JP2003250543A - Method for preparing fusion gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for obtaining a fusion gene in extremely high efficiency without restriction of the kind of the gene of a fusion target, producing a fusion gene group having various fusion forms and/or producing a fusion gene capable of exhibiting desired properties or new properties, and a means for producing a useful substance by the means. <P>SOLUTION: A vector for preparing the fusion gene contains a nucleic acid to which at least two kinds of fusion target polynucleotides are linked through a gene associated with host growth. The method for preparing the fusion gene comprises using the vector. The fusion gene is obtained by the preparation method. A fusion polypeptide is encoded with the fusion gene. The method for producing the useful substance comprises using the fusion polypeptide. A kit for preparing the fusion gene is a kit for carrying out the preparation method and contains the vector and/or a host having function inconsistent with the function of the polypeptide encoded with the gene associated with the growth of the host on the vector. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to means for preparing fusion genes. More specifically, the present invention provides a method for preparing a fusion gene, a fusion gene preparation vector and a fusion gene preparation kit used therefor, which can obtain a fusion gene from two genes that are fusion targets and have different origins. The present invention relates to a fusion gene obtainable by the method, a fusion polypeptide encoded by the fusion gene, and a method for producing a substance by the fusion polypeptide.

[0002]

2. Description of the Related Art Currently known gene fusion methods are
The method is classified into a) a method performed in vivo using biological functions of Escherichia coli, yeast, etc., and b) a method performed in vitro using a thermostable DNA polymerase.

As the method a), for example, sugar
(Satoh) et al. [Biochemistr
y), 38, 1531-1536, (1999)] and Kim et al. [Journal of Biological Chemistry.
(J. Biol. Chem), 269, 28724-28731, (1994)] and Mukaihara et al. [Genetics, 145, 563-572 (1997)]. The methods described may be mentioned.

The method of Kim described above is a plasmid in which two genes A and B having high homology are arranged and which can be replicated in a host (E. coli, yeast, etc.), and the plasmid is used as gene A. Cleavage between B and B to obtain a linear fragment, and using the linear fragment obtained above to transform the host, the genes A and B This is a method in which a fusion gene is formed by homologous recombination between the two, and a plasmid which is recircularized and can be replicated in the host is selectively obtained. However, the method of Kim et al. 1) has a low frequency of recircularization by homologous recombination in the host.
(2000 colonies per 1 μg of DNA), 2) It is not convenient in terms of sample preparation because several μg of DNA is required per transformation, 3) Genes A and B Since the fusion of is due to homologous recombination,
Since the fusion points of genes are unevenly distributed in the region having the highest homology between both genes, it is difficult to obtain various fusion genes.

The method of Mukaihara et al.
This is a method using Escherichia coli having a mutation in the single-stranded DNA binding protein gene (ssb). According to the method of Mukaihara et al., High gene fusion efficiency can be obtained. However, in the method of Mukaihara et al., For example, because gene fusion cannot be discriminated due to life or death of the host, the gene to be fused and the β-galactosidase gene (lacZ) are fused, and the expression of β-galactosidase activity is used as an index for fusion. It is necessary to detect the formation of genes. Furthermore, when the gene to be fused and lacZ are fused, it is necessary to partially modify the gene to be fused. Therefore, in the method of Mukaihara et al., 1) it is essential that the gene to be fused is a gene that is transcribed and translated in E. coli, and other genes, for example, gram-positive bacteria, phages, fungi, plants , Animal-derived genes need to be modified so that they can be transcribed and translated in E. coli, 2) it is difficult to evaluate the activity of the fusion gene itself, 3) the gene product is lethal to the host The gene is
It has the drawback of being unsuitable as a gene to be fused.

On the other hand, examples of the method b) include the method described in US Pat. No. 6,132,970 [Stemmer et al.]. Since the method of Stenmer et al. 1) uses a thermostable DNA polymerase, a replication error such as base substitution may occur at a high frequency in the process of replicating a gene. 2) It is necessary to transcribe / translate the fusion gene in the host and select the fusion gene based on the activity of the product.
A gene whose gene product is lethal to the host is not suitable as a fusion gene.3) It is possible to separate the fusion gene obtained from the fusion gene that serves as a template for replication in vitro. Since it is difficult, it is difficult to eliminate the contamination of the gene to be fused. 4) The reaction conditions of thermostable DNA polymerase differ depending on the nucleotide sequence of the gene to be fused, and the fusion pattern also changes depending on the condition settings. However, there is a drawback that the convenience is poor.

[0007]

The present invention is not limited to the types of genes to be fused, obtains a fusion gene with extremely high efficiency, creates a fusion gene group with various fusion modes, and / or desires. It is an object of the present invention to provide a means capable of creating a fusion gene capable of exhibiting the above-mentioned property or a novel property, and a means for producing a useful substance thereby. Specifically, the first object of the present invention is to provide a fusion gene preparation vector capable of obtaining a fusion gene group with high efficiency and various fusion patterns. A second object of the present invention is to obtain a fusion gene group with high efficiency and diverse fusion patterns, to obtain a fusion gene without causing a replication error in the sequence of the obtained fusion gene, and to obtain the fusion gene. The present invention provides a method for preparing a fusion gene, which makes it possible to easily produce a fusion gene and / or to create a novel fusion gene that does not exist in nature without being limited by the type of gene to be fused. . A third object of the present invention is to provide a fusion gene which has various fusion patterns and does not exist in nature. A fourth object of the present invention is to provide a fusion polypeptide which can exhibit various functions that can be produced from the fusion gene. further,
A fifth object of the present invention is to provide a method for producing a substance using the fusion polypeptide, which can achieve desired efficiency, conditions and the like. Further, a sixth object of the present invention is to provide a kit for preparing a fusion gene, which enables the method for preparing the fusion gene to be carried out simply and efficiently.

