JP3096225B2 - Method for expressing function of gene DNA - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for expressing gene DNA functions. More specifically, the present invention relates to a gene DNA held in a cloning vector.
A simple and reliable transfer of the gene into an expression vector to express a large amount of its gene function. This method can be used in a wide range of industrial fields that aim to produce large amounts of various useful substances by genetic engineering,
This is useful in academic research fields related to elucidation of functions.

[0002]

2. Description of the Related Art Conventionally, specific genes have been isolated from various species of organisms, cloned, and their structures and functions have been investigated in detail. In addition, the functions encoded by the genes have been identified in cultured cells. Attempts to produce useful substances and the like in large quantities by freely expressing them have been widely made. In recent years, PCR (Polymerase Cha
The spread of the in reaction) method has made it possible to easily amplify a small amount of gene fragment to an amount sufficient for subsequent genetic manipulation.

[0003] In such a large expression of a gene,
Plasmid D is used as a genetic engineering material for cloning a specific gene DNA isolated and / or amplified and further expressing this DNA clone in a host cell.
Various vectors using NA, phage DNA and the like and host systems thereof have been developed. That is, when cloning a gene DNA, a vector (cloning vector) capable of reliably retaining the inserted gene DNA and amplifying a large amount in a host by its self-replication ability.
Is used. When the function of the cloned gene DNA is to be expressed in a large amount in the host, a structure matching the genetic information expression system of the host, such as having a promoter mechanism for enhancing the function expression efficiency, is used. The provided vector (expression vector) is used.

[0004] Therefore, in the case of expressing a large amount of the function of the cloned gene DNA, a procedure for cutting out and fragmenting the gene DNA held in the cloning vector and inserting it into an expression vector is required. For this reason, conventionally, a plurality of DNA fragments are prepared by cutting a cloning vector with one or more restriction enzymes, and these DNA fragments are fractionated by, for example, gel electrophoresis.
Gene DN of interest using DNA fragment size as an index
The A fragment was selected to insert and bind to the expression vector.

[0005] However, a great deal of time and labor was required for the separation of DNA fragments and the selection of target fragments by such gel electrophoresis. Further, gene DNA amplified by the PCR method has a limit in the size that can be amplified by the PCR method.
It is often an A fragment, and it has been extremely difficult to accurately separate such minute fragments by gel gel electrophoresis.

[0006] The present invention has been made in view of the above circumstances, and solves the problems of the conventional method to solve the problem of the gene DNA stored in a cloning vector.
It is an object of the present invention to provide a new method which can transfer a gene into a gene in a large amount by transferring the gene into an expression vector without requiring a special separation step.

[0007]

Means for Solving the Problems The present invention solves the above-mentioned problems by transferring gene DNA held in a cloning vector having a drug resistance gene to an expression vector having the same kind of drug resistance gene. (1) Restriction enzyme A
(2) cleaving one site of the drug resistance gene sequence of the cloning vector with another restriction enzyme B, (3) ) At least the action region of the drug resistance gene is decomposed and removed by a DNA degrading enzyme that recognizes the cleavage site, and both ends of the cleaved cloning vector are blunt-ended. (4) Cloning using restriction enzyme A that recognizes both ends of the gene DNA These DNA fragments are inserted and bound into an expression vector cleaved with (5) restriction enzyme A, and (6) transformant cells into which this expression vector has been introduced are selected using drug resistance as an index, By culturing the transformed cells, it is possible to express a large amount of gene DNA functions. It provides a function expression method of gene DNA to symptoms.

Hereinafter, the method and principle of the present invention will be described in detail with reference to FIG. (1) Insert a gene DNA having a recognition sequence for an arbitrary restriction enzyme A at both ends into a cloning vector cleaved by the same restriction enzyme A. Such gene DNA can be easily obtained as a PCR product amplified using cDNA or genomic DNA of the target gene as a template. That is, an oligonucleotide having a recognition sequence for restriction enzyme A on the 5 'side and having the same translation frame as the expression vector after the target gene DNA is inserted into the expression vector is used as a PCR primer. By amplifying the gene DNA by PCR, DNA fragments having a recognition sequence for restriction enzyme A at both ends can be obtained easily and in large quantities. The gene DNA is c
It may be directly isolated from a DNA library or a genomic library. In such a case, if necessary, one or both base pairs of the target DNA fragment cut out from the library clone are modified by deletion or addition by a known method, and the recognition sequence of the restriction enzyme A is added to both ends. Can be held.

