JPS60221094A - Incidence vehicle - 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 interferon, the partial amino acid sequence Cys-la-Trp-Glu-V
al -Val -Arg -8Ia -Glu -1
1e -Net -Arg-3er, and without a preceding sequence, 165-1
66 amino acids, or if methionine is attached to the N-terminus of the first amino acid of said interferon, contains a DNA sequence consisting of a sequence encoding a polypeptide having 166-167 amino acids. A replicable expression vehicle characterized in that:

First, the background of the present invention will be described. Human leukocyte interferon (LelF) was initially isolated from l5aacs and L
Indenmann discovered and prepared it as a very coarse precipitate (Proc, R, Soc.

8147.258-267 (1957): tl, S.
P.

3,699,222). Long-continued efforts to purify and characterize it have led to the preparation of a relatively homogeneous leukocyte interferon derived from leukocytes obtained from normal or leukemic donors.
). These interferons are a group of proteins known to have the ability to confer a virus-resistant state on their targets. Additionally, inkferons act to inhibit cell proliferation and modulate immune responses. These properties have promoted the clinical use of leukocyte intanoeron in the treatment of viral infections and malignant tumors.

Leukocyte interferon has been purified to essentially homogeneity (Rubinstein et al., Proc. Natl., A.
cad, sci.

U, S, A, 76, 640-644 (1979):
Zoon et al., supra, 76.5601-5605 (19
79)) were reported to have molecular weights ranging from about 17,500 to about 21,000. The specific activity of these preparations is very high, 2 x 108 to 1 x 109 units/■ protein, but the yield from cell culture methods is appallingly low. However, with advances in protein sequencing technology, partial amino acid sequences have been determined [1000, 5cienc, etc.].
e 207.527 (1980): Levy et al., Pro.
c, Natl, Acad, Sci.

U, S, A, 77.5102-5104 (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 inkferons vary markedly in amino acid composition and sequence according to type, with amino acid homologies as low as 80% in some cases.

Although isolation from donor leukocytes has yielded sufficient quantities of homogeneous leukocyte interferon for partial characterization and limited clinical studies, it alone is insufficient for large-scale clinical trials and extensive clinical studies. It is simply insufficient to provide the interferon needed for its prophylactic and/or therapeutic use. Indeed, current clinical trials using human leukocyte-derived interferon for antitumor and antiviral purposes use crude preparations (lower than 1%) and do not produce sufficient quantities even at impractical prices. 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 by microorganisms of an extremely diverse variety of useful polypeptides. 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): Gocdel et al., Na
tLIre281.544-548 (1979)).

More recently, 1] engineered DNA technology has enabled the production of proinsulin and thymosin alpha-1;
In addition, several authors have reported on the collection of DNA encoding human leukocyte inkferon and the production of the resulting protein that controls Ij7eron activity (Na (Iata et al., Nature).
284, 316-320 (1980);
et al., Genelo, 1-10 (1980): ran+
Guchr et al., 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 plasmid DNA includes information necessary to reproduce the plasmid in daughter cells (
i.e., “replicon”) and, usually,
There is information on one or more selection characteristics, for example, in the case of bacteria, resistance to antibiotics. This information allows host cell clones containing the plasmid of interest to be recognized and preferentially grown into selective media. Plasmids are useful because they contain one type of restriction endonuclease or another type of restriction endonuclease, each of which recognizes a different site on the plasmid DNA.
Il, IJ Ilj! This means that the plasmid is specifically cut and cleaved by "oxygen." After that, a foreign gene or gene fragment is inserted into the plasmid by joining either the ends of the cut site or the reassembled ends in contact with the cut 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 cell can be transformed without developing. By doing so, recombinant plasmids containing foreign genes can be produced in large quantities. Furthermore, if the foreign gene is inserted correctly with respect to the portion of the plasmid that governs the transcription and translation of the encoded DNA information, the resulting expression vehicle will contain the actual polypeptide sequence encoded by the inserted gene. It can actually be used in a process called production or expression.

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 is overlaid by an ``operator'' region and the linked promoter-
Becomes an operator. Operators are DNA molecules recognized by so-called repressor proteins, which play a role in regulating the likelihood of transcription initiation at specific promoters.
This is A array. RNA Bolimera-1 moves along the DNA and transcribes the information contained in the coding sequence from its 5' end to its 3' end into messenger RNA, which then carries the amino acid sequence encoded by the DNA. translated into polypeptides. Each amino acid is encoded by a nucleotide triplet or "codon." These are located in what is referred to as the "structural gene" for the purposes of the present invention, that is, the part that codes for the amino acid sequence of the product to be expressed. After binding to the promoter, the RNA polymerase first The ribosy, transcribes the nucleotide that covers the mu binding site and then sends the translation initiation or pa initiation signal (usually ATG
Then, the Metsusenja RNA obtained is ALJG)
, then transcribes the nucleotide codons present in the structure B1 Buddha itself. A so-called stop codon is transcribed at the end of the structural gene. After that, the polymerase
Even though additional sequences of Methsenji A7-RNΔ may be formed, they remain untranslated by the ribosome because of the presence of the stop signal.

Ribosomes normally bind to specific junction sites on metsenger RNA in bacteria where the 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 the host cells and harvesting the product from other bacterial proteins by appropriate purification methods.

We believe 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 a highly effective method for providing large amounts of leukocyte interface. I thought it was a method. 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 Shiramen method genes of the present invention consists of the following steps.

(1) Synthesis using a partial amino acid sequence of a human leukocyte interface purified to homogeneity, containing codons as a collection representing all possible nucleotide combinations that can encode the partial amino acid sequence. A set of NA probes is drawn.

(2) Complementary DNA from induced metsenger RNA
A bacterial colony bank containing A(cDNA) is prepared. Another radiolabeled induced mRNA hybridizes with plasmid CDNA from this bank. The hybridized mRNA is eluted and tested for translation to inkferon in oocyte analysis. Thus, the plasmid DNA from the clones [shown to induce interferon activity] is tested for hybridization to the probe prepared as in (1) above.

(3) In parallel to the step (2) above, another transformed colony bank is prepared using cDNA derived from the induced mRNA in the plasmid. The probe of (1) was used to induce the synthesis of radiolabeled 1-tree cDNA for use as a hybridization probe. The synthetic probe hybridized with the induced n1 RNA as a template and was expanded by reverse transcription.
Generate radiolabeled cDNA. We further investigated the hybridization of the radiolabeled cDNA obtained in this way from the colony bank.
Confirm the presence of a full-length interferon-encoding gene. The predicted partial-length gene fragments obtained in (1) and (2) can themselves be used as full-length gene probes.

(4) The full-length gene obtained above contains a leader (
1 leader) sequences, if any, and tailored using synthetic NA to allow proper positioning in the expression vehicle with respect to the initiation signal and ribosome binding site of the microbial promoter. Ta. 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 as a probe for other partially homologous leukocyte inkferon species by hybridization.

By applying the DNA techniques outlined above, a group of homologous leukocyte ink ferons (glycosylated and (not)
It has become possible to produce microbially with high yield and high purity. These interfacial mortars are suitable for animals and humans.
can be directly expressed, harvested, and purified to levels suitable for use in viral and malignant tumor diseases. A group of interfaerons thus expressed were shown to be effective in in vitro tests and, as the first microbially produced leukocyte interferons, were also shown to be effective in in vivo tests for the first time. It was shown.

As used herein, the term "mature leukocyte interferon" refers to a microbially (e.g., bacterially) produced inkferon that lacks glycosyl groups. The mature leukocyte inknoeron of the present invention is a naturally occurring Therefore, this "mature" polypeptide has methionine (encoded by ATG) as the first amino acid in its sequence. The microbial host, on the other hand, processes the translation product by removing the first methionine. Mature leukocyte interferon, along with its normal non-leader junction proteins, Once expressed, the conjugate can be specifically removed in the extracellular or intracellular environment (see British Patent Publication Nα2007676A). It can be produced as a conjugate with a signal'' peptide. 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 in mRNA translation of the human leukocyte interferon genome.

Specific leukocyte inkferon proteins are defined herein by determined DNA genes (Tables 2 and 4) and deduced amino acid sequences (Tables 3 and 5). For these particular interferons, in fact for all groups of leukocyte interferon proteins included here, there are natural allelic variations.
ariation), and can occur between individuals. These mutations appear as amino acid differences throughout the sequence or as deletions, substitutions, insertions, inversions, or additions of amino acids in the sequence. LeIF A to LeIF
For J, such allelic variations are included within the scope of the labeling or definition thereof, and are therefore included within the scope of the present invention.

Next, the tables and figures will be explained.

Table 1 ■) Protein Ala-Glu-I 1e-Met-Ar
g Complementary 3' CTT TAG TACGC (DNA primer ■ Trif0synpef0tide (D-13) ---His-Glu-Met-11e-Gln---
Table 3 AIl EEFD QFqKA I VLHE ! II
Q FN14 T10 20 30 40! SA LL F ELQqjQDE Q””ju
(70000 LL LL 222 2nd
M M y soyeeeeeeee (!l C5C!5 (!
5 (!11 0LJ LLI LAJ LAJ TOTOTO l-)-+00 C10''''j j,-n (j C
J CJ L) +j Otsu Otsubako (L): 331 z Φ
c500ro 匡匡”匡−+ ＜＜＜＜＜〉〉””” O
Ij, IOαα ＜ ＜ ＜ ＜''''j j L
) (J L) L) (J , J j "">>>>>>
> (/l 入t/>Riri 22222">""j
LL LL 1. LLLLLL LLLLLLLL 匡)-c
L(1) C/l <<<<B ” u , J , J
,J"""zu" ΣJ J J ：＞ ：＞ ：＞
)) <<<<("u""" Q+t/l t/l) injection-ΣΣΣΣΣ ΣΣHw + + w m+ ++
＋ ＋く工H+′）Oく工H TSυ く工H ツ0”””
” Tohito I-LIJOOOO Lopi C pi ○ pi 0 and C pi υ1C pi C pi C pi O pi Cr0C
rC70匡■γCpiαLLLLLLLLLL""""
and 0 and 01: 000> and 0('J>>>>>
＞ j ``''''＜ ＜ ＜ ＜ z z z z
zj ``j'''')-)-)-)-1- ψ)'s ``confirmation 000 C10L/) C/) l/l M L/IL
IJ田LIJIJJ1) 匡”1”匡”yzzzz
/1ψ Norime ΣΣΣΣΣ Ij'1ααααα ,, J j '',, J ΣΣΣΣΣ )-)-)-)-)-Hita LLI LIJ LIJLL
Il, LILAJLIJLIJ<<<<<)-LIJ
LIJ 'we LIJ ααααα〉〉Σ＞＞＞
＞ ＞ ＞ ＞ ΦΦΦ（j （j ＞ ＞ ＞ ＞〉〉〉〉〉IJ
JLLIl, LILIJ-CJ L) (J CJ (
J t/) t/) t/l's <<<<<
＞ ＞ ＞ ＞ ＞1, LJLl, ILA, ILIJl
) NZZOZO LJLAJLIJzzzz
2 yΣI-ΣH kuworkH1'')Okuworkt-1''') L) Table 1 shows T-1
and T-13, which are isolated from human leukocytes and purified to homogeneity. All possible mRNA sequences encoding these peptides and the corresponding DNA sequences are shown. The letters J-0Δ, T, G, C and U contain the bases adenine, thymine, guanine, cytosine and uracil, respectively. Indicates the nucleotide.

The letter N is the nucleotides A and G.

Indicates either C or U. The polynucleotide is 5
It is written to be read in the direction from ' (left side) to 3' (right side). and double strands (“'d, s,”)
When referring to DNA, the order is reversed for the lower or non-coding strand. Figure 1 shows possible Le
This is an autoradiogram showing no hybridization between If' 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 interfaces, respectively labeled “A” through “l-1”. ). 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. L included
As shown in the third column A line of Table 2, the entire gene length to eIF lacks one codon that is present in the other codons. A 5' 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, eight LeEs shown by the nucleotide sequence
Comparing F protein sequences. ItlPAC-t, t1
8Commission on Biochemica
The abbreviation of one letter proposed by Nomenclature is used. That is, alanine is A, cysteine is C, aspartic acid is D, glutamic acid is E, phenylalanine is F, glycine is G, histidine is H, isoleucine is I, lysine is K, and leucine is 1-, medionine is M, asparagine is N,
Proline is represented by P, glutamine is represented by Q, arginine is represented by R, serine is represented by S1 threonine by king, valine is represented by V, tryptophan is represented by W, and tyrosine is represented by Y. Numbers indicate amino acid positions. S indicates no signal peptide. 165
The dash at position 44 in the LclF A sequence, which is an amino acid sequence of
It is inserted to align with the 66 amino acid sequence. The sequence of LelF E is defined ignoring the extra nucleotide in its coding region (position 187 in Table 2). The asterisk indicates the stop codon at the interface.

Amino acids common to all L131Fs (except for the pseudogene LelFE) are also shown. The underlined residues are amino acids that are also present in human fibroblastic interferon.

