JPH07102134B2 - Site-directed mutagenesis vector - Google Patents

Description

Detailed Description of the Invention

(Field of industrial application) The present invention relates to a vector effective for a site-specific mutation method used in genetic engineering. (Prior art) In recent years, a mutation method by a genetic engineering method has become an important tool because a mutant strain and a mutant gene can be obtained more accurately and more frequently than a mutation method by a conventional genetic method. . The mutation by this genetic engineering method is a method of introducing a desired mutation into a certain DNA sequence, and molecular biology,
It has become one of the most effective means for research such as protein engineering research. The structure of a protein can be artificially altered by manipulating the DNA sequence of the coding region, so it is possible to modify a protein found in nature, for example, an enzyme with effective properties by changing the structure of the enzyme. Is becoming. The characteristics relate to the function of the enzyme such as pH of action of protein, stability, heat resistance, Km value, and protease resistance. By changing these properties, there is a possibility that the enzyme can be modified to meet the design goal, and its potential is expected. In addition, gene expression is carried out through the processes of transcription, translation, and RNA processing.However, if it is considered that the nucleotide sequence is changed sequentially at the DNA level, the degree of expression may change. This is an important and indispensable method for improving the sex and studying the expression mechanism. In addition, the site-directed mutagenesis method is widely used in molecular biology and genetics, such as searching for active sites of physiologically active substances such as enzymes and peptides and elucidating the secretory mechanism of physiologically active substances. A typical procedure for site-directed mutagenesis consists of the following items. (See Fig. 1) (1) The DNA fragments to be mutated are single-stranded DNA and double-stranded DNA.
Clone into a vector that can be present at. (2) Hybridize an oligonucleotide synthesized by complementary synthesis except for a base portion that causes a mismatch in one region to be mutated to a circular single-stranded recombinant DNA. (3) Next, using the complementary single-stranded oligonucleotide as a primer, filling-in is performed with DNA polymerase, and the newly synthesized strand is ligated to the 5'end of the oligonucleotide by ligation. (4) Completely complementary double-stranded DN except for the intended mutation point
A is transformed into E. coli and replicated to grow a phage clone having a parent DNA and a phage clone having a mutant DNA. In this way, the phages derived from the two types of DNA were adjusted, mutant phage clones were isolated, and
Create A. In this case, the probability that each DNA-derived phage is generated is theoretically calculated to be 50%. But in fact the mutation
At present, DNA has a very low yield. (Problems to be Solved by the Invention) In the conventional site-directed mutagenesis method, a phage DNA, that is, a vector derived from M13 phage is used as a vector, but it has the following disadvantages, and this method is efficient. It was pointed out that it cannot be used for a long time. That is, since this vector is derived from a phage, a large DNA
Fragments, for example DNAs of 5 Kb or more, have the drawback of being difficult to insert. Further, it has been pointed out that such a phage vector is apt to be mutated in the replication process peculiar to the phage. (Means for Solving the Problems) The present inventors have made possible a vector that has improved these drawbacks, that is, (1) a mutant DNA can be collected with high probability, and (2) a large-sized DNA can be inserted. is there. (3) The present invention has been achieved by succeeding in producing a vector having these advantages such that the recombinant vector is not mutated during replication. That is, the present invention is a plasmid / phage fusion vector (pBSM13 +) containing a gene encoding ampicillin resistance.
