JP2528124B2 - Reverse transcriptase expression plasmid and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a reverse transcriptase expression plasmid having extremely high reverse transcriptase expression activity and a method for producing the same.

[Prior Art] In the present day, the expression of proteins by genetic engineering techniques and the development of biotechnology utilization techniques such as protein engineering have been vigorously developed. In this genetic engineering, cDNA (RNA
The production of DNA having a nucleotide sequence complementary to is important for the purpose of obtaining intron-free DNA.

When producing this cDNA, the cDNA is synthesized from messenger (m) RNA having the genetic information of the target gene product (protein) by reverse transcriptase (synthesis of RNA-dependent DNA). Has been widely used.

[Problems to be Solved by the Invention] Therefore, in the present invention, as a result of earnest research to find an expression plasmid of a reverse transcriptase having high expression activity of such an important reverse transcriptase, the present invention has been achieved. It is a thing.

[Means for Solving the Problems] That is, the present invention is intended to provide a novel reverse transcriptase expression plasmid and a method for producing the same. The purpose is to provide TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL according to the present invention.
IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL
LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTV
LDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTL
FDEALHRDLADFRIQHPDLILLQYXDDLLLAATSELDCQQGTRALLQTLG
NLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPR
QLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIK
QALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKK
LDPVAAGWPPCLRMVAAIAVLTKDAGNVTMGQPLYILAPHAVEALVKQPP
DRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGL [where A is alanine (Ala), C is cysteine (Cys), D
Is aspartic acid (Asp), E is glutamic acid (Glu),
F is phenylalanine (Phe), G is glycine (Gly),
H histidine (His), I is isoleucine (Ile), K is lysine (Lys), L is leucine (Len), M is methionine (Met), N is asparagine (Asn), P is proline (Pr).
o), Q is glutamine (Gln), R is arginine (Ar)
g), S is serine (Ser), T is threonine (Thr), V
Is a valine (Val), W represents tryptophan (Trp) and Y represents tyrosine (Tyr)], and a reverse transcriptase expression plasmid expressing a reverse transcriptase having an amino acid sequence represented by the following amino acid sequence: 223nd X from N terminus
Is a single amino acid selected from the group consisting of L (leucine), I (isoleucine), A (alanine), F (phenylalanine) and R (arginine), and a reverse transcriptase expression plasmid, and The method for producing a reverse transcriptase expression plasmid (first method), which comprises the steps of

(A) Isolate the gene encoding reverse transcriptase from the plasmid pKP1.

(B) Having two multicloning sides, having a relay nucleotide sequence in which three kinds of stop codons are inserted between the two multicloning sites, and both ends of the DNA fragment isolated in (a) above. A vector that matches the frame of
A DNA fragment is separated using the same restriction enzyme as the restriction enzyme used in the above-mentioned (a) separation operation.

(C) The DNA fragments obtained in (a) and (b) above are ligated.

Of the restriction enzyme cleavage sites possessed by the plasmid obtained in (c) above, the one located on the N-terminal side of the DNA fragment is cleaved, the cut end is filled with polymerase, and the frame is shifted by ligating again. To obtain the desired plasmid.

(E) The plasmid obtained in (D) above is introduced into E. coli to obtain white transformant colonies.

(F) A plasmid DNA prepared from this transformant,
Site-specific gene conversion was performed using oligo DNA synthesized by removing the excess amino acid sequence on the N-terminal side of the reverse transcriptase and the nucleotide sequence corresponding to the excess amino acid sequence derived from the vector to obtain the excess amino acid sequence. A mutant plasmid having the corresponding nucleotide sequence removed is prepared.

(G) The mutant plasmid pKP is obtained by introducing the mutant plasmid obtained in (f) into E. coli and performing transformation to obtain blue colonies from white colonies.
A transformant introduced with _po1- N is obtained.

(H) Plasmid pKP _po1 − prepared from this transformant
A mutant plasmid pKP that synthesizes N and an oligo DNA having a stop codon inserted at the C-terminal side of the reverse transcriptase and produces a reverse transcriptase containing no extra amino acid sequence at both ends in the same manner as (f) above. _po1 − Create NT1.

(I) The mutant plasmid obtained in (h) above is introduced into Escherichia coli for transformation, and white colonies are obtained from the blue colonies to obtain a transformant into which the mutant plasmid has been introduced. .

In addition, in the method for producing a reverse transcriptase expression plasmid (second method) of the present invention, a mutant reverse transcriptase expression plasmid may be produced by further adding the following steps (a) to (g). it can.

(A) A DNA fragment containing a reverse transcriptase region is cut out from the plasmid pSH1.

(B) It has two multicloning sites, has a relay base sequence in which three kinds of stop codons are inserted between the two multicloning sites, and is separated in (a) above.
The DNA fragments are separated using the same restriction enzymes as those used in the above-mentioned separation operation (a) for the vector in which the frames at both ends of the DNA fragment match.

(C) The DNA fragments obtained in (a) and (b) above are ligated.

(D) E. coli is transformed with the solution obtained in (c) above to obtain blue transformant colonies.

