JPS60227694A - Method for producing a polypeptide containing the amino acid sequence of human mature leukocyte interferon - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the field of recombinant DNA technology, i.e. recombinant DNA technology used for the production of polypeptides.
Related to law.

In particular, the present invention relates to mature human leukocyte interface.
The method of the present invention relates to a method for producing a polypeptide containing a partial amino acid sequence Cys-^1i
-Trp -Glu -Val -Vat -Arg
-8la -Glu -11e -Net -8rg
-Sep and without preceding sequence, 165-166 amino acids, or mebuonine attached to the N-terminus of the first amino acid of the interferon. If so, 16
An engraving of the specification characterized in that it has 6 to 167 amino acids (no change in content).Culture of a microorganism transformed with a microorganism expression vehicle that expresses and replicates this polypeptide to produce the polypeptide. Developing cells that express the lj1 polypeptide
! This method is characterized by collecting the polypeptide. .

First, the background of the present invention will be described. The leukocyte interface [1,elF] was initially introduced into the eye IJ'' tux (l5aacs) and the Lindenmann (l i
(Proc.

R, Soc, > 81 /l 7.25B-267 (1
957): U.S. Patent Portion +, s, P,) 3,699.2
22). Efforts to purify and characterize each individual have continued for a long time and have culminated in the preparation of a relatively homogeneous leukocyte protein derived from leukocytes obtained from normal or leukemic sources. German Patent Publication No. 2.9/17.134
).

These interferons are a group of proteins known to have the ability to confer a 1:1 viral resistance state on their target cells. In general, inkferon inhibits cell proliferation and immunosuppression. It is encouraged.

The white sphere interferon is purified to an essentially homogeneous state and is expressed as
aq.

Brok, S1~le, Akad, →”-I, Yoneda (proc
.

Na mouth 88 cad, sci, 11. s 8,) 7 6
, 640-646 (1979) near oon
etc., Improvement, 76.5601-5605 (1979)
) with a molecular weight ranging from about 17.500 to about 21.000. I was reported. The specific activities of these preparations are
L, extremely high, 2×10 to 1×109 units/
mU rlj white matter, but the yield from cell culture methods is extremely low1. However, advances in protein sequencing technology have led to the determination of partial amino acid sequences [zone (Z)
oon) QQ, Science 207.
527 (1980); Levy (1-evy) Q'r
, Blog, SuI~le, Acad.

4 Noi, US 1) (Proc, Hat l, 8c
ad, sci, Il, S, ^,)77゜5102-51
04 (1980)). Glysylation of various leukocyte interferons is not completely clear at present, but differences in glycosylation between I-J and N1 transcriptions (no changes in content) have been observed between the scales. cannot explain the distribution of molecular weights. (Instead, leukocyte ink ferons vary widely in amino acid composition and sequence depending on the type, with amino acid homology sometimes lower than 80%.

Donor's white blood cells J: Rinomin 1l111 and partial '/, i
Characteristics (1) and limited (1) for clinical research
-min (i la (7)), which gave a homogeneous white-faced interferon (), but it alone has not been tested in large-scale clinical trials and in a wide range of studies [11 Indeed, current clinical trials using human leukocyte-derived intafrons for antitumor and antiviral purposes use crude (less than 1%) preparations, and It has not been possible to produce enough kinat even at unreasonable prices, and research in a wide range of areas has been delayed.

However, with the advent of recombinant DNA technology, it has become possible to control the production of a wide variety of useful polypeptides by microorganisms. Already, there are bacteria that have been modified using this technology to produce polypeptide chains such as somatostatin, the A, J, and B chains of 1-insulin, and human growth hormone. 7--6-1 = Cleaning of the specification (no change in content).

Itakura etc., Suiens (Sc)
) 198, 1056-1063 (19
77); Goedel (! (Idol) et al., Nature) 281.

5544-548 (1979)). More recently,
I) The NΔ technology enables the production of proinsulin and sigma bumosine alpha 1, and in addition, the human leucoglobin t1 allows for the collection of DNA encoding the human leukocyte induron. and the resulting protein with leukocyte interferon activity? Nagata et al. reported on the production of f.
re) 284.316-320 (1980); Nante i et al., Aene Yooi, 1-10 (1980): Taniguji
cbi) et al., Nature 285,
547-5 A9 (1980)).

The laboratory of recombinant l') NA technology is the plasmid, which is a non-chromosomal loop of double-stranded DNA that is present in bacteria and other microorganisms and often exists in multiple copies in the cell. Plasmid-8 = Detailed engraving (no change in content) The information encoded in the DNA includes the information necessary to reproduce one lasmid in the daughter cell.
and, usually, there is information on one or more selection characteristics, for example, in the case of bacteria, resistance to antibiotics. This information allows the selection of targeted plasmids. This makes it possible to recognize and preferentially grow host cell clones containing plasmid r) NA in the soil for selection.
This means that the plasmid is specifically cleaved by one type or another of restriction endonucleases U or ``limiting oxygen'' that each recognize the different sites on the plasmid. By connecting both ends of the site or the reconstituted end to the cut site,
A foreign gene or gene fragment is inserted into a plasmid. I) NA recombination is carried out outside the cell, but the resulting "recombinant" plasmid is introduced into the cell by a method called transformation, and by growing the transformed cell, Recombinant plasmids containing foreign genes can be produced in humans. Furthermore, if the foreign gene is inserted correctly with respect to the portion that governs the transcription and translation of the encoded DNA information within the plasmid,
The resulting expression vehicle is actually used for the actual production of the polypeptide sequence encoding the inserted gene, a process referred to as expression.

Expression is initiated in a region known as the protease, which is recognized and bound by RNΔ polymerase. In some cases, for example, it may be useful in the practice of the present invention.
``Promoter J:'' In such cases, the promoter region overlaps with the ``operator'' region, forming a promoter-operator bond. The operator is an I)NΔ sequence that is recognized by so-called repressor proteins, which serve to regulate the likelihood of transcription initiation in specific B1] motors. RNΔVolimera-1?
moves along the DNA and transcribes the J:reφ information contained in the ]-do sequence from its 5' end to its 3' end into the messenger RNA, which then transfers the encoded amino acid to ON. The sequence is translated into the following polypeptide. Each amino acid is encoded by a nucleotide triplet or "codon." These are present in what is referred to as the "structural gene" for the purposes of the present invention, that is, the part that codes for the amino acid sequence of the product to be expressed.After binding to the promoter, the RNA polymerer U first , transcribes the nucleotide encoding the ribosome binding site and then generates a translation initiation or ``start signal'' (usually A l' G,
The resulting medullary 17 RNA transcribes the A LJ G nucleotide, followed by the WJ7am gene itself. A so-called stop codon is transcribed at the end of the structural gene. Thereafter, the polymerase may form an additional sequence of mesenger RNA, and the -C remains untranslated by the ribosome because of the presence of the stop signal.

Ribosomes normally bind to specific junction sites on the medullary r-RN, A in bacteria where lllRNA is being formed. This results in an encoded polypeptide that begins with the translation initiation signal and ends with the stop signal 11- described above.

The sequence encoding the ribosome junction (fl) is A LJ
properly positioned with respect to the G start codon, and all of the remaining ]tons are in the interface (in pl+as
If the initiation codon is followed in e), the desired product will be produced. The unobtained product is then lysed from the host cell.
1. Other bacterial protein products can be obtained by appropriate purification methods.

We have shown that the use of transgenic DNA technology (i.e., inserting interferon genes into a microbial expression vehicle and expressing them under the control of microbial gene regulatory elements) has been shown to provide large amounts of white-globulin intefarons. b I thought it was an effective method. Although it lacks the characteristic glycosylation properties of humans, it may be used in the treatment of a wide range of viral and neoplastic diseases.

The first method of collecting Shiramen method genes of the present invention consists of the following steps.

(1) Using the partial amino acid sequence of leukocyte interferon purified to a homogeneous state, human 12
- Construct a set of synthetic NA probes containing codons as a collection representing all possible nucleotide combinations U encoding the amino acid sequence.

[Complementation from Metsuhinge V-RNΔ with 21Ml 1)N
Prepare "Ronnie Bank", a bacterium that produces A (cDNA). Another radiolabeled inducible m1lN8 is hybridized with plasmid cDNAΔ from this bank. The hybridized mRNA is eluted and tested for translation to interferon in oocyte analysis. In this way,
Plasmid DNA from colonies shown to induce protein activity is tested for hybridization to the protein protein produced as described in (1) above.

(3) In parallel with the culm of (2) above, another transformation bank is prepared using the CD N A derived from the mRNA induced in the plasmid. (1)
The probe was used to induce the synthesis of radiolabeled single-stranded cDNA Δ for use as a hybridization probe. Synthetic probe [1-B] is an induced mR using a template.
NA hybrids are formed and expanded by reverse transcription,
Generate the induced label cDNA for llI. We further investigated the clones from the second bank that were hybridized against the 19-year-old radiolabeled CD N A (full-length interface [1]).
- Confirm the presence of the gene. (1) And (2) makes it 4!
The gene fragments whose partial lengths are determined can also themselves be used to generate full-length genes.

(4) The full-length gene obtained above is obtained by removing any leader sequences that may interfere with the expression of the mature polypeptide in the microorganism, and
Using a synthetic NA, the 4tf gene was synthesized to allow proper positioning in the expression vehicle with respect to the starting signal and ribosome binding site of the microbial promoter.
T was raised. The expressed interferon was purified and
Confirm its properties and measure its activity.

(5) Intafni [Ron gene] prepared by the above method
14 - The fragment itself can be used to probe other partially homologous leukocyte interferon species by hybridization.

By applying the recombinant DNA techniques outlined above, the homologous leukocyte interferon (gly)sylation of 111Y as a mature polypeptide without the corresponding preceding sequence or a portion thereof can be expressed in a ligneous manner. It is now possible to produce microbially in high yields and with high purity. These interfaces can be directly expressed, harvested, and purified to levels suitable for use in animal and human viral infections and malignant tumor diseases. A group of intanolons thus expressed were shown to be effective in in vitro tests,
As the first leukocyte interface produced microbially, it was also shown to be effective in in vitro tests.

The term "mature white interfacial interferon" as used in the literature refers to a microbially (e.g., bacterially) produced interferon that lacks glycosyl groups. It's true. The mature white polypeptide of the present invention is expressed immediately from the translation initiation signal (ATG) immediately preceding the first amino acid of the natural product. Therefore, this ``mature'' polypeptide is Mebuonine (encoded by 8TG) as the first amino acid in
It is made of goldstone, but its wood properties are not affected. How to use, the microorganism host processes the translation product to produce the first medium A.
Can remove nin. The mature leukocyte interface [-1] can be expressed together with conjugation proteins other than the normal leader, which can be specifically removed in the extracellular or intracellular environment (UK Patent Publication No. 20076768). Finally, the mature inkferon transports the conjugate to the cell wall of the microorganism.
As a conjugate with a 'signal' peptide, -(11-) can be produced. Signol is processed and released from the upper wall, and the mature polypeptide is secreted.

