JPS60221093A - Dna arrangement - 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, namely
It concerns the methods used in A technology and the products obtained by these methods.

More specifically, the present invention provides a polypeptide comprising the amino acid sequence of human mature leukocyte inkferon, the partial amino acid sequence Cys-Ala-Trp-Glu
-Val -Vat -Arg -Ala -Glu
-Ilc-Het-Arg-3er and without a preceding sequence, 165
-166 amino acids and amino 1Asp, Glu or Val at position 114, or if methionine is attached to the N-terminus of the first amino acid of the interferon, then 166-167 amino acids; and the amino acid Asp at position 115.
, Glu or Val.

First, the background of the present invention will be described. Human leukocyte interface a (LelF) was initially identified in l5aacs and L
Indenmann discovered and prepared it as a very coarse precipitate (PrOC,R,SOC.

8147.258-267 (1957); υ, s, p
.

3.699.222>. Efforts to purify and characterize it were continued for a long time and led to the preparation of a relatively homogeneous leukocyte interferon derived from leukocytes obtained from normal or leukemic donors (German Patent Publication Nα2.947.1).
34). These interferons are a group of proteins known to have the ability to confer a state of virus resistance on their target cells. In general, interferon is a cell's i! ! It mediates reproduction and regulates the immune response. These properties have promoted the clinical use of leukocyte interferons in the treatment of viral infections and malignant tumors.

Leukocyte inkferon is purified to an essentially homogeneous state (RubinsLein et al., Proc. Natl. A.
cad, sci.

U, S, A, 76, 67IO-644 (1979);
Zoon et al., supra, 76.5601'-5605 (19
79)) were reported to have molecular m ranging from about 17,500 to about 21,000. The specific activities of these preparations are extremely high (2 x 10 to 1 x 10 units/ml
Although it is a protein, the yield from cell culture methods is extremely low. However, with advances in protein sequence technology, partial amino acid sequences have been determined (Zoon et al., 5Cie.
nC8207, 527 (1980): Levy et al.
Proc, Natl, Acad, 5ciU, S, A, 7
7.5102-5104 (1980)).

Although the glycosylation of various leukocyte interferons is currently not completely clear, differences in glycosylation between species alone cannot explain the observed molecular weight distribution. Instead, leukocyte internoelones vary markedly in amino M composition and sequence according to type, with amino acid homology sometimes lower than 80%.

Although isolation from donor leukocytes has yielded sufficient m, homogeneous leukocyte interferon for partial characterization and limited clinical studies, it alone is insufficient for large-scale clinical trials and extensive prophylactic studies. and/or totally insufficient to provide the interferon needed for therapeutic use. Indeed, current clinical trials using interferon derived from human leukocytes for antitumor and antiviral purposes.
x ＼ Vice 1 meeting I Mm t If L% ”'r
k> I/1 It has not been possible to produce sufficient quantities even at impractical 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 by this technology to be able to produce polypeptide chains such as somatostatin, the A and B chains of human insulin, and human growth hormone.

(Itakura et al., 5science 198.105
6-1063 (1977): Goeddel et al., Na
ture281.544-548 (1979)). More recently, recombinant DNA technology has enabled the production of proinsulin and thymosin alpha-1, and some authors have also described the collection of DNA encoding human leukocyte inkferon and the resulting reported on the production of a protein with leukocyte inkferon activity (Naljata et al., Nature 284).
．． 316-320 (1980): Nantei et al.
Gene 0.1-10 (1980); Taniguc
hi et al., Nature 285, 547-549 (19
80)).

The tools of recombinant DNA technology are plasmids, which are non-chromosomal loops of doublet DNA that are present in bacteria and other microorganisms and often exist in multiple copies throughout the cell. The information encoded in plasmid DNA includes information necessary to reproduce the plasmid in daughter cells (
(i.e., a "replicon") and, usually, one or more selection characteristics, such as resistance to antibiotics in the case of bacteria. This information allows the target plasmid to be Plasmids are useful because they recognize clones of host cells that contain plasmid DNA and preferentially grow them in selective media. This means that the plasmid is specifically cleaved using different types of restriction endonucleases or limited oxygen. After that, both ends of the cut site,
A foreign gene or gene fragment is inserted into the plasmid by joining the reconstructed ends in contact with the cleavage site. DNA recombination can be carried out outside the cell, but the resulting recombinant plasmid is introduced into the cell by a method called transformation, and the transformed cells are allowed to grow to produce foreign genes. recombinant plasmids can be produced in large quantities containing the
If inserted correctly with respect to the portion of the encoded DNA information within the plasmid that governs transcription and translation, the resulting expression vehicle is referred to as the actual production or expression of the polypeptide sequence encoded by the inserted gene. can actually be used in the process.

Expression is initiated at a region known as the promoter, which is recognized and bound by RNA polymerase. In some cases, such as the tryptophan or "trp" promoters which are advantageous in the practice of the present invention, the promoter region may include
The "operator" regions overlap and combine to form a promoter-operator. The operator is a DNA sequence that is recognized by so-called repressor proteins, which serve to regulate the WA sequence of transcription initiation at a particular promoter. RNA polymerase moves along the DNA and converts the information contained in the coding sequence into its 5'
The messenger RNA is transcribed from end to 3' end, which is then translated into a polypeptide having the amino acid sequence encoded by the DNA. Each amino acid is coded for by a nucleotide triplet or a "codon". These are part of what, for the purposes of the present invention, is referred to as a "structural gene", i.e. codes for the amino acid sequence of the product to be expressed. It exists in the part that does. After binding to the promoter, the RNA polymerase first transcribes the nucleotide that encodes the ribosome binding site and then transcribes the translation initiation or "start signal" (usually ATG, which becomes AUG in the resulting messenger RNA). , transcribes the nucleotide cotons present in the structural gene itself. A so-called stop codon is transcribed at the end of the structural gene. Thereafter, even though the polymerase may form an additional sequence of mesenger RNA, the presence of the stop signal prevents it from being translated by the ribosome.

Ribosomes normally bind to specific junction sites on metsenger RNA in bacteria where mRNA is being formed. This results in an encoded polypeptide that begins with the translation initiation signal and ends with the stop signal described above.

If the sequence encoding the ribosome binding site is properly positioned with respect to the AUG initiation codon, and all remaining codons follow the initiation codon in phase, the desired product will be produced. The resulting product can be obtained by thawing host II cells and recovering the product from other bacterial proteins by appropriate purification methods.

We demonstrate that the use of recombinant DNA technology (i.e., inserting interferon genes into microbial expression vehicles and expressing them under the control of microbial gene regulatory elements) is an extremely effective way to provide large amounts of leukocyte interface. I thought. Although it lacks the glycosylation properties characteristic of human-derived materials, it may be used in the treatment of a wide range of viral and neoplastic diseases.

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

(1) Using a partial amino acid sequence of a human Shiromen method interface purified to a homogeneous state, it contains codons as a collection representing all possible combinations of nucleotides that can encode the partial amino acid sequence; Construct a set of synthetic NA probes.

(2) Complementary DNA from induced metsenger RNA
Prepare a bacterial colony bank containing (cDNA). Another radiolabeled inducible RNA is hybridized with plasmid cDNA from this bank. The hybridized lRNA is eluted and tested for translation to interferon in oocyte analysis. Plasmid DNA from colonies shown to induce interferon activity is thus tested for hybridization to the probe prepared as in (1) above.

(3) In parallel with step (2) above, prepare another transformed colony bank using the cDNA derived from the III RNA induced in the plasmid. The probe of (1) was used to induce the synthesis of radiolabeled single-stranded cDNA for use as a hybridization probe. The synthetic probe hybridizes with the induced mRNA as a template and is expanded by reverse transcription to induce the -
Generate radiolabeled cDNA. Clones from the colony bank hybridized to the thus obtained radiolabeled CDNA are further examined to confirm the presence of the full-length interferon-encoding gene. (
1) Also, the predicted partial-length gene fragments obtained in (2) can themselves be used as full-length gene probes.

(4) The full-length gene obtained above contains a leader (1) that may prevent microbial expression of the mature polypeptide.
using synthetic NA to allow for the removal of the Eader) sequence, if any, and proper positioning in the expression vehicle with respect to the microbial bromotor initiation signal and ribosome binding site. It was raised. The expressed interferon is purified, its properties are confirmed, and its activity is measured.

(5) The inkferon gene fragment itself prepared by the above method can be used to generate other partially homologous leukocyte interface proteins.
It is used for hybridization probes of various species.

By applying the recombinant DNA VL technique outlined above, q) a group of homologous leukocyte interfaces (glycosylated, do not have)
It has now become possible to produce microbially with high yield and high purity. These interferons can be directly expressed, harvested, and purified to levels suitable for use in animal and human viral and malignant eel ulcer diseases. A group of interferons thus expressed have been shown to be effective in in vitro tests and
It was the first leukocyte interferon produced microbially and for the first time to be shown to be effective in in vivo tests.

``Mature leukocyte interferon'' used in the present MA
The term refers to microbially (e.g., bacterially) produced interferons that lack the glycyl group.

The mature leukocyte interferon of the present invention is the first natural product.
The translation initiation signal (A
TG). Thus, although this "mature" polypeptide contains methionine (encoded by ATG) as the first amino acid in its sequence, its properties are essentially unaffected. On the other hand, the microbial host can process the translation product to remove the initial methionine. Mature leukocyte inkferons can be expressed together with conjugation proteins other than the normal leader, which can be specifically removed in the extracellular or intracellular environment (see GB 2007676A). Finally, mature InkfeO can be produced as a conjugate with a microbial "signal" peptide that transports the conjugate to the cell wall. The signal is processed and released in the cell wall, and the mature polypeptide is secreted.

"Expression" of mature leukocyte interferon refers to the production by bacteria or other microorganisms of inkferon molecules that do not contain glycosyl groups or preceding sequences, resulting directly from mRNA translation of the human leukocyte interferon genome.

Specific leukocyte interferon proteins are defined herein by determined DNA genes (Tables 2 and 4) and deduced amino acid sequences (Tables 3 and 5). Natural allelic variations exist for these particular inkferons, in fact for all groups of leukocyte interferon proteins included here.
r+atron) exists and can occur between individuals.

These mutations manifest as amino acid differences throughout the sequence or deletions, substitutions, insertions, inversions, or additions of amino acids within the sequence. For each of the ink ferons of interest in Suimei, labeled LeIF8 to LeIFJ, such allelic variation is included within the scope of the labeling or definition thereof and therefore within the scope of the present invention. Suppose it is included.

Next, the tables and figures will be explained.

Table 1 Protein Ala-Glu-I 1e-Met-Ar
g Trip, Apep, Do (T-13) ---His-Glu-Met-I 1e-Gln--
-Sl 510 520 s2] LelF G LelF HM A L P F S L M M A
L V V L S CK S S CS L G
CN LAll MA FL VLSKS 5GCLA
ll EEFD QjjqKA I VLlE QQ
FN1!0120 LelFA VGVTETPLMKEDSILAVRK
YFQRITLLelF8 1/GvIESPLMYE
DSILAVRKYFQRITLLelF CV G
V E E T P L M N E D S I L
A V R ni YFQRITLLelF D E RV
G E T P L M N V O S I L A
V K K Y F RRI T LLelFE V
GVEETPLRNVDSILAVRKYFqRLTL
l−+ccur:++ccTo+Mvunc, IfAV
KKYF(IRITLlo 20 30 40 TH5L RRL L OM Is SCL Yudan HO
Dan Dan QLFT SSA LL F ELQQNDE
Q'-N-u +-<<<<)-)-<<< LLJCr (r Cr Oααboα
Tadashi << Todoku αα Boba
L""J"-〇〇〇〇〇の paste ψli 222 k2 Eeeee: f: ROΦ0ΦO Tototototo 10 mouths 0CI Cr (7C70Cr L LL匡LLLQ+ψdai a8 to Kuchida-ichiichi'" JJJLIJLLJIJJLAJ-〇口20 Cl port sizeoCICr 1laJ C7-(J
(J (J L) (j l eL cLm (LΦ
(!lc!3Φ0 HI匡-匡H 〉”JJ-〇LA, IOααののI-1+ +−+ ええ代工工（I）のののI−１＋＋＋ αααばα yy'we y'1. 00RoroO QOshi>'knee 22M M'1 tail.

(1) C/l of l'') j, J w''''j
u, J-I (J(JωCJ(J〉〉shi〉〉 ivl
Me ψψ”>”, J u HILL LL LL LL”>
＞ ＞ JCL eL l yu, -+ ＜ ＜ ＞ ＜ Target's t/)LI1〉Σ
2ΣΣ -++ -+ ``Σ-''-ce lαboαα ``J''-BazC! 5 tJ rJ <t/)L/)L/) of ΣΣΣΣΣ LL LL LL LL L ('J Cr Cr C
CJ "u cyl>LL>>:)-)-)-)-)-)-田. 0 Mouth OJ”-”H1-LH-N-×yyny laJ LaJ LLI LLI LAJ Ct Qe
C1ce C1bi) Q” Ql: Qe 11!)
-1-+ )-)-)-Cr (7Cr Cy (7c
eαααα>> 1/) l/) lha 匡, LL匡匡匡-”””L LL LL LL LL ＞ ＞
>Shu 2 ReαHΦgeyy×--M M7MM Near 1
．． 0laJMMMsol tAu LJJ LAJ L
LI Oe oe o: CI Ce ooC7'i
C7u ``u''j 〜し＞＞＞＞−``j''
"j" u j "<<<<< 22222Q) )-
1-1/)のの−”””)−)−)−)−)−1,
1JLtJ CrCrC7l-1-H-1 (1) LA
(') (1) l/)oCr LLJ LAJ L
AJ Target's ``匡-1匡': Ie S': Ruru ni: I Oc: + oCs oL/'I 1/) (/l
t/) (1) <<<< (LAJ LIJ
LAJ LAJ LIJLL”1. J.K.” ((1:
”C(:('w:2 z:2 z (a)LQψmeψv
ΣΣΣΣΣ riαIIα3Iγ1/
) <II L/) l/l t/) ””””Σ:
E :E :E Σ02OOO+II:LL:LL:L
ayu -111-M M LIJ LJJ LIJ +
-)-)-1-)-LLI LIJ LIJ LaJ
laJr-1-t)-)-)-LIJ LLILaJ
LLJ Ll <<<< (:L/1Q)LI1shi)υ
li)-LIJ LiJ >do b” cpiCpiCpi. P0 and LL LL LL L 匡 ME〉Σ＞＞＞＞
＞＞＞J”””C! II (JC!7 QI
L) 〉:＞ )+ :＞ )−≧2222 〉〉〉
〉 −U田LIILLICyC7cy cy (r l
-I Hl-11-11-1(J (J (J
(JCr 00 Cr Cr O>>>>>>
l (L Q+CL IIJ 1.LIL+J LLI
IJJLAJ LIJ LI LJ LJ 4 'l
'l 'l 'lχeeeeee'"""yyyya -"""0ozOOLIJLLILAJLIJLJ,
Ishi:>:>:>:> z2:Z :Z :Z
Father: E ) − ΣH< :t: H”') CJ (Eng.
”) CJ (knee) tJ Table 1 shows T-1 and T-1
Shown are two amino acid sequences common to all types of interferons, isolated from human leukocytes and purified to homogeneity, called 3. All possible mRNA sequences encoding these peptides and their corresponding DNA to do
Sequence not shown. The letters A, T, G, C, and U indicate nucleotides containing the bases of adenine, thymine, guanine, sil-cine, and uracil, respectively. The letter N is the nucleotides A and G.