(E) After synthesizing an oligo DNA having an out-of-frame sequence of the plasmid pBB9 and the DNA fragment containing the reverse transcriptase region obtained in (d) above, site-specific gene conversion is performed to prepare a mutant plasmid. Using this solution, Escherichia coli was transformed, and white colonies were obtained from blue colonies to obtain a transformant into which the mutant plasmid pBBD7 was introduced.

(F) Of the nucleotide sequences in the mutant plasmid pBBD7 obtained in (e) above and the DNA containing the reverse transcriptase region, the codon encoding the 223rd valine in the amino acid sequence was changed to leucine, isoleucine, or alanine. , Phenylalanine, and arginine are converted into a codon encoding one amino acid selected from the group consisting of, and an oligo DNA in which the frame has been restored is prepared. Thus, a transformant introduced with the mutant plasmid pBBDX (X is leucine, isoleucine, alanine, phenylalanine, or arginine) is obtained.

(G) The plasmid pBBDX obtained in the above (f) (X is leucine, isoleucine, alanine, phenylalanine,
(Or arginine) is treated with a restriction enzyme to purify a DNA fragment, which is then ligated with the mutant obtained by the first method treated with the same restriction enzyme as above to obtain a DNA solution. A transformant colony is obtained by transforming E. coli, and then the plasmid pKP-T1X (X is leucine, isoleucine, alanine, phenylalanine, or arginine) is obtained.

In general, proteins expressed by plasmids, such as reverse transcriptase, have extra amino acids attached to their binding sites during the construction of the plasmid. In this case, according to the present invention, in order to express the reverse transcriptase in a pure form, the restriction enzyme Hind I at the front part is used.
II, Pst I, Sal I, Acc I, Hinc II, BamH I, Sma I, EcoR I linkers with multiple cloning sites that have the nucleotide sequence of each cleavage site (hereinafter, polylinker), intermediate sequence (must be, 3 types of stop codons), a polylinker in the rear part, and a vector constructed by connecting with lacZ 'are used to easily express a protein in pure form by performing site-specific gene conversion with synthetic DNA. Can be made.

And other structures of this vector are those having an ampicillin resistance gene (Ampr), a replication initiation signal (Ori) (this is called pKIS vector), and other structures as described above and in addition to the conventional pUC vector, Some have the intergenic region (IG) of M13 (this is called pKIS1
Called vector).

The above vector contains 3 types of frame polylinkers (front part) x 3 types of frame polylinkers (rear part) x 2
Since there are 18 types of cloning sites (there are also reverse cloning sites), it is extremely easy to use pure vector (reverse transcription) by using a vector series constructed by combining these 18 types of vectors. enzyme)
Can be prepared.

In this vector, the multi-cloning sites A and B in FIG. 1 have the nucleotide sequences shown in Table 1. The base sequences are as shown in Table 2.

In addition, as an example, of the pKIS vectors, pKIS801 / pKIS
Table 3 shows the characteristic parts of the structure of 10801.

EXAMPLES Hereinafter, the present invention will be described based on examples.

First, a method for producing the vector used in the present invention will be described.

Construction method of pUC1090 1) Using plasmid pUC9 (sold by Takara Shuzo Co., Ltd.) 10 μg with restriction enzymes EcoR I (50 units) and Hind III (50 units)
After reacting at 37 ℃ for 1 hour, the degradation products were separated using 0.7% agarose gel electrophoresis and Hind I was digested from the EcoR I cleavage site.
The polylinker fragment (30 base pairs) up to the II cleavage site was cut out from the gel and treated with phenol for purification.

2) After reacting 10 μg of plasmid pUC119 (sold by Takara Shuzo Co., Ltd.) with restriction enzymes EcoRI (50 units) and Hind III (50 units) for 1 hour at 37 ° C, the degradation products were electrophoresed on 0.7% agarose gel. Separated from the EcoR I cleavage site by Hi
A fragment (about 3,100 base pairs) containing no polylinker up to the nd III cleavage site was excised from the gel and purified by treating with phenol.

3) 0.01 μg of the polylinker fragment obtained in 1) and 2)
0.1 μg of the polylinker-free fragment obtained in (1) was mixed, and ligation reaction was performed for 12 hours at 4 ° C. using T4 DNA ligase (2 units).

4) The reaction product obtained in 3) was used to transform E. coli JM83 strain (ar
a, △ (lac-proAB), rpsL, φ80, lacZ △ M15) were used for transformation, and then L agar medium (composition: trypton per 1 ml) containing 1.5% of agar as a recipient and 100 µg of ampicillin per ml. 10g, yeast extract 5g, salt 10g, pH 7.5)
Then, the mixture was applied to and cultured overnight at 37 ° C. to obtain some colonies (transformants).

5) One of the colonies was 100 μg of ampicillin per ml
The mixture was transferred to 10 ml of L liquid medium containing the cells, shake-cultured at 37 ° C. for 12 hours, and plasmid DNA was separated by the alkali-SDS method to obtain plasmid pUC1090.

Method for constructing pUC1080 Among methods for constructing pUC1090, pUC8 (sold by Takara Shuzo Co., Ltd.) is used as the plasmid DNA of 1) and the plasmid of 2).
By using pUC118 (sold by Takara Shuzo Co., Ltd.) as DNA, the plasmid pUC1080 was prepared through the same procedure.

