JP2014113106A - Method for producing avian myeloblastoma virus reverse transcriptase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing avian myeloblastoma virus (AMV) reverse transcriptase by using a gene recombinant, which enables abundant expression of AMV reverse transcriptase by the recombinant.SOLUTION: The problem is solved by culturing the recombinant for 72 hours or longer.

Description

  The present invention relates to a method for producing avian myeloblastoma virus reverse transcriptase using genetic engineering techniques.

  Reverse transcriptase, discovered as an essential factor for retrovirus growth, has RNA-dependent DNA polymerase activity that synthesizes DNA using single-stranded RNA as a template (reverse transcription), and is essential for cDNA synthesis, etc. It is used as a genetic engineering reagent and enzyme for genetic diagnosis.

  Avian myeloblastoma virus (AMV) reverse transcriptase, one of the reverse transcriptases, is a heterodimer consisting of an α chain with a molecular weight of about 63 kDa and a β chain with a molecular weight of about 95 kDa, and is known as the ASLV family. . Of these, the α chain is formed from the β chain by proteolytic processing (Non-patent Document 1).

  AMV reverse transcriptase is reported to be expressed in E. coli or eukaryotic insect cells (Patent Document 1). There is also a report that it is expressed in E. coli by co-expression with chaperone (Patent Document 2). However, in order to industrially produce a large amount of AMV reverse transcriptase, it is necessary to express it in a larger amount than the methods described in Patent Documents 1 and 2.

Special table 2002-534125 gazette JP 2002-315584 A

Le Grice S. F. J. et al. , Reverse Transscriptase, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 163 (1993)

  An object of the present invention is to provide a method capable of expressing a large amount of AMV reverse transcriptase by a recombinant when producing avian myeloblastoma virus (AMV) reverse transcriptase using the gene recombinant. There is.

  The present inventors expressed a large amount of AMV reverse transcriptase using a transformant obtained by transforming E. coli with an expression vector containing a polynucleotide encoding avian myeloblastoma virus (AMV) reverse transcriptase. As a result of intensive studies on a method capable of achieving the above, the present invention has been completed.

  That is, in the first aspect of the present invention, AMV reversal is achieved by culturing a transformant obtained by transforming Escherichia coli with an expression vector containing a polynucleotide encoding avian myeloblastoma virus (AMV) reverse transcriptase. A method for producing a coenzyme, wherein the culture time of the transformant is 72 hours or more.

  A second aspect of the present invention is the method according to the first aspect, wherein the expression vector contains an inducible promoter, and the culture temperature after addition of the inducing agent is between 10 ° C and 30 ° C. is there.

  The third aspect of the present invention is the method according to claim 1 or 2, wherein the E. coli is any one of E. coli MV1184, E. coli W3110, E. coli GM31, E. coli HB101, and E. coli JM101.

  Hereinafter, the present invention will be described in detail.

  In the present invention, the polynucleotide encoding AMV reverse transcriptase (hereinafter also referred to as AMV reverse transcriptase gene) is a gene that can produce AMV reverse transcriptase by transcription into mRNA and translation of the transcribed mRNA. Say. The total length of the AMV reverse transcriptase gene originally possessed by AMV is 2574 bases, and its nucleotide sequence is shown in SEQ ID NO: 1. The AMV reverse transcriptase gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1 is a gene encoding the AMV reverse transcriptase β chain, but the AMV reverse transcriptase also has a lower α chain than the β chain. The nucleotide sequence consists of 1716 bases on the 5 ′ end side of the nucleotide sequence shown in SEQ ID NO: 1 (SEQ ID NO: 2). The amino acid sequences of proteins translated by these genes are recorded in public databases (for example, National Center for Biotechnology Information). As an example, SEQ ID NOs: 135 (AMV reverse transcriptase β chain) and 136 (AMV reverse transcription) Enzyme α chain).

