer was 50 mM Tris-10 mM EDTA.
It was dialyzed to obtain high molecular human DNA. Human DNA is a restriction enzyme Sa
After partial digestion with u3A I, sucrose density gradient centrifugation performed about 15-20
A kilobase-sized Sau3A I DNA fragment was prepared. After cutting the lambda phage vector Charon 28 DNA with BamHI,
Fractions containing the left end fragment and the right end fragment of Charon 28 were collected by sucrose density gradient centrifugation and collected by ethanol precipitation.

DNA fragments at both ends of Charon 28 and human 15-20 kilobase Sau
After ligating the 3A I fragment with T ₄ DNA ligase, in vitro packaging was performed by the method of Enquist and Stanberg (L. Enquist and N. Sternberg (1979) Methods in Enzymology, 68, 281). , Escherichia coli LE392 was used as a host to form plaques of recombinant phage. Next, plaque hybridization method (Benton, W.
D. , Davis, RW (1977) Science, 196, 180), to select a recombinant phage clone having the LT gene. As a probe, oligonucleotides ATGACACCACCTGAACGT, TCTACTCCCAG having sequences present in the LT gene are used.
GTGGTC and ACTGTCTTCTTTGGAGCC (these sequences correspond to the amino acid sequences -34 to -29, 75 to 80 and 163 to 168 of LT: Gray, PW et al. (1984) Nature, 312, 7
Page 21) by the phosphotriester method (Miyoshi, K. et al. (198
0) Nucleic Acids Research, vol. 8, p. 5507), and 5'-OH was labeled with [γ- ³² P] ATP and T4 polynucleotide kinase and used.

Eleven strains of forage clones which hybridize with all three kinds of synthetic DNA probes used from about 600,000 recombinant forage clones were obtained. One of the phage clones 4-1 was digested with various restriction enzymes, subjected to agarose electrophoresis, transferred to a nitrocellulose filter, and then subjected to Southern hybridization (Southern hybridization) using three kinds of synthetic DNA probes. , EM (1975) Journal of Morel Biology (J.Mol.Bio
l.) 98, 503), BamH I 4.2Kb, EcoR
The I 2.3 Kb and Sma I 2.7 Kb fragments hybridized with the three kinds of probes, and it was revealed that the DNA sequence encoding the amino acid sequence of LT was contained in these fragments. Also, the DNA of several phage clones
As a result of restriction enzyme analysis, the LT chromosomal DNA sequence and the restriction enzyme recognition sites of adjacent sequences were mapped as shown in FIG.

Example 2 LT gene subcloning The DNA of phage clone 4-1 was cleaved with the restriction enzyme BamHI, and the resulting 4.2 Kb fragment was transformed into plasmid pUC9 (Vieira, J.
And Messing, J (1982) Gene, 19: 259.
It was inserted into the BamHI site to construct pLTB4.2 (Fig. 2).

Commercially available pUC9 primer CAGGAAACAGCTATGAC,
Amino acid of AGTCACGACGTTGTA (above Takara Shuzo) and LT-2
Oligomers corresponding to 3 to -28, 75 to 80, 163 to 168 ACCCTTGGGAGGAAGAG, TCTACTCCCAGGTGGTC, ACTGTCTTCTTT
Determination of nucleotide sequences by the dideoxy method using GGAGCC as a primer (Wallace, RB et al. (1981) Gene, 16:21
Page) for pLTB4.2. As a result, the 5'side of LT is And the 3'side has It was found to have the sequence of

Example 3 Construction of pSVeSma I LT, pSVpTKLT, pSV2LLT and pSV3LLT Sequence of SV40 early gene promoter region and LT chromosome
PSVeS, a plasmid having a sequence connected to the DNA sequence
ma I LT is pLTB4.2, pSV2gpt and pSV3gpt (Mulligan, RC
And Berg, P. (1980) Science, 209, 1422) as a starting material, and were produced by the procedure shown in FIGS. 3 (a) and 3 (b).