Next, a method for preparing pUC1091 will be described.
Speaking of the relationship between 90 and pUC1091, pUC1092, the pUC1090 polylinker portion is sandwiched, one base is deleted at the front and two bases are deleted at the back is pUC1091, the polylinker portion of pUC1090 is sandwiched, PUC1092 has two bases deleted at the front and one base deleted at the back. Also, pUC1080, pUC1081, pUC10
The relationship of 82 is the same as above.

Method for creating pUC1091 pUC1091 was created in the following steps.

1) Preparation of single-stranded DNA from pUC1090 2) Synthesis of oligonucleotide lacking one base from the front of polylinker site 3) 1) Preparation of mutant using 2) 4) Single-stranded from mutant Preparation of DNA 5) Synthesis of oligonucleotide lacking 2 bases from the rear of polylinker site 6) Preparation of remutant using 4) 5) (pUC109
1) -1) Preparation of single-stranded DNA from pUC1090 pUC1090 was transformed into E. coli MV1184 strain (ara, △ (lac-pro), str
A, thi, (φ80, lacZ △ M15), △ (srl-recA) 306 :: Tn10
(Tet ^r ), F ′: TraD36, proAB, lacI ^q ZΔM15) were used for transformation.

Transformants containing ampicillin at 100 μg / ml 2
× Inoculate 10 ml of YT liquid medium (composition: 16 g of tryptone, 10 g of yeast extract, 5 g of salt, pH 7.6) per 1 ml of the medium at 37 ° C.
Time shaking culture was performed.

A helper phage solution (10 ¹⁰ pfu / ml) derived from M13K07 was added to 100 μl of the culture obtained in the above, and the mixture was allowed to stand at 37 ° C. for 30 minutes. % Of 2 × YT liquid medium, and cultured at 37 ° C. for 24 hours with shaking.

Take 1 ml of the culture solution and remove the cells by centrifugation.
By adding 200μ of 0% PEG6000 and 2.5M NaCl mixed solution and centrifuging, the phage particles were precipitated, and ethanol precipitation treatment was performed to separate and purify single-stranded DNA, and finally 50μ of TE solution (10mM Tris-HCl, 1m
M EDTA, pH 8.0 solution).

 The yield was about 2-10 μg.

-2) Preparation of Synthetic DNA Containing Mutated Portion An oligonucleotide having the sequence No. 9-1-5 shown in Table 4 was synthesized using a DNA synthesizer, and 37 ° C. using T4 DNA kinase (2 units). Reaction for 1 hour at
The 5'end portion was phosphorylated to obtain a primer DNA.

-3) Preparation of mutant having mutation in front of polylinker 1 μg of single-stranded DNA obtained in -1) and 20 pmol of primer DNA obtained in -2) were mixed at 60 ° C. for 30 minutes.
Continue to incubate at 37 ℃ for 30 minutes and anneal, then dA
TP, dCTP, dGTP and TTP were added so that the final concentration was 0.5 mM, and 25 pmol of ATP was added, and Klenow (KLENO
W) Reaction with enzyme (4 units) and T4 DNA ligase (1 unit) at 37 ° C for 3 hours, including mutation part 2
Single-stranded DNA was synthesized.

Next, a part of the reaction product (about 0.1 μg) was transferred to Escherichia coli JM83.
After transformation using the strain, the recipient bacterium was 1.5% agar, 100 μg of ampicillin per ml, and 0.005 g of X-gal.
% Of L-agar medium and cultured at 37 ° C. for 24 hours, and white colonies were selected from the colonies that appeared.
The appearance rate of white colonies was about 5 to 10%.

Then, white colonies were treated with ampicillin at 100 ml / ml.
The cells were inoculated into 10 ml of L liquid medium containing μg, shake-cultured at 37 ° C. for 12 hours, and plasmid DNA was recovered by a conventional method.
The recovered plasmid DNA was transformed again using Escherichia coli MV1184 strain, and the recipient bacteria were agar 1.5%, ampicillin 100 μg / ml, X-gal 0.005% and IPTG 1 m.
Stained on a L-agar medium containing M, cultured at 37 ° C for 24 hours, selected white colonies, and added 100 μl of ampicillin per ml.
The cells were transplanted into 10 ml of an L liquid medium containing μg, cultured with shaking at 37 ° C. for 12 hours, and plasmid DNA was isolated by a conventional method.

-4) Preparation of mutant single-stranded DNA A single-stranded DNA having a mutation was obtained from Escherichia coli MV1184 strain having the mutant plasmid obtained in -3) by using the same method as in -1). .

-5) Preparation of synthetic DNA having mutation at rear part of polylinker Using the same method as in -2), prepare synthetic DNA having base sequence No. 9-2-5 in Table 4 and phosphorylate it. DNA was created.

-6) Preparation of remutant Using the same method as in -3), Japanese chain DNA was synthesized from the single-stranded DNA obtained in -4) and the primer DNA obtained in -5), Escherichia coli JM83 strain. A blue colony was obtained by carrying out transformation with. From this the plasmid DN
A was collected and transformed again with Escherichia coli MV1184 strain to make a blue colony appear. By recovering the plasmid DNA from this, plasmid pUC109 with a synthetic 3 base pair deletion at two positions before and after the polylinker part was recovered.
Got one.

