WO1984001583A1

WO1984001583A1 - Novel vector

Info

Publication number: WO1984001583A1
Application number: PCT/JP1983/000276
Authority: WO
Inventors: Tadatsugu Taniguchi; Haruo Sugano; Tatsunari Nishi; Moriyuki Sato; Seiga Ito
Original assignee: Kyowa Hakko Kogyo Kk; Japan Found Cancer
Priority date: 1982-10-08
Filing date: 1983-08-24
Publication date: 1984-04-26
Also published as: DE121569T1

Abstract

A vector containing genetic information necessary for starting transcription and translation and a signal of starting translation (ATG or GTG), which is suitable for incorporating a peptide-coding DNA segment and effectively producing the peptide, a process for its preparation, and its utilization.

Description

Ming nuclear damage New vector

Technical field

The present invention relates to a novel plasmid expression vector, its »method and use.

M.

More specifically, the present invention relates to a vector containing the necessary information (ATG or GTG) necessary for the start of transcription and the start of art translation, and which encodes a polypeptide. A suitable expression vector for efficient expression of polypeptides by incorporating a DNA fragment to be expressed, a method for producing the same, and use thereof.

Background technology

In recent years, research and technology for genetic recombination have been developed, and its application to the industry has been made possible.How to incorporate foreign genes into brassmid vectors and integrate them It is an important challenge to synthesize polypeptides encoded on exogenous genes within the host territory. Various measures have been devised so as to make foreign host genes available in host cells, especially in large bacteria, with great ease. First, in order to increase the efficiency of transcription, ac systems and trp systems are used as promoters, which are distant information necessary for the start of transcription, and the efficiency is as low as i. For this purpose, recombinants have been created in which the distance between the ribosome binding site and the translation initiation signal (mainly ATG) is ».

In the latter example, the use of a special `` synthetic DNAJ to serve as a middle-ranking agent for attaching the foreign gene downstream of the promoter with the ATG, which is the initial proposition of translation, was used. Use of DNase Aftl * enzymes such as polymerase I, S1 nuclease, and BAL-31 nuclease, or waiting for a specific nucleotide sequence. Therefore, the distance between the ribosome binding site and the cable translation start signal must be increased, which is inconvenient. ¾ Disclosure of light

The present inventors have sought to remedy such difficulties of the known Brassmid vector by using a Brassmid vector which has an ATG under the promoter SD (a convenient site) and which can be cut in a manner that leaves a trap ATG. Ββ, and the present invention has been completed.

Hereinafter, the present invention will be described in detail.

The expression vector of the present invention is a promoter that is information necessary for transcriptional supply, and a ribosome binding site that is information necessary for initiation of β-priming (* synthesis of large β bacteria). Downstream of the Dalgarno sequence, hereafter referred to as the SD sequence), contains the beginning of the translation (ATG or GTG), which is placed in an appropriate deer sculpture. 'Wffi enzyme that leaves the (3' — protruding end) as a truncated form

(Sphl, EcoRV, Kpn I, Sac I, etc.).

As the promoter of the expression vector of the present invention, any promoter having promoter activity can be used. Preferably, for example, a promoter and a tributofolactone of lactose operon are used. A blower motor with an operon and a motor with a lipophase phosphatase operator can be used. A starting point is ATG or GTG. S DK column and the script! The base of M is 2 to 20 bases

In the case of (p), β translation is possible.

The jgfil sequence of the Wffi enzyme, which leaves the 3 'one-protruding end * as a truncated form following the initial sequence, is, for example, the overlapping sequence of ATG and SphI. ATGCATGC, ATGATATC with duplicated ATG and Eco RV sequences, ATGGTACC with duplicated ATG and Kp η I ft ATGGTACC, jgftK doubled with ATG and Sac I ATGAGCTC, etc. .

As an example of the vector that Umezo also has, the translation of the W-feed signal ATG and the sphl cleavage site (GCATGC) overlapped ^ A schematic diagram of the structure around the motor and the SD sequence is shown in Fig. 1. In addition, cleavage of the above-mentioned W-limited element that allows the site of the control S enzyme to overlap the translation initiation signal ATG Figure 2 shows the shape.

The fact that a brassmid vector such as that described in the present invention can be formed will be clearly and sharply exemplifying the case of a tributane-fan promoter of a microorganism.

The genetic information required for the initiation of transcription and translation according to the present invention is not limited to those derived from nuclear vesicles, but can also be applied to those derived from yeast cells such as yeast and commercial animals. However, for the sake of clarity, the following description focuses on Escherichia coli, a prokaryotic organism whose transcription and translation mechanisms have been elucidated in detail. Will be described.

The supply of DNA containing the trp promoter is trp-portable. ◆ Brassmid carrying the promoter p KYP 10 (Patent Application No. 56-212 13 193 US Application No. 45 2 290, No. 1 in Europe

(P. KYP10 and pKYP100 are referred to in Reference Examples 1 and 2 for the production method of pKYP10 and pKYP100). pKYP100_ (indicated by the number in Fig. 6, the same applies hereinafter) was cut at the C1aI site immediately downstream of the SD sequence of the trp L gene (see Fig. 6). After cleavage at the SphI site present in the tetracycline resistance gene of ρ Κ Υ Ρ Ο Ο Ο, large plasmid DNA fragments were subjected to agarose gel electrophoresis. Purify accordingly. On the other hand, it has a structure that facilitates rapid binding to this DNA fragment, and has a translation initiation signal (ATG or GTG) downstream of the SD sequence with an appropriate distance divider. Following the initiation signal, two types of oligonucleotides such that there are restriction sequences that leave the 3 'protruding end as a truncated form (the two recombine with each other) Thus, the desired expression vector 11 is obtained by synthesizing <10> having a similar base sequence and linking the purified plasmid DNA with the fragment. Conditions of reaction necessary for the above ¾ recombinant brass Mi de created Ru der as follows: _β

