JP2824433B2 - Novel hybrid promoter and foreign gene expression vector incorporating the same - Google Patents

Description

The present invention relates to a novel promoter obtained by fusing a chicken β-actin gene promoter with another gene, and a vector for expressing a foreign gene using the promoter.

BACKGROUND OF THE INVENTION AND PRIOR ART With the advance of genetic recombination technology, the production of useful substances utilizing genetic recombination has been rapidly advancing in recent years.
When a foreign gene is expressed using a gene recombination technique, an appropriate host cell and an expression vector having a corresponding promoter for expressing the foreign gene are used.
Until now, microorganisms such as Escherichia coli and yeast that are easy to handle have been widely studied as expression host cells, but it has been confirmed that such microorganisms have some limitations in the expression of foreign genes. In recent years, expression systems using higher animal cultured cells such as animal cells as expression host cells have been actively studied.

As a known expression system using animal cells as a host, many systems using animal virus gene promoters and animal cell gene promoters have been reported. As an example of the former, SV40 gene promoter, adenovirus major late gene promoter, hepatitis B virus gene promoter and the like are used. Examples of the latter include thymidine kinase (tk) gene promoter, metallothionein gene promoter, interferon gene promoter, immunoglobulin gene promoter and the like.

Among the above promoters, in particular, the SV40 early gene promoter, the SV40 late gene promoter and the adenovirus major late gene promoter are known to have strong promoter activity, but the productivity is not enough to be used for industrial production. However, there is a demand for the development of stronger promoters and high expression vectors.

In such a situation, the present inventors previously constructed a foreign gene expression vector using the chicken β-actin gene promoter, incorporated various foreign genes into it, and examined the effects. It has been found that even when a foreign gene is incorporated, the target gene product can be expressed at a much higher level than a conventional promoter (Japanese Patent Application No. 63-157569).

To date, as an expression system related to the β-actin gene promoter other than chicken, a method for producing a protein using a human β-actin gene promoter has been known (P. GUNNING et al .: Pro. Natl. Acad. Sci.USA, 84,4831
-4835, 1987 and Takeo Kakunaga et al .: JP-A-62-262995.
issue). However, according to these reports, the promoter activity is about 1.7 times as large as that of the SV40 early gene promoter, which is not practically satisfactory.

Recently, there has been a report comparing the strength of the human β-actin gene promoter activity using various host cells (Gene 65, 135-139, 1988). According to this, the human β-actin gene promoter showed significantly stronger activity in mouse-derived cells than the SV40 early gene promoter, but in human-derived cells and monkey-derived cells, the activity was lower than in the SV40 early gene promoter. It shows that.

In contrast, the nitrile β-actin gene promoter has a promoter activity at least 5 to 10 times that of the well-known SV40 early gene promoter, and has a mouse-derived cell (eg, L Cells) as well as hamster-derived cells (such as CHO cells),
It has been previously confirmed by the present inventors that African green monkey-derived cells (such as COS cells) and other animal cells also have a strong promoter activity.

Objectives of the present invention The inventors of the present invention have conducted studies on the improvement of the chicken β-actin gene promoter for the purpose of developing a stronger promoter and an expression vector using the same. By combining with other genes, we succeeded in constructing a novel hybrid promoter having stronger promoter activity. Expression of a foreign gene using this novel hybrid promoter revealed that expression levels several to 10 times higher than those of the original chicken β-actin gene promoter were found, and the present invention was completed. I came to.

That is, an object of the present invention is to provide an unprecedented and powerful novel hybrid promoter capable of highly expressing a foreign gene in an expression system using animal cells, and an expression vector using the same.

Structure and Effect of the Invention The chicken β-actin gene promoter used in the present invention is a gene fragment having the nucleotide sequence shown in FIG. Such a chicken β-actin gene promoter has also been previously cloned by TAKost et al. (Nucleic Acids Research 11, No. 23,828
7-8286, 1983).

The chicken β-actin gene promoter used in the present invention has a high G (guanine) C (cytosine) content as a whole and a TATA box (ANN. RES. BIOCHEM., 50 , 349-3).
83,1981) and CCAAT box (Nucleic Acids Research,
8 , 127-142, 1980).

