WO1985004673A1

WO1985004673A1 - Novel dna and its use

Info

Publication number: WO1985004673A1
Application number: PCT/JP1984/000181
Authority: WO
Inventors: Masaharu Senoo; Haruo Onda; Koichi Igarashi
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 1984-04-10
Filing date: 1984-04-10
Publication date: 1985-10-24
Also published as: JPS61181380A

Abstract

A novel DNA is prepared in which a DNA fraction having a structural gene which code a peptide containing an antibody recognition site and a DNA fraction having a structural gene which code human interleukin 2 peptide are linked to each other. This novel DNA makes it possible to produce a polypeptide in which the two peptides are linked to each other by transforming host microorganism with the DNA and culturing the transformed microorganism.

Description

Details

New DNA and its uses

Technology

The present invention has a DNA having a structural gene encoding a peptide including an antibody recognition site and a structural gene encoding a peptide of human interleukin 2; DNA, a plasmid incorporating the DNA, a transformant transformed with the plasmid, binding of a peptide containing an antibody recognition site to a peptide of human interleukin 2 The present invention relates to a polypeptide obtained and a method for producing the polypeptide using the transformant.

One back view-technique-art

Peptides containing an antibody recognition site, for example, immunoglobulin E (hereinafter sometimes abbreviated as Igβ) are responsible for important biological reactions such as allergic reactions. That is, it is known that an allergic reaction is induced by the binding of Ig bound to the toxin antigen to sensitized obese proboscis and basophils (Immunological Review JJ ^, 10). 9 (19778 ". Therefore, it is considered to use an Ig £ 1 molecule excluding the antigen-binding site in order to suppress the Argallgi reaction. However, various factors caused by Ig in vivo are considered. There are still many unresolved points about the reaction of this.One of the reasons for this is that it is not possible to supply enough humans.

As a method for producing Ig, a method of fractionating and purifying from the culture supernatant of established human myeloma cells having the ability to produce human Ig has been proposed. It is difficult to obtain a large amount of I at low cost because of the low cost. Therefore, a technique was developed that could obtain a large amount of Ig by culturing a transformant transformed with a plasmid incorporating the 1 ^ structural gene using genetic engineering technology. "Nucleic Aci-

OM? R ds Researc 11, 719 (1983; *, Nucleic Acids Research 11, 3077 (198 three).

—Interleukin 2 (hereinafter sometimes abbreviated as IL-2). Previously it was called T cell growth factor (TCGF). J is a lymphokine produced by a T cell stimulated with a lectin-Paro antigen or the like [Sciense 1993, 1007 197 6) Immunological Review 51, 25 7 (1998) 0)]. IL-12 not only allows T cells to be expanded while retaining their functions at the mouth of the mouth to maintain the long-term age, but also promotes the mitogen response of thymocytes to date ( It has the activity to restore the antibody-producing ability of splenocytes of costimulator-null mice to Τ cell-dependent antigens (: τ cell replacement factor-1) and to promote the differentiation and proliferation of killer cells (killer factor> killer cell). The journal o 丄 immunology 1 2 3, 2 9 2 8 (1 9 7 9

Immunological Review 5 1, 2 5 7 (199 8 0 l _o

However, it is difficult to produce tortoise using natural IL-12, and it is a plasmid that incorporates the human IL-12 structural gene using genetic engineering technology. By culturing the transformed transformant, a large amount of human IL-2 can be obtained [Nature 302, 305C1893 ^; Biochemical and Biophysical Research Community- ions vol. 1 09, yfa2, p. 363 C 1983; Nucleic Acids Research II, 4307 [1993]] _o

Signs of the light

The present invention ligates a DNA having a structural gene encoding a peptide containing an antibody recognition site to a human interleukin 2 (a DNA having a structural gene encoding a peptide of human IL-12). DNA, said DN A plasmid into which A has been integrated, a transformant transformed with the plasmid, a polypeptide obtained by binding a peptide containing an antibody recognition site to a peptide of human IL- ¹² , and the transformation The present invention relates to a method for producing the polypeptide using a body.

MRNAs encoding human IgE or human IL-2 alone have already been isolated and identified, and the expression of these genes has also been attempted independently. Nucleic Acids Research 11, 719 ( 1, 3077 (1993;; Nature 302, 305 (1 983)) A powerful, heterologous protein with two different immunological or biological activities. Obtaining a complex of the above has not been known before.

The present inventors have produced a complex of a heterologous protein of human I and human IL-2 using a genetic engineering technique, and found that the complex has the immunogenicity of human IgE. It has been found that it has both the antigenic properties of human IL-1 and human IL-12, and as a result of further research based on this, the present invention was completed.

That is, the present invention provides (1) a DNA having a structural gene encoding a peptide containing an antibody recognition site and an I) NA having a structural gene encoding a human IL-2 peptide, DNA linked in the open reading frame, (2), DNA having a structural gene encoding a peptide containing an antibody recognition site, and DNA having a structural gene encoding a human IL-2 peptide (3), DNA having a structural gene encoding a peptide including an antibody recognition site, and human IL- ^2. A transformant transformed with a plasmid incorporating DNA, which is obtained by joining DNAs encoding structural peptides encoding the same peptides in the same reading frame, (4), antibody recognition site And peptides containing With IL- ² peptide ( ⁵ ) a DNA having a structural gene encoding a peptide containing an antibody recognition protein and a DNA having a structural gene encoding a peptide of human IL-2, The transformant transformed with the plasmid into which the linked DNAs are aligned and ligated together is cultured in a culture medium, and the peptide containing the antibody recognition site and human IL-12 are cultured in the culture. This is a method for producing the polypeptide, comprising producing and accumulating a polypeptide which is bound to the polypeptide, and collecting the produced polypeptide.