Digestion of DNA with elementary DNA is usually 0.1 to 100 // g of DNA in 2 to 200 millimoles (preferably 10 to 40 millimoles) of Tris Hanki (116.0 ~ 9.5, preferably 117.0-8.0), 1-150 millimoles of NaC £, 2-20 millimoles (preferably 5 ~: 10mi) 0.1 to 300 units (preferably 1 to 3 units of enzyme per 1 μg of DNA) in MgC £ 2 (Preferably between 32 and 38) at 15 minutes to 24 hours. The reaction is usually stopped at 55 to 75 5 (preferably 63 to 70¾) for 5 to 30 minutes! It is also possible to use a method to inactivate W erythramine with a wipe. · Synthetic oligonucleotides can be prepared using the ethyl phosphate method [HG Khorana et al: J. Mol. Biol. , Vol. 72, p. 209 (1972)], Linmen Triestel method [R. Crea et al .: Pro. Natl. Acad. Sci. USA 75, 5765 (1978)], Or the phosphorite method (: MD

Matteucci et al .: J. ΑΜ Chen Soc. 103 Street, p. 3185 (1981).

To phosphorylate the synthesized oligonucleotides, 2 to 200 millimoles (preferably 10 to 70 millimoles) of tris-hydroxy acid (pH 6.0 to 9.5, preferably pH 7.0 to 8 * 0), 3 to 20 millimoles (preferably 4 to 10 millimoles) Mg C £ 2, 1 to 10 millimoles Using 0.1 to 100 units of T4 ribonucleotide kinase in a molar dithiothreitol at 20 to 40 (preferably at 35 to 38) Perform the reaction at 2 o'clock from the 5 min. When transferring DNA fragments, 2 to 200 millimoles (preferably 10 to 70 millimoles) of tris (H 6.0 to 9.5, preferably Ρ 7.0 to 8.0), 2 to 20 millimoles (preferably 5 to 10 millimoles) Mg C £ ₂ , 0.1 to 10 millimoles (preferred) Or 0.5 to 2 millimoles>, 1 to 50 millimoles (preferably

Ο ΡΙ Are from 1 to 37 (preferably from 3 to 20) using 0.1 to 10 units of T4 DNA ligase in 5 to 10 millimoles of dithiothreitol. ) For 15 minutes to 72 hours (preferably 2 to 20 hours).

The plasmid expression vector as described in the present invention can be produced by the above method.

Also, instead of cutting pKYP100 with C1aI in the Keizo method described above, cut it at the Hind fll site immediately downstream of the ClaI site, and then use SphI. After cutting, synthetic nucleotides can be donated. Also, a sequence that is not present on pKYP100 as a «restriction enzyme» sequence that overlaps with the translation initiation signal (for example, Kp nl

X

: GGTACC, SacI: GAGCTC), etc., are used to synthesize two types of oligonucleotides having a base sequence that completely contains the g »sequence, and to obtain pKYP10. It is only necessary to enter the C1aI site or HindHI site at 0 and the IHJ site at the BamHI site or Sa £ I site.o

PKYP100 is a positive conductor of pKYPIO. ρ Κ Υ Ρ Ι t is a trp- and π-motor-containing plasma disclosed in Japanese Patent Application No. 56-213 1393. The construction method of pKYP100 to pKYP100 is as follows.

As shown in Fig. 5, first, the plasmid pKYP101_ was digested with HhaI, and the approximately 180 bp DNA fragment _ containing the trp promoter was then purified. Refine by claramide gel electrophoresis. The DNA fragment _ was allowed to act on a large-scale bacterial DNA polymerase I · Κ1 enow fragment to remove the 3 'protruding Sue Yas generated by Hha I digestion. After this change, digest with Hindffi and purify β-DNA fragment 1 of about 100 bp containing trp promoter by polyacrylamide gel electrophoresis. On the other hand, after digestion of the plasmid pBR322_ with Ec0RI, the repair reaction of large cocoon DNA polymerase I was used to digest it by EcoRI digestion. Flatten the resulting sticky end

I will do it. Next, after digestion with HindHI, large plasmid DNA fragments are purified by low melting point agarose gel electrophoresis. Next, the DNA fragments J_ and! _ And 5 '— Immobilized Xhol linker (pCCTCGAGG) II is rapidly ligated with T4 DNA ligase to obtain the plasmid pKYPlOOJ_.

In the above-described method for producing the vector of the present invention, a method for producing a plasmid expression vector using an enriched * cleavage site immediately below the SD sequence was described. Even if such a cleavage site does not exist, the target expression vector can be folded, even if the cleavage site of the element existing upstream of the promoter is used, the promoter and the ribosome combination can be used. Synthesize oligonucleotides containing the K-sequence of all three bases, the site and the translation start symbol (ATG or GTG), and apply them to plasmid vectors such as pBR322. It can be cloned, and the Trib-to-Fan promoter of Dairan orchid was used as a promoter, a necessary element for transcription initiation. Use a large bacterial promoter other than the van pi motor. It can be. In addition, if the foreign gene to be expressed * is a box of an organism other than a large microorganism, a promoter with a high level of competence can be used. When using a promoter of an organism other than Escherichia coli, it is necessary to replace the clay base sequence containing the information necessary for initiation of translation with a base sequence corresponding to the ribosome binding site of Escherichia coli.

The expression vector obtained by the above procedure is as follows: (1) After digestion with «Johnson (Sphl, EcoRV, Kpnl, SacI, etc.), (1) the resulting 3'- Polymerase I · Κ1 Use the 3'-5 'exonuclease activity of the enow fragment to remove it, convert it to a flat end δβ, and incorporate the foreign gene into it (see Fig. 3). ), (2) Homozygous 'Polymer' block (Poly dA, PO) using terminal deoxynucleotidyltransferase on the resulting 3'-protruding end SS D T, poly d G, and poly d C), and the complementary homopolymer block is added. By incorporating the added exogenous gene (see Fig. 4), the exogenous gene can be used immediately after the start of fertilization. By culturing the host microorganism using the obtained recombinant, the foreign gene can be expressed very efficiently.