The DNA region from G at position -909 to G at position -7 shown in Fig. 1 is considered to be a gene region (intron) that is deleted (spliced) after being transcribed into messenger RNA. Can be

The hybrid promoter of the present invention constructed using such a chicken β-actin gene promoter is a hybrid promoter in which a second promoter is incorporated in the intron region of the chicken β-actin gene promoter. As used herein, the second promoter refers to a promoter other than the chicken β-actin gene promoter, and furthermore, a promoter that functions in animal cells. Preferably, a promoter derived from a virus capable of infecting animal cells, particularly preferably the SV40 early promoter, the LTR of Rous sarcoma virus (RSV), and the like are used for the construction of the hybrid promoter. Such a novel hybrid promoter of the present invention has a strong promoter activity even when compared to the original chicken β-actin gene promoter.
Furthermore, it has a surprising promoter activity of several to ten times.

The hybrid promoter as described above can be obtained by incorporating a second promoter into the intron region of the chicken β-actin gene promoter,
At this time, it is preferable that the translation initiation codon (ATG) is incorporated into the nucleotide sequence from the downstream (3 'end side) of the site incorporating the second promoter to the splicing acceptor so as not to exist. A specific position for incorporating the second promoter is most preferably a Mbo II site present in the chicken β-actin gene promoter.

As described above, in the hybrid promoter in which the second promoter has been incorporated into the intron region of the chicken β-actin gene promoter, and in a vector using the same, the 3 ′ end of the chicken β-actin gene promoter is It is preferable to use a gene fragment having at least C (cytosine) at position -5 shown in FIG. Usually, in the properties of the promoter, it is said that removing the sequence up to several tens of base pairs upstream of the original structural gene start codon ATG has little effect on the promoter activity, but it is used in the present invention. In the case of the chicken β-actin gene to be obtained, a region up to C (cytosine) 5 bases upstream of the original initiation codon ATG is left as a promoter region, and this is used for constructing the hybrid promoter of the present invention, whereby the foreign gene of interest can be obtained. The present inventors have confirmed that the expression can be further enhanced.

Further, the present invention removes all genes including the downstream (3 'end) splicing acceptor sequence from the middle of the intron region of the chicken β-actin gene promoter, and includes other splicing acceptor sequences. There is also provided a hybrid promoter characterized by connecting a gene. As the foreign gene having such a splicing acceptor sequence, for example, a gene fragment having a splicing acceptor sequence contained in a rabbit β-globin gene is used. Thus, by converting the splicing acceptor sequence of the chicken β-actin gene promoter to that of another gene, it becomes possible to further increase the activity of the chicken β-actin gene promoter beyond the original promoter activity. .

Thus, when a gene containing another splicing acceptor is fused to the chicken β-actin gene promoter, the downstream β-actin gene promoter gene is removed from the middle of the intron region of the β-actin gene promoter. To which a gene containing another splicing acceptor sequence is fused.
The site is mentioned as the preferred site.

The basic structure of the exogenous gene expression vector of the present invention is characterized in that it has a novel hybrid promoter improved from the above-mentioned chicken β-actin gene, and can incorporate a foreign gene downstream of the hybrid promoter. With appropriate restriction enzyme cleavage sites. It is sufficient that such a restriction enzyme cleavage site has a recognition site by one restriction enzyme, but it is also possible to have a recognition site by two or more restriction enzymes to facilitate incorporation of various foreign genes. Further, in order to efficiently express the foreign gene of interest, the expression vector of the present invention incorporates a polyadenylation sequence so as to be located downstream thereof when the structural gene to be expressed is incorporated. It is preferable to incorporate an appropriate marker gene for cloning.

In addition, such an expression vector of the present invention has a gene derived from an Escherichia coli plasmid to facilitate cloning in Escherichia coli. Such E. coli plasmid-derived genes include ori for replication in E. coli,
Suitable genes that can serve as selection markers during cloning, such as ampicillin, include drug resistance genes against tetracycline and the like.Also, as such a gene, a gene derived from plasmid pBR322 is often used. pBR322 replication origin (ori)
It is preferable to remove toxic sequences (Nature, 293 , p79-81, 1981) that are close to and inhibit replication in host cells.