  An example of the AMV reverse transcriptase gene in the present invention is a gene obtained by converting an AMV reverse transcriptase gene comprising the nucleotide sequence shown in SEQ ID NO: 1 or 2 using an E. coli codon. Here, “converted using an E. coli codon” means an E. coli rare codon present in mRNA transcribed from an AMV reverse transcriptase gene containing the nucleotide sequence set forth in SEQ ID NO: 1 or 2, ie, E. coli. Is used to convert a codon with a low usage frequency into a codon with a high usage frequency in E. coli without changing the amino acid encoded by the codon. The level of codon usage in E. coli can be estimated by analyzing a genomic gene with reference to a public database (for example, Codon Usage Database on the website of Kazusa DNA Laboratory). Specifically, as codons frequently used in E. coli, AGA, AGG, CGG, CGA are used for arginine (Arg), AUA is used for isoleucine (Ile), CUA is used for leucine (Leu), and GGA is used for glycine (Gly). However, CCC can be exemplified for proline (Pro). In this way, the AMV reverse transcriptase gene converted to a codon that is frequently used in Escherichia coli can be transcribed so that AMV reverse transcriptase expression on an industrial scale using E. coli can be performed. Therefore, it is preferable. Conversion using an E. coli codon is possible by converting a region (codon) of a nucleotide sequence corresponding to the codon (transcribed to the codon). The nucleotide sequence can be converted using a conventionally known mutagenesis method such as the site-directed mutagenesis method. In particular, a DNAWorks method (Nucleic acid Res., 30, 43, 2002) combining synthetic oligonucleotides and PCR is an example of a preferable conversion method using E. coli codons. This method synthesizes a full-length gene by synthesizing an oligonucleotide group consisting of several tens of bases and assembling the synthetic oligonucleotide by PCR.

  When an AMV reverse transcriptase gene converted using an E. coli codon is obtained from an AMV reverse transcriptase gene comprising the sequence described in SEQ ID NO: 1 or 2, an E. coli rare codon present in the nucleotide sequence described in SEQ ID NO: 1 or 2 ( may be converted into codons frequently used in E. coli, or a part thereof (preferably 50% or more, more preferably 80%, more preferably 90% or more) may be converted. . As a preferred example of the AMV reverse transcriptase gene in the present invention, the AMV reverse transcriptase β chain gene containing the nucleotide sequence set forth in SEQ ID NO: 1 is converted using an E. coli codon, and the nucleotide sequence set forth in SEQ ID NO: 3 is included. Examples thereof include a gene and a gene containing the nucleotide sequence shown in SEQ ID NO: 4 obtained by converting an AMV reverse transcriptase α chain gene containing the nucleotide sequence shown in SEQ ID NO: 2 using an E. coli codon. In the AMV reverse transcriptase gene according to the present invention, an oligonucleotide encoding a tag peptide for facilitating analysis / purification on the 5 ′ end side or the 3 ′ end side, or for promoting secretory expression in E. coli. An oligonucleotide encoding the signal peptide may be added. Specific examples include a gene (SEQ ID NO: 5) consisting of a sequence in which an oligonucleotide (atg) encoding methionine is added to the 5 ′ end of the nucleotide sequence shown in SEQ ID NO: 3, or the nucleotide sequence shown in SEQ ID NO: 4. And a gene (SEQ ID NO: 6) having a sequence in which an oligonucleotide (atg) encoding methionine is added to the 5′-terminal side.

  In the present invention, the AMV reverse transcriptase gene may be a gene containing a sequence substantially homologous to the nucleotide sequence of the gene as long as a protein substantially the same as the AMV reverse transcriptase can be expressed. The gene may contain a sequence that can hybridize with the nucleotide sequence of the gene under stringent conditions. Here, “a protein substantially identical to AMV reverse transcriptase” specifically means three enzyme activities of AMV reverse transcriptase (reverse transcriptase activity, DNA polymerase activity, ribonuclease H (RNase H) activity). One or more of them refers to a protein having an activity equal to or higher than the AMV reverse transcriptase originally possessed by AMV. Specifically, it is translated from a gene containing a nucleotide sequence having at least 75% homology (preferably 85%, more preferably 95% or more) with the nucleotide sequence of the AMV reverse transcriptase gene originally possessed by AMV. Any protein can be used. Here, “stringent conditions” refer to conditions in which DNAs are less likely to form double strands as compared with normal conditions. Specifically, 50% (v / v) formamide at 42 ° C. , 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, or 75 mM sodium citrate Can do.

  In the production method of the present invention, the above-described AMV reverse transcriptase gene is inserted into an appropriate expression vector, and the transformant obtained by transforming Escherichia coli using the gene is cultured. The AMV reverse transcriptase gene may be inserted into the expression vector by genetic engineering at an appropriate position of the vector to be inserted. The appropriate position herein may be freely determined within a range that does not destroy the replication function of the expression vector, a desired antibiotic marker, or a region related to transmissibility. Moreover, when inserting in an expression vector, you may add the oligonucleotide which has a promoter function, for example. Examples of the promoter that functions in E. coli include lac promoter, trc promoter, and T7 promoter. Of these, the trc promoter is preferred because it allows easy regulation of transcriptional activity. The expression vector to be inserted is not particularly limited as long as it is stably present in E. coli to be transformed and can be replicated. Specific examples of expression vectors include pTrc, pUC, pBR, pET, and broad host range (Broad-Host-Range) plasmid vectors known as plasmid vectors that stably exist and replicate in E. coli. be able to. Among them, the pTrc99a vector can be exemplified as a preferable plasmid vector in that it has a trc promoter.

