JPS59192091A - Method and composition for developing compitent eukaryote gene product - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

BACKGROUND OF THE INVENTION (1) Field of the Invention In the industrial production of proteins encoded by specific genes, such as genes encoding hormones, coagulation factors, viral proteins, insulin, interferon, etc., viral sexual, eukaryotic and other RNA-1 levels
Select and separate the NA and convert this sequence in the form of DNA into D
υ recombinant DNA is obtained by ligation to the NA vector, and this recombinant DNA is introduced into host cells capable of expressing these genes. This vector will confer on the host cell the phenotypic traits used for isolation or cloning. Gene products are isolated from cell culture by conventional techniques.

Traditionally, efforts in this field have generally been based on improving microorganisms as host cells for expressing genes of interest. Indeed, bacteria are often chosen as hosts because they can be grown rapidly, in large quantities, and at low cost. foreign DNA using vectors such as plasmids, cosmids, viruses, etc.
A can be easily introduced into bacterial cells.

However, the use of bacterial hosts is not always sufficient to obtain expression of mature eukaryotic proteins. Many important eukaryotic proteins are modified by glycosylation, acetylation, phosphorylation, specific protein cleavage, and other types of processing.

In many cases, post-transcriptional or post-translational modifications are essential in determining the ultimate biological properties of a protein product.

Post-translational non-translational modifications of some proteins, such as glycosylation, acetylation, phosphorylation and other modifications, are significantly different from the bacteria or cell type in which the gene product of interest is normally produced. If it occurs at all, it will not occur properly in these types of cells. Correct execution of these modifications is essential for the synthesis of fully effective gene products by molecular cloning techniques. [For example, if the gene product is a glycoprotein, such as the hemagglutinin of influenza virus, the detailed nature of the glycosylation may lead to the development of antibodies directed against this synthetic protein in humans or animals against influenza virus infection. This will affect efficiency. Furthermore, if glycosylation, acetylation or phosphorylation, or other modifications are required for protein stabilization, activation, intermediate intracellular transport or secretion, the precision of these modifications may depend on these genetically engineered It is essential for the practical utility of the produced tongue and spear products.

Because of the drawbacks mentioned above in using bacteria for the synthesis of complex gene products, many attempts have been made to introduce DNA encoding specific eukaryotic tongues into eukaryotic cells. It's here. The DNA is co-transformed into suitable mutant cells deficient in the production of specific enzymes such as thymidine kinase or hypoxanthine phosphoribosyltransferase.
It can be introduced by Transformed cells can then be selected by their ability to proliferate, ie, to produce the enzyme, by culturing the cells in a selective medium that does not contain the enzyme product. Alternatively to this method, drugs or selective media such as methotrexate, neomycin,
Cells can be positively co-transfected by adding genes that make them selectively resistant to other substances. Although many of these attempts had some degree of success,
In most cases, the yield of mature gene product was very limited. In order to maximize the yield of expression of a fully effective gene product of interest, the host expression unit system used should consist of an expression vector derived from a cell type or organism capable of synthesizing said fully effective gene product, and Preferably, the expression vector directs the synthesis of said gene product in the same or similar cell type or organism as the host.

Most extensively studied. To get an overview, what is Asburner? Ashburner andBo
(1979) Ce1l 17: 24
See 1-254. When cells or organs of Drosophila, which are normally at about 25°C, are exposed to heat treatment at 35°C to 37°C, a group of heat shock genes are activated and most of the genes active at 25°C are no longer transcribed. Seven genes have a molecular weight of 22,000
Encodes polypeptides ranging from 0 to 84,000 gluton. During heat treatment, these heat shock polybedetides are almost exclusively synthesized and account for 10% of total cellular protein after 8 hours (Arrigo, Bee, (A
(1979) Ph, D, T
hesis, University of Schneve).

During heat treatment of Drosophila cells, polysome-bound mRNA
Many of the A encode heat shock proteins (Mackenzie et al., 1975).
72: 1117-1121: Milault (Mlra
ult) et al. (1978) Co1d Spring H
Arbor Symp, Quant.

Riol, 42: 819-827).

All seven Drosophila heat shock protein genes have been cloned. Ligua 7ri (Liva)
k) et al. (1978) PNAS 75: 5613
~5617: 5chedl et al. (19
78) Cel114°921-929; Flave (Cr
aig) et al.

(1978) Cell 16: 575-588:
Ho Imgren et al. (197
9) Ce1l 18: 1359-1730: Wadsworth et al.

(1980) PNAS 77: 2134-213
7 Nichols (1980)
PNAS 77: 5390-5393;, t'Elmy et al. (1981) Ce
See ll 23: 261-270. Several genes have been sequenced. Karchi and Dorok (Ka
rch and Torok ) (] 980) N
ucleicAcid Re8. 8 : 3] 05
~3] 22: and Ignora and Flave (Ingo
(1982) PNAS79
: 2360-2364° All eukaryotes appear to have heat shock genes. Kelly and Schlenonger
(1978) Cell 1
5:1277-1288. Many of the heat shock genes appear to be conserved across a wide range of species, and the Dronphila heat shock genes have been shown to be present in mouse cells (Corses et al. (1981) PNAS 78).
038-7042), in frog cells (Voellmy and Rungg
(1982) PNAS 79: 1776
~1780), and in monkey cells (-! Luham (Pe
(1982) Cel130:517-52
8) It was shown that it is transcribed. Uniform! The non/nohitonyok gene region and the herpes simplex virus thymidine kinase gene region are also transcribed in these heterologous cell lines. There is no evidence to suggest that protein products of these genes are formed. Pelham, H- and Bienz, M.

M, ) (1982) pp. 43-48 r Heat 5ho
ck from Bacteria to Man,
'/Uresinger, Asvarner and Tisieres (Ti
5si'eres ) edition + Cold Spring Ha
rbour Press Nicholses et al. (1982) 2
Pages 7-34 + Heat 5hock from Ba
cteria t.

Man, Schlesinger, Asperner and Tisieres, eds., Co1d Spring Harbor Pr.
ess.

SUMMARY OF THE INVENTION A DNA construct is provided comprising a heat shock gene-derived regulatory element associated with a gene of interest, which enables expression of the gene of interest in both prokaryotic and eukaryotic cells. More specifically, homologous expression units and expression host cells are used to efficiently synthesize a fully effective gene product.
A combination is used and the properties of this homologous system are the same or similar to those of the cell type or organism that normally produces the gene product.

DESCRIPTION OF SPECIFIC EMBODIMENTS Methods and compositions for regulated expression of genes in eukaryotic hosts are provided. This invention is particularly useful for expressing mammalian genes in mammalian hosts, typically in host cell cultures similar or identical to the host in which the genes are naturally expressed. In this method, post-transcriptional and post-translational transformation of the gene product is achieved, thus improving the competency of the gene product produced.
will be guaranteed.

A DNA sequence comprising an inducible expression control region from the eukaryotic heat shock protein (hsp) gene is used. Such control regions include, for example, sequences that are induced by high temperature and are responsible for both transcriptional and translational control of the activity of hsp genes, such as the promoter region (R
(NA polymerase recognition and binding site), part 1 - and possibly further include heat shock tongue gene control sequences modified to allow constitutive expression of downstream coding sequences. The control region may further contain other portions of the translated and untranslated regions (including 3' untranslated sequences) of the hsp gene involved in transcriptional and/or translational control. Furthermore, the 3' untranslated region (f Ianking) located next to the structural gene (
That is, it can also contain sequences derived from non-coding portions of the gene of interest. Conveniently, the structural region as well as 5′
The complete hsp gene, including the and 3' flanking regions, can be used. In this case, the gene of interest will be inserted in the middle of the structural region, and upon translation a fused tongue-spear will be produced. Next, the target protein is recovered according to a conventional method.

In this specification, heat shock tongue/spear (ht4
p) The term gene refers to the complete genetic locus responsible for the inducible expression of the heat-shocked spear, specifically the 5' regulatory sequence upstream of the structural region, the mRNA
It contains the structural region encoding the A transcript (including the untranslated leader sequence) and the 3' flank region downstream of this structural gene.

Heat shock protein genes can be obtained from most higher eukaryotes. Certain heat-shocked insects are affected by fruit flies (Drosophila).
), have been identified in mice, frogs, monkeys, and humans. Heat shock genes suitable as an isolated source for the control regions of the present invention can be obtained from the above-mentioned or other eukaryotes. Although heat shock genes obtained from one species can be expressed in other species and even in prokaryotes, in general, those that most favor the expression of heat shock regulatory elements at both the transcriptional and translational levels. For this purpose, it is preferable to obtain the same or similar biological cell types as those used for expression of genes used commercially. for example,
Constructs containing genes under the control of Dronphila heat shock elements can be most advantageously expressed in Drosophila cells in culture.

7 present in Drosophila in the experimental part described below.
The inducible expression control region obtained from the heat shock protein gene encoding the gluton heat shock protein in E. coli (E. coli), CO3J monkey cells, and Xenopus oocytes. was used to express E. coli β-galactosidase. This same region was f'l in C08I cells.
influenzae was used to control the expression of the magglutinin gene.

By combining the inducible expression control region of the invention with the extrachromosomal replication system of a given host, an expression vector for that host can be obtained.

and will result in regulated transcription and translation of the inserted gene. The vector further contains selective markers in bacterial and eukaryotic host cells, a prokaryotic replication system that allows cloning of the vector in prokaryotic hosts, and other 1) NA regions of interest. can do.

Alternatively, the expression control region can be linked to the desired structural gene and the resulting DNA construct can be directly injected into host cells. Such direct transfer methods include injection of DNA into the nucleus (Cappechi (1999)).
80) Cell 22: 47'l-488), and calcium phosphate precipitation (Wigler et al. (1979) Cell 16: 777-785)
) or DEAE dextran (McCutchan, J, H,
) and Pogano, J., Nis, (Pogano J,
S, ) (1968) J, Na, t, Cancer I
n+qt, 41:351-357).

As mentioned above, the DNA i constructs of this invention can contain different portions of the hsp gene.