In addition, as a host cell that expresses the foreign gene of interest
When cells that produce SV40 large T antigen, for example, COS (African green monkey kidney-derived) cells are used, the expression vector described above may be further enhanced by incorporating a replication origin (eg, SV40 ori) that functions in animal cells. It is possible to express foreign genes well.

Furthermore, in order to increase the expression efficiency of the foreign gene, the dihydrofolate reductase (DHFR) gene can be incorporated into the expression vector of the present invention, or it can be transformed together with the DHFR gene expression plasmid during the transformation. You can also. In this case, it is desirable to use a DHFR gene-deficient cell as the host cell, and by adding methotrexate to the medium, the gene in the transformant can be amplified to obtain a higher expression level. Such a technique for increasing the expression efficiency using the DHFR gene is a known technique, but if it is applied to the expression vector of the present invention, the expression efficiency can be dramatically improved.

Because β-actin is present in various animal cells,
This expression system utilizing the β-actin gene promoter is not only highly efficient, but also has a wide host range, and thus can be said to be an expression system with extremely high industrial applicability.

The expression vector for animal cells of the present invention is an expression vector for animal cells capable of extremely high expression never before,
It is intended to provide a novel exogenous gene expression system which can be sufficiently used in industrial-level production.

Hereinafter, as an example showing that the expression vector using the hybrid promoter of the present invention is actually very useful, the following preparations were carried out using the β-galactosidase gene of Escherichia coli and the hepatitis B virus surface antigen (HBsAg) gene. The present invention will be described in more detail with reference to Examples. The preparation examples show the construction of an expression vector using the original chicken β-actin gene promoter for comparative experiments, and the examples show the construction of an expression vector using the hybrid promoter of the present invention and examples of its application.

Various operations for handling phages, plasmids, DNA, various enzymes, Escherichia coli, cultured cells, and the like in the examples were performed with reference to the following magazines and books.

1. Gene manipulation experiment method, Yasutaka Takagi, edited (1980), Kodansha 2. Gene manipulation manual, Yasutaka Takagi, edited (1982),
Kodansha 3. MOLECULAR CUONING A LABORATORY MANUAL.TM
ANIATIS et al. (1982), COLD SPRING HARBOR LABOR
ATORY. 4. METHODS IN ENZYMOLOGY, Volume 65, L. GROSSMAN, et al. (1980), ACADEMIC PRESS The following abbreviations were used in the examples.

CAT: chloramphenicol acetyl transferase lacZ: β-galactosidase (β-gal) structural gene Preparation Example (1) Preparation of chicken β-actin gene promoter region Exon 1 and 1 of chicken β-actin gene
Plasmid pAZ1037 [Natur containing the intron and part of the second exon followed by the CAT gene fused thereto
a, 314 , pp. 286-289 (1985)] and the restriction enzyme Nco I (NEB # 19).
NcoI digested in 3) recognizes the sequence of 6 base pairs shown in FIG. 2, and its cut surface is a sequence with a protruding 5 ′ end as shown in FIG. In the present invention, when used as an expression vector, the following operation was performed for the purpose of correctly functioning the splice site between the first intron and the second exon of the chicken β-actin gene. The digested DNA was treated with a modifying enzyme S1 nuclease (Takara # 2410A) to remove only the 5 'overhanging site and its adjacent one base pair. The reaction is 30
mM sodium acetate, pH 4.6, 100 mM NaCl, 1 mM ZnSO ₄ solution 80
In μ, 10 μg of the sample DNA was treated with 150 units of S1 nuclease at 37 ° C. for 1 to 4 minutes.