FIG. 2 shows a restriction endonuclease map of eight types (A through 11) of LelF cloned CD NAs. Hybrid plasmids were generated using the dC:dG pooling method (Gocdel, D, V, et al., Natur
e287, 411-416 (1980)).

The CDNA insert can then be cut using Pst. The terminal line of each CDNA insert shows no flanking homopolymerized dC:dG tails. Pvul[.

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.

Figure 3 shows the direct microbial synthesis of mature LelFA.
The construction of the gene to be coded is shown. Restriction sites and remainders are indicated (such as "'PstI"). "'b,p," indicates a base pair.

Figures 4 and 5 show restriction maps of the two gene fragments used to express the LeIF 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 indicates a stop in the corresponding DNA sequence, and a hyphen indicates a deletion or omission in the sequence.

Next, a practical example for carrying out the present invention will be shown.

8. Microorganisms used In the described practice, two types of microorganisms, namely υ,
[, C01iX17 described in S, P, 4.190.495
76 and E. coli -12294 strains <end 8,
tl+i-, hsr-, hsm+, British Patent Publication NQ
2055382) is used. respectively, tl
+c American Type Culture
Co11ection, ATCCNα315
37 and 3144.6 have been E. recombinant DN
A's experiment is based on National In5tit.
It was carried out in accordance with the applicable guidelines for ute of lcalth.

A most advantageous embodiment of the invention is strain E as defined above,
colix 1776 and 294, as well as other known E. coli strains such as E. coli and other microbial strains; Most of th
e^merican Type Culture Co
11ection (8TCC), and are available at known microbial depositories.

See also German patent 264443″2. These other microorganisms include Bacillus, e.g.
us 5ubtilis and S. enterobacteriaceae such as S.
almOnella typl) imurium 5 erratid marcescens and uses a replicating plasmid capable of expressing foreign gene sequences. Yeast such as Saccharomyces ce
M. revisiae may also be advantageously used for the production of interferon protein by expressing the gene encoding interferon under the control of a yeast promoter.

B, 'La1F mRNA raw material and purified Le
IF mRNA can be obtained from human leukocytes.

Usually the 5enda i virus or Newcastle virus, as described in German patent NQ2947134
The method uses white blood cells obtained from patients suffering from chronic osteogenic leukemia, which have been induced to produce interferon with a disease virus. Particularly useful raw materials used in the present work are:
The cell line, designated J-Runi G-1, was derived from a patient with acute osteogenic leukemia. This cell line is oeff
ler, It, P, and Go Ide, D,
W.

5science 200.1153 (1978).
ark Memorial Institute > 1
640 plus 10% heat-inactivated FC3 (fetal bovine serum), 2
5mH liver PE5M1j liquid (N-2-hydroxyethyl-
piperazine-N'-2-ethanesulfonic acid) and 50
μ9/Id grows easily on gentamicin-containing medium;
Can be subcultured (1 to 3 splits) twice a week.

Cells can be frozen by adding 10% dimethyl sulfoxide to the growth medium described above. KG-1 goes to the 8m
ican Type Culture Co11ect
ion (8TCCNIlcRL8031).

KG-I Ml cells were prepared using the method described by Rubinstein et al. (PrOC).

Hat I, Hecad, Sci, I1. s, eight, 76
, 640-644 (1979)), 5Onda
i or Ncwcastle. Cells were harvested 5 hours after induction, and RNA was extracted using the guanidine diocyane-1-guanidine hydrochloride method (Chir
gwin et al., Biochemistry±8.5294
-5299 (1979)). RNA from uninduced cells was similarly prepared. Oligodeoxythymidine (dT)-poly(A) m
128 fractions of RNA were obtained. The method is described by Green et al.
(8rch, Biochem, Biophys, 1 7
2, 74-89 (1976)) and 0kuy
u+++a etc.(8rcltBiocheI11.Bio
phys, 188, 98-104 (1978)) was used. This mRNA was analyzed in African frog oocyte analysis ((ayaI ier i et al., Proc, N
atl, ^cad, Sci, υ, S, A, 74.328
7-3291 (1977)) 8000-10.00
Interferon titer of 0 units/microgram is shown.

Preparation of a colony bank containing the C3LelF cDNA sequence using 5 μg of IRNA using standard methods (Wickens
et al., J. Biol. Chem, 253.2483-24.
95 (1978) Onibi Gocdel et al., Natur
e281.

544-548 (1979)), double-chain CD
N A fi: Prepared. The cDNA was fractionated according to size by electrophoresis on a 6% polyacrylamide gel, with materials ranging in size from 500 to 1500 b.p.
Or+o was obtained by electroelution. 10 of this cDNA
Chang et al., Nature 27
5.

617-624 (7978n, tailing (ta) with deoxyguanosine (dG) remaining at Pst 1 site
iling) L, t470na plasmid pB
When annealing with R322, Bolivar et al., Gen
e.7.

95-113 (1977)>, using this, E, c
olix 1776 was transformed. CD N A n
About 130 tetracycline-resistant and ampicillin-sensitive transformants were obtained for Q.

In a second similar experiment, for cDNAnO, ca.
00 tetracycline-resistant and ampicillin-sensitive E. coli strains and 294 transformants were obtained. In this case, size-fractionated cDNA I material ranging from 600 to 1300b, Il, was collected by electroelution and used for tailing in dC.

0. Preparation of synthetic oligonucleotides and their use Knowledge about the amino acid sequences of several tryptic fragments of human leukocyte inkferon
This allowed the design of synthetic deoxyoligonucleotides complementary to various regions of NA. Two tryptic peptides were chosen, T143 and T13. This is because their amino acid sequences require only 12 and 4 undecamers to be synthesized to account for all possible coding sequences (Table 1). Four sets of deoxyoligomethreotide probes were synthesized for each sequence. That is, three each (T-IA).

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

D) Contains the A ribonucleotide of J. The complementary deoxyoligonucleotide 11 base chain shown was chemically synthesized by the phosphotriester method (Crea et al., Pro.
c, Nat l, to cad, sci, U, s, to 7
FΣ, 5.765-5769 (1978)). ■-1
In the 3 series, 4 individual probes were prepared. As shown in Table 1, as 4 pools of 3 probes, 1
Two T-1 probes were prepared.

12 T-1 probes prepared as 4 pools of 4 individual probes and 3 primers each of the T-13 series were radiolabeled single heavy stranded CD N A for use in hybridization probes. was used to induce the synthesis of Shisei mRNA A is 12S from Sendai-derived KG-1 microorganism (8000 units IFg/μm)
Total poly(A>mRNA) derived from RNA or uninduced leukocytes (<1 C unit/μ9). 32p-labeled cD
NA was added under known reaction conditions (Noyes et al., Pr0C,N
atl, 8cad, sc i.

tl, s, A, 76.1770-1774 (1979
)) was prepared from these primers.

20118 Tris-11cI (pH 8,3>,
201IIHKCI, 8n+HHoCI2.30mH
Reactions were carried out in 60 μm volumes in β-mercaptoethanol. This reaction consisted of 1 μg of each primer (i.e., 12 μy total for the T-1 series, T-1
For the 3 series, a total of 4 μ3 and 2 μU of “induced” 128-fraction mRNA (or uninduced poly(
A) 10 μ9 of mRNA, d^TP at 0.5 mH.

dCTP, dTTP, 200 μCi (a 32
p) dCTP (8mersham.

2-3000Ci/m mole> and 6C unit reverse transcription search (Bethesda I?escarch La
boratories).

The product was separated from unbound label by gel filtration on a 10 dsephadex G-50 column, treated with 0,3 N Nao++r for 30 min at 70°C to degrade RNA, and neutralized with 11C1. Hybridization is performed using Ka (atos, etc., Nucleic Ac1d R
es.

7.1541-1552 (1979).

E., loan [1-[30 of 500 E. coli strains, 12 strains, 294 transformed strains (
Birnboim et al., NuC1e! 1
CACidS Res, E, 1513-1523 (1
979) each rapid plasmid isolation procedure was used. The grown DNA samples were denatured and loaded on nitrocellulose filters in triplicate according to the method of Kafatos et al. cited above.

Three sets of nitrocellulose filters containing 500 plasmid samples were a) induced cDNAΔ primed with primer T-1 Sera1-;

b) T-13 primed induced tlcDNA and C) hybridized with uninduced 5CDNA prepared using both sets of primers. A clone was considered positive if it hybridized more strongly to one or both of the induced cDNA probes than to the entire uninduced probe. Select 30 Pa-positive "clones (1)Ll-1)L30) from 500,
Further analysis was conducted.

F, identification of clone p131-pL39, LclFT
The i gene fragment was used as a separation probe for the Kaneko fragment plasmid (No. 104), and 32p-labeled 13 mRNA (L
Il Lanhaug et al., Biochemistry
, 15, 1858-1865 (1976)) using the Qpunstein and Logness colony hybridization method (proc.

Natl, Acad, Sci, U, S, ^, 72.39
61-3965 (1975)), E. colix
1776 transformed strains were screened. Unlabeled mRNA from uninduced cells was mixed 200:1 to probe to compete with uninduced mRNA present in the 32p-labeled preparation. Labeled 11RN
Hybridization of A should occur preferentially to colonies containing the induced sequence. Three groups of transformants were obtained.

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

(a 10% had a significantly lower degree of hybridization than 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 interferon-specific sequences by analysis by specifically hybridizing inkferon mRNA with plasmid DNA. First, tetracycline (20 p'J/ml), diaminopimelic acid (100 μg/rd), thymidine (20 pg/II
dl) OJ: M with added d-biotin (1 μg/-)
Each of 60 robust colonies (group 1) was grown individually in 100 m of 9 medium. For 1 liter of M9 medium, NaIIPO(69), ll2
Contains PO4 (3g), NaC14 (0.5g) Miyo and N114CI (1g>),
After autoclaving, 1# of sterile IHHQSO4
Ii! and 10 d of sterile 0.01 HCaC12.

Ten cultures were pooled and described by Clewell et al., Bio.
chemistry9.4428-440 (1970
) from 6 pools as described in plasmid I) NΔ
Louis) ll1111. t-ku plasmid I'INA
10μ9 of the pool was digested with Hindlll Te, denatured and DBM (diazobenzyloxy+++et
hyl) was covalently bonded to the paper.

One micron of mRNA from the induced cells was hybridized to each filter paper. Hybrid Shina (XmR
NA was removed by washing. The specifically expressed mRNA was eluted and translated in Xenopus oocytes. All 6Illil pools were negative in this analysis. From the 59 eccentric colonies (group 2), 5 pools of 10 colonies each and 1 pool of 9 colonies were prepared, plasmids were prepared from these pools, and the plasmids were prepared as described above at a significantly higher level. hybridized to interferon mRNA. To identify specific interferon cDNA clones, plasmid DNA was prepared from 9 out of 10 colonies and examined individually. Out of 9 plasmids, 1I) 2 (No.
, 101 and Nα104) are ink feron w+RN
A well above background levels. A unique 8QI Tl restriction fragment containing 260b, p, was isolated from plasmid Nα104. This can be used as ■ay+or, etc., Biochim, Biophy
s,^cta442.324-330 (1976) and in situ colony screening method (Grunstcin and Hognes).
s, Proc, Natl, Sci, U, S, 8, V2.
3961-3965 (1975)) by E, co
Four hundred li294 transformants were used as probes for individual screening.

Nine colonies (pL31-IL39) were identified that hybridized to varying degrees with this probe.

Furthermore, using the labeled 260b, I) fragment,
Similarly, 4000 E. coli 294 transformants were individually screened. We identified 50 colonies that hybridized well with this probe. One is LelF GVJT piece, one is LclF
The If fragment is divided into two fragments, one called LeIF'llll (
apparently an allele of LelF H). The resulting hybrid plasmid is pLeTF11″
etc.

6. Isolation and sequence of the first full-length LelFm gene
- Plasmid DNA was prepared from a total of 39 potential LeIF c DNA clones. And Kaf
The same 260b, tl, and DNA probes were used for rescreening by hybridization of atO3, etc. (cited above). This probe and three plasmids (pL4
, pL31, pL34) gave a very strong hybridization signal, and four (pLl3, pL30
.

pL32. pL36) was moderately hybridized, and three (1)L6, 1)18, IILl4) were weakly hybridized.

The 39 potential LeIF cDNA
Each of the primers) was probed directly with hybridized probes. As complete base-base pairing is required to give a detectable hybridization signal,
The conditions for hybridization were selected (Wallace et al., Nu
cleic Ac1d Res.

β, 3543-3557 (1979)), that is, 3
Plasmid DNA was extracted from nine clones using the standard supernatant method (cleared+ysate procedure).
, Clewell et al.) and Biorad
It was purified by Agarose^-50 column chromatography. A sample (3 μg) of each preparation was collected from Ecol?
The ring was opened by treatment with I, denatured with alkali, and spotted onto two separate di]-cellulose filters. The amount is 1.5 μq per spot. Ka'f quoted above
See the literature such as atO8. Each of the synthetic deoxyoligonucleotide primers and primer pools
(γ32P) was phosphorylated with ATP as follows.