This configuration includes at least a 5' control region carrying the promoter, control sequences such as operators, activators ~, cap signals l), , J? It will contain additional DNA IJ-Goo sequences responsible for seam junction-enhancing jig nano and other signals, as well as transcriptional and translational control. The DNA construct may further contain the protein-coding sequence portion of the hsp gene, resulting in the production of a fusion protein when the foreign gene to be expressed is inserted downstream of the hsp sequence. If fusion tongues are not desired, it may be necessary to remove the hsp coding sequences from the isolated hsp DNA by conventional methods, such as enzymatic restriction and exonuclease digestion.

It may be desirable to retain at least a portion of the hgp structural gene downstream from the natural translation initiation codon. In this case, a fusion protein containing the amine-terminal amino acid sequence of heat shock tongue 9 is obtained. If such a fusion tanno-spear is produced, a selective cleavage site can be introduced to release the precursor tanno-spear? It would be desirable to be able to separate the target tank spear from the container. See US Pat. No. 4,366,246 to Riggs, which teaches how to introduce such cleavage sites.

The expression control region of the invention will be combined with a terminator for complete transcriptional control of the inserted structural gene. Although the inserted structural gene may carry its own terminator sequence or any other suitable terminator sequence, the terminator can conveniently be obtained from the heat shock gene itself.

Extrachromosomal replication systems may also be used. Suitable replication systems include str/l//l/ (5truhl) et al. (197
9) PNAS 73: includes an autonomously replicating sequence as described in ] 471-1475, and a 2 μm plasmid for replication in yeast. Mammalian replication systems are no! Hopaviruses such as simian virus 40 and bovine papilloma virus; adenoviruses; avian retroviruses such as avian sarcoma virus; and mammalian retroviruses such as monkey white m4 virus.

In addition to any eukaryotic replication system, it is advantageous to provide a prokaryotic replication system to allow cloning of the vector in a bacterial host. This allows the vector to be propagated in large quantities in a well-characterized bacterial system prior to transformation of a eukaryotic host. Suitable prokaryotic replication systems are well known and include cilasmids such as pBR322, pRK 290. C
o7LE7, and bacteriophages such as λdv. Prokaryotic replication systems necessarily contain an origin of replication that can be recognized by the prokaryotic host and will usually contain one or more markers for selecting transformants in the prokaryotic host. Such markers include biocide resistance, toxin resistance, and the like. As an alternative to this method, supplementation that allows growth of the auxotrophic host in a selective medium is recommended.
ion) can be used. Such techniques are well known in the art and need not be described further.

Typically, the markers used will be different for selection in prokaryotic and eukaryotic hosts. A variety of dominantly functional markers are useful in selecting transformed mammalian cell lines.

These usually comprise specific genes which, upon expression, confer new drug resistance on mammalian cells in an appropriate selective medium. Specific markers include the bacterial xanthine-da-anine phosphoribosyltransferase gene, which can be selected in media containing mycophenolic acid and xanthine (Mulligan et al.
1981) PNAS: 2072-2076). Mouse cD encoding dihydrobormate reductase
Transformants with vectors carrying the NA fragment were selected for use in aminopterin-containing media (Subraman et al. (1981) Mo1.
Ce1l Bjol.

1:854-861): and a bacterial plasmid specifying an aminglycoside phosphotransferase that inactivates the antibacterial action of neomycin-kanamycin derivatives, which is toxic to most complemented mammalian cell systems. selected to use a medium containing the derivative G418 (Colpère-Galavin).
re-Garapin) et al. (1981) J, Mo1.
Biol,] 50: 1-14).

A change in the copy number of the gene expression unit introduced into the host cell and the close association of one of the amplifiable genes, e.g. the mouse dihydrofolate reductase gene, result in integrated copies of the gene expression unit. It is clear that the numbers are similarly amplified by inducing amplification of related amplifiable genes and adjacent DNA. For example, dihydroformate reductase cDNA and E in Chinese ovary cells,
Co-amplification of coli XGPRT (xanthine-guanine phospholidecyltransferase) gene
plification) (Langold, Gee (
Rungold, G. et al. (1981) J, M.
ol, andAppl, Genetics,
1, 165-175), in which both the expression control region of the heat shock protein gene and the eukaryotic replication system are located on a suitable prokaryotic plasmid. inserted into. The method and order of insertion is not critical, and it is only necessary that the resulting vector possess an active replication system in prokaryotic and, if necessary, eukaryotic hosts.

A DNA construct containing a Dronphila heat shock gene control segment operably linked to a structural gene of interest, used to synthesize the product of said gene in Dronphila cells or cultured cells closely related to Drosophila. can do. Since the structural 9 genes are placed under the transcriptional and optionally translational control of heat shock control elements, for example
When raised to 2°C, the expression of structural genes can be enhanced.

Through such induction, most of the newly generated mRNA will be derived from the above-mentioned structural gene.

Now, if the structural gene is a human gene encoding a tongue/grip that requires complex processing, the expression vector is preferably derived from a replication element, a marker gene, a human heat tongue gene, in addition to the aforementioned structural gene. It comprises a heat shock control element. This construct may be advantageously introduced into cultured human cells by the method described above to produce the product of the human gene described above. If necessary, the recipient human cell will be selected on the basis of its ability to correctly express the fully processed gene product.

A wide variety of structural genes can be introduced into the vectors of this invention to produce a variety of gene products, such as polysaccharides, such as enzymes, proteins, hormones, novel tongue structures, and the like. I can do it.

Example The following example is described as Example 911.

a 650 base pair (bp) DNA break from one of the two Drosophila 70 and Dalton heat shock tanz4' genes, containing heat shock gene transcriptional control elements;
The complete sequence leader sequence, translation initiation signal and sequence encoding the first few amino acids of Dalton's heat shock protein in Drosophila 70 were added to plasmid 132E3 (Skedl et al. (1978) Ce1l 1).
4: 921-929 (see Figure 1). Plasmid 132E3 contains two complete genes for Dalton's heat-shocked sled in 70 and the previously isolated subclone, prosmid 51 (Karch et al. (1981)).
J, Hekol Biol.

148: 219-230) is plasmid 132E3
Contains a fragment W of the first heat shock gene.

This fragment was isolated by digestion of cilasmid p51 with the restriction endonucleases Bgt1 and Lux.

The top of Figure 1a is a representation of a portion of plasmid 132E3 showing the two genes encoding the 70 Dalton heat shock protein (bold segments) and several identified restriction sites. The lower part of Figure 1a shows the previously described 65 bounded by the 5au3A cleavage site.
Represents a portion of p51 containing a Bgtll-Bam HI segment containing a 0 bp fragment.

Figure 1b is a more detailed representation of the portion of interest contained in p51 with additional restriction sites indicated, and two 5
The position of the 650 bp between the au3A sites is also shown.

FIG. 2 shows DNA sequence data for J strands contained in the 650 base pair fragment described above. The extent of this sequence is indicated by the location of the 5au3A restriction site (GATC). At the same time, )(ba(.

Restriction recognition sites for XhoT and Pst■ are shown, and the positions of transcription and translation initiation sequences are indicated by arrows 1a and 1b, respectively.
Indicated by The 650 bp fragment was added to plasmid pMc
1403 at a site controlling the expression of the test gene in this plasmid.

Plasmid T) MC14o 3 (Ca5
aJaban) et al. (1980) J, Bacter
iol, 971-980) is plasmid pBR322
(Polyvar (Bot+var) et al. (1977) Gene
2:95-113), which contains the sequence encoding the first seven amino acids of β-galactosidase and f<(.6,1□, a) from 5' to the β-galactosidase tannoξ coding region. ,-2-,ヮ7,
Two,. 5□ Contains a complete E, co-IJ 1-ac oscillation except for the translation initiation codon (1 site, translation initiation codon). Therefore,
MC carrying plasmid pMC1403]061 (Cazadapan et al. (1980) J, Mo1. Biol 1
38: 179-207) E, coli tac
The strain does not produce any β-galactosidase. pMc14
The polylinker at the 5′ end of the Aac sequence in 03 (
4 Rf + Sma I + BarnHI) is β-
Allows introduction of foreign DNA sequences upstream from the galactosiguse coding region. β by inserting a segment containing a functional promoter and RNA leader sequence.
- Galactosidase activity occurs. The amino terminus of β-galactosidase is not essential for its enzymatic activity (Muller-Hilby, and Kania, J.
r-Hill B, and Kan! a, J,)(
1974) Nature249: 561-563
) is something to be aware of. Therefore, all that is required is that the inserted promoter/RNA leader sequence be able to read correctly in frame for the incomplete β-galactosidase coding region. Therefore, we added the 650bp segment to plasmid pM
It was ligated into the Bam Hf site in front of the incomplete β-galactosidase gene of C1403. The new recombinant plasmid thus obtained (see Figure 3) was transformed into pRv15
It is called. The entire procedure for this plasmid construction is shown schematically in FIG. In Figure 3, the upper left (Figure 31L) is 5au
Figure 3b shows a portion of the above cilasmid 51 containing the 650 bp fragment 2 which has been excised using BA and inserted into the Bam'FIT site of plasmid pMc1403 shown above. It is represented by a bold line with the number 3 in the diagram of Figure 3b taken. It also contains the structural gene for ampicillin resistance. Plasmid p51 was digested with 5au3A and shown on the left in Figure 3c. Excise the aforementioned 650 bP fragment, designated by the number 2; this fragment was purified from the cibolyacrylamide dal by electroelution (
(See Figure 5C). This fragment was added to pMC1403 with BamH
It was inserted into one site in either direction. This ligation mixture was then used to transform the tae strain of E. coli MC1061. Transformants were treated with ampicillin and Xgat (β- is a substrate for lactosidase and produces an identifiable colored product after cleavage by the enzyme).
Xgal is 5-bromo-4-chloro-3-indolyl-
(Miller, J.).
,)Experiments in Mo1ecula
r Genetics r pp-47~55 r C
o1d Spring Harbor, N, Y,
1972).

Plasmid pMc1403 does not produce β-galactosidase activity in this assay, whereas derasmid pMc1403 does not produce β-galactosidase activity in this assay.
RV 15 and many other transformants produced by the method described above were found to produce substantial amounts of β-galactosidase as measured by the color change produced on Xgat plates.