The site-directed mutagenesis vector (pKMN + shown in FIG. 5 containing two amber mutation codons from the Dde I site (2715) to the Hpa II site (2752) of the gene region encoding ampicillin resistance of Escherichia coli. ) And the gene region encoding ampicillin resistance of the plasmid / phage fusion vector (pBSM13-) containing the gene encoding ampicillin resistance from the Dde I site (2715) to Hpa I
A site-directed mutagenesis vector (pKMN-) shown in FIG. 6, which contains two amber mutation codons up to the I site (2752). Is. In the present invention, a commercially available pBS vector (Stratagene
(Manufactured by K.K.) at the Nar I site of the restriction enzyme
The 454 nucleotide fragments up to the a I site were inserted and the synthesized vectors pBSM13 + and pBSM13− (Stratagene) were used as starting materials. This vector contains an intergenic region derived from bacteriophage M13 (ATCC 15669-B1) and is a plasmid / phage fusion vector. This vector is characterized by a small number of nucleotide sequences (3204b
p), it has the advantage of being able to insert a gene of large size (5 Kb or more), and has the disadvantage of the M13 vector, that is, the instability of mutation during replication is not observed. When E. coli carrying this vector was infected with M13 helper phage, single-stranded DNA was produced in good yield. Since single-stranded DNA can be collected in this way, single-stranded mutant DNA can be produced by mutagenesis with helper phage and oligonucleotides. However, this vector has a drawback that it is not easy to specifically discriminate mutant DNA from parent single-stranded DNA produced simultaneously. The vector of the present invention is a vector capable of rapidly selecting a forage clone having a mutant DNA in a host by improving this vector and rapidly producing a mutant. As described above, in the conventional site-directed mutagenesis using a phage vector, an amber mutation-introduced vector, that is, an oligonucleotide incorporating a mutation to be created is annealed, and an enzyme is used to transform a hybrid DNA that is filled in and ligated. And when I duplicated it,
Mutant phage clones supE ^- strains, can replicate grow either supE ⁺ strain, parental phage clones vectors mechanism capable of replicating grow only in supE ⁺ strains have been used. As described above, this vector is a phage vector, and has the disadvantages that it cannot insert large-sized DNA and that it shows unstable replication. The site-directed mutagenesis vector of the present invention has a plasmid containing M1
An amber mutation is introduced into a plasmid / phage fusion vector having an intergenic region of phage 3 and it encodes ampicillin resistance as a region for introducing an amber mutation in the plasmid / phage fusion vector (pBSM13 +, pBSM13-). It focuses on genes and introduces mutations without impairing their function. For example, paying attention to 37 bases from the Dde I site (2715) to the Hpa II site (2752) of the gene region encoding ampicillin resistance, an amber mutation was introduced by the method described below. The nucleotide sequence of this DNA fragment (Dde I-Hpa II) is shown below. In order to introduce an amber mutation into this region, a DNA fragment having the following nucleotide sequence was synthesized. As shown in the nucleotide sequence of the synthetic DNA fragment, the two TTG codons in the DNA fragment from the Dde I site to the Hpa II site of the pBSM13 vector were changed to CTA. As a result, it is changed to a stop codon by changing from TTG-encoded asparagine to CTA. This is because the Sup (-) strain does not have a special tRNA that functions to take up glutamine corresponding to the amber codon UAG, so the peptide chain ends there, the protein function is lost and it cannot be produced,
While the strain cannot grow, the Sup (+) strain has the characteristic of being able to grow. By this mechanism, mutant phage clones and parental phage clones can be distinguished. In other words, in the process of creating a site-specific mutant, the DNA of the mutant phage clone does not contain an amber mutation because it is derived from the plasmid / phage fusion vector (pBSM13 (+), (+) chain of pBSM13 (-)).
The DNA of the parental phage clone will contain an amber mutation. This is an amber S phage clone with parental DNA.
Amber mutations are rescued by infection with up host and culturing, but growth by Sup (-) strain is not possible. On the other hand, a mutant phage clone that does not have an amber mutation is Sup
Only mutant phage clones can be specifically selected because they can grow by infecting and culturing the strain. The vector of the present invention has T7 RNA promoter and T3 RNA promoter as polylinker sites (EcoR I, Sac I, Sma I, BamH).