In the present specification, a peptide containing an antibody recognition site is human IgE, and a polypeptide in which this is bound to a human IL-12 peptide is referred to as immunogleukirg (Immunogleukirg EC22; It may be simply abbreviated as IGL-EC22.

Examples of the peptide having an antibody recognition site in the present invention include a peptide having an anti-human immunoglobulin E antibody recognition site. Examples of the peptide containing the anti-human Ig antibody recognition site include a peptide containing all or a part of the human IgE chain. The portion of the human IgE heavy chain is one of the peptide chains constituting the human IgE molecule, and the molecule is composed of one pair of H chains and one pair of L chains. It has been known. Therefore, examples of the structural gene encoding a peptide containing an antibody recognition site include a structural gene encoding a peptide containing all or a part of a human IgE H chain.

Examples of structural genes encoding peptides containing all or a part of the Ig chain include, for example, Japanese Patent Application Laid-Open No. 59-44399, Cell vol. 29, 691 (1989) 2, Nucleic Acids Research 1, 719 [1 983], Nucleic Acids Research 11, 3077 198 3), EMBO The European Molecular Biology Organization Journal l_, 655 (1992) and the like.

The human IL-2 peptide of the present invention refers to a peptide having human IL-2 activity. As long as it has the activity, it may be a peptide shorter than human IL-2.

Examples of the structural gene encoding the peptide of the human IL-2 include, for example, Japanese Patent Application No. 225,079 (Showa 58, 1983) Date application :), Japanese patent application No. 235638 (Showa 58 (1980), filed on February 13, 1980), Nature vol. 302, p. 305 (1 983) , Biochemical and Biophysical Research Com- muni cations vol 109, 2, p.363 (1 9 82 ^, Nucleic Acids Research ll, 4307 (1 9 8 3 J) are described, for example:. DNA force; mentioned are _c above The DNA encoding the peptide of human IL-2 described in Japanese Patent Application No.

⁵ 'QQGQQGQGQQGGQQGGGA CACTCTC TTAATCACTACTGACAG AACC

S 1

TCAACTOCTQCCACA ATG TAG AGG A∑Q CAA CTC C G TCT TGC

S20 I

ATT GCA CTA AGT CTT GCA. CTT GTC ACA AA AGT G A CCT ACT TCA. AGT TCT ACA. AAG AAA ACA. GAG ΟΊ CAA CTG GA.G CAT TTA CTG CTG QAT TTA CM} ATG ΑΊ TTG AAT GGfV ATT

40

AAT AAT TAC AAG AA CCC AAA CTC ACC AQG A G CTC ACA ΊΤΤ AAG Ί Τ TAC ATG OCC AAG AAG GCC ACA GAA CTG AAA

GAT CTT CAG TOT CTA GAA GAA GAA. CTC AAA CCT CTG GA.G

80

GAA OTG CTA AAT ΓΓΑ OCT CAA AGO ¹ AAA AAC TTT GA.C ΊΤΑ AGA COG AQG GA.C TTA A∑C AGC AAT AC AAC (3ΓΑ GTT

100

CTG GAA CTA AAG GCA TCT GAA ACA. ACA TTG ATG TG GAA TAT GOT GAT GAG ACA. GC AOC ΑΊΤ GTA GAA TTT CTG AAC 120

AGA. TGG ΑΤΓ ACC TTT TGT CM AGC ATC A C TQ ACA CTG

ACT TQ ^ TlAOTAAarGCT CCCACTTAAAACA ATCAQQCC TC ATTT ATTEAAA Ar TAAOTT ACai CCGCCCCCCC ³

Among, codon L～l 3 in the nucleotide sequence represented by 3 1) NA (V) can be mentioned _c this; DNA (V), that in the ⁵ 'terminal ATG or the formula codon S. 1 to S 2 0 May be present, and preferably has TAA. TGA or TAG at the 3 'end, and particularly preferably TGA.

The above DNA (V) is represented by Formula 1

X-Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu Gin

20

Leu Glu Hi s Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met

40

Leu Thr Phe Lys P e Tyr Met Pro Lys Lys Ala Thr Glu

& o

Leu Lys His s Leu Gin C s Leu G u Glu Glu Leu Lvs Pro

'

Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe

80

His Leu Arg Pro Arg Asp Leu He Ser Asn He Asn Val

10 o

lie Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Gl Thr Ala Thr He Val Glu Phe

120

Leu Asn Arg Trp lie Thr Phe Cys Gin Ser lie He Ser

133

Thr Leu Thr

(In the above formula, X represents Met or hydrogen. It encodes a peptide represented by the following formula. Further, it encodes a peptide of human IL-2 described in Japanese Patent Application No. 58356/1983. 1) NA is the formula

