JPH06315392A - Method for producing apolipoprotein - Google Patents

Abstract

PURPOSE:To obtain an apolipoprotein in a high producing amount by culturing a CHO transformed with a specific expression vector containing a gene coding human apolipoprotein. CONSTITUTION:CHO cell is transformed with an expression vector containing a gene coding an eucaryote promotor; a gene coding a human apolipoprotein or human apolipoprotein-like substance of the formula; a splicing sequence DNA having polyadenyl site, 5' splice junction, intron and 3' splice junction. This transformed animal cell is cultured to provide the objective apolipoprotein E or apolipoprotein E-like substance.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing apolipoprotein in animal cells by recombinant DNA technology.

[0002]

2. Description of the Related Art The main lipids present in plasma are
Examples include cholesterol, phospholipids, triglycerides and free fatty acids, of which the former three are present in association with proteins. This lipid insoluble in water is solubilized-
The protein complex is called lipoprotein and forms various weights depending on the ratio of lipid to protein. It is considered that this lipoprotein has a spherical particle-like structure, nonpolar triglyceride and cholesterol ester exist in the core, and polar phospholipids and free cholesterol form a surface layer together with the protein.

The protein contained in this lipoprotein is called apolipoprotein, and 10 or more kinds are currently known. Apolipoprotein is not only required to make lipoproteins, but also plays an important role in the metabolism of lipoproteins. Apolipoprotein E is known as one of the apolipoproteins. It is known that this apolipoprotein E serves as a recognition target when various cells in the body take up lipoprotein via its receptor.

Furthermore, E2, E3, and E4 have been found as major subclasses of this apolipoprotein E. While E3 is derived from a normal person, E2 and E4 are
It is found in patients with type III hyperlipidemia and is considered to be an abnormal type (The Journal of Bi
Logical Chemistry, Vol. 25
5, No. 5,1759-1762,1980).

Those who lack this apolipoprotein E3 or have E2 or E4 often have hyperlipidemia and also arteriosclerosis. In such a case, the normal function of apolipoprotein E can be restored by administering normal E3 to the patient.
Further, apolipoprotein AI is known as one of the apolipoproteins. It is known that this apolipoprotein AI has a function of activating LCAT (lecithin-cholesterol-acyl transferase) which is an enzyme that carries the fatty acid at the β-position of lecithin to free cholesterol and converts it into cholesterol ester. Has been.

When this apolipoprotein AI is abnormal, various lipoprotein metabolism abnormalities are caused, and for example,
Hyperlipidemia often leads to arteriosclerosis. In such a case, the normal function of apolipoprotein AI can be restored by administering normal AI to the patient.

[0007]

It is an object of the present invention to provide a method for efficiently producing apolipoprotein in animal cells by recombinant DNA technology.

[0008]

The gist of the present invention is to provide a eukaryotic promoter,
A gene encoding the human apolipoprotein or human apolipoprotein-like substance shown in SEQ ID NO: 1 in the sequence listing;
Splice sequence D consisting of polyadenylation site and 5'splice site, intron and 3'splice site
There is provided a method for producing apolipoprotein E or an apolipoprotein E-like substance, which comprises culturing CHO cells transformed with an expression vector containing NA.

To explain the present invention in detail, the expression vector of the present invention comprises a eukaryotic promoter; a gene encoding an apolipoprotein or an apolipoprotein-like substance; a polyadenylation site and 5'splice site, intron and 3'splice site. Containing splice sequence DNA. The DNA fragment containing the gene encoding apolipoprotein E can be isolated as follows.

(A) For example, D used in the present invention
NA fragment of human apolipoprotein E3 encoding D
The DNA fragment containing NA is prepared by the following method. Namely, human liver slices, small intestinal epithelial cells,
Homogenize macrophages in blood or kidney sections with guanidine isothiocyanate, and separate total RNA by CsCl equilibrium density gradient ultracentrifugation (Chi
rgwin et al., Biochemistry, 18, 52.
94-5299, 1979). Then, this is purified by oligo (dT) cellulose column chromatography by a conventional method to isolate poly (A) -containing RNA (mRNA
material).

