JPH05211871A - Method for mutational transduction of designated range - Google Patents

Abstract

PURPOSE:To provide a method for readily transducing various mutation in a range designated in high frequency, to produce a mutational polypeptide from a mutant DNA obtained by this method and to provide a method for identifying an epitope of monoclonal antibody using the same polypeptide. CONSTITUTION:This method is the transduce base-substituted type mutation by designating DNA in a range wherein deoxynuclesoide triphosphate having low base pair specificity such as deoxyinosine is present in DNA synthase chain reaction. A mutant polypeptide is produced from a transformant transformed with DNA obtained by this method. This mutant polypetide prepared by this method using at least part of DNA of antigen protein gene of monoclonal antibody ad a template DNA and cross reaction between the prepared mutational polypeptide and the monoclonal antibody is measured to identify the epitope of the monoclonal antibody.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an isolated double-stranded DN.
Provided is a new technique for frequently introducing a variety of base substitution mutations in a specified region on A (gene, gene group). Further, the present invention relates to a method for producing a mutated polypeptide from a transformant having a mutation-introduced DNA obtained by the method of the present invention. in addition,
The present invention relates to a method for identifying an epitope of a monoclonal antibody using the above mutant polypeptide.

[0002]

2. Description of the Related Art Conventionally, (1) a desired mutant is selected from naturally occurring mutants and mutants produced by treatment with general mutagenizing agents such as ultraviolet rays and alkylating agents. (2) Alternatively, by incorporating a synthetic oligonucleoside, a mutation is specifically introduced at a relatively high frequency into one base designated on the oligonucleoside. (3) A technique of introducing a base substitution by a single-stranded DNA fragment (D. Shortle (Sho
rtle) et al, Proc. Natl. Acad. Sci
-USA, Vol. 77, No. 9, p5375-53
79, (1980)), but it is rarely used. Up to now, there is no method for introducing various mutations into all the bases in a specific region of DNA at high frequency with a simple operation as in the present invention.

[0003]

In the selection method from natural or induced mutants described in (1) above, the mutation rate is high and usually 1
Since it is 0 ⁻⁴ to 10 ⁻⁵ or less, a large number of specimens must be tested, which requires a huge amount of time and labor. In addition, it takes time and labor to analyze whether a mutation has been introduced into an expected gene and where in the gene the mutation has been introduced. The designated point mutation method using the oligonucleoside of (2) above has a high mutation introduction rate, and it is easy to confirm that the mutation has been introduced at the designated base. However, if the effects of mutations are not predicted, it is not possible to introduce mutations into a gene encoding a protein usually made up of hundreds of amino acids to make substitutions for all amino acids and examine the properties of each. Not realistic.

The region-directed mutation method using the D loop described in (3) above is close to the object of the present invention in that relatively diverse mutations can be introduced into a designated region of DNA. However, single-stranded DNA having a nucleotide sequence in the designated region must be prepared from a restriction enzyme fragment or by synthesis. Also,
When obtaining a restriction enzyme fragment from DNA, it is affected by the presence or absence of a restriction enzyme cleavage site, while the synthesis by the current technology is limited to 80 bases at the longest. In this way, it is not easy to isolate or synthesize the single-stranded DNA necessary for designating the region, and only the C-to-T substitution of the four types of DNA bases (A, T, G, C) Points that cannot be replaced, D
To make a loop, other enzymes such as RecA protein,
It is rarely used because of the drawbacks that it requires the aid of reagents, the operation is complicated, and that the D loop formation rate has a strong influence on the mutation acquisition rate and the reproducibility is poor.

The object of the present invention is to provide a highly specific region-specific method, which was not possible by the selection method from natural or induced mutants of (1), and the designated point mutation method of oligonucleosides of (2). Various mutations that were impossible with
A new method that eliminates the problems of base substitution mutations targeting bases other than C, which were not possible with the D-loop region-directed mutation method of (3), and the problems of operation complexity and reproducibility of method (3). It is to provide "regional mutation introduction technology". Another object of the present invention is to provide a method for producing a mutated polypeptide using a transformant having a mutated DNA.

Another object of the present invention is to provide a method for identifying an epitope of a monoclonal antibody using a mutated polypeptide. Table 1 summarizes the comparison between the method of the present invention and the above methods (1) to (3).

[0007]

[Table 1]

[0008]

DISCLOSURE OF THE INVENTION The present invention is directed to a DNA synthesizing chain reaction method in which DNA is synthesized in the coexistence of deoxynucleoside triphosphate having a low base pair specificity. To introduce a base substitution mutation.

Furthermore, the present invention is characterized in that a mutated polypeptide is produced by using a transformant transformed with the mutation-introduced DNA obtained by the above method, and the produced polypeptide is collected. To a method for producing a mutated polypeptide.