Indicates either C or (J. The polynucleotide is
It is written to be read from 5' (left side) to 3' (right side). Then, two small chains (“d, s,”
) When referring to DNA, the order is reversed for the lower or non-coding strand. Figure 1 shows possible L
This is a radiogram showing the hybridization of eIF plasmid and 32p-labeled synthetic deoxyoligonucleotide.

Table 2 shows the nucleotide sequences (coding strands) of eight gene fragments isolated as candidates for the expression of leukocyte interferons, respectively labeled "A" to "H1+". Regarding LelF,
The ATG translation start codon and stop triplet are underlined. The stop codon or 3' untranslated region following the stop codon is shown. Included Lel
The entire gene length for F lacks one codon that is present in the other codons, as shown in column 3, line A of Table 2. A 5' untranslated region precedes the leader sequence. Fragment E, which remains isolated, lacks the complete preceding sequence of the leader. However, the postulated mature LelF
Contains all genes for E.

Fragment G, which remains separated, does not have a complete coding sequence.

In Table 3, 8 Lel shown by the nucleotide sequence
Comparing F protein sequences. IUPAC-LUBCo
Mission on Biochemical N
Omenclature's suggestion of omitting one character is used. In other words, alanine is A and cysteine is C.
So, aspartic acid is D, glutamic acid is E, phenylalanine is F, chrysin is G, and histidine is 1.
'', isoleucine is 1, lysine is K, leucine is 1, methionine is M, asparagine is N, proline is P, glutamine is Q, arginine is R, serine is S1, threonine is King. , valine is represented by V, tryptophan by W, and tyrosine by Y. Numbers indicate amino acid positions. S indicates no signal peptide. The dash at position 44 in the 165 amino acid LelF A sequence is inserted to align this LelF^ sequence with the 166 amino acid sequence of the relaxed LelF. Le
The sequence of IFE is defined ignoring the extra nucleotides in its coding region (position 187 in Table 2). The asterisk indicates the stop codon at the interface. The amino acids common to all LeIFs ( Punido Doden LelF
(excluding E) are also shown. The underlined residues are amino acids that are also present in fibroblast interferon.

FIG. 2 shows [OI] 8 types of cloned cDNA (A
to H). Hybrid plasmids were generated using the dc:dG tailing method (Go
eddel, mouth, v0, etc., Nature287, 41
1-416 (1980)).

The cDNA insert can then be cut using PstI. The terminal line of each cDNA insert shows no flanking homopolymerized dc:dG tails. PvulI.

Restriction sites for EcoRI and BIJI [are also shown. The dark region indicates the coding sequence of mature LelF. The shaded area is the signal peptide coding sequence. White areas are 3' and 5' noncod sequences.

FIG. 3 shows the construction of a gene encoding direct microbial synthesis of mature LeIF A. Restriction sites and remainders are indicated (“Pst I” etc.). llb, pH indicates base pairs.

Figures 4 and 5 show restriction maps of the two gene fragments used to express the LelF B mature leukocyte interface. The codon sequences shown are the coding strand ends generated by digestion with the restriction enzyme Sau 3 a for the two cases shown.

Tables 4 and 5 show the DNA and amino acid sequences of five LeIF proteins, including types I and J (abbreviations are the same as in Table 3).

In Table 5, an asterisk (t for the corresponding DNA sequence)
In the following, preferred embodiments for carrying out the invention are not shown.

8. Microorganisms used In the described implementation, two types of microorganisms were used:
s, p, 4.190.495 description (7) E, C01
iX1776 and coli strain -12294 (endo8, tlli'', hsr-, hslIl+, described in British Patent Publication Na2055382).

respectively, the American Type Cu
Deposited in 1ture Co11ection, 8TC
C No. 31537 and 31446 are attached. All recombinant DNA experiments were carried out by National
It was performed in accordance with the appropriate guidelines of the Institute of Health.

The most advantageous embodiments of the present invention include strain E as defined above,
colix 1776 and E, coli K-12
E. coli and other microbial strains, including strain 294 as well as other known E. coli strains such as E. coli B, many of which are
the^merican Type Culture
Co11ection (ATCC), and are deposited and available at known microbial depositories. I would also like to see German patent 2644432. These other microorganisms include Bac i I lus e.g.
illus 5ubtilis and other enterobacteria such as 5alIIlonella typhimuriu
A replicating vine plasmid is used that is capable of expressing heterologous gene sequences. Yeast such as Saccharomyces
ceravisiae may also be advantageously used to produce inkferon proteins by expressing the gene encoding the interface under the control of a yeast promoter.

B, Raw material and purified LclFmRNA A
RNA can be obtained from human leukocytes.

Usually the 5enda i virus or sewcastte virus is used as described in German patent Nα2947134.
The method uses white blood cells from patients with chronic osteogenic leukemia that are induced to produce interface with a disease virus. The raw materials used in the work of Suimei II, which are particularly useful, are:
The Hill line, designated G-1, was derived from a patient with acute osteogenic leukemia. This hill line is oeff
ler, If, P., and Golde, D.W.
, .

5science 200.1153 (1978), RPHI (Rosewall
Park Memorial Institute)
1640 plus 10% heat-inactivated FC3 (fetal bovine serum)
, 25 mHill: PES buffer (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid)
and gentamicin-containing medium at 50 gg/Irtl and are subcultured (1 to 3 splits) twice a week.

Cells can be frozen in the growth medium described above with 10% 0% dimethyl sulfoxide. G-1 is the^mer
ican Type Culture Co11ect
ion (ATCCNQCRL8031).

G-1 cells were prepared using the method described by Rubinstein et al.
Proc.

Natl, 8 cad, sci, U, s, A, 7 6,
640-644 (1979)),
It was induced to produce 5endai or Newcastle. Cells were harvested at 5 hours of induction vL.
NA was prepared by the guanidine thiocyanate-guanidine hydrochloride method (Chirgwin et al., Biochcmistryl
8, 5294-5299 (1979)). RNA from uninduced cells was similarly prepared.

The 12S fraction of poly(A) mRNA was obtained using oligodeoxythymidine (dT)-cellulose chromatography and sucrose gradient ultracentrifugation. The method is
Green et al. (Arch, Biochem, Biop
hys, 172, 74-89 (1976)) and ohuyuma et al. (Arch.

Biochem, BiophVS, 188, 98-
104 (1978)) was used. This mRNA was analyzed by African frog oocyte analysis (Cavalier
i etc., Proc, Natl, Acad, Sci, tl,
S, 8, 74. 328'l-3291 (1977))
indicates an ink feron titer of 8000-10.000 units/microgram.

C. Preparation of a colony bank containing the LOIF cDNA sequence.
et al., J. Biol, Chem, 253, 2483-
2495 (1978) and Goedclel et al., Na
ture281.

544-548 (1979)), a double-stranded CDN
A was prepared. CDNA was fractionated according to size by electrophoresis on a 6% polyacrylamide gel.
A material 23 Or+g of size 1500 b,p was obtained by electroelution. 100 ng of this CDNA was tailed with deoxycytidine (dC) residues.
Hang et al., Nature275.

617-624 (1978), tailing (ta) with deoxyguanosine (dG) residues in the Pst engineering site.
iling ) L/ta4 7011g (7) BOliVar et al.
Gen02゜95-113 (1977)), which was used to transform [, colix 1776]. CD
About 130 tetracycline-resistant and ampicillin-sensitive transformants were obtained for NA no.

In a second similar experiment, for CDNAn(+, approximately i
A total of 12 294 E. coli strains were obtained which were resistant to tetracycline and sensitive to ampicillin. In this case, in the range of 600 to 1300b,p,
Size fractionated cDNA material was collected by electroelution and used for dC'c tailing.

D. Preparation of synthetic oligonucleotides J. Knowledge about the amino acid sequences of several tryptic fragments of human leukocyte interferon, LeIF mRNA.
This made it possible to design synthetic deoxyoligonucleotides complementary to various regions of A. Two tryptic peptides T1 and T13 were chosen. The reason is that their amino acid sequences are identical to all possible coding sequences (Table 1
), it is not only necessary to synthesize 12 and 4 undecamers. Four navy blue deoxyoligomethreotide probes were synthesized for each sequence. That is, three each (Ding-IA).

B, C, D) or one (T-13A, B, C.

Contains the oligonucleotide of D). The complementary deoxyoligonucleotides shown, 11 base chain length, were chemically synthesized by the phosphotriester method (Crea et al., Pro.
c, Nat I, ^cad, sci, U, s, A,
75, 5765-5769 (1978)). ■−
In the 13 series, four individual probes were prepared.

As shown in Table 1, as 4 pools of 3 probes,
Twelve T-1 probes were prepared.

12 T-13 series prepared as 4 pools of 4 respective probes and 3 primers each
The -1 probe was used to direct the synthesis of radiolabeled single heavy strand CDNA for use in hybridization probes. Template 11 RNA was derived from Sendai-induced KG-1 cells (8
Total poly(
A) 111 RNA. 32p-labeled cDNA,
Known reaction conditions (Noyes et al., Pr0C, Natl,
ACad, SCi.

U, S, ^, 76.1770-1774 (1979
)”) was prepared from these primers.

2018 Tris-11cI (pH8,3),
20 IO2KCI, 8o+HHgCl2.301+1
8 β-Merka 11 - Reactions were carried out in 60 μm volume in ethanol. This reaction consisted of 1 μg (
In other words, for the T-1 series, the total is 12μ9,
4 μg total for the T-13 series), 2 μ9 of “induced” 12S fraction IRNA (or 10 μ9 of uninduced poly(A) II RNA), 0.5 mH d^
T.P.

dCTP, dTTP, 200, l:i (a32p
) dCTP (^mersham.

2-3000Ci/m mole) and 60 units of reverse transcriptase (Bethesda Re5earch Lab
oratories) There is.

The product is 101scphadex (!I)G-5
Separate unbound label by gel filtration on a Q column and incubate with 0.3N Na01l at 70°C for 30 min.
RNA was degraded and neutralized with HCI. For hybridization, KafatO3 etc., Nuclcic Ac1
dRes.

7.1541-1552 (1979).

E., clone [1-L30, 16500 E. coli strains, 12 strains, 294 transformed strains (
To prepare 1 μg of plasmid DNA from each of the
The material was denatured and loaded onto a nitrocellulose filter in triplicate according to the method cited above by Niafatos et al.

Three sets of nitrocellulose filters containing 500 plasmid samples were a) induced CDNA primed with the T-1 set of primers.

b) ■-13 primed induced 1 cDNA and C) hybridized with uninduced cDNA prepared using both sets of primers. A clone was considered positive if it hybridized more strongly to one or both of the induced CD NA probes than to all of the uninduced probes. Select 30 positive clones (pLl-pL30) from 500,
Further analysis was conducted.

F, Identification of clone pt31-pL39, separation of plasmid (No. 104) containing the LeIF gene fragment using 32p-labeled 11RNA (LillF) as a probe.
Enhaug et al., Biochemistry, 15.1
858-1865 (1976)) using Grun
Stein and HOQneSS colony hybridization method (Proc.

Natl, Acad, Sci, υ, S, A, 72.39
61-3965 (1975)), E. colix
1776 transformed strains were screened. Non-inviting SM
Unlabeled 1 RNA from the cells was mixed 200:1 to the probe to compete with the uninduced mRNA present in the 32p-labeled preparation. labeled mRNA A
Hybridization of should occur preferentially to colonies containing the induced sequences. Three groups of transformants were obtained.

(1) 2 to 3% of colonies hybridized very strongly to 32p-mRNA.

(a 10% had a significantly lower degree of hybridization than the above group (1).

(3) The remainder showed no detectable culm hybridization signal.

Positive colonies (Group 1 and Group 21) were examined for the presence of interferon-specific sequences by analysis of specifically hybridizing interferon mRNA with plasmid DNA. Zyclin (20pg/Id), Diaminopimeline I
I (100μg/IJfri), thymidine (20pg/r
Each of the 60 robust colonies (group 1) was grown individually in 100 id of M9 medium supplemented with d-biotin (1 μl/Inf!) and d-biotin (1 μl/Inf!). For 1 liter of M9 medium, Na IIPO (6#)
, containing It2PO4 (3 g), NaC14 (0,5 g) and 8114CI (1 g), and after autoclaving, 1a of sterile IHHIISO4.
i! and 10#li of sterile 0.0IHCaC12! Add.

Ten cultures were pooled and described by Clewell et al., Bio.
chelistry9.4428-440 (197
0) Plasmid DN from 6 pools as described
A was isolated. 10 u of each plasmid DNA pool
9 G, t, HindllI Te cut, denature, DB
It was covalently bonded to M (diazobenzyloxymethyl) paper.

1 μl of purified mRNA from induced cells was hybridized to each filter paper. mRNA that does not hybridize
was washed and removed. specifically hybridized lRNA
was eluted and translated in African frog oocytes. All six pools were negative in this analysis.

From the 59 weakly positive colonies (group 2), 5 pools of 10 colonies each and 1 pool of 9 colonies were prepared, plasmids were prepared from these pools, and plasmids were prepared as described above. , hybridized to interferon III RNA A. To identify specific interferon CDNA clones, plasmid DNA was prepared from 9 out of 10 colonies in a pool and examined individually. Two of the nine plasmids (Nα101 and Nα104) bound interferon sRNA well above background levels. Plasmid N (from L 104, a unique 8gI I [
The silicate fragment was isolated. This is described by Taylor et al., Bi
ochim, BiopHys, ^cta442.32
1-330 (1976), and in situ colony screening method (Grt+n
5tein and llogness, Proc, N
at l.

Sci, U, S, A, 72.3961-3965 (1
975)), 400 E. coli 294 transformants were used as probes for individual screening.

Nine colonies (+)131-11139) were identified that hybridized to varying degrees with this probe.

Furthermore, 4000 E. coli 294 transformants were individually screened in the same manner using labeled 260b, l), fili pieces. Fifty colonies that hybridized to this probe to a species degree were identified. One contains the LelF G fragment, and one contains the LelF G fragment.
tl fragments, one containing a fragment designated LclF III (apparently an allele of LelF II). The resulting hybrid plasmid is “pLelF
11°' etc.