Preparation method of pUC1092
This was performed according to the example of making 1091. The changes are as follows.

The nucleotide sequence of (1) -2) is designated as No. 9-1-3 in Table 4.

The nucleotide sequence of (2) -5) is designated as No. 9-1-5 in Table 4.

As a result, a mutated plasmid pUC1092 lacking a total of 3 bases, 2 bases at the front and 1 base at the back of the polylinker portion, was obtained from the plasmid pUC1090.

Method for constructing pUC1081 and pUC1082 In the same manner as described above, mutant plasmids pUC1081 and pUC1082 were constructed by shifting the polylinker portion by one base and two bases based on the plasmid pUC1080.

 The changes are as follows.

(1) For both pUC1081 and pUC1082, pUC1080 was used as the plasmid of -1).

As the synthetic DNA of (2) -2), pUC1081 has No.8-1-5 in Table 4 and pUC1082 has No.8-2-5.
Was used.

No. 8-1-3 in Table 4 for pUC1081 and No. 8-1-5 for pUC1082 as synthetic DNAs of (3) -5)
Was used.

As described above, the basic plasmids pUC1090, pUC1080 and the prepared mutant plasmids pUC1091, pUC1092, pUC1081
Table 5 summarizes the relationship between pUC1082 and pUC1082.

Table 4 Synthetic DNA Nucleotide Sequence Table DNA sequence containing a mutated portion (9-1-5) 5'GCCAAGCTTGCGTAATCATG3 '(9-2-5) 5'AGCCAAGCTTCGTAATCATG3' (9-1-3) 5'ACGACGGCCAGAATTCCCGG3 '(9-1-5) 9-2-3) 5'CGACGGCCAGGAATTCCCGG3 '(8-1-5) 5'CCGGGAATTCTAATCATGGT3' (8-2-5) 5'CCGGGAATTCAATCATGGTC3 '(8-1-3) 5'ACGGCCAGTGAAGCTTGGCT3' (8-2-3) 5'CGGCCAGTGCAAGCTTGGCT3 ' Construction of pKIS1 vector series -1) Separation of fragments 10 μg of plasmid pUC1090 was reacted with restriction enzymes Hind III (50 units) and Aat II (4 units) for 1 hour at 37 ° C. to decompose the degradation products by 0.7% agarose electrophoresis. Separated from the Hind III cleavage site containing the polylinker moiety by Aat
The fragment up to the II cleavage site (about 1000 base pairs) was cut out from the gel and purified by treating with phenol.
1 μg of L fragment was obtained.

Similarly, using the restriction enzymes EcoR I (50 units) and Aat II (4 units), a DNA fragment (about 2200 base pairs) from the EcoR I cleavage site containing the polylinker moiety to the Aat II cleavage site 109
2 μg of 0-R fragment was obtained.

Hereinafter, plasmids pUC1091, 1092, 1080, 1081, 1082
By performing the same operation using
91-R, 1092-L, 1092-R, 1080-L, 1080-R, 10
A total of 12 81-L, 1081-R, 1082-L and 1082-R fragments were obtained. The combination of each fragment, the plasmid to be cleaved, and the enzyme to be cleaved is as follows.

-2) Example of preparation of joint sequence The upper and lower chains of the joint sequence shown in Table 6 were each synthesized using a DNA synthesizer.

1 μg of the upper chain and 1 μg of the lower chain were mixed, kept at 70 ° C. for 30 minutes, allowed to stand in a room, and gradually cooled to carry out annealing to obtain double-stranded DNA.

-3) Combination of L and R fragments and joint sequence 0.1 μg of 1090-L fragment obtained in -1), 1
The 090-R fragment (0.2 µg) and the joint sequence (0.01 µg) obtained in -2) were ligated using T4 DNA ligase (2 units) by a conventional method. After the reaction solution was transformed using Escherichia coli JM83, the recipient strain was 1.
5% agar and 100 μg of ampicillin per ml were applied to an L agar medium, and cultured at 37 ° C. for 24 hours to allow transformed colonies to appear.
It was transplanted to 10 ml of L liquid medium containing g and cultured at 37 ° C. for 12 hours with shaking. After that, plasmid DNA was separated from the culture solution by a conventional method to obtain a plasmid pKIS10900.

Hereinafter, by changing the combination of fragments,
pKIS10901, 10902, 10910, 10911, 10912, 10920, 1092
1, 10922, 10800, 10801, 10802, 10810, 10811, 1081
A total of 18 pKIS1 vector series of 2, 10820, 10821 and 10822 were created.

The complete pKIS1 vector and fragment combinations are as follows.

Construction of pKIS vector series -1) Construction of pKIS900,901,902,910 911,912,920,921,922 After reacting 5 μg of plasmid pUC9 with restriction enzyme Pvu II (24 units) for 1 hour at 37 ° C, separation was performed by 0.7% agarose electrophoresis. , Does not contain polylinker moiety
A DNA fragment (about 2300 base pairs) was excised from the gel and purified. In addition, pKIS109 series (ie pKIS10900,10901,109
02,10910,10911,10912,10920,10921,10922) 5 μg each
With the same restriction enzyme Pvu II (24 units) at 37 ° C
After reacting for a period of time, separation was carried out by 0.7% agarose electrophoresis, and a DNA fragment containing a polylinker portion (about 300 base pairs) was purified from the gel.