``Expression'' of a mature leukocyte intaflon includes a glycosyl group or a preceding sequence that directly results in IIIRNΔ translation of the leukocyte intfJlon genome to a human.
- refers to the production of interferon molecules in bacteria or other microorganisms that do not have

The specific white globule interferon proteins are defined herein by the determined DNA genes (Tables 2 and 4) and the genetically defined amino acid sequences (Tables 3 and 5). These particular intaf Jlon proteins, and indeed all groups of leukocyte intaf Jlon proteins included here, have natural allelic 1lfl gene mutations (alleli
c variation) exists and occurs among individuals. These mutations appear as amino acid differences throughout the sequence, or deletions, substitutions, insertions, inversions, or additions of amino acids within the sequence. Each interfron of interest herein, labeled 1, elFA to 1
．． For eIF J, such allelic variation is included within the scope of the labeling or definition thereof, and therefore:
Assumed to be within the scope of the present invention.

Next, the tables and figures will be explained.

17- Table 1 Protein Ala-Glu-I 1e-Met-Ar
g:) -C------------T-(D-10) Trif 0 Shinpef 0 Died (D-13)---tli s-G 1
u-Me L-I 1e-G 1n---G---C
--------(D-130) Japanese Patent Application Sho GO-22769
4 (7) Special opening RBO-227694 (8) <+flL)QljIJ, Ijir<-L)Id J
x-(5-engine chain-1 to 27-S-(7C7C7σ-engine σασ Cf ■-,,2□,.

CL CL (L Cw Q-eL l:L Q-CL
1 σC7001Cr C7(7C)CIC)OOC
ICIC) C) <><<<<<d==eeee: I:
==':X LLJ LIJ-LLJ LJJ LL
J uJ uJαααααα α 匡１ ”-u-１Σ
-10C) 00 C) C10C)l O0CI C
I C10CI ClzozozoΣy× N z z z
z 'z z z z Gate-C-C---1''-C111ΣucJL)Ll)-CJ LJ Ul 0σ
σσ000 (7-OtsueLαOtoyu Oto -Shi 1111zoαααααα2 and φUsu ψ2 and αct ■α■ α 2---I~-1ΣΣΣΣΣΣ
Σ Σ NLL, I Shin x × Zo × He 0σ0・C7σ
α σ C Aro OLL+00-〇-Kuku■ku→＜＜」
-C111- u u-C1 Σ -C111111111
−−1ω−t←L/'l C1+φ←−Σ−−Σ−1
Σ LJ uJ C7LLI-α-1 [F] + uJ
uJ Car u-mu J --LLI C10zozony
NXny g φrewa0= αcI! :Ctαα Cpi ICpi CpiαCpiαCpicy 111111-I L/ICr: α匡αα匡α 1, I ψ-LLI! C2: Z:2: ”-zz+ −
+ + +-ZokutoCl L/'l L/'l +HaL/'+(IiIα -J LL -C-Lw -L-C---- LLI IiVl-Iiiφ A -ctααα=ααα C1 ΣΣΣΣΣΣΣΣ Σ u= −−= −−−−= −1 LAJ w LLI LIJ LLI LLJ LIJ
LIJ w l<((<(<((−αααIIαα= C1, J>>:>>>>>>>>>1>>
N>(C>>>>>→Kukuhi<<+-nee uni] Yuzugo>-C11 -σσσC7Cl Cr Cr CrVl φ φ v> :l: :C'X:, = 工=
Engineering=! Zozo×Zon! -C1---CJ)
＞ LJ zu :＞ ＞ ＞ ＞ −＞ )n1
/l Good L/'l Q, Iψψ-ψψi C1111111111-- ＞)＞＞＞＞＞→(((＜((-〉-n) one>
LLJ0σeL C7(7Cyσ〉〉ΣΣ Σ Σ σ
000σσσσ −Σ−１ −Σ 匡１し−−１１１ −−−− −−−I CyOσay x (7cr c
rku＞I want to go! z2zzzzz wwu-w t, w w +, + RoroΦmouth■mouthCΦ)
-)-ψni I/I α + C100-CI 00-
C1 --LLIL+1LI+Kai--1((<((
((Ll, IuJuJLaJ)LaJL+JLJψ−Σ
ΣΣΣ Σ Σ ΣILLIL1. ILI-I-σ--
- ωCJ
Ku ro, V day λel + o + yu' ko 0゜
Lol! :'l ``1 N'w: cf: Nnny x 7 1- LIJ LLJ 1.a, I-n-n-n-n-to←+1-←Σ-1-Shi 111''''I J M-)>Bow-2 )-n2;)+1 z-C1111-11tl-l-l-1-humanhuman--10ααααααααα1 01 00F 0 C((700(7 ＞Shito＞ ＞ ＞ ＞ ＞ 1 LIJI MM Sozon ! = OL: Ce Ct !1 and Cpi yCpi Cpi
~->>>>>>>> ) > (( − 0000 mouths O Roro Lo He αl ΣΣΣΣαΣΣΣ Σ LL, 1LIJ−−−−−− cyl ++ 0−CLQ, (L(L(LILCL Q
-O←-1,L, IΦ-111-〉〉〉〉〉〉〉〉〉〉〉 LLI Rororo αCΦC口LLI 1 ＞ ＞ ＞ LIJ ＞ ＞ ) ＞et
co u CI LJJ W (D: SI 510 A MALTFALLVALLVLSCHMALPFS
LMMALVVLSC I M A L S F S L M A V L
V L' S YJ MAR,5FSLLl=IVV
LV'LSYCMALSFSLLMAVLVLSY 030 CI S V L HE M ENG Q' Q T F,
N L F 5100 11C 20110 050 7,0, 8C190 TEDSSAAWI:QSLLEK, FSTEIYQ2
0 130 ” 140 A YLKEKKYSPCAWEVVRHY”LMEK
KYSPCA E, VVREI EI 24 150' 160 4RSF'5FSTNLKKGtRRKDjIR5LS
, FSII'LQKRLRRKD Table 1 shows the number of human leukocytes J: 11! +
The two amino acid sequences common to all types of interfaces have been purified to homogeneity. All possible n+RNΔ sequences that contain these bevels
15 and the corresponding DNA Δ sequences are shown below: A, T, G, C
The letters ``and 1'' indicate nucleotides containing the bases adenine, bumin, gon, cy1-sine, and uracil, respectively. The letter N indicates nucleotides A, G.

C and (J) 71' The polynucleobutes are rounded and written as reading in the direction of 5' (left) and 3' (right 1110), and the two small strands (“
d, s, ") When accepting a DNA, the order is reversed for the lower J: or non]-do strand. Figure 1 shows:
Figure 2 is an O1 radiogram showing hybridization of a potential 10I plasmid with a 32p-labeled synthetic deAxyA oligonucleotide.

Table 2 shows 11 A I+ to ``1-do'', respectively.
The symbols up to (=J, leukocyte intf 1 [1
The nucleobuty sequences (]-domains) of eight genetic variants isolated as candidates for the expression of 25-specification (no changes in content) are shown. For each 101" translation initiation codon and termination codon of ΔTG
1-1 to Libretz 1- are underlined. The 3' untranslated region following the stop or stop is shown. As shown in the △ line in the third column of Table 2, all genes of LeIF^ included in the gene 1 are missing one ]ton, which is present in Haku's ``ton.'' A 5' untranslated region precedes the leader sequence. Fragment with minute H [
There is no complete 41 leading sequence of the leader. However, it contains the entire gene for the postulated mature tenE.

Fragment G, which remains isolated, has a complete ]-code sequence.

Table 3'c shows the nucleotide sequence j:
eII protein protein comparison. IIIPAC-1-
Turtle IB=r Mitsushi'n A Biochemical Nomencarb\p -(Con+n+1ssion on
Biochemical
One letter omitted according to the suggestion of In other words, alanine is 8, cysteine is 0, and aspartic acid is D.
So, glutamic acid is F, phenylalanine is F, glycine 4J G'
c.

Histidine is llr.

26-1- Iso1-1 Isine is ■, Lysine is K, Leucine is 1
-C1 methionine is M, asparagine is N,
Phosphorus is [), glutamine is Q, arginine is R,
Hilin is represented by S1, threonine is represented by T, valine is represented by V, tryptophan is represented by W, and dirosine is represented by Y. Numbers indicate amino acid positions. S indicates a signal peptide. 165
The amino acid sequence of 1,cl
It is inserted to align with the 66 amino acid sequence. The sequence of LelF E is defined ignoring the extra nucleotide in its ]-do region (position ie'1187 in Table 2). Star rF'l C1, showing the terminal 11 Nicodon in the interface. To all of you (
The seven amino acids (excluding pseudogene 1, eH) are also shown. The underlined residues are amino acids that are also present in the human lineage bud inkferon.

Figure 2 shows 1. alF ``]-n cl) N A no 8
Restriction endonuclease maps of several types (from 8 to 1-1) are shown. The hybrid plasmid was produced using the dC:dG theory 27-copy of the specification (no changes to the contents), the link method (J Chill (Gocddel, mouth v) tongue, nave 1/- (Nature) 287, 411-416 (
1980)).

The cDNA insert is then cut using pstT. The line at the end of each cDNΔ insert indicates the flanking homopolymerized dC:dG tails. Pvt+ l[.

Restriction sites for EcoR and Bjll are also indicated. The edge region shows the needle sequence of the mature 1e layer. The shaded area is the signal peptide sequence. The white parts are 3' and 5' non-cord locations 511.

Figure 3 shows maturity 1. el "Direct microbial synthesis of" -
The construction of the gene to be coded is shown with the 1° restriction site and the remainder indicated (such as "'Psti"). "b,p," indicates a base pair.

Figures 4 and 5 show restriction maps of the two apical fragments used to express the 1clrB mature white-globulin interferon. This is the end of the chain produced by digestion with the enzyme Sau3a.

Tables 1 and 5 show 28 types, including types I and j.
- -28-1 - In Table 5, the W mark indicates a stop codon in the corresponding ONΔ sequence, and the hyphen indicates a deletion or G-7 in the sequence. , which shows advantageous embodiments for implementing the invention.

8. Microorganisms Used In the described implementation, two microorganisms are used, F. coli X 1776 and E.

-]su (E, coli) K-1229/1 strain (]-nd A, thi-, hsr-, hsn+", British Y
[described in Publication No. 2055382] is used. The American Type Cu1tu Collection
re Co11ection) and ATCCN
0.31537 and 31/146 are attached. I) All of NA's experiments were carried out by the National Institutes of Health.
5 posture of 1leath) (D applicable-
Reel 29 - Reprint of specification (no change in content) This was carried out in accordance with the Idline.

A most advantageous embodiment of the invention is the strain E. coli x 1776 as defined above.