That is, pSV3gpt was cut with Hind III and the largest DNA
The fragment was circularized with T4 DNA ligase to generate pH I. Then p
The Pvu II site of HI was changed to a Sal I site using a Sal I linker to prepare pH II. In addition, the Hind III site of pH II
Introduce Sma I site using nd III-Sma I adapter,
pHSma I was prepared. pHSma I was cleaved with Sal I and EcoR I to obtain pSV2
After cleaving the BamHI site of gpt with BamHI, DNA polymerase I
The pS I prepared by blunting with (Klenow) and circularizing with T4 DNA ligase was also cleaved with Sal I and EcoR I, and ligated with a DNA fragment containing the ampicillin resistance gene using T4 DNA ligase.
I made pSVeSma I. Then pLTB4.2 is cut with Sma I and L
A DNA fragment containing the T gene sequence was obtained and p
It was introduced into SVeSma I to prepare pSVeSma I LT.

The Sal I linker and Sma I adapter used had d (dGGTCGACC) and d (pAGCTCCCGGG) sequences, respectively. As the DNA polymerase I, Klenow fragment was used.

PSVpTKLT, a plasmid having a sequence in which the LT gene is connected to the promoter region sequence of herpes simplex virus type 1 thymidine kinase, is pLTB4.2, pH.
SV106 (McKnight, SL and Gabis, ER (1980) Nucleic Assists Research, 8: 5931) and pSVeSma I
Was used as a starting material and was manufactured by the method shown in FIG.
That is, the LT gene BamHI-SamI fragment contained in pLTB4.2 was inserted into the BglII-SmaI site of pHSV106 to prepare pHSVLT. Next, from pHSVLT, LT gene Ba with TK promoter was added.
The mHI-SmaI fragment was introduced into the BamHI-SmaI site of pSVeSmaI to prepare pSVpTKLT.

In addition, pSV2LLT and pSV3LLT, which are plasmids having a sequence of the LT gene connected to the late gene promoter region of SV40, were prepared by using pLTB4.2, pSV2gpt and pSV3gpt as starting materials and by the method shown in FIGS. 5 and 6. It was made. That is, the LT gene SmaI-SmaI2.5Kb fragment contained in pLTB4.2 was ligated to the PvuII site of pSV2gpt to prepare pSV2LLT. Next, psV2LLT is partially cut with BamHI and pSV3gpt is cut there.
The T-antigen gene BamH I fragment contained in pSV3LLT was prepared.

Example 4 Introduction of pSVeSma I LT, pSVpTKLT, pSV2LLT and pSV3LLT into cultured cells and production of LT In order to examine expression of LT gene contained in the expression vectors pSVeSma I LT, pSVpTKLT, pSV2LLT and pSV3LLT, various animal cultures were conducted. Method of Wigler et al. (1977
(Y.) Cell, Vol. 11, p. 223). The DNA sequence described in each claim of the present application is the third DNA sequence.
It is the sequence of the BamHI-SmaI fragment shown in Figure (b). Cells (2 × 10 ⁵ cells) in which the pramised-calcium phosphate coprecipitate was previously grown in Eagle MEM medium containing 10% bovine neonatal serum.
Cells / 3ml medium / 6cm diameter culture dish), renew the medium 15 hours later, continue the culture, and LT contained in the medium 48 hours later
Was measured by the cell-killing effect of targeting L929 cells (Ruff, MR and Gifford, GE (1981) Lymphokines,
Volume 2, p. 235). That is, 2 × 10 ⁴ in 96 holes multi-touch.
After culturing in cells / well / 100μ medium for 1 day, remove the culture solution,
Accinomycin D 1 μg / ml Samples diluted with Eagle MEM medium containing 5% fetal bovine serum to various concentrations were 100
μ was added, and the denaturing and lethal effect of cells was measured 20 hours later.
The LT1 unit was the concentration that gave a lethality of 50%. PSVeSma I LT, pSVpTKLT, pSV2LLT or p
Expression of LT was observed in all cultured cells into which SV3LLT was introduced. The expression of pSV3LLT was higher than that of pSV2LLT in all cultured cells.