After treating the DNA with S1 nuclease in this way,
The single-stranded DNA portion is modified by the action of a modifying enzyme T4 DNA polymerase (Takara # 2040A), and the 5′-terminal phosphorylated synthetic DNA pCCAAGCTTGG (pHind III linker, NEB # 1)
050) together with T4 DNA ligase (Takara # 2011A). Escherichia coli HB101 strain was transformed with the obtained DNA solution, and a single colony was separated. The plasmid DNA in the transformant was recovered, and the restriction enzyme Hind III (NEB #
104) and digested with Nar I (NEB # 191), and subjected to 6% acrylamide gel electrophoresis. First, a clone in which a DNA fragment of an appropriate size was detected was selected, and the DNA fragment was transferred to a phage vector M13mp19 (NEB # 400). −19) Sal I−Kpn I
The clone was cloned into the site, the nucleotide sequence near the Hind III site was determined by the dideoxy method (PRO NAS, 74 , 5463-5467, 1977), and the target clone was screened.

The plasmid clone p28 thus obtained is
As shown in FIG. 4, the nucleotide sequence from the splicing site of the original chicken β-actin gene to the structural gene was retained, and the gene 3 ′ from the start codon ATG was removed, and a new Hind III site was newly added. Has been introduced.

(2) Construction of Expression Vector pAc-2 Incorporating Chicken β-Actin Gene Promoter Plasmid pSV2-cat [Mol.Cel containing a splicing site for SV40 early transcription and a polyadenylation signal
l. Biology, 2, p1044-1051 (1982)]
(Takara # 1070A), and after repairing the ends with T4 DNA polymerase, a phosphorylated HindIII linker was added to T4D.
It was bound by NA ligase. Add this to Hind III
And BamHI (NEB # 136), and 6% acrylamide gel electrophoresis was performed to extract a HindIII-BamHI fragment of about 900 bp. Next, p28 constructed in (1) was replaced with the restriction enzyme Hind.
It was digested with III and BamHI and dephosphorylated by the action of calf small intestine alkaline phosphatase (Takara # 2250A). This and the aforementioned 900 bp HindIII-BamHI fragment were ligated and circularized with T4 DNA ligase, and the plasmid pAc-
2 (see FIG. 6) was obtained.

(3) β-galactosidase expression plasmid pAc-lacZ
Construction of plasmid pCH110 [Pharmacia # 27-4508] containing the entire region of the lacZ gene encoding β-galactosidase.
-01] was digested with restriction enzymes Hind III and BamHI, and about 3.8 kbp of Hind III-BamH was analyzed by 1% agarose gel electrophoresis.
I fragment was prepared. This fragment also contains a splicing site and a polyadenylation site of the SV40 early gene transcript.

The plasmid p28 constructed in the above (1) was replaced with the restriction enzyme Hin.
It was digested with dIII and BamHI and dephosphorylated using calf intestinal alkaline phosphatase. The plasmid pAc-lacZ was constructed by adding the approximately 3.8 kb Hind III-BamHI fragment obtained by the above-mentioned treatment and ligating it with T4 DNA ligase.

Example 1: chicken β-actin gene promoter and SV
Preparation of Expression Vectors: pAS-lacZ and pAS-2 Using Hybrid Promoter with 40 Early Promoters The plasmid pSV2-cat used in Preparation Example (2) was digested with restriction enzymes Pvu II (Takara # 1076A) and Hind III. ,
An approximately 340 bp Pvu II-Hind III fragment containing the SV40 early promoter was prepared by 6% acrylic gel electrophoresis. The end of this Pvu II-Hind III fragment was T4
The DNA polymerase repaired and phosphorylated Xba I linker was attached by T4 DNA ligase. Restriction enzyme Xba
After digestion with I (Takara # 1093A), an approximately 350 bp XbaI-XbaI fragment was prepared by 6% acrylic gel electrophoresis. Next, plasmid pSAc-2 (Japanese Patent Application No. 63-1575)
No. 69) was digested with restriction enzymes Xba I and Pvu II, and an Xba I-Pvu I fragment of about 1.6 kbp was prepared by 1% agarose gel electrophoresis. This is called restriction enzyme Mbo II (NEB # 1)
After digestion at 48) and repairing the ends with T4 DNA polymerase, the phosphorylated XbaI linker was ligated with T4 DNA ligase. Then, restriction enzymes Xba I and Hind II
Digest with I and perform 1% agarose gel electrophoresis.
A kbp Xba I-Hind III fragment was prepared.

