JPH0330678A - Dna compound coding luciferase and manifestation vector containing the same compound - Google Patents

Abstract

NEW MATERIAL:A DNA compound coding luciferase of Cypridinacea. USE:Producing a reagent for detecting various substances by taking advantage of chemical emission reaction. PREPARATION:For example, Cypridinacea is pulverized in liquid nitrogen, the fine powder is subjected to guanidine thiocyanate/cesium chloride method, whole cell RNA is extracted, poly(A)RNA is purified and separated by oligo(dT) cellulose column chromatography method. A cDNA library is constituted by using the mRNA by a conventional procedure, the cDNA library is screened by using complementary oligonucleotide probe by plaque hybrid method to select a clone containing DNA coding luciferase of Cypridinacea. A plasmid is extracted from the clone and treated with a restriction enzyme to give a DNA compound coding luciferase.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to the marine crustacean, the sea firefly.
la hilgendorfii, formerly Cyprid
tna hilgendorrii)
An appropriate host cell is transformed using a DNA compound encoding Cypridina luciferase, an enzyme that catalyzes the luminescent phenomenon, an expression vector containing the DNA compound, and the expression vector. The present invention relates to a method for producing Cypridina luciferase, which comprises culturing.

[Prior Art and Problems to be Solved by the Invention] Luciferase is an enzyme that catalyzes chemiluminescent reactions observed in various biological species. This luminescent reaction is a reaction in which the substrate luciferin is oxidized to oxyluciferin by the enzymatic action of luciferase in the presence of oxygen, and is represented by the following formula.

Luciferin + O1 → Oxyluciferin + CO1 The photoreaction mechanism differs depending on the species, and some require nucleotides such as ATP as cofactors.

It is also known that closely related species can interact with each other.
It has extremely high substrate specificity, and although it reacts with luciferin and luciferase obtained from the same species, it is said that, in principle, it does not cross-react with enzymes and substrates derived from different species.

As described above, the substrate specificity of luciferase (luciferin) is extremely high and its luminescence is sensitive, so it is possible to detect and/or quantify various substances using this enzyme-substrate chemiluminescent reaction. it is conceivable that.

In general, enzymatic reactions are highly sensitive due to their substrate specificity and require mild reaction conditions, so they are applied in various fields. For example, horseradish verxidase, which catalyzes oxidation reactions in the presence of hydrogen peroxide, is one of the most widely used enzymes.

In particular, this enzyme is important for the analysis of substances that can be detected through reactions involving the generation of hydrogen peroxide.

Although other useful enzymes have been extracted and isolated, many more usable enzymes are needed in various fields to meet diversifying purposes. Therefore, if a method for stably supplying luciferase, an enzyme that catalyzes a luminescent reaction, is established, new uses for this enzyme will be developed.

By the way, luciferase has a number of factors in expressing its catalytic activity.
Auxiliary substances other than oxygen and luciferin (trace amounts of ATP
Some require nucleotides (such as nucleotides) and others do not. Luciferase, which belongs to the former category, is used to detect auxiliary substances (trace amounts of ATP). On the other hand, enzymes belonging to the latter category, such as Cypridina luciferase, require no substances other than oxygen and a substrate (in this case, Cypridina luciferin), so the reaction is simple, and therefore has a wide range of applications and is highly useful. It is assumed that. In order to promote development research and practical application of sea worm luciferase in the field of application, a stable supply of highly pure sea worm luciferase is required. However, as with enzymes derived from other organisms, the extraction, isolation and purification of enzymes from organisms requires much time and expense, making it difficult to meet the above demands. Therefore,
It is desired to establish a simple and efficient method for producing Cypridina luciferase.

[Means for Solving the Problems] In view of this situation, the present inventors focused on producing Cypridina luciferase by genetic engineering,
The cDNA encoding the enzyme was cloned, and the DNA
The A sequence was determined. Next, the DNA obtained in this way
We constructed an expression vector containing the compound, expressed the expression vector in mammalian cells, and succeeded in secreting Cypridina luciferase into the culture medium.