50 pmoles of oligonucleotide and 100p
100p (732P) ATP (New
England Nuclear, 2500 Ci/
m mole) of 5QmH,
Combined in 30 μm of a mixture of 10 mH HiJC12 and 15+++H β-mercaptoethanol. 2
of T4 polynucleotide kinase and incubate at 37°C.
After 30 min at 22p-labeled primer, 10 rrt
l 5ephadex 50 ka 54 top. Purified by uromatography. 1106 cp Prarai'? −
Hybridization was performed using T-13C or 3x166 cpm primer pool T-IC.

Hybridization was performed using 6XSSC (IXSSC=0.15HNaCI) according to the method cited above by Δallace et al.
, 0.0158 <1-lium citrate, pt+7,2
), 10x Denhardts (0.2% bovine blood m-albumin, 0.2% polyvinylpyrrolidone, 0.2% F
icoll) solution for 14 hours at 15°C. Filters were washed three times for 5 minutes at 0°C in 6xSSC. It was dried and exposed to X-ray film for two times. The results are shown in FIG. 1 for p-primer pool T-13G and primer-TIC.

Plasmid DNA from clone 104 hybridized frequently with primer pool T-IC and primer T-130. However, no detectable hybridization was observed with other undecamers. As shown in Figure 1, how many of the 39 possible LelF plasmids (pL2.4, 13.17.20.30,
31°34) hybridized with both of these probes. However, restriction analysis shows that one of these plasmids: 1) 131 is 260b, p, internal B
g1 [also contained fragments. As a result of digesting p[31 with Pst engineering, the size of the cDNA insert was approximately 1000 b,
It was p.

p[31 Pst ■ Sequence of the entire insert using Haxam
-Gilbert chemical methods (Methods EnzyI)
lol, 65. , 499-560 (1980)
) and dideoxy chain termination method (Smltl+, Hc
tl+ods Enzymol, 65, 560-5
80 (1980)'). At that time, 5au3
af! Six pieces were subcloned into the 813 vector as J5. The DNA sequence is shown in Table 2, 'A'. A reading frame was suggested.Then, including the play or signal peptide,
The entire eIF amino acid sequence is shown. First ATG
The Ill translation start 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 responsible for secretion of mature LeIr 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 1) LelF encoded by L31 as "'LelF8''. From the sequence data ("A") of 83, tryptic peptides T1 and T13 of LelF
It can be seen that they correspond to 45=149 and 57-61, respectively. The actual DNA found in these two regions
The coding sequences are those represented in primer pool TI-C and primer 713-C (see Table 4).

11. Direct generation of mature leukocyte ink feron A (LelFA) The procedure for direct expression of LelFA A as a mature interferon polypeptide is unique in that it involves the combination of synthetic (N-terminal) and complementary DNA. The lch method used for hormones (Goeddel et al., Natur
This is a modification of e281.544-548 (1979).

As shown in Figure 3, the 5au3a restriction site conveniently lies between codons 1 and 3 to LeIF. Two synthetic deoxyoligomethreotides were designed containing an ATG translational start, repairing the codon for amino acid 1 (cysteine), and creating a new ECORI sticky end. These oligos 7- are 34b of p[31, p, S
au3a-Ava II fragment. arise 4
The 5b,p product was ligated to two additional DNA fragments resulting in the 865b,D, synthetic-natural hybrid gene. It encodes LelFA, EcoRI and PstI
It is sandwiched between restriction sites. This gene is Eco
Between the R engineering and Pst ■ sites, pBR3
22 and designated plasmid pLe[FΔ1.

? , construction of a tryptophan regulatory element (containing the E. coli trp promoter, operator and trp leader ribosome binding site but lacking the ATG sequence for translation initiation) Plasmid ρG old contains the deletion ΔLE 1413 in E. coli - Responsible for the liptophan operon (old oz.
Zari et al., J. Bacteriology 33
, 1457-1466 (1978)). Therefore, t
the first six amino acids of the rp leader and trp E
Approximately the last 1/3 of the polypeptide (hereinafter collectively referred to as L
E') and a fusion protein containing the entire trpD polypeptide. This is all tr
It is under the control of the p promoter-operator system. 20 μ3 of this plasmid was digested with the restriction enzyme PVu I. This cuts the plasmid at five sites. This gene fragment is T-EcoRI! J>
h -(+)CATGAAITCATG (7) composed of self-complementary oligonucleotides),
Atotori et al., an ECORI cleavage site for cloning into a plasmid containing the EcoR site. I) 20 μ9 of the DNA fragment obtained from G.
51-phosphorylated synthetic oligonucleotide pcATGA
In the presence of AT1cATG, add 20μI T4 DNA ligase buffer (20mHTris pH 7,6,0,5ml
The cells were treated overnight at 4°C with 10 units of T4 DNA ligase in ATP, 10i + HHoC12, 5mM dithiothreitol). 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 coR■ ends were separated using 5% polyacrylamide gel electrophoresis (hereinafter referred to as PAGE), the three larger fragments were first stained with ethidium bromide, the fragments were positioned using ultraviolet light, and the gel I separated it by cutting out the more targeted part. Each gel fragment (
300μl 0.1xTBE) into the dialysis bag and 1ffl 0.1xTBE buffer (TBEII!) into the dialysis bag.
1i14. tlo, 8SF of Ir1s base, 5.5 g of boric acid, 0.099 Na2EDTA in 1 liter of 8
Electrophoresis was carried out at 100 V for 1 hour in a 100 V solution (contained in 20 ml). An aqueous solution was collected from a dialysis bag, extracted with phenol, extracted with chloroform, adjusted to a sodium chloride concentration of 0.2M, and DNA was collected in ethanol precipitated condensate. EcoR: [The trp promoter-operator-containing gene with 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 pBRH1 (Rodrigue Z et al., Nuc
leicAcids Res, Dan, 3267-3287
(1979)) expresses ampicillin resistance and contains a tetracycline resistance gene. However, since there is no promoter associated with it,
It does not develop its resistance. This plasmid is therefore tetracycline sensitive. By introducing a promoter-operator system into this EcoR site, the plasmid can be made resistant to tetracycline.

DBRIII was digested with EcoR:[, the enzyme was removed by phenol extraction and chloroform extraction, and ethanol precipitated and suspended in water. Product N present in separate reaction mixtures
The A molecule was merged with each of the β DNA fragments obtained above and ligated with the 4 DNA ligapi as described above. Using the DNA present in the reaction mixture, standard methods (Ilershfieldcl et al., Proc. Natl.
, Δcad, Sci, U, S, A.

71.3455-3459 (1974)) by DN
One 12 strains of 294 were transformed into E and C01t capable of taking up A, and the bacteria were transformed into 20μ9/#li! LB (Luria-Bertani) plates containing 5 μg/le of ampicillin and 5 μg/le of tetracycline were inoculated. Several tetracycline-resistant colonies were picked, plasmid DNA was isolated, and the presence of the desired fragment was confirmed by restriction enzyme analysis. The resulting plasmid is pB
It is called RHtrp.

EcoR■ and Bam1l of hepatitis B viral genome
1 Digestion product was collected by a conventional method, and plasmid pG11
EcoR of 6: [and cloned into the Bam1 I site. Goeddcl et al., Nature 281, 54
4 (1979)), and the plasmid pH is 32. Plasmid pH332 was cut with Xba I, phenol extracted, chloroform extracted, and ethanol precipitated.

Precipitation was performed using 0, 1 mW dTTP and 0.11II.
30μ containing HdCTP! Polymerapi buffer (5
0mM potassium phosphate pH 7,4,7IIIHHgC1
1 μIE in 21 mH β-mercaptoethanol),
coli DNA polymerase ■, x+enow fragment (Boehringer-Hannheim),
It was treated 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 dT are incorporated to give an end with two overhanging 5' nucleotides. This linear residue of plasmid lllll332 (phenol and chloroform extraction followed by ethanol precipitation and water) was cut with EcoRI. large plasmid fragment,
The smaller EcoR:[-Xba I fragment was separated by PAGE and separated by electroelution. pHs 32 (
This DNA fragment from 0.2 μg) was ligated to the EcoRI-Taq I fragment of the tryptophan operon (approximately 0.01 μg) from pBRHtrp under conditions similar to those described above.

+7) pH332 (7) fragment above with IEcoRI-r
In this method of ligation to the aq I IFi piece, although not a complete Watson-Crick base pair, the Tag
The overhanging end of Xba I is ligated to the remaining overhang of Xba I.

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 and 3 rtd
1LB (Luria-Bertani) and the plasmids were isolated. Six of these colonies were found to have Xba I sites that were regenerated by DNA repair and replication by E. coli.

It was also found that these plasmids were cut with both EcoRI and HDaI to give the expected restriction fragments. One plasmid, designated plrpl4, was used to express a foreign polypeptide as discussed below.

Plasmid I) HGHI O7 (Goeddel et al., N
atLIre281.544 (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
Although lacking the sequence codon, it contains the ATCtJl translation initiation codon. This gene is 10μpHGH10
7, EcoRI treatment followed by E as described above.
, E. coli DNA polymerase I K10nOW fragment and isolated via dTIP and dATP treatment.

The ethanol precipitated plasmid after phenol and chloroform extraction was treated with BaIIIHl.

The human growth hormone (HGH) m gene-containing fragment is PAG
E, followed by separation by electroelution. The resulting DNA fragment lacks the tetracycline promoter-operator system but also contains the first 350 nucleotides of the tetracycline resistance structural gene;
When subsequently cloned into an expression plasmid,
Plasmids containing the insert can be identified by restoration of tetracycline resistance. Since the EcoR end of the fragment was filled with the Ienow volume rapi method, the fragment had one blunt end (formerly unt end
) and one sticky end. This ensures correct orientation when later inserted into an expression plasmid.

Expression plasmid pTrpl4 was then prepared to receive the l-IGH gene-containing fragment prepared above. That is, pTrpl 4 was digested with Xba I,
The resulting sticky ends are labeled with dATP, dTTPldGTP
and dCTP using the Ienow volumerase method. 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 is similar to 1-I as described above.
This made it possible to recombine with the G l-1 gene-containing fragment in the correct orientation.

HGH gene fragment and I) TrDl 4ΔXba-BaI
The llH1 fragment was merged and ligated to each other under conditions similar to those described above. The filled Xba I and Eco RI ends are blunt-ended ligated, and the Xba I site and E
Both coRI sites were reconstituted.

Filled Xba I Filled EcoRI 1lG
II3i gene origin Xba I EcoRI This construct also creates a tetracycline resistance gene. Plasmid EIHGtfl O7 is 1-IGH
Since tetracycline resistance is expressed from the promoter (lac promoter) located upstream of i Butsuko,
This construct, designated pHG11207, 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 μg/ml tetracycline.

Plasmid pHC11207 was EcoR1 digested and tr
p promoter-contains operator and trp leader ribosome binding site but ATG for translation initiation
The 300b,P, fragment lacking sequence was collected by PAGE followed by electroelution. This [)NA% piece is pLe
Cloned into EcoRI site into IF. An expression plasmid containing the modified TRP reouton described above (an E. coli TRP operon in which the attenuator sequence has been deleted to regulate high expression levels) is sufficient to suppress the promoter-operator system. can be grown to a predetermined level in a medium supplemented with tryptophan. The desired product can then be expressed by removing the tryptophan and removing inhibition of the system.

To be more specific, with reference to Figure 3, 250μ9
Plasmid pL31 of was digested with pst I and 1000
b, The insert of P was separated by gel electrophoresis on a 6% polyacrylamide gel. Approximately 40 μ3 of insert was electroeluted from the gel, divided into three portions, and further digested as follows. a) A 16μ sample of this fragment was partially digested with 40 units of B (71μ) for 45 min at 37°C and the reaction mixture was purified on a 6% polyacrylamide gel.
Approximately 2 μg of fragments of b, El, were collected. b) 1000
b, p, Another sample (8 μg) of Pst I insert was added to A
Digested with va It and DI II. After gel electrophoresis, 1 μg of the above 150b, D fragment was obtained.

C) 100 ob, p.

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

Ato electrophoresed on a 10% polyacrylamide gel,
34b, l of approximately 0.25 μ9 (101) mole)
).

Got a piece. Two indicated deoxyoligonucleotides, 5'-dAATTCATGTGT (Fragment 1)
and 5′-dGATCACACATG (fragment 2
) was synthesized by the phosphotriester method. Fragment 2 was phosphorylated as follows. , 200 μm (approximately 40 pmole) (
7P) A T P (Amers1+am, 5000
Ci/mllote) was dried and 5QmH Tri
s-11cI (pH 8), 10mM HgCl2.
15 mH Constructed from β-mercaptoethanol, 10
The suspension was resuspended in 30 μm of a solution containing 0 pmole of DNA fragments and 2 units of T4 polynucleotide kinase. 3
After 15 min at 7°C, 1 μM of 10 mM ATP was added;
The reaction was allowed to proceed for an additional 15 minutes. The mixture is then heated to 70°C
Heat for 15 minutes at
a Merged with the Ava II fragment.

2018 Tris-IIcI (al17.5),
10mM) 1ocl, 10all dithiothreitol, Q, 5mM ATP and 10 units T4'DN
Ligations were made between 5 Fi in 50 μm of A ligase at 4°C.