[0008]

That is, the present invention is
[1] A fusion gene preparation vector comprising a nucleic acid to which at least two kinds of polynucleotides to be fused are linked via a gene related to host growth (referred to as a marker gene), [2], [1] ] The method for preparing a fusion gene, which comprises using the vector for preparing a fusion gene according to [3], [3] a fusion gene obtained by the method for preparing a fusion gene according to [2] above, [4] above [3]
A fusion polypeptide encoded by the fusion gene described in [5], [5] a method for producing a substance characterized by using the fusion polypeptide described in [4] above, and [6] preparation of the fusion gene described in [2] above. A kit for carrying out the method, comprising: (a) a vector for preparing a fusion gene according to the above [1], and / or (b) a gene associated with the growth of a host on the vector (referred to as a marker gene). And a host having a function contradictory to the function of the polypeptide described above.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a drug acts on a wild-type ribosomal protein to exhibit drug sensitivity, while
Mutation in the gene encoding the wild-type ribosomal protein (hereinafter referred to as wild-type ribosomal gene) produces a mutant ribosome in the host, and the mechanism of exhibiting the drug resistance is high in the formation of the fusion gene due to deletion. The surprising findings of the present inventors that they exhibit sensitivity and high selectivity for cells having a fusion gene, and a plasmid in which the wild-type ribosomal gene and two genes to be fused are arranged before and after the wild-type ribosomal gene. Based on the surprising finding of the present inventors that a fusion gene with the gene to be fused can be efficiently obtained by selecting a drug using the above method.

The fusion gene preparation vector of the present invention contains a nucleic acid in which at least two kinds of polynucleotides to be fused are linked via a gene related to the growth of the host (referred to as a marker gene). There are features. Therefore, the fusion gene preparation vector of the present invention exhibits an excellent effect that it is possible to prepare a fusion gene group having various fusion patterns with high efficiency. In addition, since the fusion gene preparation vector of the present invention exhibits high selectivity for the formation of the fusion gene, it has been difficult to prepare the fusion gene in the past, and it has a lethal property and function to the host. Polynucleotides encoding the peptides can also be fusion targets. In the fusion gene preparation vector of the present invention, the sequence in which two types of fusion control polynucleotides are linked via the marker gene may be a sequence having a structure repeated twice or more.

The fusion gene preparation vector of the present invention includes, for example, a fusion target polynucleotide A, a marker gene C, and a fusion target polynucleotide B having a continuous sequence homologous to the polynucleotide A. A vector containing a nucleic acid consisting of and a nucleic acid arranged in the order of A-C-B (hereinafter, also referred to as "nucleic acid A-C-B"); The fusion target polynucleotides a ₁ , a ₂ ... And the marker genes α, β ... Are a ₁ -α-a ₂ -β ...
Examples include vectors containing nucleic acids arranged in this order. Here, the marker genes α, β ...
* Is a different gene, respectively.

The polynucleotide to be fused, for example, the polynucleotides A and B, encode a polypeptide having any property and function as long as the polynucleotides have a homologous and continuous sequence. Good, but not particularly limited, for example, interleukins, lymphokines, hormones, peroxidase, esterase, lipase, immunoglobulins, hormone receptors, polynucleotides encoding polypeptides having various physiological activities such as polymerase, phospholipase, nuclease, protease. , Lysozyme, Ced, and the like, and polynucleotides encoding polypeptides having lethal properties and functions for the host.

Further, it is desirable that at least two kinds of polynucleotides to be fused, for example, the polynucleotide A and the polynucleotide B to be fused have homology as a whole or identity in a specific region.

In the present specification, the term "whole" means that at least two kinds of polynucleotides to be fused are aligned with each other, for example, the polynucleotides A and B to be fused are properly aligned. It means that the entire comparison target area in the case of being included is also included. That is, when comparing at least two types of fusion target polynucleotides, for example, fusion target polynucleotide A and polynucleotide B, a sequence gap may be present. Here, the alignment is performed by, for example, the homology alignment algorithm of Needleman et al. [J. Mol. Biol., 48, 443 (1970)].
And so on. In the present invention, proper state alignment can be achieved by various kinds of software based on an algorithm suitable for sequence alignment such as the aforementioned algorithm. The software is not particularly limited, but examples thereof include BLAST. The BLAST is, for example, a web page http: // www. ncbi. nlm. nih. g
ov / BLAST / and sub sites linked to it.

In the present specification, the term "specific area" is used.
Is a partial sequence that is the same between at least two types of polynucleotides to be fused, for example, at least 2
A region having a nucleotide length, preferably at least 3 nucleotides, more preferably 10 nucleotides or more, and even more preferably 30 nucleotides or more can be mentioned. From the viewpoint of gene fusion efficiency, the size of the specific region is preferably 10 nucleotides or more.

In the present invention, at least two kinds of fusion target polynucleotides, for example, homology in the case where the fusion target polynucleotide A and the polynucleotide B are properly aligned, is the homology in terms of gene fusion efficiency. At least 10%, preferably 15% or more, more preferably 20% or more, more preferably 6
It is desirable to be 5% or more, and more preferably 80% or more. Specifically, at least two types of fusion control polynucleotides, for example, in each of the fusion target polynucleotide A and polynucleotide B, a region in which at least 3 consecutive nucleotides have the same length,
Examples of the fusion target used in the present invention include polynucleotides that have at least 10 contiguous nucleotides having the same length and have a homology of at least 30% over the entire sequence aligned by BLAST. The polynucleotide is not limited to such an example.