From this mRNA raw material, the method of Okayama and Berg (Molecular and Cellular) was used.
A cDNA library is obtained according to Biology, 2, 161-170, 1982). That is, pBR3
The hybrid plasmid of 22 and SV40 is used to obtain the vector primer and oligo (dG) tail linker. Reverse transcriptase is allowed to act in the presence of this vector primer and the above mRNA to synthesize cDNA, which is then digested with the restriction enzyme Hind III, and then cyclized using the above linker. Then, the mRNA portion is replaced with DNA to obtain a cDNA fragment-containing plasmid.

Then, Escherichia coli and the like are transformed into ampicillin-resistant strains by using the conventional method. Then, as a probe, the apolipoprotein E amino acid sequences 218-222

[0013]

Embedded image Met-Glu-Glu-Met-Gly

A synthetic oligonucleotide containing a part or all of the nucleotide sequence corresponding to or a synthetic oligonucleotide containing at least the nucleotide sequence is used to select a clone containing a sequence complementary thereto. From the clones thus obtained, a clone having a plasmid in which the longest cDNA is inserted is selected by cutting with an appropriate restriction enzyme.

The cDNA fragment obtained from the clone is obtained by the method of Maxam and Gilbert (Method).
in Enzymology, 65, 499-56.
0, 1980) determines the base sequence.

As an example of the DNA fragment used in the present invention, a DNA encoding apolipoprotein E3
The nucleotide sequence of the DNA fragment containing the amino acid sequence and the amino acid sequence deduced therefrom are shown in SEQ ID NO: 1 in the sequence listing.
The NA fragment is not necessarily required to have the same nucleotide sequence as this, and if the substance encoded by the DNA contained in the DNA fragment is an apolipoprotein E-like substance having the same physiological activity as apolipoprotein E. ,
It may be a base sequence in which a part of the base sequence is replaced or deleted, or a base is added by oligonucleotide site-directed mutagenesis, restriction enzyme treatment, use of synthetic DNA fragment, and the like. In addition, abnormal subclass E2 or E
The DNA fragment corresponding to 4 can be obtained by using a liver slice derived from a patient having E2 or E4.

(B) A DNA fragment containing a DNA encoding apolipoprotein AI is prepared by the following method. That is, human liver slices, small intestinal epithelial cells, macrophages in blood, kidney slices and the like are homogenized with guanidine isothiocyanate to obtain Cs.
Isolate total RNA by Cl equilibrium density gradient ultracentrifugation (Chirgwin et al., Biochemistry, 1).
8, 5294-5299, 1979). Then, this is purified by oligo (dT) cellulose column chromatography by a conventional method to isolate poly (A) -containing RNA (mRNA raw material).

From this mRNA raw material, the method of Okayama and Berg (Molecular and Cellular) was used.
A cDNA library is obtained according to Biology, 2, 161-170, 1982). That is, pBR3
The hybrid plasmid of 22 and SV40 is used to obtain the vector primer and oligo (dG) tail linker. Reverse transcriptase is allowed to act in the presence of this vector primer and the above mRNA to synthesize cDNA, which is then digested with the restriction enzyme Hind III, and then cyclized using the above linker. Then, the mRNA portion is replaced with DNA to obtain a cDNA fragment-containing plasmid.

Then, Escherichia coli and the like are transformed into ampicillin-resistant strains by using the conventional method. Then, as a probe, amino acid sequence 108-112 of apolipoprotein AI

[0020]

Embedded image Trp-Gln-Glu-Glu-Met

A clone containing a sequence complementary thereto is selected using the synthetic oligonucleotide corresponding to. From the clones thus obtained, a clone having a plasmid in which the longest cDNA is inserted is selected by cutting with an appropriate restriction enzyme. The cDNA fragment obtained from the clone was obtained by the method of Maxam and Gilbert (Methods in Enzymology,
65, 499-560, 1980), the base sequence is determined.