The above-mentioned tax statement describes a large-scale bacterium, which is most elucidated at the molecular level, but the present invention is not limited to the large-scale bacterium, but is not limited to the large-scale bacterium. It can be used for eukaryotic bran vesicles such as rich mothers and higher animals. That is, the structure downstream of the translation initiation signal is left as it is, and instead of the aforementioned bacterial promoter and large] »other nuclei such as Bacillus subtilis»跑, and if the genetic information necessary for the replication and maintenance of the plasmid in the prokaryotic bran vesicle is added, the vector that is effective for the expression of the plasmid is effective for other boxes. Can be built. In the case of Shinpuyuan, the mechanism of transcription and translation is not elucidated in detail. Instead of the ribosome binding site, it replaces »the genetic information required for translation initiation, and also provides the necessary genetic information to maintain and replicate the plasmid vector in the eukaryotic cell Then, it is possible to construct an effective vector for eukaryotic cells.

Simple sharps of the drawing

FIG. 1 is an example of the vector of the present invention, and is a schematic diagram of a translation initiation signal ATG and an SphI cleavage site (GCATGC) overlapping each other.

FIG. 2 shows a cut form of restricted dross which can be used in the vector of the present invention. FIG. 3 shows a step of incorporating a foreign gene into a vector of the present invention using a DNA polymerase I · Κ1 enow fragment.

FIG. 4 shows a step of incorporating an exogenous gene into the vector of the present invention using a terminal transfase.

FIG. 5 shows a process of forming a brassmid pKYP100. In the figure, J_ is a plasmid p KY P 10 and 2 is about 180 including the trp promoter.

OMPI WIPO b DNA fragment, _ is a DNA fragment of about 100 bp carrying trp promoter, 丄 is XhoI linker, _ is plasmid pBR322, and pBR322 is DNA. The fragment, 1_, indicates the plasmid ρ Κ Υ Υ Ι Ο Ο.

Fig. 6 shows the process of constructing the brassmid pTrS3. In 0, _L is about 3.82K of the plasmid pKYP10 and J_ is about pKYP10. b fragment, 10 indicates a double-stranded DNA paired with two kinds of synthetic oligonucleotides, and 11 indicates a plasmid pTrS3.

Fig. 7 shows the construction process of pKYP-5.

FIG. 8 shows a construction process of Ρ Κ Υ Ρ—10.

FIG. 9 shows the construction process of pFJ-123.

FIG. 10 shows a process of constructing pFM-9.

Fig. 11 (A) shows the construction process of pKYP-11. (Β) indicates 逮 Κ Υ 1-12 chair arrest.

FIG. 12 shows the construction process of pLE-3.

BEST MODE FOR CARRYING OUT THE INVENTION

The creation method of the vector of the present invention and its usage will be explained to the jurisdiction with examples.

Example 1

Construction of a plasmid pTrS3 having a translation initiation signal ATG and an SphI cleavage site downstream of the trp promoter and ribosome binding site:

From pKYP100 created in Reference 2, pTrS3 is obtained as follows. 5 g of PKYP 101 to 10 mM MT ris — EC & (pH 7.5), 7 mm MM g C £ ₂ , 6 mm M 2 Total amount including 1 mol of ethanol 5 units of Cal I (Boehringer's Mannheim KK) were added in a reaction solution of £ / £ and reacted at 37 t: 2 o'clock. Then, at 65, 5 minutes After terminating the reaction by thermal treatment, the reaction temperature is reduced to 100 mM MT ris-HC £ (pH 7.5), 70 mM

WIPO Mg C £ 2. 1. OMN a C £, 60 m M 2 — Melkabutanol and 16 jt of water and 7 units of SphI (Boehringer ♦ And the reaction was carried out at 37 c for 2 hours. 6 Stop the reaction by heat treatment for 5 minutes in 5 and then perform low-point agarose gel electrophoresis (see ow-Gel 1 ing-Tempesatile Agarose Gel Electrophoresis>

LGT-AGE) [Lars Hieslander: Analytical Carno, 'Io Chemistry I, Vol. 98, p. 305 (1979)], shows a large brassmid DNA fragment J_

(Approximately 3.82 Kb). Next, two kinds of talents

5 '-C G A T A A G C T A T G C A T G-3' and 5 '-C A

TAGCTTAT—3 ′ was synthesized by the phosphoric acid triester method. After 5 ′ monophosphorylation of these two synthetic oligonucleotides, 10 mM M Tris — so that the concentration of each is 20 M.

H C £ (pH 7.5) v l O O m M Na C £, mix in l M M E D T A, 10 minutes at 65¾, 120 minutes at 37 * c, then at room temperature

The person was left for 120 minutes, and then paired. As shown in the figure below, the pair of two moths D N A

p C G A T A A G C T A T G C A T G

T A T T C G A T A C p

It has a plum structure that can be linked to the sticky daughter produced by C1aI digestion or SphI digestion, and that, after linkage, the C1aI or SphI cleavage site is reformed. The combination of the two synthetic oligonucleotides was combined with the purified plasmid DNA fragment described above. _ Was added and the two were allowed to quiescent using T4DNA ligase. That is,

The two synthetic oligonucleotides, pCGATATAGCTCATGCCATG and CATAGGCTTAT, were each paired in 1-comomolar amounts, and about 0.15 jug of the purified plasmid DNA fragment 1 was added.

20 m MT ris-HC £ (pH 7.6)> 10 m MM g C £ ₂ ^

In a total reaction volume of 20 μ £ containing 1 OmM dithiothreitol and 0.5 mM ATP, add 0.5 units of T4DNA ligase (Takara Shuzo Co., Ltd.).

OMPI The binding reaction was performed at 4 o'clock at 16 o'clock.