Next, 0.2 μg of the above-described pUC9-derived DNA fragment and pKIS109
0.05 μg each of the DNA fragments derived from the series were mixed, ligated with T4 DNA ligase (2 units) at 4 ° C for 12 hours, and sequentially transformed with E. coli JM83 strain to obtain plasmid DNA from the transformant. Sequentially separated and plasmid pKIS9
We got 00,901,902,910,911,912,920,921,922.

-2) Creation of pKIS800,801,802,810 811,812,820,821,822 In -1), pKIS108 was replaced with pKIS108.
Series (ie pKIS10800,10801,10802,10810,10811,
10812,10820,10821,10822), the plasmid pKIS800,801,802,810,811,
Got 820,821,822.

 Specific examples using the above vector will be described.

(Reference example) It encodes the reverse transcriptase of mouse leukocyte virus.
Plasmid pKP1 containing approximately 2.3 kb (kilobase pairs) of the pol gene
[("Proceeding National Academy Science (PNAS),
USA "vol.82, pages 4944-4948, 1985) and cloned Sac I-Hind III (2.3 kb) of the plasmid pSH1
Recombined into pUC18] 2 μg was added to the restriction enzyme EcoR I 10
Unit and Hind III 12 units, treated at 37 ℃ for 1 hour, 0.7
% After agarose electrophoresis, approximately 2. containing reverse transcriptase gene.
A 3 kb DNA fragment was cut out from the gel and treated with phenol for purification. In addition, 2 μg of pKIS811 was added to the restriction enzyme EcoR I (10 units) and Hind III (12 units) to
It was treated at 1 ° C. for 1 hour, and a DNA fragment of about 2.7 kb was similarly recovered. 0.1 µg of the obtained 2.3 kb DNA fragment and 2.7 kb DNA fragment
Mix 0.1 μg and add 16 with T4 DNA ligase (2 units).
It was treated at ℃ for 12 hours. E. coli JM83 using this DNA solution
The strain was transformed, and the recipient cells were spread on L agar medium containing agar 1.5%, ampicillin 100 μg / ml, and X-gal 0.005%, and cultured overnight at 37 ° C. Obtained. This transformant was transformed with L liquid medium to 37
Culturing was performed at 12 ° C. for 12 hours, and a plasmid DNA was prepared by the alkali-SDS method to obtain pKPB.

2 μg of pKPB is restriction enzyme EcoRI (10 units), Klenow enzyme (2 units), and final concentration of 0.1 mM each of dATP, dGTP and dCT.
A mixture of P and TTP was added, treated at 37 ° C for 1 hour, treated with T4 DNA ligase (2 units) at 16 ° C for 12 hours, and then E. coli.
It was similarly introduced into JM83 strain. A white transformant colony was inoculated into an L liquid medium and cultured at 37 ° C. for 12 hours, and then a plasmid DNA was prepared by the alkali-SDS method to obtain pKPW.

An extra amino acid sequence on the N-terminal side of the reverse transcriptase was removed from pKPW by site-specific gene conversion using synthetic DNA.

5 μg of pKPW was added to the restriction enzymes Pvu II (18 units) and Ndel (2
(0 unit) at 37 ° C. for 1 hour, and 0.7% agarose gel electrophoresis was performed to recover a DNA fragment of about 2.2 kb. Also, pKPW 5
µg of restriction enzyme Sca I (18 units) and dephosphorylating enzyme BAP
(0.2 units) at 37 ℃ for 1 hour, similarly 5kb
The DNA fragment was recovered. The obtained 5.5 kb and 5 kb DNA fragments were mixed in an amount of 0.6 μg each, and 50 pmol of the 5′-phosphorylated synthetic DNA (40 bases) shown in Table 7a was mixed to obtain 100 mM NaC.
l, 6.6 mM Tris-HCl (pH 7.6), 8 mM MgCl ₂ , 1 mM β-mercaptoethanol solution, treated at 100 ° C. for 3 minutes and then at 30 ° C. for 30 minutes. This DNA solution 17.2μ, 5mM
dNTP (dATP, dGTP, dCTP, TTP) 7μ, 10mM ATP3.6
μ, Klenow enzyme (4 units), T4 DNA ligase (1
(Unit) and added and treated at 25 ° C. for 3 hours. Using this DNA solution, Escherichia coli JM83 strain was transformed to obtain a clone pKP pol-N forming a blue colony on L agar medium containing ampicillin and X-gal. This mutant was obtained with a frequency of about 5%.

pKP pol-N (a plasmid encoding a reverse transcriptase and β-galactosidase fusion protein) was added to synthetic DNA (7th plasmid).
A site-specific gene conversion using Tables b and c) introduced a stop codon at the C-terminus (T1 and T2), which appears to be the pure frequency of reverse transcriptase.

The plasmid pKP pol-N 5 μg was added to the restriction enzyme Sca I (18
Unit) and dephosphorylating enzyme BAP (0.2 units) at 37 ° C. for 1 hour, and similarly a DNA fragment of about 4.9 kb was recovered.

