JP2001204474A - Cloning vector using green fluorescent protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new cloning vector capable of easily discriminating the presence or absence of the insertion of an extraneous DNA (insert). SOLUTION: This is a cloning vector that contains a DNA sequence encoding a green fluorescent protein(GFP) and a regulatory sequence controlling the expression of GFP, and has a cloning site in a DNA region encoding a helix structure.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a cloning vector using green fluorescent protein (GFP), and more particularly to a cloning site in a DNA sequence encoding GFP, and insertion of an insert into the cloning site. A cloning vector whose presence or absence can be determined by the presence or absence of fluorescence emission.

[0002]

2. Description of the Prior Art T having a thymidine at both 3'-termini.
-The extension vector (T-vector) has 3 'A-ov
erhang has been used as a useful tool for direct cloning of PCR products because it can be easily ligated to the PCR product (Mead et al., 1991, B
io / Technology 9, 657-663). All T-vectors reported to date utilize a blue-white screening system, which uses pUC (Sh
ort et al., 1988, Nucleic Acids Res. 16, 7583-760
0) and pBluescript (Yanisch-Perron et al., 1985,
Gene 33, 103-119) and other ordinary cloning vectors for E. coli. This blue-white screening system utilizes the inactivation of α-complementation of β-galactosidase in order to distinguish recombinant from non-recombinant, and is used as a chromogenic substrate for β-galactosidase. 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) is required. However, since the transformant may show an intermediate color between white and blue, it is sometimes difficult to distinguish a colony having a plasmid having an insert from a colony having no insert, especially when the insert is short. Was.

Green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria exhibits autofluorescence without the need for cofactors and / or substrates. GF
Since the P gene was cloned (Chalfie et a
l., 1994, Science 263, 802-805), GFP has been studied extensively and many applications have been studied (Tsien, 1998, A
nnu. Rev. Biochem. 67, 509-544). GFP, when fully folded, has many proteases, heat (65 ° C).
Robust and very stable against 1% sodium dodecyl sulfate or 8M urea (Cubitt et a
l., 1995, Biochem. Sci. 20, 448-455). According to X-ray crystallography (Ormo et al., 1996, Science 273, 1392-1
395; Yang et al., 1996, Nature Biotech. 14, 1246-1
251), the structure of GFP is cylindrical with 11-chain β-barrel and one short α-helix, inside which the fluorophore is located. This structure protects the fluorophore from quenching.

Have constructed pGreenscript A, a cloning vector using green fluorescent protein (GFP) as an indicator of the presence or absence of an insert (Inouye).
etal., 1997, Gene 189, 159-162). The multiple cloning site of this vector is located between the promoter / operator region and the GFP gene. pGreenscript
When A was used, 1.0 kb DN
It was possible to distinguish between the case where the A fragment was inserted (non-fluorescent on UV irradiation and white in sunlight) and the case where it was not inserted (green fluorescent on UV irradiation and yellow on sunlight).
However, with pGreenscript A, it was not so easy to determine the color of the colony, and it was difficult to accurately and simply determine whether or not an insert had been inserted. More recently, it has been reported that when a mutated GFP in which a mutation is introduced into the amino acid sequence of GFP is used, the fluorescence intensity upon excitation at 488 nm is increased (Ito et al., 1999,
Biochem. Biophys. Res. Commun. 264, 556-560). However, there is still a need to develop a novel cloning vector that can accurately and easily determine the presence or absence of insertion of an insert into a cloning site in a vector, particularly a T-vector suitable for cloning a PCR product. Was.

[0005]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the prior art.
That is, an object of the present invention is to provide a novel cloning vector that can easily determine the presence or absence of insertion of a foreign DNA (insert). Further, the present invention relates to P
The problem to be solved was to provide a novel T-vector capable of easily cloning a CR product.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, even if a short fragment, a foreign DNA fragment (insert) is converted into a β-sheet or When inserted at a site corresponding to the α-helix, due to sensitivity to structural perturbations (Abedi et al., 1998, Nucleic Acids Res. 26, 623-6
30), GFP was found to be non-fluorescent. Furthermore, the present inventors have shown that the efficiency of discriminating between fluorescent and non-fluorescent colonies was GFPuv4 (Ito et a
l., 1999, Biochem. Biophys. Res. Commun. 264, 556-5
60) was found to be improved by using a GFP mutant. The present invention has been completed based on these findings.