As the above-mentioned "gene related to the growth of the host", a gene capable of responding to a drug or a foreign invader, a polynucleotide encoding a polypeptide capable of responding to the drug, and an assimilation essential for the growth of the host Genes related to sex,
Examples thereof include a polynucleotide encoding a polypeptide that is essential for assimilation for host growth. In addition, in the present specification, the “gene related to the growth of the host” is also simply referred to as a marker gene.

The drug or foreign invader is not particularly limited as long as it inhibits the growth of the host, but is not limited to
[Escherichia coli and Sarmoella
nella), 2nd edition, 2532-2572, published by ASM Press (1996)], such as streptomycin, specthiomycin, rifampicin, nalidixic acid,
Phage etc. are mentioned. From the viewpoint of selectivity, streptomycin and spectomycin are desirable.

Examples of the marker gene include a polynucleotide encoding a polypeptide sensitive to the above drug (hereinafter referred to as "drug sensitivity marker gene"), rpsL, rpsE, rpoB, gyrA and the like. From the viewpoint of selectivity, a drug sensitivity marker gene is preferable.

In the present specification, the term “(is) sensitive to (drug)” means that when the drug sensitivity marker gene is introduced into an appropriate host, it can grow in the host in the presence of the drug. The expression of the property of disappearing or the property of suppressing growth.

The drug susceptibility marker gene includes
For example, rpsL, rpsE (hereinafter, referred to as “wild-type ribosomal gene”), which are genes encoding wild-type ribosomal proteins, and the like can be mentioned. From the viewpoint of sensitivity and selectivity of detection in forming a fusion gene, Type ribosomal genes are preferred.

As the wild-type ribosomal gene, for example, for streptomycin, ribosomal S1
RpsL gene encoding 2 subunits [Genebank accession number: AF312717 and AAG30937], and specthiomycin, rpsE gene encoding ribosomal S5 subunit [Genebank accession number: AAC76328], etc. Is mentioned. In the present invention, the rpsL gene and the rpsE gene are preferable from the viewpoints of detection sensitivity and selectivity when forming a fusion gene.

The fusion gene preparation vector of the present invention comprises a nucleic acid to which at least two kinds of polynucleotides to be fused are linked via a marker gene, and any vector capable of replicating in an appropriate host, such as a plasmid, It consists of phages and viruses. Therefore, for example, one of the polynucleotides to be fused, the marker gene and the other polynucleotide to be fused, and any plasmid that can be replicated in a suitable host can be used in Molecular Cloning Laboratory Manual Third Edition [Zanbruk ( Sambrook) et al., Molecular Cloning: A Labora
tory Manual 3rded., Cold Spring Harbor Laboratory
The fusion gene preparation vector of the present invention can be constructed by ligation by a conventional genetic engineering method described in, for example, Issuance, (2001)].

The "at least two polynucleotides to be fused" are preferably arranged in the same transcription and translation directions.

The "appropriate host" may be any host having a function that is contrary to the function of the polypeptide encoded by the marker gene, and may be either a prokaryotic cell or a eukaryotic cell. Specifically, examples of the host include Escherichia coli, yeast, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella, and the like. From the viewpoints of simplicity and safety, Escherichia coli and yeast are preferable.

[0026] Examples of the "host having a function contradictory to the function of the polypeptide encoded by the marker gene" include, for example, when the marker gene is the drug-sensitive marker gene, the drug corresponding to the marker gene is Hosts that are resistant to the same are mentioned. Specifically, when the marker gene is the wild-type ribosomal gene, a host having a gene encoding a drug-insensitive ribosomal protein (referred to as drug-insensitive ribosomal gene) is desirable. More specifically, the drug-insensitive rpsL gene, the drug-insensitive rpsE gene, the drug-insensitive rpoB gene, the drug-insensitive gylA gene and the like can be mentioned.

In the present specification, the term "drug-insensitive ribosome gene" refers to a polynucleotide having a sequence in the wild-type ribosome gene that has a mutation that inhibits the action of a drug on the ribosome. For example, in the case of the drug insensitive rpsL gene, the gene bank
(GenBank) Accession number: AF312717 Amino acid sequence encoded by the nucleotide sequence shown in [GenBank
(GenBank) Accession number: AAG30937]
Substitution of Lys for Arg at amino acid number: 43, Gene Bank
(GenBank) In the base sequence shown in the accession number: AF312717, one having a mutation in the substitution of A for G at base number: 128 and the like can be mentioned.

From the viewpoint of obtaining a fusion gene group of various fusion modes with higher efficiency, the above-mentioned host has a mutation with an increased deletion frequency, specifically, for example, single-stranded DNA.
A host having a mutation in the binding protein gene is preferable,
A host having the ssb-3 mutation, which is one of the aryl mutations in the single-stranded DNA binding protein gene, is more preferable. Specifically, Escherichia coli MK1019 strain is suitable. The E. coli MK1019 strain is Escherichia
Named and displayed as coli MK1019, accession number: FERM
P-18742, National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center (Postal code 305-8566)
It has been deposited on February 27, 2002 at 1-chome, 1-chome, 1-chome, Tsukuba-shi, Ibaraki prefecture, 6th central).

Vectors that can be replicated in a suitable host include, for example, pBR322 and pUC19 when the host is Escherichia coli.
, PHSG396 (Takara Shuzo), pHSG399 (Takara Shuzo), pCY
Examples include conventional plasmid vectors such as C184 and derivatives thereof, and examples include conventional plasmid vectors such as pYAC derivatives when the host is yeast. In addition to the commonly used vector, a vector constructed from an appropriate origin of replication, cloning site, etc. may be used as long as it does not impair the object of the present invention.

The fusion gene preparation vector of the present invention provides a method for preparing a fusion gene. The fusion gene preparation method of the present invention is characterized by using a fusion gene preparation vector. Therefore, according to the preparation method of the present invention, without being limited to the type of gene to be fused, without causing a replication error in the sequence of the obtained fusion gene, high efficiency, and a variety of fusion modes, It exhibits an excellent effect that a novel fusion gene group that does not exist in nature can be easily produced.
The preparation method of the present invention can be applied to protein functional analysis, substance production, medical fields, and the like.