The present invention will be described in detail below. In the present invention, in order to specify a region of double-stranded DNA, known DNA synthase chain reaction (PCR: DNA Polym
erase-Chain Reaction Figure 1) method is used. In the PCR method, a region on the double-stranded DNA sandwiched by the same nucleotide sequences as the oligonucleosides called two primers is (1) denatured (single-stranded) of the double-stranded DNA, and (2) single-stranded with the primer. This is a reaction for geometrically amplifying by repeating a series of reactions in which the DNA is doubled by annealing with a strand DNA and (3) double stranding by a DNA chain extension reaction by a DNA synthase.

The PCR method originally had a problem when it was used for DNA structure analysis in that there were many errors in the incorporation of bases, but in the present invention, this was used in reverse (FIG. 2). That is, an error in the incorporation of a base results in a base substitution type mutation. In order to further increase the error in the incorporation of the base, a system of DNA chain elongation reaction by a DNA synthase,
An appropriate amount of deoxynucleoside triphosphate having low base pair specificity is added. As a result, the present invention succeeded in introducing a variety of region-specific mutations, which are region-specifically designated by a primer, at a high frequency of 10 ^-2 .

The feature of the mutagenesis method of the present invention is that PCR is performed.
In the DNA chain extension reaction (synthesis reaction) of the method, deoxynucleoside triphosphates having low base pair specificity are allowed to coexist with four types of deoxynucleoside triphosphates.
Examples of deoxynucleoside triphosphates having low base pair specificity include 2'-deoxyinosine-5'-triphosphate,
5-bromo-2'-deoxyuridine-5'-triphosphate, N ⁴ -. Aminoshichijin 5'-triphosphate (Takahashi et al. (1988) Mol.Cell.Biol 8, 347
-352), or 5-methylcytosine-5'-triphosphate that naturally substitutes for thymine after being incorporated into DNA.

Particularly, 2'-deoxyinosine-5'-triphosphate (hereinafter referred to as deoxyinosine triphosphate) is preferable from the viewpoint that an unspecified base substitution type mutation can be introduced. Also, 5-bromo-2'-deoxyuridine-5'-triphosphate can mainly convert AT base pairs to GC base pairs. N ⁴ - Aminoshichijin-5'-phosphate and 5'-methyl cytosine 5'-triphosphate G
-C base pairs can be changed to AT base pairs. The coexisting amount of these deoxynucleoside triphosphates can be appropriately determined in consideration of the degree of base pair specificity of the deoxynucleoside triphosphate to be used, the desired amount of mutation introduced, and the like. For example, in the case of deoxyinosine triphosphate,
For example, it is suitable to set it in the range of 0.2 μM to 200 μM.

The PCR method may be carried out by using the conventional conditions as they are, but from the viewpoint of increasing the mutation rate,
Contrary to the conventional condition setting, it is preferable to set the DNA chain extension reaction temperature to a relatively high temperature and to use a DNA synthase having low base pair specificity. From this point of view, the use of Taq DNA synthase is preferable.

The thus-mutated DNA having the mutation is placed in an appropriate vector using a restriction enzyme or the like by a conventional method to construct a DNA library, and an appropriate host is transformed. The resulting transformant is used to produce a mutated polypeptide by a conventional method. However, since the transformant is a mixture that produces both a normal polypeptide and a mutated polypeptide, the mutated polypeptide is collected from the produced polypeptide by a conventional method. As a collection method, a usual method for separating and purifying a polypeptide can be used as it is, and when the polypeptide has an antigenicity, a method utilizing a cross-reaction with a monoclonal antibody can be adopted.

An example of the method of the present invention will be described with reference to FIG. (1) Primer 1 and primer 2 having sequences at both ends of a region containing a target site (in the example, the entire RecA gene)
Using Taq DNA synthase (Taq DNA polyzyme) in the presence of deoxyinosine triphosphate (dITP).
PCR by merase). Deoxyinosine triphosphate is added to promote DNA synthesis errors (ie, mutations). (2) A restriction enzyme fragment (a C-terminal 93 amino acid region of RecA protein in the example is coded in the example) having a region to be mutated from the obtained mutant DNA by treatment with an appropriate restriction enzyme (EcoRI in the example). (EcoRI fragment) having a base sequence of (3) The obtained restriction enzyme fragment is incorporated into an appropriate vector DNA, cells are transformed with the DNA, and the gene having the desired mutation is selected from the obtained transformants. Vector selection and mutation selection methods are devised according to the purpose. Here, R described in the examples is used.
The identification of the antigenic determinant site of the ecA protein is described.