That is, according to the present invention, a DNA region containing a green fluorescent protein (GFP) -encoding DNA sequence and a regulatory sequence controlling GFP expression, and encoding a GFP β-sheet structure or α-helix structure, A cloning vector having a cloning site is provided.

[0008] Preferably, as the DNA sequence encoding GFP, a DNA sequence encoding a mutant GFP whose fluorescence intensity is higher than that of wild-type GFP is used. More preferably, as a DNA sequence encoding GFP, 6
The fourth amino acid is Leu, the 65th amino acid is Thr, the 99th amino acid is Ser,
The amino acid at position 153 is Thr, the amino acid at position 163 is Ala, and the amino acid at position 208 is Leu.
Is used.

[0009] Preferably, the cloning site is in a DNA region encoding a β-sheet structure region of GFP.
More preferably, the cloning site is at the fourth of the β-barrel.
Located on the strand. Preferably, the cloning site is a restriction enzyme site that forms a blunt end. Preferably, the regulatory sequence is a promoter capable of controlling the expression of GFP in E. coli. The cloning vector of the present invention preferably further has an origin of replication. The cloning vector of the present invention preferably further contains an antibiotic resistance gene.

[0010] The cloning vector of the present invention preferably contains green fluorescent protein (GFP), a lac promoter, an origin of replication derived from pBR322, and an antibiotic resistance gene, and encodes a GFP β-sheet structural region.
It has an EcoRV site as a cloning site in the NA region. An example of the cloning vector of the present invention is a cloning vector having the nucleotide sequence of SEQ ID NO: 1.

According to another aspect of the present invention, the cloning site of the above-described cloning vector of the present invention is cleaved with a restriction enzyme, and thymidine (T) is added to both 3 'ends of the obtained linear double-stranded vector. And a linear cloning vector obtained by combining

According to still another aspect of the present invention, there is provided an insert into a cloning site of a cloning vector of the present invention, which comprises determining the presence or absence of fluorescence of GFP generated by a host transformed with the cloning vector of the present invention. And a method for determining the presence or absence of the insertion of the. Preferably, in the present invention, the insert is a PCR product.

According to still another aspect of the present invention, there is provided a recombinant vector obtained by inserting an insert into the cloning site of the above-mentioned cloning vector of the present invention. Preferably in the present invention the insert is P
It is a CR product.

According to still another aspect of the present invention, there is provided a transformant obtained by transforming a cloning vector of the present invention or a recombinant vector of the present invention into a host. Preferably, the host is selected from the group consisting of bacterial cells, yeast cells, fungal cells, insect cells, nematode cells, plant cells or animal cells.

[0015]

DETAILED DESCRIPTION OF THE INVENTION Green Fluorescent Protein (GFP)
Is a kind of jellyfish, Aequorea Victoria
victoria) (luminescent jellyfish). GFP at Equalia Victoria
Is aequorin stimulated by calcium
Absorbs the energy produced by and emits green light. When excited by near-ultraviolet or blue light, GFP expressed in prokaryotic and eukaryotic cells emits strong green fluorescence. The amino acid sequence of GFP is known (Pracher, DC, et al., (1992) Gene, 111, 229-233). Since amino acid residues are usually encoded by a plurality of DNA triplets, there are many DNA sequences encoding the amino acid sequence of wild-type GFP or mutant GFP.
The GFP coding sequence in the cloning vector of the present invention may be any of these DNA sequences.

In the present invention, a mutant GFP can be used. In particular, it is preferable to use a mutant GFP whose green fluorescence intensity is higher than that of a wild-type GFP. Examples of such a mutant GFP include one or more (for example, 1 to several, specifically 1 to 30, preferably 1 to 20, more preferably 1 to 10) in the amino acid sequence of wild-type GFP. To 10 (particularly preferably 1 to 5) amino acids having an amino acid sequence in which a deletion, substitution, insertion and / or addition has been made. In the present specification, GFP is intended to include all mutant GFPs having green fluorescence.