In addition, a β-lactosidase-encoding derasmid construct such as pRV15 was used as a radiolabeled p51-derived 650 bp Sau as a hybridization probe.
3A fragment (see Fig. 4b) or the 2 kbp Xba [gene fragment (Fig. 1a, 4d) from plasmid 123E3
Grunstein (Gru
colony x hybridization assay (Grunstein and Hognes)
++) (1975 PNAS 72:3961~
3966). These fragments were processed using the nick translation method (Manlat f, x, (Manlat
is) et al. (1975) PNAS 72: 118
4). As seen in Figures 4a and 4b, all selected transformants hybridized with both radioactive probes, demonstrating the presence of both DNA sequences in the recombinant plasmid, such as pRv 15. It was done.

The presence of a 650 bP fragment containing the Dalton heat shock gene control element in Dronphila 70 was confirmed by restriction analysis (sections 5a, b and C).

Plasmid pMC1403 contains EeoR) and CyV■
has a unique restriction site for XhoI or Xb
It does not have the a1 site. However, plasmid p51
The di-3A 650 bp fragment from has no site for prime. In contrast, a recombinant plasmid such as pRV15 should contain specific sites for all four enzymes mentioned above (see Figure 3). If the structure shown for pRV 15 in Figure 3 is correct, restriction of recombinant DNA from a plasmid such as pRV 15 with Xhoi and 3aj or Two interruptions of 6.5-7 kbp should be generated. However, plasmid pMC1403 should only be linearized. Figure 5a shows a copy of the DNA fragment prepared with the above-mentioned restriction enzymes after electrophoresis on an agarose scale.
The structure of VI 5 was confirmed. Take the results again in the 5th
b Shown in figure. In Figure 5b, Si21 +
Digestion with either ho I or 4 h I was shown to linearize pRV 15, further confirming the structure shown in Figure 3.

In addition, pRV 15 is Bam HI of pMc1403.
650 bp3a of plasmid p51 pushed into the site
If it contains a u3A fragment, the 650 bp Sau 3A fragment could be recovered by digestion of recombinant DNA such as pRv 15 with the restriction enzyme 5au3A. This case is indicated by an arrow in lane 7 of FIG. 5C. Furthermore, the identification of the 650 bp excised fragment was confirmed by Xba l I! or its restriction by Xhol. Lanes 5 and 6 of Figure 5C
checking ...

Evidence that the Sau 3 A 650 bp fragment and the β-galactosidase coding sequence are in the orientation shown for pRv15 in FIG. 3 is shown in FIGS. 6a and 5. In Figure 6a, the correct orientation is illustrated and 650b correctly oriented for galactosidase gene 12.
l) Fragments of 150 bp and 220 bp are expected to be excised from the fragment, respectively. In Figure 6a, arrow 11 indicates the direction of transcription of the heat shock control element. The above prediction was verified by restriction group analysis shown in Figure 6b.

In Figure 6b, EcoRI and Xba i as evidenced by electrophoresis on 5% acrylamide glue.
An approximately 150 bp fragment was released by double digestion of Ii:
A 220 bp fragment was released by double digestion of coR( and Xho(). These experiments revealed that plasmid pRV
15 was confirmed to have the desired directional control and code elements shown in FIG. The positions of the nodes of sizes 515, 220 and 154 bp are indicated by correspondingly numbered arrows.

Hybrid plasmid pRV described in the previous section
15 and other identical isolates constructed were Xga
Substantial amounts of β-galactosidase gold are produced, determined by examining the color change on the t7'' rate. β-galactosidase is produced in E. coli under the control of Drosophila heat shock control elements, and β-galactosidase gold is produced in E. coli under the control of Drosophila heat shock control elements. existence,
Tan A' newly synthesized using 358-methionine
This was unambiguously demonstrated by immunoprecipitation of E. coli tongue and spear extracts containing plasmid pRV15 after radiolabeling the cells.

The immune serum against authentic E. coli β-galactosidase yielded a molecular weight of approximately 120.00 from the above protein extract.
A 0 dalton polypeptide was precipitated.

Details of the construction and expression of pRV l 5 are described below. Below in the margin A, the construction of plasmid pRV 15 10 μs of plasmid p51 (Figure 1a, bottom)
The incubation buffer for this digestion and the other digestions described below is
Restriction enzyme supplier (New England Biola)
bs Cat, 1982) for 4 hours at 37°C.

DNA concentration during digestion with Sau3A was 40μ
It was Vml. This digestion preparation was mixed with 1x TBE (1 part water to 10.99 parts Tris base, 5.5 g boric acid,
0.939 NIL2EDTA) native 5 in buffer
Electrophoresis was performed on polyacrylamide gel. pBR3
22 Haki 3A digests were electrophoresed in parallel and 6
50 bp 51kariu3A bromotor fragment (2
) was used for the identification of Figure 50). DNA fragments were visualized using ethizoompromit' (EtBr). 50th
The region containing the bromotor fragment, indicated by the arrow in the figure, was excised from the glue and the fragment was electroeluted in a dialysis membrane. Electroelution l x 200v in TBE buffer
It was conducted for 5 hours. This eluate was mixed with 15-gon recondated Co.
Collected in a rex tube and ethanol precipitated overnight at -20°C. This precipitate was centrifuged (Sorvall,
30 i#, r 10.00Orpm + 88340
- dried in a freeze dryer,
μt TE buffer (10mM TriS, HCL,
pH 7.5.

resuspended in lrnNINa2EDTA). The DNA was then extracted twice with TE-saturated phenol,
Extracted twice with ether. This solution was then converted into (Past
5 ephadex G 75 mini-column (in EUR Bit). The DNA in the column bath was ethanol precipitated twice, dried and resuspended in 20 μt TE buffer. A 10 μl aliquot of the appropriate digestion buffer was incubated at 37° C. for 1 hour. Partially digested DNA was electrophoresed on a 5-way polyacrylamide gel. Since the bromotor fragment contains only the Xba 1 site of Gerasmi l'p51, the isolated segment for this experiment was 6
It was proved to be a 50 bp'Sa3A fragment.

4 units of Grasmid pMC1403 of lAg) II
was used for 30 minutes at 37°C.

Digested DNA was extracted three times with phenol and three times with ether. This DNA was further purified by two subsequent evaporation precipitations. The K-let DNA was dried and then resuspended in 20 μt TE buffer.

Aliquots of 10 μt containing the 650 bpf lomotor fragment and 10 μt containing the digested pMC1403.
Combine the µt aliquot solution and then add a total volume of 25
pt (New England Biolabs 1
982Cat, an excess of T 4 DNA (+) in InkyPage 7g buffer) recommended by Mr.
Incubation was carried out overnight at 4°C. The ligation mixture was then incubated with 8 units of Bam, HI for several hours at 14°C. Then CaC42-treated g, rlJM
Q, 51 nl aliquots of C1061 were transformed.

Then an aliquot of this transformation suspension) (0,1 mJ
), 10 μg/wtl ampirin and 40 μm
Placed on LB agar containing 17ml xga+. After incubation overnight at 37° C., blue colonies were observed. Approximately 80 β-galactonase-producing coloviruses were isolated. The presence of the p51 Sau 3A7' lomoter fragment in this recombinant was demonstrated by tern/utain colony hygrotization (Fig. 4b). This hybridization
A 7-yeon globe was prepared as follows: p51-
Affected 3A glomotor fragments were isolated as described above. 5
0 μg of 132E3 was incubated with 50 units of Xb for 2 hours at 37°C.
Digested using al. This digested preparation was separated on 0.9 thiagarose dal. A 2 kbp 70 Dalton heat shock protein gene fragment was electrophoretically eluted from this gel and purified as described above. The latter fragment and the p 51 Sau 3 A fragment were then P-labeled by nick transray to a specific activity of 2×10 8 cpm/μI. The two globes were then denatured and hybridized to two nitrocellulose filters containing the DNA of 80 selected transformants and the DNA of pMC1,403 (Fig. 4 a r b). Both globes hybridized strongly with all 80 recombinants. This suggests that all recombinants contained the 650 bp promoter fragment. clone 5.1
5+25+35+45 and 55 were selected for the next study. A small amount of DI was prepared from each of these clones (Davis et al. (1980) M
etbods in Enzymology + Grogsmann and Mo1
dave), editor 65°404-414). Furthermore, these DNAs and pMc1403 DNA were compared by restriction digestion and electrophoresis on a 0.9% agarose scale (Figures g5a, b). The results showed that restriction of the recombinant plasmid of pRV 15 with 5ail and 1jXbaI and 5atl produced two fragments of 4 and 6.5-7 kbp. In this way, the Grasmid pRV shown in Figure 3d
15 grooves were confirmed.

B, Bacteria containing the Torosophila heat/Yock-hybrid Grasmid pRV15 in L. coli and other isolates constructed as described in the previous section were determined by examining color changes on the Xgat scale. The determined fC produces a substantial amount of β-galactonase. E under the control of the Drosophila Heaton Yock control element
The presence of β-galactonase protein produced in E. coli was also detected by radioactively labeling the newly synthesized tongue with 358-methionine and then using fluorasmid pR.
Immunoprecipitation of E. coli tongue and mother extract containing V15 (Bromly et al. (1979) J, V
irol.

u:86-93). True E, coli β-
Immune serum against galacto/dase clearly precipitated a polypeptide with a molecular weight of about 120,000 Daltons from the protein extract.

Gerasumi t'pRV15 (50-100μ, j7),
Digested with excess 5aAI and Eco Rl.

The two restriction fragments obtained were then separated on a 0.851a looseglue. A 7 kb fragment containing the hybrid gene but not vector sequences was electroeluted and
Purification was performed by gel filtration on ephadex G75. This fragment was then injected with an excess of T 4 DNA in a total volume of 25μ as described above to allow ring formation.
- I keebated with I. This ligated fragment was synthesized by Voellmy and Lanno, y-(Voellmy and
dRungger) (1982) PNAS79:
1776-1780 into the oocytes. Following grain incubation for 6-20 hours at 20°C, the oocytes were treated with α-5.
2P-GTP was injected twice and the oocytes were
Heat treated at 7°C for 2 hours or kept at 20°C for the same time.