Furthermore, in the present invention, a very small DNA of about 15 base pairs, which could not be targeted at all by the conventional method which requires a selection step by gel electrophoresis,
The fragments can also be subjected to a functional expression system. Other requirements are not particularly limited as long as the cloning vector has a drug resistance gene in common with the expression vector used in the subsequent step, and bacterial and / or animal cells such as plasmid DNA and cosmid DNA are used. A general-purpose cloning vector using as a host can be used. The resistance gene can be a resistance gene for widely used ampicillin, tetracycline, kanamycin, and the like.

[0010] Insertion of a gene DNA into a cloning vector can be carried out according to a known method. (2) One site of the resistance gene sequence of the cloning vector into which the gene DNA has been inserted is cleaved with another restriction enzyme B. The restriction enzyme B recognizes one site in the resistance gene sequence of the cloning vector and cleaves it, and selects a restriction enzyme such that this recognition site is not present in or near the gene DNA sequence. (3) DN recognizing the cleavage site by restriction enzyme B
A cloning vector D which has been decomposed and removed at least by the action region of the resistance gene with
Both ends of NA are blunt-ended.

[0011] The DNA degrading enzyme can be appropriately selected in consideration of the affinity with the restriction enzyme B. In addition, it is preferable to decompose and remove all of the resistance genes with this decomposing enzyme, but leave the gene DNA intact. (4) The above cloning vector DNA is fragmented with restriction enzyme A. By this treatment, both ends of the gene DNA fragment are cleaved by restriction enzyme A,
Two DNA fragments separated from the A fragment are obtained. The gene DNA fragment is cleaved at both ends by restriction enzyme A to form protruding ends, whereas the DNA fragment has protruding ends at one end and blunt ends at the other end. (5) an expression vector cleaved by restriction enzyme A,
The above DNA fragments are mixed, and each DNA fragment is inserted and bound to an expression vector.

At this time, the DNA fragments that can be inserted and bound into the expression vector are a gene DNA fragment having protruding ends at both ends and a DNA fragment + in which both blunt ends are connected to each other to form protruding ends. . However, since the probability that a DNA fragment + is formed by blunt-end ligation is extremely low, most of the DNA fragments actually inserted into an expression vector are gene DNA fragments.

As the expression vector, a general-purpose vector such as a plasmid DNA or a cosmid DNA using bacteria and / or animal cells as a host can be used in the same manner as the above-mentioned cloning vector. (6) The above-described expression vector is introduced into an appropriate host cell, the transformant cells are selected using the resistance to the drug as an index, and the transformant cells are cultured to express a large amount of the gene DNA function.

That is, when the expression vector into which the DNA fragment is inserted and bound in the above (5) is introduced into a host cell, a small number of the host cells may have the DNA fragment +. However, this DNA fragment +
Has lost its drug resistance function, and therefore it is possible to reliably select only the desired transformant cells by using the drug resistance as an index.

By culturing the transformed cells by a known method, the function of gene DNA (for example, protein production) can be expressed in a large amount. Hereinafter, the method of the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.

[0016]

EXAMPLE Gene DNA of 300 base pairs or less was transferred from a cloning vector to an expression vector according to the method of the present invention as follows. First, a cDNA clone encoding human fibronectin (EMBO J., 4, 1
755-1759, 1985) as a template and an oligonucleotide primer (28-mer) having a recognition sequence for the restriction enzyme BamH1
And PCR products (285 bp fragment and 207 bp fragment) were prepared using PCR and Taq polymerase.

The PCR product was used as shown in FIG.
Cloning vector p containing ampicillin resistance gene (Amp) and kanamycin resistance gene (Kan)
It was inserted into the BamH1 cleavage site (5 'side of LacZa gene) of CRII (manufactured by Invitrogen). The pCRII was transfected into cells, and ampicillin and X-gal
The transformed colonies were selected by culturing in the containing medium. The presence or absence of the inserted PCR product was confirmed using the above PCR primer as a probe.