As a method for obtaining a mutant GFP gene,
Any method known in the art such as a chemical synthesis method, a genetic engineering method, and a mutagenesis method can be used. Obtaining an available GFP gene, based on which a mutant GF
P DNA can be obtained. Mutagenesis into a gene can be performed in many ways. For example, the method can be carried out using a method of bringing into contact with a drug serving as a mutagen, a method of irradiating ultraviolet rays, a genetic engineering technique, or the like.
Site-directed mutagenesis, which is one of the genetic engineering techniques, is useful because it can introduce a specific mutation at a specific position, and is useful in Molecular Cloning, A Laboratory Manu.
al, Second Edition, Cold Spring Harbor Laboratory
Press (1989), Current Protocols in Molecular Biolo
gy, John Wiley & Sons (1987-1997) and the like.

Examples of suitable mutant GFPs for use in the present invention include, for example, Ito et al., 1999, Biochem. Biop.
hys. Res. Commun. 264, 556-560
P mutants, specifically, GFPuv (F99
S, M153T, V163A), GFPuv2 (F99
S, M153T, V163A, S208L), GFPu
v3 (F99S, M153T, V163A, F64L,
S65T), and GFPuv4 (F99S, M153
T, V163A, F64L, S65T, S208L) (in the above, parentheses indicate amino acid mutations relative to wild-type GFP; for example, F99S indicates that the 99th amino acid is replaced by F to S). .
Among the above mutant GFPs, GFPuv2 (F99
S, M153T, V163A, S208L), GFPu
v3 (F99S, M153T, V163A, F64L,
S65T), and GFPuv4 (F99S, M153
T, V163A, F64L, S65T, S208L) are more preferable, and GFPuv4 (F99S, M153T,
V163A, F64L, S65T, S208L) are particularly preferred.

The cloning vector of the present invention is GFP
Encoding a β-sheet structure or α-helix structure of
It has a cloning site in the A region.
The DNA region encoding the β-sheet structure of GFP corresponds to amino acids 11 to 23, 25 to 3 in natural wild-type GFP.
8, 40 to 48, 90 to 101, 103 to 114, 11
8-128, 143-154, 160-171, 175
188, 197 to 210, 215 to 228 (11-st
rands) and encodes an α-helix structure
A region is amino acid number 56-72 in natural wild-type GFP.
(1-strand) (Ormo et al., Science, 27
3, 1392-1395 (1996)). The vector GFPuv described above
In the case of No. 4, since there are 24 amino acids at the N-terminus, the DNA region encoding the β-sheet structure has amino acid numbers 35 to 47, 49 to 62, 64 to 72, 114 to
125, 127-137, 142-152, 167-1
78, 184-195, 199-212, 221-223
4, 239-252 (11-strands), and the DNA region encoding the α-helix structure is a native wild type GF
It is present at amino acid numbers 80 to 96 (1-strand) in P. The cloning site is preferably located in the DNA region encoding the β-sheet structural region, and particularly preferably
-Located on the fourth strand of the barrel. The type of restriction enzyme site constituting the cloning site is not particularly limited.
However, the cloning vector of the present invention can be suitably used as a T-vector, in which case a restriction enzyme site (for example, BsrB
I, HpaI, RsaI, ScaI, SfoI, SwaI, etc.) are preferably used as the cloning site. The cloning site (ie, restriction enzyme site) can be introduced into the GFP gene by using the above-mentioned recombinant gene technology. It is necessary to make a selection so that green fluorescence is impaired when DNA) is introduced. Preferred cloning sites include Ec
An oRV site can be mentioned. For example, an EcoRV site can be introduced into a nucleotide sequence encoding the 98th amino acid of GFP by mutagenesis of the nucleotide sequence.

The cloning vector of the present invention is GFP
And a regulatory sequence that controls the expression of A regulatory sequence means any regulatory sequence capable of expressing GFP in a host, and includes a promoter, an enhancer, and the like. The regulatory sequence is incorporated into the vector so as to be functionally linked to the GFP. More specifically, the regulatory sequence is located at a position capable of controlling the expression of the DNA sequence encoding GFP (usually upstream of the GFP sequence). ) And orientation in the vector. This regulatory sequence induces transcription of the DNA sequence encoding GFP.