I, Xba I, Sal I, Hinc II, Acc I, Pst I, Sph I, Hin
It is located on both sides of d III). Therefore, there is an advantage that a gene that has undergone site-specific mutation can be immediately synthesized and translated by T7 RNA polymerase T3 RNA polymerase to produce a protein, and the change in function due to mutation can be investigated. Since it has many polylinker sites, it has a feature that foreign genes can be easily inserted. The vector is called pKMN
They were named (+) and pKMN (-). Next, the procedure for constructing pKMN (+) and pKMN (−) is shown. (1) Aat II from pBSM13 (+) and pBSM13 (-)
Preparation of (3140) -Pst I (924) (989bp) First, a restriction enzyme was prepared to prepare the region containing the transgenic region of pBSM13 (+) and pBSM13 (-) to M13.
Double digest with Aat II and Pst I,
The digestion product was subjected to agarose gel electrophoresis to separate and purify a DNA fragment having a length of 989 bp. pBSM13 (+), and pBSM
The restriction map of 13 (-) is shown in Figs. Ast II
The reaction conditions for double digestion for adjusting the (3140) -Pst I (924) (989 bp) fragment are as follows. The above mixture was reacted at a temperature of 37 ° C. for 2 hours. (2) Aat II (3139) -Hpa II from pBSM13 (+)
(2752) (388bp), Dde I (2714) -Bal I (2338) (3
77 bp), Bgl I (2337) -Pst I (925) (1413 bp) adjustment Each DNA fragment for constructing pKMN (+) and pKMN (-) vectors from pBSM13 (+) was adjusted with each restriction enzyme. Double digestion was performed and the decomposed product was subjected to agarose electrophoresis and separated and purified from agarose. The DNA fragment shown in FIG. 4 was prepared by this method. (3) Synthesis of Hpa II (2751) -Dde I (2715) fragment Hpa II (2751) -Dde I of pBSM13 (+) and pBSM13 (-)
In order to add an amber mutation to the (2715) region, the following DN
The A fragment was synthesized according to a conventional method. (4) Construction of pKMN (+) and pKMN (-) of the present invention In order to construct vectors pKMN (+) and pKMN (-) having an amber mutation from the fragments prepared in the above process, a mixture of each fragment was treated with T4 DNA ligase. It was ligated at 16 ° C for 24 hours. After ligation, the reaction solution was used as a commercially available E. coli BMH71-1.
Transformed into 8MUTS (Sup (+)). pKMN
Three clones were obtained from the reaction liquid for making (+), and two clones were obtained from the reaction liquid for making pKMN (-). The plasmid obtained from this clone was transformed into E. coli MK30-3 (Sup (-)), and the production of the clone was tested. However, none of the produced clones were found, and the five vectors had an amber mutation. confirmed. Furthermore, in order to confirm the orientation of the transgenic region, each plasmid was prepared from the transformant. Prepare the prepared plasmid with restriction enzyme Nae I
It was an expected result that the direction was confirmed by performing double digestion with Pvu I and BamH I, and electrophoresing the degradation product to check the length of the fragment. The prepared pKMN (+) and pKMN (−) are shown in FIGS. 5 and 6. Next, an example in which a site-specific mutation is carried out in a nucleotide sequence encoding a physiologically active substance using the site-specific mutation vector of the present invention is shown. Example 1 By mutating two bases of a nucleotide sequence of a DNA of about 6000 bp (hereinafter referred to as γII-1A) encoding a rat brain membrane protein, Arg at position 1315 of the protein was changed to Gln, and
I tried to change Arg at position 1319 to Gln. In order to determine whether such mutation was successful, one base was mutated to introduce a site cleaved by the restriction enzyme Nsv I. Each step is shown below. (1) Preparation of pKMN (−) Inserting Foreign Gene γII-1A γII-1A was inserted into the polylinker site of pKMN (−) of the present invention by a conventional method to give a recombinant plasmid pKMN (−) (γ
II-1A) was inserted. E. coli JM
The recombinant plasmid pKMN (-) (γI
(2) Preparation of ssDNA of pKMN (−) (γII-1A) pKMN (−) (γII-1A) / JM101 was conditioned on pKMN (−) (γII-1A) / JM101 (Bactotryptone: 16g / 1, yeast extract: 10g / l, NaCl: 15g / l, pH7.6) and helper phage R4 when grown to 0.D. ₆₀₀ = 0.3
08 was added to moi20, and horizontal tilt shaking culture (37 ° C, 1 hr) was performed. Based on this, further add 50 ml of medium (2
xYT, ampicillin 100 μg / ml, kanamycin 100 μg / m
l, 0.01% thiamine). After culturing, centrifuge and add 20% polyethylene glycol 6000 and 2.