Total RNA prepared from these oocytes, or total RNA prepared from oocytes containing no foreign DNA,
Grasmid pMc 1403 Sat 1/Eco
Hybridized to a Southern plot of RI digestion. Oocytes that did not contain the hybrid gene did not produce measurable amounts of RNA complementary to the β-galacto/dase gene. Both heat-treated and untreated oocytes containing the pRV 15 fragment contained approximately the same amount of β.
- It was found that galacto/dase RNA is produced.

In another 7 Leeds experiment, unlabeled RNA
was isolated from oocytes containing the hybrid gene that had been heat treated for 2 hours or incubated at 20°C for the same time in the presence or absence of low concentrations of α-amanitin (Bonirmy and Lansher (1982) PNAS
79: 1776-1780). This oocyte RNA
Bromley et al. (1979) J, Virol, 31
: Labeled by reverse transcription and hybridized to pMC1403 Southern plot as described p. 86-93. Again, transcription of β-galact7dase was performed in RNA from heat-treated and untreated oocytes. α-Amaniten inhibited transcription of the hybrid gene at both temperatures, indicating that all transcriptional activity observed was based on RNA polymerase B (i.e., ■).

The use of Grasmid as indicated at 520 in the plasmid pRV15 and the Grasmid psVod (Mello 7
(Mellon) et al. (1981) Cel127:279
~288). Plasmid 520 Saito m (Gluzman (1981) Cel12
3-2 175-182) can be efficiently stored,
This plasmid 520 allows rapid analysis of transcriptional regulation. The construction of Grasmid 520 is illustrated in FIG.

Grasmid pRV 15 (FIG. 7a, face-centered representation) is digested with -di-concave-■ and then with hi-1, and the resulting 650 bp Drosophila regulatory element 2 present in pRV 15 and A 7 kbp fragment containing lac gene fragment 3 derived from pMC1403 (no. 7b
Figure) was isolated by electrophoretic elution from preparative agarose dal.

1 lasmid psVod (Figure 7c) was digested with polymerase I (I) and the cohesive ends were digested with DNA 7]'?limerasecle/
-(Klenow) fragment 7 and finally this DN
A was digested with -3a7I. In Figure 7C, the number 1
0 indicates a deletion site in the pBR322 sequence of lkb that is claimed to inhibit replication in eukaryotic cells. The cut out psVod fragment (7th d.
Figure) isolated from pRvl, 5 1. ．． 7′C7 kbp fragment. Then, using this ligation mixture, E, coli M
C]061 was transformed. The structure of Grasmid 520 is shown in Figure 7e. In this figure, the details of the 650bp segment are also NυI, μso ■ and pst
1 is displayed with restriction sites. Another restriction site (P*u[
) is also shown, and this region is located between 60 and 70 b shown in Figure 2.
This corresponds to the CAGCTG sequence of p. heart! ■The importance of the parts will be explained later. Plasmid 520 is shown as a circle in Figure 8a. In Figure 8a, segments a, y and 2
represents the gene of the lae operon. Number 11 is SV4
The fragment containing the 0 starting point is shown.

Transformants were plated on xgILl-ambinlin as described above, and blue transformants were isolated that appeared to contain Gerasmid 520. From the structure shown in Figure 8a, Grasmid 520 was , when digested with the combination of restriction enzymes listed below, should produce fragments of the following sizes: This 520 plasmid, upon digestion, has Fragments of the correct size were generated. Additional fragments prepared by digestion with different combinations of enzymes serve as controls for interpretation of the above digestions.

Grasmid p81 is a 650 bp heat/p520
Genes encoding eukaryotic tongue and haunches are placed so that grinding is consistent with the yock control elements.

As starting material for this construction, vector PAT15
3/Pvu I[/Human influenza hemagglutinin gene inserted into the Pvu 1 site of 8 (
Grasmid A containing 1775 bp, number 20) /
PR8/ms/34 (Mount 5ina
i) Clone No. 4.76 from Dr.G.

Brownlee) (University of 0
xford rDepartment of Path
Obtained from U.

Grasmid containing the hemagglutinin (HA) gene (see insert 20, FIG. 9) was digested with μ>m Hl and the ends were filled with DNA polymerase Kl-fragment. ,H
Following digestion with ind II, the hemagglutinin gene fragment was purified on an agarose gel. Insertion 20 contains the complete deuteranase coding sequence, the &extensive leader segment, and 40 bp of 3' untranslated sequence. H
The A gene-containing fragment was inserted into the grasmid vector psVod and 1 rasmid PR81 was prepared as follows:
The psVod DNA was digested with Sat I, the ends were filled in as above, and the I) NA was further digested with Hin.
Digested with dDl (see structure in Figure 9b). Kite Fragment 20, and psVod's SaAl/danind I
[[The digest was incubated with T in the presence of BamHI and 5ail.
4 [)] are ligated together using NA ligase and PR8
1 (see Figure 9c) was obtained. This ligation mixture was then used to transform E. coli IJ C600.

Transformants were analyzed for the presence of pR81-like grasmids (for predicted structure, see Figure 9c) as follows: Grun/Utaincolonino hybridisenyon was isolated using the method described previously for pRV l 5 (see Figure 9c). (See Figure 4).

Probe for the nick-translated progenitor gene (p
A/PR8/34/ms/4.76k
PR8
1-like transformants (see Figures 9d and 90). Figures 9d and 98 show autoradiographs of eight colonies. Figures 9d and 9e show that this DNA globe hybridizes to the majority of transformants. psVod-containing colonies indicated in the figure by arrows are used as negative controls.

Furthermore, PR81-like glasmid was determined by restriction analysis as shown in Figure 9f. In Figure 90, PH
The structure shown for 10 is confirmed by the formation of DNA fragments of the sizes listed below after digestion with the restriction enzymes listed below.

Plasmid fragment size kbp Enzyme PR used
813,2, 1,5-1.6 α-IPR8/34
3.8.1.3 Ikari R1psVod
3.3 Eco RIPR814, 5dan order dlIl/To4I PR8/34 3.3, nd for 1°7~1.8
lll/average 11psVod 2.7, 0.6~
0.7 Hind m /SaAl The results given in Figure 9f indicate the presence of said fragment.

A summary of the structure of plasmid PR84 is shown in Sections 10a to 1.
Shown in Figure 0e. Briefly, Grasmid PR84 places the Tomoe gene under the control of the expression control region of the aforementioned Dronphila heat shock gene. The hybrid gene itself is placed under the replication control of Grasmid pSVod.

Grasmid 52 with Pvu It and Nco'l
Digestion of 0 (Fig. 10a) resulted in the formation of two fragments approximately 1 kbp in length. One of them contains part of the SV40 origin of replication sequence and a 650 bp heat-no-yock regulatory element containing 400 bp of 5'-untranscribed sequence and 60 bp of RNA leader sequence. The two 1 kbp fragments were isolated by electrophoresis on a preliminary gel of 1°2 low melting agarose.

Grasmid PR81 (Figure 10e) was digested with dllll, its ends were filled with DNA polymerase cle/- fragments, and the DNA was injected into 3V4 in this grasmid.
N with a specific cleavage site located within the 0 origin of replication sequence
Digested using coI (see Figure 10d). The resulting PR81 fragment was isolated from Grasmid 520.
p fragment and use this ligation mixture to create E, coIJ
C600 was transformed. The resulting transformants contained a plasmid such as PR84, the predicted structure of which is shown in Figure 10e.

A more detailed structure for PR84, including a partial [)NA predicted sequence, is shown in Figure 10f in the form of a Grasmid single strand sequence representation. As shown in Figure 10f, PH84 is located at the 5' end, followed by the 25
0 bp origin of replication segment.

346 bp sequence from pug 322, originally 650
400b contained in the bp heat/yock control sequence
p Dronphila 5' non-transcribed sequence (p51; p13
2E3゜Karch et al. (1981) J, M
ot, Biot148:219-230), Drosophila 65 bp hsp gene RNA IJ-Goo fragment (base pairs numbered 1-65), 8 base pair linker
segment, the hemagglutinin (HA) RNA leader segment (base pairs numbered 1 to 32), the HA/glucal peptide coding segment (base pairs numbered 33 to 83), and the 翫 protein coding segment (base pairs numbered 84 onward).
p).

This rather complex architectural scheme chosen for Grasmid PR84 was confirmed by restriction digest analysis of the parental plasmid used, the results of which are shown in Figure 11a. The following restriction sites were predicted for this grasmid: PH10:1 site, 1 site for dln, and 1 site in the SV40 origin of replication sequence.

PR8/ms/ 34: I Nco I site, HA
There are no sites in the gene.

520: INcoi site, several P in lac operon
vu n site, a pair of 1 kbp Neo l/P
vu II fragments, one of them origin-hsp
Contains the 707' lomoter fragment.

The compatibility of these predictions with the structure of these grasmids shown in the structural scheme is shown by the group in Figure 11a.
This is illustrated by the results of the restriction analysis shown by the turn.

Two l kbp-μKo1/Pvu II fragments were clearly identified and are indicated by arrows in Figure 11 (lane 17).

Additionally, characterization of PR84-like grasmids was performed using the previously described Grundstein Colonies procedure (see Figure 4). In this case, (ngrasmid p5 containing drosophila hsp regulatory elements)
The Ssu 3 A/Xho 1 fragment nonic transrayed globe (approximately 108 cpm/μ
g) was used. The results are shown in FIG. 11b.

Several positive colonies identified in this manner were expanded for preparation of small amounts of each plasmid as described above, and a series of restriction analyzes were performed. The glue thus obtained is shown in Figures 1e and d. As predicted from the structure of PR84 shown in Figure 10,
The expected parent turn of the digested preparation formed by the digestion is one 405 bp Xhol fragment and a very large fragment. Figure 11c shows the results of this analysis and the Grasmid clones in lanes 2, 4.6 and 8 have the expected pattern compared to the Hinf I standard digestion pattern of psVod. These glasmids were selected for the next analysis and they were labeled with Xho I/
Is it possible to use Neo I or Zendo 1/N-1?
r Digested. The results obtained are shown in Figure 11d. 10th
As shown in the figure, 7′? : The expected fragment sizes prepared from the map are approximately 1 fragment of 700 bp and 1 fragment of 405 bp and 1 large fragment when using Hol/Neo I, and 1/N + fast ■
was used, one fragment of 650 bp and one large fragment. Figure 1 id demonstrated the validity of these predictions for all grasmids analyzed.