An example of the DNA fragment used in the present invention is shown in Japanese Patent Application No. 59-216988. However, the DNA fragment used in the present invention is not necessarily required to have the same base sequence, and the DNA fragment DNA included
Is encoded by apolipoprotein A-
Apolipoprotein AI having the same physiological activity as I
As long as it is a substance like the apolipoprotein E-like substance, it may be a base sequence in which a part of the base sequence is substituted or deleted, or a base is added by the same method as described above.

The expression vector of the present invention transcribes the apolipoprotein gene obtained as described above (hereinafter, a gene encoding an apolipoprotein or an apolipoprotein-like substance may be referred to as "apolipoprotein structural gene"). Contains a eukaryotic promoter in a controllable position.

As such a promoter, SV40
Early promoter, SV40 late promoter, apolipoprotein E gene promoter, apolipoprotein A
-I gene promoter, heat shock gene promoter (Proc. Natl. Sci. USA, 78 70
38-7042 (1981), promoter of metallothionein gene (Proc. Natl. Sci. USA,
77 6511-6515 (1980)), HSVTK
Promoter, adenovirus promoter (Ad 2
Major late promoter (Ad 2 MLP promoter), retrovirus LTR (Long Term)
internal repeat) and the like, but SV40
Promoters and promoters of the metallothionein gene are preferred.

The expression vector of the present invention also contains a 5'splice junction d.
onor site, intron, and 3'splice junction access.
splice sequence DNA consisting of ptor site)
(A common nucleotide sequence is found around the exon-intron junction site and the same junction site, and the intron region always starts with 2 bases of GT (donor site) and ends with 2 bases of AG (acceptor site). That is, the so-called GT / AG rule holds).

One or more such splice sequence DNAs may be present in the expression vector of the present invention, and the position thereof may be upstream or downstream of the apolipoprotein structural gene.

[0027] Specific examples of the splice sequence DNA, in exon 2 derived from Bam HI- Pvu II fragment containing exon 2 and exon 3 of rabbit β- globin gene from Bam HI site of the exon 3 Eco RI
DNA fragment up to the site (Science, 26 339)
(1979)), promoters of the metallothionein gene, exons 1, 2 and 3 and introns A and B.
Of the mouse metallothionein-I gene containing
kb DNA fragment (Proc. Natl. Acad. S
ci. USA 77 6513 (see 1980)). Also,
The 5'and 3'splice sites do not have to be derived from the same gene; for example, a sequence in which the 5'splice site contained in adenovirus DNA and the 3'splice site derived from the Ig variable region gene are linked can be used. .

The cloning site for the apolipoprotein structural gene is as follows.
The Bam HI site and the Bgl II site in exon 1 of the mouse metallothionein gene can be used. The expression vector of the present invention further contains a polyadenylation site. The polyadenylation site is located downstream of the apolipoprotein structural gene. Specific examples of the polyadenylation site include those derived from SV40 DNA, β-globin gene or metallothionein gene. Also,
β-globin gene polyadenylation site and SV40
The polyadenylation site of DNA may be linked.

The expression vector of the present invention may have a dominant selectable marker that enables the selection of transformants.
The transformed animal cells of the present invention can be selected by the double transformation method even if there is no selection marker in the expression vector. Examples of such a selection marker include a DHFR gene that confers MTX (methotrexate) resistance, and a transformation t in a HAT medium.
Herpes simplex virus (HSV) tk gene allowing selection of k ^- strains. Aminoglycoside 3'from E. coli transposon Tn5 conferring resistance to 3'-deoxystreptamine antibiotic G418.
-A phosphotransferase gene, a bovine papillomavirus gene that enables morphological distinction by multilayer growth, an aprt gene, and the like.

In order to select animal cells transformed with the expression vector of the present invention by the double transformation method, a plasmid or other DNA containing the above-mentioned gene serving as a selection marker is transformed with the expression vector. The transformed cells can be selected by the above-mentioned phenotypic trait by the expression of the selectable marker after transformation. When the expression vector of the present invention contains a plasmid fragment having a replication origin derived from bacteria such as Escherichia coli, it is advantageous because it can be cloned in bacteria. Such plasmids include pBR322 and pB
Examples include R327 and pML.