Using the recombinant plasmid DNA obtained in this manner,

HB 1 0 1 strain was form K transformation, the resulting A down-bi Shi Li down resistance, the bra scan Mi de DNA having the shape R transformed strain Te preparative Rasa Lee click Li down sensitive (A p ^R T c ^s) Min «fine». These plasmid DNAs are converted into EcoRI,

X h 0 I, P st t l (or more, Takara Shuzo g), C la I, S p h I

(Above, Behringer's Mannheim GmbH) digested with a limited amount of drunk to form the desired brassmid vector pTrS311. And confirmed. Furthermore, the Maxam 'Gilbert method [AM Maxa 麵 et al .: Proc. Natl. Acad. Sci.] Confirmed that the base K sequence of the DNA from the Clal site to the SphI site of pTrS3 was ATCGATAAGCTATGCATGC. Vol. 74, p. 560 (1977)]. The large II strain that harbors pTrS3 was reported to the Institute of Microbial Industry and Technology (Ibaraki, Japan) on September 29, 1977 by: (Escherichia coli) ITr S — 3 FERMP — 6753, deposited on July 26, 1983, transferred to Kunitaka Deposit and given FERMBP — 328.

Actual »Example 2.

An interface with an extra methionine attached to the N-terminus (abbreviated as IFN—).

Using the pit vector pTrS-3 created in Jeongjeon example 1, the IFN- with an additional ffl of methionine added before the N-terminal methionine of IFN- A recombinant brassmid that realizes a conductor was obtained as follows.

1 0 g of p T r S- 3 to 1 O m MT ris -. HC £ (ρ H 7. 5), 5 0 m M NaC £, 7 m MM g C £ 2 6 m M 2- menu Luke Bed In a total of 40 reactions *, including ethanol, 10 units of SphI (Boehringer's Mannheim ») were added, and the mixture was reacted at 37 ° C for 2 hours. After the reaction, perform the extraction of ethanol and hologram and the extraction of ethanol.

OMPI Was. The precipitated DNA fragments on the total amount 4 0 £ of 5 0 m MT ris -. HC £ (p H 7. 6), 7 m MgC £ 2 1 0 m M 2 one main Rukabu preparative ethanolate Lumpur, 0.2 5 mM M d ATP, 0.25 mm M d CTP, 0.25 mm M d GTP, 0.25 mm M d TTP, followed by 4 units of large-scale DNA polymerase I · Kienow fragment (Bethesda 'Research' Laboratories) was added, and the mixture was reacted at 15 for 2 hours. After the reaction, phenol and ethanol were extracted and ethanol was precipitated. A total of 30 を of the precipitated DNA fragment was treated with 30 1 of 100 M MT ris-EC £ (pH 7.5), 50 mM Na C £, 7 mm M g C £ ₂ , 6 mM 2 — Melkabute It was dissolved in ethanol, added with 16 units of Ec0RI (Takara Shuzo), and reacted at 37 for 2 hours. After digestion with EcoRI, a small plasmid DNA fragment (about 130 bp) was purified by low-point agarose gel electrophoresis.

Next, the recombinant plasmid pLE—3 having IFN—gene

5 μg 1 O m MT ris-HC £ (pH 7.5), 7 mm MM g C £ ₂ .

10 units of C £ aI (manufactured by Boehringer Mannheim) were added in a total volume of 30 μM reaction solution containing 6 mM M2 ethanol solution, and

37-C. The reaction was performed for 2 hours.

The structure around the C £ a I site of p LE — 3 is as follows.

C £ a I

AAA | AAGGGTATCGATGAGCTAC--TTTTTCCCATAGCTACTCGAT GSD Met Ser Tyr m

MM g C £ 2.1 .0 mm M 2 — Melkabutanol, dissolved in 0.25 mm M d ATP, 0.25 mm M d TTP, resulting in a mass of 12 units »Add bacterial DNA polymerase I (New Englnd Biolabs) and add 37 t H reaction for 30 minutes at

As a result of this reaction, the 5 'overhang generated by digestion with CaI was flattened by the 5'-3' exonuclease activity of DNA polymerase I, as shown below. End »

C £ a I

T A T A

In this reaction, dATP and dTTP prevent DNA base I from extruding in the 3'-5 'direction from the DNA molecule divider by the 3'-5'exonuclease activity of DNA polymerase I. It was added for In this reaction, the ratio of obtaining a flat daughter like the above is about 2% to 20%.

After the above reaction, extraction of phenol and black mouth and extraction with ethanol were performed. The precipitated DNA fragments on the total amount 3 0 mu £ of 1 0 O m MT ris -. HC £ (p H 7. 5), 5 0 m MN a C £, 7 m M g C £ 2 6 m M 2- main After dissolving in lukabutanol, adding 16 units of EcoRI (Takara Shuzo Fold) was added, and the mixture was reacted at 37 for 2 hours.

After digestion with Ec0RI, a large plasmid DNA fragment (approximately 5.1 Kb) was purified by low melting point agarose gel electrophoresis. The about 130 bp DNA fragment (about 40 ng) containing the trp promoter, SDK sequence and ATG codon obtained in this way and about 5.1 Kbp containing the IFN-9 gene are obtained. b DNA fragment (about 1 5 0 ng) of 2 0 m MT ris -. HC £ (p H 7. 6), 1 0 m MM g C £ 2 1 0 m dithio Threshold Level I DOO Lumpur, 0. One unit of T4 DNA ligase was added to the reaction solution with a total volume of 20 # containing 5 m MATP, and the reaction was carried out at 4 for 20 hours.

Using the recombinant plasmid DNA obtained in this manner, E. coli HB101C H. Boyer et al .: J. Molec. Biolo., 41 59 (1969)] The resulting A p ^R T c ^s p FJ brass mi de DNA with traits耘換strain shown in FIG. 9 was separated膽精folds Ri by the usual method of - give the 1 2 3.

p FJ — 1 2 3 movements are restricted by the restriction alcohol Eco RI, X ho I, P st I, B am HI (both from Takara Shuzo) and C £ al (from Boehringer Mannheim) After digestion by agarose gel, confirmation was made by agarose gel electrophoresis.