Also, 5 μg of pKP pol-N was treated with restriction enzymes Kpn I (50 units) and Hind III (24 units) at 37 ° C for 1 hour,
Similarly, a DNA fragment of about 2.9 kb was recovered. 4.9kb and 2.9kb
0.6 µg of each DNA fragment, and 50 poml of 5'-
Using the phosphorylated synthetic DNA (Table 7c), the mutants of the white transformants and pKP pol-N were treated in the same manner as above.
Got T2.

 The above manufacturing process is shown in FIG.

The reverse transcriptase activity of the obtained clone was measured.

 First, a method for measuring reverse transcriptase activity will be described.

For each clone, the one introduced into Escherichia coli JM103 strain was used. Escherichia coli carrying each plasmid was shake-cultured in an L liquid medium at 37 ° C for 12 hours, and 0.2 ml of the culture solution was added to an M9 liquid medium (composition: disodium phosphate 18 g, potassium 1 potassium 3 g, ammonium chloride 1 g, Salt 0.5g, magnesium sulfate 1mM, calcium chloride 0.1mM, pH 7.
4) (+ 0.2% casamino acid + 0.2% glycerol + 5μg / m
Thiamin) (10 ml) and shake-cultured at 30 ° C. for 1 hour, 50 μm of 200 mM IPTG was added, and the mixture was further shake-cultured at 30 ° C. for 3 hours. Soak each culture in ice for 1 hour,
▲ OD ⁶⁶⁰ ₁₀ ▼ was measured respectively. Next, each sample was diluted with M9 medium to an OD ⁶⁶⁰ ₁₀ of 0.2, and then 0.1 ml was collected in a microcentrifuge tube.The cells were collected by centrifugation and buffered (50 mM Tris). Hydrochloric acid, 0.5 mM ED
The cells were washed with TA, 0.3 M NaCl, pH 7.5), and the cells collected by centrifugation were stirred in 8 µ of the same buffer solution, and then 1 µ of 10 mg.
/ ml lysozyme solution was added and treated at 0 ° C for 15 minutes. Further, 1% Triton X-100 and 1 mM of 10 mM dithiothreitol (DTT) were added, and the mixture was treated at 0 ° C for 10 minutes to prepare each crude extract. 90 μl of reaction solution (30 μg / ml poly rC (dG _12-18 ) or poly rA (d
G _12-18 ), 20 μM dGTP or TTP, 1000 CPM / pmol α−32P−
dGTP, 0.5 mM MnCl ₂ or MgCl ₂ , 50 mM Tris-HCl, pH 8.
3, 20 mM dithiothreitol, 60 mM NaCl, 0.1% NP-4
0) was added and the mixture was treated at 25 ° C for 30 minutes. After completion of the reaction, 30μ of the reaction solution was spotted on a DE-81 disc and 2xSSC
(Composition: 0.3M NaCl, 0.03M sodium citrate) Treatment with 200ml for 5 minutes 3 times, 59.5 with 200ml of 99.5% ethanol.
After treatment for minutes, it was air dried.

The dried DE-81 disc was put into a vial, and the count was measured using the Cherenkov effect with a scintillation counter.

The Cherenkov effect is a phenomenon in which electromagnetic waves or light are emitted when fast charged particles pass through a transparent medium and the speed is higher than the speed of light in the medium.

Reverse transcription activity was observed in pKP pol-N as compared to the clone containing only the vector. For pKP pol-NT2,
The activity was further increased.

Furthermore, the C-terminal (T1) predicted from the amino acid sequence of the reverse transcriptase of cauliflower mosaic virus was subjected to site-specific gene conversion using synthetic DNA similarly shown in Table 7b.

The obtained clone pKP pol-NT1 showed higher activity than pKP pol-NT2, as shown in FIG.

(Example 1) Next, a method for preparing an expression plasmid for a mutant reverse transcriptase of Moloney murine leukemia virus and its reverse transcription activity will be described.

In the Moloney murine leukemia virus reverse transcriptase, a mutation was inserted in the amino acid sequence having the highest homology with other retroviruses. As a result, those showing activity similar to natural ones, those showing activity not only in manganese but also in magnesium, and those having higher activity than natural ones were obtained.

(Construction method) A plasmid having a region containing the amino acid sequence with the highest homology to the reverse transcriptase of Moloney murine leukemia virus
pBB9 was prepared. Clone pSH1 containing reverse transcriptase region
("Proceeding National Academy Science (PNAS), US
A ”vol.82, page 4944-4948, 1985) 5 μg with restriction enzyme B
It was treated with amH I (20 units) at 37 ° C. for 1 hour, a 0.3 kb (kilobase pair) DNA fragment was cut out using 0.7% agarose electrophoresis, and purified by treating with phenol.
On the other hand, pKIS9002 μg was added with the restriction enzyme BamHI (10 units) to 37
After treating at ℃ for 1 hour,
The 2.7 kb DNA fragment was purified. 0.
0.1 μg each of 3 kb and 2.7 kb DNA fragment of pKIS900 was mixed, and T
Ligation reaction was carried out at 4 ° C. for 12 hours using 4DNA ligase (unit). A part of the reaction mixture was transformed with Escherichia coli JM83 strain, and the recipient cells were transformed with 1.5% agar and ampicillin 100μ.
Apply to L agar medium containing g / ml and X-gal 0.005%, and
Cultivation was performed overnight at 7 ° C. to obtain blue transformant colonies.