(3-1) The EcoRI fragment was Lambda
A DNA library is prepared by incorporating the gtll-expressing phage vector into the EcoRI cleavage site. (3-2) Escherichia coli Y1090
The strain is used as a host to form plaques of Lambda phage in the library. (3-3) Transfer the protein expressed in plaque to two membranes. Anti-R for proteins transferred to each membrane
The cross-ability of the ecA protein monoclonal IgG with the ARM191 antibody or the ARM193 antibody is tested by a color reaction by the enzyme antibody method. (3-4) Phages are recovered from plaques that intersect with only one antibody and used as mutant candidates.

By the way, the present inventors needed a method for identifying the epitope of an anti-RecA protein monoclonal class G antibody (IgG) on the amino acid sequence. That is, according to the conventional method, it was found that there are three anti-RecA protein IgG epitopes between the ^260th amino acid (Phe ²⁶⁰ ) of the RecA protein consisting of 352 amino acids and the C-terminus, but shorter protein fragments are those epitopes. It did not cross with any of the antibodies and could not be positioned more precisely. However, there was another data that those IgGs have different antigens from each other. So those I
It became necessary to identify the epitope of gG on the amino acid sequence.

Therefore, the present inventors have found a method for identifying the epitope of a monoclonal antibody using the above-mentioned mutagenesis method. That is, another aspect of the present invention is a method for identifying an epitope of a monoclonal antibody, which comprises DNA of at least a part of a gene of an antigen protein of the above monoclonal antibody.
The present invention relates to the above method, wherein a polypeptide mutated by the above-mentioned method is prepared using as a template DNA, and the cross-reactivity between the obtained polypeptide and the above-mentioned monoclonal antibody is measured.

The identification method of the present invention will be described with reference to the above anti-RecA protein monoclonal G-class antibody (IgG).
To introduce various amino acid substitution mutations between Rhe ²⁶⁰ and C-terminal of ecA protein, and test which amino acid changes the antigenicity to each IgG by the presence or absence of cross-reactivity with monoclonal antibody This allows precise localization of the epitope.

[0021]

INDUSTRIAL APPLICABILITY In the present invention, a method of selecting from naturally occurring mutants and mutants produced by treatment with general mutagenizing agents such as ultraviolet rays and alkylating agents (first method)
Highly specified region-specific method that was not possible with, and designated point mutation method using oligonucleoside (second method)
Various mutations that were not possible with, and further with base substitution mutations that target bases other than C that were not possible with the region-directed mutation method using the D loop (third method).
The problem of complexity and reproducibility of the operation which was in compliance with the law was eliminated.

The present invention makes it possible to identify an epitope on a polypeptide or protein very easily. In addition, promotion of research and development by utilizing the present invention in a wide range of basic science and applied technical fields such as identification of active site on protein and enzyme, enhancement of enzyme function by modification of enzyme activity, specificity conversion, analysis of gene function, etc. Can be achieved.

[0023]

Example: Monoclonal Ig against RecA protein
G, ARM191 antibody, ARM193 antibody, MAb15
The identification of the epitope for 6 will be described as an example. These 3
It was known that each of the two anti-RecA protein IgGs has an epitope between the ^260th amino acid (Phe ²⁶⁰ ) and the C-terminus, but none of the polypeptide fragments in this range crossed these IgGs by conventional methods. Could not identify the epitope.

Example 1 The mutation-introducing region is between Phe ²⁶⁰ and the C terminus, but it happens to occur at the restriction enzyme E at the codon position of Phe ^260.
Since there is a cleavage site for coRI, a mutation was introduced by PCR targeting the entire RecA gene in consideration of its use for other purposes. That is, the translation initiation codon (AT
G) containing primer 1 (5'-ATGGCTATCG
ACGAAAAACAA-3 ') and primer 2 (5'-) having a complementary sequence in the vicinity of 80 base pairs downstream of the translation stop codon.
GAATTCTGTCATGGCATATCCTT-
3 ') was synthesized. On the other hand, double-stranded DNA obtained by linearizing the plasmid pBEU14 cloned with the RecA gene with a restriction enzyme BamHI was used as a template for PCR.

The PCR reaction was carried out by using 1 μM each of primer 1 and primer 2, template DNA of about 3 pM, 200 μM
Each of four deoxynucleoside triphosphates (dAT
P, dTTP, dGTP, dCTP), 200 μM deoxyinosine triphosphate (dITP), 0.025 unit /
μl Taq DNA synthase, 1.5 mM MgC
l ₂ , 50 mM KCl, 0.001% gelatin, 1
It was carried out in 0 mM Tris-HCl buffer (pH 8.3). One PCR cycle is (1) 94 ° C., 1 minute double-stranded DNA denaturation step, (2) 37 ° C., 2 minutes primer-single-stranded DNA annealing step, (3) 72 ° C.
It consists of a 3 minute DNA chain extension reaction. This cycle was repeated 25 times. The correlation between deoxyinosine triphosphate concentration and mutant acquisition is shown in Table 2. If the concentration of deoxyinosine triphosphate is higher than 200 μM, the DNA amplification reaction may be inhibited.