(5 -AGT TCT ACA AAG AAA ACA CAG CTA CAA CTG One

GAG CAT TTA CTG CTG GAT TTA CAG ATG ATT TTG AAT GGA ATT AAT AAT TAG AAG AAT CCC AAA CTC ACC AGG ATG CTC ACA TTT AAG TTT TAG ATG CCC AAG AAG GCC ACA GAA CTG AAA CAT CTT CAG TGT CTA GAA GAA GAA CTC AAA CCT CTG GAG GAA GTG CTA AAT TTA GCT CAA AGC AAA AAC TTT CAC TTA AGA CCC AGG GAC TTA ATC AGC AAT ATC AAC GTA ATA GTT CTG GAA CTA AAG GGA TCT GAA ACA ACA TTC ATG TGT GAA TAT GCT GAT GAG ACA GCA ACC GTA GAA TTT CTG AAC AGA TGG ATT ACC TTT TGT CAA AGC ATC ATC TCA ACA CTG ACT-Y

(3; c i)

[Where X represents a codon or hydrogen other than AGC, AGT, TCA, TCC, TCG, TCT, TAA, TAG and TGA, and Y represents a codon or a hydroxyl group]. DNA having the above formula (VH is the formula

Ζ-Ser Ser Thr Lys Lys Thr Gin Leu Gin Leu Glu His Leu Leu Leu Asp Leu Gin Met lie Leu Asn Gly lie Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gin Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gin Ser Lys Asn Phe His Leu Arg Pro Ar As Leu lie Ser Asn lie Asn Val He Val Leu Glu- Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr lie Val Glu Phe Leu Asn Arg Trp lie Thr Phe Cys Gin Ser He He Ser Thr Leu Thr C \ lf)

[Wherein, Z represents an amino acid residue other than Ser or hydrogen].

Α Regarding VI, the codon indicated by X is a codon encoding a amino acid constituting a part of νιπ (where X is Ser) Any of L, which is not a translation termination codon and a translation termination codon, may be used, and may further have one or more codons encoding an amino acid at the 5 'end thereof. Is particularly preferably ATG.

The codon represented by Y may be any translation termination codon or any codon that encodes an amino acid that forms part of a polypeptide having substantially the same activity as human IL-2. And may further have one or more codons encoding an amino acid at the ³ 'end thereof When the codon represented by Y is a codon other than the translation termination codon (and further at the ³ ' end thereof) (Including one or more amino acid-encoding codons) preferably has a translation termination codon at the end.The codon indicated by Y may be TAA, TAG or T More preferred, and particularly preferred is TGA.

Regarding the polypeptide, the amino acid residue other than Ser represented by Z may be an amino acid residue that is part of a polypeptide having substantially the same activity as human IL-12. Any of these may be used, for example, the amino acid residues shown in Table 1 (excluding Ser), and further on the N-terminal side thereof, for example, the amino β group shown in Table 1 or these amino acid residues May be included. Particularly, Ζ is preferably Met or. A DNA having a structural gene encoding a peptide containing an antibody recognition site and a DNA having a structural gene encoding a peptide of human IL-2 are ligated in the same reading frame. The ligation may be performed according to a known method. For example, one T3 DNA ligase is used to ligate one 3OH end of NA to the 5 P end of the other DNA in the presence of ATP.

It is preferable that a linker is interposed between the two genes. As the linker, for example, EcoRI linker (pCTAGAATTCTAG) and the like can be used, and those available from New England Biolabs (USA) and other commercially available linkers can be used. For example, the linker can be ligated by terminating the gene with blunt ends using Escherichia coli polymerase I (large fragment or Si nuclease), and linking the terminus with the terminus of the linker using T4 DNA ligase. It is necessary to digest with a restriction enzyme to make it sticky to remove overlapping linkages between linkers, etc. The type of restriction enzyme varies depending on the type of linker, for example, EcoRJ Linker The restriction enzyme used is EcoRI, and the resulting sticky ends are used to ligate the two genes together using T4 DNA ligase to obtain one DNA via a linker.

It is preferable to link a promoter upstream of the DNA linking the two structural genes.

The order of the two structural genes to be linked downstream of the promoter is such that the structural gene encoding the peptide containing the antibody recognition site downstream of the promoter and then the human IL-12 structural gene are used. Alternatively, it is ligated downstream of the promoter so as to be a structural gene for human IL-2 and then a structural gene encoding a peptide containing an antibody recognition site. The above

《OMPI ′ The order of connection of the two members other than the mouth motor is not particularly limited, but it is essential that the promoter is connected at the most upstream position. The promoter includes, for example, tryptophan synthesis (trp promoter s' motor, recA promoter-lactomer promoter, etc., and trp promoter is particularly preferable.

As a method for linking the promoter, for example, a translation initiation codon ATG is added to the 5 terminus of the gene to be expressed using an appropriate linker, and this is linked to the 3 'terminus of the promoter. As the enzyme used at this time, T ligase is generally used, and the treatment method is the same as described above. It is desirable that an appropriate restriction enzyme recognition site is provided downstream of the promoter.

In order to express DNA in which the two genes are linked to each other in a host microorganism, a plasmid is used as a vector, and the plasmid is incorporated into the plasmid. As a plasmid as the vector, for example, pBR32 2 [Gene 2_, 95 (1977)]:! Derived from CollEI is the most commonly used force ^ other plasmids. Any of those that can be expressed efficiently can be used, for example, ptrp781 and ptrp771 incorporating a triptophan synthesis promoter. In order to incorporate the thus obtained into plasmid, first, the 1NA of plasmid is treated with an appropriate restriction enzyme to form a linear form. Ν Connect A with T ligase.