G. Isolation and Sequencing of the First Full-Length LelF Gene Plasmid DNA was prepared from 39 potential LelF c DNA clones overall. And to ar
The same 260b, p, DNA probe was used for rescreening using the hybridization procedure of atos et al. (cited above). This probe and three plasmids (pL4,
IIL31, +1134”) gave a very strong hybridization signal, and four (pLl3, pL
30.

DL32.1)136) is moderately hybridized and 3
(pL6, pL8, pLl4) were weakly hybridized.

The 39 potential LeIF cDNA recombinant plasmids also contained a 32p-labeled synthetic undecamer (
Each of the T-1 primer boule or each of the T-13 primers) was probed directly with the hyligation probe. Hybridization conditions were chosen such that complete base-base pairing was required to give a detectable hybridization signal (Wallace et al., Nucl.
eic Ac1d Res.

Dan, 3543-3557 (1979)). In other words, 3
Plasmid DNA was extracted from nine clones using the standard supernatant procedure.
, Clcwell et al., supra) and Biorad
Purified by Agarose A-507J 7 b chromatography. A sample (3μ) of each preparation was prepared using [co
R: Treated with [ to open the ring, denatured with alkali, and blown into two separate nitrocellulose filters.

1.5 μg per spot. +t in the above quote
See the literature by Aratos et al. Each of the synthetic deoxyoligonucleotide primers and primer pools was phosphorylated by 2 (γ P)ATP.

50 pmole of A ribonucleotide and 1o.

pmole's (732P) ATP (New
England Nuclear, 2500 Ci/
m mole), 50 mH of Tris-tlcI,
The 22p-labeled primer was combined in 30 μM of a mixture of 10 + nH H (ICI2 and 15 mH β-mercaptoethanol). 2 units of T4 polynucleotide kinase was added and after 30 min at 37 °C the 22p-labeled primer was incubated at 10 rtdl.
Purified by tenochromatography on a 5ephadex'G-50 column. Hybridization was performed using 106 cpm of primer T-13C or 3×166C11m of primer full T-IC.

Hybridization was carried out using 6XSSC (IXSSC=0.15HNaCl) according to the method cited above by Wallace et al.
, 0.0158 <sodium citrate, p1147.2
), 10x Denhardts (0.2% bovine serum albumin, 0.2% polyvinylpyrrolidone, 0.2%
The mixture was hybridized in a Ficoll solution for 14 hours at 15°C. The filter was washed three times with 6XSSCXSSC intermediate. It was dried and exposed to X-ray film. Result is,
FIG. 1 shows the case of 32p-primer pool T-13G and primer-T-IC.

Plasmid DNA from clone 104 hybridized significantly with primer pool T-IC and primer T-130. However, no detectable hybridization was observed with other undecamers. As shown in Figure 1, some of the 39 possible e1F plasmids (pL2, 4, 13, 17, 20.30,
31°34) hybridized with both of these probes. However, restriction analysis showed that not one of these plasmids, pL31, was identified as 26011.11. Inside B
Digestion of p[31 with Pst I revealed that the size of the cDNA insert was approximately 1000.
b, p, were.

The entire Pst I insert of pL31 was sequenced using HaXam
-Gilbert chemical methods
ol, 65, 499-560 (1980)) and dideoxy chain termination methods (Smith, Methods
Enzymol, 65, 560-580 (19
80)). At that time, the 5aU3 a fragment was 81
3 vector. The DNA sequence is shown in "A" in Table 2. Information on the available vine protein sequences, the range of known LeIF molecular weights, and the relative presence of stop triplets in the three possible reading frames suggested a suitable translational reading frame. The entire LeIF amino acid sequence, including the prey or signal peptide, was then shown. First AT Gil! The translation initiation codon is present at the 60th nucleotide from the 5' end of the sequence, and the TGA termination triplet is present 188 codons later. There are 342 untranslated nucleotides at the 3' end followed by a poly(A) sequence.

The postulated signal peptide (presumably involved in secretion of mature LeIF from leukocytes) is 23 amino acids long. The calculated molecular weight of the 165 amino acids that constitute mature LeIF is 19.390. We designated this LelF encoded by p[31 as "LelFA." From the sequence data (“A′”) in Table 3, tryptic peptides T1 and T13 of LelF B are 145-1 to LelF.
49 and 57-61, respectively.

The actual DNA coding sequences found in these two regions are primer pool TI-C and primer T1.
3-C (see Table 4).

11. Direct expression of mature leukocyte interferon A (LeIFA) The procedure for directly expressing LeIF A as a mature inkferon polypeptide is a synthetic (N-terminal)
and the method used for human growth hormone (GOe (Idel et al., Nat.
This is a modification of ure2B1, 544-548 (1979).

As shown in Figure 3, the 5au3a restriction endonuclease site is conveniently located between codons 1 and 3 of LelFA. Contains the ATG translation initiation codon, restores the codon for amino 1G1 (cystine), and EcoRI! To! Two synthetic deoxyoligonucleotides were designed that included the creation of a new i-coat end. These oligomers, pL31 (7
) 34b, p, Sau3a-Ava II fragment. The resulting 45b,ll' product was ligated to two additional DNA fragments and 865b,,p,synthesized-
It was made into a natural hybrid gene. It encodes LelFA and is flanked by EcoRI and PStI restriction sites. This gene contains Ecoll I and Pst
It was inserted into pBR322 between the I sites and named plasmid pLelF^1.

2. Tryptophan regulatory element (E, col i trp
Contains promoter, operator and trp leader ribosome binding site but ATGd for translation initiation
The transversely constructed plasmid pGHIG of the E, colit-lyptophan operon containing the deletion ΔLE 1413 (formerly Ozzari et al., J, Bacterioloc+V13
3, 1457-1466 (1978)). Therefore, the first six amino acids of the trp leader and t
A fusion protein is expressed that contains approximately the last third of the rp E polypeptide (hereinafter collectively referred to as LE') as well as the entire trp Q polypeptide. This is all under the control of the trp promoter-operator system. 20μ of this plasmid was added with restriction enzyme Pvu.
Digested with I. This cuts the plasmid at five sites. This gene fragment is then ligated with an EcoRI linker (consisting of an oligonucleotide self-complementary to pCATGAA-T'TC8TG) to provide an EcoRI cleavage site for later cloning into a plasmid containing an ECORI site. . D obtained from pG old
20 μ9 of 1 piece of NAll, 51 of 200 pmole
- Phosphorylated synthetic oligonucleotide A to pc to TG to TT
In the presence of C^Ding G, 20μ114 DNA ligase buffer (20mHTris pit 7.6.0.5mHAT
P, 1 oi14NoCI2,51118 dithiothreitol) with 10 units of T4 DNA ligase at 4°C overnight. The solution was then heated to 70° C. for 10 minutes to stop the ligation. The linker was cut by EcoRI digestion and E
The fragments with coRI ends were separated using 5% polyacrylamide gel electrophoresis (hereinafter referred to as PAGE), and the three larger fragments were first stained with tidium bromide, and the positions of the fragments were determined using ultraviolet light. The target part was separated from the gel by cutting it out. Place each gel fragment (300 μI O, IXTBE) into a dialysis bag;
And 0.1XTI3E buffer (TBE buffer is 10
．． 8g Tris base, 5.5g boric acid, 0.099
of Na2EDTA in 1 liter of H2O) at 100V for 1 hour. Collect aqueous solution from dialysis bag, extract with phenol, extract with chloroform, and extract with 0.2M
Sodium chloride m degree, DN in ethanol precipitated condensate water
A was collected. The trp promoter-operator-containing gene with EcoR■ sticky ends was identified by the procedure described below. Briefly, a fragment was inserted into a tetracycline-sensitive plasmid, which was made tetracycline resistant by insertion of a promoter-operator.

Plasmid pBR old (Rodriguez et al., NLI
CIeiCAcids Res, 6. 3267-32
87 (1979)) expresses ampicillin resistance and contains a tetracycline resistance gene.

However, since there is no promoter associated with it, it does not express its resistance. This plasmid is therefore tetracycline sensitive. By introducing a promoter-operator system into this EcoRI site, the plasmid is made resistant to tetracycline.

pBRtlI was digested with EcoR, the enzyme was removed with phenol extraction, chloroform extraction, and ethanol precipitated into water. Product NA present in separate reaction mixtures
The molecules were combined with each of the three DNA fragments obtained above and ligated with T4 DNA ligase as described above. Using the DNA present in the reaction mixture,
Standard methods (HerSMield et al., Proc, Na, Ac
ad, Sci, u, s, ^.

DN by L Yu, 3455-3459 (1974))
One 12 strains of E. coli 294 were transformed into E. coli capable of taking up A, and the bacteria were treated with L B containing 20 ug/d of ampicillin and 5 μg/d of tetracycline.
(Luria-Bertani) plates. Select some tetracycline resistant colonies,
Plasmid DNA was isolated and the presence of the desired fragment was confirmed by restriction enzyme analysis. The resulting plasmid was called pBRHt.
It is called rp.

EcoRI and Ba1ll of the hepatitis B viral genome
■Collect the engineered product using a conventional method and use the plasmid pGH.
Goedde et al., Nature 281, 54.
4 (1979)), plasmid pH 332. Plasmid pus 32 was cut with Xba I, phenol extracted, chloroform extracted, and ethanol precipitated.

The precipitates were 0, 1n+HdTTP and 0.11IHd.
30μm polymerase buffer containing CTP (50a
+H potassium phosphate pH7,4,7mHNoCI2゜1
1μIE, co
li DNA polymerase■, lenow fragment (
Boehringer-Hannheim) at 0°C.
for 30 minutes and then at 37°C for 2 hours. This treatment fills in two of the four nucleotides complementary to the 5' overhang of the Xba I cleavage site.

5'CTAGA-5'CTAGA-3' T-TC
T- Two nucleotides dC and six are incorporated to give an end with two overhanging 5' nucleotides. This linear residue of plasmid pu832 (extracted with phenol and chloroform followed by ethanol precipitation and taken up in water) was cut with EcoRI. The large plasmid fragment was
It was separated from the smaller [COR■-xbaI fragment by GE and separated by electroelution. pH332 (0.2 μg)
This DNA fragment was isolated from pBB under conditions similar to those described above.
10trp]・Liptophan Oberon (approx. 0
．． 01u! ? ) (7) ECORI-TaQ I l
It was connected to a li piece.

In this method of ligating the pH 332 fragment described above to the EcoRI-Taq I fragment, the overhanging end of Taq I is ligated to the remaining overhang of Xba I, although not in full Watson-Crick base pairing.

A portion of this ligation mixture was used to transform E. coli 294 cells, heat treated, and plated into LB plates containing ampicillin. Select 24 colonies, 371-
B (Luria-, Bertani) and the plasmids were isolated. Six of these colonies
was found to have an Xba'1 site regenerated by DNA repair and replication by E. coli.

-TCTAGA-, -TCTAGA- -AGCTCT--AGATCT- These plasmids contain EcoIII and Hpa
It was also found that it was cleaved by both of the following, giving the expected restriction fragment. One plasmid, designated pTrpl4, was used to express foreign polypeptides as discussed below.

Plasmid ptlGH107 (Goeddel et al., Na
tllre281.544 (1979)) is composed of 23 amino acid codons generated from a synthetic NA fragment and 163 amino acid codons obtained from complementary DNA generated by reverse transcription of human growth hormone messenger RNA. Contains the gene for human growth hormone. This gene is the “precedent” of human growth hormone
This gene lacks the sequence codon but contains the ATC translation initiation codon (10μI) HGHI O
7, EcoRI treatment followed by E as described above.
, E. coli DNA polymerase Klenow fragment and was isolated via dTTP and d^TP treatment. BaIIH! Processed with.

The human growth hormone (HGH) 311 gene-containing fragment is P
Separation was by AGE followed by electroelution. Generated DN
Although the A fragment lacks the tetracycline promoter-operator system, it also contains the first 350 nucleotides of the tetracycline resistance structural gene and therefore contains the insert when subsequently cloned into an expression plasmid. Plasmids can be identified by restoration of tetracycline resistance. Fragment EcoRI
The ends were filled in with the Ienow polymerase 1 method, so this fragment had one blunt end.
end ) and one sticky end. So,
This ensures correct orientation when later inserted into an expression plasmid.

The expression plasmid pTrp14 was then transformed into H
Fragments containing cut bamboo shoots containing the GH'ii gene were prepared.

That is, pTrpl4 was digested with XbaI, and the resulting sticky ends were digested with dATP, dTTP, and dGTPJ.
The Klenow polymerase 1 method using 5-Yohi dcTP was used. After phenol and chloroform extraction and ethanol precipitation, the resulting DNA was treated with Bam HI and the resulting large plasmid fragments were separated by PAGE and electroelution. The fragment derived from pTrpl 4 has one truncated end and one sticky end, and contains the HGH
This enabled recombination with the gene-containing fragment in the correct orientation.

HGH gene fragment and 1) 1rI114ΔXba-Ba
The mH1 fragment was merged and interconnected under conditions similar to those described above. The filled Xba I and EcoRI ends are blunt-ended ligated to the Xba I site and
Both oRI sites were reconstituted.

Filled Xba I Filled EcoRI IIG
I1m gene origin Xba I EcoRI This construct also creates a tetracycline resistance gene. Plasmid EllG11107 expresses tetracycline resistance from the promoter (lacj promoter) located upstream of the HGH gene, so the pH
This ift construct, designated GH207, allows expression of the tetracycline resistance gene under the control of the tryptophan promoter-operator. The ligation mixture was then transformed into E. coli 294 and colonies were selected on LB plates containing 5 μ9/mfl of tetracytalin.

Plasmid pHGI+ 207 was digested with EcoRI and t
rp promoter - contains operator and trp leader ribosome binding site but AT for translation initiation
The 300b, P, fragment lacking the G sequence was collected by PAGE followed by electroelution. This DNA1! The Ii piece was cloned into the [:CORI site of pLelFA.

An expression plasmid containing the modified trp regulon described above (an E. coli trp operon in which the attenuator sequence has been deleted to regulate high expression levels) has a sufficient amount of trp regulon to suppress the promoter-operator system. They can be grown to predetermined levels on tryptophan-supplemented media. The desired product is then expressed by removing the tryptophan and deregulating the system.

To be more specific, with reference to Figure 3, 250μ3
Plasmid pL31 was digested with Pst I and 1000
b, The insert of P was separated by gel electrophoresis on a 6% polyacrylamide gel. Approximately 40 μ7 of insert was electroeluted from the gel, divided into three portions, and further digested as follows. a) A 16 μg sample of this fragment was partially digested with 40 units of 1 μg of B for 45 min at 37°C and the reaction mixture was
% polyacrylamide gel. desired 670b
, p, fragment 2μ9 was collected. b) 1000 b
, p, Pst ■ Another sample (8 μg) of the insert was
Digested with II and B (II If). After gel electrophoresis, 1μ of the above 150b,p fragment was obtained.