Specific examples of the plasmid vector used for the expression vector of the present invention include SV40 early promoter, Bam HI site as a cloning site, and splice sequence DN derived from rabbit β-globin gene.
A, pKCR (Proc. Natl. Aca) containing the polyadenylation site from the rabbit β-globin gene, the polyadenylation site from the SV40 early region and the origin of replication from pBR322 and the ampicillin resistance gene.
d. Sci. USA 78,1528 (1981) refer), was replaced by pBR322 moiety pBR327 portion of pKCR, PKCRH2 introduced with Hind III site at the Eco RI site present in the exon 3 of rabbit β- globin gene (see Nature 307 605) ,
P containing the BPV gene and the metallothionein gene
BPVMT1 (Proc. Natl. Acad. Sc
i. USA 80 398 (1983)) and the like.

The cloning site of the apolipoprotein gene in the above-mentioned plasmid was pKC.
Bam HI site of R, Hin dIII sites of PKCRH2, include Bgl II site in exon 1 of the metallothionein -I gene PBPVMT1. Examples of animal cells transformed with the expression vector of the present invention include CHO cells.

The expression vector of the present invention can be transferred into animal cells by a transfection method, a microinjection method or the like. As a transfection method, Ca-PO ₄ (Virology 52,456-
467 (1973)) is the most common.

Culturing of animal cells transformed by transfer can be carried out in a suspension medium or a fixed medium by a conventional method. As the medium, MEM, RPMI1640, etc. are generally used. The produced protein can be separated by a conventional method.

[0035]

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to the following examples unless it exceeds the gist. In the examples, treatments with restriction enzymes, modification enzymes, etc. were carried out by the manufacturers and sellers of these reagents (Takara Shuzo Co., Ltd., New England).
Biolabs).

Reference Example 1 (Preparation of DNA fragment containing structural gene encoding apolipoprotein E) (1) Human liver slices were crushed with liquid nitrogen, and then guanidine isothiocyanate aqueous solution was added to homogenize.
The resulting homogenate was subjected to the method of Chirgwin et al. (Biochemistry, 18, 5294-529).
9, 1979), total RNA was isolated by cesium chloride equilibrium density gradient ultracentrifugation. Then, this was purified by oligo (dT) cellulose column chromatography by a conventional method to isolate poly (A) -containing RNA, and
A raw material was used.

(2) On the other hand, the method of Okayama and Berg (Mo
regular and Cellular Biol
, 2, 161-170, 1982), pB
A hybrid plasmid of R322 and SV40 was used to obtain the vector primer and oligo (dG) tail linker. That is, 400 µg of a hybrid plasmid of pBR322 and SV40 (map unit 0.71-0.86) was digested with a restriction enzyme Kpn I containing a bovine serum albumin at 37 ° C for 4 hours.

Then, DNA was recovered by ethanol precipitation by a conventional method, dissolved in a buffer containing dTTP, added with terminal deoxynucleotidyl transferase, and reacted at 37 ° C. for 30 minutes to give a restriction enzyme Kpn I. After adding about 60 dT tails to the digested end of DNA, DNA was recovered by ethanol precipitation. Then, this DNA was digested with a restriction enzyme Hpa I in a buffer containing bovine serum albumin (37 ° C., 5 hours). The larger DNA fragment was purified by agarose gel electrophoresis and the glass powder method (Vogelste) was used.
in et al., Proc. Natl. Acad. Sci. U.
S. A. , 76, 615-619, 1979). Then, this DNA was oligo (dA) at 0 ° C.
The solution was applied to a cellulose column, eluted with water, and then recovered with ethanol to obtain a vector primer having an oligo (dT) tail.