]S(F, coli) K-12 strain-29-1- Not only 294 but also other known E.]S(E, C01)
i) Strain (hereinafter referred to as F, COI+), for example [, col
iB, co++, and other microbial strains, many of which are part of the 1F American type CaldiA7 collection (the At
nerican l'ypc Culture Co1
1ection) (ATCC), and are available at known microbial depositories. I would also like to see German patent 2644432. These other microorganisms include Bacillus, such as Bacillus 5ubtiliS, and other enteric bacteria, such as Salmonella typhimurium.
(Salmonella typhin+urium)
and Serratia marcescens (Serratia m.
Arcescens), which uses a replicable plasmid to express the foreign gene sequence. Yeast, for example, Saccharomyt'! Sacchae
romyces ccrevisiae) t3 also,
Expressing the gene encoding interferon under the control of a yeast promoter. He is 11 in IJ construction and can be right-handed.

B, raw materials and purification of lefUmRNA A 1, eIF
mRNA can be collected from human leukocytes.

Usually, the Sendai virus or the Newcastle disease virus is induced to produce interferon as described in German Patent No. 294.7134. (no change) White blood cells from patients with chronic osteogenic leukemia are used. A particularly advantageous raw material used herein is a reruline designated KG-1 derived from a patient with acute osteogenic leukemia. This keel line is KocHIer (11.l'), Onibi Gorde (G
Olde, D. L.) Science
e) 200.1153 (1978).
ark Memorial Institute ))
1640 plus 10% heat inactivated FC8 (live fetal surface clearing)
, 25 mHIl [PES buffer (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid)
It grows easily on medium containing gentamicin at 50 II 9 /mQ and can be subcultured (1 to 3 splits) twice a week.

GJ cells were frozen in the above growth medium with 10% 0% dimethylsulfo-4-hydride. sell. KG-1 is located on the 1st floor American Type Caldier] Collection (the
American Type Culture Co1
1ection) (ATCCN [1CRI-8031)
It has been deposited in

31- Copy of specification (no changes in content) 6-1 cells are produced by Rubinstei.
n) Methods described in block, natle, akado, 1 no "i, etc.
Rice 1) (Proc, Natl, Hecad, Sci, 1
1. s, 8,) ■.

640-6/44 (1979)), Sendai (
5enda i) or 2u, -castle (Newc
astle )-311- Reprint of specification (no changes to the contents) Leukocyte interaction-1-[]n+nR using disease virus
induced to produce NA. Cells were harvested 5 hours after induction, and RNA was extracted using the guanidine diocyanate method (Ct + irgw).
in), etc., Biochemist
ry) j 8.529/I-5299 (1979)
). RNA from induced/induced cells was prepared in the same way.1. a
3 J: using a sucrose gradient t-m centrifugation,
1q of 12S fraction to poly(△)mRN was collected. The lj method is
Green IJ -:/ (Green) etc.
Kumi, Pai Office (^rch, Biochem, Bi
ophVS, ) 17λ.

7'1-89 (1976)) Onibi Ari] 17 (0k
uyu+t+a) etc.
) 18 B-, 98-104 (1978)). This +11RNΔ is African mega-
■ - Cellular analysis (Cavali IJ)
eri) etc., block, s1~le.

Akad, Rii, Rice (Fl <1'roc, Hatl,
Eight cad, sc, i.

32- Engraving of specification (no change in content) 11, s, 8) 74. 3287-3291 (1
977)) indicates an interferon titer of 8000-10, 000 units/microgram 1\.

-32-1- Reprint of specification (no change in content) Preparation of 5 μ9 ml bank containing C, LclF CD NΔ sequence using standard method Likens (Wi
ckcns), etc., Jitoi, Bai Aru, Riichimi, (,1
゜Biol, Chem, 2! :) 3, 2483
-2495 (1978) OJ, Gocdde
l ) etc., Nature (NaturO) 281.5
44-4518 (1979)), two single-stranded cDNΔ
was prepared. The cDNA was fractionated according to size by electrophoresis on a 6% polyacrylamide gel.
00 to 1500b, rl size material II 2301
10 was obtained by electroelution. 100 of this cDN△
Nave A/-(N
ature) 275.617-624 (1978),
Tailing LJ with a deoxyguanosine (dG) residue at the Pst site
When annealing with plasmid pBR322 of O, llolivar et al.
2.95-113 (1977)), using this, E, c
olix 177633--transformation of the specification (no changes in content). Approximately 130 transformants were obtained for cDNA An1) that were icrin resistant and ampicillin sensitive.

-33-1- In a second similar experiment, for cDNA no.
About 1,000 transformants of E. coli K-12 strain 294, which were ampicillin-sensitive, were obtained with about 1,000 Te1hera1nyclin-resistant F1. In this case, fractionated CDNAjA ranging from 600 to 1300 b,p was collected by electroelution and used for chilling with dC.

D1 Preparation of Synthetic A Rigonucleobids and Their Uses Knowledge about the amino acid sequences of some 1-ribusin degradation fragments of leukocyte interferon are found in Left”
This made it possible to design synthetic deoxyoligonucleotides complementary to various regions of mRNA. Two 1-helipsin peptides were chosen, T1 and T13. The truth is that their amino acid sequences are all possible ]-
This is because only 12 and 4 undecamers need to be synthesized to describe the deA4-thioligonucleotide sequence (Table 1).3 For each sequence, four sets of deA4-thioligonucleA diprobes were synthesized. Three each (T-IA.

34- Engraving of specification (no change in content) B, C, D> or one (1--13Δ, B, 0°1)
) containing A-rigomecleodide. The complementary thiolnidio9gonucleotide 11 base chain sequence shown is phospho1
It was chemically synthesized by the ester method (Crea).
), etc., ``1ri, nato) Lt, lkado, 4ji.

USli, 1 (Proc, Natl, ^cad, sci,
I1. s, A, ) 75, 5765-E5769
(1978)). For the -13 series, four individual probes were prepared. As shown in Table 1, 12 -[-1 probes were prepared as 4 pools of 3 probes.

Prepared as 4 pools of 4 respective probes a3 and 3 primers each of the r-13 series/-212
The T-1 probe was used to direct the synthesis of synthetically labeled single-stranded CD NA for use in hybridized probes. Template mRNA A is G for Rendai induction.
-1 from 1 cell (800011i IF activity/μg)
2SRNΔ or all poly(A) mRNA produced in uninduced leukocytes (<10 units/μg). 32p-labeled cDNA was subjected to known reaction conditions fl (Noyes).
), etc., Broc., Nat. 35- Clarification of the specification (no change in content) Le, Acad., V., United States (Proc. Nat I,
Acad, Sci.

11,s,,A,>70,000,1770-177/I (1
979))-65-1- was prepared from these primers using the engraving of the specification (no changes in content).

20°C Old Ris (1'ris) (hereinafter referred to as 74ris) -11CI (pH 8,3), 20mHKCl,
81118 8 (IcI2.30mHβ-mercapl-
I3 reactions were carried out in 60 μm volumes in ethanol.

This reaction was performed using 1719 of each primer (TSJ:SU, T-
For the 1 series, a total of 12μ, for the -13 series a total of /lf1g), and 2/1g of ``induced'' 12S fraction mRNΔ (or uninduced poly(A
) mRNA3000 Ci/m mole) and 60
Unit Reverse Transcriptase (Bethesda Reverse Transcriptase)
boratories) ).

The product was 10mQ Sephadex (5ephadex
■)■ (Hereinafter referred to as 5cphadcx 'C-! Separate the unbound labels by gel filtration on a JUG-50 column, treat with 0.3N Na leaf at 70℃ for 30 minutes, and RN
A was decomposed and neutralized with 11C1. Hybridization is carried out by afatos, etc., Nucleic Acids Res.
, 15111-1552 (1979).

Identification of [Clone Ill'j 2] 130 - 36-1 - Copy of specification (no change in content) From each of 500 E. coli K-12 strains and 294 transformed strains (see item 0), 1 of plasmid DNA
Birnbo to prepare 11 g.
im) Tongue, Nucl
cic Ac1ds Res, ) 7, 1513-
1523 (1979) was used. Each I) NΔ trial profit is transmuted, Kapha 1 hess (af
3 replicates were applied on a Lorel Rose filter according to the method described in [al:os] et al.

Nitrorel [l-
The three sets of filters were primed with a) 7 primers 1--1 of Sera 1: I,: Cl) #C1) N Δ .

+1) 1-13 primed derived cDNA and C) hybridized with 1-1 cDNA prepared using both sets of primers. A clone is considered positive if it hybridizes more strongly to one or both of the induced CDNΔbu[]-bu than to the entirety of the uninduced CDNAbu[1-bu. (No change in content) I thought about it. Out of 500 samples, 30 positive samples were selected and further analyzed.

[, Identification of clones p1.31-pL39, LelF
Isolation of a plasmid (Nα104) containing a gene fragment ``1-plasmid and 32p-label induction + 11RNA'' Biochemistry (1,1llenhauo et al.) 15,
1858-1865 (1976)), Grunstin (6runstcin) and Bognes (+
10 (lness)' Ronnie hybridization method (Proc. Na.
Mouth, Acad, Sci, 11. s, A,
)-72, 3961-3965 (1975)'') were screened for transformants of r.colix 1776. The unlabeled l11 RNA of 9 J mm of uninduced cells was compared to the uninduced +ll RNA present in the 32p-labeled preparation.
To compete with A, it was mixed at a ratio of 200:1 to B[1-B]. Hybridization of labeled n1RNA contains the induced sequence OJ
This should occur selectively in colonies that are Three groups of transformants were obtained.

(1) 2 to 3% of colonies contain 320-+11 RNA
In non-38- +2110%, the rate of hybridization was significantly lower than in Group J (1) above.

-38-1- Reprint of specification (no changes in content) (3) The remaining samples showed no detectable hybridization signal.

For positive I11] [groups (1) and (2), interferon n1 RNA is linked to plasmid DNA.
The presence of sequences specific to the ink protein was examined by hybridization analysis. First, de-lariiculin (20ug/rd), diaminopimeline M (100μ5/mQ), and thymidine (2
0 μ”: I / mQ) 83 J: 100 of N918 with addition of Bi d-BiA′tin (lttq/mQ >
In m12, each of the 60 strong Yang 111] Ronnie (group 1) was developed individually I! Ta. M9 medium is 1 liter
About ~le, Ha IIPO4 (6g), ll2P
O4(3!1), NaC1((), 5'I
) a3 (Includes IN114 C1 (1g))
Fi L, sterile after autoclaving
No4 1#11! OJ: Sterile 0.01 HCaCl
Add 10 mQ of 2.

The 10 J8 nutrients were pooled and added to the Clewel
l), etc., Biochen+1s
try) 9.

The librasmid DNA was separated into six pools as described in J.D./42B-440 (1970). Each 39- Copy of specification (no change in content) -3' of plasmid DNA boolean? -1- Reprint of the specification (no changes to the content) Purified n+RNA from
The I It rl was hybridized to each filter paper. Non-hybridized mRNA was removed by washing. The specifically hybridized mRNA was eluted and translated in Xenopus oocytes. In this analysis, 6 pools were J-all negative. Five pools of each iQ]I]nee and one pool of 9 ml r'lnee were prepared from 59 weak positive (Q) mouths (2nd group), and plasmids were prepared from these pools. , investigated as above.