That is, the present invention provides a DNA compound encoding Cypridina luciferase.

The present invention also provides an expression vector containing the DNA compound.

The present invention also provides a method for producing Cypridina luciferase, which comprises transfecting a host cell using the expression vector, culturing it, and recovering Cypridina luciferase from the culture medium.

The chemiluminescent reaction between Cypridina luciferase and the substrate Cypridina luciferin has been well studied [Harvey (
Harvey, E., N.), Am., J.
P hysiol.

42318-341. 1917 and Johnson (J
ohnson, F.

H,) et al., Methods Enzymol, 57
331-364. 1978].

The reaction is shown by the reaction formula below.

Represents dioxetanone intermediate oxyluciferin R3=-CH(CH,)CH,CH. )In addition,
λ□ is 460 nm.

Screening of Cypridina luciferase cDNA,
Nucleotide sequence determination, expression vector construction, host cell transfection and culture were performed using methods known in the art. The outline is shown below.

Literature [Tsuji (F, I, ) M
ethos Enzymol.

(57, 364-372, 1978)], Cypridina luciferase was partially purified using an affinity column and completely purified. When this sample was directly subjected to the Edman degradation method, the amino acid could not be assigned. This means that the amine 7 group of the N-terminal amino acid of the peptide is blocked. Therefore, purified Cypridina luciferase was digested with endopeptidase, and the resulting peptide fragments were subjected to Edman degradation to determine the amino acid sequence. Next, design an oligonucleotide probe using the appropriate moiety, and
NA synthesizer (Applied Biosystems)
I nc,,M.

de1381A) using the phosphoramidite method (pho
sphoramidite method) [Caruthers.

M,H,)+5ynthesis and App
lications of DNA and RNA,
Edited by Ninarang (SA) (Aca
demic Press, New York),
pp, 47-94r1987].

On the other hand, sea fireflies (Tsuji, supra) collected in the Chiba area were ground into fine powder in liquid nitrogen, and this powder was processed using the guanidine thiocyanate/cesium chloride method [Chigwin (Chigwin)].
, J., M.) et al., BiocheIlistry
L8. 5294-5299.1979] to extract total cellular RNA. Then, oligo(dT) cellulose column chromatography [Aviv,
H,) et al., Proc, Natl, Acad,
Sci, USA 69.1408-°1412
．． [1972] to purify poly(A) RNA, and J. Morishita et al.
Biol, Chem, (262,3844385
1, 1987).

This cDNA library was subjected to plaque hybridization using the above oligonucleotide probe [Benton and Davis (Benton, W.).

D., & Davis, R.W.), 5cienc.
e 196. 180-182°1977]. Two clones, VL16 and VL18, were selected from the eight positive clones and a restriction enzyme map was created. Clone L16 encodes the N-terminal side of luciferase, and VL13 encodes the C-terminal side, and they have an overlapping region of 830 nucleotides (the second
Figures and C). Therefore, these two fragments were digested with EcoR1, the obtained fragments were subcloned, and 7-Dea
za DNA sequencing kit (Takara Shuzo), and [
α-31P] dcTP (222TBq/imol) (N
Dideoxynucleotide chain growth termination reaction using Sanger (England Nuclear)
Ger, F.) et al., Proc, Natl.
Acad, Sci, USA 74.5463
-5487°1977 and Cheno (Chen, E.
The nucleotide sequence was determined by Y. et al., DNA4゜165-170.1985]. The restriction enzyme map of the full-length cDNA is shown in Figure 2a, and the nucleotide sequence and deduced amino acid sequence are shown in Figure 3. This deduced amino acid sequence completely matched the amino acid sequence determined by the Edman degradation method described above.

Next, an expression vector was constructed using full-length cDNA clones constructed from clones VL16 and VL18 (see FIG. 2d). Expression vectors may be either prokaryotic or eukaryotic. Those skilled in the art are well aware of methods for selecting a suitable starting vector, inserting a DNA compound encoding a desired peptide into it, and constructing an expression vector for the peptide. In this specification, expression vectors that can be expressed in mammalian cells are exemplified because the products are secreted into the culture medium and are therefore easy to process. Plasmid pRS, an exemplary vector of the present invention
V V L contains cDNA encoding Cypridina luciferase downstream of the Rous sarcoma virus long terminal repeat promoter. This plasmid pRS V V L is used to transfect host cells in a conventional manner.