C) 1000b, p.

16μ9 of the pieces were treated with 5au3a and Ava II.

After electrophoresis on a 10% polyacrylamide gel,
Approximately 0.25 μg (10 pmole) of the 34b,p fragment was obtained. the two indicated deoxyoligonucleotides,
5′-dAATTCATGTGT (fragment 1) and 5′-dGATCACACATG (ffi fragment 2
) was synthesized by the phosphotriester method. Fragment 2 was phosphorylated as follows. 200μm (approximately 4011m01
8) (732P) A T P (Amersham,
5000 Ci/mmole) and 6 nmH
Tris-11cI (pH 8), 10111H HgCl2.15ml 14β-mercaptoethanol was resuspended in 30 μl of a solution containing 100 pmole of the DNA fragment and 2 units of T4 polynucleotide kinase. After 15 min at 37°C, 1 μm of 10 mM AT
P 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 5
'-OH fragment 1 and 10 pmol e of 34 b
, p, merged with the Sau 3 a Ava II lli piece.

20mM Tris-HCI (al17.5), 1
0mMHgC12, 10ml (dithiothreitol, 0
．． 5 mHATP and 10 units of T4 DNA ligase
Ligation was carried out in 0 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, I), 3 of the product
0n(] (1pmole), 150 b, p, A
0.5 μg (5 μm
ole) and 670b, 11. BgI n-PSt
1 μg (2 μmole) of the I fragment. 20
Ligation was performed at 20° C. for 16 hours using T4 DNA ligase. The ligase was inactivated by heating at 65°C for 10 minutes. The mixture was digested with EcoRI and Pst to remove gene polymers. The mixture was purified by 6% PAGE. 865 of about 20no (0.04pmole)
b, p, The product was separated. Half of this! (10ng)
, pBR322 (0.3 μg) with EcoRI and P
It was inserted between the st I sites. E. coli 2
94 gave 70 tetracycline-resistant, ampicillin-sensitive transformants. Plasmid DNA was isolated from 18 of these and digested with EcoRI and PStI. Of the 18 plasmids, 16 were 865 b, p, long EcoRI-Pst.
It had an I fragment. One of these pLelF
1 μ9 of AI was digested with EcoRI, and E. coli
300 b, +1. as prepared above, with a trp promoter and a trp leader ribosome binding site.
The EcoRI fragment (0.1 μg) was ligated. Transformants containing the trp promoter are Grunstein-
Identified using a 3”P-trp probe in combination with the 11ogness colony screening method.trp
The asymmetrically located xba1 site in the fragment
This allowed the identification of recombinant products in which the p promoter was oriented in the direction of the m gene to LelF.

1. In vitro and in vivo active IF to LelF
Extracts for analysis were prepared as follows. One culture was grown in L broth containing 5 μg/d of tetracycline to give an A55 value of approximately 1.0 and then diluted into 25 d of M9 medium containing 5 μg/d of tetracycline.

When A35o reached 1.0, a 10 d sample was collected by centrifugation, and the cell mass was dissolved in 15% sucrose, 50mt4Tri
5-HCI (llH8,0), 50mM EDTA
1#117. Lysozyme of immy was added, and after 5 minutes at 0°C, the cells were disrupted by sonication. The samples were centrifuged for 10 minutes (15,000 rpm), and the inkferon activity in the supernatant was determined by cytopathic effect (CP
E) was determined by inhibition analysis compared to standard LeIF. To measure the number of IF molecules per cell, 4X
A LeIF specific activity of 108 units/mg was used.

As shown in Table 6, clone pLelF A trp 25, in which the trp promoter was inserted in the desired orientation, showed high activity (2.5×10 8 units/1). As shown in Table 7-
[, coli K-12 strain 294/pLe
The IF generated by IF Atrp25 behaved similarly to the human LeIF preparation. Stable to treatment with p112,
Neutralized by rabbit anti-human leukocyte antibody. The apparent molecular weight of this interfron is approximately 20,000. Clone pLelF A tri) 25 has been deposited with the American Type Culture Collection (ATCC).

Inkferon's in vivo effects require macrophages and natural killer (NK) 111 cells. And in vivo effects appear to involve stimulation of these cells. So, E.

Interferon produced by E. coli294/DLelF A 25 has antiviral activity in cell culture assays, but may be ineffective in infected animals.

Furthermore, non-glycosylated Le produced by bacteria
The in vivo antiviral effects on IF may be different from that of glycosylated LelF, which is derived from human "buffy coat" leukocytes. We therefore compared the biological activity of bacterially synthesized LeIF A (2% purity) with buffy coat LeIF (8% purity) in a lethal dose of encephalomyocarditis virus (EMC) infection of squirrel monkeys. Mitaku table 8)
.

Table 6 Interfaeron activity of E.coli extracts Table 7
1: Comparison of activity between C0Ii294/pLelF A 25 extract and standard 1, c I F'' E.coli294/pLelF described by Mr. 6
250,000 units per d of extract of A trp 25 was diluted 500 times in minimum essential medium to give a specific activity of 500 units/d. White blood cell ink noelon mark $ (Wa old ey
InStitlJte) was titered in advance against the N1)l leukocyte interferon standard, and the titer was also 5.
It was diluted to a final concentration of 00 LJ/d. 1d amount is 1N11
I) N2 in CI, incubated for 52 hours at 4°C, neutralized by addition of NaOH, and IF activity was determined by standard CPE inhibition assay. 25 μ of 500 units/d sample (untreated)
The cells were incubated with 25 μl of rabbit anti-human leukocyte induron for 60 minutes at 37°C, centrifuged at 12.0 OOX for 5 minutes, and the supernatant was analyzed.

Table 8. Antiviral effects of various LeIF preparations on EMC virus infection in squirrel monkeys. All monkeys were male (average body weight 7131 kg).
I have no MC virus antibodies. In the colander, 10100x
LD5oE virus (measured in mice) was infected intramuscularly. Control infected monkeys died at 134,158,164 hours post-infection.

106 units ink feron treatment 4 hours before infection, 2.23.29.48.72.168 and 240 hours after infection.
It was administered intravenously at 10:00. Bacterial leukocyte interferon was column chromatographically fractionated from the lysate of E. coli 294/1) LelF^25, and had a specific activity of 7.4X1.
06 units/~protein. Control bacterial proteins were E,
E. coli294/I) corresponding column fractions from BR322 lysates doubled the total protein content. Leukocyte interferon standard is chromatographically 32X1
The 5endai virus-induced ink feron from normal human buff ecoat'' cells was purified to a specific activity of 0.06 units/lny protein.

Control monkeys showed progressive coma, loss of balance,
The animals showed flaccid hind limbs, paralysis, and lachrymal drainage starting approximately 8 hours before death. Inkferon-treated monkeys did not suffer from any of these abnormalities, were always active, and showed no symptoms due to viremia. One control monkey that did not show viremia until 4 days later died (164 hours post-infection); EMC was found during the inspection on the 21st.
It did not produce antibodies against the virus.

Show that it can be done. This is because the contaminating proteins are completely different between bacteria and buffy coat preparations. Furthermore, these results indicate that glycosylation is not required for the in vivo anti-inflammatory activity of LelFA.

J. CDNA for other leukocyte ink ferons
Isolation of DNA from a fully characterized LelF A CDNA-containing plasmid. It was cut with Pst I, separated by electrophoresis, and labeled with 32p. The resulting radiolabeled DNA was used as a probe and the -GrunStO
An additional transformed strain of E. coli 294 obtained in the same manner as in Section 0 above was screened by the in situ and hooness in situ colony screening methods. We isolated "Ronnies" that hybridized to the probe to varying degrees. Plasmid D from these colonies
Plasmid DNA from NA and the 10 hybridized colonies shown in Section G above was isolated by Pst I digestion and characterized in three ways. First, these p
The st fragment was characterized by restriction endomeclease digestion pattern using the enzymes Bgl II, Pvu II and EcoRI.

% analysis was performed for at least eight different types (Lcll' A, LelF B, LelF C,
LelF D.

LeIF E, LeIF F, LeIF G and L
Enabled classification of old eIF. This indicates the approximate location of various restriction breakpoints with respect to the previously known leading and coding sequences of LeIF A.

Among these, LeIF D is believed to be the same as that reported by Nagata et al. in Nature LH, 316-320 (1980).

Second, some of these DNAs were tested for their ability to selectively remove LeIF mRNA from Poly A-containing KG-1111 RNA using the hybridization selection analysis described by C1eveland et al. in Ce1l 20.95-105 (1980). Investigated by. 1. eI[^,
B, C and [ were positive in this analysis. Third, insert these Pst fragments into expression plasmids and create E. co
li 294 was transformed with the plasmid and the fragment was expressed. All expression products believed to be preintaf10in were positive in the CPE assay for itaferon activity, except for the LelF fragment, which was marginally positive.

In addition to the above, all of the LeIF types listed above have been sequenced.

In the second mature leukocyte interferon (LelF B)
An isolated fragment pLel[A25 containing the gene for mature IQIF B] was isolated from pLel[A25, and the fragment with the comotor operator, the ribosome binding site and the beginning of LelFA (=B > The remaining part of the sequence was ligated to pL4 (containing the LelF B Pst terminal gene shown in FIG. 2, respectively).

According to the sequence in Figure 4, approximately 950b, 11. To obtain a Sau 3 a-Pst I fragment of 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) From Sau 3 a to EcoR: [to 110b
b, p, .

b) 132b, p. from IEcoRI to Xba.

c) >700b from Xba to PSt, l).

2. Fragments (1a) and (1b) were ligated and cut with Xba and BolII to create Sau3a and Xb
Prevented self-polymerization via the a-terminus (related Sau
The 3a site is within the BQIl[site: Jll[cleavage left a Sau3a sticky end]. 242b, p.
, the fragments were separated.

pst I and B (111
I cut it with. A fragment of approximately 950 b, p from Sau 3 a to Pst i (4th diagram) was isolated. This fragment is not common to LelFA: 1. It contained part of the ell BJ gene.

4, a fragment (approximately 300 b, l) containing the trp promoter-operator, ribosome binding site, ATG initiation signal and cysteine codon of LeIF A (formerly ndll to Sau3
(up to a) were isolated from pLelF25.

5, about 3600 from Pst I) to lindll
b, p.

The fragment was isolated from pBR322. It encodes replin and tetracycline resistance, but does not contain ampicillin resistance.

6 Triple ligate the fragments obtained in steps 3.4 and 5 and use the resulting plasmid to incubate E. coli K-12 strain 294.
was converted.

Convertible stocks were screened by mini-screening (Birnboim et al., N.
uclcic Ac1ds Rcs, E, 1513-1
523 (1979)), and plasmid samples were
Digested with RI. The digest gave three fragments, which were characterized by: 1) EcoR-nee EcoRI trp promoter fragment; 2) I) internal EcoR-Ec of L4;
oRi flIIH fragment; and 3) I) L4 protein translation initiation signal-IEcoRI fragment.

For CPE analysis, bacterial extracts from the clones prepared as described above were typically approximately 10 x 106 for A350-1.
Unit/j! of interferon activity. One representative loan prepared in this way is E. coli.
294/1) LelF B trD 7. This original version has been deposited with the AC CC.

[, yet another mature leukocyte interferon (LelFC)
Infection of OrHI and J Additional full-length gene fragments containing other LclF types can be placed into expression vehicles for tiller and expression, as in LelFA. The N-terminal amino acid codon that was lost due to the removal of the preceding sequence is synthesized by
A convenient method is to replace the A fragment by ligation, but whether the restriction site is sufficiently close to the mature intenocontyle to determine whether the complete restriction site is present by conventional methods is This will be known from the detailed sequencing. If that doesn't work, you can use the following operation. In short, the fragment containing the preceding sequence is cleaved exactly before the point at which the sequence begins for the first amino acid of the mature polypeptide. exactly 1 double-stranded DNA in the area surrounding that point
Converting to heavy strand DNA: 2. Convert the single strand DNA into single strand DNA by positioning the 5' end of the primer on the opposite side of the nucleotide that binds to the desired cleavage site in the single strand region generated in step 11). Hybridize complementary lines.

3. The part of the second strand removed in step 1 in the 3' direction from the primer is restored in a reaction with DNA polymerase in the presence of adenine, thymine, guanine and cytosine containing deoxynucleotide triphosphates.

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

A short synthetic NA carrying the translation initiation signal ATG at the 3' end of the coding strand is then ligated, for example by blunt end ligation, to the gene tailored for the resulting mature interferon, and this gene is inserted into an expression plasmid and placed under the control of a promoter and associated bosomal binding sites.

Gene fragments encoding Le[FC and LeIF D were suitably sequenced for direct bacterial expression in a manner similar to that used in section 2 above. As a strategy for the expression of these additional leukocyte interferons, in each case 1.1 from pLelF^25. clF
An approximately 300 b,p fragment (formerly ndln to Sau3a) containing the trp promoter-operator, ribosome binding site, ATG initiation signal and cysteine tons of A is used. In contrast, gene fragments from other inf10 genes encoding the respective amino acid sequences beyond the first cysteine, which is common to all, are ligated. E. coli K-12 strain 294 was transformed using each of the obtained plasmids. The connections to form each gene are as follows.

Isolate the following fragments from LelF C pLelF 'C: (a) S
35b, p from au3a to 5au96.

(b) >900b from Sau 96 to Pst I
, I) (C) As in section K(4) above, pLeI
A fragment of approximately 300b, p, from F^-25 (1lindl
l -5au3 a) is separated.

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

(1) Connect (a) and (C). Brill II
, cut with tlind ■, approximately 335b, +1. Separate the products.

(2) Perform triple ligation with (1) +(b) +(d) and transform E. coli with the resulting plasmid.

A representative loan prepared in this manner is [, coli
K-12 strain 294/pLelF ctrp 3
It is 5. This clone has been deposited with the ATCC.

LeIF D pLelF Separate from D as follows: a) sa
35b from u 3 a to Ava II, 1) b) A
vaI [150b from Bgl II, pc) B
Approximately 700b from gl II to Pst I, l]+)L
From eIF A25, isolate the following fragment: d) old n
300b from dnl to Sau 3 a, 11, pBR
322 to isolate the following fragment: e) old ndl [[from Pst I to about 3600b, p.

(1) (a) + (b) are ligated and cleaved with Bgl I [185b,p, product (1) is purified.

(2) (1) Concatenate +(d) and form old ndI[[,B
(III) and purify the product (2) by approximately 500 b,p.

(3) (2) + (c) ligate (e) and transform coli with the resulting plasmid.

Representative clones prepared in this way were [,co
il K-12 strain 294/pl-elF D tr
i) It is 11. This clone has been deposited with the ATCC.