Specifically, the preparation method of the present invention comprises (a)
Transforming the host with the fusion gene preparation vector to obtain a transformant expressing a polypeptide encoded by the marker gene on the vector; and (b) in the step (a) The obtained transformant is cultured in the presence of a drug corresponding to the marker gene, and the presence or absence of growth in each medium is used as an index to express a function that is contrary to the function of the polypeptide encoded by the marker gene. Examples thereof include a step of obtaining a cell, and (c) a step of obtaining a nucleic acid containing a fusion gene from the cell obtained in the step (b).

In the preparation method of the present invention, first, the above-mentioned vector for preparing fusion gene is used to transform the above-mentioned host to obtain a transformant expressing the polypeptide encoded by the marker gene on the vector. Obtain [referred to as step (a)].

The transformation in the step (a) is
Although it depends on the type of host used and the like, it can be performed by a conventional method described in, for example, the above-mentioned Molecular Cloning Laboratory Manual, 3rd edition. Examples of the transformation method include an electroporation method, a calcium phosphate method, a conjugation transfer method and the like.

According to the step (a), cells having the fusion gene preparation vector as a transformant expressing the polypeptide encoded by the marker gene can be obtained.

Then, the transformant obtained in the step (a) is cultured to obtain cells expressing a function contrary to the function of the polypeptide encoded by the marker gene [referred to as step (b)].

In the step (b), the culture conditions of the transformant can be appropriately set depending on the host to be used and the expression rate of recombination ability in the host. For example, when Escherichia coli MK1019 strain is used as a host, for example, LB medium [composition: 1% polypeptone, 0.5% yeast extract, 1%
NaCl] at 37 ° C. for 2 days.

In the above-mentioned (b), cells expressing a function that is contrary to the function of the polypeptide encoded by the marker gene correspond to the transformant obtained in the above-mentioned step (a) corresponding to the marker gene. It can be cultivated in the presence or absence of a drug, and can be selected using the presence or absence of growth in each medium as an index [referred to as step (b ')]. For example, when the marker gene is a drug susceptibility marker gene, by selecting cells that grow in both the medium in the presence and absence of the drug, the polypeptide encoded by the marker gene A cell expressing a function that is contrary to the function is obtained.

Then, a nucleic acid containing the fusion gene is obtained from the cells obtained in step (b) [referred to as step (c)].

The nucleic acid containing the fusion gene can be obtained from the cells obtained in step (b) by a conventional nucleic acid isolation method described in the above Molecular Cloning Laboratory Manual, Third Edition.

The formation of the fusion gene was carried out, if desired, by a conventional nucleotide sequencing method, using an oligonucleotide specific to each of the polynucleotides to be fused,
Nucleic acid hybridization method or nucleic acid amplification method, by using a marker gene such as β-galactosidase,
It can be done as appropriate.

Further, the method for preparing the fusion gene of the present invention comprises:
More efficiently by using a fusion gene preparation vector, and / or (b) a fusion gene preparation kit containing a host having a function contradictory to the function of the polypeptide encoded by the marker gene on the vector, It is possible to easily create a fusion gene group having various fusion modes and exhibiting desired properties or novel properties. Such kits are also included in the present invention. The fusion gene preparation kit of the present invention may contain a medium suitable for culturing a host, various reagents used for gene recombination, a host suitable for expressing a fusion polypeptide, and the like. When the marker gene is the drug sensitivity marker gene, it may contain a drug corresponding to the marker gene.

The fusion gene obtained by the method for preparing a fusion gene of the present invention has various fusion modes and does not exist in nature, and is applicable to the creation of new proteins, substance production, medical fields and the like. Be expected. Further, the fusion polypeptide encoded by the fusion gene of the present invention,
The fusion modes are diverse and do not exist in nature, and are expected to be applied to the substance production and medical fields. Such fusion genes and fusion polypeptides are also included in the present invention.

The fusion polypeptide encoded by the fusion gene of the present invention is expressed by a conventional protein expression method, and a conventional protein purification method (for example, salting out method,
Ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc.).

Upon expression of the fusion polypeptide from the fusion gene of the present invention, the fusion gene is integrated into a conventional expression vector or the like, the obtained recombinant vector is used to transform an appropriate host, and the obtained trait is obtained. The fusion polypeptide of the present invention can be obtained from the transformant by a conventional method. The fusion polypeptide may be secreted extracellularly depending on the host and vector used, and in such a case, the purification may be performed from the culture supernatant in the same manner. The transformant may be cultured under optimal conditions for expression of the fusion polypeptide, such as medium composition, medium pH, culture temperature, culture time, and induction conditions.

Hosts that can be used to obtain the fusion polypeptide include, for example, E. coli HB101, C600, JM109.
Bacteria such as; yeast such as Saccharomyces cerevisiae;
Animal culture cells such as L cells, COS cells, CHO cells, sf9
Insect culture cells and the like.

Used to Obtain Fusion Polypeptide
As the vector, for example, an inducible promoter,
Appropriate factors such as selectable marker gene and terminator
You may have a plasmid vector, phage vector
And virus vectors. Large as a host
When using Enterobacter, vectors include pUC18 and pUC19.
 , PBR322, pKK223-3 (Pharmacia)
Sumid vector; λZAP ^TMII, λgt11, etc.
Kuta. When yeast is used as the host,
Examples of actors include pYEUra3. As a host
When using cultured insect cells, the baculovirus vector
ー etc. Using animal culture cells as a host
In this case, pKCR can be used.