Transfection methods and host cells can be selected as appropriate, but in the present invention, the calcium phosphate method (G
raham, F., L. et al., Virology
52. 456467, 1973 and Wigle
r, M et al. Ce1l 14.725-731.197
8), monkey COS cells [Gluzm
an, Y, ), Ce1l 23. 175-182
．． 1981° (7XIO')] was transferred. After incubation with a medium suitable for expression of Cypridina luciferase, the medium was collected, cells were harvested, and cell extracts were prepared.

The Cypridina luciferase activity of the culture medium and cell extracts thus obtained was determined by Mitchell Hasti.
Measured by ngs photometer [Miteh
el I, G, W, ) et al., A nal, B
iochem, 39. 243-250].

As a result, only a small amount of Cypridina luciferase activity was detected in the cell extract, but clear transferase activity was detected in the medium (Fig. 4). This luminescence was extremely sensitive, and a signal clearly higher than the background was detected using 5 μQ in medium 10−.

As described above, according to the present invention, Cypridina luciferase can be easily produced by genetic engineering.

DNA encoding firefly luciferase according to the present invention
Now that the nucleotide sequence of the compound has been determined, those skilled in the art can readily produce firefly luciferase using known means in genetic engineering. Many starting materials (expression vectors containing promoters, etc.) and host cells for constructing expression vectors suitable for expressing the DNA compound encoding the Cypridina luciferase of the present invention are known, and the promoters exemplified herein - The same effects as the method of the present invention can be achieved by selecting a system other than the host system. For those skilled in the art, suitable promoters -
Methods are known for selecting a host system, constructing an expression vector, transforming host cells with the resulting expression vector, culturing, and isolating the product. Accordingly, those skilled in the art will appreciate that the present invention is not limited to the exemplary plasmid, pRSVVL, but includes any expression vector suitable for the expression of a DNA compound encoding the Cypridina luciferase of the present invention. It will be readily appreciated that suitable promoters for such other expression vectors include the following.

That is, promoters for expression in animal cells include monkey 5V4Q virus promoter, cytomegalovirus promoter, adenovirus promoter, and AIDS promoter.
Viral promoters, silkworm polygon virus promoters, etc. At this time, mouse NIH3T3 cells, C12
7 cells, L cells, hamster CHO cells, human HeLa
Cells, silkworms, etc. can be used as host cells. Further, as expression promoters in prokaryotic cells, λ phage PL promoter, Tac promoter, T7 promoter, lac promoter, etc. are known, and expression can be performed using E. coli as a host. Furthermore, it is also possible to express Cypridina luciferase using yeast or Bacillus subtilis as a host using a suitable promoter.

EXAMPLES The present invention will be explained in further detail by way of Examples below.

Example 1 Cloning of Cypridina cDNA 1, purification and sequencing of Cypridina luciferase Method of Tsuji (Tsuji, F., 1.)
ds Enzymol, (57, 364-372, 19
78) Cypridina luciferase partially purified according to
A tributamine affinity column (Pierce Chemical) equilibrated in 30% ethylene glycol 10.17M NaCl (pH 7.2) was used.
Stepwise elution was performed with 210.07M Tris-HCC (pH 7,2). Then, after concentration by ultrafiltration, p-aminobenzamidine affinity column (P
It was purified to homogeneity under similar chromatographic conditions using 1erce Chemical).

Purified Cypridina luciferase was subjected to sodium dodecyl sulfate/gradient polyacrylamide gel electrophoresis. Gradient device 0-15% fast gel (PhastGelX
When the sample was analyzed using a Fast Gel Silver Staining Kit (Pharmacia) and the protein was observed using a Fast Gel Silver Staining Kit (Pharmacia), it showed a single band of Mr68,000.