Phage was precipitated by adding 5 M to make it. After centrifugation (8000 rpm, 15 minutes), the precipitate was suspended in 600 μl of TE buffer. X10 High Buffer 70μl, DNase
70 μl of a mixture of RNase and RNase was added and incubated at 37 ° C. for 1 hour. After completion of the reaction, phenol treatment and chloroform treatment were repeated twice, NaCl was added to the aqueous layer, the mixture was left at -20 ° C and centrifuged, and the precipitate was collected. 300 μl of TE buffer was added to the precipitate to dissolve it, and ssDNA (15 μg) was dissolved. Phenol treatment and ethanol precipitation were performed to recover the precipitate.
An ssDNA solution was prepared by adding 0 μl of distilled water. (3) Preparation of pBSM13 (−) γII-1A with γII-1A inserted pBSM13 (−) and pB prepared from γII-1A by a conventional method
SM13 (−) γII-1A 15 μg was digested with restriction enzymes Nsi I and Mlu
I cut it. Further, after alkaline phosphatase treatment, acrylamide gel electrophoresis was performed. After electrophoresis, the DNA was extracted from the gel to prepare the NsiI-MluI fragment pBSM13 (−) (γII-1A) of linear DNA. (4) Preparation of mutation primer Amino acid of gene encoding rat brain membrane protein, that is, Ar
The following primers were prepared in order to change g (1315) → Gln and Arg (1319) →→ Gln. At the same time, in order to select the mutation from the third codon T → C of Phe (1320) for the purpose of selecting mutations only by analysis with a restriction enzyme, a primer (lower strand) having the following DNA sequence was prepared. In this case, Phe (1320) remains unchanged and does not change to another amino acid. ・ The marked sites are mismatched bases. When DNA is synthesized using the lower strand as a template

[] Has the following sequence, and a new Nsp V site is created at the underline. The above-mentioned primer (oligomer 56mer) used for mutation was labeled by the following method. Primer (12 pmol) 2 μl Kinase buffer 0.5 μl (γ- ³² P) -ATP 0.5 μl T4 polynucleotide kinase 0.5 μl 3.5 μl The above mixture was reacted at 37 ° C. for 30 minutes. 10 mM ATP (cold) 0.5 μl T4 polynucleotide kinase 0.5 μl 1.0 μl Further, the above mixture was added and reacted at 37 ° C. for 30 minutes. (5) Construction of Gapped Duplex DNA (A) Hybridization of dsDNA and ssDNA Vector containing the dsDNA fragment generated in (3), that is, Nsi I
PBSM13 (-) (γII-1A) containing -Mlu I fragment
0.6 μg and pKMN (−) (γII-1A) prepared with (2)
1.5 μg of ssDNA of was hybridized as follows. The above mixture was prepared and heated at 100 ° C. for 3 minutes and then incubated at 65 ° C. for another 5-10 minutes. After this, it was frozen at -20 ° C. The reaction solution of the sample (1) lacks the double strand of Nsi I-Mlu I fragment-pBSM13 (−) (γII-1A), and the reaction liquid of Msi I-Mlu I fragment-pBSM13 (−) (γII-
It was confirmed by agarose electrophoresis that the ssDNAs of 1A) and pKMN (−) (γII-1A) were hybridized. (B) Annealing of Mutant Primer The hybridization reaction solution prepared in (5)-(A) and the labeled mutant primer prepared in (4) were annealed as follows. Hybridization reaction solution 8 μl Labeled mutagenic primer 1.5 μl H ₂ O 0.5 μl 10.0 μl The above mixture was prepared, heated at 65 ° C. for 5 minutes, and then slowly cooled to room temperature for annealing. (C) Polymerization and ligation (5)-(B) -prepared annealing DNA
The polymerization and filling-in were performed as follows. Distilled water 23 μl x10 Filling buffer 4 μl Klenow fragment 1 μl T4 DNA ligase 2 μl 40 μl After reacting the above mixture at room temperature for 45 minutes, 0.5M EDTA (p