The promoter may be any promoter as long as it can function in the host cell. For example, tr
p promoter (P trp), lac promoter (P lac), P _L promoter, P _R promoter and P _SE promoter,
Promoters derived from Escherichia coli, phage and the like, SP01 promoter, SP02 promoter, penP promoter and the like can be mentioned. Further, artificially designed and modified promoters such as a promoter in which two P trps are connected in series (P trp × 2), a tac promoter, a letl promoter, and a lacT7 promoter can also be used.

Examples of promoters that function in yeast include PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1, gal10 promoter, heat shock protein promoter,
Promoters such as MFα1 promoter and CUP1 promoter can be mentioned. Examples of promoters that function in animal cells include cytomegalovirus (human CM
V) promoter of the IE (immediate early) gene, SV40
Early promoter, retroviral promoter,
Examples include a metallothionein promoter, a heat shock promoter, and an SRα promoter. Further, an enhancer of the IE gene of human CMV may be used together with the promoter. When another host is used, a regulatory sequence (promoter or the like) that functions in the host can be appropriately selected.

The cloning vector of the present invention preferably contains an origin of replication. For example, when the host is Escherichia coli, an origin of replication derived from pBR322 can be selected, and when the host is yeast, ARS and the like can be selected.

The cloning vector of the present invention preferably contains an antibiotic resistance gene. By including the antibiotic resistance gene in the vector, untransformed host cells (which do not carry the vector) can be eliminated by culturing the transformant after the transformation operation in an antibiotic-containing medium. The type of the antibiotic resistance gene is not particularly limited, and examples thereof include an ampicillin resistance gene and a tetracycline resistance gene.

The cloning vector of the present invention can be produced as appropriate by those skilled in the art. For example, it can be produced by ligating a modified GFP gene to a commercially available cloning vector. Methods for introducing a cloning site into the GFP gene or introducing a mutation that enhances fluorescence intensity are known to those skilled in the art, and are specifically as described above in the present specification. In addition, mutant GFP
For a method for producing a vector having a gene, see Ito et al.
al., 1999, Biochem. Biophys. Res.Commun. 264, 556
The cloning vector of the present invention can also be produced according to the method described in the above-mentioned document.

The cloning vector of the present invention can be preferably used as a T-vector. A T-vector is a vector having one base of thymidine (T) at both 3 'ends of a double-stranded DNA having blunt ends.
It is a vector useful for directly cloning CR products. When the cloning vector of the present invention is used as a T-vector, a cloning site having a blunt end (for example, an EcoRV site) is treated with a restriction enzyme,
After forming a linear double-stranded DNA having blunt ends,
T (thymidine) may be bound to both 3 'ends.
The binding of thymidine to the 3 'end can be carried out by incubating with a suitable DNA polymerase such as Taq polymerase in a suitable buffer containing dTTP.

The present invention further provides a transformant obtained by transforming a host with the above-described cloning vector of the present invention or a recombinant vector having an insert inserted into the cloning site of the cloning vector. The type of host used in the present invention is not particularly limited, but is preferably selected from bacterial cells, yeast cells, fungal cells, insect cells, nematode cells, plant cells or animal cells.

As bacterial cells and fungal cells, Escherichi
genus a, Corynebacterium, Brevibacterium, Bacillus
Genus, Microbacterium, Serratia, Pseudomonas, A
genus grobacterium, Alicyclobacillus, Anabaena, An
acystis, Arthrobacter, Azobacter, Chromatium
Genera, Erwinia, Methylobacterium, Phormidium, R
genus hodobacter, Rhodopseudomonas, Rhodospiri11um
Genus, Scenedesmun, Streptomyces, Synnecoccus,
A microorganism belonging to the genus Zymomonas and the like can be mentioned, and a microorganism belonging to the genus Escherichia is preferable. Escheri
Specific examples of the genus chia include, for example, Escherichia coli XL1
-Blue, XL2-Blue, DH1, DH5α, MC1000, KY3
276, same W1485, same JM109, same HB101, same No49, same W3110,
NY49, MP347 and NM522.