The results are shown in Figure 1. In the figure, lane 1 is a low molecular weight marker manufactured by Pharmacia, and lane 2 is Cypridina luciferase purified by affinity chromatography (
50r+9). Molecular weights of marker proteins are indicated in bovine rhodalton.

120 μ9 of the purified protein was treated with trypsin (Boe
Endopeptidase digestion was performed using lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.) or arginyl endopeptidase (Takara Shuzo).

This digest was subjected to reverse phase chromatography using a C1a-protein-peptide HPLC column (Vydac) to isolate each peptide fragment. Then, a gas phase protein sequencer (Applied Biosys
stems Inc., Model 477 A
) The N-terminal amino acid sequences of the undigested luciferase and purified peptide were determined by the Edman degradation method.

2. Preparation of lllRNA and cDNA library Niokamaya A sea firefly (Tsuji, supra) collected in Chiba Prefecture was immediately frozen in liquid nitrogen. This sea firefly (ostacods) (weight 59) was processed using an ultraturrax homogenizer.
omogenizerXJ anke & Kunke
1) in liquid nitrogen. This fine powder was prepared using the guanidine thiocyanate/cesium chloride method [thiguine (C
higwin, J., M.) et al., Biochemi
Total cellular RNA was extracted by subjecting the cells to Stry 1g, 5294-529L 1979. Then oligo(dT
) Cellulose column chromatography method [Av
iv, H,) et al., Proc, Natl, Acad,
Sci, USA 69, 1408-1412.1972
] Bo1ba A) RNA was purified. This mRN
A cDNA library was constructed using the method of Morishita, K., except that double-stranded EcoR1-digested cDNA was size-selected by two rounds of purification using low-melting agarose (BioRad). Method [J, Biol, Chem, (262,3g
44-3851. I followed 1987.

3. Screening of cDNA clones 1. A portion of the peptide fragment prepared in T hr-Me is a peptide sequence with minimal codon ambiguity.
t-G 1u-A 5n-L eu-A 5p-G I
It had yGln-Lys. Using this, deoxyinosine was included in three locations with high codon degeneracy [Ohtsuka, E. et al., J. Biol.
, Chera, 260°2605-2608.19
[85], designed the following complementary oligonucleotide probes: 5° (T/C) TT (T/C) TG I CC (A/
G)TCI AGGTT (T/C)TCCAT I
GT 3' Additionally, a second complementary probe having A instead of G at position 15 was synthesized. Oligonucleotide probes were synthesized using a DNA synthesizer (Applied
Biosystems Inc., Model
381 A) by the phosphoramidite method [Caruthers, M.

H,) + 5ynthesis and Appli
cations of DNA and RNA, Na
Rang, S.A., ed., (Academic Pres.
s, New York), p9.47-94.
1987].

This oligonucleotide probe was combined with T4 polynucleotide kinase (Takara Shuzo) and [γ-”P]ATP (
222T Bq/*mol, New England
Specific activity:
5-6xlO"cpIIl/pmot), obtained 2
Plaque hybridization using a mixture of probes [Benton and Davis (Benton, W, D,
& Davis, R, W, )+5science 1
96.180-182.1977], the Cypridina cDNA library prepared at 2° was screened. That is, 1X10@ recombinant phages were screened using this probe. For hybridization, lower the temperature to 28 °C and filter at 37 °C.
C-? ? Except for washing with S S C (0,15M NaCQ, 15aM sodium citrate, pH 7,0).
The method of Wahl, G., M. et al. [Pr.
oc, Natl, Acad, Sci, USA (76, 3
683-3687, 1979)]. Eight positive clones were isolated from the library, among which
Clone V with insert lengths 1, 2 kb and 1.5 kb
L15 and clone VL18 were selected for further analysis and restriction enzyme maps were created. Restriction enzyme maps of these clones are shown in FIG.