Fragment containing LeIF F LeIF F contains telF B and L
Direct expression can be achieved by rearrangement that takes advantage of the fact that amino acids 1 to 13 of eIF F are completely homologous. From the above pHGH207, ps
via t i and Xba i digestion, then about 105
By separating the 0b and +1 fragments, a tl'p promoter-containing fragment (a) with appropriate sequence ends is obtained. The second fragment (b) consists of plasmid 1) l-IKYl
The larger of the fragments resulting from Pst I and B (11I) digestion of o. and contains all of the tetracycline gene except the combined promoter. Fragments (a) and (b) are joined by a four-ligapi enzyme, and the product is treated with Xba I and B(]l). The fragment (C) containing the trp promoter-operator and the tetracycline and ampicillin resistance genes is obtained by preventing diminization from occurring.

Fragment (d) of approximately 580 b, p is A of pLelF F.
Obtained by digestion of va II and Bgl II. This is 1. Contains codons for amino acids 14 to 166 of elF l:.

Fragment (e) (49b, p,) is the
ba I and Ava I [obtained by digestion. Fragment (e
) encodes amino acids 1 to 13 of LelF F.

Fragments (c), (d) and (e) are triple ligated in the presence of T4 ligase. The cohesive ends of each fragment ensure that the composite plasmid is properly circularized and that the t1-lacycline resistance gene, together with the mature LelF F gene, is under the control of the trp promoter-operator. Bacteria transformed with the desired plasmid can then be selected on tetracyclin-containing plates. Such preparations have been deposited with the ATCC.

1. eIF H The complete [eu H3H gene can be arranged as follows to express it as a mature leukocyte inkferon.

1. Plasmid pLelF H was transformed into HacI [and Rs
ai digestion to separate the 816b,p fragment spanning from signal peptide amino acid 10 to the 3' non-coding region.
IIt.

2. Denature this fragment and inject it with DNA polymerase l(I).
(KInow et al., Proc, Naguchi, Acad, Sci.

Synthetic deoxyribo-oligonucleotide primer 5 was prepared by
'-dATG TGT AAT CTG Processing is performed using repair composition.

3. Cleave the resulting product with Sau 3 a to separate the 452b,p fragment representing 1 to 150 amino acids.

4. Digest pteu H with au3a and Pst I, separate the resulting 500b, l) fragment, and obtain gene f containing the code from amino acid 150 to the end of the coding sequence.

5. The fragments separated in steps (3) and (4) are ligated to create fragment 1 166 met cys asp stop ATG TGT...H----GAT TGA-...
- Obtain pst l5au 3 a. This encodes 166 amino acids of LeIFH.

6. Digest pLeIF A trp 25 with Xba I, make blunt ends using DNA bolimerer t'I, and digest the product with Pst I. The large fragment obtained can be separated and ligated with the product of step (5) to form an expression plasmid. This can be used to transform E. coli K-12 strain 294 or other host bacteria to express mature 1, oIUH.

The face λ of the human genome constructed by LeIF I Lawn et al.
CI+aron 4 A recombinant library (Cel+
.

1 Ch., 1157 (1978)) as cited above.
The method of n et al. and Maniatis et al., Ce1l 1 branch.

687 (1978) for leukocyte interferon. Approximately 500,000 plaques were screened using the radioactive 11LelF probe derived from CD N A clone tel'A. Six 1.ell' genomic clones were selected. Following rescreening and plaque purification. One of these clones, λHLelF2, was selected for further analysis.

Using the methods described above, other probes can also be advantageously used to isolate other LeIF clones from the human genome. These can then be used to construct other leukocyte interferon proteins according to the invention.

1. 2000 b of clone λHLelF 2, I)
, EcoRI fragment in pBR at the EcoR]: site.
325. The obtained plasmid LelF I was cut with EcoRI, 2000b, p.
The fragments were separated.

This 2000 b, I), the first thiooligonucleotide dAATTCTGCAG (IEco
RI-Pst I converter). The resulting product was cleaved with Pst I to give a 2000b, p, fragment with Pst I. This was cut with 5au95. one pst I and one 5au96
The 1100b,p, fragment with the ends was isolated.

2. Convert plasmid pLelF Ctrp 35 to Pst
Digested with I and Xba I. Large pieces were separated.

3. Small XbaI from pLcTF Ctrp 35
-Pst I fragment was digested with Xba■ and 5au96r.

The 40 b, p, xba I-Sau 96 fragment was isolated.

4. The fragments separated from steps (1), (2) and (3) were ligated to form a pLelFI trp 1 expression plasmid. This clone has been deposited with the ATCC.

LelF J 1, plasmid pLelF J contains a 3.8 kilobase Hindl[l fragment of human genomic DNA containing the LeIF J gene sequence. From this plasmid 7
60 b,p,DdeI-RsaIlli fragments were isolated.

2. Plasmid I) LeIF B trl) 7 was cut with old ndll and Ode I, and the 340b, p, old ndl [-06e 1 fragments were isolated.

3. Plasmid pBIt322 was cut with Pst 1, incubated with DNA polymerase 2, made blunt ended (Klenow fragment), and then digested with old ndll. A large (approximately 3600b,p) fragment was isolated.

4. The fragments separated in steps (1), (2) and (3) were ligated to create the expression plasmid pLel[J trp
It was set to 1. This clone has been deposited with the ATCC.

L dish 1 The leukocyte ink feron content in the bacterial extract was increased as follows.

1. Polyethylene-imine precipitation. Here, interface 1
Most of the cellular proteins, including protein, remain in the supernatant.

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

3. Ammonium sulfate pellets were mixed with 0.06M potassium phosphate, 10 mHTris-flcI, pH 7.2
Suspended in 25 mM Tris-HCI, pH 7
, 9. Intaf10in activity remains in solution.

4. Adjust the above supernatant to pH 8.5 and perform cloning gelatinization on a DEAE-cellulose column (25 mH
0 to 0.2M N in Tris-HCI, pH 8.5
elute with a linear gradient of aCl).

5.Cibachrome Blue-A gar
Adsorb to ose or hydroxylapatite and elute with 1.58KCI or 0.2M phosphate (as necessary).

6. Size the molecules using a 5ephadex G-75 column.

7, p+I5. Perform cation exchange chromatography on CM-cellulose in 2511IH ammonium acetate. Develop with an ammonium acetate gradient (up to 0.2M ammonium acetate).

The method described above yields a product with >95% purity.

The material may also be purified by size exclusion frontography, reverse phase (RP-8) high pressure liquid chromatography, or affinidi cloning on immobilized anti-interferon antibodies.

Alternatively, the material from step 4 above can be
SScientific American 243.
66 (1980), plus 0.2 M acetic acid, 0.1% TI'1tO
It can be eluted with n and 0.15H NaCl.

In another advantageous embodiment, the leukocyte intanoeron produced in the above procedure can be purified in the following step.

1. Grind the frozen cell mass containing the expressed leukocyte inkferon manually or with a suitable grinding device. Partially dissolved cells were adjusted to pH 7.5-8.0,
1M Tris, 1Q% (W) Sucrose, 0.2
HNaCI, 5mM EDTA, 0.1iHPMSF
and 10-110-1O0IIIHH-containing buffer in 4 volumes. The suspension is kept at approximately 4°C.

The suspension was passed through a homogenizer at approximately 6 oopst;
Then run it a second time under 1000psi.

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

2. Slowly add polyethylene-imine to the homogenate to a concentration of about 0.35% and leave for about 30 minutes. Remove solids 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 approximately 1/10 of the original volume.

If particulate matter or cloudiness 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/hr (eg, 25-40 mhr on a 2.6 tJ diameter column). This column was then injected into approximately 10 column volumes of 20m HTris-HCI, Dll.
7.5-8.5 (HaCl (0,5M) and Tr
iton X-100 (<0.2%) or equivalent surfactant-containing). After washing, add 0.15 to this column.
HNaCI and Triton X-100〈0.1
%) or equivalent surfactant is passed through about 10 column volumes. This column was used as Triton X-1.
0 (0,1%) or equivalent containing surfactant
, eluted with 2Ml acid. Protein peak from monoclonal antibody column (measured by LIV absorption or other methods)
Collect 1NHa leaves or 1. p with OM Tris base
Adjust l+ to approximately 4.5.

4. After adding the pooled ink feron peak to an appropriate pH value such as ammonium acetate, wash the column with an equilibration buffer until the UV absorption of the effluent becomes irritating, and The condition is such that almost no protein is eluted from the sample.

The column was then washed with 25mM ammonium acetate 10.12
M sodium chloride or a combination that gives the best recovery of interferon is eluted to give a lyophilized cake with satisfactory appearance and solubility.

The monoclonal antibodies in this advantageous embodiment described above include 5tachcl in, Proc, Natl, Ac
ad, Sci.

It can be prepared by the method described in U.S.A., 7th Edition, 1848-52 (1981). Monoclonal antibodies are purified and covalently coupled to AffiSat-10 as described below.

Preparation and purification of monoclonal antibodies from ascites fluid 1IB
Each of five alb/c mice received a hybrid y-? taken midway through the logarithmic reproductive phase. (hybridoma
) 5-10×10 cells were seeded. Approximately 5×10 6 viable cells obtained from the ascites fluid produced by the mice were inoculated intraperitoneally into each of 10 or more mice. This ascites fluid was applied repeatedly to each mouse (2 to 4
times) were collected. Transfers and harvests were performed from one group of mice to the next group up to three times. Ascites fluid collected after each transfer was pooled.

Cells and debris were removed from the ascites fluid by low-speed centrifugation (500, -1000×g) for 15 minutes. Then 18.0
Centrifugation was performed at 0 rpm for 90 minutes. The supernatant was frozen and stored at -20°C. After melting, 35. OOOrp
Fibrin and particulate matter were further removed by centrifugation for 90 minutes at m. Batches of ascites fluid from each transfer were tested for specific antibody activity by solid phase antibody binding assays (5taehel in et al., cited above) and pooled if satisfactory.

The protein concentration in the pooled solution was determined using the approximation that 1 Illg of protein gives an absorbance value of 1.2 in 28 On 1Il in a 1.0 cm thick cuvette.

Ascites fluid with high concentration of antibodies has 30-35 my protein/Ill+! Contains. This is 4-7■ specific antibody/
Corresponds to d. This liquid was dissolved in PBS (0.01M sodium phosphate, I)117.3.0.15M NaCI)
Dilute with 10 to 12#1g/d! The protein concentration was set to

100 #11 each of diluted solutions! To this, 90 iie of ammonium sulfate solution saturated at room temperature was added at 0° C. with vigorous stirring. The suspension was kept in water for 40 to 60 minutes. Then, it was centrifuged at 4° C. and 10,000 rpl for 15 minutes. The supernatant was removed by straining, and the liquid was thoroughly drained.

The protein mass was dissolved in 0.02M Ir1sHCI (pH 7,9
Dissolved in >10.04M NaCI (Buffer I). Add the protein solution to 100 volumes of buffer 1 at room temperature.
Dialysis was performed for 6 to 18 hours. During this time, the buffer was exchanged at least once.

15. dialysis solution. The mixture was centrifuged at OOOrpm for 10 minutes to remove insoluble matter. Approximately 30 to 35% of the first m of total protein in ascites was collected as estimated from absorbance values at 280 nm.

A solution containing 30-4011 rfl of protein for 1 d was then treated with DEA equilibrated with 3 uffcr I.
Added to E-cellulose column. A column bed volume of at least 1.0 OId was used for each gram of protein added. N in 0.02M Tris tlcl pH 7,9
The antibody was eluted from the column by changing the NaCl concentration linearly from 0.04 M to 0.5 M NaCl at 11 degrees. The peak fractions from 0.06 to 0.1M NaCl were pooled, and an equal volume of saturated ammonium sulfate solution (room temperature) was added, precipitated and centrifuged, and concentrated. 0.2M protein mass
Na1lCO3 (pH approx. 8.0) 10.3M NaC
1 (Buffer II) Dissolved at room temperature, same buffer (
Dialyzed against 3 changes). The dialyzed solution was 20.0
The mixture was centrifuged at 00xy for 15 minutes to remove insoluble matter. Adjust the protein concentration to 20 to 25 m'J/mf with buffer ■! Closed.

Preparation of immunoadsorbent 8ffigel-10 (Bio Rad Labor
atories.

Richmond, Calif.) was washed three times with water-cooled isopropanol and then three times with water-cooled distilled water on a glass filter. Gel slurry (cold water towel approx. 5
0%) was transferred to a plastic duplex and briefly centrifuged 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, the gel was centrifuged and buffer solution (0.1M N811CO310,
The cells were washed twice with 15M NaCl) 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.
The mixture was mixed with ethanolamine/ICI (p)l 8 '') and treated at room temperature for 60 minutes by rotating the long axis in a circular shape.

Wash the gel slurry with PBS to remove the reactant, and store at 4°C.
In the presence of 0.02% (W/V) sodium azide, P
Stored in BS.

N. Parenteral Administration LeIF can be administered parenterally to patients in need of anti-tumor or anti-viral therapy. It may also be administered to patients exhibiting an immunosuppressed state. Dosages and rates of administration will be similar to those currently practiced clinically for substances of human origin. For example, a purity of greater than 1% at about (1-10)

Essentially homogeneous bacterial LeI in a form for parenteral administration
Suitable dosage forms of F include, for example, 2 x 108 units/
Specific activity of ll1g (7) LelF3mg is 25mf! of 5N human serum albumin, pass this solution through a biological filter, and aseptically divide the filtered solution into 100 bottles, each containing 6 x 1
06 units of pure interferon. It is desirable to keep these bottles in a cool place (-20°C) before use.

The compounds of this 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 [Re, W. Hartin.
m! n(]jOn'S PIlarmaCeujICa
As described in 15sciences.

This is cited as a reference in this specification. In these compositions, an effective amount of an intaf-10 protein is combined with a suitable vehicle into a pharmaceutically acceptable composition suitable for effective administration to a host.

One advantageous mode of administration is parenteral administration.