  The host in the present invention is E. coli, preferably E. coli MV1184, E. coli GM31, E. coli HB101, E. coli JM101, E. coli W3110, and more preferably E. coli W3110. In addition, E. coli mutant strains that have been subjected to mutation treatment by a conventionally known means such as chemical substances such as nitrosoguanidine and ethyl methanesulfonate, ultraviolet rays, radiation, etc. may be used.

For a method for transforming E. coli using an expression vector, see, for example, Method in Enzymology, 216, p. 469-631, 1992, Academic Press and Method in Enzymology, 204, p. 305-636, 1991, Academic Press.
The transformant obtained by the above-described method may be cultured in, for example, an LB (Luria-Bertani) medium supplemented with necessary nutrient sources. For the purpose of selecting and cultivating a transformant containing the expression vector, a selection agent based on the structure of the expression vector may be added to the medium. For example, when using an expression vector having an ampicillin resistance gene, ampicillin or carbenicillin may be added to the medium. In addition to carbon, nitrogen, and inorganic salt sources, an appropriate nutrient source may be added to the medium. Further, one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate and dithiothreitol may be added. The culture temperature may be, for example, in the range of about 20 ° C. to 40 ° C., preferably in the range of 35 ° C. to 38 ° C., more preferably in the vicinity of 37 ° C. The pH may be in the range of 5.9 to 8.1, and preferably in the range of 5.9 to 7.1.

  When an AMV reverse transcriptase is produced by culturing the transformant obtained by the method described above, if the expression vector contained in the transformant contains an inducible promoter such as trc promoter, the culture is started. Occasionally, an inducer may be added to express AMV reverse transcriptase, or after culturing to the extent that good AMV reverse transcriptase productivity is obtained, an inducer is added to express AMV reverse transcriptase. You may let them. Preferably, the inducer is added during the growth period in which the turbidity (absorbance at 660 nm) of the culture solution shows about 0.5 to 20.0, more preferably in the growth period in which the turbidity shows 1.0 to 10.0. An example is a method in which the guidance operation is performed, followed by culturing. What is necessary is just to select and use the induction | guidance | derivation agent to add suitably according to the promoter to be used. For example, when the inducible promoter is a trc promoter, IPTG (isopropyl-β-thiogalactopyranoside) can be exemplified as an inducing agent to be added. The addition concentration of IPTG may be appropriately selected from the range of about 0.1 to 10.0 mM in the final concentration, but preferably in the range of 1.0 mM to 10.0 mM, more preferably in the vicinity of 5.0 mM. is there. In the production method of the present invention, it is preferable to lower the culture temperature after the induction operation (after the addition of the inducer) to a temperature lower than the optimal culture temperature of Escherichia coli, specifically in the range from 10 ° C to 30 ° C, More preferably, it is the range from 15 degreeC to 25 degreeC, More preferably, it is about 25 degreeC.

  The culture time in the present invention is 72 hours or longer, which is longer than the general culture time of E. coli, preferably 100 hours or longer, more preferably 100 hours to 170 hours, most preferably 100 hours. 150 hours.

  When AMV reverse transcriptase is extracted from the culture medium of the transformant obtained by the above-described method, an extraction method may be appropriately selected depending on the form of expression. For example, when the expressed AMV reverse transcriptase accumulates in Escherichia coli, the bacterial cells may be collected by centrifugation or the like, and then disrupted and extracted by an enzyme treatment agent or ultrasonic disruption. Although various proteins are mixed in the extraction step, AMV reverse transcriptase can be efficiently precipitated and recovered by coprecipitation with nucleic acid by adding streptomycin hydrochloride or polymin P. Furthermore, AMV reverse transcriptase can be purified with high purity by applying chromatography such as ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and affinity chromatography alone or in combination. As an example of AMV reverse transcriptase purification using chromatography, after purifying AMV reverse transcriptase using hydrophobic interaction chromatography into which a polypropylene glycol group has been introduced, ion exchange chromatography using a phosphocellulose carrier is used. By further purification, the AMV reverse transcriptase α chain, the AMV reverse transcriptase β chain, and the AMV reverse transcriptase α chain β chain heterocomplex can be separated and purified.