4. Determination of the nucleotide sequence of Cypridina luciferase As shown in Figures 2 and C, clone L16
and clone VL18 each have a single internal EcoR
There is one site and an overlap of 830 nucleotide sequences. The EcoR1 fragments of both these clones were pUC
3 was subcloned. [a-”P]dCTP (222TBq/zmol) (
Dideoxynucleotide chain growth termination reaction using Sanger (New England Nuclear)
nger+ F, ) et al., Proc, Nat.
1. Δcad, Sci, U 5A74, 5463
-5467, 1977 and Chen, EY,
) et al., DNA 4. 165-170. 1985)]
As a result of nucleotide sequence analysis, clone V
It was found that L16 encodes the N-terminal portion of luciferase and clone VL18 encodes the C-terminal portion. The restriction map of the full-length Cypridina luciferase cDNAΔ constructed from these clones is shown in Figure 2a.
The nucleotide sequence and deduced amino acid sequence were added to the third
As shown in the figure. In FIG. 2a, the shaded area is the putative signal sequence. In FIG. 3, the numbers above each column are the nucleotide positions, and the numbers below each column are the amino acid positions. Horizontal arrows represent regions with identical amino acid sequences to those obtained by endopeptidase digestion. Sequences compatible with N-glycosylation are boxed and the polyadenylation signal AAT
AAA is underlined.

The molecular weight of Cypridina luciferase calculated from this sequence is 62,171 Daltons, and it contains an open reading frame of 1665 nucleotides that encodes a protein of 555 amino acids. This open reading frame contains the amino acid sequence used to construct the oligonucleotide probe. Furthermore, the amino acid sequences of the other seven peptides determined by Edman degradation completely matched the amino acid sequences deduced from the nucleotide sequences.

A translation start codon starting from the 16th nucleotide
Hit the TG codon. The nucleotide sequence surrounding this putative start codon is consistent with the consensus sequence CC(A/G)CCAUGG, which is characteristic of many eukaryotic mRNAs [Kozak (K.

zak, M.), Ce1l 15.1109-112
3.1978]. The N-terminal amino acid sequence has many characteristics of a signal sequence to play the biological role of luciferase as a secreted protein.
Heine (von Heljne, G.), Eur.
J, Biochem, 133°17-21.19
83]. Note that the blocked N-terminus was predicted by the method described below.

Amino acid residues 186 and 40 were also detected by luciferase positive staining by periodic acid-Schiff reaction.
Two N-glycosylation sites (A 5n-X −
Ser/Thr). Although the sequence A 5n-Pro-3et exists at residue position 258, this sequence is generally not efficiently glycosylated [Marshall, R, D, Ann, R.
ev, Biochem Niji, 673-702. 1
972]. When N-glycosidase F acts, the size of the luciferase molecule decreases by 2000-3000 daltons, but no such decrease is observed with digestive enzymes specific for the 0-glycosylation site. These results demonstrate that Cypridina luciferase is N-glycosylated, the theoretical molecular weight of the polypeptide estimated from the nucleotide sequence, gel electrophoresis (Figure 1), gel filtration, and sedimentation equilibrium analysis (Tsuji et al. B 1oche+++1
stry 13.5204-5209. (1974) show that the difference with the molecular weight of the native protein is related to the absence or presence of carbohydrate moieties.

The amine terminus was derived from the structure of luciferin and the structure of enzymes that catalyze bioluminescent reactions in other marine organisms, which have similar reaction mechanisms. Jellyfish (7
victoria) and directly compared its bioluminescent reaction [Johnson
, F., H.) et al., Methods Enzymo
l, 57. 271291, 1978].

Jellyfish emit light due to the calcium-binding protein aequorin, which is excited in the presence of calcium ions and emits light. Aequorin is a complex of apoaequorin (apoprotein), coelenterazine, and oxygen molecules; when aequorin binds calcium ions, a conformational change occurs, converting the protein to oxygenase, and then an intramolecular reaction oxidizes coelenterazine. be done. The emitter in this reaction is coelenterazine in an excited state bound to apoaequorin [ShiIIlomura.

0, ) et al., Tetrahedron Lett, N
o, 31. 2963-2966, 1973].