The reaction was stopped by adding 1 μl of H8.0) and incubated at 65 ° C. for 10 minutes. The x10 filling buffer has the following composition. 625 mM KCl 275 mM Tris-HCl (pH 7.5) 150 mM MgCl ₂ 20 mM DTT 0.5 mM ATP 0.25 mM of 4 dNTPs After each reaction, agarose gel electrophoresis was performed and it was confirmed that filling was performed. (D) Transformation into E. coli Supressor E ⁺ strain 30 μl of filling-in DNA reaction solution, 20 μl of x10 chloramphenicol, 150 μl of water, E. coli SupE ⁺ competent cells were mixed and spread on an agar plate. Then, the mixture was incubated overnight to grow colonies, and the formation of a total of 38 colonies was observed. (E) Separation of DNA from colonies Twelve clones were collected from 38 colonies, and plasmids were extracted and purified to obtain 100 μl of DNA solution. (F) Transformation into E. coli SupE ⁻ strain The DNA of each clone prepared in (5)-(E) was transformed into E. coli E ⁻ strain MK30-3 by the following method. DNA 3 μl Chloramphenicol (x10) 50 μl Combitent cell 53 μl 106 μl The above mixture was applied to an agar plate to grow a colony. The number of grown colonies was as follows. (G) Separation of DNA and confirmation of mutation by restriction enzyme Nsp V Pick up colonies from each plate with a toothpick
Was extracted (PEG precipitation method) to prepare a DNA solution. It was adjusted
5 μl of the DAN solution was cleaved with the restriction enzyme Nsv I, and the digestion solution was subjected to agarose electrophoresis to examine the cleavage property with the restriction enzyme Nsp V. As a result, 9 clones out of 12 clones were Ns
It was cut with p V and 3 clones were not cut. The DNA of 9 clones was sequenced by the Sanger method to confirm that it was mutated, and Arg (1315) → Gl
It was confirmed that a mutation occurred in n, Arg (1319) → Gln. (Effects of the Invention) The site-directed mutagenesis vector of the present invention allows mutant DNA to be collected with a high probability and allows large-sized DNA insertion.
It has the advantage that the replicating recombinant vector is not mutated. By using such a vector for partial specific mutation, it is possible to artificially change the structure of the protein to modify it into a protein having effective properties. In addition, by sequentially changing the nucleotide sequence, it is possible to study the mechanism of gene expression and the like, and the uses thereof are diverse.

[Brief description of drawings]

 FIG. 1 shows a typical operation procedure of the site-directed mutagenesis method. FIG. 2 shows a restriction map of pBSM13 (+). Fig. 3 shows a restriction map of pBSM13 (-). FIG. 4 shows three fragments of pBSM13 (+). FIG. 5 shows a restriction enzyme map of pKMN (+) of the present invention. FIG. 6 shows a restriction enzyme map of pKMN (−) of the present invention.

Claims (2)

[Claims] 1. Two amber mutation codons from the Dde I site (2715) to the Hpa II site (2752) of the gene region encoding ampicillin resistance of a plasmid / phage fusion vector (pBSM13 +) containing a gene encoding ampicillin resistance. 5. The site-specific mutation vector (pKMN +) shown in FIG. 2. Two amber mutations from the Dde I site (2715) to the Hpa II site (2752) of the gene region encoding ampicillin resistance of a plasmid / phage fusion vector (pBSM13-) containing a gene encoding ampicillin resistance. A site-directed mutagenesis vector (pKMN-) shown in FIG. 6, which contains a codon.