Transformant colonies were transferred to L liquid medium (ampicillin was
10 ml (containing 100 μg per liter) was inoculated and cultured with shaking at 37 ° C. for 12 hours to isolate plasmid DNA pBB9 by a conventional method.

After treating 2 μg of pBB9 with restriction enzyme Sca I (18 units) and dephosphorylating enzyme BAP (0.2) unit at 37 ° C. for 1 hour,
A DNA fragment of about 3 kb was purified using 0.7% agarose electrophoresis. In addition, 2 µg of pBB9 was treated with restriction enzymes EcoR I (20 units) and Pst I (20 units) at 37 ° C for 1 hour, and then the same operation was performed to purify a 2.7 kb DNA fragment. Was subjected to site-specific gene conversion.

0.6 μg of each of the above DNA fragments and 50 pmol of 5'phosphorylated synthetic DNA (shown in Table 8a) were treated at 100 ° C. for 3 minutes and then annealed at 30 ° C. for 30 minutes.
DNA solution 17.2μ to 5mM dNTP 7μ and 10mM ATP 3.6μ,
Klenow enzyme (4 units) and T4 DNA ligase (1 unit) were added and treated at 25 ° C for 3 hours. Using this DNA solution, Escherichia coli JM83 strain was transformed, and white colonies of transformants were obtained on L agar medium containing X-gal and ampicillin. This transformant colony was inoculated into 10 ml of L liquid medium and cultured at 37 ° C. for 12 hours, and plasmid DNA (pBBD7) was isolated by a conventional method.

2 μg of pBBD7 was treated with restriction enzyme Sca I (18 units) and dephosphorylating enzyme BAP (0.2 unit) at 37 ° C. for 1 hour and then 0.7
The 3 kb DNA fragment was purified using% agarose electrophoresis. In addition, 2 μg of pBBD7 was added to the restriction enzyme EcoR I (20 units)
, And Pst I (20 units) were treated at 37 ° C. for 1 hour, and a 2.7 kb DNA fragment was purified by the same procedure. These 3kb
Site-specific gene conversion was performed by the above method using 0.6 μg of each of the DNA fragments of 2.7 kb and 2.7 kb and 50 pmol of 5 ′ phosphorylated synthetic DNA (shown in Table 8b). Twenty-four blue colonies of the obtained transformant were inoculated, plasmid DNA was isolated by a conventional method, and then transformed into Escherichia coli JM83 strain.

Blue colonies were inoculated on 24 liquid plates in 24 plates, and after culturing, plasmid DNA (pBB
X) was separated. Each DNA was subjected to DNA sequencing by the dideoxy method to confirm the converted base sequence and amino acid.

2 μg of each amino acid-converted plasmid was treated with restriction enzyme BamHI (10 units) at 37 ° C. for 1 hour.
The 0.3 kb DNA fragment was purified using 0.7% agarose electrophoresis. In addition, the natural reverse transcriptase expression plasmid pKP po
l-NT1 (valine) 5μg with BamHI (50 units)
After treatment at 37 ° C for 1 hour, the same operation was performed to purify a DNA fragment of about 4.7 kb. This 4.7 kb DNA fragment and the 0.3 kb DNA fragment containing each amino acid mutation are respectively
0.1 μg of each mixture was mixed, and ligation reaction was performed for 12 hours at 4 ° C. using T4 DNA ligase (2 units each).

E. coli JM83 strain was transformed with these DNA solutions, and transformant colonies were obtained on L agar medium containing ampicillin and X-gal. These were inoculated into L liquid medium, shake-cultured at 37 ° C. for 12 hours, and plasmid DNA was isolated by a conventional method.

 The above manufacturing process is shown in FIG.

These are pKP-T1L (leucine), pKP-T1I, respectively.
(Isoleucine), pKP-T1A (alanine), pKP-T1F
(Phenylalanine), pKP-T1R (arginine), pKP
-T1K (lysine), pKP-T1D (aspartic acid), pKP-
T1N (asparagine), pKP-T1M (methionine), pKP-
T1G (glycine).

The reverse transcription activity was measured for these clones.
The results are shown in Table 8.

[Effects of the Invention] As described above, according to the method for producing a reverse transcriptase expression plasmid of the present invention, a reverse transcriptase expression plasmid having extremely high reverse transcriptase expression activity can be obtained.

[Brief description of drawings]

FIG. 1 is a gene map of an example of a vector used in the present invention, FIG. 2 is a process chart showing an embodiment of the production method of the present invention, and FIG. 3 is another embodiment of the production method of the present invention. The process chart and FIG. 4 are graphs showing the reverse transcription activity.