  The production method of the present invention produces AMV reverse transcriptase using a transformant obtained by transforming Escherichia coli with an expression vector containing a polynucleotide encoding avian myeloblastoma virus (AMV) reverse transcriptase. In doing so, it is possible to express a large amount of AMV reverse transcriptase by setting the culture time to 72 hours or longer, which is useful for industrial production of AMV reverse transcriptase.

The nucleotide sequence of the AMV reverse transcriptase β chain gene (SEQ ID NO: 1, bottom) originally possessed by the avian myeloblastoma virus and the nucleotide sequence of the AMV reverse transcriptase β chain gene designed in Example 1 (SEQ ID NO: 3, This is a result of comparison with (upper). The nucleotide sequence of the AMV reverse transcriptase β chain gene (SEQ ID NO: 1, bottom) originally possessed by the avian myeloblastoma virus and the nucleotide sequence of the AMV reverse transcriptase β chain gene designed in Example 1 (SEQ ID NO: 3, This is a result of comparison with (upper). It is a figure which shows the structure of plasmid vector pTrcAMVB. It is a figure which shows the structure of plasmid vector pTrcAMVA. It is a figure which shows the structure of plasmid vector pTrcAMVBwild. It is a figure which shows the structure of plasmid vector pTrcAMVAwild. It is the electrophoresis (SDS-PAGE) photograph which shows the difference in the expression level in colon_bacillus | E._coli by the difference in a plasmid vector. It is the figure which showed the difference in the expression level by the difference in Escherichia coli strain which is a host. It is a figure which shows the change of the turbidity during culture | cultivation, and the change of AMV reverse transcriptase production amount. In the figure, black circles indicate changes in turbidity during culture, and white circles indicate changes in AMV reverse transcriptase production.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this Example is for describing one Embodiment of this invention, and does not limit this invention.

Example 1 Sequence design of avian myeloblastoma virus (AMV) reverse transcriptase gene The AMV reverse transcriptase β chain gene (AMV codon) consisting of the nucleotide sequence set forth in SEQ ID NO: 1 was converted using an E. coli codon. The AMV reverse transcriptase β chain gene consisting of the nucleotide sequence set forth in SEQ ID NO: 3 was designed by the DNAWorks method. The results of comparing the nucleotide sequence (SEQ ID NO: 1) of the AMV reverse transcriptase β chain gene (AMV codon) with the nucleotide sequence (SEQ ID NO: 3) of the AMV reverse transcriptase β chain gene (E. coli codon) are shown in FIGS. Shown in The amino acid sequence of the AMV reverse transcriptase β chain translated by the gene consisting of the sequences shown in SEQ ID NOs: 1 and 3 was the same (SEQ ID NO: 135), but the nucleotide sequence homology was 74%.

Example 2 Production of AMV Reverse Transcriptase Gene (1) In order to produce the AMV reverse transcriptase β chain gene composed of the nucleotide sequence shown in SEQ ID NO: 3 designed in Example 1, 120 kinds of oligonucleotides composed of nucleotide sequences were synthesized.
(2) In order to produce the AMV reverse transcriptase β chain gene consisting of the nucleotide sequence shown in SEQ ID NO: 3 from the oligonucleotide synthesized in (1), the following two-step PCR reaction was performed.
(2-1) Prepare reaction solutions shown in Table 1, heat treatment at 94 ° C. for 5 minutes, first step at 94 ° C. for 30 seconds, second step at 62 ° C. for 30 seconds, first step at 72 ° C. for 1 minute. The first step PCR was carried out by performing 25 cycles of 3 steps and finally reacting at 72 ° C. for 7 minutes. In addition, the DNA mix in Table 1 is a solution obtained by collecting 120 parts of oligonucleotide solutions (concentration: 50 pmol / μL) each having the sequence described in SEQ ID NOs: 7 to 126 and mixing them.

（２−２）表２に示す反応液を作製し、９４℃で５分間熱処理後、９４℃で３０秒間の第一ステップ、６５℃で３０秒間の第二ステップ、７２℃で１分間の第三ステップを２５サイクル行ない、最後に７２℃で７分間反応させることで二段階目のＰＣＲを行なった。なお、ＰＣＲプライマーとして配列番号７（５’−ＡＴＧＡＣＧＧＴＧＧＣＧＴＴＡＣＡＣＣＴＧ−３’）および配列番号１２６（５’−ＴＴＡＧＧＣＡＡＡＣＡＧＣＧＧＡＧＡＧＧＣＴＴＣＧＴＣＣＴＴＣＴＴＣＧＴＣＡＣＴＴＣＧＴＣＴ−３’）に記載の配列からなるオリゴヌクレオチドを用いた。

(2-2) Prepare reaction solutions shown in Table 2, heat treatment at 94 ° C. for 5 minutes, first step at 94 ° C. for 30 seconds, second step at 65 ° C. for 30 seconds, first step at 72 ° C. for 1 minute. The second step PCR was performed by performing 25 cycles of 3 steps and finally reacting at 72 ° C. for 7 minutes. In addition, the oligonucleotide which consists of a sequence | arrangement of sequence number 7 (5'-ATGACGGGTGCGGTTACACCTG-3 ') and sequence number 126 (5'-TTAGGCAAACAGCGGAGAGGCTTCGTCCCTTCTCTCTCTCTGCTCT-3') was used as a PCR primer.