Of the six pools tested, one (KIO) hybridized to interferon mRNA at levels significantly above background levels in each test. Plasmid DNA from 9 out of 10 colonies was pooled to identify specific infu1-lon CD DNA clones.
A was prepared and examined individually. Two of the nine plasmids (No. 101 and N0104) were
1 [1] was bound to mRNA at well above background levels. Plasmid No., from 10/l, 2
6011.11. A unique Bgl IT restriction fragment was isolated containing J. This is a tiller (layer)
'8, Biochemistry.

Bioffice. Acta (Biochim, Biophy
s, Acta)/142.32/l-330 (197G
) and label it with 32p using the method of
Screening method (Grunstei et al.
n andllogncss), ``1ri, s1~le,
Cy, USA (Proc.

Natl, Sci, 11. S, 8) 72. 3961
-3965 (1975)) [, coli 294
400 (II, I) of the convertible strain were used for individual screening of bu[-1-zo.
31-rl139) was identified.

Roughly labeled I 260 b, using a LIli piece, make it like fi-ll, /l OO0 F, col
i294 transformants were screened individually. We identified 50 hybrids covering the probe and the balance rod. One is 1. one elFG fragment, one clF If fragment, one LclF II
Fragment designated I 41- (apparently an allele of 1elF II) (no changes in content). The resulting hybrid plasmid is “'p1.c
It was named as IF11'' etc.

-41~1- Reprint of the specification (no changes to the contents) G. Isolation and sequence determination of the first full-length 1eIF gene, resulting in 39 potential Lelto cDNA clones J; Re-plasmid DNA was prepared. Then, the same 260b, 11. Rescreening was performed using a DNA probe. This probe and three plasmids (rl171.I) L31, pl-3
4) gives a very strong hybridization signal, and 4 (
r+1.13, pl30. pl-32, I) L3
6) was moderately hybridized, and 3 (pl6,
pl, 8, pl-14> were weakly hybridized.

The 39 potential LelF cDNA recombinant plasmids also directly hybridized 11 p-label synthetic undecamers (each of the 1-1 primer pools or each of the T-13 primers).
-1-B was used for the search. The hybridization conditions were chosen such that complete base-base pairing was required to give a detectable hybridization signal (Wallas).
lace), etc., Nucleitsu 42 - Engraving of Specification (no change in content) 42-1 - Engraving of specification (no change in content) 1979-3543-3557 (1979')). Next,
39 glue [Cone 31: Librasmid DNA,
Standard supernatant method (cleared +ysate prO
cedllro; Clcwell et al., supra);
d^garosc) Purified using 8-50 column chroma 1 to graphie. A sample (3 μl) of each preparation was
Treated with oRl: to open the ring, and modified with alkali (IIS1↓
Spora 1 to 2 were placed on two separate 21-IT+relulose filters. 1.51.1 Sl for 1 spot
Doriru. Cuff 71~s quoted above (Kafatos)
See the literature such as J:. Each of the synthetic thio:1-thioligonucleotide primers and primer pools (
γ32P) was phosphorylated with -']) as follows.r
) Opmolc (D-A oligonucleotide Oyohi 10
011mole of (732P)Al-P(2)--England Nicole Claire-(New EnglandN
uclear), 2 F500Ci/m mole), 50 mH Tris-IIcI, 10 mJl NO
CI2 rice was combined with 15 mH of β-mercap 1 in a mixture of 1-tanol. After adding 43- medium 14-bolynucleotide A kinase and 30 minutes at 37°C, the 32p-labeled primer was
10mLl? Fadex (5cphadex■) G-
50 Calamus paste = 46-1- Engraving of specification (no change in content) Purified by romatography. 106 cpm plarai? -T-13G or GJ: 3 X 166Cpln (
1)-, f Hybridization was performed using primer pool T-IC.

Hybridization was carried out using 6 X S S C (I X
S S (', =0, 15HNaCl, 0.0158
<E/VM]-1 helium, al17.2), 1
0XDenl+ardts (0.2% bovine serum albumin, 0.2% polyvinyl vinyl 1]ridone, 0.2% ric
141.1.ol l) in solution at 15°C. It became a hybrid. Filter is 6 x S S C110℃
I washed it three times for 5 minutes. It was dried and grained onto X-ray film. The results are shown in Figure 1 for 32p-purine-pool T
-13C and primer-T-IC.

Plasmid I)NΔ from clone 104 hybridized significantly with primer pool T-1G and primer ■-13G. However, there was no detectable culm hybridization with other undecamers. As shown in Figure 1, some of the 39 possible LeIF plasmids 44- Description (no change in content) (t+12./1.13.17.20, 30.31°3
4) hybridized with both of these probes. However, restriction analysis shows that one of these plasmids (), p131 is 260b, p, internal not I
It also contained a t fragment. As a result of digesting p131 with Pst I, the size of the cDNA insert was approximately 10,001), 1)
,Met.

The sequence of the entire Psl, I insert at t+1.31 was sequenced from Maxam Gilbert (Haxan+-Gi 1bcrt).
Chemical methods (Methods)
Enzymol, ) 65.

/l 99. -560 (1980)) and dideoxy chain termination method (Smith, Method Enzymol (1980)) and dideoxy chain termination method (Smith,
SmitllHet, l+ods Enzymol,
) 55, 55 Q-580 (1980)). At that time, the 5au3a fragment had been subcloned into the 813 vector. , l') NA sequences are shown in Table 2.
A''. Information on the available protein sequence, the range of known 1.eIF molecular weights, and the relative presence of stop 11-repletion 1 in the three possible reading frames suggests that The translation reading frame was determined.Then, the complete sequence of the 101F amino acid including the signal peptide was shown.The first ATG IA translation. Start't
It is present at the 60th nucleotide from the 5' end of sequence -45-1-, and is located at the 18th nucleotide.
At 8 ml, there is a TGΔ terminal 1 triplet. At the 3' end, there are 3712 non-rA translation nucleotides, followed by a poly(A) sequence.

The postulated signal peptide (presumably involved in the secretion of mature 1. e IF from leukocytes) is a 23 amino acid sequence. 165 constitutes mature LeIF
The intermolecular reduction candy for amino acids is 19.390. We hereby pl. 31]
- The coded +-err' 1. From the sequence data ("'A") in Table 3, tryptic peptides T1 and T13 of LeIF B correspond to 145-1'19.13J: and 57-61 of LelFA, respectively. The actual DNA sequences found in these two regions are those represented by primer pool King t-Cd5 J: and primer T13-C (see Table 4).

11. Direct expression of mature white sphere Intafron A (IelFA)■ 46- Transcription of specification (no change in content) LeIF8 is directly expressed by mature Intafron A (IelFA) 1! The procedure for this is the synthesis (N-terminus) and the combination of complementary DNAs 1! The method of human growth hormone containing Il+ (Gocdel)
) et al., Nature 281.

544-548 (1979).

The 5au3a restriction endonuclease site shown in FIG. 3 is conveniently located between 1 and 3 of LcTFA. We defolded two synthetic deoxyA oligonucleotides containing the ΔTG translation initiation initiator, restoring the ton for amino 11 (cysteine), and creating a new coRI sticky end. Shilko. These A ribomers are p131 3
/1 b, p, sau 3 a-8 va H fragment. The resulting 45b,p product is two additional DNA
865b, 11. It was made into a synthetic-natural hybrid gene. This is done by ]-doing l ell^ and
It is flanked by pst T restriction sites. This gene was inserted into pBR322 between the FcoRI and Pst sites, and the plasmid was inserted into the plasmid.

2, 1-! J'71-/an regulatory element ([g, col
i trFl promoter, operator and trp
Construction of the plasmid ρG (containing the leader ribosis binding site but lacking the ΔT G sequence for translation initiation) contains the deletion Δ1F1413 [
, colil ~ responsible for the ribtophane operon (myo amount f
(formerly ozzari), etc., di, bacterio [l, 1, BactcriolooV) 133, 1
/I 57-1466 (1978)). Therefore, t
The first six amino acids of the rp leader and trp E
Express a fusion protein containing the a3 J: final third of the polypeptide (hereinafter collectively referred to as 11') as well as the entire trp() polypeptide. This is all under the control of the trp promoter-operator system. This plasmid 20μ9 was added with restriction enzyme P
VLI]'[ was converted to H1j. This cuts the plasmid at five sites. This gene fragment is then attached to an EcoRI linker (pC8').
CATG was ligated with a self-complementary oligonucleotide), and later cloned into a plasmid containing a coRI site.
This is used as the EcoRi cleavage site for conjugation. 20 μm of the DNA fragment obtained from pG old was added to 200 pmole.
5'-phosphorylation of synthetic A oligonuclease to pc
In the presence of A A 1'1' CA l' G, 20
μm King, r) NA Rigapi buffer (201118Tri
S pl+ 7.6.0.5mHATP, 10mHH
Treated overnight at 4°C with 10 units of T4r) NΔ ligase in an oC12,5mH chamber. The solution was then heated to 70° C. for 10 minutes to stop the ligation. The linker was cut with EcoRI digestion, and the fragments with EcoRT ends were separated using 5-percent polyacrylamide gel electrophoresis (hereinafter referred to as PAGE). The three largest fragments were first digested with tidium bromide. The fragments were stained, positioned using ultraviolet light, and separated by cutting out the target section from the gel. Each gel fragment (300μI
O, IXTBE) into a dialysis bag, and 0.1
X Ding BF buffer (T8F buffer has an Ir1s of 10.89
Base, 5,5 q Noho M, 0.09 g N'a
Electrophoresis was carried out at 100V for 1 hour in 2 FDT A (contained in 1 liter of 1-120) for 1 hour. Collect the aqueous solution from the dialysis bag, extract phenol,
DNA was collected in 1" small lumen extraction, 0.2M sodium chloride solution, and ethanol precipitation. IcoR
The trp promoter-operator-containing gene with I sticky ends was identified by the procedure described below. That is, a break was inserted into the De1-razaicline-sensitive norasmid, which resulted in tetracycline resistance 11 due to the insertion of a promoter-operator.

Plasmid pBR111 (Rodriguez
ez), etc., Nucleitsk Acid Research (Nuc
leic^cids Res,)6.3267~328
7 (1979)) expresses ampicillin resistance and contains the tetral no-italin resistance gene.

However, since there is no promoter associated with it, it does not express its resistance. This plasmid is therefore tetracycline resistant +47-. This EcoRI
The plasmid is made resistant to tetracycline by introducing the [Pl] motor-operator system into the site.

50- Reprint of specification (no change in content) pBRllI was digested with ECORi and the enzyme was extracted with phenol (50-1-1) and extracted with water. The product NA molecules present in the separate reaction mixtures were Each of the three DNA fragments obtained in
It was ligated with Δligase. D present in the reaction mixture
Using N△, Muku semi-method (Barcy) field (1
lershf i e l d) et al.

No. 1-R, Acad, R., USA (Proc, Nat.
l, AcadSci, 11. s, 8,) 7 1.
3455-3459 (197II) ) NiJ:SuDN
F with the ability to get into A.