[Brief explanation of the drawing]

FIG. 1 is an orthoradiogram showing possible hybridization of the 1,eH plasmid with a 32p-labeled synthetic deoxyoligonucleotide. Figure 2 shows eight types of LelF cloned cDNA (A
to 11) are shown. FIG. 3 shows the construction of a gene encoding direct microbial synthesis of mature LelFA. Figures 4 and 5 show restriction maps of the two gene fragments used to express telF B mature leukocyte internoelon. Agent Akira Asamura Drawing (j・t, (1°・j each unchanged) 32P-T
-13c Probe - Figure 1 Female hard-eyed mountain Q OLLI L Q Construction procedure amendment (method) September 1999 ΔΔ Date Mr. Commissioner of the Japan Patent Office 1, Incident indication Showa ζ 2 Patent Application No. 2 ζ Mi 9-1 B No. 2, Name of the invention DA/A layer and group 3. Relationship with famous case for amendment Patent applicant 4, agent 5, date of amendment order More particularly, the present invention relates to a polypeptide comprising the amino acid sequence of human mature leukocyte inkferon, comprising the partial amino acid sequence Cys-8la-Trp-Glu
-Val -Vat -Arg -8Ia -Glu
-11e -) 1et -Arg-3er and without a preceding sequence, 16
5-166 amino acids and the amino acid Asp, Glu or Vat at position 114, or if methionine is attached to the N-terminus of the first amino acid of the interferon, 166-167 amino acids and an amino acid ^S at position 115
It relates to a DNA sequence characterized in that it consists of a sequence encoding a polypeptide having p, Glu or Vat. First, the background of the present invention will be described. Human leukocyte interferon (LelF) was first identified by Isaacs (l5a
acs) and Lindenmann (Lindenmar)
Ill) was discovered and prepared as a very coarse precipitate by [Brock, Earl, Soci, (Pro.
c. R,Soc,)B 1 47. 258-267 (1
957): U.S. Patent (u, s, p,) 3.699.222). Efforts to purify and characterize it have been ongoing for a long time, leading to the preparation of a relatively homogeneous leukocyte ink feron derived from leukocytes obtained from normal or leukemic donors (German Special Publication No. 2.947.1).
34. ), these interferons are a group of proteins known to be responsible for their ability to confer a virus-resistant state to cells within the plug. In addition, inkferon inhibits cell proliferation and suppresses immune responses.
It can act like a clause A. These properties have promoted the clinical use of leukocyte inkferons in the treatment of viral infections and malignant tumors. Leukocyte interferon has been purified to an essentially homogeneous state (Rubinstein et al., PrOC.
, 640-644 (1979):Zoon
et al., Id., 76° 5601-5605 (1979)) were reported to have molecular weights ranging from about 17.500 to about 21,000. The specific activity of these preparations is very high, between 2x10 and 1x1098 units/m9 protein, but the yield from the Ill cell culture method is appallingly low. However, with advances in protein sequencing technology, partial amino acid sequences have been determined (zones).
Science 207
, 527 (1980) Levy et al., Blok, Natl., Akad, Zai. United States (Proc, Natl, Acad, Sci, U,
S, 8,)77゜5101-5104 (1980))
. Although the glycosylation of various leukocyte inkferons is currently not completely clear, differences in glycosylation between species alone cannot explain the observed molecular weight distribution. Instead, leukocyte ink ferons vary markedly in amino acid composition and structure according to type, with amino acid homology in some cases lower than 80%. Although isolation of donor leukocytes has yielded a homogeneous leukocyte ink feron of sufficient size for partial characterization and limited clinical studies, it is not sufficient for large-scale clinical trials and extensive clinical studies. It is simply insufficient to provide the inkferon needed for prophylactic and/or therapeutic use. Indeed, current clinical trials using human leukocyte-derived inkferon for antitumor and antiviral purposes use crude (less than 1%) preparations. Even at impractical prices, it has not been possible to produce sufficient quantities, which has delayed RILL research in a wide range of areas. However, with the advent of recombinant DNA technology, it has become possible to control the production by microorganisms of a wide variety of useful polypeptides. 1-This technology has been modified to make it possible to produce polypeptide chains such as somatostatin, the A and B chains of human insulin, and human growth hormone. It has been reached. (Itakura, etc., Xience (Sc)
i ence ) 198.1056-1063 (1
977); Godel et al., Nature 281. 54.4-548 (1979)). More recently, recombinant DNA technology has enabled the production of proinsulin and thymosin alpha-1, and some authors have also developed DNA encoding human leukocyte interferon.
We report on the collection of A and the resulting production of a protein with leukocyte interferon activity (
Nature such as Nagata
e) 284, 316-320 (1980); Nantei et al., Gene
1 death. 1-10 (1980): Taniguchi
) etc., Nature 285.547
-549 (1980)). The tools of recombinant DNA technology are plasmids, which are non-chromosomal loops of doublet DNA that are present in bacteria and other microorganisms and often exist in multiple copies throughout the cell. The information encoded in the plasmid DNA includes the information necessary to reproduce the plasmid in the daughter 11 [11 cell (i.e., "replin"]) and, usually, one species or more than one species. For example, in the case of bacteria, there is information about the selection properties of antibiotics.This information allows us to recognize host cell clones containing the plasmid of interest and prioritize them in the selection medium. Plasmids are useful because they can be specifically developed by one type or another of fri11 restriction endonucleases, or ``restricted oxygen,'' each of which recognizes a different site on the plasmid DNA. This means that it can be severed. After that, both sides of the cutting site 3 LelFHMALPFSLMMALVVLSCKSSC
5LGCNLSQTH5LNNAll MA FL V
LS to S! GCL TH5LAAll EEFD
QFqKA I VLIIE! IQ jjNI4 T
55゜20 10 40 IRL L QM Is S! ,L 0R)IDF danqI LL F ELQqNDE Q 'L#lWjVVKAj1MHKO1koL)K111Jh
) shows the nucleotide sequence (]-do chain) of ILRRKE. For each LeIF, the ATG translation start codon and stop triplet are underlined. The stop codon or 3' untranslated region following the stop codon is shown. As shown in the third column A line of Table 2, the entire gene length of LelF A included lacks one codon that is present in other codons. 5′
An untranslated region precedes the leader sequence. Fragment E, which remains isolated, lacks the complete preceding sequence of the leader. However, it contains the entire gene for the postulated mature LelFE. Fragment G, which remains separated, does not have a complete coding sequence. In Table 3, 8 Lel shown by the nucleotide sequence
Comparing F protein sequences. IUPAC-LtlB Commission on Biochemical Culture
The abbreviation of one character proposed by alNOIIIQnClature) is used. In other words, alanine is A, cysteine is C, aspartic acid is D, glutamic acid is E, phenylalanine is F, glycine is G, and histidine is 1. ) etc., Nature 287.411-416 (1
980)). The CD N A insert can then be cut using Pst I. The line at the end of each CD N A insert is the homo chassis dC: (IG detail shown, P
vu II. EcoRI and B1111 restriction sites are also indicated. The dark region indicates the coding sequence of mature LelF. The shaded area is the signal peptide coding sequence. White areas are 3' and 5' noncod sequences. Figure 3 shows the construction of a gene encoding direct microbial synthesis to mature LelF. Restriction sites and remainders are indicated ('PstI', etc.). ttb, p, rr indicate base pairs. Figures 4 and 5 show the two genes used to express telF B mature leukocyte inkferon. Restriction maps of the fragments are shown. The codon sequences shown are the ends of the DNA chain produced by digestion with the restriction enzyme Sau 3 a for the two cases shown. amino acid(
The abbreviations are the same as in Table 3). In Table 5, an asterisk indicates a stop codon in the corresponding DNA sequence and a hyphen indicates a deletion or gap in the sequence. A. Microorganisms Used In the described implementation, two types of microorganisms are used, viz.
coli ([, coli) X 1776 and E. ] pickpocket, coli) K-12294 strain (end A,
thi-, hsr-, hsm+, British Patent Publication N. 2055382) is used. respectively, the
American Type Karuji A7- Collection (t
bc American Type Culture
Co11ection), ArCCNo.
31537 and 31446 are attached. Recombinant D
All of NA's experiments are conducted at National Institute 1.
He of Health (Nationai In5titu)
It was carried out in accordance with the applicable guidelines of TE of 11Earth). A most advantageous embodiment of the invention is the strain E. coli (E. coli) x 1776 and E. coli as defined above. Not only E. coli strain 294 but also the known E. coli ([, col
i) Strain (hereinafter referred to as E, C01i) For example, E, col
I will discuss E. coli and other microbial strains, including B. many of these in the American Type Culture Collection.
rican Type Culture Co11ec
It has been deposited and is available at known microbial depositories, such as the National Microbial Repository (8CC). I would also like to see German patent 2644432. These other microorganisms include Bacillus 1: e.g.
Bacillus 5ubtilis
and other intestinal bacteria such as Salmonel la typhimurium.
m) and Serratia maru I? Lens (Serrati)
a marcescens) and uses a replicable plasmid capable of expressing foreign gene sequences. Yeasts such as Saccharomyces and Levisiae
aromyces cerevisiae) may also be advantageously used for the production of interferon proteins by expressing the gene encoding inkferon under the control of the yeast vector motor. B, LelF IIIRN A raw material J: and purified Le
IF mRNA can be obtained from human leukocytes. Typically, leukocytes from patients with chronic osteoporotic leukemia induced to produce inkferon with Sendai virus or Newcastle disease virus are used, as described in German Patent No. 2947134. . Particularly advantageous raw materials used in the work herein are:
This is a cell line called KG-1 derived from a patient with acute osteogenic leukemia. This cell line is known as Koffler (
KocNler, 11. P, ) and Golde (Gol
dc, 0.14. , ) Science (SCi13n
Ce) 200.1153 (1978), [Rosewall Park Memorial Institute RP (formerly RP)
eIIlorialInstitute ) ) 16
40 plus 10% heat-inactivated FC3 (fetal bovine serum), 2
5 mHHEPES mild ma (N-2-hydroxyedyrubiperazine-N'-2-ethanesulfonic acid) and 5
Cells were frozen by adding 10% dimedyl sulfoside to the growth medium described above, which readily grows in medium containing 0 μg/Id gentamicin and can be subcultured twice a week (1 to 3 splits). sell. In G-1, the American Type Fuji Collection (the
An+erican Type Culture Co
11ection) (8 TCC No. cRL 8031
) has been deposited at KG-1 cells are Rubinstei
Proc, Natl, Acad, Sci, IJ, etc.
, S, ^,)76゜640-644 (1979))
using Rendai or Newcastle disease virus,
FC A cells induced to produce leukocyte inkferon RNA were harvested 5 hours after induction and RNA was extracted using the guanidine thiocyanate-guanidine hydrochloride method (Chirgwin et al., Biochemistry Tech. 8,5294). -52
99 (1979)). RNA from uninduced cells was similarly prepared. Poly(A > ml of N
123 fractions of A were obtained. The method is Green
) etc. (Ark, Biochem, Pi Office (^rch
, Biochem, Biophys, ) 172. 74-89 (1976)) and Okuyu
ma ) etc. (arch, biochem, pie office, (A
rcb, Biochcm, Biophys, >188
．． 98-104 (1978)) was used. This mRNA is African frog oocyte 1fIIIF!
Analysis (Cavalieri et al., Bloch, Natl. 11, S.A.) 74.3287-3291 (197
7)) indicates the interferon titer in 5ooo-io, ooo units/microgram. C, LeIF cDNA sequence] Preparation of a knee bank Using 5μ9 mRNA, standard method Likens (Wi
J. Biol, Chem, 253, 2483-2495.
(1978) and Gocdel et al. -(Nature) 281.544. -54
8 (1979)), 2Vfi chain CD N A was prepared. CD N A was fractionated according to size by electrophoresis on a 6% polyacrylamide gel, and 230 ng of material ranging in size from 500 to 1500 b,p, was obtained by electroelution. A chunk of 1100n of this cDNA was tailed with a deoxycytidine (dC) residue (Chano et al., Nature CNature)27
5.617-624 (1978), tailing (t
ailino) L/ta4, 70 nQ of plasmid pB11322 and polyvar (13o
Gene 2.95-
113 (1977)), and using this, E. colix
1776 and ICoCDNAn! About 130 tetracycline-resistant and ampicillin-sensitive transformants were obtained for If. B, C, D) or one (T-13A, B, C. D) oligonucleotide. The complementary deoxyoligonucleotides shown, 11 base chain length, were chemically synthesized by the small subtriester method (Crea et al., Broc, Natl, Acad, Sci. ,
U, s, 8, ) 75.5765-5769 (1978
)). For the -13 series, four individual probes were prepared. As shown in Table 1, 12 -110-bums were prepared as 4 pools of 3 probes. ■ -13 series of 4 respective probes and 4 pools of 3 primers each. 12 T prepared
-110-b was used to direct the synthesis of radiolabeled single heavy chain cDNA for use in hybridized probes. Template +11RNA A is G-1 for Sendai induction
12S R from cells (8000 units I 'activity/μ9)
Total poly(A) mRNA derived from NA or uninduced leukocytes (10 units/μ3). 32p-label CD
Let N A be the known reaction conditions (Noyes)
etc., Broc, Natl, Acad, Sai, US (Proc,
Natl, ^cad, Sci. U, S, A, >76.1770-1774 (1979
)) was prepared from these primers. 20 mH1-Tris (hereinafter referred to as TriS) -11cI (pH 8,3), 20 mHKCl,
8nrH14ocI2.30 mH β-mercaptoethanol was used for the 60 μm reaction. This reaction consisted of 1 μg of each primer (i.e. 12 μg total for the T-1 series and 4 μg total for the T-13 series), 2 μg of the “interrogated” 12S fraction m
RNA (or uninduced poly(A) mRNA3000
Ci/m mole) and 60 units of reverse transcriptase [Bethesda Research Laboratory
Research Laboratories))
It is. The product is 107! 5ephadex
■)■ (5ephaaexr shown below) Gel wave passage on the G-50 column allows the unbound label to be separated by 1 min, 7 min.
RNA was degraded by treatment with 0.3N Na leaf for 30 minutes at 0°C and neutralized with 11C1. Hybridization is performed by Nucleic Acid Res, 1
541-1552 (1979). [, Identification of clones pL1-pL30, 500 E,
coli K-12 strain 294 Kataga convertible strain (see item 0)
To prepare 1 μ9 of plasmid DNA from each of the
1ds Rcs,) 7, 1 5 1 3 -152
The rapid plasmid isolation procedure of 3 (1979) was used. Each DNA sample was denatured and aratos
), etc., in triplicate onto 21 helocellulose filters. 311 of the nitrocellulose filters containing 500 plasmid samples were a) induced CDNA primed with a let of primer T-1. b) Inducible CD NA primed with D-13 and C) 1-1.1 prepared using both sets of primers. hybridized with cDNA. Clones were considered positive if they hyperinhibited more strongly against one or both of the induced CD NA probes than against all of the uninduced probes. Out of 500 clones, 30 'positive' clones +111-pL30) were selected for further analysis.F, Identification of clone 1)131-1)139, LelF
I gene fragment Kaneko fragment plasmid (No. 104) separation probe 32p-labeled 1 mRNA Lillenhaug et al., Biochemistry (Biochemistry, ) 15.1858-
1865 (1976)) and Gransdein (1976)).
GrLlnStein) and Hogness (tlogn
colony vibridization method (Proc, Natl, USA (Proc, Natl, 8ca)
d, sci, U, s, ^,)72.3961-3965
(1975)) to screen for transformed strains of E. colix 1776. To compete the unlabeled mRNA from uninduced cells with the uninduced IRNA A present in the 32p-labeled preparation, the probe was
They were mixed at a ratio of 0:00 to 1. Hybridization of labeled 111 RNA should occur preferentially to colonies containing the induced sequence. Three groups of transformants were obtained. tl)':]D2O)2 to 3% is 32p mRNA
A1c was very strongly hybridized. In 12110%, the degree of hybridization was significantly lower than in the above group (1). (3) The remaining samples showed no detectable hybridization signal. Positive colonies (Groups (1) and (2)) were examined for the presence of Inkferon-specific sequences by analysis of specifically hybridizing Inkferon mRNA with plasmid DNA. First, tetracycline (20μ9/rtdl>, diaminopimeline M (100μg/d), thymidine (20μCl/d>
and d-pyotine (M9j with 1μ9/#Ii added)
Each of the 6011ij robust colonies (group 1) was grown individually over 100 m in 8 locations. M9 medium is
For 1 liter, Ha211PO4 (6g), K
1111!! of sterile I HHgSO4 and sterile 0
．． Add 10 m of OI HCaCl2. The 10 cultures were pooled and cultured using Clewell.
), etc., biochemistry (Biochemistry)
) 9. 4428-440 (1970), plasmid DNA was isolated from the six pools. 10 ug of each plasmid DNA pool was cut with Le&, old ndllI, denatured, and covalently bound to DBM (diazobenzyloxymethyl) paper. 1 μg of Vi mRNA from the induced cells was hybridized to each filter paper. I don't vibrid
RNA was removed by washing. The specifically hybridized mRNA was eluted and translated in Xenopus oocytes. All six pools were negative in this analysis. From 59 weakly positive colonies (group 2), 5 pools of 10 colonies each and 1 pool of 9 colonies were prepared, and plasmids were prepared from these pools and examined as described above. Of the six pools tested, one (K1
0) vibridized to interfae O111 RNA at levels significantly above background levels in each test. To identify specific Inkferon CDNA clones, plasmid DNA was prepared from 9 out of 10 colonies in a pool and examined individually. Two of the nine plasmids (No, 101 and Nα104) bound interferon 111 RNA well above background levels. From plasmid Nα104,
Unique BglIILll containing @260b, p,
The restricted fragment was isolated. This is called Taylor
etc., biochemistry. Biochim, Bioph
ys, ^cta) 442.324-330 (1976
) and labeled with 32p using the in situ colony screening method (Grunste et al.
in and1+01JneSS), Block, Nattle,
Zai, USA (Proc. Hall, Sci, U, S, 8, > 72. 3961
-3965 (1975)), 400 E. coli 294 transformed strains were used as probes for individual screening. 9 colonies (pL31-pL39) that hybridize to this probe to a balanced degree
was identified. Additionally, 4000 E. coli 294 transformants were individually screened in the same manner using the labeled 260 b, t+, fragment. Fifty colonies that hybridized to this probe to a species degree were identified. One is LeIF G fragment, one is LeIF
H fragments, one containing a fragment designated LeIF old (apparently an allele of LeIF If). The resulting hybrid plasmid was designated as "pLelF11" or the like. 6. Isolation and Sequencing of the First Full-Length LelFJ Gene Plasmid DNA was prepared from a total of 39 potential LelF cDNA clones. Then, by the hybridization operation of Afatos et al. (cited above), the same; 260b, +1. DNA pu-
Rescreening was performed using This probe and three plasmids (pL4, pL31, pL34)
gives a very strong hybridization signal, with 4 (1)
Ll3. +1130.1)L32. p136) was moderately hybridized, and three (pL6, pL8,
1) Ll 4 ) was weakly hybridized. The 39 potential LeIF cDNA recombinant plasmids also contained 32p-labeled synthetic undecamer (
Each of the T-1 primer pools or each of the T-13 primers) was probed for hybridization. Hybridization conditions were chosen such that complete base-base pairing was required to give a detectable hybridization signal (Wallace
)? ,,Nucl
eic Ac1d Res, )dan, 3543-355
7 (1979)). In other words, plasmid DNA was extracted from 39 clones using the ex semi-supernatant method (cleared).
1ysate procedure;
and purified by Biorad Agarose 8-50 column chromatography. A sample of each preparation (3μ
g) was ring-opened by treatment with EcoRI, denatured with alkali, and spotted onto two separate nitrocellulose filters. 1.5 μg per spot. See Kafatos et al., cited above. Each of the synthetic deoxyoligonucleotide primers and primer boules were phosphorylated with (γ32P)ATP as follows: 50 pmole of oligonucleotide and 100 pmole of (732P) )A
TP (Ne L-England New Flare-(Ne
wEr+olandNuclear), 2500C
, i/m mole) and 5QmH Tris-tlc
2 units of T4 polynucleotide kinase were added and after 30 min at 37°C.
It was purified by chromatography on a 0 dt phadex (5 ephadex) G-50 column. Hybridization was performed using 1106C11 primer T-13C or 3x 166 cpm (7) primer boolean T-IC. Hybridization was performed using 6XSSC (IXSSC=0,
15HNaCI, 0.0158 <sodium citrate,
pH7,2), 10x Denhardts (0,2
% bovine serum albumin, 0.2% polyvinylpyrrolidone,
Hybridization was performed in a 0.2% Ficoll solution at 15° C. for 14 hours. The filter was washed three times with 6XSSCXSSC intermediate. It was dried and exposed to X-ray film. The results are shown in Figure 1.32p-primer boule T-13
The case of primer-C and primer-T-IC is shown. Plasmid DNA from clone 104 hybridized significantly with primer Boolean T-IC and primer -13G. However, no detectable hybridization was observed with other undecamers. As shown in Figure 1, some of the 39 potential telF plasmids (pL2.4.13, 17.20, 30.3
1°34) hybridized with both of these probes. However, restriction analysis revealed that pL31 was 260 b, l), internal B (1
1 [also contained fragments. As a result of digesting pL31 with Pst I, the size of the cDNA insert was approximately 1000 b.
It was p. The entire Pst I insert of p[31 was sequenced using Maxam-
Haxam-Gilbert Chemical Methods (14methods Enzymol)
zymol,) 6 branches. 499-560 (1980)) OJ: Method for terminating dideoxy chains (Smith, Method of Enzymol (Smi)
th Methods Enzymol,) 65,
560-580 (1980)). that time,
5au3a [11 pieces were subcloned into 813 vector and yielded +3. The DNA sequence is shown in A'' in Table 2. Information on available protein sequences, known L