For the transformation, a method suitable for the host to be used may be used, and examples include the above-mentioned transformation method and DEAE-dextran method.

When the host is Escherichia coli, the expression product may form an insoluble inclusion body. In this case, commonly used protein solubilizers, for example,
By obtaining a solubilized protein using urea, guanidine hydrochloride or the like, and performing a refolding operation using a dialysis method or a dilution method, a preparation containing the desired fusion polypeptide can be obtained. If desired, this preparation can be further purified by various chromatographies to obtain the desired fusion polypeptide.

The fusion polypeptide of the present invention can also provide a method for producing a substance by utilizing its properties, functions, activities and the like. According to the substance production method of the present invention,
Since a fusion polypeptide suitable for substance production can be used, it exhibits an excellent effect that desired efficiency, conditions, etc. can be achieved and a desired substance can be obtained.

In the method for producing a substance of the present invention, a fusion gene encoding a fusion polypeptide capable of achieving a desired efficiency and conditions and obtaining a desired substance is prepared by the above-mentioned method for preparing a fusion gene, A substance can be produced by obtaining the fusion polypeptide as described above and using the fusion polypeptide under conditions suitable for producing the desired substance.

[0051]

The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.
In addition, unless otherwise specified, various operations in this example are described by, for example, molecular cloning.
Laboratory Manual 3rd Edition [Zambrok (Sambroo
k) et al., Molecular Cloning: A Laboratory Manual3rd e.
d., Cold Spring Harbor Laboratory, (2001)] and the like.

Example 1 Two flagellin genes fljB and fl of Salmonella typhimurium
Using iC as the gene to be fused, a fused gene was prepared as follows.

(1) Preparation of fljB gene cassette Genomic DNA of Salmonella typhimurium SJ2353 strain was digested with SalI and EcoRI.
It was digested with and a DNA fragment was obtained. The obtained DNA fragment is
It was inserted between the SalI and EcoRI sites on plasmid pBR322. Escherichia coli EJ2081, which is a flagellin gene-deficient strain, was transformed with the obtained plasmid, and the obtained transformant was transformed into a soft agar medium [composition: containing 0.3% agar.
LB medium (composition: 1% polypeptone, 0.5% yeast extract,
1% NaCl)], and cultured at 30 ° C. for 24 hours. A transformant that spreads and spreads in the soft agar medium is selected by considering it as a transformant whose motility is restored, and a plasmid pC containing the fljB gene is selected from the transformant by a conventional method.
B105B was isolated.

Then, from the pCB105B, a StuI of 1.8 kb was prepared.
-Cut out a fragment containing the promoter of the fljB gene and the full-length ORF (1518 bp) as an EcoRI fragment.
It was inserted into 396 (Takara Shuzo) to construct pBZ302.

A fragment of the fljB gene was excised from pBZ302 as a 1.7 kb NspI fragment. The obtained fragment is
PBZ110 was constructed by inserting it into the SphI site of plasmid pUC119. According to the pBZ110, the promoter of the fljB gene
A fragment containing the ORF positions 1 to 1431 (hereinafter fljBΔ87
Can be excised as a 1.7 kb XhoI-SalI cassette.

(2) Preparation of fliC gene cassette Genomic DNA of Salmonella typhimurium SJ2353 strain was digested with HindIII to obtain a DNA fragment. The obtained DNA fragment was inserted into the HindIII site on the plasmid pHSG399. Escherichia coli EJ2081 which is a flagellin gene-deficient strain was transformed with the obtained plasmid, and the obtained transformant was inoculated on the soft agar medium and cultured at 37 ° C. for 24 hours. A transformant whose motility was restored was selected on the soft agar medium, and a plasmid pHI301 containing the fljC gene was isolated from the transformant by a conventional method.

As a 2.2 kb ScaI-HindIII fragment, a fragment containing the 3'-half half region of the ORF of fliC was excised from pHI301, and the ends were blunted with Klenow fragment to obtain a DNA fragment. The obtained DNA fragment was ligated with a BamHI linker and then inserted into pHSG397 to obtain pCF1.
Build 07. According to the pCF107, the ORF of the fliC gene
Of the gene containing the entire region from the 604th position to the latter half (Δ603
fliC) can be excised as a 2.2 kb SalI-XhoI cassette.

(3) Streptomycin sensitivity (wild type rp
sL gene) Preparation of cassette Escherichia coli wild strain W3110 strain genomic DNA was transformed into SacII and BamHI
It was digested with and then the ends were blunted with Klenow fragment to obtain a DNA fragment. The obtained DNA fragment was inserted into the SmaI site of plasmid pKK223-3 (Pharmacia) to obtain a recombinant plasmid. Using the obtained recombinant plasmid, Escherichia coli HB10 having the rpsL20 mutation on the chromosome
One strain was transformed. The Escherichia coli HB101 was rpsL
The Strr phenotype is resistant to streptomycin due to the 20 mutations. Therefore, for the obtained transformant,
By culturing at 37 ° C for 24 hours in each of the LB medium and the LB medium containing streptomycin, a bacterium that exhibits a streptomycin-sensitive phenotype is selected, thereby,
A plasmid pNC121L containing rpsL was obtained.

The above-mentioned plasmid pNC121L is a plasmid inserted downstream of the tac promoter in the direction in which the rpsL gene is expressed. According to the plasmid pNC121L,
A fragment containing the Ptac-rpsL gene can be excised as a 1.2 kb SalI cassette.

(4) Construction of fusion gene preparation vector pLC210 From the pCF107 obtained in (2) above, Δ603fliC was cut out as a 2.2 kb SalI-XhoI cassette, and SalI on pBZ110 obtained in (1) above was excised. A pCF110 was constructed by inserting between the site and the XhoI site. In pCF110, the two flagellin genes fljB and fliC are arranged in tandem in the same direction.