One 12 strains of E.coli 294 were transformed, and this bacterium was transformed into
20fl! 7 / mQ of ampicillin rice 5
II 9/m (L containing l detraliicrin
11 (1uria-Rθ口aV1!) Play 1-
was inoculated. Some Te1 Herariikrin Resistance 11]
The plasmid 1') was isolated and the presence of the desired fragment j) was determined by restriction enzyme analysis.The resulting plasmid was called DBRII trp1J.

Hepatitis B viral genome EcOlII and Bam1
The plasmid 41 product was collected by a conventional method, and the plasmid 1
G11-- Ward 1 - Copying of the specification (no changes to the contents) The FCORI and BamllT sites of
281.544 Lasmid ρIIs 32 was cleaved with XbaI, phenol extracted, 1-1[I]form extracted, and ethanol precipitated. 1 precipitate is 0.1 ml dTTP 8
3 and Q, 30 containing 1mt4+l CT P
ttl polymerase buffer (50mt+potassium phosphate lz pH7, 4,7ntHH (IcI2° (Boehringer-Hanheim)
nheim) for 30 min at 0°C, then 2 min at 37°C.
Time place P1! Shita. With this treatment, 5 of the Xba I cleavage site
'2 of 45 4 nucleotides complementary to the overhanging end
The quantity is filled.

5'CTAGA-5'CTAGA-3' T-TC
T--Two nucleotides dC and dT are incorporated to give an end with two overhanging 5' nucleotides. This linear residue of plasmid puS 32 (phenol and chloroform extraction followed by ethanol precipitation and water) was cleaved with EcoRI. The large 52--b'l-1-1 52-1-EcoRI-Xba I fragment was separated by electroelution. This [')NA fragment from pl-1332 (0,2 μq) was purified from pB old It,
tryptonoane oberon derived from rp (approximately 0.01μ
g) was ligated to the EcoRI-Taq I fragment.

The above fragment of DtlS 32 was purified with EcoRI-Taq.
In this method, the overhanging end of Taq"f is ligated to the remaining overhang of Xba I, although it is not a perfect Watson-Crick base pair ().

A portion of this ligation mixture was used to transform r.coli 294 cells, heat treated, and plated into 7-ampicillin-containing LB plates. Select 24 colonies, 3 d
The plasmid was isolated after growth in LB (Iuria-Bertani). Six of these] colonies were found to have an X, ba I site that had been regenerated by DNA repair and replication by [:, coli.

53 - Copying of the specification (no change in content) -T C-1-A G A ”” -T C T A G
△--8G C-'0--A G AT CT-
These plasmids are EcoIII I and 1lpa
One plasmid, designated IC6pTrp14, which was cleaved at both ends to give the expected restriction fragment, was used to express the heterologous polypeptide as discussed below.

Plasmid rllllGI+107 (Goed
del) etc., Nave X7- (Naturc) 'L8
1. 5441.1979) is a synthetic I)N△cut) 23 amino acids produced from 1] and the complementary DNAΔJ produced by reverse transcription of the human human hormone metsenger RN△. 1 631t! +] Amino acids] Ton J: 1 to 1 to 1 to 1 to J
It contains lυ genes. This gene contains an ATCwI translation initiation codon, although it lacks a G-J ton of the ``preceding'' sequence of the 1=1.1 hormone. This gene was treated with 10 u pHGH107J: [<:oRJ treatment, followed by [] as described above.

colil)NΔvolimer~ze■klenow(ni+eno
w) (hereinafter referred to as KlenOW'?I) fragment and dTT
, P and dATP 54- Reprint of specification (no change in content) IJ, phenol and dATP [1[1
Holm draw 5/1. -1- After extraction, ethanol precipitate the plasmid into Bamtl
I″c treated.

The human growth hormone (1-IGH) l gene-containing fragment was separated by PAGE followed by electroelution. The resulting DNA fragment lacks the tetracycline promoter--A" bellator system but contains the first 350 nucleotides of the tetracycline icrin resistance
When subsequently cloned into an expression plasmid, plasmids containing the insert can be identified by restoration of tetracycline resistance. Since the EcoRI ends of the fragment (1) were filled with the old 0nOW polymer 1iT method, this fragment has one blunt end and one sticky end. Therefore, it can be inserted into the expression plasmid after 4 Correct orientation is ensured even when the

Expression plasmid p? rpl 4 was then prepared to receive the 1-IGI-1 gene-containing rice cake pieces prepared above. That is, plrpL4 was digested with xba I, and the resulting sticky ends were extracted with dATP, dTTP% dG 1
'P and dCIP were loaded using the 55-lenow polymerase method. After phenol and chloroborum extraction and ethanol precipitation, the resulting DNA was treated with Baml-II and the resulting large plasmid fragments were separated by PAGE and electroelution. The pTrpl4 fragment has one truncated end and one sticky end, and contains the 1-I G
This made it possible to recombine with the +-1 gene-containing fragment in the correct orientation.

HGHI gene fragment and pTrpl AΔXba-Ba1
l-l 1 fragment is the 41f fragment under similar conditions as above.
L phase! Connected to 1. Satisfied Xba ■OJ: BiE
The coR■ ends were ligated with blunt ends to reconstitute both the Xba I and J: and FcoRJ sites.

Filled Xba I Filled FcoRT IIG
II31 gene origin Xba I EcoRI This construct also creates a te]-racycline resistance gene. Plasmid pHG11107 is l-I G
The promoter (lac
This construct, designated pHGt1207, allows expression of the Tei~laryclin resistance gene under the control of the toptonoan promoter operator. Then,
Transform E. coli 294 with the ligation mixture and 5μ9/
Ronnie was selected on the LB plate containing mQ of tetracycline.

Plasmid pHGll 207 was ECORi digested and t
rp promoter - contains the operator and the trp leader ribosome binding site, but A for translation initiation
300b, P, fragment lacking the rG sequence, 1”AG
E was collected by subsequent electroelution. This 1) NA
The fragment was cloned into the EcoRI site of pl,elFA. The above modified TRP regulon
) (attenuated to regulate to increase expression level)
An expression plasmid containing an Ig (oli trp operon) with the -ta sequence deleted is incubated to a predetermined level in medium supplemented with a sufficient amount of tributonoan to suppress the promoter-operator system. The desired product is then expressed by removing the tryptophan and uninhibiting the system.

More specifically, with reference to Figure 3, 250 I
Ig plasmid p131 was digested with PstI and 1
The ooob,p, insert was separated by gel electrophoresis on a 6% polyacrylamide gel. Approximately 40,719 inserts were electroeluted from the gel, divided into three portions, and further digested as follows. a) Add a 16 μg sample of this fragment to B! for 45 minutes at 37°C. 11 m was partially digested at position 4011j, and the reaction mixture was purified on a 6% polyacrylamide gel. A fragment of 2μ9 of the desired 670b,p was collected. b
) Another trial r1 of 1000 b, p, Pst I inserts
(8 μg) was digested with Ava IT and B(]fl[. After gel electrophoresis, 1μ9 of the above 150b,p fragment was obtained.C) 1000b,I).

16μ of the pieces were treated with 5au3a and Ava fl.

Ato electrophoresed on a 10% polyacrylamide gel,
Approximately 0.25μg (10u+ole) of 34. b, I)
.

Got a piece. The two indicated de-4th thioligomethreotides, 5'-dAAT1c^TGTGT (fragment 1) and 58-Description (no change in content) 5'-dG8 to C8 to ACATG (fragment 2), are phosphorylated. Synthesized using the triester method. Fragment 2 was then phosphorylated. , 200/l (approximately 40 μmole) (
γ32P) A T r) (Amersham (8 mcrs
ham), 5000 Ci/mmolc) and 60 mH Tris-11cI (pH 8), 10
H of l1ltl (lc12.15mHβ-mercaptoeta/-)tt, D of 100 pmole
NA fragment Onii 2 units of 14 polynucleotide Kina-1
! Resuspend in 30 μl of containing solution. After 15 minutes at 37°C, 1 flI of 10 mH ATP was added and the reaction was allowed to proceed for an additional 15 minutes. The mixture was then heated at 70°C for 15 minutes and 100 pmole of the 5'-0f-1 fragment was added to the
:bi1Qpm018 34 b, rl, 5all 3
a AVa If fragments/j. 20mHTris
llCI (DII7, 5), 10m14 HoC
I2.10m1t Jiji A Slaytall, (1,5m)l
50 of ATP and 10 units T4DNA Rigger V
Ligation was carried out in μm for 5 hours at 4°C. The mixture was electrophoresed on a 6% polyacrylamide gel and the 45b,p product was collected by electroelution. 45b,p, 30n of product
o (1pIIlole), 0.5 μg (5 μmole) of the 15059-Specification (no change in content) b, p, Ava TI--59-1--
k(F670b, tl, BQI IT-Pstl fragment combined with 1 μg (2 pHole) [IL, t=.2
2 using T4 DNA IJ Gar-1 at position 011i
Connection treatment was carried out for 16 hours at 0°C. The ligature was inactivated by heating at 65° C. for 10 minutes. The mixture is [coRI and I
' The gene polymer was removed by digestion with St I. The mixture was purified with 6% PAGE. Approximately 20 μg (0,
04pmole) of the 865b,D product was isolated.

Half of this (10no) was added to pBf1322 (0,3
pg) between the EcoRI and Pst site. When E. coli 294 is transformed, 70
7 tetracycline-resistant and 7 nubicillin-sensitive transformants were obtained. From 18 of these, plasmid D
NA was isolated and digested with IEcoRI and PstI. Of the 18 plasmids, 16 were 865 h;
+3, long EcoRI-Pst I fragment. One of these, 1μ of pLelF AI, was added to [COR
■ A 300 t), rl, EcoRI fragment as prepared above containing the F, coli trp promoter and trp leader ribosome binding site.
- Linked to the engraving of the specification (no change in content) (0,111g). The transformer containing the trp motor is the Gransdain - Small Gunnes (
Grunsteinilogncss)-10 was identified using a 32Ptrp probe in combination with the Nice screening method. The asymmetrically located Xl)a I site in the trp fragment indicates that the trp promoter
This allowed the identification of recombinant products oriented in the direction of the lFA gene.

1. In vitro and in vivo activity of 1elFA I [
Analysis 1. 1: An extract of melon was prepared as follows. 1
8i & 1 mQ of 5μg/#11! Contains 11% of tetracycline [V1A550 grown in broth
value of approximately 1.0 and then 25 #ll of M9 medium containing 511'J/mfl of tetracycline! diluted in

10In when Δ550 reaches 1.0! ! A sample of
r i 5-1-1cI (DI+8.0>, 50m1
Suspended in 1 mQ of 4EDTA. 1 ml of lysozyme was added, and after 5 minutes at 0°C, the cells were disrupted by sonication. The sample was heated for 10 minutes (15,0OOrDIIl
)-61 = Copy of the specification (no changes to the contents) Centrifuge the intaf Jlon activity f1 in the supernatant to cells-6.
1-1~ For inhibition analysis of denaturing effect (CPF), standard 1. eH
It was measured in comparison with. To measure the number of IF molecules per cell (+), 4X” [18 units/my (DL
eIF specific activity was used. - As shown in Table 6, clone pLelr^trp25, in which the trpf11 motor was inserted in the desired orientation, had high activity (
2.5 x 108 units/1). As shown in Table 7, E. coli K-'I 2 strain 294/pLel
The production of F^trp25 I[is Hii~1. It behaved similarly to the eH preparation, was stable to treatment with nrll+2-C, and was neutralized by anti-leukocyte antibodies. This interface [con's molecule m(,1 about 20.000
It is. Clone plan trl125 has been deposited in the American Type Culture V~ Collection (Δ-1-CC).