Next, the plasmid pAc-lacZ constructed in Preparation Example (3) was used.
Was digested with restriction enzymes XbaI and HindIII, and an approximately 5.8 kbp XbaI-HindIII fragment was prepared by 1% agarose gel electrophoresis.
The ndIII fragment was mixed, digested with the restriction enzyme XbaI, ligated and circularized with T4 DNA ligase, and the plasmid pA
c-lacZ-XbaI was constructed.

The plasmid pAc-lacZ-XbaI was digested with the restriction enzyme XbaI, mixed with the above-mentioned approximately 350 bp XbaI-XbaI fragment, and ligated with T4 DNA ligase to give a plasmid.
pAS-lacZ was constructed.

Next, the plasmid pAc-2 suitable in Preparation Example (2) was digested with restriction enzymes HindIII and BamHI, and a HindIII-BamHI fragment of about 900 bp was extracted by 1% agarose gel electrophoresis. It contains the splicing site of the SV40 early promoter and a polyadenylation signal.

The plasmid pAS-lacZ was then replaced with the restriction enzyme Hind II.
After digestion with I and BamHI, a HindIII-BamHI fragment of about 3.7 kbp was prepared by 1% agarose gel electrophoresis. This about 3.7 kbp Hind III-BamHI fragment and the above about 900 bp Hind III-BamHI fragment were mixed and ligated using T4 DNA ligase to give plasmid pAS
-2 (FIG. 7) was constructed.

Example 2: chicken β-actin gene promoter and RS
Expression plasmid using hybrid promoter with V-LTR promoter; Preparation of pAR-lacZ and pAR-2 Plasmid pRSV-cat [Pro. Natl. Aca
d. Sci. USA, 79 , 6777-6678 (1982)], and the restriction enzyme Nru I
(NEB # 192) and Taq I (NEB # 149), and a Nru I-Taq I fragment of about 340 bP was prepared by 6% acrylic gel electrophoresis.

The end of this Nru I-Taq I fragment was repaired with T4 DNA polymerase, and a phosphorylated Xba I linker was added to T4 DNA.
It was bound by ligase.

After digestion with the restriction enzyme XbaI, an approximately 350 bP XbaI-XbaI fragment was prepared by 6% acrylic gel electrophoresis.

Next, the plasmid pAc-lacZ-XbaI constructed in Example 1 was used.
Was digested with the restriction enzyme XbaI, and the above-mentioned about 350 bp XbaI-XbaI
The plasmid pAR-lacZ was constructed by mixing with the fragment and ligating with T4 DNA ligase.

Next, plasmid pAR-lacZ was replaced with restriction enzymes HindIII and Bam.
Digest with HI and run 1% agarose gel electrophoresis.
A 3.7 kbp Hind III-BamHI fragment was prepared.

This approximately 3.7 kbp Hind III-BamHI fragment and the approximately 900 bp Hind III-BamHI fragment prepared in Example 1 were mixed and ligated using T4 DNA ligase to construct plasmid pAR-2 (FIG. 8). .

Example 3 Preparation of Expression Plasmids Using a Hybrid Promoter of Chicken β-Actin Gene Promoter and Rabbit β-Globin Gene: pAG-lacZ and pAG-2 Exon 3 from the middle of the second exon of rabbit β-globin gene Plasmid pKCR [Pro.Na
USA, 78 , 1527-1531 (1981)] (Fig. 9).
Was digested with the restriction enzyme Apa I (NEB # Apa I),
After repair with DNA polymerase, the phosphorylated Xba I linker was ligated with T4 DNA ligase. After this, digest with restriction enzyme EcoR I and DNA polymerase I Large Fragm
The end was modified with ent (NEB # 210). A pHind III linker (NEB # 1022) was ligated using T4 DNA ligase, digested with restriction enzymes XbaI and HindIII, and an approximately 90 bp XbaI-HindIII fragment was prepared by 6% acrylic gel electrophoresis. . This contains the splicing acceptor site of the second intron of the rabbit β-globin gene.