Transfection of eukaryotic cells with DNA from Grasmid PR847. I did J-n. CO8 cells (Gurraman (1981) Cell 23:175-182) are suitable cells for conducting such experiments, and rapid expression analysis of gene composition (Guteing and ThumpC
Getting and Sambrook
(1981) Nature 293:620-625)
It was chosen because of its usefulness. After transfection, the cells were labeled with methionine (3
5s) at 37°C or 42°C. The contents of the cells were then infected with PR8/ms influenza virus (Dr. J.
, 5kehel) + M, R, C, Laborato
rieg.

The assay was performed by immunoprecipitation using a rabbit antiserum raised against (a gift from Mill Hill, London U.). The complexes purified with Tanori A-5 epharose ■ were finally subjected to electrophoresis on a polyacrylamide gel and identified by fluorography. The results of this analysis indicate that true glyconated hemagglutinin (Elder et al. (1979) Virology 9
5:343-350) and indistinguishable high-yielding tan.
It showed the presence of gore (Mr=75,000). Synthesis of hemagglutinin occurs at 37°C and 42°C.
Synthesis also occurred to a very low extent.

Any transfected DNA other than pR84Lu
We did not observe such specific polygutides in immune complexes other than those using anti-PR8 antiserum.

The construction and expression details of the eukaryotic expression vector are described below.

A. Structure of cilasmid 520 50 μg of cilasmid pRV15 was first mixed with 50 units of S
digested with mal for 2 hours at 37°C and then with 50 units of DARI I for 2 hours at 37°C (New
Buffer recommended by England Biolabs). This digest was then electrophoresed on a 09% agarose scale. The region containing the 7 kb lac fragment was excised from the cage and the DNA was electroeluted (200 V,
6 hours).

DNA in the eluate was collected by ethanol precipitation. The fragments were then dried and resuspended in 200 μl of TE buffer, extracted twice with phenol and ether, and then passed through the small 5 ephadex ■ G75 column described above. DNA in the column eluate was collected by ethanol precipitation.

10 μs Grasmid psVod for 2 hours at 37°C.
Digested with single BamHI. This digested DN
A was purified by three phenol extractions, three ethyl extractions and two ethanol precipitations. The dried DNA was then resuspended in TE buffer and:C
015mM deoxyadenosine triphosphate (d
ATP), deoxycytidine triphosphate (dCT
P), deoxyguanosine triphosphate (dG
TP) and deoxythymidine triphosphate) (
dTTP) with several units of DNA polymerase Cournot fragment for 1 hour at 6-7°C. The DNA was repurified by repeated phenol and ether extractions and ethanol precipitation. Next, the purified DNA was heated at 37℃C2 using 10 units of lotus.
I spent time. This digested preparation was again purified as described above. Next item 7kbp NopRV 15 Sma I
/Sal I fragment and the psVod fragment were incubated overnight (in a molar ratio of 10:1) at 14°C with excess T4DNAIJ gauze. This ligation mixture was used to transform Danzo IJMC1061. Sma
1 and Ram HI were included in this ligation mixture.

Transformants were treated with 10 μg/ml ampicillin and 4
Plated on LB plates containing 0 μl/fnl of X'gal. A blue transformant was isolated. DNA from such recombinants was converted to Skedl et al. (1978) Ce
1114:921-929 as described above. Recombinants were identified by restriction analysis.

B, Plasmid PR81 III'j, Professor Degene Lee, University of Oxford
, U, K. Plasmid A/I) R8/ obtained from
50 μg of Clone No. 34, 4.76, was digested with 30 units of UdI11 and Hao1 (1) at 37° C. for 2 hours. This digest was electrophoresed on a 0.85% agarose scale. This ndlTI-sucmal hemagglutinin gene fragment was purified by electroalkalinity as described above. An aliquot of this DNA was nick-translated and probed to identify similar recombinants by PH10 and Grunstein colony hybridization. (The specific radioactivity of the glove was about I
Q8cpm/μg).

50 μl of A/PR8/34/4.76 was digested with 50 units of Bam HI for 2 hours at 37°C. This D
NA was extracted three times with phenol, extracted with ether and precipitated with ethanol. The dried DNA was then resuspended in 25 μl of TE buffer and mixed with several units of DNA d = limerase Cournot fragment and Q, 3 mM of all four deoxynucleotide triphosphates in a total volume of 100 μl. Incubated for 90 minutes at 7°C. The DNA was then purified by phenol and ether extraction as described above. Following ethanol precipitation, the DNA was dried and then resuspended in 25 μl of TE buffer. This was then digested with 54 units of ind Ill for 1 hour at 37°C.

Digested DNA was purified by phenol extraction.

10 μg of plasmid psVod (Mellon et al. (198
1) Cell 27:279-288) for 30 units of 5
Digested with alt for 1 hour at 37°C. The DNA was purified by phenol extraction as described above, followed by 0.5 mM dATP, dCTP.

Incubate with several units of Cournot fragment in 100 μl in the presence of dGTP and dTTP for 1 hour at 37°C. After repurification by phenol extraction, this DNA was further purified at 37°C using 25 units of Hjndll.
for 1 hour and purified by phenol extraction.

A/PR8/34 digest (15 μg) and psVod
The digest (2μIi,) was mixed with excess T in a total volume of 30μβ.
4 DNA +) was incubated overnight at 14°C using gauze. Several units of BamHI and 5ail were included in the ligation mixture. This ligated DNA was purified by phenol extraction as described above. Then add this to 6
Digestion was performed with 1 unit of Bam HI for 1 hour at 37°C, followed by incubation for 1 hour at 37°C with 12 units of Homo1. Using an aliquot of this reaction mixture, CR
Ct2-treated E. coli C600 was transformed. Transformants were isolated on L8 grates containing 10 μm 1 /at ampicillin. Approximately 350 clones were generated and analyzed by colony hybridization using the nick-translated World III-Ba dish Hl hemagglutinin gene fragment as a globe. Approximately 80-90% of these clones were found to contain the hemagglutinin gene insertion.

Seven positive clones were expanded and DNA was prepared from them. These DNAs were purified by restriction digestion and electrophoresis on 0.9 thiagarose glucose.
/PR8/34 and pSVod.

Digested recombinant using predicted fragment size R1 3.2kbp+ 1.5-1.6
kbpA / PR8/34 3.8 kbp'
, 1.3 kbps Vod 3
．． 3 kbp digested recombinant with ndllJ and 5ali 4.5 kbp A/PR8/34 3.3 kbp, 1.7-1
．． 8kbpps Vod 2.7kbp,
Five of the seven recombinants 0.6-0.7 kbp analyzed showed the expected digestion pattern. PR81 is
One of these five clones.

PR81 DNA by Skedl et al. (1978) Cel114
: Prepared according to the method of 921-929,
It was further analyzed by restriction enzyme digestion.

C,! The constructed p520 DNA (75 μg) of the lasmid PR84 was digested with 25 units of H'' for 1 hour at 37°C, followed by 2 hours of digestion at 37°C with 20 units of Ncal. This digested product is 0.05
Electrophoresis was performed on a 1st and 2nd column low-lysis agarose gel containing μ9/ml of EtBrf. A region containing two l kbp BeguI/S1 fragments, one of which is the hsp70 gene promoter fragment, was excised from the dal. The agarose portion and 10 volumes of TE buffer were heated to 65° C. for 10 minutes, and the DNA was then extracted with an equal volume of phenol. Furthermore, phenol/
After extraction once with chloroform and three times with ether, the DNA was precipitated twice with ethanol.

PR81 DNA (10 μg) was added to 15 units of multiple ndll.
The DNA was digested with a few units of Cournot fragment and 0.5 mM of all four deoxyribonucleotide triphosphates at 6-7°C.
and incubated for 1 hour. After repurification, the DNA was digested with 10 units of μl I for 90 minutes at 37°C and repurified by phenol extraction.

An aliquot of the l kbp Soheki1/PN4 fragment of p520 (0.25 μg) and an aliquot of the PR81 fragment (005-0.1 μg) was incubated overnight at 14°C with excess T4 DNA ligase. did.

This mixture was used to transform E. coli IJ C600. Approximately 600 ampicillin-resistant transformants were isolated and nick-translated as a globe with p51.
Shira 3A/μtol promoter fragment (approximately 11080
(Fig. 11b).

The 10 positives were propagated and r from these clones
) NA was prepared. Aliquots of these DNAs were
HO) and analyzed on a 5-tipolyacrylamide gel.

Due to the presence of the XhoI fragment and one soybean, hsp70
The presence of recombinant r)NA of both the Dalton gene promoter and the hemagglutinin gene was well established. Four of the ten clones tested showed the expected restriction pattern (two fragments; one large, approximately 5 k
bp, and one 400 bp) (Anchor 11C and d figures).

By additional digestion, our initial analysis k JI (f
L'- did. PR84 is one of four grasmids with the correct structure.

D, Expression experiment using plasmid PR84 in CO8-I cells (Gluzman (1981) Cell
1 23:175-182) in Dulbecco MEM containing 10 ml of newborn calf serum.
After storage in culture medium, subculturing one day before transfection, and culturing for an additional day, cultures containing approximately 10 6 cells per 6 cm diameter Vetri dish were obtained. Transfection of cells was performed as described below.

Remove the medium from the cell culture at room temperature and store the culture at 5 m
The cells were washed twice with 1 liter of phosphate buffered saline (PBS). Subsequently, 1 ml per dish of the following preparation was added.

1. DEAE Dextran (500μg)
and Dulbecco's MEM medium containing chloroquine (200I4) but no serum.

2 Same medium as 1 except containing an additional 10 μg of cilasmid 520 DNA.

3 Same medium as 1 except containing an additional 10 μg of cilasmid PR84 DNA.

Transfections were performed on 5 dishes of cells in each well of the three conditions.