In addition, mini-F plasmid pTN1105 (Kawasa
ki Med. J. 12: 163-176, 1986) of 2.7 kb including ori1
The XhoI fragment was removed and recircularized to construct pTN1102.

Then, from the pCF110, fljBΔ87 and Δ60
A fragment containing 3fliC was excised as a 3.9 kb XhoI fragment and inserted into the SalI site of the plasmid pTN1102 to construct a plasmid pLF106.

Then, fljBΔ87 and Δ603fli of pLF106
1.2 kb Ptac-r derived from pNC121B at the SalI site between C and
The psL gene cassette was inserted as a SalI fragment to construct a fusion gene preparation vector pLF110.

The fliC gene on pLF110 was ORF
Since it lacks the first half region of the 5'side, according to pLF110, fl
The fusion gene occurring between the jB and the 3'half region of the fliC ORF can be mainly isolated. When the gene fusions between fljB and fliC are in the same reading frame, the two defective flagellin genes form one complete flagellin gene. When a complete flagellin gene is formed, the motility of the Escherichia coli flagellin gene-deficient strain carrying this gene is restored on the soft agar medium, and therefore it can be discriminated.

(5) Preparation of fusion gene using fusion gene preparation vector Using the plasmid pLF110 obtained in the above (4), streptomycin resistance, flagellin gene deficiency, single-stranded DNA binding protein gene (Ssb) wild-type Escherichia coli MK1018 strain and streptomycin resistance, lacking flagellin gene, single-stranded DNA
Escherichia coli MK10 having ssb-3 mutation in the binding protein gene
Each of the 19 strains was transformed and seeded in LB medium. The obtained colonies were inoculated on an LB medium and the medium containing streptomycin, and it was confirmed that the transformant exhibited streptomycin sensitivity.

Then, the above transformant was cultivated with shaking in an LB liquid medium at 37 ° C. for 12 hours, the culture solution was applied to an LB medium containing streptomycin, and statically cultured at 37 ° C. for 2 days.

The appearance frequency of the colonies that had regained streptomycin resistance and had grown in the above medium was measured. In the wild-type E. coli MK1018 strain, the frequency of appearance was 0.6 per 10 ⁶ colonies. Motility was restored in 10% of the streptomycin-resistant colonies that appeared.

Therefore, in the wild-type Escherichia coli MK1018 strain, the formation frequency of the fusion gene having the correct reading frame was calculated to be 0.06 per 10 ⁶ colonies. In contrast, single-stranded DNA
In the E. coli MK1019 strain having the ssb-3 mutation in the binding protein, 240 streptomycin-resistant colonies appeared per 10 ⁶ colonies. Motility was restored in 90% of the streptomycin-resistant colonies that appeared, and the formation frequency of the fusion gene with the correct reading frame was calculated to be 216 per 10 ⁶ colonies in the Escherichia coli MK1019 strain having the ssb-3 mutation. It was

A shaking culture of the transformant of the fusion gene preparation vector pLF110 was simultaneously applied to LB medium containing no streptomycin, and static culture was carried out at 37 ° C. for 2 days. In each of the MK1018 strain and the MK1019 strain, recovery of motility of the obtained colonies was examined on the above soft agar medium. Tested for both MK1018 and MK1019 strains
None of the 200 colonies recovered motility, and the fusion gene could not be obtained without selection with streptomycin.

Example 2 Vector pLF1 for preparing fusion gene used in Example 1 above
E. coli DB-1 strain having a mutR mutation was transformed with 10. Transformants that became sensitive to streptomycin,
After culturing with shaking in an LB liquid medium at 37 ° C for 12 hours, the culture was applied to an LB medium containing streptomycin and statically cultured at 37 ° C for 2 days.

The appearance frequency of the colonies that had regained streptomycin resistance and had grown in the above medium was measured. As a result, in the Escherichia coli DB-1 strain having the mutR mutation, the appearance frequency was 20 per 10 ⁶ colonies.

Example 3 By the same procedure as in Example 1, colonies resistant to streptomycin were obtained. A plasmid was extracted from the obtained colony and the sequence of the fusion flagellin gene was DN
The sequence of the fusion point of the fljB gene and the fliC gene was confirmed by the determination with the A sequencer.

As a result, no replication error in the gene sequence of the fusion gene was detected.

Further, the nucleotide length of the homologous region between the fljB gene and the fliC gene and the fl on the extracted plasmid
The distribution of the points where the gene fusion actually occurred between the jB gene and the fliC gene was calculated using the wild-type MK1018 strain and ssb-3 for ssb.
The comparison was made with the MK1019 strain having a mutation. The results are shown in Table 1.

[0075]

[Table 1]

As shown in Table 1, when the MK1018 strain was used, a fusion gene could not be obtained when the homologous region had a length of 11 nucleotides or less, but when the MK1019 strain was used, the homologous region was 3 It can be seen that a fusion gene can be obtained even with a nucleotide length.

Example 4 Phospholipase derived from actinomycetes as a gene to be fused
Two genes (DDBJ accession numbers: AB062136 and AB05873), which are D genes, were arranged in tandem on pACYC184, and the tac promoter (Pt
The fusion gene preparation vector pLC111 was constructed by inserting the wild-type rpsL gene, which is a streptomycin-sensitive gene expressed under the control of ac).

Using the plasmid pLC111, the Escherichia coli MK1018 strain that is streptomycin resistant and wild type with respect to the single-stranded DNA binding protein gene (ssb) and the streptomycin resistant and ssb- Escherichia coli MK1019 strain having 3 mutations was transformed and seeded in LB medium. The obtained colonies are
The LB medium and the medium containing streptomycin were seeded, and transformants exhibiting streptomycin sensitivity were selected.