The mixture was electrophoresed on a 6% polyacrylamide gel, and the 45b,p product was collected by electroelution. 45t
1. D, 30no (1pIIlote) of the product was combined with 150b,p, 0.5u9 (5μmole) of the Ava IF-hl IF fragment and 670b,p,
1 μg (2 μmote) of BgI n-pst I fragment
) merged with Ligation was performed at 20°C for 16 hours using 20 units of T4 DNA ligase. The ligase was inactivated by heating at 65°C for 10 minutes. The mixture contains EcoRI and P
The gene polymer was removed by digestion with stI. The mixture was purified by 6% PAGE. Approximately 20r+(J (0,0
4pmole) of 865b, l), the product was isolated. Half of this <10 ng) was added to pH 1122 (0.3μ
g) between the EcoRI and pstI sites. Transformation of E. coli 294 yielded 70 tetracycline-resistant, ampicillin-sensitive transformants. From 18 of these, plasmid D
NA was isolated and digested with EcoRI and PstI. Of the 18 plasmids, 16 were 865t1.1
), had a long EcoRI-Pst IIi piece.

EC of 1 μ8 of one of these pLelF At
300 tl as prepared above, digested with ORI and carrying the E. coli trp promoter and trp leader ribosome binding site, 11. [: ligated to C0RI fragment (0.1 μg). A transformant containing the trp promoter was GrunSteill-1+00ne
ss: 10 In combination with the knee cleaning method, 3
It was identified using a 2Ptrp probe. The asymmetrically located Xba I site in the trp fragment allowed the identification of recombinant products in which the trp promoter was oriented in the direction of the LelFA gene.

1. In-human O and in-vivo active IF of LelFA
Extracts for analysis were prepared as follows. 1 ml of the culture was grown in broth containing 5 μg/d of tetracycline to give an A35o value of approximately 1.0 and then grown in M9 medium 2 containing 5 μg/d of tetracycline.
Diluted in 5d.

When A 550 reached 1.0, a 10 m sample was collected by centrifugation, and the cell mass was diluted with 15% sucrose and 50 mHTri.
5-HCI (pH 8,0), 501118EDT
It was suspended in 11d of A. After adding 1 m9 of lysozyme and incubating at 0°C for 5 minutes, cells were disrupted by sonication.

The samples were centrifuged for 10 minutes (15,000 rpm:
CPE) in comparison to standard LeIF. To measure the number of IF molecules per cell,
A LeIF specific activity of 4×10 8 units/litre was used.

As shown in Table 6, D-type tlLelF A trp 25 with the trp promoter inserted in the desired orientation showed high activity (2.5 x 108 units/1). As shown in Table 7, E, colt K-12 strain 294/pLe
IF generated by Atrp25 behaved similarly to the human tel standard. Stable to treatment with p112,
Neutralized by rabbit anti-human leukocyte antibody. The apparent molecular weight of this interferon is approximately 20,000. Clone pLelF A trp 25 has been deposited with the American Type Culture Collection (ATCC).

Inkferon's in vivo effects require macrophages and natural killer (NK) III follicles. And the in vivo effect appears to involve stimulation of these IIl follicles. So, [.

Colt294/I) Interfae O produced by LelF A 25 has antiviral activity in cell culture assays but may be ineffective in infected animals. Furthermore, the in vivo antiviral effects of non-glycosylated LelF produced by bacteria may be different from that of glycosylated LelF derived from human "buHy coat" leukocytes. We therefore compared the biological activity of bacterially synthesized LeIF8 (2% purity) with buffy coat LelF (8% purity) in lethal encephalomyocarditis virus (EMC) i infection of squirrel monkeys. I tried it (Table 8).

Table 6. Inkferon activity of E.coli extract Table 7. Activity comparison between E.coli extract and standard LeIF
Extract of A trp 25 - 250,000 units per diluted 500 times with minimal essential medium, 500 units/#I
i! The specific activity of Leukocyte interferon standard (14
ad 1ey Institute) in advance at NIH.
Titer against leukocyte interferon standard,
Yasu 500LJ/at! +7) Dilute to final concentration. 1dlffl was adjusted to 1) 82 with 1HHCI, incubated at 4°C for 52 hours, neutralized by adding NaOH, and
F activity was measured with a standard CPE11fl stop assay. 500
Unit/II! 1! 25 μm of sample (untreated)
m rabbit anti-human leukocyte interferon at 37°C.
Incubate for 0 minutes, centrifuge at 12,000×g for 5 minutes, and analyze the supernatant.

Table 8. Antiviral effects of various LeIF preparations on EMC virus infection in squirrel monkeys (squirrel monkeys). All monkeys were male (average body weight 713 J), and before infection,
Does not have EMC virus antibodies. For monkeys, 100×L
D5° EMC virus (measured in mice) was infected intramuscularly. Control infected monkeys had 134.158.164 after infection.
Died in time.

106 units Inkferon treatment was administered intravenously 4 hours before infection, 2.23.29.48.72.168 and 240 hours after infection. Bacterial leukocyte interferon was a column chromad fraction from a lysate of E. coli294/DLelF^25 and had a specific activity of 7.4 x 106 units/per protein. The control bacterial protein was E, col
i294/pBR322mWl product.

The total protein content was doubled in the corresponding column fractions.

The white blood cell ink feron standard is clontogella fi,
It was a 5enda i virus-induced infusin from normal human buffed Ecoat'' cells purified to a specific activity of 32 x 106 units/■ 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 exhibit any of these abnormalities and were constantly active and showed no symptoms of viremia. One control monkey that did not show viremia until day 4 died later.
However, after death (164 hours after infection), high titer lesions were detected in the heart and brain. Inkferon-treated monkeys did not produce antibodies against the EMC virus when tested 14 and 21 days post-infection.

Show that it can be done. This is because the contaminating proteins are quite different in bacteria and in buffered food preparations. Furthermore, these results indicate that glycosylation is not required for the in vivo anti-inflammatory activity of LeIF A.

J, CD N for other leukocyte ink ferons
Isolation of A DNA from a fully characterized LelF A cDNA-containing plasmid was cut with Pst I, separated by electrophoresis, and labeled with 32p. The resulting radiolabeled DNA was used as a probe and
An additional transformed strain of E. coli 294 obtained in the same manner as in Section 0 above was screened by the in situ colony screening method of n and logness. Colonies that hybridized to the probe to varying degrees were isolated. Plasmid DNA from these larvae and from the 10 hybridized colonies shown in Section G above was isolated by Pst I digestion and characterized in three ways. First, these Pst 117i pieces were treated with the enzymes Bol If, Pvu T
It is characterized by a restriction endonuclease digestion pattern using L and EcoRI.

The analysis is shown in Figure 2 - at least eight different types (LelF A, LelF B, LelF C,
LelF DlLelF E, LelF F, Lel
FG and LelF H). This indicates the approximate location of the various restriction breakpoints with respect to the previously known leading and coding sequences of LelFA.

One of these, LelF D, was reported by Nagata et al. in Nature 284.316-320 (1980).
It is believed that this is the same as what was reported.

Second, for some of these DNAs, LelF mRNA A
C1cvland for its ability to selectively remove
et al. Ce1l 20.95-105 (1980). LclF
A, B, C and [ were positive in this analysis. Third, these Pst Ili pieces were inserted into expression plasmids and E. coli 294 was transformed with the plasmids to express the fragments. 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 LOIF types listed above have been sequenced.

K, second mature leukocyte interferon (LelFfi)
An isolated fragment containing the gene for direct valve maturation LelF B of ) promoter-operator, ribosome binding site and a fragment with the beginning of telFA (=8) 3m Buddha was isolated from pLelF A25 and the remaining B sequences were isolated in the expression plasmid. combined with parts. pL 4 (second
The LOIF B pst terminal '1M3M Buddha shown in the figure is shown in the content diagram, respectively.

According to the sequence of Fig. 4, the Sau 3 a-pst 1 fragment of approximately 950 b,p relative to the Pst I fragment has one or more intervening Sau 3 fragments.
Since there is a restriction site, several steps are necessary. In other words, the next J: unaru.

1. The fragments marked with were isolated.

a) 110b from Sau 3 a to EcoRI
b, p, :b) 132 from EcoRI to Xba
b, p.

c) >700b from xba to PSt, ρ.

2. Fragments (1a) and (1b) were ligated and the relevant Sa
The u3a site is within the BglII site: l1011
[Cut left Sau3a sticky ends). 242
b, p, fragments were isolated.

Pst I and [jgl
Cut with II. A fragment of approximately 950 b, p from Sau 3 a to pst I (fourth box) was isolated. This fragment is common to LelF Bi, which is not common to LelF A.
Contains parts of Buddha.

4, about 300 b, I), fragment (llinclm to Sau
3a) were isolated from pLelF25.

5, about 3600b from PSt I to Hindnl,
p.

The fragment was isolated from IIB R322. replicon and encodes tetracycline resistance, but does not contain ampicillin resistance.

6. Fragment numbers obtained in steps 3.4 and 5 are triple ligated and the resulting plasmid is used for E. coli K-12 strain 29.
4 was converted.

Convertible stocks were screened by mini-screening (Birnboim et al., N.
ucleic Ac1ds Res, 7.1513-
1523 (1979)), and the plasmid samples were Ec
oR]: Digested with. The digestate gave three fragments,
Their characteristics are: 1) EcORI -EC0RI tr
p promoter fragment; 2) pL4 internal EcoRI −
EcoRJ fragment: and 3) pL4 protein translation initiation signal-Ecall engineering fragment.

For CPE analysis, bacterial extracts from the clones prepared as described above were typically approximately 10 x 10
It gave an inkferon activity of 6 units/1. One representative loan prepared in this way is [, coli
294/ pLelr B trp 7. This clone has been deposited with the ATCC.

[, yet another mature leukocyte inkferon (LelFC)
1D, F, II, ! and J) Direct Expression Additional full-length gene fragments containing other LelF types can be placed into an expression vehicle for tiller expression, as in telF A. The N-terminal amino acid codon that was lost due to the removal of the preceding sequence is synthesized by
The presence of a restriction site sufficiently close to the first amino acid codon of the mature interferon type to determine whether the convenient method of replacing it by ligation of A fragments can be determined by conventional methods of determining whether the complete sequence It will be seen from the decision. If that doesn't work, you can use the following operation. In short, the fragment containing the preceding sequence is cleaved exactly before the start of the codon for the first amino acid of the mature polypeptide. In other words, 1 In the region surrounding that point, 2 positive strands of DNA are 1
Converting to heavy chain DNA: 2. Place the 5' end of the primer on the opposite side of the nucleotide that binds to the desired cleavage site in the 7c1 heavy chain region generated in step (1), and convert into single-strand DNA. Hybridize complementary primers.

3. Recovering the portion of the second strand removed in step 1, in the 3′ direction from the primer, in a reaction using a DNA polymerase in the presence of adenine, dimine, guanine, and cytosine-containing deoxynucleotide tribosphates. let

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 to the gene tailored for the resulting mature inkferon, for example by blunt end ligation, and this gene is inserted into an expression plasmid and placed under the control of a promoter and associated bosomal binding sites.

In the same manner as used in section above, LelF Ca3
The gene fragments encoding and LelF D were suitably sequenced for direct bacterial expression. As a strategy for the expression of these additional leukocyte interferons, in each case LeI from pLelF A25
A fragment of approximately 300 b, p, containing the trp promoter-operator, ribosome binding site, ATG initiation signal and cysteine codon of F A (llindl
l[ to Sau 3 a). In contrast, gene fragments from other inkferon genes encoding the respective amino acid sequences beyond the first cysteine, which is common to all, are combined. 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 fragment from LeIF C pLelF C: (a) Sa
35b, l) from u3a to 5au96.

(b) >900b from Sau 96 to pst I
, p.

(C) As in section K(4) above, pLelFA-
25, about 300 b, I), a fragment of (tlind[l-
Separate 3au3 a).

(d) Divide the fragment of about 3600b,p in the above K(5) term into 11111.

sex! (1) Connect (a) and (C). Bgl f
1, tlind■ to separate the product of approximately 335 b,p.

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

A typical loan prepared in this way is E. coli.
K-12 strain 294/1) LeIF ctrp
It is 35. This clone has been deposited with the ATCC.

LeIF D pLelF Separate from D as follows: a) Sa
u 3 a to Ava I [35b, 11°b)
B from Avall! 150b up to III II, 11
゜c) B(+1 I[From Pst I about 700b
, p.

Isolate the following fragment from pLelF A25 = d)
HindI [from sau 3 a to 300b, l)
.

Isolate the following fragment from pBR322: e), 1I
indI [[from PSt I approximately 3600b, p.

Concatenate Kamei (1) (a) + (b) and create B(II II
Cut at 185b, +1. Purify product (1).

(2) (1) Connect +(d), 1lindll,
B (cut with II I [and about 500 b, I),
Purify product (2).

(3) (2) +(c) +(e) are ligated and the resulting plasmid is used to transform E. coli.