Examples of yeast cells include Saccharomyces cerevisae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullonias, and Saccharomyces cerevisae. Schwanniomyces a11uvius) and the like. Animal cells include Vero cells, HeLa cells, CV1 cells, Namalva cells, COS1 cells, COS7 cells, CHO cells, as well as various vertebrate, invertebrate, and mammalian cells. As insect cells, Sf9, Sf21 which are ovarian cells of Spodoptera frugiperda
(Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company
ndCompany), New York (New York), (1992)), Tric
Hoplusia ni ovary cells, eg, High5 (manufactured by Invitrogen) can be used.

A method for introducing a cloning vector or a recombinant vector into a host can be appropriately selected depending on the type of the host and the like. For example, calcium phosphate method, protoplast method, electroporation method,
The vector can be introduced into a host by appropriately selecting a spheroblast method, a lithium acetate method, a lipofection method, a microinjection method, or the like.

By using a transformant into which a vector has been introduced by the above-described method, it is possible to determine whether or not the vector has an insert at the cloning site by determining the presence or absence of green fluorescence. That is, the present invention provides a method for determining whether or not an insert has been inserted into a cloning site of a cloning vector of the present invention by determining the presence or absence of GFP fluorescence emitted by a host transformed with the cloning vector of the present invention.

Since the cloning site of the cloning vector of the present invention exists in the region of the GFP gene, when the insert (foreign DNA) is inserted into the cloning site, the expressed protein changes in the protein structure due to the presence of the insert. No longer emits fluorescence.
On the other hand, a vector containing no insert exhibits the fluorescence originally retained by GFP, and thus a transformant carrying such a vector emits green fluorescence. Therefore, by determining the presence or absence of green fluorescence, it can be determined whether or not the insert has been inserted into the cloning site of the vector in the transformant. The present invention will be specifically described by the following examples, but the present invention is not limited to the examples.

[0033]

EXAMPLES Example 1 Preparation of T-Expansion Vector (also referred to as T-vector) EcoR digested to blunt end on GFPuv4 gene
To create a V site (see FIG. 1A), a point mutation was made to pGFPgcn4 (ItIt) with the gene for GFPuv4.
o et al., 1999, Biochem. Biophys. Res. Commun. 26
4, 556-560)).
FPTA was manufactured. SEQ ID NO: 1 shows the nucleotide sequence of pGFPTA. The T-vector is described by Marchuk et al.
uk et al., 1991, Nucleic Acids Res. 19, 1154). Plasmid pGFPTA was replaced with EcoRV
And in buffer (67 mM Tris pH 8.8, 2.5
mM MgCl ₂ , 16.6 mM (NH ₄ ) ₂ SO ₄ , 0.45% TritonX-100,
0.2 mg / ml gelatin and 2 mM dTTP), Taq polymerase (Greiner, 2 units / μg plasmid DNA /
(50 μl) at 72 ° C. for 3 hours. The T-vector obtained by desalting using a gel filtration column (NICK column, Pharmacia) was used for the following cloning.

FIG. 1 shows the plasmids pGFPTA and T-
2 shows the construction of the vector. FIG. 1A shows a plasmid map of pGFPTA. EcoR of GFPuv4 gene
The V site was prepared as follows. Forward primer (for
5′-GAAGGTTATGTAC which is a ward primer)
AGGAACGCAC GATATC T-3 '(SEQ ID NO: 2) and 5'-TAGTTTG which is a reverse primer
PCR using TACTCGAGTTTGTG-3 ′ (SEQ ID NO: 3) (94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 1 second)
(15 cycles per minute). The amplified fragment was digested with prs using BrsGI and XhoI sites.
It was cloned into GFPgcn4.

FIG. 1B shows the pGFPTA Eco.
The sequence around the RV site is shown. Number of the amino acid position of GFP from the N-terminus (top) (where the numbering refers to wild-type G
FP numbering), the amino acid sequence of GFP (middle), and the base sequence (lower). The arrow is EcoR
The V cleavage site is shown. FIG. 1C shows the sequence around the end of the T-vector prepared in Example 1. T in bold is T
Represents thymidine added by aq polymerase.
The shade is the 5'-F used to sequence the inserted PCR product.
Shows the sequence of the ITC primer.