Example 5 Production of LT in normal medium and serum-free medium BHK-21 (C-13) medium introduced with pSVeSma I LT, pSVpTKLT or pSV3LLT in Example 4 was mixed with 10% fetal bovine serum, 25
μg / ml mycophenolic acid, 250 μg / ml xanthine in M
The medium was replaced with EM medium 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 cultured in a MEM medium containing 5% fetal calf serum (FCS) and a MEM medium containing no fetal calf serum for 24 hours,
The LT activity contained in the medium was measured. As shown in the table below, the isolated cell lines produced LT with or without serum.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a recombinant phage DNA obtained by cloning a chromosomal DNA fragment containing the LT gene, FIG. 2 is a schematic diagram showing pramised pLTB4.2, and FIG. 3- (a) is a plasmid pSVeSmaI prepared. Schematic diagram, Fig. 3- (b) is a schematic diagram of preparation of plasmid pSVeSma I LT, and Fig. 4 is plasmid pS.
Fig. 5 is a schematic diagram of preparation of VpTKLT, Fig. 5 is a schematic diagram of preparation of plasmid pSV2LLT, and Fig. 6 is a schematic diagram of preparation of plasmid pSV3LLT. In FIG. 1, E shows the recognition site of the restriction enzyme EcoR I, and B shows the recognition site of the restriction enzyme BamHI. 2-6, Sma I, Ava I, BamH I, Acc I, Sca I, EcoR
I, Sac I, Hind III, Pvu II, Sal I and Bgl II represent the recognition sites of the respective restriction enzymes. HuLT is human LT gene, pUC9 is a region derived from vector plasmid pUC9, Amp ^γ is ampicillin resistance gene, Ecogpt is guanine phosphoribosyl tranferase gene of E. coli, SVe is SV40 virus early gene promoter region T-ag is SV40 T-antigen gene, pTK is the thymidine kinase promoter region, T
K represents a thymidine kinase gene, and (Pvu II / Sma I) represents a combination of the restriction enzyme Pvu II cleavage site and the Sma I cleavage site.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location C12P 21/02 L 9282-4B (72) Inventor Hajime Kawarada 2183-2, Hiraoka-cho, Kakogawa-shi, Hyogo Prefecture (72) Inventor Kiyoshi Watanabe 41, 5-15, Matsugaoka, Akashi-shi, Hyogo (56) References JP 56-160993 (JP, A) Nature, 312, P. 721-724 (1984) "Genetic recombination practical technology second collection" (March 15, 1957) Science Forum, P. 33-34 Nature, 209, P.I. 1422 ~ 1427 (1980)

Claims (11)

[Claims] 1. On the 5'side And the 3'side has DNA sequence in which the human lymphotoxin chromosomal DNA sequence characterized by the following restriction enzyme cleavage map and the promoter region sequence of a gene having constitutive expression that functions in animal cultured cells are connected. 2. The promoter region is the promoter region of SV40 early gene or thymidine kinase gene of herpes simplex virus (HSV-I).
The DNA sequence described in the item. 3. The DNA according to claim 1, wherein the promoter region is the promoter region of SV40 late gene.
Array. 4. A DNA sequence having the same D sequence as the SV40 T antigen gene sequence.
The DNA sequence according to claim 3, which is present on the NA sequence. 5. A DNA sequence having plasmids pSVeSmalLT and pSVpTKL.
The DNA sequence according to any one of claims 1 to 4, which is a DNA sequence of T, pSV2LLT or pSV3LLT. 6. On the 5'side And the 3'side has And a DNA sequence in which the human lymphotoxin chromosomal DNA sequence characterized by the restriction enzyme cleavage map below and the promoter region sequence of a gene that has a constitutive expression functioning in animal cultured cells are connected. Animal cultured cells transformed with the DNA. 7. The animal cultured cell according to claim 6, wherein the cell is derived from a mammal. 8. The animal culture cell according to claim 6, wherein the cell origin is human. 9. The animal cultured cell according to any one of claims 6 to 8, wherein the cell is a cell transformed with SV40. 10. The cells are CHO-K1, Vero, Wl-26 VA4, BHK-2.
The animal cultured cell according to claim 6, which is 1 (C-13) or L929. 11. On the 5'side And the 3'side has And a DNA sequence in which the human lymphotoxin chromosomal DNA sequence characterized by the following restriction enzyme cleavage map and the promoter region sequence of a gene that has a constitutive expression functioning in animal cultured cells are connected. A microorganism containing a DNA having.