The large cocoon HB101 strain having the recombinant plasmid pFJ-123 was named IFJ-123, and the productivity of interferon was determined by the following method.

Dissolve this strain in an LG medium (10 g of tripton, 5 g of rich mother extract, 5 g of NaC, 5 g of glucose and 1 g of water, and adjust the pH to 7.2 with NaOH). At 37 and at 18:00. The culture solution 0.2 * 1 was added to the 10 MCG medium (Na ₂ HP 04 0.6%; KH ₂ P 0 0.3%, Na Ce 0.5%; NH ₄ C £ 0.1%, glucose 0.5%, casamino acid 0.5%, Mg S04 4 lmM, thiamine hydrochloride 4 # g / Bl, pH 7.23, inoculated at 30 to 4-8 After the incubation, add 10 pg Z ml of indoleacrylic acid (hereinafter referred to as IAA), which is an inducer of the tributophane gene, and add it for 5 to 12 hours. Culture was started.

The culture is arrested at 8,00 rpm for 10 minutes, collected, and washed with 30 mM NaC £, 30 mM MT ris -HC £ (pH 7.5) buffer solution. The washed cells are mixed with 1 ml of the above buffer solution, and 200 g of lysozyme and 0.25 MEDTA (ethylenamine acetate) are added. After standing for 0 minutes, the cells were frozen and »thawed three times to disrupt the cells. This was arrested at 1,500 rpm for 30 minutes, and the upper stomach was collected. The amount of interferon in the supernatant was quantified according to the method of armstronging [JA Armstrong: Appl. Microbiol. \, 723-725 (1971)]. However, the virus is Vesicular Stoline ati Us Virus, and the animal »跑 is the human amniotic membrane» Wish Ce from the vesicle Was used.

Lee printer-safe b down activity of a result of the measurement, the strain is 3 X 1 0 ³ units

It produced £ interferon.

Of the plasmid (PFJ-123) held by the IFJ-123 strain

The nucleotide sequence around the SDS sequence and ATG was determined by the Maxam-Gilbert method.

A A G G G T A T C G A T A A G C T A T G A T G

S D

Was found to be.

The large cocoon strain, which houses PFJ—123, was sent to the Institute of Biotechnology, Institute of Biotechnology, Japan on January 24, 1988 as Escherichia coli IFJ-123 FERMP-6882. Has been deposited.

FIG. 9 illustrates the process of forming the above-mentioned PFJ-123.

Λ O.

Expression of amino acid-deficient IFN- 耪 N-terminal conductor:

In fact, using the expression vector pTrS-3 created in Example 1, a recombinant plasmid expressing IFN-derivatives lacking some amino acids of the N-terminus of IFN- And get it as follows.

15 JU g of ρ LE — 3 is 10 mm MT ris — HC £ (pH 7.5), 7 mm MM g C έ 2, 6 mm M 2 Includes one-but-one ethanol Whole amount

20 units of C £ aI (Boehringer Mannheim, Inc.) were added in the reaction mixture of 10 and reacted at 37 for 2 hours. A total of 3 precipitated DNA fragments were treated with 1 Om MT ris-HC £ (pH 7.5).

— 0. Dissolved in SmMEDTA. Use this DNA solution 6

. (DNA weight of about 3 g> and蓁習water 1 3 # £ and鑀衝solution (6 0 m MC a C £ 2, 6 0 m MM g C £ 2 3 MN a C £, l OO m MT ris - After mixing HC £ (pH 8.1), 5m MEDTA) 5 ^ /

0.5 units of Exonuclease BAL 3 1 (New England Biolabs Was added and the mixture was reacted at 30 "C for 3 minutes. In this reaction, a 1 to 30 bp DNA arrowhead was cut in the direction of £ from Cp aI Sue Yasushi.

It was stopped by the extraction of knoll, followed by extraction of holocalm and ethanol.

Precipitation was performed. Transfer the precipitated DNA fragments to a total volume of 20

T ris-H C £ (pH 7.5), 50 mM Na C £, 7 mM M g

C6 2.6 mM 2-dissolved in ethanol, and 8 units

Of EcoRI (Takara Shuzo Co., Ltd.) was added, and the mixture was reacted at 37 ° C. for 2 hours.

After digestion with Ec0RI, low »point agarose gel electrophoresis was performed.

The large plasmid DNA fragment (approximately 5.1 Kb) was purified.

Approximately 5.1 Kb of DNA containing the IFN- gene thus obtained

The fragment (about 150 ng) and the trp promoter made in SW in Example 2,

Approximately 130 bp DNA fragment with SD sequence and ATG codon

(Approximately 40 ng) with 20 mM Tris-H C (H 7.6), 10

mM M g C £ 2.1 l O mM dithiothreitol, 0.5 mM ATP

1 unit of T4DNA ligase (Takara)

(Manufactured by Shozo Co., Ltd.), and the binding reaction was carried out at 4 for 20 hours.

Using the recombinant plasmid DNA obtained in this way, the bacteria

HB 1 0 1 strain was transformed by Rikatachi ϊί a conventional method, the shape of the resulting A p ^R T c ^s

»Separate and purify the plasmid DNA of the transformed strain by a conventional method.

The pFM-9 shown in the figure was obtained.

The structure of pFM-9 is restricted to restriction enzymes EcoRI, XhoI, Pstl,

After digestion with Bam HI and C £ aI, agarose gel electrophoresis was performed.

Confirmed by the movement.

Large strain waiting for recombinant plasmid pFM-9

This was named ninth, and the productivity of the interface pin was determined in the same manner as in Example 2.

At that time, it produced 8 x 10 square units / interferon.