Representative clones prepared in this way were E, co
il K-12 strain 294/1) LelF D tr
It is pll. This clone has been deposited with the ATCC.

Fragments containing LclF F LeIF F can be tailored for direct expression by rearrangement that takes advantage of the complete homology of amino acids 1 to 13 of LclF B and LeIF F. From the above pt-IGH2,07, a trp promoter-containing fragment (a
). The second fragment (b) is the plasmid pHKY1
vine as the larger of the fragments resulting from 0 Pst I and 8g+1i digestions. Fragment (a) contains about half of the gene encoding ampicillin resistance; fragment (b) contains the remainder of the gene and also contains all of the tetracycline gene except the associated promoter. Fragments (a) and (b) are ligated by T4 ligase and the product is treated with Xba I and B (11II to prevent diphonization, t+”p promoter-operator and tetracycline and ampicillin resistance. Fragment (C) containing the sex gene.

Fragment (d) of approximately 580b, Il, is A of pLeIF F.
Obtained by digestion of va II and hl II. It contains codons for amino acids 14 to 166 of LeIF F.

Fragment (e) (49b,p,) is 0LelF B(7
) Xba I # and Ava I [obtained by digestion. Fragment ((3> encodes amino M1 to 13 of LeIF F.

Fragments (C), (d) and (e) are triple ligated in the presence of T4 ligase. The cohesive ends of each fragment ensured that the composite plasmid was properly circularized and that the tetracycline resistance gene, along with the mature LelF F gene, was under the control of the trp promoter-operator, allowing for transformation with the desired plasmid. Bacteria can be selected on tetracycillin-containing plates.

Such preparations have been deposited with the ATCC.

The complete LclF H LelF Il gene can be arranged as follows for expression as a mature leukocyte interferon.

1. Plasmid pLelF 11 was subjected to Hae II and Rsa I digestion to remove the signal peptide amino 11.
81 eb, p, extending from 10 to 3' non-coding region.
Separate the i pieces. -2. Denature this fragment and add it to DNA polymerase
enow fragment (KIenOW et al., Proc, Natl
, ^cad, Sci.

U, S 8, - (Me 151.1.68 (1970)
), the synthetic deoxyribo-oligonucleotide primer 5'-dATG TGT AAT CTGTCT
Process the repair composite using .

3. The resulting product was cleaved with Sau 3 a to reveal 1 to 150 amino acids, 452 b, p, [! Separate the li pieces.

4.3au3a and Pst I 1' 1) LelF
H was purified and the obtained 500b, p, fragment was separated,
A gene encoding the sequence from the 150th amino acid to the end of the coding sequence is obtained.

5 The fragments separated in steps (3) and (4) are ligated to form fragment 1 166 1et Cys aSp stop ATG TGT--GAT TGA--
・-- Obtain Pst l5au 3 a. This loads 166 amino acids of LeIFH.

6. Digest 0LelF A trp 25 with XbalF, make blunt ends using DNA polymerase I, and digest the product with Pst I. The resulting large arai can be isolated 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 Lcl'II.

The face λ of the human genome constructed by LeIF I Lawn et al.
Charon 4 A recombinant library (Cell.

1 Ch., 1157 (1978)) as cited above.
The method of n et al. and Haniati S et al. on Ce1l 1.

687 (1978).
Screened for zero. CDNA clone L
Approximately 500,000 plaques were screened using the radioactive LeIF probe derived from eIFA. Six LeIF genomic clones were selected. Following rescreening and plaque purification, one of these clones, λIILelF2, was selected for further analysis.

Using the methods described above, the Sacral probe can also be used advantageously to isolate other LeIF clones from the human genome. These can then be used to produce other leukocyte infusin proteins according to the invention.

1. 2000b, ll of clone λHLelF 2,
The EcoRI fragment was inserted into pBR32 at the EcoR■ site.
5 was subcloned into. The resulting plasmid Le
lF I was cleaved with EcoR, and the 2000b, p, and 2000b fragments were separated.

This 2000 b, p, EcoRI fragment was supplemented with deoxyoligonucleotide dAATTCTGCAG (EcoRI
- pst I converter). The resulting product was cleaved with Pat I and had less than 20
00b, p, fragment. This was cut with Sau 96 Te. A 1100b,p, fragment with one Pst and one 5au96 end was isolated.

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

3. Small XbaI from pLelF Ctrp 35
-pst I fragment was digested with Xba I and 5au96.

Isolation of 40 b, p, Xba I-sau 96 fragments 1. tc.

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

clFJ1, plasmid pLcIFJ contains a 3.8 kilobase 11indl fragment of human genomic DNA containing the LelFJ gene sequence. The 760 b, p, Dde I-Rsa I fragment was isolated from this plasmid.

2. Plasmid pLelF B trp 7 to 1Ii
ndi [and DdeIr cleavage, 340b, p, old n
The dl[[-0de I fragment was isolated.

3. Cut plasmid pBR322 with PStI,
Incubate with DNA polymerase, make blunt ends, <K Ienow 1g piece), and cut into 1 piece with 1 piece.
dlnr was digested. A large (approximately 3600 b, l) fragment was isolated.

4. The fragments separated in steps (1), (2), and (3) were ligated to create the expression plasmid 11LelFJ t
ri) was set to 1. This clone has been deposited with the ATCC.

The content of leukocyte protein in H3 purified bacterial extract was increased in the following manner.

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, 10IIIHTris-tlcl, pH
7,2, suspended in 25 mHTTris-llCl,
Dialyze against pH 7.9. Intaf10in activity remains in solution.

4. Adjust the above supernatant to pH 8.5 and perform clontogelatology on a DEAE-cellulose column (251n
HTris-HCI, 0 to 0.2M in pH 8.5
Elute with a linear gradient of NaCl).

5, Cibachrome 31ue -A galo
Adsorb to se or 11 hydroxyla Datite and elute with 1.58 KCI or 0.2 M phosphate (as necessary).

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

7. Perform cation exchange chromatography on CM-cellulose in ammonium acid at 25 mHI at pH 15.0. 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 used for size exclusion chromatography, reverse phase (RP-8>high pressure liquid chromatography).

or purified by affinity chromatography on immobilized anti-interferon antibodies.

Alternatively, the material from step 4 above can be
n, 5cientific American 24
3.66 (1980), plus 0.2 M acetic acid, 0.1% Trit.
on and 0.15HNaCl t-elution t, ur.

In another advantageous embodiment, the leukocyte infusin produced by the procedure described above can be purified in the next 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 l'ris, 1Q% (w/v) Suf0-su,
0.2H NaCI, 5mMEDTA, 0.1mHP
Suspend in 4 volumes of buffer A containing MSF and 10-1001118 8 (lcI2). The suspension is kept at approximately 4°C.

The suspension was passed through a homogenizer at approximately 6000 pSi;
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. Solids are removed by centrifugation or filtration. At this stage, adjust the temperature or! , done quickly enough to keep the supernatant (filtrate) below 10°C. Supernatant (filtrate)
Concentrate by ultrafiltration to about 1/1 of the original volume. If you notice particulate matter or cloudiness, remove it with a suitable filter such as a micropore membrane.

3. The clarification solution is flowed directly onto the monoclonal antibody column at 5-8 cm/hour (eg, 25-40 rrtl/hour on a 2.6 cIn diameter column). Next, this column was added to a 2011+8 Tris with a capacity of about 10 columns.
-HCl, pH 7,5-8,5 (NaCl (0,5M
) and Triton X-100 (0.2%) or equivalent surfactant). After washing, the column was treated with 0.15H NaCI and Triton X-10.
0 (0.1%) or about 1 containing equivalent surfactant
Pass 0 column volumes of solution through. Use this column with Triton
Elute with 0.2M # acid containing X-100 (0.1%) or equivalent surfactant. Protein peaks (measured by UV absorption or other methods) from the monoclonal antibody column were collected and treated with 1N NaOH or 1.0M Tris.
Adjust pl+ to about 4.5 with base.

4. Transfer the pooled ink feron beak to a suitable buffer such as ammonium acetate pH 4,5 (50mM).
Add to Latman CM 52 cellulose or equivalent ion exchanger equilibrated with

After loading, the column is washed with an equilibration buffer until the UV absorption of the effluent becomes irritating, so that almost no protein is eluted from the column.

The column was then washed with 25mM ammonium acetate 10.12
M Sodium Chloride or Inkferon is eluted with a combination that gives the best recovery and yields a lyophilized cake with satisfactory appearance and solubility.

The monoclonal antibodies in this advantageous embodiment described above are 5ta (!1IQIin etc. are Proc, Natl, A
cad, sci.

U, S, A, 78.1848-52 (1981) D. Monoclonal antibodies are purified and covalently coupled to Affigcl-10 as described below.

Preparation and purification of monoclonal antibodies from ascites fluid 11B
Five alb/c mice were each inoculated with 5-10×10 6 hybridoma cells taken at mid-log phase. Approximately 5 x 106 viable cells obtained from ascites fluid produced by mice were
Or more than 10 mice each were inoculated intraperitoneally. Repeat this ascites fluid for each mouse'' (2 to 4 times)
Collected. Mice from one group of mice were replaced and harvested up to three times in the next group. Ascites fluid collected after each transfer was pooled.

Treat with low speed centrifugation (500-1000x9) for 15 minutes,
The 11ll cysts and debris were removed from the ascites fluid. Then 18
．． Centrifugation was performed at OOOrpm for 90 minutes. The supernatant was frozen and stored at -20°C. After melting, 35. OQQ
Fibrin and particulate matter were further removed by centrifugation at rpm for 90 minutes. A batch of ascites fluid from each transfer was tested using solid phase antibody binding assays (5taehelin et al.,
The specific antibody activity was tested according to the cited reference above) and pooled if satisfactory.

The protein concentration in the pooled solution is 1 m! The measurement was carried out using an approximation method in which the protein j exhibits an absorbance value of 1.2 at 280 rim in a 1.0 cm thick cuvette.

Ascites fluid with a high concentration of antibodies contains 30-35 my protein/rd. This is 4-7 my specific antibody/d
corresponds to This liquid was dissolved in PBS (0, OIM Notrium Phosphate, l)I+7.3.0. 15M NaC1
) to give a protein concentration of 10 to 12 m9/mfl.

To each 100 ml of diluted solution, 90 d of ammonium sulfate solution saturated at room temperature were 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 10.00 rpm for 15 minutes.

The supernatant was removed with a spatula, and the liquid was thoroughly drained. 0.02M TriSllcl (pH 7,9) 10
．． 04M NaCl (Buffer Engineering). The protein solution was dialyzed against 100 volumes of buffer ■ for 16 to 18 hours at room temperature. During this time, the buffer was exchanged at least once.

The dialysis solution was centrifuged at 15.00 Or11111 for 10 minutes to remove insoluble matter. Approximately 3 of the initial amount of total protein in ascites
0 to 35% was estimated from the absorbance value at 28 Or+m.

A solution containing 30-'401n9 of protein for 1 d was then applied to a DEAE-cellulose column equilibrated with 3 uHcr I. A column bed volume of at least 100 d was used for each gram of protein added. 0.0
NaCl in 2M Tris HCI pH 7,9 to 0.
NaCl linearly from 04M to 0.5M NaCl 1 degree
Antibodies were eluted from the column at varying concentrations. 0.0
6 to 0. Pool the peak fractions of IM NaCl,
An equal volume of saturated ammonium sulfate solution (room temperature) was added, and the mixture was centrifuged for precipitation and concentrated. Protein mass in 0.2M Na1lCO
3 (pH about 8.0) 10, 3M NaCl (buffer ■)
and dialyzed against the same buffer (three changes) at room temperature. 20. The mixture was centrifuged at OOOXg for 15 minutes to remove insoluble matter. The protein concentration was adjusted to 20 to 25 mg/mfl with buffer ■.

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 tube and briefly centrifuged to sediment. The supernatant was removed with an aspirator, and the packed gel was mixed with an equal volume of Q of the 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 diluted with buffer ■ (0°I M Na1lCO3/
The cells were washed twice with 0.15M NaCI) to remove unbound antibodies. When the washing solution was combined and the U(L protein was measured, more than 90% of the antibody was bound to the gel.

To fill the unreacted area, add an equal volume of 0.1M gel.
Mix with ethanolamine/llCl (1)H8),
The mixture was treated at room temperature for 60 minutes while rotating the long axis in a circular motion. The gel slurry was washed with PBS to remove the reactant, and the gel slurry was washed with PBS in the presence of 0.02% (w/V) sodium azide at 4°C.
Stored in S.

Parenteral Administration LelF can be administered parenterally to patients requiring anti-tumor or anti-viral therapy. It can also be administered to patients who are not immunosuppressed. Dosage and administration regimes will be similar to those currently practiced clinically for substances of human origin. For example, 1% at approximately (1-10)XIO6 units/day
In cases of higher purity, it can amount to, for example, 5×10 7 units/day.