The relative presence of stop triplets between the eIF molecules and the three possible reading frames suggested a suitable translation reading frame. The entire LeIF amino acid sequence, including the prey or signal peptide, was then shown. 1st A
The TG translation initiation codon is unique to human growth hormone in that the manipulations to avoid direct expression of the sequence LclFA as a mature ink ferron bolibbutide involve the combination of synthetic (N-terminal) and complementary DNA. The methods used (Goeddel et al., Nature
) 281. 544-548 (1979). As shown in Figure 3, the 5au3a restriction endonuclease site conveniently lies between codons 1 and 3 of Lcl', which contains the ATG translation initiation codon and contains amino acid 1 (cystine). Two synthetic deoxyoligonucleotides were designed that repaired the opposing codons and created a new EcoRI sticky end. These A ribomers contained 34 b, D,
It was ligated to the Sau 3 a-^va II fragment. The resulting 45b,p product was ligated to two additional DNA fragments, creating 865b,+1. It was made into a synthetic-natural hybrid gene. It codes for LelF A, EcoRI and ps
It is flanked by tI restriction sites. This gene is
Between the EC0RI and Pst I sites, p
It was inserted into BR322 and set to 1 into plasmid pLelF. 2. Construction of a tryptophan regulatory element (E. coli trp promoter, containing the operator and trp leader ribosome binding sites but lacking the ATG sequence for translation initiation) Plasmid Le Gold contains the deletion ΔLE 1413 Zuru E, coIN-responsible for liptophan oberon (Hiozzari et al., J. Bacteriology (J, BaCteriOIO (IV) 133, 14
57-1466 (1978)). Therefore, trp
A fusion protein is expressed that contains the first six amino acids of the leader and approximately the last third of the trp E polypeptide (hereinafter collectively referred to as LE') as well as the entire trp Q polypeptide. This is J
It is under the control of the rp promoter-operator system. 20 μJ of this plasmid was digested with restriction enzyme Pvu II. This cuts the plasmid at five sites. This gene fragment is then attached to an EcoRI linker (
pCATGAATTC^TG (consisting of self-complementary oligonucleotide 1), and then Eco
Cloned into a plasmid containing an RI site. Collect the aqueous solution from the dialysis bag, phenol extraction, chloroform extraction,
A 0.2M sodium chloride concentrate was used, and DNA was collected in ethanol-precipitated condensate. t with EcoRI sticky ends
The rp promoter-operator containing gene was identified by the procedure described below. Briefly, a fragment was inserted into a tetracycline-sensitive plasmid, which was made tetracycline resistant by insertion of a promoter-operator. Plasmid pBR111 (Rodriguez
uez) etc., Nucleitsk Acid Research (Nu
cleic Acids Res, ) 6.3267-3
287 (1979)) expresses ampicillin resistance and contains a tetracycline resistance gene. However, since there is no promoter associated with it, it does not express its resistance. This plasmid is therefore tetracycline sensitive. By introducing a promoter-operator system into this ECoRI site, the plasmid is made resistant to tetracycline. pBllllll was digested with EcoR*, the enzyme was removed by phenol extraction, chloroform extraction, and ethanol precipitated into water. Product N present in separate reaction mixtures
The A molecule was merged with each of the three DNA fragments obtained above and ligated with T4 DNA ligase as described above. Using the DNA present in the reaction mixture, standard methods (such as Hershficld, etc.)
Block. Natl, Akado, Sai, USA (Proc, Natl,
Acad, Sci, U, S, A, ) 71,
3455-3459 (1974)) was transformed into [coli K-12 strain 294, which has DNA uptake ability.
The bacteria were treated with 20 μ9/d ampicillin and 5 μ9
/d tetracycline containing L B (Luri
a-Berta (old) plates. Select some De1~racycline resistant colonies and add plasmid D
The NA was isolated and the presence of the desired fragment was confirmed by restriction enzyme analysis. The resulting plasmid (referred to as IBRHtr+1) contains EcoRI and Bam1l of the hepatitis B viral genome.
The I digestion product was collected by a conventional method, and the plasmid pGII
6 into the EcoRI and BamHI sites of Goeddel et al.
re) 281.544 (1979)), plasmid pHs 32. Plasmid pH332 was cut with Xba I, phenol extracted, chloroform extracted, and ethanol precipitated. Precipitate with 0.1 mHdTTP and 0.1 mHdCTP
30 μm polymerase buffer (50 mN potassium phosphate I) containing H7,4,7 mHNoC12° (Boehringer-Hannheim)
Heim) at 0°C for 30 minutes and then at 37°C for 2 hours. This treatment fills in two of the four nucleotides complementary to the 5' overhang of the xbaI cleavage site. 5'CTAGA-5'CTAGA-3' T, -T
CT - Two nucleotides dq and dT are incorporated to give an end with two overhanging 5' nucleotides. This linear residue of plasmid DII332 (phenol and chloroform extraction followed by ethanol precipitation and water) was cleaved with EcoR■. The large plasmid fragment was
AGE, smaller -TCTAGA-u-TC:TA
GA--AGCTCT--AGATCT- These plasmids contain EcoRI and 1lpa I
It was also found that both were cut to give the expected restriction fragments. One plasmid, designated pTrpl4, was used to express foreign polypeptides as discussed below. Plasmid pHG11107 (Goedde
l ) etc., Nature 281.5
44 (1979) is a human growth hormone consisting of 23 amino acid codons generated from a synthetic NA fragment and 163 amino acid codons obtained from complementary DNA generated by reverse transcription of human growth hormone messenger RNA. Contains genes for. This gene is
Although it lacks the codons of the human growth hormone "leading" sequence, it contains the ATC translation initiation codon. This gene was derived from 10 μptlGI1107,
Following that, [. coli DNA polymerase I Klenow (1enOW)
) (denoted below as lenow) and dTTP
and separated through d^TP treatment. Phenol and phosphoric acid 5'-dGATCACACATG (fragment 2) was synthesized by the phosphotriester method. Fragment 2 was phosphorylated as follows. 200 μm (approx. 40 pmole)
(732P) ATP (Amersham
m), dry 5000Ci/lImol(3), 5Q+nH Tris 1lcl (pit 8),
The DNA fragments were resuspended in 30 μm of 10 mH HgCl2.15IIIHβ-mercaptoethanol containing 100 pmole of DNA fragments and 2 units of T4 polynucleotide kinase. After 15 min at 37°C, 1 μm of 1
0 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, i o o
5'-01- of pmo+e (fragments 1 and 10Di
ole's 34 b, p, sau 3 a Ava II
Merged with fragments. 20mHTris-11cI (pl
-17,5), 10 fflHH+IC+2.10mM dithiothreitol, 0.5m)l ATP and ligated in 50μm of 10 units T4 DNA ligase for 5 hours at 4°C. The mixture was electrophoresed on a 6% polyacrylamide gel and the 45b,p product was collected by electroelution. 4
5b, p, 30no (1 tunole) of product
was ligated to 150b,p,Ava II- (0.1 μg). Transformants containing the trp promoter are Grunstein-Bognes
stein-hogncss): l+1 using 32P-trp probe in combination with the knee cleaning method. Established. Asymmetrically located X in the trp Ii piece
The ba I site is located at the trp promoter
This enabled the identification of recombinant products oriented in the direction of the i gene. 1. In vitro and in vivo active IF of telFA
Extracts for analysis were prepared as follows. 11 d of the culture was treated with L containing 5 μg/rd of tetracycline.
grown in broth to give an A35o value of approximately 1.0, then 5 μg/ml! M9 containing tetracycline of
Medium 251Id! diluted in A5. When o reached 1.0, a sample of 10 d was collected by centrifugation, and the cell mass was diluted with 15% sucrose and 50 mHTri.
5-)-1cI (Dl18.O), 50m14E
Suspended in 11 Idl of DTA. 1 ml of lysozyme was added, and after 5 minutes at 0°C, the cells were disrupted by sonication. The sample was centrifuged for 10 minutes (15,000 rpw+), and the interferon activity in the supernatant was determined by cell table 7 E, c.
oli294/pLelFA25 extract and standard LelF
Activity comparison with ``E, coli294 /I)LelF described in person 6
A tri) 250.000 units per batch of 25 extracts were diluted 500@ in minimum essential medium to give 500 units/d.
The specific activity of Leukocyte Ink Feron Standard [Wadley Institute
e) ) was previously prepared with the NIH leukocyte interfunron standard, titered, and also diluted to a final concentration of 500 U/d. One volume was brought to DI+2 with 1 NII Cl, incubated for 52 hours at 4°C, neutralized by addition of NaOH, and IF activity was measured by standard *CPE inhibition assay. 25μI of 500 units/d sample (untreated)
rabbit anti-10100XLD5oE virus (measured in mice)
was infected intramuscularly. Control infected monkeys were infected at 134.
158. Died at 164 hours. 106 units interferon treatment was administered intravenously 4 hours before infection, 2.23.29.48.72.168 and 240 hours after infection. Column chromatographic fractionation from the lysate of bacterial leukocyte interferon, E. coli294/pLelF^25, with a specific activity of 7.4 x 106 units/mfl protein. Control bacterial proteins were E, c
The total protein amount was doubled with the corresponding column fraction from the oli294/pBR322@lysate. The leukocyte inkferon standard was prepared using normal human "Batufico-1" cells purified by chromatography to a specific activity of 32 x 106 units/m9 protein.
) was a virus-induced interface. Control monkeys showed progressive coma, loss of balance,
The animals showed flaccid hind limbs, paralysis, and lachrymal drainage starting approximately 8 hours before death. Interferon-treated monkeys did not show any of these abnormalities and were always resistant to Grunstine.
The above 0
Additional E. coli 2 was added as in Section 1.
94 convertible strains were screened. Colonies that hybridized to the probe to varying degrees were isolated. Plasmid DNA from these colonies and plasmid D from the 10 hybridized colonies shown in Section G above,
NA was isolated by Pst I cleavage and characterized in three ways. First, these PSt fragments were treated with the enzyme Bgl
II, Pvu fl and EcoRI were characterized by restriction endonuclease digestion patterns. This analysis shows that at least eight different types (Le
IF A, telF B, LelrC, LelrD
, LelF E, LelF F, LelF G and LelF II). This indicates the approximate location of the various restriction breakpoints with respect to the previously known forward (j and coding sequences) to LclF. One of these, LelrD, was reported by NaQata et al. Nature
284.316-320 (1980). Second, for some of these DNAs, LeIFmRNA
Cleveland (C
1evcland) etc. are cells, λ''1.
95-105 (1980). LclF8, BIC and F were positive in this assay. Third, these pst fragments were inserted into expression plasmids and E. E. coli 294 was transformed with the plasmid and the fragment was expressed. Expression products believed to be preinkferon were all positive in the CPE assay for itaferon activity, except for the LeIF F fragment, which was marginally positive. In addition to the above, all of the LclF types listed above have been sequenced. K, second mature leukocyte interferon (LelF B
The sequence of the isolated fragment containing the gene for directly expressed mature LelF B of ) shows that the first 14 nucleotides of types A and B are the same. Therefore, the trD-promoter-operator, 112 strains 294 at the ribosome junction.
was converted. Convertible stocks are screened using mini-screening (Birnboim).
Boim), etc., Nucleitsu Acid Research (Nu
cleic Ac1ds Res,)ヱ, 1513-1
523 (1979)), and the plasmid samples were
Digested with oRI. The digest gave three fragments, which were characterized by: 1) EcoRI-EcoRI trp
Promoter fragment: 2) Internal EcoR■-Ec of pL4
oll I fragment; Onibi 3) l) L4 protein translation initiation signal-EcoR■ fragment. For CPE analysis, bacterial extracts prepared as described above or prepared as described above are typically extracted at approximately 10X at A5,o-1.
106 units/! 7j gives interferon activity. One representative loan prepared in this way is E, c
oli294/ pLelF B trp 7. This clone has been deposited with the ATCC. [, yet another mature leukocyte idetafuron (telFC)
, D, F, II, I and J). Additional full-length gene fragments containing other LelF types include:
As in LelFA, it can be tilled and placed in an expression vehicle for expression. The mature tracycline resistance gene, together with the mature LelF F gene,
p promoter-operator, so that bacteria transformed with the desired plasmid will
Can be selected on tetracycline-containing plates. Representative clones thus prepared include E, coli K
-12 strain 294/pLelF F trl) 1. This clone has been deposited with the ATCC. elF H Complete LelF IDE! In order to prevent Butsuko from being expressed as a mature leukocyte interferon, it is arranged as follows. 1. Plasmid pLelF H was transformed into Hae II and R
Upon sa I digestion, the signal peptide amino acids 10
The 816b,p, fragment extending from to the 3' non-coding region is isolated. 2. This fragment was denatured and treated with DNA polymerase Ie.
Now Fragment (Klenow et al., Proc, Natl, Cy, US (Proc, Natl, ^
cad. Sci., U.S., 168 (1970)), the synthetic DeAxyriboA oligonucleotide primer can be released and ligated with the product of step (5) to form an expression plasmid. This is E. coli K-12 strain 294
or used to transform other host bacteria into mature elFII
can be expressed. eIF I Human genome face λ Charon 4 A Ill recombination library constructed by Lawn et al. (Cell, 15.1157 (197
8)) by the method of Lawn et al. cited above and the method of Haniatis et al.
) 1 branch. 687 (1978) for leukocyte inkferons. cDNA clone L
Approximately 500,000 plaques were screened using the eIF A-derived radioactive LelF probe. Six LeIF genomic clones were selected. Following rescreening and plaque purification, one of these clones, λ1ILelF2, was selected for further analysis. Using the methods described above, other probes may also be used advantageously to separate other LeIF clones from the human genome. These then remain in the supernatant as most of the cellular proteins, including tahuerone according to the invention. 2. Ammonium sulfate fraction. Here, interferon is precipitated from a solution of 55% saturated ammonium sulfate. 3. Suspend ammonium sulfate pellets in 0.06 M potassium phosphate, 10 mHTris-11cI, pH 7.2, and add 25 mHTris-IIcI, pH 7.
Dialyze against 9. Inkferon activity remains in solution. 4. Adjust the above supernatant to pH 8.5 and perform chromatography on a DEAE-cellulose column (25 mH
Tris-IIcI, pH 8.5 from O to 0.2M
Elute with a linear gradient of N, aCl). 5. Cibach Blue-Agarose (Cibach
rome BIue -Agarose) or hydroxyl apatite (hyaroxy+apat;te
) and elute with 1.58 KCI or 0.2 M phosphate (actual m if necessary). 6. 5ephadex G-75
Separate molecules by size using columns. 7,91+5. Q2511IN C in ammonium acetate
Perform cation exchange chromatography on M-cellulose. Develop with an ammonium acetate gradient (up to 0.2M ammonium acetate). The method described above yields a product with >95% purity. This material can also be purified by size exclusion chromatography, reverse phase (RP-8) high pressure liquid chromatography, or affinity chromatography on immobilized anti-Inkferon antibodies. Alternatively, the material from stage 111si4 above may be prepared by Milstein, Scientific American.
an) 243.66 (1980), plus 0.2 M acetic acid, 0.
1% Triton and 0.15HNaC
It can be eluted with l. In another advantageous embodiment, the leukocyte interferon produced in the above procedure can be purified in the following step. 1. Crush the frozen cell mass containing the expressed leukocyte interferon manually or with a suitable crushing device. Partially melted cells were adjusted to pH 7,5-8,0, 0,1.
M Tris, 10% column with approximately 10 column volumes 20m+4 Tris-11C1, ptl 7.5-
8.5 (NaCl (0,5M) and Triton (1'
riton) X-100 (0.2%) or equivalent surfactant-containing). After washing, add 0 to this column.
, 15 HNaCl and Triton
Approximately 10 column volumes of solution containing X-100 (0.1%) or equivalent surfactant are passed through. This column was coated with Triton X-100 (0.1%).
Or elute with 0.2M acetic acid containing an equivalent surfactant. Protein peak from monoclonal antibody column (U
(measured by V absorption or other methods) and 1N Na
1, adjust DH to approximately 4.5 with OM Tris base. 4. Transfer the pooled inkferon peaks to a suitable buffer such as ammonium acetate pH 4,5 (50111
) Whatman equilibrated with 1)
Add to CM 52 cellulose or equivalent cation exchanger. After loading, the column is washed with an equilibration buffer until the UV absorption of the effluent reaches 1ζ, and almost no protein is eluted from the column. The column was then washed with 25mM ammonium acetate 10.12
The lyophilized cake is eluted with a combination that maximizes the recovery of M-lium chloride or interferon, and has a satisfactory appearance and solubility. The monoclonal antibodies in this advantageous embodiment described above are described by Staehelin et al. in Proc, Natl.
l. 8 cad, sci, u, s, 8, > 78. 1848
-52 (1981). Monoclonal antibodies are purified and covalently coupled to Affigel-10 as described below. Preparation and purification of monoclonal antibodies from ascites fluid 11f
Each of the five tBalb/c mice was given a logarithmic growth of 11
! ] Hybrido-V (hybrido
ma) 5-10X106 cells were seeded. Each of 10 or more mice was inoculated intraperitoneally with approximately 5 x 10 6 viable cells obtained from the ascites fluid produced by the mice. This ascites fluid was applied repeatedly to each mouse (2
4 times). Transfers and harvests were performed from one group of mice to the next group up to three times. Ascites fluid collected after each transfer was pooled. Treat with low speed centrifugation (500-1000Xg) for 15 minutes,
Cells and debris were removed from the ascites fluid. Then 18.00
Centrifugation was performed for 90 minutes at Orpm. Freeze the supernatant at minus 2
Stored at 0°C. After melting, 35. OOOrFl
Fibrin and particulate matter were further removed by centrifugation for 90 minutes at m. Batches of ascites fluid after every 8 transfers were tested using a solid phase antibody binding test (Stohelin).
The specific antibody activity was tested according to the above-mentioned literature, and if it was satisfactory, it was pooled. The protein concentration in the pooled solution was 1.2 at 2801 m for 1 ml of protein in a 1.0 cm thick cuvette.
It was measured using an approximation method that shows the absorbance value of . Ascites fluid with a high concentration of antibodies contains 30-35 ms protein/d'. This is a 44-7In specific antibody/-
corresponds to This liquid was diluted with PBS (0.01M sodium phosphate, pr17.3.0.15M NaCI) to give a protein concentration of 10 to 12 trty/raft. To each 100 ml of diluted solution, 90 ml of ammonium sulfate solution saturated at room temperature was added at 0° C. with vigorous stirring and the 7jo suspension was kept in water for 40 to 60 minutes. The mixture was then centrifuged at 10,000 rp+n for 15 minutes at 4°C. The supernatant was filtered off and the liquid was thoroughly drained. Protein mass was added to 0.02M Tristlcl (all 7.9
) Dissolved in 10.04M NaCl (buffer ■). Add the protein solution to 100 volumes of buffer at room temperature.
Dialysis was performed for 6 to 18 hours. During this time, the buffer was exchanged at least once. 15. dialysis solution. Centrifugation was performed at OOOrpm for 10 minutes to remove insoluble matter. Approximately 30 to 35% of the first whistle of the total protein in the ascites was collected as estimated from the absorbance value at 280 nm. A solution containing 3030-40 protein per component was then applied to an I) EAE-cellulose column equilibrated with Buffer I. A column bed volume of at least 100 d was used for each gram of protein added. 0.02M Tris Cl pH 7,
The antibody was eluted from the column by linearly changing NaCl 1 degree from 0.04M to 0.5M NaCl 1 degree. The peak fractions from 0.06 to 0.1 M NaCl were pooled and added to an equal volume of saturated ammonium sulfate solution (room m
) was added, precipitated, centrifuged, and concentrated. 0.2M protein mass
HaHCO3(1)II approx. 8.0) 10.3M NaC
1 (buffer ■) and dialyzed against the same buffer (three changes) at room temperature. Dialyzed solution at 20.000X
The mixture was centrifuged at 9° C. for 15 minutes to remove insoluble matter. Increase protein concentration from 20 with buffer ■. It was set as 2FIF/d. Preparation of immunoadsorbent Affigel-10 (Bio Lab Laboratories, Richmond, California)
o tlad Laboratories, Rich
Mo., CalNornia) was washed three times with water-cooled isopropanol on a glass filter, and then three times with water-cooled distilled water. The gel slurry (approximately 50% cold water) was transferred to a plastic duplex and briefly centrifuged 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, the gel was centrifuged and buffer I[[(0, I M Na1lCO310, 15M
NaC1) t"2 was dissolved in 5N human serum albumin, the solution was passed through a biological filter, and the filtered solution was aseptically divided into 100 bottles, each containing 6 bottles, suitable for parenteral administration. ×10
Contains 6 units of pure ink feron. 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. Suitable vehicles and their formulation are described in Remington's Pharmaceutical Sciences (ReIIlinQtOn'SP) by [W, Hartin].
harllaCeLltiCalSCienCeS)
As stated in This is cited as a reference in this specification. In these compositions, an effective amount of inkferon protein and a suitable amount of vehicle are combined into a pharmaceutically acceptable composition suitable for effective administration to a host. One advantageous mode of administration is parenteral administration.