Claims (2)

(57) [Claims] 1. TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGG
MGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILV
PCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG
LPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRL
PQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYXDDLLLAATSELDCQQ
GTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETV
MGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGP
DQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGNVTMGQPLVILAPH
AVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPE
EGL [where A is alanine (Ala), C is cysteine (Cy
s), D is aspartic acid (Asp), E is glutamic acid (Glu), F is phenylalanine (Phe), G is glycine (Gly), H is histidine (His), I is isoleucine (Ile), and K is lysine (Ile). Lys), L is leucine (Len),
M is methionine (Met), N is asparagine (Asn), P
Is proline (Pro), Q is glutamine (Gln), R is arginine (Arg), S is serine (Ser), T is threonine (Thr), V is valine (Val), W is tryptophan (Tr).
p) and Y represent tyrosine (Tyr)], which is a reverse transcriptase expression plasmid expressing a reverse transcriptase having an amino acid sequence represented by the following formula, wherein 223rd X from the N-terminal is L ( Leucine), I (isoleucine), A (alanine), F
A reverse transcriptase expression plasmid, which is one amino acid selected from the group consisting of (phenylalanine) and R (arginine). 2. TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGG
MGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILV
PCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG
LPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRL
PQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYXDDLLLAATSELDCQQ
GTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETV
MGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGP
DQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW
RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGNVTMGQPLYILAPH
AVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPE
EGL [where A is alanine (Ala), C is cysteine (Cy
s), D is aspartic acid (Asp), E is glutamic acid (Glu), F is phenylalanine (Phe), G is glycine (Gly), H histidine (His), I is isoleucine (Il).
e), K is lysine (Lys), L is leucine (Len), M is methionine (Met), N is asparagine (Asn), P is proline (Pro), Q is glutamine (Gln), and R is arginine (Arg). ), S is serine (Ser), T is threonine (Th
r), V is valine (Val), W is tryptophan (Trp)
And Y represents tyrosine (Tyr)], and in the amino acid sequence, the X at the 223rd position from the N-terminal is L (leucine), I (isoleucine), A (alanine), F ( A method for producing a reverse transcriptase expression plasmid that expresses a reverse transcriptase, which is one amino acid selected from the group consisting of phenylalanine) and R (arginine), comprising the following steps: A method for producing a plasmid, (a) A DNA fragment encoding a reverse transcriptase separated from a gene encoding the amino acid sequence in which X is V (valine) using a restriction enzyme, two multi-cloning sites and the two Vector having a repeat base sequence having 3 types of stop codons inserted between the multiple cloning sites, and the frames at both ends of the DNA fragment match The step of ligating with a DNA fragment separated using the restriction enzyme to obtain a plasmid, (b) of the restriction enzyme cleavage sites of the plasmid,
A step of cleaving a DNA fragment located on the N-terminal side of the DNA fragment encoding the reverse transcriptase, filling the cut end with a polymerase, and then ligating again to obtain a plasmid in which the frame is shifted, (c) (b) It corresponds to a plasmid prepared from a white transformant colony obtained by introducing the plasmid obtained in 1. into Escherichia coli, an extra amidic acid sequence on the N-terminal side of reverse transcriptase, and an extra amino acid sequence derived from the vector. Site-specific gene conversion using oligo DNA synthesized by removing the base sequence to obtain a mutant plasmid pKP _po1- N in which the base sequence corresponding to the extra amino acid sequence is removed, (d) (c) PKP _po1 -N the resulting PKP _po1 -N prepared from transformant colonies blue obtained by introducing into E. coli
And an oligo DN with a stop codon inserted at the C-terminal side of the reverse transcriptase
Site-specific gene conversion using A to obtain a specific plasmid pKP _po1- NT1 that does not contain extra amino acid sequences at both ends. (E) The pKP _po1- NT1 obtained in (d) is transformed into E. coli. Step of preparing pKP _po1- NT1 from the white transformant colony obtained by introduction, (h) DNA fragment encoding reverse transcriptase isolated from plasmid pSH1 using a restriction enzyme, and two multi-cloning sites And a vector having the intermediary nucleotide sequence having three types of stop codons inserted between the two multi-cloning sites and having the same frame at both ends of the DNA fragment separated from pSH1. A step of obtaining a plasmid by ligating with a DNA fragment separated using an enzyme, (p) A plasmid pB prepared from a blue transformant colony obtained by introducing the plasmid obtained in (h) into Escherichia coli B9 and a step of site-specific gene conversion using oligo DNA having an out-of-frame sequence of a DNA fragment containing the reverse transcriptase region to obtain mutant plasmid pBBD7 Plasmid pBB7 prepared from white transformant colonies obtained by introducing pBBD7 into Escherichia coli
And, in the base sequence in the DNA containing the reverse transcriptase region, the codon encoding the 223rd valine in the amino acid sequence is one amino acid selected from the group consisting of leucine, isoleucine, alanine, phenylalanine, and arginine. A step of obtaining a mutant plasmid pBBDX (X is leucine, isoleucine, alanine, phenylalanine, or arginine) by performing site-specific gene conversion using the oligo DNA in which the frame has been restored to its original codon, Plasmid pBBD prepared from a white transformant colony obtained by introducing the pBBDX obtained in
A step of ligating the DNA fragment isolated from X with a restriction enzyme and the DNA fragment isolated from pKP _po1- NT1 prepared in (e) using the restriction enzyme used to cleave pBBDX to obtain a plasmid, The plasmid pKP-T1X (X) was isolated from a transformant colony obtained by introducing the plasmid obtained in
Is a step of preparing leucine, isoleucine, alanine, phenylalanine, or arginine).