（３）ＰＣＲ反応終了後、０．９％アガロース電気泳動により設計通りのサイズに相当するＤＮＡバンドを確認し、当該ＤＮＡバンドから市販の試薬（ＱＩＡｑｕｉｃｋ Ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔ：キアゲン社）を用いてＤＮＡを抽出した。
（４）抽出したＤＮＡの５’末端を市販の試薬（ＴａＫａＲａ ＢＫＬ Ｋｉｔ：タカラバイオ社）を用いてリン酸化し、制限酵素ＳｍａＩで消化したｐＴｒｃ９９ａプラスミドベクターに挿入後、当該ベクターを用いて大腸菌ＪＭ１０９株（タカラバイオ社）を形質転換した。調製したプラスミドベクターｐＴｒｃＡＭＶＢ（図３）は制限酵素解析およびヌクレオチド配列決定（実施例３）を行なった。プラスミドベクターｐＴｒｃＡＭＶＢのヌクレオチド配列を配列番号１３１に示した。
（５）配列番号４に記載のヌクレオチド配列からなるＡＭＶ逆転写酵素α鎖遺伝子をＰＣＲ反応により作製した。ＰＣＲ反応は、鋳型としてｐＴｒｃＡＭＶＢを、ＰＣＲプライマーとして配列番号７および配列番号１２７（５’−ＴＴＡＡＴＡＣＧＣＴＴＧＡＡＡＣＧＴＧＧＣＣＴＧＧＣＴＡＴＣＣＧＣＣ−３’）に記載の配列からなるオリゴヌクレオチドを、それぞれ用いた他は、（２−２）に記載の方法と同様な方法で実施した。
（６）ＰＣＲ反応終了後、（３）から（４）に記載の方法と同様な方法で、形質転換体を取得し、プラスミドベクターｐＴｒｃＡＭＶＡを調製した。調製したプラスミドベクターｐＴｒｃＡＭＶＡ（図４）は制限酵素解析およびヌクレオチド配列決定（実施例３）を行なった。プラスミドベクターｐＴｒｃＡＭＶＡのヌクレオチド配列を配列番号１３２に示した。
（７）ＡＭＶ逆転写酵素β鎖遺伝子（ＡＭＶコドン）を含むプラスミドを保持した大腸菌クローン（ＡＴＣＣ ３１９９０）からＰＣＲ反応によりＡＭＶ逆転写酵素β鎖をコードする遺伝子を作製した。ＰＣＲ反応は、鋳型としてＡＴＣＣ３１９９０の培養抽出物を、ＰＣＲプライマーとして配列番号１２８（５’−ＡＴＧＡＣＴＧＴＴＧＣＧＣＴＡＣＡＴＣＴＧＧＣＴＡＴＴＣＣＧＣＴＣ−３’）および配列番号１２９（５’−ＴＴＡＴＧＣＡＡＡＡＡＧＡＧＧＧＣＴＣＧＣＣＴＣＡＴＣ−３’）に記載の配列からなるオリゴヌクレオチドを用いた他は、（２−２）に記載の方法と同様な方法で実施した。
（８）ＰＣＲ反応終了後、（３）から（４）に記載の方法と同様な方法で、形質転換体を取得し、プラスミドベクターｐＴｒｃＡＭＶＢｗｉｌｄ（図５）を調製した。プラスミドベクターｐＴｒｃＡＭＶＢｗｉｌｄのヌクレオチド配列を配列番号１３３に示した。
（９）ＡＴＣＣ ３１９９０からＰＣＲ反応によりＡＭＶ逆転写酵素α鎖をコードする遺伝子を作製した。ＰＣＲ反応は、鋳型としてＡＴＣＣ ３１９９０の培養抽出物を、ＰＣＲプライマーとして配列番号１２８および配列番号１３０（５’−ＴＴＡＡＴＡＣＧＣＴＴＧＡＡＡＧＧＴＧＧＣＴＴＧＧＣＴＡＴＣＴＧＣＣ−３’）に記載の配列からなるオリゴヌクレオチドを用いた他は、（２−２）に記載の方法と同様な方法で実施した。
（１０）ＰＣＲ反応終了後、（３）から（４）に記載の方法と同様な方法で、形質転換体を取得し、プラスミドベクターｐＴｒｃＡＭＶＡｗｉｌｄを調製（図６）した。プラスミドベクターｐＴｒｃＡＭＶＡｗｉｌｄのヌクレオチド配列を配列番号１３４に示した。