Example 2 Green-White Screening and Insertion Check A 100 bp PCR product with a random sequence was used as an insert, which is commonly used in evolutionary molecular engineering (Ellington et al., 1990, Nature 34
6, 818-822; Eli et al., 1999, J. Biochem. 125, 790
-794). The PCR product was ligated to the T-vector prepared in Example 1, and the resulting recombinant vector was used to transform Escherichia coli strain DH5α. This transformant was incubated at 37 ° C. for 19 hours on LB medium supplemented with ampicillin (75 μg / ml). The fluorescence of the transformed colonies on the plate was observed under sunlight. To confirm that the non-fluorescent clone had the insert, a clone plasmid was prepared in small quantities and digested with XhoI or MluI and BsrGI. These sites are located on either side of the EcoRV site. Restriction digests were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining, or 6%
Inspection was performed using polyacrylamide gel electrophoresis and silver staining. The insert was sequenced using a DNA sequencer (DSQ2000-L, Shimazu) using either of the two primers (FIG. 1C) labeled at the 5 ′ end with fluorescein isothiocyanate (FITC).

Example 3 Comparison of Discrimination Efficiency with β-Galactosidase System As an insert, 60b located next to the primer was used.
A 100 bp PCR product with p random sequences was used (Eli et al., 1999, J. Biochem. 125, 790-79).
Four). Original TA cloning as β-galactosidase system
A ning kit (Invitrogen) was used. The cloning method described by the manufacturer was followed. After incubation at 37 ° C for 19 hours (GFP system) or after incubation at 37 ° C for 18 hours and storage at 4 ° C for 18 hours (β
-Galactosidase system), the color of the colonies was identified. Inserts were tested by a PCR modified method using colonies directly as templates. The fragment containing the insert was
PCR amplification using both C-labeled sequencing primers (FIG. 1C) (94 ° C. for 30 seconds, 55 ° C. for 30 seconds and
30 cycles of 1 minute at 72 ° C). The amplified fragment was electrophoresed on a 1.5% agarose gel and detected using an image analyzer (Molecular Imager FX, Bio-Rad).

Results of Examples: Colonies expressing GFPuv4 could be clearly distinguished from non-expressing colonies under normal light (FIG. 2A). The GFPuv4 gene is
Under the control of the lac promoter. After digestion of plasmid pGFPTA with EcoRV, the expected single band was observed on 0.8% agarose gel electrophoresis. The T-vector was then prepared as described in Method 1 above. In order to examine the performance of the T-vector of the present invention, the results of cloning using the T-vector
-Compared to the results for the galactosidase system. 100 bp PCR with random sequence as test insert
The product was used, which is commonly used in evolutionary molecular engineering (Ellington et al., 1990, Nature 346,
818-822; Eli et al., 1999, J. Biochem. 125, 790-79
Four). When this fragment is inserted into a T-vector, no frameshift occurs and the primer region does not contain a stop codon. The transformed colony containing this T-vector is shown in FIG. 2B. Transformant colonies had only two colors (green fluorescence and white). Transformants with green fluorescence appeared with a probability greater than 0.2, depending on the number of cycles of freezing and thawing of the T-vector. On the other hand, there were many colors of β-galactosidase colonies. Few white colonies appeared (data not shown).

Next, the correlation between the color of the clone and the presence of the insert was examined. The results are shown in Table 1 below. In a fluorescent green-white screening system, a randomly selected 1
All 14 fluorescent clones did not contain the insert (data not shown). On the other hand, the non-fluorescent clone
There was a 4% chance of including an insert. In the blue-white screening system, clones classified into three types of blue, white and neutral colors were randomly examined. However, some of these three categories of colonies, even though blue, had inserts.

[0040]

[Table 1]

To confirm whether a fluorescent green-white screening system could be used even with longer inserts, a 0.9 kb PCR product was cloned. The color of this transformant was also clearly classified into only two colors, green fluorescence and white. Non-fluorescent clones contained the insert with a 99% probability (only 4 out of 339 clones did not contain the insert). The insert was sequenced using the 5'-FITC primer. These primer sites are Eco
Located on both sides of the RV site (FIG. 1C). Both the 100 bp and 0.9 kb inserts could be sequenced without problems, as in the β-galactosidase system.