Next, the plasmid pAc-lacZ-Xba constructed in Example 1 was used.
I was digested with restriction enzymes XbaI and HindIII, and about 7 kbp of XbaI-HindIII was digested by 1% agarose gel electrophoresis.
Fragments were prepared. In addition to this, about 90 bp Xba I-
Hind III fragments were mixed and ligated with T4 DNA ligase to construct plasmid pAG-lacZ.

This plasmid pAG-lacZ was replaced with restriction enzymes Hind III and B
digested with amHI and electrophoresed on 1% agarose gel.
An approximately 3.3 kbp Hind III-BamHI fragment was prepared.
This about 3.3 kbp Hind III-BamHI fragment and the about 900 bp Hind III-BamHI fragment prepared in Example 1 were mixed and ligated using T4 DNA ligase to construct plasmid pAG-2 (FIG. 10). did.

Example 4: Expression plasmid pAcS-lacZ with SV40 ori,
pAcS-2, pARS-lacZ, pARS-2, pAGS-lacZ and pAGS
The plasmid pSV2-cat used in the preparation (2) was digested with restriction enzymes Hpa I (NEB # 105) and Hind III,
After repair with 4 DNA polymerase, it was ligated and circularized with T4 DNA ligase to prepare plasmid pS-1 (FIG. 11).

This plasmid pS-1 was digested with the restriction enzyme SphI, the ends were repaired with T4 DNA polymerase, and
After adding the mHI linker (NEB # 1021), the plasmid was ligated with T4 DNA ligase and ligated to form a plasmid pS-2 (FIG. 12).
Was prepared.

This plasmid pS-2 was digested with the restriction enzyme BamHI,
% Acryl gel electrophoresis, about 350 bp BamH I-BamH
I fragment was obtained. Among them, SV40ori and SV40
Contains the polyadenylation signal of early transcription.

Next, the plasmid pAc-lacZ constructed in Preparation Example (3) was used.
Was digested with the restriction enzyme BamHI, and the above approximately 350 bp BamHI-BamHI was digested.
The I fragment was added and ligated to circularize with T4 DNA ligase to construct plasmid pAcS-lacZ.

Further, the plasmid pAc-2 constructed in Preparation Example (2) was digested with the restriction enzyme BamHI, the above-mentioned approximately 350 bp BamHI-BamHI fragment was added, and the resultant was ligated and circularized with T4 DNA ligase to give the plasmid pAcS -2 (FIG. 13) was constructed.

Further, the plasmid pAR-lasZ constructed in Example 2 was digested with a restriction enzyme BamHI, and the above-mentioned about 350 bp BamHI-BamHI
The fragment was added and ligated circularized with T4 DNA ligase to construct plasmid pARS-lacZ.

Similarly, plasmid pAR-2 constructed in Example 2 was used.
From plasmid pARS-2 (FIG. 14), and from plasmid pAG-lacZ constructed in Example 3,
S-lacZ was constructed, and plasmid pAG- constructed in Example 3 was constructed.
From plasmid 2, plasmid pAGS-2 (FIG. 15) was constructed.

Example 5: Production of β-galactosidase by an expression plasmid using an improved chicken β-actin gene promoter In a 100 mm circular dish, 1 × 10 10 COS-cells or L-cells
⁶ cells / dish were seeded, and various plasmids (pCH110, pAS-lacZ, pAR-lacZ,
pAG-lacZ, pAcS-lacZ, pARs-lacZ, pAGs-lacZ) DNA
Each 20 μg was introduced into these host cells.

After the calcium phosphate-DNA gel was brought into contact with the cells for 24 hours, the cells were further cultured in 10% FCS-DME for 24 hours. After detaching the cells from the dish by trypsin treatment and pelleting by low-speed centrifugation, FT buffer (250 mM sucrose, 10 mM
(ris.HCl, pH 7.5, 10 mM EDTA) 200 μl, and freeze-thaw was repeated three times, followed by centrifugation, and the supernatant was recovered.