(3) After completion of the PCR reaction, a DNA band corresponding to the designed size is confirmed by 0.9% agarose electrophoresis, and DNA is extracted from the DNA band using a commercially available reagent (QIAquick Gel extraction kit: Qiagen). Extracted.
(4) The 5 ′ end of the extracted DNA is phosphorylated using a commercially available reagent (TaKaRa BKL Kit: Takara Bio Inc.), inserted into a pTrc99a plasmid vector digested with the restriction enzyme SmaI, and then E. coli JM109 is used using the vector. A strain (Takara Bio Inc.) was transformed. The prepared plasmid vector pTrcAMVB (FIG. 3) was subjected to restriction enzyme analysis and nucleotide sequencing (Example 3). The nucleotide sequence of the plasmid vector pTrcAMVB is shown in SEQ ID NO: 131.
(5) An AMV reverse transcriptase α chain gene consisting of the nucleotide sequence shown in SEQ ID NO: 4 was prepared by PCR reaction. (2-2) except that pTrcAMVB was used as a template for the PCR reaction, and an oligonucleotide having the sequence described in SEQ ID NO: 7 and SEQ ID NO: 127 (5′-TTATATACGCTTGAAACGGTGCCCTGGCTATCCGCC-3 ′) was used as the PCR primer. The same method as described was carried out.
(6) After completion of the PCR reaction, a transformant was obtained by the same method as described in (3) to (4), and a plasmid vector pTrcAMVA was prepared. The prepared plasmid vector pTrcAMVA (FIG. 4) was subjected to restriction enzyme analysis and nucleotide sequencing (Example 3). The nucleotide sequence of the plasmid vector pTrcAMVA is shown in SEQ ID NO: 132.
(7) A gene encoding the AMV reverse transcriptase β chain was prepared by PCR reaction from an E. coli clone (ATCC 31990) carrying a plasmid containing the AMV reverse transcriptase β chain gene (AMV codon). In the PCR reaction, a culture extract of ATCC 31990 was used as a template, and an oligonucleotide consisting of the sequence described in SEQ ID NO: 128 (5′-ATGACTGTTGCGCTACATCTGCCTTCCCGCTC-3 ′) and SEQ ID NO: 129 (5′-TTATGCAAAAAGAGGGCTCGCCCTCATC-3 ′) as PCR primers. Other than using, the same method as described in (2-2) was performed.
(8) After completion of the PCR reaction, a transformant was obtained by the same method as described in (3) to (4), and a plasmid vector pTrcAMVBwild (FIG. 5) was prepared. The nucleotide sequence of the plasmid vector pTrcAMVBwild is shown in SEQ ID NO: 133.
(9) A gene encoding AMV reverse transcriptase α chain was prepared from ATCC 31990 by PCR reaction. The PCR reaction was carried out using a culture extract of ATCC 31990 as a template, and an oligonucleotide having the sequence described in SEQ ID NO: 128 and SEQ ID NO: 130 (5′-TTATATACGCTTGAAAGGTGGCTTGGCTATCTGCCC-3 ′) as a PCR primer (2- It implemented by the method similar to the method as described in 2).
(10) After completion of the PCR reaction, a transformant was obtained by the same method as described in (3) to (4), and a plasmid vector pTrcAMVAwild was prepared (FIG. 6). The nucleotide sequence of the plasmid vector pTrcAMVAwild is shown in SEQ ID NO: 134.

Example 3 Sequence Confirmation The nucleotide sequence of the AMV reverse transcriptase gene inserted into the plasmid vectors pTrcAMVB (FIG. 3) and pTrcAMVA (FIG. 4) prepared in Example 2 was converted into a commercially available reagent kit (Big Dye) based on the chain terminator method. Terminator Cycle Sequencing FS read Reaction kit (PE Applied Biosystems) was used for a cycle sequence reaction and analyzed with a fully automated DNA sequencer ABI Prism 3700 DNA Analyzer (PE Applied Biosystems). In addition, 120 kinds of synthesized oligonucleotides were appropriately used as sequencing primers. As a result, it was confirmed that the nucleotide sequences of the DNA inserted into pTrcAMVB and pTrcAMVA were as designed.