The in vivo effects of Intaf1t]n include MacHino2
-di and normal killer (NK) cells are required. The effects of So-C in vivo appear to include stimulation of these cells. So, E.

In 62-tafuron, produced in E. coli 294/DLelF825, exhibits antiviral activity in cell culture assays, but may be ineffective in infected animals. Furthermore, the in vivo antiviral activity of non-glysylated LelF produced by bacteria is tl-”/';':z
'--]-t (buffy coat) ``It may be different from the glycosylated LelF derived from white blood cells. Therefore, the biological activity of bacterially synthesized LelF^ (2% purity) was transferred to squirrel monkeys. In a lethal dose of encephalomyocarditis virus (FMC) infection,
Table 8) for comparison with -1-LeTF (8% purity).

Table 6 Intafton activity of E. coli extracts 63
- Reprint of specification (no change in content) Table 7 [, coli29 /I /r11. ejF A
25 Extract standard 1, eI [* Which active 1'l comparison tree Listed in Table 6 (7) [, CO1 + 294 / rllel
F A trtl 250.0 per mQ of extract of 25
00 units was diluted 500 times with minimum essential medium to give a specific activity of 500 units/d. Leukocyte Intaf 1 ton standard [Wadley I
n5t ute)) was pre-titered against the N I +-1 leukocyte interferon standard and also diluted to a final concentration of 500 U/trdl. 1
mQFrk was set to 0112 with 1NllClCl and 52 at 4°C.
At time, the ink was coated and neutralized by the addition of NaOl-1, and IF activity was measured using a standard CPF II fl-stop assay.

25 μm of 500 single If//tnQ test n (untreated)
64-1-25 ul of 4J anti-oxidant 64-1-Human white surface method interface 10 at 37°C for 60 minutes in Ink Covey 1, 12. Centrifuge at OOOxg for 5 minutes and analyze the supernatant.

Table 8 IE M C of Squirrel t-key (Lysdell)
Antiviral effects of various LeI [preparations] against viral infections. All Leels are Ht (average weight 713 g>) and do not have EMC virus antibodies before infection. is 100Xt[5oEMC virus (measured in mice)
Intramuscular infection was avoided. λ1 irradiation control is 1 infection 1
Died between 34.158.16411S.

106 Interfacial] treatment was carried out 4 hours before infection, 11t 2.23.29.48.72.168 and 2.
It was administered intravenously at 40 hours. Bacterial leukocyte ink 7
Tron is r, coli29 /l /111clF
Column chromatography fractionation from the melt of A25 showed a specific activity of 7.4 x 106 single B/m9 protein. Control bacterial proteins were r, coli294/pBR322
Equivalent column fractionation of the lysate doubled the total protein content. The leukocyte interferon standard was purified by chromatography to a specific activity of 32 x 106 units/m'J protein, and was purified by chromatography to a Sendai-fils-induced interface from normal IC1 cells. 1] It's hot.

Control samples showed progressive coma, loss of balance, flaccid hind limbs, paralysis, and lachrymal discharge beginning 8 hours before death; intephron-treated mice showed that these abnormalities were not present. He was always active and showed no symptoms due to viremia.

One monkey in the control group, which did not show viremia until day 4, died at the latest (infected monkeys did not produce antibodies against the EMC virus when tested 14 and 21E1 post-infection).

Show that it can be done. This is because the protein content of bacteria is quite different from that of bacterial protein preparations. Furthermore, these results show that 1. We show that glycylation is not required for eHA in vivo resistance to active bamboo.

J. CDNAs for other white-faced interferons
Separation of DNA from a fully characterized leHA cDNA-containing plasmid was cut with Pst, separated by electrophoresis, and labeled with 32p. Using the resulting radiolabeled DNA as a probe, the in situ colony screening method of Grunstein and Gunnes (+10(]n4!33)) was used. An additional transformant strain of coli 294, obtained as in Section C above, was screened and one was isolated that hybridized to the probe to a different extent. Plasmid I') N A O J: and 10 hybridizations shown in section G above] Plasmid D' from Ronnie
NA, Pst, was separated into one cleavage and characterized in three ways. First, these PSt fragments were characterized with a restriction endonucleolytic pattern using enzyme B (111, PVII IT and σ [coll T]).
Jlz20 This analysis is based on at least 8
Different types of species (1-clF 8, I-eTF 8.
1. clF C11-elU D, LelF F, 1c
lF F, 1. OTF G and 1. all'If)
classification. This [,1] shows the approximate positions of the various restriction breakpoints with respect to the heretofore known leading sequences of LerF A and J: and]-do sequences.

The worst part of these things is 1. elF'D is Nagata (Na
oata ) etc. are nature (Nature) 284
．． 316-32068-Engraving of the specification (no change in content) It is believed to be the same as reported in (1980).

68-1- KG-1 thin k RNΔJ containing LeeΔ, 1. elFm
Regarding the ability to selectively remove RN△, Cleveland et al.
95-1(:)5 (1980) and examined by hybridization selection analysis/C01,eH^,B,
C and [(Mako's analysis showed positive 1. Third, these pst fragments were inserted into an expression plasmid and 1:.

E. coli 294 was transformed with the plasmid to express the fragment. The expression products that can be expressed by Plaintough Engineering, except for the LeIF F fragment, are barely expressed
CPE analysis for itafelone activity was positive. In addition to the above, the layout 111 was determined for all of the above 101 [types].

K, Second Mature White Surface Method Ink Feed [1 N (LOII-
8> directly expressed mature 1eft containing the gene for J-rumin 11I
The sequence of the I fragment shows that the first 14 nucleotides of types A, J, and B are the same. So, trp −
A fragment containing the promoter-operator, ribosome junction 9-113J: and the start of the LeIF8 (-B) gene was isolated from pLcTr825 and ligated with the remainder of the B sequence in an expression plasmid. pL
4 (1 and eTF B Pst terminal genes shown in FIG. 2 are shown in the diagram, respectively).

According to the arrangement of Fig. 4x, Pst I cut) OJ for t
To obtain the Sau 3 a-Pst I fragment of: 950b, p, one or more intervening Sau
Several steps are required because of the presence of the 3a restriction site. In other words, it becomes as follows.

1. The following fragments were isolated.

a) 110b from Sau3a to FcoRI
b, p, :l+) [132 from coRJ to Xba
b, l).

c) >700b, p from Xba to Pst.

2 Fragments (1a) and J: and (1b) were ligated and cleaved with Xba and +3(III to prevent self-polymerization via the Sau 3 a and Xba ends (associated Sa
The u3a site is B (=70- in the 11II site; l1oll cleavage left a Sau3a sticky ↑11 end). 242b, p, fragment 11111 L,
Ta.

The products of (2) and (1C) were ligated together again with Pst II and Bgt IT to prevent self-polymerization.
I cut it with. 5alt 3 a to Psi I (
Figure 4) A fragment of approximately 950b,p was isolated. This break is not common to LelF8. 1. It contained part of the erF B gene.

Approximately 300 b, p, fragment containing the trp promoter-operator, ribosome binding site, ATG initiation signal and cysteine] of LeIF A (IIindII
3a) were isolated from pLejF25.

Approximately 36001 from Pst J) to 1indlff
1.11, the fragments were separated using I]BR322. It is loaded with replenium and tetracycline anti-11, but does not contain ampicillin resistance.

Triple connect the cross sections obtained in steps 3.4 and 5,
The resulting plasmid contains 1. E.coli 71-Craft of specification (no changes in content) K-12 strain 294 was converted.

Convertible stocks are screened using mini-screening (Birnboim (B i
rnl)oim)etc., Nucleic acid linage (Nucleic 8cids Res,) 7
, 1 5 1 3 -1523 (1979))
The plasmid samples were digested with rcallT. Digestion gives 3 pieces! , Second, their characteristics are 1.
2) the internal EcoR■-EcoR■ fragment of Ill-4; and 3) the protein translation initiation jig length Ru4coR',l fragment of 4.

CP [For analysis, bacterial extracts from clones prepared as described above are typically approximately 10×
It gave an interferon activity of 106 medium/1. This J
: One representative sea urchin prepared from sea urchin,
I', coli294/ plell B trp 7
It is. This link has been deposited with the ATCC.

1. Yet another mature white ball interferon ('Le
IF+-: , D, r, II, Ionibi,'') Additional full-length gene fragments containing the other L(31F types) were added to the tiller as well as telF8 and Ij. 72- 72-1- The N-terminal amino acid that was removed and was lost due to the removal of the preceding sequence ~
The question is whether a restriction site exists sufficiently close to the first amino acid of the mature interferon type to determine whether the convenient method of synthesizing and replacing it by ligation of an NA fragment can be used. This will be known from complete sequencing using conventional methods. If that doesn't work, you can use the following operation. In short, the preceding sequence-containing fragment is cleaved exactly before the beginning of the sequence for the first amino acid of the mature polypeptide. In other words, 1. Change double-stranded DNA to single-stranded DNA in the region surrounding that point: 2. Convert double-stranded DNA to single-stranded DNA in the region surrounding that point: 2. Add methreotide to the region of the single-stranded chain generated in step (1) on the opposite side to the target cleavage site. A complementary primer of single-stranded DNA is hybridized so that the 5' end of the primer is positioned.

73-3 The part of the second strand removed in step 1 in the direction 3' from the prine is converted into ON'A in the presence of adenine, thymine, guanine and cytosine containing deA' coconut nucleobuty triphosphate. Recover by reaction using polymerase.

4. Digest any remaining single-stranded DNA that protrudes beyond the point to be cut.

A short synthetic NA carrying the translation initiation signal A-TG at the 3' end of the coding strand is then ligated to the resulting gene tailored for the mature interferon, e.g. by blunt end ligation, and This gene is inserted into an expression plasmid and placed under the control of a promoter and its associated bosol\ binding sites.

In a manner similar to that used in Section 1 above, the genetic clone encoding <RTIgt;leI[C</RTI> and <RTIgt;telFD]</RTI> is suitably sequenced for direct bacterial expression. As a strategy for the expression of these additional leukocyte interferons, in each case pl, eIF approximately 300b, l) containing a G-initiating jig pull and a cisjin]ton.
, the cut j', (lIindl[l to Sau 3 a
) is used. In contrast, gene fragments from other inktuconilon genes encoding the respective amino acid sequences beyond the first cysteine, which is common to all, are combined. R. coli K-12 strain 294 was transformed using each of the obtained plasmids.

The connections to form each gene are as follows.

Shiri. ! ] pleatCJ: Ri separate the following fragments: (a) Sa
35b from u 3 a to Sau 96, I).

(I+) >900 from Sau 96 to pst I
b, I) (C1 As in section K(4) above, pl,
Approximately 300b, p fragment from eIF A-25 (llin
dll-5au3 a).