On the other hand, 100 μg of a hybrid plasmid of pBR322 and SV40 (map unit 0.19 to 0.32) was digested with a restriction enzyme Pst I in a buffer containing bovine serum albumin (37 ° C., 1 hour and a half).
Then, the DNA is recovered, dissolved in a buffer containing dGTP, added with terminal deoxynucleotidyl transferase, reacted at 37 ° C. for 20 minutes,
15 dG tails were added. The recovered DNA, restriction enzyme Hin in a buffer containing bovine serum albumin dII
It was digested with I (37 ° C., 1 hour), and then subjected to agarose gel (1.8%) electrophoresis to give small oligo (d
G) Tail linker DNA was extracted and collected.

(3) Okayama and Berg's method (Molec
ural and Cellular Biology
2, 161-170, 1982) to obtain a cDNA library. That is, Tris-HCl (pH
8.3), MgCl ₂ , KCl, dithiothreitol,
To an aqueous solution containing dATP, dTTP, dGTP and [ ³² P] dCTP, 30 μg of the mRNA obtained in (1) above and 10 μg of the vector primer obtained in (2) above were added, and the mixture was incubated at 37 ° C. in the presence of reverse transcriptase. The reaction was carried out for 20 minutes to synthesize a plasmid-cDNA: mRNA, which was precipitated with ethanol and recovered in the form of a pellet. The pellets were coated with CoCl ₂ , dithiothreitol, poly (A), [
Dissolve in a buffer containing ³² P] dCTP and terminal deoxynucleotidyl transferase,
The reaction is carried out at 10 ° C for 10 minutes, and 10 to 1 of dCMP is added per end.
Five residues were added. Then, the recovered oligo (d
C) Tail plasmid cDNA: pellets containing the mRNA was dissolved in a buffer containing bovine serum albumin, 37 ° C. with the restriction enzyme Hin dIII, and digested 1 hour by ethanol precipitation, Hin dIII digested oligo (dC) tail cDNA: The mRNA plasmid was recovered. This is the oligo (dG) tail linker DNA obtained in (2) above.
Was dissolved in a buffer solution containing the above, incubated at 65 ° C. for 2 minutes, kept at 42 ° C. for 30 minutes, and cooled to 0 ° C.
Then β-NAD (nicotine adenine dinucleotide)
The presence, in addition to E. coli (E. Coli) DNA ligase and incubated overnight. After that, dATP, dT
TP, dGTP, dCTP, β-NAD, E.I. coli
DNA ligase, E. E. coli DNA polymerase and E. coli . E. coli RNase H was added and the mixture was incubated at 12 ° C. for 1 hour and then at room temperature for 1 hour, then cooled to stop the reaction and obtain the desired cDNA fragment-containing plasmid.

(4) Then, E. coli HB101 was transformed with this plasmid by a conventional method. Then, as a probe, the apolipoprotein E amino acid sequences 218-222

[0042]

Embedded image Four kinds of synthetic oligonucleotides corresponding to Met-Glu-Glu-Met-Gly

[0043]

[Chemical 4]

Using the method of Hanahan et al. (Ge
ne, 10, 63-67, 1980), and a clone containing a sequence complementary thereto was selected. About 5 out of 100,000 transformants
0 clones were selected, 14 of which were treated with several restriction enzymes and 3 of them
It was found that two clones had a common restriction enzyme recognition site. The largest clone among them, pYAE10, was selected, and the base sequences of this clone were selected as Maxam and Gil.
It was determined by the method of Bert.

FIG. 1 shows a restriction enzyme digestion map prepared for determining the nucleotide sequence of the above clone (ATG: translation initiation codon, TGA: translation termination codon). In addition, the base sequence of the above DNA fragment and the amino acid sequence expected therefrom are shown in SEQ ID NO: 1 of the sequence listing (the number of amino acid residues: 31).
7). This DNA fragment corresponds to apolipoprotein E3 derived from a normal person.

Example 1 (Expression of apolipoprotein E in CHO cells) (i) Removal of poly G and poly A portions of apolipoprotein cDNA (FIG. 2) Plasmid pYAE10 obtained in Reference Example 1 was Hin d
A 1.7 kbp DNA fragment containing a DNA encoding apolipoprotein E was obtained by digestion with III and Acc I. Then, this DNA fragment was treated with Bal31 exonuclease, digested with Hin fI, the smooth-ended with T4DNA polymerase, T4DN a Hin dIII linker
A ligase was added in the presence. This was digested with Hin dIII, was introduced into the Hin dIII site of plasmid pBR322, yielding plasmid PBR322HAPE.