From the cell extract 200μ thus obtained to 10μ
Was used for measurement of β-gal activity. β-gal activity can be determined by ONPG (o-NITROPHENYL-β-D-GAL
ACTOPYRANOSIDE) by examining the change in absorbance at 420 nm with color development. The plasmid pCH110 using the SV40 early promoter [Pharmacia # 27-4508]
Table 1 shows relative values when the absorbance of the cell extract of COS-cells or L-cells transformed according to the formula [-01] was defined as 1.

As is clear from the above table, the plasmid having the lacZ gene incorporated into the vector using the hybrid promoter of the present invention has a very high expression level as compared with the vector using the original chicken β-actin gene promoter. Indicated.

Example 6: Production of HBsAg by expression plasmid using improved chicken β-actin gene promoter (1) HBsAg expression plasmid pAS-HBs, pAR-HBs, pAG-
Construction of HBs, pAcS-HBs, pARS-HBs and pAGS-HBs Plasmid pAS101 having a yeast inhibitory acid phosphatase promoter and capable of producing HBsAg by transforming yeast (JP-B 61-55951) ( Ten
μg) is reacted with XbaI (10 u) at 37 ° C. for 4 hours and electrophoresed on a 6% agarose gel. 1.3Kb including HBs gene
Is cut out of the agarose gel, put into a dialysis tube, and electrophoresed again. After the DNA is eluted from the gel fragment, only the DNA solution is taken out of the dialysis tube, and the DNA is extracted by ethanol precipitation. HBs extracted
1 μg of the DNA fragment containing the gene was ligated to T4 DNA polymerase (1
Incubate at 37 ° C for 30 minutes in u). DNA is extracted by phenol treatment and ethanol precipitation.

Next, the six types of plasmids pAS-2, pAR-2, pAG-2, pAcS-2, and pARS-2 constructed in Examples 1 to 4 described above.
And pAGS-2 were each digested with a restriction enzyme SalI, and then dephosphorylated by the action of calf small intestine-derived alkaline phosphatase. Then, a HBsAg expression plasmid pAS-HBs, pAR-HBs, pAG-HBs, pAcS- HBs, p
ARS-HBs and pAGS-HBs were constructed.

A reference expression plasmid pSVES-HBs for evaluating the expression level of HBsAg was constructed as follows.

In the same manner as described above, the plasmid pAS101 was digested with the restriction enzyme XboI, and an about 1.3 kbp DNA fragment containing the HBsAg gene was extracted by acrylamide gel electrophoresis.

On the other hand, a plasmid having the SV40 early gene promoter
1 μg of pKSV-10 (Pharmacia # 27-4926-01) was added to Bgl
Incubate at 37 ° C for 1 hour with E (1u). DNA is extracted by phenol treatment and ethanol precipitation. The extracted DNA is treated with T4 DNA polymerase in the same manner as described above. 500 ng of the HBs gene fragment whose ends were changed to blunt ends by the T4 DNA polymerase reaction and pKSV-1050 ng were treated at 4 ° C. with T4 DNA ligase (1 u).
Incubate for 12 hours. This reaction solution and Escherichia coli HB101 are transformed. The plasmid was extracted from the transformant, and pKSV-
The plasmid pSVES-HBs having the HBs gene inserted into 10 was obtained.

(2) Expression of HBsAg in COS cells 1 × 10 ⁵ cells /
The COs-cells in the wells were seeded, and 5 µg of each of the following plasmid DNAs was introduced by the DEAE-Dextran method as usual. Cell Phect Tr
Using an ansfection Kit (Pharmacia), the culture supernatant three days after DNA introduction was collected, and HBsAg activity was measured using an HBsAg detection kit “Austria II” (manufactured by Dynabot) (Table 2).

As a result, all of the expression plasmids using the improved chicken β-actin gene promoter had higher HBsA than pSVES-HBs using the SV40 early gene promoter.
g activity.