Claims (14)

[Claims] (1) A polypeptide comprising the amino acid sequence of interaeron of human mature leukocytes, which has a partial amino acid sequence Cy
s-hela -Trp -Glu -Val -Val
-8rg -Ala -Glu -11e -Net
7Arg-3er, and without a preceding sequence, it has 165-166 amino acids and the amino acid ^sp, G at position 114
lu or Vat, or if methionine is attached to the N-terminus of the first amino acid of the interferon, the polypeptide has 166-167 amino acids and has amino acid 1llAsp, Glu or Vat at position 115. A DNA sequence characterized in that it consists of a sequence encoding a peptide. (2) The polypeptide is human leukocyte interferon A
The DNA of claim 1 which is (LclF A)
array. (3) The polypeptide is human leukocyte interferon B
The DNA of claim 1 which is (LelF B)
array. (4) The polypeptide is human leukocyte interface 0inC (
A DNA sequence according to claim 1, wherein LelF C>. (5) The polypeptide is human leukocyte ink pheond
(LelF D)
array. (6) The polypeptide is human leukocyte inkferon F
DN of claim 1 which is (1,elF F)
A array. (7) The polypeptide is human leukocyte interferon H (
The DNA of claim 1 which is LelF 11)
array. (8) The polypeptide is human leukocyte interferon I
(LelF I)
array. (9) The polypeptide is human leukocyte interferon J
(LeIF J)
array. (10) The DNA sequence according to claim 1, which is a DNA sequence resulting from allelic variation of the DNA sequence according to any one of claims 2 to 9. (11) The DNA sequence according to claim 1, which is a DNA sequence resulting from an allelic variation of the DNA sequence encoding LclF8. (12) The DNA sequence according to claim 1, which is characterized by a polypeptide that differs from LelFA in only one amino acid. (13) The DNA sequence according to claim 1, which is characterized by a polypeptide that differs from LclFA in the amino acid at position 23. (14) LelF A in that it contains Arg at position 23
The DNA sequence of claim 1 encoding a polypeptide different from .