This strain was established on January 24, 1983 by

Escherichia coli I F-9 F E R M P-6 8 8 3

Has been deposited. The recombinant plasmid carried by this strain was

O PI, Call one nine,

The construction process of the above-mentioned SB pFJ-123 is shown in Fig. 10H. Next, the SD of the plasmid pFM-9 held by the bacteria IFM-9

The nucleotide sequence around the S row and ATG was determined by the Maxam-Gilbert method.

AA G G G T A T C G A T A A G C TA T G GA TT C

S D

Thus, ρFM-9 was found to contain a gene coding for IFN-, which lacks the amino acid »CSer Tyr Asn Leu Leu from the N-terminal end of the 5th amino acid.

Reference example 1.

Construction of a plasmid vector pKYPIO with a trp promoter:

(a) Tribute fan type 煑 Introductory phage

The λ ptrp phage c 1857trpED 10 [hereinafter abbreviated as trpBD GFMiazzari et al .: J. Bacterid. 133, pp. 14457 (1978)] is a large strain of JA1904! : F-, A ', r _K ', m, Δ trp E ₅ .

1 eu6, J, Carbon Becc ^ binant Molecules p. 3oo (1977) Raven Press]

By culturing JA194 (λtrpED) for 30 minutes at 42 ° C, the trpED phage was attracted, and the phage lysate was recovered. The cesium chloride equilibrium density gradient arrest method from this phage solution (Yukawa et al.

"Chemicals of Nucleic Acid I" The phage was purified according to Tokyo Chemical Dojinsha P. 54-61, 19743.

Then, from this phage, phenol treatment and kuguchi-holm treatment were performed according to Yamakawa et al.'S method [& Acid Chemistry I, Tokyo Kagaku Dojinsha, p. 62-65, 1974]. And purified.

(b) Closing the trp promoter to the plasmid

The cloning of the trp operon from λ trp ED phage DNA is performed as follows: _β, ie, 8 g of trp ED DNA 20 mm Tris HC (pH 7.5), 75 mm MNa C £, 10 mm MM g C £ 2, 5 * M 16 units EcoR I in dimethyls

(Takara Shuzo Co., Ltd., same hereafter) and 16 units of Hindm (Takara Shuzo Co., Ltd., same hereafter) were added and the mixture was digested at 37 t: for 2 hours. On the other hand, pBR32 5DNALg of brasmid was similarly digested with 2 units of EcoRI and 2 units of Hindm (final volume 30 // £). Then at 65,

The reaction was stopped by heat treatment for 5 minutes, and the digested DNA solution (15 #) was combined with each other, and a final concentration of 50 OM ATP and T4 DNΑ ligase (5 units, New England Biolab) ) Was added and the reaction was performed for 18 hours at 4.

Large using a mixture of brassmid obtained in this way) »Bacterial C600 SF strain [Cameron et al .: Broch. Null Academie, The Observation of Science, Vol. 72, p. 3416 (1975)], using a known method [SN Cohen et al .: Floatings of the Transformed in accordance with 'National Accademia' Ob 'Science, Vol. 69, pp. 2110 (1972), and resistant to ambicilin

(A p ^R), to obtain a transformant耘換strain having te preparative La Size Lee click Li down resistance (T c ^R> and clients B ram full Eniko Lumpur sensitive (C m). The cormorants yo this Brassmid DNA was hybridized from the transformant strain, which was obtained as a bacterial strain, and named pKYP-1.

Brassmid p KYP — l is digested with Taq I and EcoRI to contain a tributary fan promoter and an SD sequence. 2.6 Kb (kilobase: same hereafter) The DNA fragment was purified by agarose gel electrophoresis. The 2.6 Kb DNA fragment is cloned into a known vector, PBR322, by the method shown in FIG. That is, 8 units of PBR 3 2 2 plus 2 units of Taql (Takara Shuzo «), 10 m MT ris — HC (pH 8.4), 6 mm MM g C £ 100 m MN a C £, 6 mm M 2-The reaction was carried out for 60 minutes with 45 in a 100-volume reaction solution containing mercaptoethanol. T aq Melting point agarose gel electrophoresis after nodal digestion with I [L. ア Lars Wies lander: Analytical 'no, * iochemistry 9 Volume 8, page 35 (1979) :), purify the 4.36 Kb DNA fragment, add this DNA (about 1.5 g) to 3 units of EcoRI, After complete digestion by reaction at 37 for 3 hours, a DNA fragment of about 4.33 Kb (about 1. Og) was obtained by low-melting point agarose gel electrophoresis in the same manner as above. Was.

Next, 12 g of PKYP-1 DNA was partially digested with 3 units of T aq I in the same manner as above, and an 8.5 Kb DNA fragment (about 2 g) was obtained by low melting point agarose gel electrophoresis. Then, the DNA was completely digested with EcoRI to purify a DNA fragment of 2.6 Kb, about 0.5 g. The 4.33 b DNA (0.2 μg) of PBR32 2 and the 2.6 Kb DNA fragment (0.25 // g) of ρ Κ Υ Ρ—1 20 m MT ris-HC £ (pH 7.6〉, 10 mm M g C £ 2, l O mm M In a 20 μβ total reaction solution containing dithiothreitol, 0.5 mM of Α Τ Ρ and 4 units of T4 DNA ligase were added, and the ligating reaction was performed at 4 * C for 18 hours. using DNA, large »fungus C 6 0 0 SF 8 strain was form K transform, resulting a p ^R, it was separated and purified the blanking la scan Mi de with the T c form ¾ transformant strain of ^R. this blanking La Six kinds of restriction of Smid DNA »Element, EcoRI, HindΠ, C1aI (Boehringer's Manheim), Hpal (Takara Shuzo IB), HincΠ ( Takara Shuzo Co., Ltd.) and Bam HI (Takara Shuzo Co., Ltd.) digested it to break the structure of brassmid and named it pKYP-5.