The cell culture was kept at room temperature for 30 minutes with occasional gentle shaking to distribute the transfection solution throughout the cell culture.

After removing the transfection solution, 5 ml per dish
Dulbecco's MEM medium containing 10% of serum was added.

Cell cultures were further incubated at 37°C for 36 hours. Half of the two cultures were incubated at 42°C for the last 4 hours of this period. After this period, the medium is removed and the culture is combined with 3-1/l L-methionine and 1
The cells were washed once with Dulbecco's MEM medium containing nascent serum of P. chinensis. 35S-methion (50zjc
A low saturated methionine medium, which was the same as above except that 7 ml of methionine (7 ml, 968 Ci/mmol) was present, was added to each plate.
Cell cultures were labeled by adding mA. Cultures were incubated at 37°C in the case of heat shock samples.
Or incubated at 42°C for 1 hour. After this period of labeling, the 35s-methionine medium was removed, the culture was washed three times with very cold PBS, and the cells were washed with 100ml of NET buffer (0, IMNaCl,
0.01 M Trig-HCt%I 7.8, 0
．． After vigorous pipetting to disrupt the outer cell membrane, nuclei and cell debris were dispersed in 0.001 MEDTA, 0.54 NP 40 ) for 5 min at 4 °C.
Removed by centrifugation at 00>1. The cytoplasmic supernatant was removed and aliquots were taken for immunoprecipitation as follows: An aliquot of the cytoplasmic supernatant (500 μl) was kept on ice, 5 μl of specific antibody was added, This solution was heated at 4°C for 2
Mix gently for an hour. Antibodies used were as follows: normal rabbit antiserum, purified E. coli antiserum raised against β-galactosidase, and J. Siekel (Dr. J.).

5hekel), MRC Laboratores,
Mill Hllll.

Antiserum raised against PR8 influenza virus obtained from London, United States. anti-HA antiserum,
Prior to use for immunoprecipitation of labeled samples, cytoplasmic extracts of unlabeled CO8 cells were used to
Preadsorption was carried out for 30 minutes in ET buffer.

Protein Δ-3epharose■ (Pharmaci
Add 50 μl of a 50% suspension (manufactured by Company A) and incubate at 4°C for 1
Continue shaking for 15,0QOX! Antigen-antibody complexes were isolated by collecting the bound complexes by centrifugation at i' for 1 minute. This tongue/grip A-
The complex on 5epharose was diluted four times with 1 ml of NET and 1 ml of RIPA solution (0, t
5M NaCl, 0°05M Tris-HCtp1
47.5, 0.02 MEDTA, I M Urea
, 1% Triton) < 100. monosodium deoxycholate (sodium deoxychola)
te)) and centrifuged. The final 5epbarose pellets were mixed with 100 μl of sample buffer (Laemmli (197)).
0) Nature 227:680-685) for 3 minutes. After centrifugation at 15,0 QOXg for 1 minute, the samples were subjected to electrophoresis on a 10% polyacrylamide gel as described by Laemmli supra.

Glue analysis by fluorography when cell cultures transfected with Grasmid PR R84 DNA were obtained and the labeled cytoplasmic extracts were immunoprecipitated with antiserum raised against the PR8 influenza virus. (La5key and Mills
Mills) (1975) Eur.

J. Biochem, 56: 335-341
) is a true glycosylated hemagglutinin (Elder et al.
1979) Virology 95: 343-35
0) and indistinguishable in size (Mr=75,000). Mr about 42,
Several possible processings to give polypeptides of 000 and 36,000 were observed in some of these experiments. The synthesis of hemagglutinin is
This occurred when labeling was performed at 37°C and to a lesser extent also occurred when labeling was performed at 42°C.

No such specific polypeptide was observed when using any transfecting DNA other than PR84 or in immunoprecipitations other than using anti-PR8 antiserum.

In a second series of the same transfection experiments, CO8 cells transfected with PR84 were heat shocked for 5 hours at 42°C and then labeled with S-methionine as described above. However, it should be kept at 37°C for 14 hours. Subsequent immunoprecipitation with anti-1 (A antiserum) revealed greater amounts of HA than in heat-untreated samples labeled under identical conditions.
Production is shown.

These results suggest that the Dronphila heat shock promoter functions better in monkey cells at a heat shock temperature than at a control temperature of 37°C. As already described using 37° C. as the heat shock temperature for NijiZZ cells,
One would expect a certain activity of this promoter at 37°C. Improved synthesis of HA when cells are returned to 37°C after a period of heat shock suggests that translational control of Drosophila heat shock promoter fragments, especially the Drosophila liposome binding site, is at least monkey-like at higher temperatures. This suggests that it does not function or has a poor function in cells of this type. In this way, the execution (promotion) of transcription is carried out in a continuous manner.
) NA sequence, although liposome binding and initiation of translation occurs optimally at 37°C with HA I
This may occur using the J&some binding site.

The present invention provides expression vectors that result in efficient transformation of eukaryotic hosts and high levels of expression within the host. Specifically, we isolated the transcription/translation control region of Dalton's heat shock protein at 70 and linked it to a replication system obtained from a monkey virus. The glycosyl glycolytic produced converted hemagglutinin.

Construction of plasmid p629 Drosophila control element/HA gene construction other than the above.
Like PR84! This was done to expand the utility of the lasmid construct. The HA gene, which is a viral gene, is rather special as a model for any eukaryotic gene that has its own 3' end signal and its own RNA IJ-G sequence;
Contains both the NA leader and the Drosophila RNA leader.

Plasmid p629 was constructed as a more general example of constructing a gene expression unit using heat shock control elements. This plasmid contained the entire region of the heat shock RNA IJ-gu in addition to the Drosophila heat shock glomotor, but it was modified to lack the first 40 amino acid codons (attached to the HA gene.
(R.

? Cilasmid p622b (obtained from R. Voellmy) was used. This plasmid contains an RNA IJ-Goo sequence (represented by the 4S1/Shadow 3A fragment of plasmid pRV 15 described above) and
Contains 450 bp of µg/gp carrying the Lonphila hsp70 promoter. p 62
2b is a derivative of the CO8 cell vector psVod.

The sequence near the Bgl11 site is shown below.

15μy of p622b (for 15 minutes) 12 units of B
and 15 μg of PR8/m
s/34 was digested with 15 units of Xhol. Digestion was for 2 hours at 37°C. The DNA was then extracted three times with phenol, three times with ether and precipitated twice with EtO). Both DNAs were resuspended in 20 μl of TE buffer and the ends were treated with several units of DNA polymerase (Cournot fragment) and 0.5 mM each of the four d
Reaction involving NTPS (total volume fl: 100 ttl
, reaction for 2 hours at 6°C). DN of both parties
A was again phenol extracted and purified as above,
Bathed in 20μ4 TE buffer. These were then individually digested with 15 units of BamI (total volume 100 μl for 2 h at 37°C). Following phenol extraction, these DNAs were collected by EtOH precipitation and dissolved in 15 μl TE buffer. .Total capacity・2
1 μl of 622b digest and 1-5 μl of P in 5 μl
Ligation of different sets with R8/mu/34R87 gold u was carried out by incubating overnight at 14 °C (enzymes: 1-2μ11T4 DNA ligase, Ne
w England Biolabs).

The ligase was inactivated at 65°C for 10 minutes and the samples were then incubated (subsequently with NewEnglan
d using conditions suggested by Biolabs)
Volume 50 μl using several units of C1a I and Siri
Digestion was carried out for 2 hours at 37°C. These DNA mixtures are then used to generate E, Co! 2MC]061 was transformed.

Approximately 300 individual transformants were further characterized using a Grunstein colony hybridization assay. Two separate sets of hybridizations were performed: the probes used were: a) p62
450 bp μ from 2b × Tagawa Katabi b) PR
The 1775 bp and dll/Bayu J (I gene fragments) from 8,4,76 were separated in a low melting point agarose gel and then eluted from these genes.These were then subjected to nick translation. (107-110
8cp/lt&;l Q6cpm/filter).

Eight colonies that hybridized to both probes were selected (Figures 120 and d) and the DNA was transferred to the miniD
Prepared from them using the NA YJ procedure.

These DNAs were extracted using 4S I or AII/E.
(! Digested using ORI and the digests analyzed on 5% polyacrylamide gels.
bp and two fragments with a size of approximately 5 kb were expected to result. 11 by Machin If/RI digestion
Fragments of 30 bps, 1100 bps and approximately 2.6 kb were expected to be obtained. Three of the eight colonies tested showed 4 turns (the exact limit) (the 12th
8 and b).

Expression experiments using plasmid p629 Transfection of CO8I cells was carried out using plasmid P629.
Expression experiments with R84 were performed in a manner similar to that described. Immunoprecipitation performed as described above precipitated one major and several minor polypeptides with molecular weights around 75,000 Daltons.
When S-methionine labeling was performed in the presence of 1 ti:i/ml tunicamycin, one predominant band (Mr = 75,000)
Only observed. This suggested that this major band was a product of glycosylation. Synthesis of HA protein occurs at 14°C at 37°C following a period of heat shock.
During the time labeling period, the chorus increased more than during the current parallel labeling period without heat shock.

Although the invention has been described in greater detail in 31.1 by way of clarity and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the claims. Probably.

In addition, the plasmid molecule V-15゜P related to this invention
Three strains of E. coli (B.
, coli) was published on February 2, 1984 in The American Type Culture Collection (TheAm
erican 'I”ype Culture Co1
1ection l.