The obtained transformant was applied to LB agar medium containing streptomycin, and statically cultured at 37 ° C. for 2 days. Then, a plasmid was extracted from the obtained bacterial cells,
The size was observed by electrophoresis.

As a result, the MK1018 strain has only the same size as compared with the original plasmid before selection in streptomycin medium, whereas the MK1019 strain
Obviously they were all small in size. That is, it can be seen that gene fusion is efficiently performed when the MK1019 strain is used.

Example 5 Preparation of spectinomycin-sensitive (wild type rpsE gene) cassette Genomic DNA of Escherichia coli wild strain W3110 strain was digested with PvuII to obtain a DNA fragment. The resulting DNA fragment is used as a plasmid
It was inserted into the SmaI site of pKK223-3 (Pharmacia) to obtain a recombinant plasmid. The resulting recombinant plasmid was used to transform Escherichia coli strain ME6097 having the rpsE mutation (which exhibits a spectinomycin-resistant Spcr phenotype) on its chromosome. , The resulting transformants were incubated at 37 ° C for 24 hours in LB medium and spectinomycin-containing medium.
By culturing for a period of time, a strain in which the spectinomycin resistance phenotype of the ME6097 strain was restored to sensitivity by the introduction of the wild-type rpsE gene was selected, and thereby plasmid pNC121E having rpsE was obtained.

The plasmid pNC121E was inserted downstream of the tac promoter in such a direction that the rpsE gene was expressed, and a gene fragment containing the Ptac-rpsE gene was inserted into a 2.4 kb SalI fragment.
It can be cut out as a cassette.

Example 6 Preparation of Kit for Preparing Fusion Gene The streptomycin drug-sensitive gene was excised with SalI from the plasmid pNC121L having the streptomycin-sensitive cassette described in Example 1, pET-12a-c and
A plasmid inserted in the SalI site of pET-22a-c (Novagen) in the direction opposite to the transcription / translation direction of the T7 promoter.
pETStr was prepared.

Furthermore, the spectinomycin-sensitive cassette described in Example 5 was excised from pNC121E with the spectinomycin drug-sensitive gene cut with SalI, and XhoI of pETStr was excised.
A plasmid pETStrSpec was inserted at the site in the reverse direction of the transcription / translation direction of the T7 promoter.

Also, a fusion gene preparation kit was prepared by packaging a host containing E. coli MK1019 strain competent cells and a fusion polypeptide expression host containing BL21 (DE3) strain competent cells. And

Thus, NcoI, BamHI and EcoR of pETStr and pETStrSpec were used as restriction enzyme sites for insertion of gene A.
I, SacI, etc. can be used. Moreover, as a restriction enzyme site for insertion of gene B, HindIII, NotI, XmaI,
XhoI, AvaI, Bpu1102, etc. can be used, and with pETStrSpec,
HindIII, NotI, XmaI, Bpu1102, etc. can be used. Furthermore, when gene D is used as the material for the third fusion gene, pETStrSpec is used as a restriction enzyme site for its insertion.
Bpu1102 can be used, and it is also possible to obtain a fusion gene of three genes A, B and D.

When the fusion polypeptide encoded by the fusion gene is expressed, the gene A is added to the initiation codon.
As long as it is inserted so that the coding frame of the gene matches, after fusion of the gene, a plasmid carrying the fused gene is extracted from the host MK1019 strain, and the host is BL21 (DE3).
Alternatively, by inducing the expression of the fusion gene by isopropyl-β-D-thiogalactopyranoside under the control of the T7 promoter, it is possible to easily obtain the fusion polypeptide. In the pET12 series of plasmids, the fusion protein is secreted outside the cells, and in the pET22 series of plasmids, the fusion polypeptide functions to secrete the fusion polypeptide outside the cells.

Example 7 Expression of Fusion Polypeptide The Escherichia coli MK1019 strain was transformed by the conventional method with the two types of phospholipase D fusion gene preparation vectors described in Example 4, and a large number of transformants exhibiting streptomachine sensitivity were transformed. Obtained. The obtained transformant was applied to a streptomycin-containing LB agar medium, statically cultured at 37 ° C. for 2 days, and a plasmid was extracted from the obtained bacterial cells.

From among the above-mentioned plasmids, those having a smaller size compared to the plasmid before selection in streptomycin medium were selected, and 5 ′ of the fusion gene was selected from them.
Genes were amplified by polymerase chain reaction using a primer having a restriction enzyme NcoI site containing a start codon at the end and a primer having a stop codon at the restriction enzyme EcoRI site.

These amplification products were digested with the restriction enzymes NcoI and EcoRI.
Digested with and the literature of Iwasaki et al. [Biotechnology Progress (Biotechnology Progress) 13
Vol. 864-868, (1997)], and was inserted between the restriction enzyme NcoI site and EcoRI site of the plasmid pETKmS2 having a kanamycin resistance gene. Then, Escherichia coli BL21 (DE3) strain was transformed with the obtained recombinant plasmid and selected on LB agar medium containing kanamycin to obtain a transformant showing kanamycin resistance.

The transformant obtained was treated with kanamycin
The culture was carried out in LB medium at 37 ° C for 16 hours with shaking, and a part of the culture was carried out by Iwasaki et al. (Biotechnology Progress Vol. 13, 864-868, (199
The culture medium for expression described in 7)) was added so that the volume became 1/100, and the mixture was cultured at 30 ° C. for 12 hours with shaking.

Thereafter, isopropyl-β-D-thiogalactopyranoside was added to the obtained culture to a final concentration of 1 mM, and the mixture was cultivated with shaking at 16 ° C. for 4 days to obtain plasmid pETKmS.
The fused phospholipase D was secreted outside the cells by the action of the secretion signal of 2 to obtain a culture supernatant showing the enzymatic activity of phospholipase D.