(d) Separate a fragment of about 3600b,p of the above K(5) term.

75-1 (1) (a) Connect OJ, and (C) 1-R. Bgl
TI, cut with 1lind■, about 335b, 11.
Separate the products.

(2) (1) + (11) (((1) and ! ~ real ligation, and transform E. coli with the resulting plasmid.

A representative 4 [clone is F, col
i K-12 strain 294/rll, eTF CtrD
It is 35. This line has been deposited with the ATCC.

LeIF D pLeIF D as follows: l1llll? l:
a) Sau 3 a to 8 Va T [35 b,
p.

b) 8 vallJ: B (150b to 11■, p.

c) Approximately 700b from Bgl II to Pst I, 1
1゜pLelF A25 J: S, next fragment *t
71-ru:d) 1Iindll[J:Sau 3
300b to a, r+.

Isolate the following fragment from pBR322: e) l1i
ndIIIJ: about 3600 b, p to spst I.

(1) (a) Ligate +(1+), cleave with BQI II, -76=185b,p, purify product (1).

(2) (1) +-(d), 1lir+rl
li, Bol II and approximately 500b, p
, purify the product (2).

(3) (2) -) Connect (C) → (e) and transform R. coli with the resulting plasmid.

Representative clones prepared in this way were F, co
il K-12 strain 294/tll, elF D t
It is rpll. This clone has been deposited with the ATCC.

Fragments containing 1eTF F, 1eiFB and Le
Direct expression can be achieved by rearrangement that takes advantage of the complete homology of amino acids 1 to 13 of IFF. From 118 G 8.207 above, Pst T and Obtain (a). The second fragment (b) is the larger of the fragments resulting from Pst I and 1ull II digestion of plasmid rlHKY10. Fragment (a) contains about half of the gene encoding ampicillin resistance; fragment (b) contains the remainder of the gene and also contains the lacycline gene other than the combined promoter. Contains all of the following. 7)-7(a) and (b) are joined by a four-way ligase, and the product is converted into Xb'a I and B
(]l m to prevent diphthongization, and t
A fragment (C) containing the cp promoter-A-belator and the genes for tetragonic icrin and ampicillin resistance v1 is produced.

Approximately 580b, fragment (d) of p is the AVt of pLeIF F
'l TI and Bgl [obtained by digestion. This is 1. Contains tons for amino acids 14 to 166 of eTF r.

Fragment (e) (49b,p,) is the X of pLelF B
Obtained by ba T and Ava If digestion. Fragment (e
) is 1. Amino acids 1 to 13 of eTF F].

Fragments (C), (d) and (e) are triple ligated in the presence of T4 rigger 1. The cohesive ends of fragments 11 and 11 indicate that the composite plasmid is properly circularized and that the neuculin resistance gene is matured. Together with the elF gene, it is placed under the control of the trp promoter-A promoter, so bacteria transformed with the desired plasmid are selected on tetracycline-containing plates. A representative clone thus prepared was E. coli K-12 strain 294/rlleH.
F trDl. This clone has been deposited with the ATCC.

1. eHII To express the complete 1elF II apical gene as a mature leukocyte interferon, the following J: urinary sequence can be arranged.

1, plasmid p1. eTF If was subjected to 1lae IT and Rsa I digestion to generate 816b, p.
, fragment 11111-iJ.

2 Denature this fragment and incubate it with DNA polymerase
now fragment (Klenow et al., Proc.
NatL, Acad.

Sci. Il. S. 8.) 65-1168 (1970)
), 79- Reprint of specification (no change in content) Synthetic deoxyribo-A oligonucleotide brimer-79-
1-5'-dATG TGr AAT CTG C" is used for repair composition.

3 The resulting product is cleaved with Sau 3 a and the 4·52b,p fragment representing amino acids 1 to 150 is separated.

pLelF II with 4Sau3a and pstI
811, and the resulting 500b,p fragment was separated,
From the 150th amino acid to after R of the coding sequence]-
Obtain the gene f to be used.

5. Connect the fragments separated in steps (3) and (4) to create fragment 1 166 mat cys asp stop AIG TGT...-←--C--GAT, T
Obtain G8-...-Pst, l5au 3 a. This is 1. , 166 amino acids of elr If.・ 6. Xbair digested DLelF 81rp 25,
1) Make blunt ends using NA polymera-1ffiI,
The product is digested with pst,I. The obtained large fragment was separated and ligated with the product of step (5) to create an expression plasmid. This is R. coli K-12 strain 294
or for transformation of other host bacteria, mature 1, eIF
'If can be expressed.

Human genome face λ Charon' 4A recombinant library (Cel+), upper 5', 1157 (19
78)), the method of Lawn et al. cited in -1-2 and the method of HaniatiS et al.
e11) 1 above.

687 (197B) were used to screen for leukocyte interferon. cDNA clone 1. Radioactivity derived from clF8 1. Approximately 500,000 blurs were screened using the eT probe. Six L e IF genomic clones were selected and analyzed. Following rescreening and plaque purification, one of these linkages λlI1
．． clF2 was selected for further analysis. Using the method described in □-, other probes can also be used to separate other 1011 molecules from the human genome. These are then converted into 81-
1- Vines used to produce other Shiromen method int protein proteins.

1 R[]-]nλ old-eIF2's 2000 b, p,
The EcoRT fragment was inserted into pBR32 at the Eco81 site.
5 was subcloned. , the obtained plasmid 1. eIF I was cut with [coRI, 2000b, p
The fragments were separated.

This 2000b,p,EcoRI fragment was added with the fourth thioligonucleotide dAAT-CTGCAG (EcoRI
-Pst, J] converter) were linked. The resulting product was cleaved with pst 2 with less than pst I
000b, 11 was broken. Cut this with 5au96
No Iko. One Pst I and one severe 5au96
A 1100b, p fragment with an end of 11 was isolated.

2. Plasmid p1. clF Ctrp 35 ps
Digested with tI and XbaT. Large pieces were separated.

3, pl, el[Ctrp 35 J: Rino's small Xb
aL-Pst J fragment with Xba I and 5alt 9
Digested in 6.

40 b, p, Xba ■-Sau 95 fragments were isolated.

4. Min-1 from steps (1), (2) and (3)
The resulting fragments are ligated and l) 1. eIFI trll 1
expression plasmid and −82=. This clone has been deposited with the ATCC.

eIF J 1, plasmid pLeTF J is 1. A protein containing a 3.810 base old ndI fragment of human genomic DNA with the eHJil gene sequence. From this plasmid 7
The 60 b, D, Dde I-Rsa I fragments were isolated.

2. Plasmid p1. elF B trp 7 to 1l
cut with indlll and Ode I, 340 b,
p, ll1ndI [[-Ddel fragment was isolated.

3 Cut plasmid pBR322 with pstT,
') Incubate with NA Bolimerer t'I, make blunt ends, (Klenow fragment), and incubate with 1lind.
Digested with lTI. A large (approximately 3600b, r+, .) fragment was isolated.

4 The fragments separated in steps (1), (2) and (3) were ligated to create the expression plasmid pleHJ trp 1
And so. This clone has been deposited with the ATCC.

The content of intafTron in bacterial extracts can be increased in the following manner.

1. Polyethylene-imine precipitation. Here, in 83
- Transcription of specification (no changes to the content) Most of the cellular proteins, including Tough T, remain in the supernatant.

2. Ammonium sulfate fraction. Here, interferon is precipitated from a solution of 55% saturated ammonium sulfate.

3. Add ammonium sulfate cum pellets to 0.06M potassium phosphate, 10 mHTris-11cI, pH 7.
, suspended in 2! , 25mHTris-IICI,
Dialyze against pH 7.9. Interfuron activity remains in solution.

4.1- Adjust the supernatant to pH 8.5 to D[ΔFE-
1? Chromatography is performed on a Rulose column (25
mHTris-11cI, pH 8.5 from O to 0.2
Elute with a linear gradient of M NaCl).

5. Bubachrome-Blue-Agarose (Cibacl)
lrome 131tie-A garose) or hydroxylapatite (hydroxylapatite)
te) and eluted with 1.58 KCI or 0.2 M phosphate (as necessary).

6. Seph/'Dex (5 OphadeX■) G-7
5 Forces 84- -84-1-- Copy of specification (no change in content) -[L]-Zoo 1- Perform cation exchange chroma 1~graph. Develop with an ammonium acetate gradient (up to 0.2M ammonium acetate).

A product with >95% purity is obtained by the method described in Section L.

This material can also be used in noise exclusion chromatography, reverse phase (Itl”-8) high pressure liquid chromatography, I1
Alternatively, it can be purified by affixing with immobilized anti-interfuron antibodies.

After cutting the grass, remove the material from step 4 above by millstaining (
Hi 1stein), Scientific)7
Scientific American
243.66 (1980), plus 0.2 M l'II acid, 0.1% 1'r: ton a
-3 and 0.15H NaCI.

Using another example of a right-handed shellfish, the leukocyte interferon produced by the above procedure is purified in the next step.

1. The frozen cell mass containing the expressed white-globulin intafron was mixed with 85--85-1-(w/V) sucrose, 0.2H NaCI, 5 mM using a suitable grinding device or by hand.
EDTA, 0.1 mHPMSF and 10-100 mH
Suspend in 4 volumes of HClCI2 containing buffer. The suspension is kept at 4°C.

The suspension is passed through a homogenizer 1f- at approximately 6000 psi and then passed a second time at less than 1000 psi.

The effluent from the homogenizer from both passes is cooled in a water bath.

2. Slowly add boliebrenimine to the homogenate to a concentration of about 0.35% and leave it for about 30 minutes. Solid matter is removed by centrifugation or filtration. This step is carried out quickly enough so that the temperature is regulated or the supernatant (filtrate) is kept below 10°C. The supernatant (filtrate) is concentrated by ultrafiltration to about 1/10 of the original volume.

If particulate matter or dirt is observed, it may be removed with a suitable filter, such as a micropore membrane.

3. The clarification solution is flowed directly onto the monoclonal antibody column at 5-8 cm/hour (eg, between 25-40 ml/uX! on a 2.6 cm diameter column). Next 8
6-11C1, all 7.574%, 5 (NaC1(
0,5M> a and 1 hem 1 hen (Triton) X
-100 (0.2%) or equivalent (containing surfactant). After washing, add 0, 15) IN to this column.
a(', l and 1-liton X -
Pass approximately 10 column volumes of solution containing 100 (0.1%) or equivalent surfactant. io This column from 1 to
Triton X-100 (0.1%)
or equivalent surfactant ('), eluted with 2MMt acid. Collect the protein peak (measured by LJ V absorption or its method) from the monoclonal antibody column,
I N Na011 or j, OM Trism M
Adjust the pH to approximately 4.5.

4 The pooled interfacial peak is
&jM For example, ammonium acetate pl+4,5 (50
1118) r-equilibrated Bowatman (Wllatm
an >, CM 52 t Rulose or equivalent cation exchanger. After addition, wash the column with the buffer for equilibration and wait until the jJV absorption of the effluent becomes irritating. The condition is such that almost no protein 87-1171 manuscript (no change in content) is eluted from the column.