A host bacterium is transformed with the thus-obtained plasmid into which DNA is ligated, and a transformant is produced.

Examples of the host bacteria include Escherichia bacteria, Bacillus subtilis, yeast, and the like, and particularly preferred are Escherichia bacteria. Said Escherichia sp. For example, E. coli (Escherichia coli DH1 strain Nature 217,

11 10—1 1 14 (1968)].

Examples of a method for transforming a host cell with the plasmid include a calcium method and a protoplast method, with the calcium method being particularly preferred.

As an example of the transformant obtained by the above method, the transformant produced in Example 1 described below was used.

ichi a co 1 i DH lZ _p GJEL 1028. The microorganism was found in the Fermentation Research Institute (IFO, 532) at 18-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Japan. The deposit was deposited on March 27 as accession number IFO 14332, and the microorganism was submitted to the Research Institute of Microorganisms and Industrial Technology of the Ministry of International Trade and Industry of Japan (FRI, 2005) in Yatabe-cho, Tsukuba-gun, Ibaraki, Japan. It was deposited on 1-chome 1-3, as accession number f, ERM J-7568 according to March 29, 1894 AD.

As a medium used for culturing the transformant, a liquid medium is suitable, in which a carbon source, a nitrogen source, an inorganic substance, and the like necessary for growth of the transformant are included. It is included. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose, and examples of nitrogen sources include ammonium salts, nitrates, corn chip-liquor, leptone, casein, meat extract, and the like. Inorganic or organic substances such as soybean meal and barley extract, and examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. Also, yeast extract, vitamins, growth promoting factor and the like may be added. In particular, a medium containing about 0.1 <¾ of calcium chloride • dihydrate and about 2% of sodium dihydrogen phosphate 'dihydrate is advantageous for production of the desired product. Conditions such as temperature, pH, and time for culturing are such that the production of the target product and its activity are maximized. In general, the culture temperature is around 20 to 40 ° C, the culture pH is from acidic to slightly alkaline, and especially around neutral, and the culture time is from 6 to 10 ° C. About an hour.

In the fermentation of the present invention, the target polypeptide is usually accumulated in the cells, so to collect the polypeptide accumulated in the culture, the cells are first collected by centrifugation or filtration. This is done by extracting the polypeptide. In order to efficiently extract the peptide, for example, ultrasonic treatment, J ′ zyme treatment, treatment with a chemical such as a surfactant, or the like is performed. ―

Purification of the polypeptide extracted in this manner can be performed by a conventional protein or peptide purification method such as ammonium sulfate precipitation, alcohol precipitation, ion exchange column chromatography, cellulose column chromatography, or the like. This is performed by applying a gel filtration method or the like.

The DNA obtained in Example 1, which will be described later, is a DNA containing a polynucleotide having a nucleotide sequence shown in FIG. Among them, the polynucleotide shown as the nucleotide sequence 9-356 in FIG. 1 is the polypeptide represented by the amino acid sequence 3—118 in FIG. 2, that is, the human Ig_E H chain. Encoding the C2 region of

The polynucleotide shown in FIG. 1 as the nucleotide sequence 369—767 is a polypeptide represented as the amino acid sequence 123—255 in FIG. 2, that is, a human. Code IL-2. These polynucleotides may have an ATG so as to match the open reading frame to the 5 'end for direct expression. In this case, a polypeptide having Met at the N-terminus is encoded.

Also, these polynucleotides are used to match the reading frame,

OMPI It may have any intervening sequence. In FIG. I, this intervening sequence is shown as the nucleotide sequence 357-368. In this case,

2 encodes a polypeptide represented as the amino acid sequence 1 19 -122.

As described above, for human IgE and human IL-2, the mRNAs encoding them have been isolated and identified by themselves, and the expression of these genes has also been attempted alone. However, as in the present invention, two or more genes are linked to form a new polypeptide having two or more different immunological or biological activities. The concept of manufacturing is never before seen. Furthermore, one polypeptide having two or more independent immunological or biological activities is rare in the natural world, and the present invention provides a novel active protein. It is the pioneer of peptide science that followed.

The polypeptide obtained by binding the peptide containing the antibody recognition site and the human IL-2 peptide obtained by the method of the present invention is useful as a reagent for purifying the human IL-2 antibody. For example, IGL-EC22 comprising the above amino acid sequence obtained by the method of the present invention has both the antigenicity of human IgE and the antigenicity of human IL-2. Therefore, IGL-EC22 was purified using an anti-human IgE antibody, and an anti-human IL-2 antibody was prepared using the purified IGL-C22. It can be used as a medium for a series of human IL-12 purification processes in which human IL-12 is purified using an anti-human IL-2 antibody. In this case, the force of Azukasuru I GL- EC ^{2 2} force directly related is when purifying Kohi Bok IL- 2 antibody; a method of making and purifying such unknown antibodies exceptional without It is greatly expected as a new technology for antibody purification. In addition, IGL-EC22 has both immunological and biological St life of human IgE involved in the allergic reaction and human IL-12 having T cell proliferating action. It is expected to have an unknown third activity. As a specific example of the purification of the human IL-2 antibody, the following method is performed. First, an anti-human Ig β antibody and agarose gel beads (Bio-Had

Create an antibody column to which Affigel-10 is bound. IGLC22 contained in the E. coli DHl / pGEL1028 extract is specifically bound to this antibody column. Next, the ascites of a rat immunized with human IL-12 or the serum of a rabbit is reacted with the above-mentioned anti-human IgE antibody column adsorbed on IGL-_EC22. Elution this in appropriate buffer, IOL- EC ^{2 2} and a mixture of human Bok丄^L-2 antibody is obtained. The mixture was I) when eluted with a salt concentration adsorbed proteins other than antibodies of the A _{beta-cellulose} column (such as manufactured by Whatman Cellulose52) human IL one 2 antibody is purified. In the present specification and drawings, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations of the IUPAC-I UB Commission on Biochemical Nomenclature or commonly used abbreviations in the relevant field. It is listed in the table. In addition, when there is an optically distinct organism for an amino acid, the L-isomer shall be indicated unless otherwise specified.