(Discussion) The T-vector of the present invention utilizes GFP insertion inactivation in order to distinguish a recombinant from a non-recombinant. The insertion site for GFP is at amino acid position 98 (the number of this amino acid residue is based on the number of wild-type GFP; see FIG. 1B). According to the structure of GFP (Ormo et al., 1996, Science 273, 1392-139).
5; Yang et al., 1996, Nature Biotech. 14, 1246-125
1), this insertion site is located on the fourth strand of the β-barrel.

To evaluate the T-vector prepared in Example 1, 0.9 kb and 10 kb
The 0 bp PCR product was used as insert. In both cases, the transformed colonies expressing GFPuv4 emitted bright green fluorescence as shown in FIG. 2A, making it easy to distinguish between green and non-fluorescent colonies even in sunlight. there were. The reason for the presence of green fluorescent colonies was that thymidine was not added to the 3'-end of the linearized plasmid (or during repeated thawing and thawing, thymidine at the 3'-end of the T-vector). Has been deleted), or has been restored to pGFPTA in the ligation step. On the other hand, some white colonies without insert were observed. It is considered that such a result is caused by the fact that GFP fluorescence disappears due to mutation, or that GFP fluorescence disappears due to insertion of thymidine into the cloning site.

10 containing a random sequence as insert
When the 0 bp PCR product was used, transformants were classified into only two color types from the fluorescent green-white screening system, and the green colonies had no insert. On the other hand, from the blue-white screening system, the transformants had various colony colors, and some blue colonies had inserts (Table 1). This means
At the normal insertion site, the GFP system was shown to be more susceptible to insertion inactivation than the β-galactosidase system.

[0045]

According to the present invention, a novel T-vector, which is particularly useful for cloning PCR products, has been developed. The T-vector of the present invention can be easily prepared. Using the MscI site located at the end of the short α-helix (Ormo et al., 1996, Science 273, 1392-1395; Yan
g et al., 1996, Nature Biotech. 14, 1246-1251),
Other T-vectors can be made. Compared to the blue-white screening method, the fluorescent green-white screening
Has two major advantages. The first advantage is that transformants containing the PCR product can be easily selected even if the PCR product is short, and the second advantage is that no cofactor such as X-gal is required at all. That is. Due to this advantage, when a clone having no insert is cultured, a clone having no insert can be easily identified by distinguishing the bright green fluorescence of the medium.

[0046]

[Sequence List] SEQUENCE LISTING <110> GenCom <120> A cloning vector using a green fluorescent protein <130> A01027MA <160> 3

<210> 1 <211> 3249 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNA constructed from DNAs with different origin <400> 1 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 60 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 120 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa 180 gcttgcatgc ctgcaggtcg actctagagg atccccgggt accggtagaa aaaatgagta 240 aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta 300 atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta 360 cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta 420 ctctgacgta tggtgttcaa tgcttttccc gttatccgga tcatatgaaa cggcatgact 480 ttttcaagag tgccatgccc gaaggttatg tacaggaacg cacgatatct ttcaaagatg 540 acgggaacta caagacgcgt gctgaagtca agtttgaagg tgataccctt gttaatcgta 600 tcgagttaaa aggtattgat tttaaagaag atggaaacat tctcggacac aaactcgagt 660 acaactataa ctcacacaat gtatacatca cggcagacaa acaaaagaa t ggaatcaaag 720 ctaacttcaa aattcgccac aacattgaag atggatccgt tcaactagca gaccattatc 780 aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcga 840 cacaatctgc ccttttgaaa gatcccaacg aaaagcgtga ccacatggtc cttcttgagt 900 ttgtaactgc tgctgggatt acacatggca tggatgagct ctacaaataa tgaattcttg 960 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt 1020 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 1080 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 1140 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 1200 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 1260 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 1320 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 1380 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 1440 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 1500 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcgg c 1560 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 1620 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 1680 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac 1740 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 1800 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 1860 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 1920 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 1980 acagatcgct gagataggtg cgtcactgat taagcattgg taactgtcag accaagttta 2040 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 2100 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 2160 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 2220 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 2280 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 2340 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 2400 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 2460 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 2520 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 2580 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 2640 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 2700 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 2760 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 2820 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 2880 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 2940 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg 3000 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 3060 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg 3120 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 3180 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 3240 gcgag gcag 3249

<210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 2 gaaggttatg tacaggaacg cacgatatct 30

<210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 3 tagttgtact cgagtttgtg 20

[Brief description of the drawings]

FIG. 1 shows the construction of plasmids pGFPTA and T-vector.