Essentially homogeneous bacterial Lel in a form for parenteral administration
Suitable dosage forms of F include, for example, 2 x 108 units/
ll1g specific activity c7) LelF3 #ISF25
d in 5N human serum albumin, and this solution was dissolved in 5N human serum albumin.
Pass the filtrate solution through a 4° physical filter aseptically at 10°C.
1 Cp per 0 bottles, suitable for parenteral administration.
The book contains 6 x 106 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. For suitable vehicles and their formulation, see [Rem by Hartin, 1].
1nqton's pharmaCeutiCal 5
As described in the following sciences. This is cited as a reference in this specification. In these compositions, an effective amount of inf10 protein is combined with a suitable amount of vehicle to form 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 autoradiogram showing potential hybridization of the LeIF plasmid with a 32p labeled synthetic deoxyoligonucleotide. Figure 2 shows eight types of LelF cloned cDNA (A
Restriction endonuclease maps of 1 to 1) 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 the LelF B mature leukocyte interneuron. Agent Akira Asamura Drawing of Western Chicken (Contents (further L) ``2P-T-13c Probe Bi- No. 10. - Drawing cO00LLI L Q Engineering Page 1 continued ■ Int, CI, ' Identification symbol Internal serial number priority claim 019EMESeptember 8th 0United States (U
S) @256204 [Phase] Inventor Sydney
Plague Power United States Le, Brussels @ Applicant Genentek, Parakeet United States - Volated Scots Point North Caldowade Terrace, New Chassis
82 California, South San Franji San Pruno Boulevard 460 Procedural amendment amendment formula) 1930/10/3] Mr. Commissioner of the Patent Office 1, Indication of the case 1989 Patent Application No. U';,! , 9-e7 No. 2, title of the invention 6/Otsu/71 3, relationship with the case of the person making the amendment Patent applicant 4, agent 5, date of amendment order drawing and sugar 1) 3. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the field of recombinant DNA technology, namely recombinant DNA technology.
It concerns the methods used in A technology and the products obtained by these methods. More specifically, the present invention provides human mature leukocyte ink
A polypeptide comprising the partial amino acid sequence Cys-hela-Trp-Glu-V
al -Val -Arg -Ala -Glu -1
1e --rg-3er, and without a preceding sequence, has 165-166 amino acids, or a methionine is present at the N-terminus of the first amino acid of the heptadon. , -- if 1'6
The background of human white face 1 feron (1) relates to a replication 1 vehicle characterized by containing an A sequence consisting of a sequence encoding a polypeptide having 6 to 167 amino acids'''-1↑.
LelF) was first discovered and prepared as a very coarse precipitate of Isaacs and Lindenman (Brock,
Procrt, soc, B147, 258-267 (1957); u, s, p
. 3.699.222>. Long-standing efforts to purify and characterize it have led to the preparation of a relatively homogeneous leukocyte interferon derived from leukocytes obtained from normal or leukemic donors (German Patent Publication No. 2, 947).
, 134 ), these noinkferons are a group of proteins known to have the ability to confer a state of virus resistance on their target cells. Additionally, inkferons act to inhibit cell proliferation and modulate immune responses. These properties have promoted the clinical use of leukocyte inkferons in the treatment of viral infections and malignant tumors. Leukocyte ink feron is produced in an essentially homogeneous state (Rubinstein et al., proc. Natl, Acad, Sci, U, S, 8, 76).
, 64.0-644 (1979); Zoon et al., supra, 76.5601-5605 (1979)) about 17,
It was reported to have a molecular weight ranging from 500 to about 21,000. The specific activity of these preparations is extremely high;
2 x 108 to 1 x 109 units/mg protein, but the yield from cell culture methods is extremely low. However, with advances in protein sequencing technology, partial amino acid sequences have been determined [Zoon et al., Science 20
7.527 (1980) Levy et al., Blok, Natl., Akad, Sai. United States (Proc, Natl, Acad, Sci, U,
S, 8,) L Yu. 5102-5104 (1980)). Although the glycosylation of various leukocyte interferons is currently not completely clear, differences in glycosylation between types alone cannot explain the observed distribution of molecule m. Instead, leukocyte interferons vary significantly in amino acid composition and sequence depending on the type, with amino acid homology sometimes lower than 80%. Although isolation from donor leukocytes has yielded sufficient quantities of 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 human leukocyte-derived inkferon for antitumor and antiviral use are using crude preparations (less than 1%) and are not producing sufficient quantities even at impractical prices. 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, etc., Science (Sc)
) 198.1056-1063 (197
7); Goeddel et al., Nature (
Nature) 28.1.544-548 (1979
)). More recently, recombinant DNA technology has enabled the production of proinsulin and thymosin alpha-1, and some authors have also described the harvesting and consequent production of DNA encoding human leukocyte interferons. reported the production of a protein with leukocyte interferon activity ()-gata (N
aqata) etc., Nature 284
. 316-320 (1980): Nantc
i) etc., Gene 10.1-10 (19
80) Taniguchi
7- (Nature)285.547-549 (1
980)). 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., “replicon”) and, usually,
There is information on one or more selection characteristics, for example, in the case of bacteria, resistance to antibiotics. With this information, host 1 containing the plasmid of interest
Clones of cells can be recognized and preferentially grown in a selective medium. Plasmids are useful because they contain one or another type of restriction endonuclease or "restriction endonuclease" or "restriction endonuclease" that each recognizes a specific site on the plasmid DNA.
! IQ FN14+0 20 10 40 H5L RRL L QM Is SCL DdanHDF
DanQIZt0 150 160 +66 E KYS CAl! ! EVVj3AEIMR5S S
Q 'L' K nucleotide sequence (coding strand)
shows. 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. A 5' 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 Left E. Fragment G, which remains separated, does not have a complete coding sequence. In Table 3, the eight Lel indicated by the nucleotide sequence
Comparing F protein sequences. IUPAC-LUB Commission on Biochemica
The abbreviation of one letter proposed by INOmlClature) 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, histidine is H, and the leg method (Goeddel, D, V, et al., Neech A7-(Nature) 287, 411-416)
(1980)). The CD N A insert can then be cut using Pst I. The line at the end of each cDNA insert shows the flanking homopolymerized dC:dG tails. Pvu[. Restriction sites for EcoRI and Bgl It 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. Figure 3 shows the construction of a gene encoding direct microbial synthesis to mature LelF. Restriction sites and remainders are indicated (“Pst I” etc.). Il b, p
, 11 indicates a base pair. Figures 4 and 5 show restriction maps of the two gene fragments used to express the LeIF B mature leukocyte inkferon. 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 indicates a termination codon in the corresponding DNA sequence and a hyphen indicates a deletion or gap in the sequence. Next, preferred embodiments for carrying out the invention are indicated. 8. Microorganisms Used In the described implementation, two microorganisms are used, namely, the microorganisms described in U.S. Pat.
coli (E. coli) X 1776 and E. coli. ] (E. coli) -12294 strain (E. coli)
5382) is used. The American Type Culture Collection (the A
merican Type Culture Co11
Deposited in ATCC No. 3153
7 and 31446 are attached. All recombinant DNA experiments were performed by the National Institute of
Health (National Intitude of
The study was carried out in accordance with the applicable guidelines of the Ministry of Health. A most advantageous embodiment of the invention is the strain E. coli x 1776 and E. coli as defined above. Not only E. coli strain 294 but also other known E. coli
i) strain (hereinafter referred to as [, coli), for example [, col
iB, C01i, and other microbial strains, many of which are included in the American Type Culture Collection.
rican Type Culture Co11ec
tion) (ATCC) and are deposited and available at known microbial depositories. I would also like to see German patent 2644432. These other microorganisms include Bacillus, e.g.
Bacillus 5ubtilis
and other enteric bacteria such as Salmonella typhimurium and Serratia marcescens.
rccscens) and uses a replicable plasmid capable of expressing foreign gene sequences. Yeast such as Saccharomyces t? L/Bishi7I (Saccha
romyces cerevisiae) can also be used to produce interferon protein by expressing the gene encoding inkferon under the control of a yeast promoter. B, Raw material of LeIF mRNAΔ and purified Lel'F
mRNA can be collected from human leukocytes. Usually obtained from leukocytes of chronic 1'l osteoblastic leukemia patients induced to produce interferon with 5endai virus or Newcastle disease virus, as described in German Patent No. 2947134. Use things. A particularly advantageous raw material used in the present work is 16 KG-1 derived from one patient with acute osteogenic leukemia.
It is a cell line called. This cell line has been identified by Fuller (in oeHler, 11.P, ) and Goldet (G.
Olde, D. W., ) Science
) 200.1153 (1978).
Memorial Institute ) ) 164
0 plus 10% heat-inactivated FC3 (fetal bovine serum), 25
mN IIEPEs buffer (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfone 1 m) J3
and gentamicin-containing medium at 50μ9/d and are subcultured twice a week (1 to 3 splits).
I can do it. Cells can be frozen by adding 10% dimedyl sulfate to the above growth medium. KG-1 is The American
Type Culture Collection (the Amer
ican Type Culture Co11ect
ion) (81CCNoCRL 8031). In G-1 cells, Rubinstei
n) Methods described in Proc, Natl, 8cad, Sci, U, etc.
Leukocyte inkferon111 RNA was induced to produce leukocyte inkferon1111 RNA using 5Ondai or Newcastle disease virus by M.S., >76°640-644 (1979). Cells were harvested 5 hours after induction, and RNA was extracted using the guanidine thiocyanate-guanidine hydrochloride method (Cherwin).
Biochemistry (C11irgwin) etc.
chemistry) 18, b 294-529
9 (1979)). RNA from uninduced cells was similarly prepared. Oligodeoxythymidine (
Poly(A) mRNA was isolated using dT)-tRulose chromatography and sucrose gradient 1 to ultracentrifugation.
12S fraction was obtained. The method is Green
) etc. (Arc, Biochem, Pioffice (Arch,
Biochen+, Biophys, > 172. 7189 (1976)) and Okuyu
ma ) etc. (arch. Biochem, Pioffice, (8arch, Biochem, Biophys, > 1
88, 98-104 (1978)) was used. This mRNA was analyzed by African frog oocyte analysis (
Cavalli (Caval 1cri) etc., BuOri, Natl. Acad, Sci. U.S.A. 74.3287-3291 (1977
) indicates an interferon titer of 8000-10.000 units/microgram. C. Preparation of a colony bank containing LeIF CD N A sequences Using 5μ7 mRNA, standard method Likens (Wi
J. Biol, Chem, 253, 2483-2 II.
95 (1978) and Goeddel (1978).
) etc., Nature 281, 544
-548 (1979)), a double antibody cDNA was prepared. CD N A was fractionated according to size by electrophoresis on a 6% polyacrylamide gel, and 500
230 n of material of size 1500 b, p, was obtained by electroelution. 1100 n of this CD N A
Deoxythymidine (dC) residues can be used to create tailing chunks (Chana) and other natural substances (Chana).
e) 275.617-624 (1978), Pst ■
Polyvar (B
Gene 2.95
-113 (197'7)), and using this, E. coli
x 1776 was transformed. About 130 tetracycline-resistant and ampicillin-sensitive transformants were obtained for cDNA no. B, C, D> or one (-r-13A, [3, C. D) oligonucleotide. The complementary deoxyoligonucleotide 11 chain length shown was chemically synthesized by the phosphotriester method.
'J, Brok, Su1 Hell, Akado, Sai. United States (Proc, Natl, 8cad, sci, U
, s 8,) 7\-5, 5765-5769 (197
8)). In the T-13 series, there are four individual
- Prepared. As shown in Table 1, 12 T-1 probes were prepared as 4 pools of 3 probes. T-13 series of 4 respective probes and 12 1 prepared as 4 pools of 3 primers each.
The --1 probe was used to induce the synthesis of radiolabeled 1-heavy CI)NA for use in hybridized probes. Template mRNA is 12S RNA from Sendai-induced KG-1 cells (8000 units I [activity/μ9) or total poly(A) mRNA derived from uninduced leukocytes (<10 units/μg). 32p-label CD N A
using known reaction conditions (Noyes et al., Proc., Natl., USA).
, Acad, Sci. U, S, A, ) 76.1770-1774 (197
9)) from these Bly Y-. 20mHt-Tris (hereinafter referred to as Tris) -11cI (11118,3), 20mHKCl
Reactions were carried out in 60 μm volumes in 2.30 mH β-mercaptoethanol. This reaction consisted of 1 μg of each primer (i.e., 12 μg total for the −“-1 series, 4 μg total for the T-13 series), 2 μq of “induced” 128-fraction mRNA (or uninduced Poly(A) mRNA3000
Ci/m mole) and 60 units of reverse transcriptase [Bethesd
a Research Laboratories)
). The product is 5 ephadex
)■ (hereinafter referred to as S e p l + ad ex) G-
Unbound label was separated by gel filtration on a 50°C column, RNA was degraded by treatment with 0.3N Na chloride for 30 min at 70'C, and neutralized with IIcI. Hybridization is carried out by a method such as Nucleic Ac1d, such as afatos.