Below margin

[Brief explanation of the drawing]

FIG. 1a is a schematic representation of a partial restriction map of a portion of Grasmid 132E3, which has two 70 Dalton's Heat Shockta Blades, 7 (hsp) genes (bold lines, directions indicated by arrows). The lower part of Figure 1a is for attending school! The corresponding partial restriction r upper diagram of lasmid 51 is shown. FIG. 1b shows a more detailed restriction map of a portion of Gerasmi'51, with additional restriction sites including two specific Sau 3 A sites, in full enlargement. Figure 2 shows hs at Dalton 70 in plasmid 51.
p, shows the complete single-stranded DNA sequence of the DNA preceding the gene. However, the sequence shown in white on a black background exists in the other 70 Dalton gene sequences. The arrangement shown in this figure was created using the method of Maxam and Norbert (M
axam and G11bert) ((1977
). PNAS 14: 560). The figure also shows some important restriction sequences and transcription and translation initiation sites (arrows). Figures g3a-3d are! A diagram illustrating the method of construction of lasmid pRV15 is provided. For more details, Figure 3a shows the glass, ? ) '51 restriction map and this plasmid i
After digestion with l'1sau3A, a fragment of approximately 650 bp (
One end of this fragment is shown in Figure 3c (left side). Figure 3b shows the restriction map of the cilasmid pMc1403, which, after restriction treatment with BamHI,
One end is completely generated as shown in FIG. 3C (right side). Figure 3c is! Figure 3d shows details of the sequence of the ends formed by restriction digestion of lasmid 51 and pMc1403. The structure of lasmid pRV15 linked to the BamH1 site of lasmid pMc1403 is shown. The .beta.-galactosidase gene and a single portion of Drosophila DNA are shown in the plasmid. Additionally, some restriction sites are also shown. The g4a diagram is shown in Figure 3d! Last middle R"v'1
5 structure, Grunstein
and Hognes (1975J PN
1 is a photograph showing the characterization of AS 72:3961 by Colony Hinolidization A.S. (a photograph regarding the morphology of an organism). This assay! Lasmid 132
2kbp Xba excised from E3 (see Figure 1c)
L@Kataka! Recanonic translation of the labeled 650 bp Sau 3Ai fragment from lasmid 51 (Figure 4b) (Maniatis)
ti s) Iii! , (1975) PNAS
72:1184) was used for actual inspection using a radioactive glove made by Nishi. Shown in Figure 3: Several transformants prepared using the mushroom growth method were tested, and some colonies of pMc1403 were included as controls. All of these colonies are indicated by arrows in Figure 4a. Figure 4b shows the same sickle colony hybridization cimin assay as above. In this case the 5au3A 650 kbp fragment from Grasmid 51 was used. Control colonies of pMc1403 are indicated by arrows. Figures 5a-50 show the Grasmid pay shown in Figure 3d.
Figure 2 shows a restriction analysis characterization of the structure of is. Fragment sizes are indicated in bp. More specifically, Figure 5a shows the electropherograms of various DNA fragments. For plasmids pMC1403, pRV15, and two similar fractions, pRV25 and pRV5, the Mignolasmi pf reparation (tebit)' (
David) Ike, (1980), Methods i
nEnzymology XGroagmann and M
D.
NA was prepared. corner? Ttf my own! Lasmid below #
DNA fragments digested and produced by IJ restriction fermentation
Electrophoresis was carried out on R0.85% acetate at 150 V for 4 hours, after which the entire cypan was visualized by ethidium gromide staining. The lane indications are as follows. Nornl pMc1403- Sau 3A/Xh
o 12 p tt -3ail/Kba13
// I/ -Sal l /EcoRI4 p
RV25-SalI/Xho15p
tr-Sal I /Xba16 1 #
-Sall/FJcoRI7 pRV15-S
all/χholV-78pRV 15-S
al I / Xba I9 tt tt -5
al) /KcoR110pRV5-Sal
j/Xho 111 /l /l -5ail
/Xba112 // l/ -5ail
/KcoRi Figure 5b is similar to Figure 5a, and all the digestion and analysis sequences were performed similarly. Next, the lane gold shows OV-1 pRV15-XhoI2tt p
-Xba) 3 tt p -5ail/Xho14 tt
p-5ail/Xba15 tttt-
3al1 6 pRV5-XhoI 7〃〃-Xbal B tt p゛-5ail/Xho1g tt
p-5alI/Xba110 ptr-5
all Figure 5C is similar to Figures 58 and 5b and relates to restriction digestion and analysis performed on fragments separated on 5 polyacrylamide groups. Somnolencephoresis was performed in 401/ for 14 hours. Lane 1 51-5au3A/Xba12 5
1-5au3A/XhoI3 51-5a
u3A4 pMC1403-5au3A5 pRV15-Sau3A/Xba
I6 pRV15-5au3A/Xho17
pRV15-5au3A 8 Diagrammatic representation of plasmid 5l-8au3A 9 pB
Diagrammatic representation J6a-6b of R322-Sau 3A is
Dalton hsp:i gene 5au3A prayer piece direction (orier
ltation). In more detail, Figure 6a illustrates the expected restriction fragment size if the required orientation of the Dronophila element and the E. coli β-galactosidase gene is achieved in Grasmid pRV15. Figure 6b is a photograph showing the results of an acrylamide dal (5%) analysis of Grasmid pay 15 and pBR322 restriction to confirm the predictions illustrated in Figures 6&. The analysis was carried out according to the method described for the case shown in Figure 5c. Lane 1 pBR322-1 (1nf I2 pRV
15- EcoRI3 pRV 15- Xba I/EcoRT4 p
RV 15- Xho I/EcoRI5 pRV 1
The positions of bands of size 517 bp, 220 bp and 154 bp are indicated by arrows, which are
Derived from the known size of the fragment derived from restriction of Grasmid pBR322 with infI. jtl (Figures 7a-7e show glasmid or !lasmid fragments after digestion with restriction enzymes, !lasmid 5
The 14 formations of 20 are schematically shown. This glasmid is la
Co(Sma I of pRV15 containing the nalon fragment)
- Sal I fragment and Drosophila 650 bp control element using BamHI - Sal I restriction sites!
It is constructed by introducing it into a lasmid plsvod. In more detail, Figure 7a shows pRV15'z: OK7 Figure c shows pSv
Figure 7b shows the 5rna I- of pRV15.
Figure 7d shows the BamHl-SaI fragment;
pSVod is shown after excision of the l l fragment; and Figure 7e shows p520. Figure i8a is a restriction diagram of Grasmid vector 520 showing the location of selected restriction sites in the pig plasmid. Figure 8b is a true sister of pRV15 by the method described in Figure 5a (see Figure 5a). A copy of the Grasmid 5200 restriction enzyme digestion is a copy of the Rosedell analysis. Lane 1-Hindl [[ 2-Xho I/EcoRI 3- Hl nd III/EcoR1-Pstl 5- Hl ndll/Pst I 6- Pat l/H1ndl/gcoR17-Pst
I/EcoRI IAB c Figure is a photograph regarding the characterization of Grasmid 520, plasmid ps't10d, pRV 15
, as well as 520-like Grasmid, ie! Lasmid 6
39°520.519. The agarose del contents of the X, Y, and z restriction digests, lfr are shown. Lane l 639 - Sal I/EcoRI25
20 -'tt tt 3519-tt tt 4516 - p tt 5) (-〃 # 6Y-tt tt 7z -tt p 8 psVod -〃p 9 pRV15- a tt lo psVod -Xho I/EcoR111pR
Digests in V15-ttp lanes 8 and 9 are incomplete. Figure 8d is! Photos further show the restriction digest of lasmid 520 compared to the digests of lasmid psVod and pRV15. Lane 1 psVod-PsuI/EcoR12psV
od-Hindlll/Sal I3 pRV15
-Patl/EcoRI4 pRV15- H1nd
ffl/Sal l5pRV15 once al I /Xb
a 16 520 - Pstl/EcoR17520
-Hind[II/Sal I8 520 -3ail
Digests in lanes 2 and 7 are incomplete. Figures 9a-90 relate to the structure of cilasmid PR81. For more information, the iigQa diagram shows Mount Sinai (Mount 5in
ai) A/PR8/ms/4.76 grasmid all shown, and from this grasmid BamHI and Hlndll
The fragment (see no. 20) was cut out using Ik;
ndl[l-8alI@piece'eft shows no psVodk; and the i9c diagram is! Lasmid PR81, namely BamHI-Hl nd III fragment 20 and Sal
The ligation product with I-HindflI digest is shown. Figures 9d and 9e are photographs of Grunstein colony hybridization analysis of PR81-like grasmids performed according to the method described for pRV15 (see Figure 4). The nick translation gloves used were A/PR8/
BamHI/H1ndl about ms/4.76
It is an IT fragment. As a control, some psvod-containing colonies are indicated by full arrows. Figure 9f is a photograph of restriction analysis of PR81-like grasmids shown by colony hybridization analysis in Figures 9d and 9e. Lane 1 filter 21. Sandals 3O-EcoR) 2
//21+sangle 44-tt ty3#22. Sample H-tttt 4 t' 22 + sample 24- tt
tt5 〃 23. Sandals IQ-# //
6 tt 23 + sungle 3l-ttpf//24. Sample 8- 〃 〃 8 pSVod -tt tt
9 PR8/ma/4.76 - //
l/io filter 21. Su/Guru 30 once kondru 111 // 211 Sanguru 44-//
112//22. Nagar 11-#p
Lane 13 filter 22. Sunsol 24-Hindl 4al114 #23. Sandals 10-〃
ri15 23. Sandals 31-//
tt16 1/24. Sandals 8-
〃 〃17pSVod -
tt p18 PR8/ms /4.76
-tt ttFigures 10a-10e relate to the construction of plasmid PR84. This Grasmid is configured as shown in the figure, and! Lasmid 520
The β-lactosidase gene in Grasmid PR81
It is made by replacing the hemagglutinin (HA) gene 20 present in the. More specifically, Figure '10a represents Grasmid 520;
The figure represents plasmid PR81'; figure 10b shows 52
Figure 10d represents PR81 after digestion with Hlndlll and Ncol; and Figure 10e (iPR81) is shown.
Represented schematically at 84'. Figure 10f shows PR84 in more detail with a single strand of base pairs containing the sequence starting at PR8/ms. Only the essential linking region between the Drosophila H8 control element and the flood initiation sequence is shown in detail. Figures 11a-11d relate to checking the resulting configuration of PH10. Figure 11a shows plasmids PR81, PR8/
ms/34, p SVo d and 520 restriction digests. Lane l PR81-Hindlll/EcoRI2
PR81-Xhol/gcoRI3PR81-Nc
oI 4 PR81-Neo I/Xho I5 PR8Fm
g/34- HindIII/EcoRI6 PR8F
ms/34- Xho I/EeoRI7 PR8Fm
lI/34- Neo 18 PR8FrIIg/34
- Ncol/Xho I9 pSVod -N
coI/5a1110 pSVod-PvuI
[/Sal 111 pSVod-Hlndl
I[/EcoR112psVod-HlnfI
fig13520 - )"vul[ Lane 14520 - PvulT/Xba11
5520-Pvull/Xho1165
20-Pvul/gcoR11752
0-PvuII/Nco1 18520 - Nco119520
- NeoT/xbaI20520'
-Neo l/XhoI Figure 11b (1) and 1st
Figure 1b (2) relates to Grunstein Colony High! Ridaizaceae analysis of PR84-like Grasmid. In this test, Grasmid 51 Sau 3A/Xh
o I Nick translation of the Drosophila control element fragment (approximately 108 cpg) was used. Figure 11c shows PR841! selected from the positive colonies identified in Figure 11b! Plasmid Xhol
Lane 1 filter 3 for 5% polyacrylamide Dal analysis of the digestion products. Sangle 12 -Xh
oI2 tt 3. p 15 (P
H10)-”3 #3. //
22/-//Leh/4filku3. Sangle 4
2 (P R84) -X upper 2■s tt 3
．． tt 45'# -I6 //
4. I19 1F -//7 〃 4
+ 〃 40' / I3 t
t 5. tl 15 n -t
l9 tt 5. tl 28
tt -ttIQ tt 5. p
39 tt - tt], 1 psVod
tt - Hlnf I standard 1st id
The figure relates to additional restriction analysis of grasmids providing the Xhol digestion protein shown in lanes 2, 4.6 and 8 of Figure 11c. Lane 1φX 174 RF -Hae
III+? 2 filters on u+5. Nangle l6-X
b a T/Nc o I3 tl 5. t
l 16-Xho I/Neo I4 ” 4
r” 19-Xba I/Neo 15t
t4. // 19-Xhol/Neo
I6 〃3+ 〃42-Xbal/NcoI7
/' L ” 42-Xhol/NcoI8
"L' 15-XbaI/Nco19
'/ 3 * // 15-Xho l/N
Oo 1 Figures 12N-12b show the special 1 damage of Grasmid p629. Figure 12a shows some candidates 62'L;/IJ-'s Grasmid 5% polyacrylamide dal analysis.
i29/7 tt 3629/6 p 4629/4 /1 5 DNA Standard Figure 12b relates to the above-mentioned 5 polyacrylamide dal analysis of all Grasmids of the 8 German Supplement 629 series. Grasmid Lane 1 629/I Xho12 629/
2 /7 3 629/3 // 4 629/4 tt 5 62915 // Lane 6 629/6 Xho17 629/
7 // 8 629/8 p FIG. 12c shows a Grunstein colonization assay using plasmid p622b or the Lanonic translation '/wsn Xho-Bgl II fragment. Figure 12d shows plasmid A/PR8/m x/3
Grunstein colony hybridization assay using Grunstein colony hybridization using nick transtorion f (indlν shouting HI, HA + silicon fragment from 4). Patent Attorney Akira Yamaguchi Masaya NishiyamaFIG. 4a FIG, 4bFIG, 5c Fig. 6Q, Fig. 70-527- Fig. 7c Fig. 7e Fig. 8Q Fig. I2 Te Figure 10e Oral? Weak divination - R84 Procedural amendment (voluntary) April 2, 1980 Kazuo Wakasugi, Commissioner of the Patent Office 1, Indication of the case 1982 Patent Application No. 020675 2, Title of the invention Competent Expression method and expression composition of eukaryotic gene products 3, relationship with the amended person's case Patent applicant name: Patel Memorial Institute 4, agent address: 10-5, Toranomon-chome, Minato-ku, Tokyo 105 , Column 6 of "Detailed Description of the Invention" of the specification to be amended, contents of the amendment (1) Page 78, line 14 of the specification, r PR-81J is amended to r629J. (2) Page 78 of the specification, line 17, “Deposited with.”
Internationally deposited under the Budapest Treaty,
No. 39597, No. 39596 and No. 395, respectively.
I was given the number 98. ” will be corrected. Procedural amendment (method) Kazuo Wakasugi, Commissioner of the Sennichi Patent Office, filed in May 1980, 1, Indication of the case, 1988 Patent Application No. 20675 and Expression Composition 3, Relationship with the person making the amendment Patent Applicant name: Patel Memorial Institute 4;
Agents (4 others) 5. Date of amendment order: April 24, 1980 (shipment date - Kake, Taka) ->
. 6 Subject of amendment - Power of attorney (2) Drawings 7 Contents of amendment (11 Attachment 9 (2) Engraving of drawings (no change in content) 8 List of attached documents - (1) Power of attorney and translation each 1 Trace (1 copy of 21 engraving drawings)