(Ii) Reconstruction of expression vector The plasmid pKCRH2 containing the SV40 promoter was transformed into H
It was digested with indIII, and a Hin dIII fragment containing the DNA encoding apolipoprotein E was introduced into the Hin dIII site, which was obtained by digesting the plasmid pBR322HAPE with Hin dIII.
PE was obtained (Fig. 3).

(Iii) CHO (dhfr ⁻ ) cells (Pr
oc. Natl. Acad. Sci. , 77,421
No. 6,1980), double-transformation into the above plasmid pKCRHAPE and plasmid pMTVdhfr (Nature, 29) containing the dhfr gene.
4,228, (1981)) by the calcium phosphate method using a DNA-calcium phosphate coprecipitate.
(Dhfr ⁻ ) cells are transfected.

After culturing for 2 days, α-MEM medium (No. 410-2000, GIBCO, USA) (-Thy
Subculture with midine, -Hypoxanthine). Clones that survived here (dhfr ⁺ )
Was isolated. When the apolipoprotein E productivity of this clone was examined by Western blotting, it was about 10 mg.
It was / L / 1 day.

Comparative Example In (iii) of Example 1, L, C127, COS cells were used instead of CHO cells, and pSV2neo [J. Mol. Appl. Genet.
1,327 (1982)], G418 as a selective medium.
A clone capable of producing human apoE was selected in the same manner except that an α-MEM medium containing 400 μg (GIBCO) was used. The results are shown below.

[0051]

[Table 1] * Value as a transient expression

CHO obtained in Example 1 as described above
It can be seen that the value of 10 mg / L / day of cells is highly efficient as compared with other animal cells.

[0053]

According to the method of the present invention, apolipoprotein protein can be obtained in a high production amount. For example, in the case of apolipoprotein E, the protein can be obtained in a form having a sugar chain.