[0093]

INDUSTRIAL APPLICABILITY The fusion gene preparation vector of the present invention has an excellent effect that it is possible to obtain a fusion gene group having high efficiency and various fusion modes. In addition, the method for preparing a fusion gene of the present invention does not cause a replication error in the sequence of the obtained fusion gene, is not limited by the kind of the gene to be fused, has high efficiency, and has various fusion modes. It has an excellent effect that a fusion gene group that does not exist in nature can be easily obtained.
Further, the fusion gene, fusion polypeptide and substance production method of the present invention are expected to be applied to protein functional analysis, substance production, medical treatment and the like. Furthermore, according to the fusion gene preparation kit of the present invention, the method for preparing the fusion gene can be easily carried out, and the fusion gene sequence can be selected without causing a replication error in the obtained fusion gene sequence. Without being limited, the present invention has an excellent effect that it is possible to obtain a fusion gene group which is highly efficient and has various fusion modes and which does not exist in nature.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Koichi Mori             7549-1 Yoshikawa, Kayo-cho, Kamibo-gun, Okayama Prefecture Okayama Prefecture             Biological Sciences Research Institute (72) Inventor Yufumi Hatanaka             7549-1 Yoshikawa, Kayo-cho, Kamibo-gun, Okayama Prefecture Okayama Prefecture             Biological Sciences Research Institute F term (reference) 4B024 AA20 BA11 BA80 CA03 CA07                       DA06 DA12 EA04 FA10 GA11                 4H045 AA10 AA20 BA41 CA11 CA15                       FA74

Claims (25)

[Claims] 1. A fusion gene preparation vector comprising a nucleic acid in which at least two kinds of polynucleotides to be fused are linked via a gene related to host growth (referred to as a marker gene). 2. At least two polynucleotides to be fused are arranged in the same transcription and translation directions.
The fusion gene preparation vector according to claim 1. 3. A polynucleotide A to be fused, a marker gene C, and a polynucleotide B to be fused having a continuous sequence homologous to the polynucleotide A.
The fusion gene preparation vector according to claim 1 or 2, which comprises a nucleic acid consisting of and a nucleic acid arranged in the order of A-C-B. 4. The fusion gene preparation vector according to claim 1, wherein the marker gene is a drug sensitivity marker gene. 5. The fusion gene preparation vector according to claim 4, wherein the drug sensitivity marker gene is a gene encoding a wild-type ribosomal protein. 6. The fusion gene preparation vector according to claim 1, which can be replicated in a host having a function that is contrary to the function of the polypeptide encoded by the marker gene. 7. A method for preparing a fusion gene, which comprises using the fusion gene preparation vector according to any one of claims 1 to 6. 8. (a) A host having a function contradictory to the function of a polypeptide encoded by a marker gene on the vector, which comprises using the fusion gene preparation vector according to any one of claims 1 to 6. To obtain a transformant expressing the polypeptide encoded by the marker gene, (b) culturing the transformant obtained in the step (a), and encoding the marker gene. 8. The method according to claim 7, further comprising the step of selecting cells expressing a function contrary to that of the polypeptide, and (c) obtaining a nucleic acid containing a fusion gene from the cells obtained in the step (b). Preparation method. 9. In the step (b), (b ′) the transformant obtained in the step (a) is cultured in the presence or absence of a drug corresponding to the marker gene,
The method according to claim 8, wherein the step of selecting cells expressing a function that is contrary to the function of the polypeptide encoded by the marker gene is carried out using the presence or absence of growth in each medium as an index. 10. The method according to claim 8 or 9, wherein the host is Escherichia coli or yeast. 11. The marker gene is the drug sensitivity marker gene, and the host is resistant to a drug corresponding to the marker gene.
The preparation method according to any one of items. 12. The preparation method according to claim 8, wherein the host carries a drug-insensitive ribosomal gene. 13. The method according to any one of claims 8 to 12, wherein the host has a mutation with an increased deletion frequency. 14. The host having a mutation with an increased deletion frequency is Escherichia coli having a mutation in the single-stranded DNA binding protein gene or yeast having a mutation in the single-stranded DNA binding protein gene. Preparation method of. 15. The host is Escherichia coli MK1019 strain (deposit number: FERMP-18742).
The preparation method described. 16. A fusion gene obtained by the method for preparing a fusion gene according to any one of claims 7 to 15. 17. A fusion polypeptide encoded by the fusion gene according to claim 16. 18. A method for producing a substance, which comprises using the fusion polypeptide according to claim 17. 19. A kit for carrying out the method for preparing a fusion gene according to claim 7.
(A) a vector for preparing the fusion gene according to any one of claims 1 to 6, and / or (b) a polypeptide encoded by a gene (referred to as a marker gene) related to the growth of a host on the vector. A fusion gene preparation kit, comprising a host having a function that is contrary to the function. 20. The fusion gene preparation kit according to claim 19, wherein the host is Escherichia coli or yeast. 21. The marker gene is the drug sensitivity marker gene, and the host is resistant to Escherichia coli resistant to a drug corresponding to the marker gene or a drug corresponding to the marker gene. Is a yeast,
The fusion gene preparation kit according to claim 19 or 20. 22. The fusion gene preparation kit according to any one of claims 19 to 21, wherein the host carries a drug-insensitive ribosomal gene. 23. The fusion gene preparation kit according to any one of claims 19 to 22, wherein the host is a host having a mutation with an increased deletion frequency. 24. The host having a mutation that increases the deletion frequency is Escherichia coli having a mutation in the single-stranded DNA binding protein gene or yeast having a mutation in the single-stranded DNA binding protein gene. A kit for preparing a fusion gene of. 25. The host is the Escherichia coli MK1019 strain (deposit number: FERMP-18742).
The fusion gene preparation kit as described.