Example 4 Confirmation of AMV Reverse Transcriptase Expression (1) Plasmid vector pTrcAMVB (FIG. 3) was used to transform E. coli JM109 strain (Takara Bio), and LB agar medium supplemented with 50 μg / mL antibiotic ampicillin was used. And cultured at 37 ° C. for 18 hours.
(2) After the culture, a transformant was obtained by selecting an arbitrary colony that appeared. The transformant obtained here was named JM109 / pTrcAMVB. In addition, it implemented similarly about pTrcAMVA (FIG. 4), pTrcAMVBwild (FIG. 5), and pTrcAMVAwild (FIG. 6), and obtained the transformant (JM109 / pTrcAMVA, JM109 / pTrcAMVBwild, JM109 / pTrcAMVAwild), respectively.
(3) The obtained transformant was inoculated into an LB liquid medium supplemented with 50 μg / mL antibiotic ampicillin and cultured at 37 ° C. When the value of OD660 nm reached about 0.5, 0.15 mM IPTG (isopropyl-β-thiogalactopyranoside) (Takara Bio Inc.) was added.
(4) Each transformant culture solution was collected at an appropriate interval from the addition of IPTG, and a protein solution was prepared using a commercially available kit (Bugbuster protein extraction Kit, Novagen). The prepared protein solution was subjected to SDS-polyacrylamide electrophoresis (SDS-PAGE) (Example 10).

  The SDS-PAGE result is shown in FIG. When an AMV reverse transcriptase is expressed by a transformant obtained by transforming E. coli using a plasmid vector containing an AMV reverse transcriptase gene, an AMV codon is used as an AMV reverse transcriptase gene (JM109 / It can be seen that the expression level of the one using the E. coli codon which is the same as the host (JM109 / pTrcAMVB, JM109 / pTrcAMVA) is higher than that of pTrcAMVBwild, JM109 / pTrcAMVAwild).

Example 5 Examination of host E. coli strain (1) Using plasmid vector pTrcAMVB (FIG. 3), E. coli JM109 strain (Takara Bio), E. coli JM110 strain (Takara Bio), E. coli JM101 strain (Agilent Technology), E. coli W3110 (ATCC 27325), E. coli SK383, E. coli KY1436, E. coli TG1 (Agilent Technology), E. coli HB101 (Takara Bio), E. coli RB791 (ATCC 53622), E. coli MV1184 (Takara Bio) ), E. coli C600 (Agilent Technology), E. coli MC4110, E. coli GM31 (NBRP ME7741), E. coli CC118, E. coli BL21, E. coli DH5 (Toyobo), or E. coli DH5α (Toyobo) After spinning, Inc.), respectively transformed, was cultured for 18 hours at 37 ° C. using a LB agar medium supplemented with 50 [mu] g / mL of the antibiotic (Ampicillin).
(2) After culture, an arbitrary colony that appeared was selected and inoculated into 2 × YT liquid medium supplemented with 50 μg / mL antibiotic carbenicillin.
(3) After shaking culture at 37 ° C. and 160 rpm, when the value of OD660 nm reaches about 0.5, the culture temperature is lowered to 15 ° C. or 25 ° C., and 5 mM IPTG (Takara Bio Inc.) is added for an additional 45 hours. Shaking culture was performed.
(4) The cultured cells were collected by centrifugation at 4 ° C. and 5000 rpm for 30 minutes, and stored frozen at −30 ° C.
(5) 5 mL / wet cell mass of buffer A (20 mM Tris-HCl (pH 7.4), 10% (w / v) glycerol, 1 mM DTT, 0.2% Triton X-100 (trade name) ), And after ultrasonic disruption, the mixture was centrifuged at 4 ° C. and 5000 rpm for 30 minutes to remove unbroken cells and obtain an extract.
(6) The activity of the extract was evaluated by a method using the TRC method (Translation Reverse-translation Concerted reaction) shown below (Japanese Patent Laid-Open No. 2003-334095).
(6-1) An RNA sample was prepared by diluting the positive standard solution attached to TRCRtest NoroW (Tosoh Corp.) five times with the negative standard solution attached to TRCRtest NoroW (Tosoh Corp.).
(6-2) Add 10 μL of the substrate reagent attached to TRCRtest NoroW (Tosoh Corporation) and 10 μL of the primer reagent attached to TRCRtest NoroW (Tosoh Corporation) to a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, SSI). The RNA sample was dispensed and 5 μL of the RNA sample prepared in (1) was added thereto.
(6-3) The mixed solution of (6-2) was incubated at 42 ° C. for 5 minutes, and then 5 μL of an enzyme solution having the following composition, which was kept warm at 42 ° C. for 2 minutes in advance, was added.