It was carried out according to the description in Res., ), 1541-1552 (1979). Identification of 500 [, rin[1-pL1-pL30] [,
E. coli K-12 strain 294 transformed strain (see section 0)
To prepare 1μ9 of plasmid DNA from each of Bil'nllOim '8,
Nucleic acid research
Ac1ds Res, ) 7 , 1 5 1 3
-1523 (1979) rapid plasmid isolation procedure was used. Each DNA sample was denatured and Kaf
Atos) et al., the sample was placed on a 3-cellulose filter in accordance with the method cited above. Three phases of J nitrocellulose filters containing 500 plasmid samples were a) induced CDNA primed with the T-1 set of primers. b) Induced cDNA primed with T-13 and C) hybridized with uninduced cDNA prepared using both primers Sera1~. A clone was considered positive if it hybridized more strongly to one or both of the induced cDNA probes than to the entire uninduced probe. 3 from 500 pieces
0 “positive” clones 1)L 1-1)L30)
were selected for further analysis. "Identification of clone pL31-1) L39, separation of plasmid (No. 104) containing LelFi gene fragment 11, 32p-label induced +++ RNA Lilenhaug et al., Biochemistry ((Biochemistry, ) 15
．． 1858-1865 (1976)') using the colony hybridization method of Grunstcin and Llogness (Proc, Natl.
l, ^cad, Sci, U, S, 8,) 52
．． 3961-3965 (1975)) by F, co
lix 1776 transformants were screened. In order to compete the unlabeled mRNA from uninduced cells with the uninduced mRNA present in the 32p-labeled preparation,
Mixed 200:1 to probe. Hybridization of the labeled 111 RNA contains the induced sequence
- It should occur selectively in J colonies. Three l!
A transformed strain of T was obtained. (1) 2 to 3% of colonies hybridized very strongly to 321)-n+ RNA. (2110% means that the degree of hybridization is 1111% above.
! It was significantly lower than 'l'. (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 interferon mRNA with plasmid DNA. First, tetralycline (20 μg/if), diaminopimeline M (
100μy, ”*>, thymidine (20μ'j/rd>
Each of the 60 robust colonies (group 1) was grown individually on 100 d of M9 medium supplemented with d-biotin (1 μ9/d). M9 medium contains 1 liter of Na IIPO (>6g), 12PO4 (3
9) , containing NaC14 (0,51 and 1n4cl(is),
- After claving, 1d of sterile 114HgS04 and 1d of sterile 0. 10nli of OI HCaCl2! Add. The 10 cultures were pooled and subjected to Froebel (Newall)
etc., biochemistry (Biocbc+n1stry)
) 9. 4428-440 (1970), plasmid DNA was isolated from the six pools. 10μ9 of each plasmid DNA pool was cut with 1lindlll, denatured and DB
M (diazobenzyloxymethyl) covalently bonded to paper I!
I-ko. 1μ9 of sticky mRNA from induced cells was hybridized to each filter paper. mR that does not hybridize
NA was removed by washing. specifically hybridized mR
NA is eluted and translated in African frog oocytes. All six pools were negative in this analysis. From 59 apparatus colonies (group 2), 1 each
Five pools with 0 colonies and one pool with 9 colonies were prepared, and plasmids were prepared from these pools and examined as described above. Of the six pools tested, one (KIO
) hybridized to Inkferon mRNA at levels significantly above background levels in each test. To identify specific interferon cDNA clones, pool plasmid D from 9 out of 10 colonies.
NAs were prepared and examined individually. Two of the nine plasmids (N(1,101 and No, 104) bound Inkferon+11 RNA well above background levels. Plasmid No. 10
4, a unique B(11
m restriction fragment was isolated and 1c0 was isolated using a Tailer (Tayl).
or) etc., biochemistry. Pioffice, Acta (Biochim, Biophy
s, 8cta) 442.324-330 (1976)
Labeled with 2 ri p according to the method of
llo(]neSS), ProcNatl, SCi, U, S, 8, 72.
3961-3965 (1975)) Nis E, col
400 of the i294 transformants were used as probes for individual screening. Nine colonies (pL31-
pL39) was identified. Additionally, using the labeled 260b,l) fragment,
Similarly, 4000 [:,C01i294 transformants were individually screened. Fifty colonies were identified that hybridized to this probe to the same degree. One contains the LelF G fragment and one contains the LeIF H fragment.
One of the fragments is called LelF old,
LeIF (which is an allele of If). The resulting hybrid plasmid was designated as pLclF11°'. G. Isolation and Sequencing of the First Full-Length LeIF3u Gene Plasmid DNA was prepared from a total of 39 potential LeIF3u cDNA clones. Then, rescreening was performed using the same 260b, p, DNA b[1-b] by the hybridization procedure of Kafatos et al. (cited above). This probe and three plasmids (pL4, pL31, pL34) gave very strong hybridization signals, four (pLl)
3. pL30. pL32. pL36) was moderately hybridized, and three (pL6, pL8, p
Ll 4 ) 7 was weakly hybridized. The 39 potential LeIF cDNA recombinant plasmids also contained a 32p-labeled synthetic undecamer (
The IC of each of the T-1 primer pools was determined using each of the T-13 primers directly as a probe for hybridization. Detectable high. The hybridization conditions were chosen such that complete base-to-base pairing was required to provide the hybridization signal (Wallace et al., Nucleic Acid Res, ).
3543-3557 (1979)). In other words, plasmid DNA was extracted from 39 clones using the standard supernatant method (c
Clewell et al., supra) and Biorad Agarose.
It was purified by A-50 column chromatography. A sample (3 μg) of each preparation was treated with EcoR■ to open the ring and
It was denatured with Arno J filter and spotted onto two separate nitro luulose filters. 1.5 per spot
Make it μ. Cuff 71 Hess (aratos) quoted above
) etc. Each of the synthetic deoxyoligonucleotide primers and primer pools (γ
32P) was phosphorylated with ATP as follows. 5 Q
pmole oligonucleotide and 100 pmol
le's (732P) ATP to New England Nuclear
, 2500Ci/m mole), 50n+H Tr
is-IC! , 10 mH HgC1, and 15mM
of β-mercaptoethanol. After adding 2 units of T4 polynucleotide kinase and 30 minutes at 37°C, the 32p-labeled primer was
0Id Cephadex (5ephadex■) G-50
Purified by on-column chromatography. Hybridization was performed using 1Q6 cpm of Blalymer T-13C or 3x166 cpm of primer pool T-10. Hybridization was performed using 6xSSC (1xSSC=0,
15HNaC1,0,0158<sodium citrate, a
l17.2), 10xDenhardts (0,2
% raw 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 pool T-13
The case of G and primer-T-IC is shown. Plasmid DNA from clone 104 hybridized significantly with primer pool T-I C and primer -13C, but showed no detectable hybridization with other undecamers.
As shown in the figure, some of the 39 isotropic Lell-plasmids (pL2.A, 13.1
7. 20. 30. 31°34) hybridized with both of these probes. However, from the restriction analysis,
Not one of these plasmids, 11131
also contained the 260b, p internal B (11■ fragment. pL
As a result of urination of 31 with Pst I, the size of the CD NA insert was approximately 1000 b1). The sequence of the entire Pst I insert of p[31 was
Gilbert (14axam-Gilbert) chemical method (Methods En 971 mol, (Methods En
zymol, ) 55. /1.99-560 (1980)) Onibijib No. 4 Nijijin Ending Method (Smith, Method en l Issel (1980))
Smitb 14methods [nzymol, )
65, 560-580 (1980)). At that time, the 5aU3a fragment was subcloned into the 813 vector and placed in C. The DNA sequence is shown in Table 2, A''. Information on the available protein sequences, known LeIF molecules, and the relative presence of stop trypsin 1 in three possible reading frames is appropriate. Then, the entire LelF amino acid sequence including the signal peptide was shown. Manipulations for direct expression as peptides are similar to those used for human growth hormone (Gocddel et al.), Nature (Na.
ture) 281. 544-548 (1979). As shown in Figure 3, the sau 3'a restriction endonuclease site is conveniently located at codon 1 of LelFA.
and 3 exist between. We designed two synthetic deoxyoligonucleotides containing ATG translation start], repairing the codon for amino acid 1 (cysteine), and creating a new EcoRI sticky end. The oligomer of p[31's 34b
, p, Sau 3a -Ava II ligation. The resulting 45b,p product was ligated to two additional DNA fragments to create the 865b,I), synthetic-natural hybrid gene. This decodes LelF A]- and EcoR:[
and PSt I restriction sites. This gene was inserted into pBll 322 between the Ecolt I and Pst I sites and the plasmid p
It was named LelF AI. 2. Construction of a tryptophan regulatory element (E. coli containing the trD promoter, operator and trp leader ribosome binding site but lacking the ATG sequence for translation initiation) Plasmid Le G contains the deletion ΔLE 1413 , responsible for the C01il-lyptophan operon (formerly Ozzari), etc., J. Bacteriolo(ly) 133, 14
57-1466 (1978)). Therefore, trp
The first six amino acids of the leader and approximately the last third of the trD E polypeptide (hereinafter collectively referred to as LE
') and a fusion protein containing the entire trll D polypeptide. Save this tr
It is under the control of the p promoter-operator system. 20μ3 of this plasmid was digested with the restriction enzyme PVLI I. This cuts the plasmid at 5 sites 1 gi. This gene fragment is then linked to an EcoRI linker (comprised of a self-complementary oligonucleotide of DCATGAATTCATG), and later
oRl: Cloned into a plasmid containing the site. An aqueous solution was collected from a dialysis bag, extracted with phenol, extracted with chloroform, adjusted to a concentration of 1 to 0.2 Mm, and precipitated with ethanol, and DNA was collected in water. The trp promoter-operator-containing gene with EcoRI sticky ends is
It was identified using 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
z), etc., Nucleitsk Acid Research (Nucl
cicAcids Res, > 6.3267-328
7 (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 can be made resistant to tetracycline. pBRll was digested with EcoRI, the enzyme was removed by 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 I) NA fragments obtained above and ligated with T4 DNA ligase as described above. Using the DNA present in the reaction mixture, standard methods (Hershfield et al., Proc. Natl., USA) were performed using the DNA present in the reaction mixture.
8cad, Sci, U, S, A, ) 71.34
55-3459 (1974)), which has the ability to incorporate DNA. One 12 strains of E.coli 294 were transformed, and this bacterium was transformed into
LB (Luria-Ber) containing 20 μ9/me ampicillin and 5 μg/d tetracycline
tani) plates. Several tetracycline-resistant colonies were picked, plasmid DNA was isolated, and the presence of the desired fragment was confirmed by restriction enzyme analysis. The resulting plasmid is designated pB old I trp. Hepatitis B viral genome [coRI and BamHI
Digestion products were collected by a conventional method, and plasmid pGII6
was cloned into the EcoR and BamllI sites of Goeddel et al.
ure) 281.544 (1979)), plasmid pH 332. Plasmid pHs 32 was cut with xba I, phenol extracted, chloroform extracted, and ethanol precipitated. Precipitation was performed using 0, 1 mHdTTP and Q, 1 mHdCT.
30μm polymerase buffer containing P (50n+M
Potassium phosphate pH 7,4,711188 (ICI2゜ [Boehringer- Mannheim
Hannheim) for 30 min at 0°C, then 37 min.
It was treated at ℃ for 2 hours. This treatment removes two of the four nucleotides complementary to the 5' overhang of the xba I cleavage site.
The quantity is filled. 5'CTAGA-5'CTAGA-3' T-TC
T- Two nucleotides dC and dT are incorporated to give an end with two overhanging 5' nucleotides. This linear residue of plasmid 1111532 (phenol and chloroform extraction followed by ethanol precipitation and water) was cut with EcoRI. humanoid plasmid fragment,
PAGE, smaller -TCTAGA--TCTAG
A--AGCTCT--AGATCT- These plasmids were also found to be cut with both EcoRI and HpaI to give the expected restriction fragments. One plasmid, designated 11Trl)14, was used to express a heterologous polypeptide as discussed below. Plasmid pHGI+107 (Goedde
l ) etc., Nature 281.54
4 (1979), 2 produced from synthetic NA fragments.
Complementary DNA generated by reverse transcription of 3 amino acids l1mm] and human growth hormone mesendi A7-RNA
It contains the gene for human growth hormone, which is made up of 163 amino acid codons derived from A. This gene lacks the codons of the human growth hormone "leading" sequence, but contains the ATC translation initiation codon.
Processing, followed by [. coli DNA polymerase I Klenow (niIe!n0
W) fragment (denoted below as lenow) and min-1 via dTTP and dATP treatment. Phenol and chloroform extraction 5'-dGATCACAC^TG (fragment 2) was synthesized by the phosphotriester method, and lC0 fragment 2 was phosphorylated as follows. 200 μl (approx. 40 pmole) (7
) (732P) ATP (7 Mersyam (Amer)
sham), 5000Ci/mmole) and 5QmH Tris-HCI (pH 8), 10m
H of H (IC + 2.15 mH β-mercaptoethanol) was resuspended in 30 μl of a solution containing 100 pmole (7) DNA fragments and 2 units of T4 polynucleotide kinase. After 15 min at 37°C, 1 μm of 1
On+H ATP was added and the reaction was allowed to proceed for an additional 15 minutes. The mixture was then heated at 70°C for 15 minutes and
) 5'-OH fragments of mole 1 and l Q plo
le 34 b, p, Sau 3 a Ava II
T! It merged with the li piece. 20mHTris-HCI (
pH7.5), 101118 8 (IC+2.10ml
The ligations were ligated in 50 μM of l dithiothreitol, 0.5 m HATP and 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. 30 ng (1 pmole) of the 45b,p product was ligated to 150b,p 8 val- (0.1 μg). Transformants containing the trp promoter are Gränstein-Hognes (GrLI