Although the substrate structures and mechanisms of bioluminescence reactions in Cypridina and jellyfish are similar to each other, there is little cross-reactivity. However, from a comparison of their amino acid sequences, two regions of Cypridina luciferase (residues 97 to 154 and residue 3)
53-411) is the amino acid sequence of apoaequorin 1.
It was found that the amino acid sequence of this region (residues 82 to 144) shows significant similarity (see FIG. 5). Figure 5 shows sea worm luciferase (a) and jellyfish (A equi
rea) with aequorin (b), and the mutation data matrix of Dayhof4 [Dayhof (DayhofT, M, O
), Schwartz.

R, M, et al. At1as of Protein 5
sequence andS structure(N a
tional Biochemical Resea
rchFoundation, Washington
, D, C,), Vol. 5. pp.

345-352.1978] are indicated by two dots (=), and similar amino acids are indicated by one dot (•). The numbers represent the position from the N-terminus of each protein. The size of the apoaequorin molecule is approximately one-third that of Cypridina luciferase, and it consists of 189 amino acid residues, and it is predicted that one or both of the similar regions of Cypridina luciferase are involved in luminescence. .

The region of Cypridina luciferase corresponding to residues 87 to 144 of apoaequorin is residues 97 to 154, and a shift of 10 amino acid residues is clearly observed. This indicates that the first 11 amino acid residues are the leader peptide required for secretion, and the N-
This suggests that the terminal is the 12th tyrosine (see Figure 3). Furthermore, the relationship between alanine and adjacent amino acid residues matches the cleavage site of the signal sequence. von Heijne et al., Eur.

J, Biochem, 133.17-21.1
983]. From the above, it is predicted that the cleavage site of the peptide is at position 11, and the amino terminal is tyrosine.

Another feature of the amino acid sequence of Cypridina luciferase is the presence of a highly cysteine-rich region at the N-terminus. In this region, amino acid residues 39 to
Nine cysteine residues are found between the 82 and 82. However, since no free sulfhydryl groups were detected in Cypridina luciferase (Tsuji et al., supra), the cysteine residues probably form intramolecular disulfide bridges.

Example 2 Construction of an expression vector containing Cypridina luciferase An expression vector was constructed by placing the full-length Cyprid luciferase cDNA under the Rous sarcoma virus long terminal repeat promoter. That is, HindI and Bgl II linkers were inserted at the 5° and 3° ends of the Cypridina luciferase cDNA, respectively.
(Takara Shuzo) was consolidated. Dr.

Plasmid pR3VL encoding firefly luciferase obtained from S. subramani [deWet, J, R, et al., Mol, Ce.
l 1. Biol, 7. The linear plasmid obtained by cutting 725-73 and the HindII[
and Bgl II fragment, Hindl[-BglI[
An expression plasmid pRSVVL containing the fragment was obtained. E. coli transformed with this plasmid, E5cheri
chia coli pRS V V L has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Entrusted mortar: June 15, 1989, accession number: FERMp-t0782)
.

Example 3 Plasmid pRSVVL c.

Transfection of S cells and expression of Cypridina luciferase (1) Transfection 10% fetal bovine serum () () in Petri dishes at 10 cy
Dulbecco (Dulbec) containing
co) modified Eagle medium (Hinaga) lozg with monkey CO
3 cells [GluzIIlan, Y,
Ce1l 23.175-182゜1981: ATCC
CRL1650] was seeded (7×108). These cells were isolated using the calcium phosphate method [Graham,
F, L, ) et al., Virology 52.456
-467, 1973 and Wigler
, M. ) et al., Ce1114, 725-731.19
78], the plasmid pR8V prepared in Example 2
vL DNA 1oμ9 was transfected. 48
After incubation for an hour, the medium was collected and the cells were harvested. Then, after repeatedly freezing and thawing the cells,
Cell extracts were prepared by centrifugation (de
Wet, J.

R1 et al., supra).

2. Measurement of Cypridina luciferase activity The Cypridina luciferase activity of the cell extract and culture medium prepared in 1. was measured using Cypridina luciferin as a substrate.