[0054]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 309 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA Origin organism name: Human sequence TCACAGGCAG GAAG ATG AAG GTT CTG TGG GCT GCG TTG CTG GTC ACA 47 Met Lys Val Leu Trp Ala Ala Leu Leu Val Thr -18 -15 -10 TTC CTG GCA GGA TGC CAG GCC AAG GTG GAG CAA GCG GTG GAG ACA GAG 95 Phe Leu Ala Gly Cys Gln Ala Lys Val Glu Gln Ala Val Glu Thr Glu -5 15 CCG GAG CCC GAG CTG CGC CAG CAG ACC GAG TGG CAG AGC GGC CAG CGC 143 Pro Glu Pro Glu Leu Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg 10 15 20 25 TGG GAA CTG GCA CTG GGT CGC TTT TGG GAT TAC CTG CGC TGG GTG CAG 191 Trp Glu Leu Ala Leu Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln 30 35 40 ACA CTG TCT GAG CAG GTG CAG GAG GAG CTG CTC AGC TCC CAG GTC ACC 239 Thr Leu Ser Glu Gln Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr 45 50 55 CAG GAA CTG AGG GCG CTG ATG GAC GAG ACC ATG AAG GAG TTG AAG GCC 287 Gln Glu Leu Arg Ala Leu Met Asp Glu Thr Me t Lys Glu Leu Lys Ala 60 65 70 TAC AAA TCG GAA CTG GAG GAA CAA CTG ACC CCG GTG GCG GAG GAG ACG 335 Tyr Lys Ser Glu Leu Glu Glu Gln Leu Thr Pro Val Ala Glu Glu Thr 75 80 85 CGG GCA CGG CTG TCC AAG GAG CTG CAG GCG GCG CAG GCC CGG CTG GGC 383 Arg Ala Arg Leu Ser Lys Glu Leu Gln Ala Ala Gln Ala Arg Leu Gly 90 95 100 105 GCG GAC ATG GAG GAC GTG TGC GGC CGC CTG GTG CAG TAC CGC GGC GAG 431 Ala Asp Met Glu Asp Val Cys Gly Arg Leu Val Gln Tyr Arg Gly Glu 110 115 120 GTG CAG GCC ATG CTC GGC CAG AGC ACC GAG GAG CTG CGG GTG CGC CTC 479 Val Gln Ala Met Leu Gly Gln Ser Thr Glu Glu Leu Arg Val Arg Leu 125 130 135 GCC TCC CAC CTG CGC AAG CTG CGT AAG CGG CTC CTC CGC GAT GCC CAT 527 Ala Ser His Leu Arg Lys Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp 140 145 150 GAC CTG CAG AAG CGC CTG GCA GTG TAC CAG GCC GGG GCC CGC GAG GGC 575 Asp Leu Gln Lys Arg Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly 155 160 165 GCC GAG CGC GGC CTC AGC GCC ATC CGC GAG CGC CTG GGG CCC CTG GTG 623 Ala Glu Arg Gly Leu Ser Ala Ile Arg Gl u Arg Leu Gly Pro Leu Val 170 175 180 185 GAA CAG GGC CGC GTG CGG GCC GCC ACT GTG GGC TCC CTG GCC GGC CAG 671 Glu Gln Gly Arg Val Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln 190 195 200 CCG CTA CAG GAG CGG GCC CAG GCC TGG GGC GAG CGG CTG CGC GCG CGG 719 Pro Leu Gln Glu Arg Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala Arg 205 210 215 ATG GAG GAG ATG GGC AGC CGG ACC CGC GAC CGC CTG GAC GAG GTG AAG 767 Met Glu Glu Met Gly Ser Arg Thr Arg Asp Arg Leu Asp Glu Val Lys 220 225 230 GAG CAG GTG GCC GAG GTG CGC GCC AAG CTG GAG GAG CAG GCC CAG CAG 815 Glu Gln Val Ala Glu Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln 235 240 245 ATA CGC CTG CAG GCC GAG GCC TTC CAG GCC CGC CTC AAG AGC TGG TTC 863 Ile Arg Leu Gln Ala Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe 250 255 260 265 GAG CCC CTG GTG GAA GAC ATG CAG CGC CAG TGG GCC GGG CTG GTG GAG 911 Glu Pro Leu Val Glu Asp Met Gln Arg Gln Trp Ala Gly Leu Val Glu 270 275 280 AAG GTG CAG GCT GCC GTG GGC ACC AGC GCC GCC CCT GTG CCC AGC GAC 959 Lys Val Gln Ala Ala V al Gly Thr Ser Ala Ala Pro Val Pro Ser Asp 285 290 295 AAT CAC TGA ACGCCGAAGC CTGCAGCCAT GCGACCCCAC GCCACCCCGT GCCTCCTGCC 1018 Asn His term 300 TCCGCGCAGC CTGCAGCGGG AGACCCTGTC CCCGCCCCAG CCGTCCTCCT GGGGTGGACC 1078 CTAGTTCAAG AAAG

[Brief description of drawings]

FIG. 1 shows a restriction enzyme cleavage map of a clone having a plasmid into which a DNA fragment containing a gene encoding apolipoprotein E has been inserted.

FIG. 2 shows a schematic diagram of the construction steps of plasmid pBR322HAPE.

FIG. 3 shows a schematic diagram of the construction steps of plasmid pKCRHAPE.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12N 15/12 ZNA 15/18 // (C12P 21/02 C12R 1:91) (C12N 5/10 C12R 1:91)

Claims (2)

[Claims] 1. A eukaryotic promoter; a gene encoding a human apolipoprotein or a human apolipoprotein-like substance set forth in SEQ ID NO: 1 in the sequence listing; consisting of a polyadenylation site and a 5'splice site, an intron and a 3'splice site. A method for producing apolipoprotein E or an apolipoprotein E-like substance, which comprises culturing CHO cells transformed with an expression vector containing splice sequence DNA. 2. The production method according to claim 1, which comprises transforming by a double transformation method.