(c) Making the trp promoter portable

The above plasmid pKYP-5 has a 1 aI site and a Hind ET site downstream of the SD sequence (1 to 20 or less), so it can be sufficiently used as a DNA introduction vector. However, p KY Ρ — 5 DNA has another force except for the C 1 a I site immediately after the SDK sequence.

OMPI The presence of the CI a I site and the fragment containing the trp promoter cut out from p KYP — 5 DNA with Eco RI and Hind ffi are slightly larger at 2.65 Kb, making it easier to use. To achieve this, a plasmid containing a smaller DNA fragment containing a tributane fan motor is constructed as shown in FIG. That is, pKYP-5 DNA was digested with H pan and Hind BI to purify a DNA fragment of about 340 bp (base pair), which was digested with C1aI and HindI to obtain pBF? As shown in Fig. 6, it was inserted into 3 2 2 to obtain pKYP-10. The structure of pKYP-10 was confirmed by agarose gel electrophoresis after digestion with restriction enzymes EcoRI, C1aI, HindM, and HpaI.

(d) The speed of the t rp

Next, with the aim of developing a brassmid vector with even stronger promoter activity, we wiped out connecting two bottles of the trp promoter. That is, as shown in Fig. 11 (A), a DNA fragment of about 340 bp containing the trp promoter described in the previous section (c) was digested with CaI and Hind ffl into pKYP-10. And obtained p KYP — 1 1. In the same way, p KYP-12 (Fig. 11 (Β)), in which trp promoters 3 偭 are connected in the same direction, was also constructed, and EcoRI, Cp aI, Hind ffl, Hpa Digestion with I confirmed the structure.

Reference example 2.

1 ^ 丫? Creation of 100:

In order to create the plasmid vector described in the present invention, as shown in FIG. 5, a more easily usable form of pKYP100_ is created.

As a supply line for DNA that enriches the trp promoter, a brassmid that returns the trp portable promoter p KYP 10 J_ (Special Tokusatsu 56--21 13 193, see Figure 5) ) Is used.

Add 50 units of HhaI (Takara Shuzo) to 50 g of pKYP10.

O? I In addition, lO mM Tris-TC 6 (pH 7.5), 7 mM Mg C £ ₂ , 6 mM 2 2 o'clock 1 <1 5% Polyacyl 'after digestion with HhaI; Lamid gel electrophoresis ft [AM Maxa Plas s Proc. Natl. Acad. Sci., 74, 560 (1977); hereinafter abbreviated as PAGE] As a result, a DNA fragment of about 180 bp containing a trp-opening motor was closely examined. This is because one of the two DMA fragments other than the DNA fragment could not be properly distributed by PAGE. To be refined. Three purified DNA fragments

(Approximately 4 jug) was added to 50 mM Tris-HC6 (pH 7.6), '7 mM MgC £ 2.10 mM M2 1-mercaptoethanol, 0.25 mM d ATP, 0.25 md CTP, 0.25 mM dGTP, 0.25 mM

DNA polymerase I'Klenow fragment (Bethesda's Research 'Laboratories S) was added and allowed to react for 152 hours. As a result of this reaction, the 3'-protruding end »generated by HhaI digestion is the DNA polymerase I · 1 enow fragment awaits 3 '-5' exonucleic acid It can be converted to a flat powder by its enzyme activity and 5'- → 3 'repair / synthesis activity. The DNA volamerase by heat & process for 72, 30 minutes

After inactivating the new piece of I-Klenow, set the NaCjg key to 50 mM with 1 MNaCp, add 8 units of HindEI (Takara Shuzo Co., Ltd.), and press Allowed to react for hours. After digestion with Hind DI, a DNA fragment of about 100 bp containing the trp promoter was separated and purified by PAGE.

On the other hand, 8 units of Eco RI (Takara Shuzo Co., Ltd.) was added to 5 g of the plasmid PBR32, and lO mM Tris-HC £ (pH 7.5), 50 mM NaC £ _{. 7 m MM g C £ 2} . the reaction solution a total volume of 2 0 containing 6 m M 2 Ichime Luca Bed Toetano Lumpur was 2 Tokiran reaction 3 7 ΐ. After the reaction, after extraction of phenol and black form and ethanol digging, a total of 20 # £ of 50 mM Tris-HC £ (pH 7.6), 7 mm MM g C £ ₂ > 6 mm 2 Melkabutanol, 0.25 mm M d ATP, 0.25 mm M d CTP, 0 25 Dissolve in 5 mM dTTP, add 8 units of large DNA polymerase I · K1 enow fragment (Bethesda Research Laboratories) and add 15 units. The reaction was performed at 1C for 2 hours. The 5'-protruding protein produced by the EcoRI digestion was changed to a flat terminal by the activity of repairing and synthesizing the DNA polymerase I · 1 enow fragment. In 72, the DNA polymerase I • Κ1 enow fragment was inactivated by heat treatment in the 30th cabinet, and then the NaC concentration was adjusted to 50 mM with 1 MNaC. Eight units of Hind HI (Takara Shuzo «) were added, and the reaction was carried out at 37 for 2 hours. After digestion with HindB, a larger plasmid DNA fragment (approximately 4.33 Kb) was purified by low melting point agarose gel electrophoresis.

The thus obtained about 100 bp DNA fragment 1 (about 50 ng) containing the trp promoter and the about 4.33b DNA fragment derived from pBR322 (about 0.2 ng) were obtained. i "g) and 50 ng of 5 'monophosphated XhoI linker (p CCTCGAGG, manufactured by Collaborative Research), and add 20m MT ris-HC £ ( pH 7.6), 10 mm MM g C £ 2 .l O m M dithylolate slat, 0.5 m MATP, containing 1 unit of T 4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) was added, and the reaction was carried out for 40 hours at 4. The recombinant plasmid DNA obtained in this manner was used to transform large bacteria HB101 the strain was transformed, resulting a p ^R, T c bra scan Mi de DNA having the transformants in ^R was separated fine ¾. this is found in the blanking la scan Mi de DNA eight kinds of restriction enzymes E co RI, X hol, H indi, H ae ΠΙ

(Both from Takara Shuzo), C1aI (Boehringer's Manheim), TaqI (Bethesda Research 'Laboratories), Rsal

DNA fragment 3 containing the approximately 100 bp trp promoter and XhoI linker 4を通ブ 0 1 0 0

WIPO Named _L.