D N A Deoxyribonucleic acid

c DNA complementary doxy !; nucleic acid

R N A Ribosuccinic acid

mRNA messenger ribonucleic acid

A Deoxyduronic acid 5 one

τ Thymidylate

G Deoxyguanylic acid c Deoxycytidylic acid

U peridylic acid

d ATP deoxy ^ adenosine triphosphate dTTP deoxythymidine triphosphate d GT P deoxyguanosine triphosphate dCTP deoxy ^ thidine triphosphate

A T P adenosine triphosphate

ED TA Ethylenediamine tetracarboxylic acid

S D S Sodium dodecyl sulfate

Gly

A 1 a

Va 1 Lin

Leu Leucine

I 1 e

Ser Serine

Th r threonine

Cy s スティン Stin

Me t Methionin

Glutamate.

As p, Snowpuff

Lys lysine

A r g Arginine

H is histidine P he feniralanin

T y r H

Tr r rrip fan

Pro Proline

A s n Asno Lagin

G 1 π Glutamine

b P base pairs

Simple explanation of the drawing

FIG. 1 shows a DNA having a structural gene encoding a peptide of human Ig ^ and a DN having a structural gene encoding a peptide of human IL-2.

Fig. 2 shows the nucleotide sequence of the DNA linked to A, Fig. 2 shows the amino acid sequence corresponding to the nucleotide sequence shown in Fig. 1, Fig. 3 shows the construction of Reference Example 1, and Fig. 4 Shows the grooves of Reference Example 2). Fig. 5 shows an embodiment

1 shows a construction diagram, "''/" / 〃,囊部represents a region encoding a peptide of human Bok IL one ^2.

—Best mode for carrying out the invention

Reference example 1.

A plasmid that expresses the polypeptide of the human IgE H chain C2 region pGETTrp818—C

The DNA 1 of plasmid pGET trp 302 constructed in ten thousand method described in Japanese JP-5 9 ^{4 4 3 9 9} No. cleaved with Clal, cut sticky ends at both ends by performing an S iota nuclease digestion Was. This DNA was purified by phenol extraction and ethanol precipitation, and then the ligated EcoRI linker dCTAGAATTCTAGs (0 ng) was ligated using T4 DNA ligase, and the EcoRI linker was ligated. To remove the mid The compound was cut with EcoRI. This DNA was religated with T4 DNA ligase to transform E. coli 294, and pGET trp 302-a was obtained from the transformed strain. the _{pGET tr P 3 0 2 1.2 kb} DNA fragment of an a that may arise from was digested with EcoRI and Pstl, was 1.2% Agarosugeru electrophoresis by Li fraction times Carabid. T The DNA fragment _{5 0 n g, EcoRI, Ps} t I digested base for expression Kuta one Ptrp 7 8 1 [Nurleic Acids Re search ll, .3077 one 3 0 85 [1 9 8 3)] in 2 0 Ong E. coli 294 was transformed by incorporation using 4 DNA ligase, and pGET trp302-b was obtained from a transformant that was sensitive to ampinline. After pGET trp 302-b was digested with the restriction enzyme Xbal, the sticky end of the resulting DNA was digested with S1 nuclease to be blunt-ended. This DNA was purified by phenol extraction and ethanol precipitation, and religated using T41) NA ligase to transform Daekyo ²⁹⁴ . PGETtrp 302-d was obtained from the transformed strain.

pGETt 卬 302-d was cleaved at the restriction enzyme ΡνιιΠ site, then cut at the restriction enzyme AvaI site, and the DNA promoted at the Aval site was fractionated. After shaping both ends of the NA chain with S1 nuclease to smooth it, EcoRI linker dCTAGAATTCTAG was bound thereto, digested with EcoRI, and religated to construct the expression plasmid pGETt 卬 818-c ( See Figure 3 :).

pGETt 卬 818-c encodes a 121-amino acid in the C2 region and a 13.000 molecular weight peptide having Leu, Glu, and Phe generated at the C-terminus by linker attachment. This plasmid has Met and Leu at the N-terminus, which are not derived from the IgE heavy chain and are encoded by the adapter.