87-1- Reprint of specification (no change in content) The column was then washed with 25nH14 ammonium acetate 10.
Elute with a combination that maximizes the recovery of 12 MJ of 1-lium or ink feron, resulting in a lyophilized cake of satisfactory appearance and solubility.

The monoclonal antibodies listed below (in parentheses) are examples of the monoclonal antibodies that Staellelin et al.
at l.

8cad, Sci, 11. s, eight, > 78. 18
4875.2 (, 19B 1.). Vine prepared by method. Monoclonal antibodies are purified and covalently bound to Affi gels (Affi! 301) ~ 10 as described below.

Ascitic fluid J: Rino Nanatsuku [1 Preparation of null antibodies Onibi purification 1
Five tlt Bal h,/c mice were each injected with hybridomas taken at mid-logarithmic growth phase.
idoma) cells were inoculated. Approximately 5 x 10 samples obtained from ascites fluid produced by mice.
One viable cell was inoculated intraperitoneally into each of 10 or more mice. This ascites fluid was collected repeatedly (2 to 4 times) from each mouse. one nT
The ascitic fluid collected was pooled from each mouse to the next group of mice up to 3 times, -88 to -88-1.

Cells and debris were removed from the ascites fluid by low-speed centrifugation (500-1000'xg) for 15 minutes. Then 1B, O
The mixture was centrifuged for 90 minutes at OOrl)m. The supernatant was frozen and stored at -20°C. After melting, 35.00 Or
The mixture was centrifuged at pm for 90 minutes to remove fibrin particles. For each transfer, which bag of ascites fluid,
Solid phase antibody binding assay (Staeheli
The specific antibody activity f1 was tested according to the above-mentioned literature, and if it was satisfactory, the antibodies were pooled.

The protein in the pooled solution is 280 rll when 1 mg of protein is added to a 1.0 ctn thick base.
The measurement was carried out using an approximation method in which the absorbance 1+fi is 1.2.

High I! ii Ascites fluid with 1st grade antibodies is 30-35
J3 containing mg protein/11118, which is /l
Corresponds to -711rfl specific antibody/mQ. P138 (0.01M sodium phosphate, pH 7.3,
diluted with 0,15M NaCl) and diluted with 10 to 12 m9/
The protein concentration of rrtl is I.

89- Reprint of the specification (no change in content) Add 100 d of each diluted solution to 89- sulfuric acid saturated at room temperature.
1- Engraving of specification (no change in content) 90 d of ammonium solution was added at 0° C. with vigorous stirring. The suspension was kept in water for 40 to 60 minutes. Then, it was centrifuged at 10,000 rpm for 15 minutes at 4°C. - Remove the supernatant with a squeeze bottle and drain the liquid thoroughly.
l 7.9) 10.04M Dissolved in 148 cl (Buffer 1). The protein solution was dialyzed against 100 volumes of buffer ■ for 16 to 18 hours at room temperature. During this time, the buffer was exchanged at least once.

The dialysis solution was centrifuged at 15,000 rpm for 0 min.
Insoluble matter was removed. Approximately 30% of the initial amount of total protein in the crotch
35% was estimated from the absorbance value at 280 nm.

A solution containing 30-40 mg of protein for 11d was then added to a DEAE-T column equilibrated with Buffer 1. A column bed volume of at least 100 ml was used for each gram of added protein. O,' 02M Tris IIC
Ipl! 7.9 NaCl in O, '04M to 0.5M
By changing NaCl111 degrees linearly with HaCl concentration,
Color 90- 90-1- I have a copy of the specification (no changes to the contents). 0.06 to 0. The 1M NaC117) peak fractions were pooled, an equal volume of ammonium sulfate saturated solution 1111 (room temperature) was added, precipitated and centrifuged, and concentrated. Protein 13M to 0.2M Hall+203(1)I+approx.8
．． 0) Dissolved in 10.3M NaCI (Ilii solution ■), filtered and dialyzed against mild vIJ solution (3 exchanges) at room temperature. The dialyzed solution was centrifuged at 20.00X9 for 15 minutes to remove insoluble matter. Protein moisture was adjusted to 20 to 25 m9/mQ with buffer ■.

Preparation of immunoadsorbent Aneugel (801 ocl > -10 (Bio Lab Laboratories, Richmond, Cali Minor)
Rad LaboratoriesXRichmon
d, California) with glass filter-L
Washed three times with water-cooled isopropanol and then three times with water-cooled distilled water. The gel slurry (approximately 50% cold water) was transferred to a Plusdec tube and centrifuged briefly to sediment.

The supernatant was removed with an aspirator, and the packed gel was mixed with an equal volume of purified antibody solution and rotated at 4° C. for 5 hours so that the long axis rotated in a circular manner. After the reaction, centrifuge the gel 91-Specification (contents unchanged) and add buffer III (0, IM Na11C0
310, 15M NaCl) r2-91-1 was washed once to remove unbound antibodies. When the washing solution was combined and protein was measured, more than 90% of the antibody was bound to the gel.

To fill up any unreacted areas, add an equal volume of 0.1M gel.
Mix with 1-tanolamine IIcI (pl(8)) and process for 60 minutes at room temperature by rotating the long sleeve in a circular motion.

The gel slurry was washed with P 13 S to remove the reactants and stored in PBS at 4° C. in the presence of 0.02% (w/v) sodium azide.

N. Parenteral administration 1elF is administered parenterally to patients requiring anti-pulmonary or anti-viral treatment. It may also be administered to patients exhibiting immunosuppressed conditions. Dosages and rates of administration will be similar to those currently practiced clinically for substances of human origin. For example, approximately (1-10) x 106 units/day is 1
For purity greater than %, for example, 5 x 107 units/
Vines that reach the sun.

Essentially homogeneous bacterial 1c in a form for parenteral administration
A suitable dosage form of LelF31119, for example, has a specific activity of 2 x 108 units/l11g (7).
-92- Pass the filtered solution through a sterile filter and aseptically divide it into 100 tubes suitable for parenteral administration; sea urchin,
Each bottle contains 6 x 10 units of pure interferon. It is desirable to keep these bottles in a cool place (-20°C) before use.

The compounds of the invention can be formulated according to known methods for preparing pharmaceutically useful compositions.

The polypeptide is then mixed with a pharmaceutically acceptable carrier vehicle. For suitable vehicles and their formulation, see Martin (E.).

Remington's Pharmaceuticals (W, Hartin)
It is a3 as described in Pharmaceutical Sciences). This is cited as a reference for the present invention. In these compositions, the interface [
] An effective amount of a protein and a suitable amount of a vehicle are combined to form a pharmaceutically acceptable composition suitable for effective administration to a host. One advantageous mode of administration is parenteral administration.

93-

[Brief explanation of the drawing]

FIG. 1 is a radiogram showing possible hybridization between the +-eu plasmid and the 32p labeled synthetic deoxyoligonucleotide. Figure 2 shows 1. Eight types of eIF cloned cDNA (
A to 1-1) restriction endonuclease maps are shown. Figure 3 shows the construction of a gene encoding direct microbial synthesis to mature 1elF. Figures 4 and 5 show restriction maps of the two gene fragments used to express the LeIF B mature leukocyte protein. Agent: Akira Asari 94- Engraving of drawings (no changes to the contents) 32P-T-1c probe 32P-T-13C probe N1 diagram Hotokai JP-A-Sho GO-227694 (40) Continuation of page 1 ■ Int, CI ,' Identification symbol Internal serial number priority claim [Phase] 198 Cape September 8 [Phase] United States (US) [Stock] 184909 ■ 198 Shi November 10 [Phase] United States (U
S) ■ 205578 [phase] April 21, 1981 [phase]
United States (US) 256204 @ Inventor Sidney Plague Force United States Nil, Prutsukusai @ Applicant Genentek, Inco United States Carbonated Co Point SaAIQ- 1- North Caldouet Terrace, Chassis 82 Zaus San Francisco, Lifornia / Brno Boulevard 460 Procedural Amendment (Method) Date of Yugetsu 2R, Showa 2θ, Mr. Commissioner of the Patent Office ■, Indication of the Case, Patent Application No. 39, Showa 7. B-Issue 3, Relationship with the case of the person making the amendment Patent applicant address 4, Agent 5, Date of amendment order Drawing (No. 113E13 8, Contents of amendment Engraving of the drawing as attached) (No change in content)

Claims (1)

[Scope of Claims] (1) A polypeptide comprising a 7-amino acid sequence of human mature leukocyte interface [1], comprising a partial amino acid sequence C
ys -Ala -Trp -Gill -Val -
Val -Arg -8la -Glu--Ile-
Net -8rj] -3Qr and without a preceding sequence, 165-16
6 amino acids, or if methionine is bonded to the N-terminus of the first amino acid of the interferon, in producing a polypeptide containing 166-167 amino acids, this polypeptide A method for producing a polypeptide, which comprises growing a culture of a microorganism transformed with a microbial expression vehicle capable of expressing and replicating the polypeptide, expressing the polypeptide, and collecting the polypeptide. (2) If the polypeptide has no preceding sequence, add sp, GILI or Va to the amino acid at position 114.
l, or methionine is the first interferon
The method for producing a polypeptide according to claim 1, characterized in that when the amino acid Asp', Gl'u or vat is attached to the N-terminus of the 115th amino acid, the polypeptide is added to the 115th position. . (3) The method for producing a polypeptide according to claim 1, wherein the polypeptide is human leukocyte interferon A'(l', cTF 8). (4) The polypeptide is human leukocyte interferon B
A method for producing the polypeptide according to claim 1, which is (fel "B). (5) The polypeptide is human 1 to leukocyte interferon 0.
(101 [C)] A method for producing the polypeptide according to claim 1. (6) The method for producing a polypeptide according to claim 1, wherein the polypeptide is fen D. (7) Polypeptide for human white skin (Interferon F
(10I [“)”) A method for producing the polypeptide of Claim 1. (8) The polypeptide is human 1 to leukocyte interface I IT
Claim 1 which is “In I” (1, eTF H)
2. Method for producing the polypeptide of section 2. (9) The polypeptide is Hi I ~ White bulb intaf Tron I
A method for producing the polypeptide according to claim 1, which is (LeIF I). (10) The polypeptide is human leukocyte interferon J
(1, elrJ) A method for producing the polypeptide of claim 1 rn. (11) The polypeptide is claimed in claims 3 to 1.
A method for producing a polypeptide according to claim 1, which is a polypeptide obtained by a pair I gene mutation of any one of the polypeptides according to claim 0. (12) The method for producing a polypeptide according to claim 1, wherein the polypeptide is a polypeptide resulting from an allelic mutation to 1eTF. (13) A method for producing a polypeptide according to claim 1, wherein the polypeptide differs from IeTF A by one or more amino acids. (14) A method for producing a polypeptide according to claim 1, wherein the polypeptide consists of LeIF 8 and 231 amino acids. (15) A method for producing a polypeptide according to claim 1, wherein the polypeptide is a fish containing Arg at position 23 and is different from 1elFA.