Reference example 3.

Construction of p L E-3:

pTu IFN / 9-5 (separated from ATCC 318979) by Hind M, digestion with DNA polymerase I (New England Biolabs) and binding reaction And shown in Fig. 12

pT u IFNH-5 was obtained. The IFN gene was cut off from pTu IFN 9H-5 and cloned into a brassmid ρ Κ Υ Ρ 112 with a trp promoter. That, ρ Κ Υ Ρ - 1 2 DNA 2 g restriction醉素C £ a I was added 4 units, 1 0 m MT ris -. . HC £ (H 7. 5) 6 m M g C £ 2 5 m After reacting at 37 for 2 o'clock in M dithiothreitol (hereinafter referred to as C £ a buffer) (total amount of 30〃 £), the Na C £ was brought to a final concentration of 10 O m In addition, the reaction was controlled at 2 o'clock with 3 7. Then, IB heating was performed at 65 ° C for 5 minutes to inactivate melamine, and a DNA fragment containing a trp promoter of about 5 Kb was purified by low-melting point agarose-gel Xiosphorophoresis. Obtained. Next, 15 g of pTuIFNH-5 DNA is digested with the restriction enzymes C £ aI and BamHI in the same manner as above, purified by low-point agarose gel electrophoresis, and DNA containing the IFN gene. Fragment (1.1 Kb) Approximately l «" g was obtained. The 2 捆 DNA fragments (5 Kb and 1.1 Kb in Fig. 12) obtained as described above mM Tris-HC £ (pH 7.5) 6 mM Mg C £ ₂ , 5 mM Diotis rail, 500; u MATP, add 4 units of T4 DNA ligase, At 18:00, the reaction was carried out at 4 o'clock in step 4. Using the resulting complex of plasmids, the HB101 strain was transformed in the usual manner, and Ap to obtain a ^R co b knee this co π separating plus Mi de from the culture medium of the knee, PLE was shown in the first 2 Figure -. 3 was obtained p LE -. 3 of Umezo is C £ al After digestion with Hind ffi and Bam HI, the resultant was digested by agarose gel electrophoresis. — Open from 3 SD IE columns (AAGG)

OMPI The row of haniki R up to the first codon (ATG) is described as “Maxam. Confirmed.

Large strains, including pLE-3, are found in Escherichia coli i L E-3 AT in the American Type 'Culture' collection (ATC C).

Deposited as CC39010.

O PI WIPO

Claims

Surrounding claims

制, a S-enzyme that has a promoter, a ribosome binding site, and a translation initiation signal, and leaves the 3 'end of the ffi hem as a truncated form following the translation initiation signal.

Expression base Kuta Gihai ¾ there is one _e

2. A patent application waiting for the start signal to be ATG or GTG

3. The expression vector according to item 1 of Jiang.

3. Characterize that the Ιύιδ enzyme is Spni, 匚 ooRV, Κηί or SacI.

The expression vector according to claim 1, characterized in that:

4. The expression vector as described in Item I of the Patent Application, which is characterized by the fact that the promoter is a promoter of Gundam Harada.

5. A motor is trp-, iac- and phage-introduced

One coater or an expression vector scope of paragraph 4 of Ru, wherein this claims to be a Bacillus subtilis promoter ₌

6. The expression vector according to claim 1, wherein the arrow motor, the ribosome tethering site, and the translation initiation signal are in the order of ~ 10.

Ding, arrow motor, liposome bonding Si position and 'translation opening signal', and 3'-thrust after cutting start signal is left as a cut form? ^ After cutting the expression vector, which contains the yeast strain system, with enzyme,

Recombinant DNA was transformed into flat-bearing elegans using DA bolimerase i (Kieno * mf) or T4D bolimerase, and a foreign harmonized gene was integrated into the flat end.

A method of transforming the king and cultivating the resulting transformant to express the extragene.

8. Recognition of a control enzyme that has a promoter, ribosome binding site and translation initiation signal, and leaves the 3 'end of the ffi mouse end as a truncated form following the translation start signal.

OMPI After cutting the expression vector containing the recognition sequence with a restriction enzyme, a homopolymer block is added to the resulting 3 'one-protruding end using terminal dextran xynucleotidyltransferase and complementary to this. A method in which a host microorganism is transformed with a crude recombinant incorporating a foreign gene to which a unique homopolymer-block has been added, and the resulting transformant is cultured to express the foreign gene.

9. The person described in paragraphs 7 or 8 of the patent application, wherein the translation start signal is ATG or GTG.

The method according to claims 7 to 8, wherein the enzyme is Spni, EcoRV, {ρη} or Sacί.

10. The method according to claim 7 or 8, wherein the bromotor is a promoter from Gundam.

2. The method according to claim 1, wherein the promoter is a tn) -type, an iac-yarn and a phage-input pi-enterobacterium promoter. 1

3. The method according to claim 7, wherein the daguchi motor, the ribosome binding site, and the translation initiation signal are those of Eukaryotic cells.

"14, Microbial cells containing the expression vector according to claim ϋ.

1, 5, Escherichia coli i T "S_3.

16 、 The Gene of Haruka Haruka is Interflon I gene or its derivative

The claim described in claim 7 characterized in that:

17. The claim is characterized in that the main microscopic substance is in the field.

7 K¾.

1 8, Escherichia coli i F J—Ί25.

1 9, Escherichia coli I M—9 b

O PI

, IPO