OMPI

—'-― .. Reference example 2

Construction of plasmid pTF1, which expresses human IL-12 peptide: (1) (i) Isolation of mRNA encoding human IL-12

Lymphocytes prepared from human peripheral blood were treated with 12-0-tetradecanoylphorone 13-acetate (ΤΡΑ) C 15 ng / m £) and concanaparin A 40 μ ^ Αιί The culture was carried out at 37 ° C. in RPMI 1640 medium (containing 10 fetal bovine serum), and IL-12 was induced. ² After 4 hours, the 1 X 10 ¹⁰ cells of human lymphocytes obtained by the induced 5 M Guanijinchio Aneto, 5% Menore force script ethanol, 5 0 JnM Tr is · HC 1 pH7.6, 1 0 mM EDTA solution After centrifugation with a Teflon homogenizer, add sodium N-lauroylyl sarconate to 4, and homogenize the mixture on a 5.7 M cesium chloride solution (5.7 M cesium chloride, 0.1 M EDTA) 6. The mixture was centrifuged, and centrifuged at 24,000 rpm for 48 hours at 15 using a Beckman SW28 rotor to obtain an RNA precipitate. 0.25? After dissolving in β-lauroy / sodium lesalconate solution, the mixture was precipitated with ethanol to obtain 10 RNA. This RNA was adsorbed to an oligo (dTJ cellulose column) in a high salt solution [0.5ΜNaC1, 10 mM Tris · HC1 pH7.6, imM EDTA, 0.3% SDS J, and poly (A) was The mRNA containing the poly (A) was eluted with a low salt solution (10 mM Tris—HC1. PH 7.6, 1 mM EDTA, 0.3 ^ SDS) to obtain 300 ig of the mRNA containing poly (A).

This mRNA was further precipitated with ethanol, dissolved in a 0.2π solution (1 OmM Tris-HCI pH 7.6, 2 mM EDTA, 0.3 ^ SDS), treated with 65 for 2 minutes, and subjected to a 10 to 35% sucrose gradient. Centrifugation (centrifugation at 20 and 25000 rpm for 21 hours using a Beckman SW 28 rotor ^ Separation :) to obtain 22 fractions. A portion of the RNA from each of these fractions was injected into the oocytes of Lactobacillus frogs, the IL-12 activity in the synthesized protein was measured, and fractions 11 to 15 (sedimentation constant 8 S ~ 15S), the activity of IL-12 was detected. The IL-2 mRNA in this fraction was about 25.

(ii) Synthesis of single chain DNA:

Using the mRNA and reverse transcriptase obtained above, 100 reaction solutions (5 / mRNA, 50 ig oligo (dT), 100 units of reverse transcriptase, 1 111 丄 1 of 1 ?, d CTP, 01 1 ^>ぉ 41 ¹ ?, 8 mM MgC 12, 50 mM KC 1, lo mM dithiothreitol, 50 mM

After incubating for 1 hour at 42 ° C in Tris-HCl pH 8.3, deproteinize with phenol and 70 with 0.1 ml NaOH. c, 20 minutes treatment to decompose and remove RNA.

Double-stranded DNA synthesis:

The single-stranded complementary DNA synthesized here was reacted in a 50/50 reaction solution (the same reaction solution as described above except that mRNA and oligo dT were not contained) to react for 2 hours at 42 ° C. Heavy chain I) NA was synthesized.

(iv) Addition of dC till:

Nuclease S 1 50 ^ of the reaction solution to this duplex DN A (: duplex DN A 0.1 M sodium acetate pH 4.5, 0.2 5 M NaC 1 , 1.5mM ZnS0 4, 6 0 Yuni' Bok of S 1 quinuclidine Reactase] at room temperature for 30 minutes, deproteinize with phenol, precipitate the DNA with ethanol, and add 50% reaction solution (double-stranded DNA) , 0.1 4 M Kako Gilles acid strength Li, 0.3 M Tris (base) ρΗ 7.6, 2 mM Jichiosurei DOO one _{le, 1 mM Co C 1 2,} 0.1 5 mM d CTP, 3 0 Yuni' preparative terminal DOO 3 minutes in translanase). C to extend about 15 deoxytidine chains at both 3 'ends of the double-stranded DNA. These series of reactions yielded about 300 ng of double-stranded DNA with a deoxycytidine chain.

(V) Cleavage of E. coli plasmid and addition of dG till:

On the other hand, the restriction enzyme Pst was added to 10 ig of E. coli plasmid pBR322 DNA.

The reaction solution 50 I ## 10 g DNA, 50 mM N a C 1, 6 mM Tris. HC1 · pH7.4, 6 mM MgC 1 2, 6 mM 2- mercaptoethanol, 100 μ / M bovine serum albumin , 20 units of Pst IJ for 3 hours at 37 ° C to cleave one Ps11 recognition site in PBR322 DNA, deproteinize it with phenol, and then conduct terminal transfection. the reaction solution La one peptidase 50 [DNA 10 ig, 0.14 M force prying oxide Li, O. SMTris • base pH 7.6, 2 mM Jichiosurei Bok _{Ichiru, 1 mM CaCl 2, 0.15 mM} GTP, 3 0 Yuni' Tota Terminal 3 minutes in Transferase :)

The DNA was allowed to act with 37 to extend approximately 1 デ of the blocking guanine chain to the three ends of the plasmid PBR 3 222 DNA.