FIG. 2 shows fluorescent and non-fluorescent E. coli under normal light. 1 shows a photograph of an E. coli DH5α colony. (A) of FIG. 2 shows pGFPgc in the promoter of GFP.
indicating a colony having n4 or a deletion mutant thereof,
FIG. 2 (B) shows the T-vector prepared in Example 1 and 10
A colony of a transformant having a ligation product with a 0 bp PCR product is shown. Both plates are ampicillin (75 μg / m
Incubated at 37 ° C. for 19 hours on LB medium supplemented with l). Green and white colonies can be more easily distinguished in the laboratory because green colonies emit light, while white colonies reflect light.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/68 C12N 5/00 A F term (Reference) 4B024 AA20 DA01 DA02 DA06 DA11 DA12 EA04 FA10 4B063 QA11 QQ07 QQ08 QQ09 QQ13 QQ43 QQ49 QR08 QR59 QR62 QS05 QS25 QS36 QX02 4B065 AA01X AA72X AA89X AA90X AB01 BA02 BA25 CA41

Claims (18)

[Claims] 1. A cloning vector comprising a DNA sequence encoding a green fluorescent protein (GFP) and a regulatory sequence controlling the expression of GFP, and having a cloning site in a DNA region encoding a β-sheet structure or α-helix structure of GFP. . 2. A DNA sequence encoding GFP,
Mutant GF whose fluorescence intensity is higher than wild-type GFP
The cloning vector according to claim 1, wherein a DNA sequence encoding P is used. 3. A DNA sequence encoding GFP,
The amino acid at position 64 is Leu, the amino acid at position 65 is Thr, the amino acid at position 99 is Ser, the amino acid at position 153 is Thr, the amino acid at position 163 is Ala, and the amino acid at position 208 is amino acid. L
The cloning vector according to claim 2, wherein a mutant GFP that is eu is used. 4. The cloning vector according to claim 1, wherein the cloning site is present in a DNA region encoding a GFP β-sheet structure region. 5. The cloning vector according to claim 4, wherein the cloning site is located on the fourth strand of the β-barrel. 6. The cloning vector according to claim 1, wherein the cloning site is a restriction enzyme site forming a blunt end. 7. The control sequence according to claim 1, wherein the regulatory sequence is a promoter capable of controlling GFP expression in E. coli.
7. The cloning vector according to any one of items 1 to 6. 8. The cloning vector according to claim 1, further comprising an origin of replication. 9. The composition further comprising an antibiotic resistance gene.
The cloning vector according to any one of claims 1 to 8. 10. Green fluorescent protein (GFP), la
c promoter, an origin of replication derived from pBR322, and an antibiotic resistance gene, and a cloning site Ec
A cloning vector having an oRV site. 11. A cloning vector having the nucleotide sequence of SEQ ID NO: 1. 12. The cloning site of the cloning vector according to any one of claims 1 to 11, which is cleaved with a restriction enzyme, and thymidine (T) is added to both 3 ′ ends of the obtained linear double-stranded vector. And a linear cloning vector obtained by combining 13. A method of inserting an insert into a cloning site of a cloning vector according to any one of claims 1 to 12, comprising determining the presence or absence of fluorescence of GFP generated by a host transformed with the cloning vector according to any one of claims 1 to 12. A method for determining the presence or absence of insertion. 14. The method according to claim 12, wherein the insert is a PCR product. A recombinant vector obtained by incorporating an insert into a cloning site of the cloning vector according to any one of claims 1 to 12. 16. The recombinant vector according to claim 15, wherein the insert is a PCR product. 17. A transformant obtained by transforming a cloning vector according to any one of claims 1 to 12 or a recombinant vector according to claim 15 or 16 into a host. 18. The transformant according to claim 17, wherein the host is selected from the group consisting of a bacterial cell, a yeast cell, a fungal cell, an insect cell, a nematode cell, a plant cell or an animal cell.