Claims (1)

[Scope of Claims] 1. The "gene expression unit" is derived from the eukaryotic heat shock tongue gene] Expression control region (a) of 0 kb or less; transcription of the expression control region (a) at least one site (b) with which the structural gene of interest (c) is associated so as to be subject to regulatory and/or translational control k
and using as "host cell system" a cell of bacterial origin, or a cell from at least one species from which said expression control region is similar to the species from which it was isolated, and in which said structural gene of interest ( c) a "combined gene expression unit-host cell system" in which the control region (a) operably linked to is present to produce the product of said structural gene; 2. The "gene expression unit-host cell system" according to claim 1, wherein the structural gene of interest is associated by insertion into at least one site of the gene expression unit. 3. The "gene expression unit-host cell system" according to claim 1, wherein the heat shock gene control region is derived from a heat shock gene from a eukaryotic organism. 4. Heat shock gene control region
3. The "gene source, unit-host cell system" according to claim 3, which is derived from the r-nosed DNA of A. ater ). 5. The "gene expression unit-host cell system" according to claim 4, wherein the heat shock gene control region is derived from one of the drugs. 6. Claim 1, wherein at least a part of the functional heat shock gene control region is a part of a synthesis origin.
"Gene Expression Unit - Host Cell System" described in Section 1. 7. The expression unit according to claim 1, wherein the expression unit further comprises at least one selectable marker linked to the expression unit allowing selection of transformed hosts.
Gene Expression Unit--Host Cell System'' 8. The self-expression unit comprises a prokaryotic replication system allowing further propagation of the expression unit, and at least one antibiotic resistance gene. "Gene Expression Unit - Host Cell System" described in Section 1. 9. "Gene expression unit-host cell system" according to claim 1, wherein said expression unit is linked to a fragment containing a eukaryotic extrachromosomal replication system. 10. Gene expression unit - host cell according to claim 1, wherein the eukaryotic heat shock gene control region is linked to a cellular or viral transcriptional enhancing element that allows constitutive expression of the gene of interest. system". 11. Gene Expression Unit - Host Cell System J according to claim 1, wherein the heat shock promoter element is linked to the complete RNA coding region of the structural gene of interest. 12. Eukaryotic heat shock gene promoter
The RNA leader segment is the protein of the target structural gene.
``Gene expression unit--host cell system'' according to claim 1, which is linked to the mother coding region. 13. The "gene expression unit-host cell system" according to claim 1, wherein the eukaryotic heat shock gene control element is linked to at least a portion of the DNA coding region of the structural gene of interest. 14. Plasmid pRV15゜1 as a novel construct
5 Plasmid PR84゜16 as a new construct,
A method for producing a gene expression-host cell combination according to claim 1, comprising introducing the expression unit into the host cell by co-transformation with a selectable marker. 17. The method of claim 16, wherein the expression unit is introduced into the host cell together with an amplifiable gene. 18. Claim 16, wherein the expression unit is introduced into the host cell by transformation or transfection.
The method described in section. 19. An expression vector comprising an expression control region derived from a eukaryotic extrachromosomal replication system, a eukaryote heat shock receptor, and at least one insertion site under the transcriptional control of the expression control region. 20. 21. The expression vector of claim 19, wherein the vector does not contain a translation start codon between the expression control region and the insertion site. 21. The expression vector of claim 19, which has a translation start codon upstream from the insertion site. The expression vector according to claim 19, which is the expression vector according to claim 19.23. An expression vector according to claim 22, which is derived from the Toshock gene. 24. Claim 19, further comprising a prokaryotic replication system allowing stable maintenance in a prokaryotic host.
Expression vectors described in section. 25. The expression vector according to claim 19, further comprising at least one selectable marker that enables the selection of the transformed host with iJ′ ability. 26. Eukaryotic extrachromosomal replication system a heat shock gene control region substantially homologous to a 650 base pair sequence that controls transcription of a 70 kiloglutenon heat shock protein in Doronphin furanogaster, and at least one component under the transcriptional control of the heat shock gene control region. Expression vector containing insertion site. 27, an expression control region derived from a eukaryotic heat shock gene and an I 5 kb containing at least an insertion site under the transcriptional control of the heat shock gene control region
DNA composition with less p. 28. The heat shock gene is 70 kilodalton heat shock tan of Doronfin melanogaster.
The DNA according to claim 27, which is characterized by being flat.
composition. 29. A method for producing a competent gene product, the method comprising linking a structural gene to an expression control region derived from a eukaryotic heat shock gene, and using a eukaryotic host suitable for expression of the structural gene. A manufacturing method comprising introducing said structural gene and control region into said host and propagating said host, thereby producing said product. 30. The structural gene and control region are linked together in an expression vector with a eukaryotic extrachromosomal replication system.
A method according to claim 29. , 31. 30. The method of claim 29, wherein the structural gene and control region are introduced into the host by co-transformation with a selectable marker. 32. The method of claim 29, wherein the expression control region is derived from a heat shock gene encoding a 70 kilodalton heat shock tough i4 gene in Drosophila melanoga W:. 33. The method of claim 29, wherein the heat shock gene is derived from a eukaryotic species different from the host. 34. The method according to claim 29, wherein the structural gene is a mammalian gene. 35. The method of claim 34, wherein the host is a mammalian cell culture. 36. The method according to claim 29, wherein the structural gene is a human gene. 37. The method according to claim 36, wherein the host is a mammalian cell culture. 38. The method of claim 37, wherein the host is a CO81 cell culture.