(vi) cDNA association and E. coli transformation:

The thus obtained synthetic double-stranded DNA o.lig and the above plasmid pBR322,0.5. In a solution consisting of 1M NaCl, 50 mM Tris-HC1 pH 7.6 and imM EDTA, the mixture was heated at 65 ° C for 2 minutes and 45 ° C for 2 hours, then cooled and allowed to associate with each other, according to the method of Enea et al. [J. 1. B iol., 96,

E. coli MM294 was transformed according to 495 C1975 :).

(vii) Isolation of plasmid containing cDNA:

In this way, about 20,000 tetracycline-resistant f strains were isolated, and each of these DNAs was immobilized on a ditrocellulose filter. Next report from Taniguchi et al. : Nature, 302, 305 (1983)] IL- 2 of Amino acid sequences based on Amino acid ^{^ 7 4~7 8 (Lys 74 -} Hi s-Leu-Gln-Cys) and Amino Acid £ 122-126 [Thr ¹²² - Phe - Met- Cys- Glu) ( ⁵ AAA CAT CTT CAG TGT ³ and ⁵ ACA TTC ATG TGT GAA ³ by the ester method [Crsa, R. et al. Proc. Natl. Acad. Sci. USA. 75, 5765 (1978)].

The O Rigonuku Rechio 50 reaction solution by using of T 4 poly quinuclidine Reochi Dokaine Ichisu against de (O Rigonuku Reochi de 0.2 O ug, 5 0 mM Tr is • HC1 Η 8.0, 1 0 mM MgC 1 2, 10 mM mercaptoethanol, 50 μ Cir- ³² ΡΑΤΡ, 3 Yuni' preparative Τ reacted with 4 Porinuku Reochi de force rice in Ichisu 1 hour 3 7 ° C, was labeled at the 5 'end with ³² P. the labeled O The lignonucleotide was used as a probe to associate with the DNA fixed on the nitrocell filter according to the method of Lawn et al. [Nucleic Acids Res., 9, 6103 (1981)], and the autoradiography was performed. Thus, four strains that responded to the above two types of oligonucleotide probes were isolated, and plasmid DNA was isolated from the cells of each of these strains by the alkaline method [Birncoim HC & Doly, J. Nucleic Acids Reagents]. , 7, 1513 (1979)]. The insert of rasmid DNA was excised with the restriction enzyme PstI, and among the separated plasmids, the one containing the fragment with the longest insert was selected, and this plasmid was designated PILOT 135-8. I named it.

(2) Construction of PlusMid PTF 1:

The plasmid PILOT 135-8 obtained in the above (1) was digested with the restriction enzyme HgiAI to obtain a 1294 bp DNA fragment. This DNA fragment was treated with T4 DNA polymerase to make blunt ends, and then EcoRI linker dTGCCAT GAATTCATGGCA was ligated using T4 DMA ligase, the ligated product was digested with EcoRI to remove the duplicated plasmid of the 51coRI linker, and this fragment was digested with the restriction enzyme PstI to obtain human IL— A DNA fragment with the translation initiation codon "ATG" added was prepared in accordance with the reading of the two genes.

This DNA fragment was ligated to the expression plasmid ptrp 781 "Nuc 1 eic Acids Research 11, .3077-3085 C 1983 JJ digested with the restriction enzymes EcoRI and Pst I using T4 DNA ligase. This reaction resulted in the trp promoter. The human IL-2 expression plasmid pTFl was constructed by matching the reading frame with the translation initiation codon downstream of-(See Fig. ^4. This plasmid was used to transform Escherichia coli DH1). As a result, a strain containing the desired plasmid PTFI was obtained.

Example 1.

Construction of IGL-EC22 polypeptide expression plasmid pGEL1028 and production of transformants:

Plasmid pGET trp818-C was digested with restriction enzymes C1al and EcoRI to obtain a DNA fragment of about ³ Kbp. In addition, the plasmid pTFi was digested with restriction enzymes EcoRI and Pst1 to obtain a DNA fragment of about 500 bp. These ^two DNA fragments were joined by T 4 DNA ligase. By this reaction, a translation initiation codon was provided downstream of the trp promoter, and an IGL-EC22 expression plasmid pGEL1028 was constructed in the same reading frame (see FIG. ⁵ ). Escherichia coli DE1 was transformed with this plasmid to obtain a transformant Ε · DH1 / GEL1028 (1FO14332, FERM P-7568) containing the plasmid PGEL1028.

Example 2. ゥ ΜΡΪ (i) Preparation of cell extract containing IGL-EC22:

E. coli containing IGL-EC 22 expression plasmid pGEL1028 E.co1iDH1 / pGJjlL 1028 C IFO 14332, FERM P— 7568) containing 20 m 1% glucose, 0.4% casamino acid M 3 in 9 media. After culturing for 4 hours at C, indoleacrylic acid was added to 30 µ / U, and culturing was further performed at 37 ° C for 3 hours. After collecting the cells and washing with saline, a 0.5 ^ lysate (10 mM Tris. HC1, pH 8.0, 10 mM EDTA 0.2 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 0.02 % Triton X lOO, 0.1 / ni lysozyme). The cells were allowed to stand for 45 minutes at 37 ° C and 2 minutes at 37 ° C for lysis. This was further gently sonicated (30 seconds) to cut the DNA of the eluted cells, and then cut at 15,000 rpm at 4 ° C (Sabal SS 34 rotor, manufactured by DuPont), 30 A supernatant was obtained by centrifugation for minutes. This supernatant was used as a cell extract.

(2) Measurement of IgE activity of bacterial cell extract:

RIST method using IgE assay kit [IgE test 'Shionogi, Shionogi & Co., Japan: Japan!] !: Radio immunosorbent test, Immunology. 14, 265 (1968)] did.

I gE activity I GL- EC 22 was found to be 42 x 10 ³ units / ^ culture.