Composition of enzyme solution:
0.12 mg / mL bovine serum albumin 2.0% (w / v) sorbitol 28.4 U / μL T7 RNA polymerase 3 μL extracted fraction or 6.4 U AMV-RT control (Life Techno)
logy)
(6-4) Subsequently, using a fluorescence spectrophotometer with a temperature control function capable of directly measuring a PCR tube, the reaction solution is reacted at 42 ° C. and simultaneously the fluorescence intensity (excitation wavelength: 470 nm, fluorescence wavelength: 520 nm) of the reaction solution is increased with time. Measured for minutes. The time when the fluorescence measurement value was 1.2 times the initial fluorescence value was taken as the detection time.

  The results are shown in FIG. Among the E. coli strains examined this time, transformants obtained by transforming DH5α strain, C600 strain, JM110 strain, MC4110 strain, TG1 strain, MV1184 strain, W3110 strain, GM31 strain, HB101 strain, JM101 strain are used. In this case, the detection time is shortened as compared with the transformant obtained by transforming the JM109 strain, and it can be seen that the AMV reverse transcriptase expression level is increased in the transformant. In particular, it can be seen that transformants obtained by transforming MV1184 strain, W3110 strain, GM31 strain, HB101 strain, and JM101 strain highly express AMV reverse transcriptase.

Example 6 Culture Production of AMV Reverse Transcriptase Among the transformants obtained in Example 5, a transformant obtained by transforming Escherichia coli W3110 strain using plasmid vector pTrcAMVB (FIG. 3) (W3110 / pTrcAMVB) was used to express AMV reverse transcriptase.
(1) Inoculate W3110 / pTrcAMVB into 100 mL of 2 × YT medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride, 0.1 mg / mL carbenicillin sodium) in a 500 mL baffled Erlenmeyer flask Pre-culture was performed by shaking culture at 30 ° C. and 130 rpm overnight.
(2) 3.25 L of the medium shown in Table 3 was placed in a 5.0 L fermentor (Sakura Seiki Co., Ltd., TFL-5), sterilized at 121 ° C. for 20 minutes, and then the preculture solution obtained in (1) was added to 32 .5 mL was inoculated and main culture was started. The culture temperature of the fermenter was set to 37 ° C., the stirring speed was set to 450 rpm, the pH was set to 5.9 to 6.1, and the pH was adjusted using a 50% phosphoric acid aqueous solution.

（３）培養開始から３時間後に発酵槽の培養温度を２５℃に下げ、０．５ＭのＩＰＴＧ水溶液を３２．５ｍＬ添加した。
（４）約１４０時間、培養を行なった。培養中は、適宜、培養液を２０ｍＬ採取し、４℃・８０００ｒｐｍで２０分間遠心分離することで、菌体を回収した。
（５）回収した菌体から実施例５（５）に記載の方法でタンパク質を抽出後、抽出液の活性を実施例５（６）に記載のＴＲＣ法により測定した。

(3) The culture temperature of the fermenter was lowered to 25 ° C. 3 hours after the start of the culture, and 32.5 mL of a 0.5 M IPTG aqueous solution was added.
(4) Culture was performed for about 140 hours. During the culture, 20 mL of the culture solution was appropriately collected and centrifuged at 4 ° C. and 8000 rpm for 20 minutes to collect the cells.
(5) After extracting the protein from the collected cells by the method described in Example 5 (5), the activity of the extract was measured by the TRC method described in Example 5 (6).

  FIG. 9 shows changes in turbidity during culture and changes in AMV reverse transcriptase production determined by the TRC method. It can be seen that by culturing for 100 hours or more, a production amount approximately twice as large as that obtained at the 50-hour culture time can be obtained.

Claims (3)

A method for producing AMV reverse transcriptase by culturing a transformant obtained by transforming Escherichia coli with an expression vector comprising a polynucleotide encoding avian myeloblastoma virus (AMV) reverse transcriptase, The method as described above, wherein the culture time of the transformant is 72 hours or more. The method according to claim 1, wherein the expression vector contains an inducible promoter, and the culture temperature after addition of the inducing agent is between 10 ° C and 30 ° C. The method according to claim 1 or 2, wherein the Escherichia coli is any one of Escherichia coli MV1184, Escherichia coli W3110, Escherichia coli GM31, Escherichia coli HB101, and Escherichia coli JM101.

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