The DNA in which the amplified mutation is introduced is Ec
It was treated with oRI and the EcoRI fragment encoding the C-terminal 93 amino acid region thereon was isolated by gel electrophoresis. This DNA fragment was incorporated into the Lambda gtll expressing phage vector at the EcoRI cleavage site to construct a DNA library. At this time, with a probability of 1/2, the LacZ gene on the vector and the portion encoding the C-terminal 93 amino acid region of RecA protein are connected in the same translation frame. E. coli (Escherichia coli) Y
1090 was used as a host and L
Ambda gtll creates plaques, the proteins expressed in the plaques are transferred to two membranes, and the proteins in the plaques are ARM191 antibody or ARM191.
Whether or not it showed crossover with the 193 antibody was identified by a color reaction by the enzyme antibody method.

When the EcoRI fragment was incorporated into the vector in the reverse orientation, it did not express the C-terminal portion of the RecA protein and therefore did not show any crossover with any IgG. When the DNA fragment having no mutation is incorporated in the correct orientation, it shows a cross with any IgG. If there is a mutation in any of the IgG epitopes,
It does not show a crossover with the IgG but shows a crossover with the other IgG (FIG. 2).

Thus, the phage in the plaque showing a crossover with only one IgG was recovered, the EcoRI fragment incorporated from this phage was recovered, and pU
It was incorporated into a C119 plasmid vector, and its nucleotide sequence was analyzed for both DNA chains. In addition, the EcoRI fragment in which the mutation was confirmed was cloned into Re on pMI996.
ARM19 of the mutated RecA protein was integrated under appropriate conditions into the EcoRI cleavage site at the C-terminal codon of the N-terminal about 260 amino acids of the cA gene.
Specific antigenicity against 1 antibody, ARM193 antibody, and MAb156 antibody (assuming the antigenicity of wild-type RecA protein to be 1) was quantified by ELISA.

The results are shown in Tables 2 to 4 and FIG.
(1) All the identified mutations were mutations in which amino acids were replaced by base substitutions, and various types of base substitutions were obtained (Tables 3 and 4). Most of the mutants had 1 base substitution, but some had 2 or 3 base substitutions (Tables 3 and 4). (2) The higher the concentration of deoxyinosine triphosphate, the higher the mutant acquisition rate (Table 2). However, as described above, when the concentration is higher than 200 μM, the DNA amplification reaction is strongly inhibited.

(3) As shown in FIG. 3, in the mutation between about 290th position and about 310th position, ARM191 antibody and M
Although the antigenicity against Ab156 antibody is impaired, ARM19
The antigenicity to the 3 antibody is normal. However, these 2
The epitopes for the two antibodies are slightly offset.
(4) It was revealed that the mutation at the amino acid position from the 330th amino acid to the 338th amino acid impairs the antigenicity to the ARM193 antibody, but the antigenicity to the ARM191 antibody and the MAb156 antibody is normal.

In FIG. 3, mutation (amino acid substitution)
Shows the antigenicity of each of the RecA proteins having a mutation in the site and each IgG to IgG. ●, ARM
Antigenicity against 191 antibody, ◯, antigenicity against ARM193 antibody, Δ, antigenicity against MAb156 antibody. The mark in the gray area indicates that no crossing is observed with that value as the limit of measurement.

As described above, other types of mutations can be easily obtained, and as a result, three types of anti-Re can be easily obtained.
The epitope of cA protein IgG could be identified at the amino acid sequence level.

[0033]

[Table 2]

[0034]

[Table 3]

[0035]

[Table 4]

[Brief description of drawings]

FIG. 1 is an explanatory diagram of a DNA synthase chain reaction.

FIG. 2 is an explanatory diagram of the region-directed mutation introduction method of the present invention.

FIG. 3 shows the relationship between the site of mutation (amino acid substitution) and the antigenicity (specific crossing ability) of each RecA protein having a mutation therein to IgG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location G01N 33/53 D 8310-2J

Claims (3)

[Claims] 1. In the DNA synthase chain reaction method, D
A method for introducing a base-substitution mutation into a DNA, wherein NA is synthesized in the coexistence of deoxynucleoside triphosphate having low base pair specificity. 2. A DN obtained by the method according to claim 1.
A method for producing a mutated polypeptide, which comprises producing a mutated polypeptide using the transformant transformed with A and collecting the produced polypeptide. 3. A method for identifying an epitope of a monoclonal antibody, which is a polypeptide mutated by the method according to claim 2, wherein at least a part of the DNA of the antigen protein gene of the monoclonal antibody is used as a template DNA. And measuring the cross-reactivity of the obtained polypeptide with the above monoclonal antibody.