From the transformant cells, plasmid DNA
Was cleaved with a restriction enzyme XmnI having a recognition sequence in the Amp gene of pCRII, and the Amp gene was decomposed and removed by a DNA degrading enzyme Bal31 that recognizes this cleavage site. The thus-cleaved pCRII was extracted with saturated phenol / chloroform, purified by ethanol precipitation, further fragmented with the restriction enzyme BamH1, and the DNA fragment was purified by phenol extraction.

Next, the expression vector pMALc2 (New England Biolabs) having the Amp gene is cleaved with the restriction enzyme BamH1 and treated with phosphatase.
The above DNA fragment was inserted here. This insertion position
The protein located at the 3 'end of the malE gene and encoded by the inserted DNA fragment is expressed by fusing with a maltose binding protein.

Then, the cells were transfected with the recombinant pMALc2 and cultured in an ampicillin-containing medium, and colonies of the transformed cells were selected. Lastly, it was confirmed whether or not the ampicillin-resistant transformant cells obtained by the above-described method actually had an expression vector retaining gene DNA (PCR product) selectively, using kanamycin resistance as an index. . That is, the PCR product (285
pCRII-derived fragments (K fragments other than bp or 207 bp fragments)
(including the an gene) is inserted into the expression vector, the host cell shows resistance to kanamycin as well as ampicillin. On the other hand, when only the PCR product is inserted, the host cell is sensitive to kanamycin because the Kan gene is not present in the expression vector.

The results are as shown in Table 1. As a comparative example, pMAL into which a DNA fragment cut out from pCRII (PCR product: containing a 285 bp fragment or a 207 bp fragment) not treated with BamH1 and Bal31 was inserted.
c2 transformants were tested in the same manner.

[0022]

[Table 1]

As is clear from Table 1, almost all of the comparative examples have Kan resistance.
While it is certain that the DNA fragment containing the Kan gene has been inserted, the colony of the host cell into which the expression vector prepared by the above embodiment of the present invention has been introduced is almost completely (100%, 94%) PCR sensitive to Kan
It was confirmed that no DNA fragment other than the product was inserted.

It should be noted that the screening was performed using the PCR primers, and that 80% or more of the expression vectors were
The presence of the product was confirmed. Very good results were also obtained for functional expression (production of fusion protein).

[0025]

As described in detail above, according to the present invention, the gene DNA of the cloning vector can be easily and reliably transferred to the expression vector without the need for complicated operations such as gel electrophoresis, and Can be expressed in a large amount. In particular, it is possible to efficiently express a small PCR product that could not be accurately separated by a conventional selection method using gel electrophoresis.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a process procedure and a principle of the method of the present invention.

FIG. 2 is a schematic diagram showing the configuration of a cloning vector and an expression vector used in Examples of the present invention and the steps for preparing each.

────────────────────────────────────────────────── ─── Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00 -15/90 C12N 1/00-5/28 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG )

Claims (3)

(57) [Claims] 1. A method for transferring gene DNA held in a cloning vector having a drug resistance gene to an expression vector having the same kind of drug resistance gene to express a large amount of its function, comprising the following steps: The gene DNA having the recognition sequence of A at both ends is inserted and bound to the cloning vector cleaved by the restriction enzyme A, (2)
One site of the drug resistance gene sequence of the cloning vector is cleaved with another restriction enzyme B, and (3) at least the action region of the drug resistance gene is decomposed and removed by a DNA degrading enzyme recognizing this cleavage site, and the cleaved cloning vector (4) Gene DNA
The cloning vector is fragmented with a restriction enzyme A that recognizes both ends of the DNA fragment, (5) these DNA fragments are inserted and bound to an expression vector cleaved with the restriction enzyme A, and (6) a transformant cell into which the expression vector has been introduced. A method for expressing the function of a gene DNA, which comprises selecting a cell line using the resistance to a drug as an index and culturing the transformed cell to express a large amount of the function of the gene DNA. 2. A gene DNA is 15 bp to 10 ⁴ methods of gene DNA functional expression Claim 1 which is cDNA or genomic DNA sequences of base pairs. 3. The method according to claim 2, wherein the cDNA sequence or the genomic DNA sequence is a DNA sequence amplified by a PCR method.