In a scintillation vial with a volume of ff120++2, the medium or cell extract obtained in step 1 above was diluted with 200mM
Diluted with Tris-HCQ (pH 7, 6) to make a total of 1
is 1.5xe, Mitchell-Hasting
s photometer [Mitchell, G.
, W.) et al., Anal, Biochell, 39.
243-250.1971]. On the other hand, Tsuji's method [Me
thods Enzymol, (57, 364-372
Cypridina luciferin prepared according to 200 J! M sodium phosphate buffer (pH 6,8
) dissolved in (-/a degree 50 nM) L, its 1.51 f2 was injected into a scintillation vial. Immediately before injecting luciferin, the shutter of the photometer was opened, the injection point was set at 0 m1n, and after 1.5++ in, the shutter was closed and the luminescence was recorded during that time.

The light intensity was measured using 14C-hexadecane light as a standard, and the light intensity was converted into the number of photons per second [Hassing
stings J, W, ) et al., J, Opt, Soc, A
m.

53, 1410-1415.・1963. the result,
Little luciferase activity was detected in cell extracts of transfected CO8 cells. However, a large amount of luciferase activity was detected in the culture medium of CO8 cells (see Figure 4). The luminescence from this culture medium was directly proportional to the volume of the test medium, and the luminescence was extremely sensitive.

That is, a signal clearly higher than the background was detected from a sample of only 5 μQ obtained from culture medium 10 of Cos cells transfected with the Cypridina luciferase expression vector. In contrast, cultures of CO8 cells that were not transfected with DNA were transfected with psVOcAT [Gorman, C, M
, ) et al., Mo1. Cell, Biol, 2
．． 1044-1051. 1982] or pR8
vL [deWet, J, R, et al., Mo1. Ce11. Biol, Z., 725-73.
7.1987] was more than two orders of magnitude weaker than the luminescence shown in Figure 4.

The above results indicate that the reconstructed cDNA is the full-length Lamibota luciferase cDNA, and that the cDNA
This shows that A also encodes a signal sequence necessary for secreting the protein.

[Action] DNA encoding the Cypridina luciferase of the present invention
The compounds can be used to express firefly luciferase in a desired host. The chemiluminescence reaction between Cypridina luciferase and Cypridina luciferin is a simple reaction that requires only oxygen molecules. However, it has high substrate specificity and the sensitivity of luminescence measurement is sensitive. Furthermore, as shown in the Examples, CO8 cells transfected with the expression vector of the present invention secrete Cypridina luciferase into the culture medium, making it extremely easy to recover the product. These facts clearly demonstrate the usefulness of the DNA compound encoding Cypridina luciferase of the present invention. Therefore, the DNA compound of the present invention is believed to be useful not only in the biomedical field but also in various fields including the environment.

[Brief explanation of drawings]

FIG. 1 is a reproduction of a photograph showing the results of sodium dodecyl sulfate/gradient polyacrylamide gel electrophoresis of purified Cypridina luciferase. FIG. 2 shows a restriction enzyme map of the Cypridina luciferase cDNA and a restriction enzyme map of the clone used to construct the full-length clone. (a) Full-length Cypridina luciferase cDNA
, (b) clone VL16, (c) clone VL1
(d) is a restriction enzyme map showing the restriction enzyme sites of No. 3, and (d) is a restriction enzyme map of the full-length cDNA constructed from clones VL16 and VL18. FIG. 3 is a schematic diagram showing the nucleotide sequence and deduced amino acid sequence of Cypridina luciferase cDNA. Figure 4 shows CO8
1 is a graph showing measurement results of the activity of Cypridina luciferase synthesized and secreted by cells. Figure 5 shows sea worm luciferase (a) and jellyfish aequorin (
b) Schematic diagram showing homology of amino acid sequences.

Claims (1)

[Scope of Claims] 1. A DNA compound encoding Cypridina luciferase. 2. An expression vector containing the DNA compound according to claim 1. 3. The expression vector according to claim 2, which is a plasmid pRSVVL. 4. A method for producing Cypridina luciferase, which comprises transfecting host cells with the expression vector according to claim 2 or 3 and culturing them.