(3) IL-1 activity of bacterial cell extract:

Human IL-12 activity was measured using the mouse TCGF-dependent cell line NKC3.

[Records of the General Meeting of the Immunology Society of Japan, Vol. 11, p. 277 (1989):] were used. That is, first, samples 50 of various concentrations diluted by two-step dilution were taken and placed in a flat-bottom microplate (manufactured by Falcon). Next, add 50 ^ ^ RPMI-1640 solution containing 3 x 10 ⁴ NKC3 cells and 10 fetal calf serum C10 ^ FCS, and in a CO2 incubator at 37 ° C,

"Well,--- The culture was performed for 20 hours. Further, after adding ³ H-thymidine 1 iCi and culturing for 4 hours, the cells were trapped in a glass filter using a cell filter (Wake Chemical Co., Japan), and the cells were washed, dried, and dried. After that, the radioactivity was measured by a scintillation counter 1. The IL-12 activity of IGL-EC22 in the sample was 16 × 10 ³ units of the culture solution.

The activity was measured based on the calculation method described in Japanese Patent Application Laid-Open No. 58-116498.

(4) Purification of 1 (31 ^ —〇22:

By the method described in Japanese JP 58 - ⁹ 6028 JP and the Anahi preparative IE monoclonal antibodies conjugated to 1 0 (Bio-Rad Inc. water-insoluble carrier Abuigeru. Kohi preparative IgE monoclonal antibody has one Abuigeru 1 0 Apply 5 π of the cell extract containing E. coli DHl pGELi 028 obtained in the previous section (3) to column ΐπ, and add PBS containing 20% dextrose (20 mM phosphate buffer, pH 6.8). After washing the column with 0.15 M NaCl 50, the IGL-EC22 adsorbed on the column was eluted from the column with 0.2 M acetic acid and 0.15 M NaCl solution 5, and the eluate was immediately neutralized. After that, it was dialyzed against PBS 1 for 24 hours at 5 ° C. By this operation, a polypeptide of IGL-EC22 with a purity of 80 or more was obtained with a recovery of about 50.

Industrial applicability ''

By culturing the transformant transformed with the plasmid into which the novel DNA ^ of the present invention has been incorporated, a polypeptide comprising a peptide containing an antibody recognition site and a human interleukin-2 peptide is bound. The polypeptide is useful as a reagent when purifying human interleukin 2 antibody.

Claims

The scope of the claims

1. Alignment of the open reading frames for DNA having a structural gene encoding a peptide containing an antibody recognition site and DNA having a structural gene encoding a peptide for human interleukin 2 Ligated; DNA.

2. The DNA according to claim 1, wherein a promoter is linked upstream of both structural genes.

3. The DNA according to claim 1, wherein a lysocar is interposed between the two structural genes.

4. The DNA according to claim 1, wherein the peptide containing an antibody recognition site is a peptide containing an anti-human immunoglobulin E antibody recognition site.

5. Alignment of reading frames for DNA containing a structural gene encoding a peptide containing an antibody recognition site and DNA containing a structural gene encoding a peptide for human interleukin 2 A plasmid containing the DNA from which it was contacted.

6. The plasmid according to claim 5, wherein the DNA is a DNA obtained by ligating a promoter upstream of the structural gene O.

7. The plasmid according to claim 5, wherein the DNA is a DNA formed by interposing a linker between structural genes.

8. The plasmid according to claim 5, wherein the peptide containing antibody recognition »is a peptide containing an anti-human immunoglobulin E antibody recognition site.

9. DNA having a structural gene encoding a peptide containing an antibody recognition site and DNA having a structural gene encoding a peptide of human interleukin 2 are ligated in the same reading frame. A transformant transformed with a plasmid having integrated therein 1 NA.

10. The claim according to claim 9, wherein the host of the transformant is a genus Escherichia. Transformants.

11. The transformant according to claim 9, wherein A is DNA obtained by linking a promoter upstream of a structural gene.

12. The transformant according to claim 9, wherein the DNA is a DNA comprising a linker between structural genes.

13. The transformant according to claim 9, wherein the peptide containing the antibody recognition site is an anti-human immunoglobulin: a peptide containing the E antibody recognition site.

1 4. A polypeptide that combines a peptide containing an antibody recognition site with a human interleukin-2 peptide.

15. The polypeptide according to claim 14, wherein the peptide containing an antibody recognition site is a peptide containing an anti-human immunoglobulin E antibody recognition site.

1 6. A DNA comprising a structural gene encoding a peptide containing an antibody recognition site and a DNA comprising a structural gene encoding a peptide of human leukin 2 are ligated together in reading frame. The transformant transformed with the integrated plasmid is cultured in a medium, and a polypeptide in which the peptide containing the antibody recognition site and the polypeptide of human interleukin 2 are bound in the culture medium. A method for producing the polypeptide, comprising producing, accumulating, and collecting the same.

17. The method for producing a polypeptide according to claim 16, wherein the host of the transformant is a genus E. coli.

18. The method for producing a polypeptide according to claim 16, wherein the DNA is a DNA obtained by linking a promoter upstream of a structural gene.

19. DNA is between structural genes! Through the car! )] The method for producing a polypeptide according to claim 16, wherein:

20. Peptide containing antibody recognition site is anti-human immunoglobulin E antibody recognition part

/ c "'

, ' 85/04673

27. A peptide containing the production position of the polypeptide according to claim 16, which is a 27-peptide.

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