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the chicken β-actin gene promoter. FIG. 2 shows the sequence recognized by the restriction enzyme NcoI. FIG. 3 shows a cut surface of the restriction enzyme NcoI. FIG. 4 shows the position of the HindIII linker incorporated downstream of the promoter in the plasmid p28 constructed in Preparation Example (1). FIG. 5 shows the nucleotide sequence of a hybrid promoter of a chicken β-actin gene promoter and a rabbit β-globin gene, which pAG-2 constructed in the Examples has. FIG. 6 shows the structure of plasmid pAc-2. FIG. 7 shows the structure of plasmid pAS-2. FIG. 8 shows the structure of plasmid pAR-2. FIG. 9 shows the structure of plasmid pKCR. FIG. 10 shows the structure of plasmid pAG-2. FIG. 11 shows the structure of plasmid pS-1. FIG. 12 shows the structure of plasmid pS-2. FIG. 13 shows the plasmid pAcS-2 structure. FIG. 14 shows the plasmid pARS-2 structure. FIG. 15 shows the plasmid pAGS-2 structure.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:91) (C12P 21/00 C12R 1:91) Examiner Mayumi Saito (56) References GENE, Vol. 48, No. 1, (1986 RECD 1987); 1-12 Proc. Natl. Acad. Sc i. USA, Vol. 85, (Jully 1988), p. 5031-5035 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 C12N 9/00-9/99 C12P 21/00-21/06 BIOSIS (DIALOG) WPI (DIALOG) )

Claims (19)

(57) [Claims] 1. A hybrid promoter for expressing a foreign gene, wherein a second promoter is incorporated into an intron region of a chicken β-actin gene promoter. 2. The hybrid promoter according to claim 1, wherein the second promoter is a gene of a virus capable of infecting animal cells. 3. The hybrid promoter according to the above (1) or (2), wherein the second promoter is a promoter derived from the SV40 gene. 4. The hybrid promoter according to any one of the above (1) to (3), wherein the second promoter is an SV40 early promoter. 5. The hybrid promoter according to the above (1) or (2), wherein the second promoter is an RTR of Rous sarcoma virus (RSV). 6. The chicken β-actin gene promoter, which is located in the middle of the intron region and downstream thereof (3 ′
3 'including the splicing acceptor sequence at the end)
A hybrid promoter for exogenous gene expression, characterized in that the gene on the end side is removed and a gene containing another splicing acceptor sequence is connected to the gene. 7. The hybrid promoter according to (6), wherein the other splicing acceptor sequence is a gene fragment having a splicing acceptor sequence contained in the rabbit β-globin gene. 8. A chicken β-actin gene promoter having a hybrid promoter in which a second promoter is incorporated in an intron region thereof, and further comprising a restriction enzyme cleavage site downstream thereof, into which a foreign gene can be incorporated. A foreign gene expression vector. 9. The vector according to (8), wherein said second promoter is a gene of a virus capable of infecting animal cells. 10. The vector according to (8) or (9), wherein the second promoter is a promoter derived from the SV40 gene. 11. The vector according to any one of (8) to (10), wherein said second promoter is an SV40 early promoter. 12. The vector according to the above (8) or (9), wherein the second promoter is an LTR of Rous sarcoma virus (RSV). 13. An intermediate part of the intron region of the chicken β-actin gene promoter and downstream thereof (3 ′ end side).
A hybrid promoter characterized by removing the gene at the 3 'end including the splicing acceptor sequence, and connecting a gene containing the other splicing acceptor sequence thereto, and further inserting a foreign gene downstream thereof. A vector having a restriction enzyme cleavage site capable of being integrated. 14. The hybrid promoter according to (13), wherein the other splicing acceptor sequence is a gene fragment having a splicing acceptor sequence contained in the rabbit β-globin gene. 15. The vector according to any one of the above (8) or (14), which has a gene derived from an Escherichia coli plasmid. 16. The vector according to (15), wherein the E. coli plasmid-derived gene is a drug resistance gene and a gene fragment containing a replication initiation region that functions in E. coli. 17. The vector according to any one of (8) to (16), further comprising an SV40 early transcription splicing site and a polyadenylation site. 18. The vector according to any one of (8) to (17), further comprising an SV40 replication initiation region (SV40ori). 19. A trait obtained by incorporating a foreign gene into the restriction enzyme cleavage site for introducing a foreign gene of the vector according to any one of the above items (8) to (18), and introducing this into an animal cell. A method for expressing a foreign gene, comprising culturing a transformed animal cell.