nStQin-110(lnO3s):] was identified using a 32Ptrp probe in combination with the Ronnie screening method. (The XbaI site located asymmetrically in the rp It column piece indicates that the trp promoter
This allowed the identification of recombinant products oriented in the direction of the F Afi gene. 1. In vitro and in vivo active IF of LelFA
Extracts for analysis were prepared as follows. One culture was grown in L broth containing 5 μg/d of tetracycline to give an A 550 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 sample of 10Id was collected by centrifugation, and the cell mass was incubated with 15% sucrose and 5QmHTri.
The cells were suspended in "flnR" in 5-HCI (pH 8,0), 50 mM EDTA. 1 my of lysozyme was added, and after 5 min at 0°C, the cells were disrupted by sonication. , OOOrpm) and centrifuged, and the inkferon activity in the supernatant was determined by cell table 7 E, coli
Comparison of activity between 294/pLelF825 extract and standard LelF* E.coli294/pLelF described by Mr. 6
250.000 units per d of the extract of 8 TRP 25 was diluted 500 times with minimum essential medium to give a specific activity of 500 units/Id. Leukocyte Interferon Standard (Wadley Institute)
) was previously prepared as the NIH leukocyte interfunron standard, titered, and also diluted to a final concentration of 5001 J/ml. 1 d volume was made into p112 with 1N11Cl, incubated for 52 hours at 4'C, and NaOH
The IF activity was measured using a standard CPE inhibition assay, and 25 μm portions of the untreated samples were measured at IC0500 units/d.
5μm rabbit anti-100x D5oEMC virus (measured in mice)
was infected intramuscularly. Control infected monkeys were infected at 134.
158. Died at 164 hours. 106 units Inkferon treatment was administered intravenously 4 hours before infection, 2.23.29.48.72.168 and 240 hours after infection. Bacterial leukocyte inkferon was column chromatographically fractionated from the lysate of E. coli 294/DLelF A 25 and had a specific activity of 7.4 x 106 units/■ protein. The control bacterial protein was E, col
The total protein content was doubled in the corresponding column fraction of the i 294 /ρBR322 melt. The leukocyte inkferon standard is a Sendai (5enda
) was a virus-induced interferon. 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.
Additional E. coli2, obtained as in Section 0 above, by the in situ 1 co-colony screening method of tein) and tlogness
94 convertible strains were screened. Colonies that hybridized to the probe to varying degrees were isolated. Plasmid DNA from these colonies and from the 10 hybridized colonies shown in Section G above.
A was isolated by Pst I cleavage and characterized in three ways. First, these Pst fragments were treated with the enzyme Bgl
, Pvul[ and restriction endomeclease digestion pattern using EcoRI. This analysis shows that at least eight different types (LelF^
, LelrB, LelF C, LelF D, Le
IF E. The classification of LelF F, LelF G and LelF If) was made possible. This is the previously known L
The approximate locations of various restriction cut points with respect to the leading and coding sequences of elF A are shown. Among these, Nobitotsu LelFD is Nagata (Nagata)
etc. is Nature 2 B 4.316
-320 (1980). Second, we investigated the ability of some of these DNAs to selectively remove [eIFmRNAΔ] from polyA-containing KG-11111 RNAΔ.
eveland) etc. are Cell- and rivers, 95
-105 (1980). LelF8, B, C and ' were positive in this analysis. Third, these Pst fl
Insert the i piece into the expression plasmid 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 Itafe-O activity, except for the LeIF FIgi fragment, which was marginally positive. In addition to the above, the sequences of all of the above LeIF types were determined. K, second mature leukocyte inkferon (LelF B)
The sequence of the isolated fragment containing the direct expression of mature telF (relative to B) shows that the first 14 nucleotides of types A and B are the same. , so the trp-promoter-operator, 112 strains 2 at the ribosome junction.
94 was converted. Convertible stocks are screened using mini-screening (Birnboim).
biom), nuclear acid research (
Nucleic Ac1ds Res, )ヱ, 151
3-1523 (1979)) and the plasmid samples were digested with EcoRI. The digest gave three rgI ha, and their characteristics were: 1) EcoRI-EcoRI
trp promoter fragment; 2) internal EcoR of DL4
I-EcoRI fragment; and 3) I) protein translation initiation signal of L4-EcoRI fragment. For CPE analysis, bacterial extracts from clones tested as described above were typically approximately 10 x 106 for A350-1.
It gave an interferon activity of unit/1. One representative loan prepared in this manner is E. coli2
94/ pLelF B trp 7. This clone has been deposited with the ATCC. [Yet another mature leukocyte ink FC
, [), F, 11, I and J) Additional full-length gene fragments containing other LelF types include:
As in LelFA, tiller and place in 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 strains 294/pLelF F trp 1. This clone has been deposited with the ATCC. LeIF H Complete LeIF Illll Kaneko mature leukocyte interface [
In order to avoid expression as a single sequence, arrange them as follows. 1. Plasmid pLeIF H was transformed into Hae [and Rs
aI digestion to isolate the 816b,p, fragment extending from signal peptide amino acid 10 to the 3' non-coding region. 2. Denature this fragment and inject it with DNA polymerase Kle.
nowi piece (Klenow et al., Proc. Natl., 8 cad. Sci, Ll, S, A, ) 65.168 (1970
)), the synthetic deoxyribo-oligonucleotide primer can be released 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 produce mature LeI
FH can be expressed. eIF I Human genome face λ Charon 4 A recombinant library constructed by Lawn et al.
8)) by the method of Lawn et al. cited above and the method of Haniatis et al.
) 1 branch. 687 (1978) for leukocyte interferon. Approximately 500,000 plaques were screened using a radioactive LelF probe derived from the CD N A clone Lef'A. Six LelF genomic clones were selected, and following cross-screening and plaque purification, these One of the clones, λII LelF 2, was selected for further analysis. Using the above method, other probes can also be used advantageously to isolate other LelF clones from the human genome. These were then used in this study. Most of the cellular proteins, including tafferon according to the invention, remain in the supernatant. 2. Ammonium sulfate fractionation, where inkferon is precipitated from a solution of 55% saturated ammonium sulfate. 3. Ammonium sulfate pellets are precipitated from a solution of 0.06 M phosphoric acid. Potassium, 10 IN Tris-IIcI, pH 7,
2, suspended in 25 mM Tris-HCI, pH 7
, 9. Interferon activity remains in solution. 4. Adjust the above supernatant to pH 8.5 and perform chromatography on a DEAE-cellulose column (25 mH
Tris-HCI 1 pHa, O to 0.2 M in 5
Elute with a linear gradient of NaCl). 5. Cibach Blue-Agarose (Cibach
rome Blue -Agarose) or hydroxylapatit
e) and elute with 1.58KCI or 0.2M phosphate (as required). 6. Sephadexo G-
Size the molecules with a 75 force ram. 7. CM in 25 mN ammonium acetate at pH 15.0
- Carry out cation exchange O mudography on cellulose. 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 chromatography, reverse phase (RP-8) high pressure liquid chromatography, or affinity chromatography on immobilized anti-interferon antibodies. Alternatively, the material from Step 4 above may be mixed with Milstein (
(formerly 1stein), Scientific American 24
3.66 (1980) and eluted with 0.2M acetic acid, 0.1% Triton and 0.15H NaCl. In another advantageous embodiment, the leukocyte interferon produced in the above procedure can be purified in the following step. 1. The frozen cell mass containing the expressed leukocyte interfaeron is crushed manually or with a suitable crushing device. Partially adjust the cells to pH 7.5-8.0/=0
．． IM Tris, 10% column with approximately 10 column volumes of 20 m14 Tris-1101, DH7,5
-8,5 (NaCl (0,5M) and Triton (T
riton) X-100 (0.2%) or equivalent surfactant-containing). Wash the column with 0.15HNaCl and Triton.
Approximately 10 column volumes of a solution containing Triton X-100 (0.1%) or an equivalent surfactant are passed through the column. Elute with 0.2M acetic acid containing the drug.The protein peak from the monoclonal antibody column (UV
(measured by absorbance or other methods) and adjust 011 to approximately 4.5 with INNa or 1, OM Tris base. 4. Transfer the pooled interferon peaks to a suitable buffer such as ammonium acetate (14,5
Whatman equilibrated at >mM
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 becomes irritating, so that almost no protein is eluted from the column. The column was then washed with 25mM ammonium acetate 10.12
Elute with a combination that maximizes the recovery of monolithium chloride or interferon to yield a lyophilized cake of satisfactory appearance and solubility. The monoclonal antibodies in this advantageous embodiment described above may be prepared by Staehelin et al.
Natl 6 Fukado, Sai, USA (Proc, Natl,
Acad, Sci, Ll, S, A, > 78.184
8-52 (1981). Monoclonal antibodies are prepared and covalently linked to Affigel-10 as described below. Preparation of monoclonal antibodies from ascites fluid
Five a+b,'c mice were each inoculated with 5-10×10 6 hybridoma cells taken at mid-log phase. Approximately 5 x 10' viable cells obtained from ascites fluid produced by mice were
Zero or more than 10 mice each were inoculated intraperitoneally. Repeat this ascites fluid for each mouse (2 to 4 times)
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. Centrifuge at low speed (500-1000xg) for 15 minutes,
Cells and debris were removed from the ascites fluid. Then 18. OO
Centrifugation was performed for 90 minutes at Orpm. Freeze the supernatant at minus 2
Stored at 0°C. After melting, 35. OOOrpm
The mixture was centrifuged for 90 minutes to remove fibrin and particulate matter. A batch of ascites fluid with 8 transfers was tested in a solid phase antibody binding assay (Staeh'elin).
), etc., the above-mentioned cited documents) to test the specific antibody activity,
If I was satisfied, I pooled. Protein'f! in the pooled solution The degree is that 1 m9 of protein is 280 nm in a 1.0 cm thick cuvette.
The measurement was performed using an approximate method that shows an absorbance value of 1.2. Ascites fluid with a high concentration of antibodies contains 30-35 ms protein/d. This corresponds to 4-7g# specific antibody/weight. Dilute this liquid with PBS (0,01M sodium phosphate, pH 7, 3,0,15M NaCl),
A protein concentration of 10 to 12 #lff/ld was used. To each 100 d of diluted solution, 90 d of saturated ammonium sulfate solution at room temperature was added at 0° C. with vigorous stirring. The suspension was kept in water for 40 to 60 minutes. Then, at 4°C, io, oo. Centrifugation was performed for 15 minutes in rpII. The supernatant was removed by rinsing and the liquid was thoroughly drained. Protein mass in 0.02M Tris
HCI (pH7,9>10.04M NaCl (mild)
fjJ′a■). The protein solution was dialyzed against 100 volumes of buffer ■ for 16 to 18 hours at room temperature. During this time, the buffer was exchanged at least once. Add the dialysis solution i5. The mixture was centrifuged at ooorp+n for 10 minutes to remove insoluble matter. Approximately 30 to 35% of the total protein in the ascites was initially collected as estimated from the absorbance value of 2BOnlll. A solution containing 30-401179 protein per ml was then added to a DEAE-cellulose column equilibrated with Buffer I. At least 100 ml (D
Column bed capacity Toshita. 0.02M Tris lIcl
0.04M to 0.5M NaCl in pH 7,9
The antibody was eluted from the column by linearly changing the 14aCl concentration every once in a while. The peak fractions from 0.06 to 0.1 M 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
Na1lC03 (all about 8.0) 10, 3M tla
cl (slow vfJ solution ■) and incubate at room temperature with the same buffer (
Dialyzed against 3 changes). 20. O
The mixture was centrifuged in OOXg for 15 minutes to remove insoluble matter. The protein concentration was adjusted to 20 to 25 μ/ndl with buffer solution ■. 111 degrees left 11 affigel (8 ffigel) -10 (Bio Laboratories, Richmond, Calipornia (Bi
o Rad Laboratories, Rich-
tond, California) was washed three times with water-cooled isopropanol and then three times with water-cooled distilled water on a glass filter. Transfer the gel slurry (approximately 50% cold water) to a plastic tube and centrifuge briefly to sediment.
! 1 [. The supernatant was removed with an aspirator, and the backed gel was mixed with an equal volume of purified antibody solution and rotated in a circular motion at 4°C for 5 vI. After the reaction, the gel was centrifuged and buffer m (0, IM Na1lCO31
0,15M NaCI) r2 in 5N human serum albumin, pass this solution through a biological filter, and aseptically divide the filtered solution into 100 bottles, each bottle suitable for parenteral administration. 6X10
1 to contain 6 units of pure interferon. It is desirable to keep these bottles in a cool place (-20°C) before use. The compounds of the invention can be formulated according to known methods for preparing pharmaceutically useful compositions. The polypeptide is then mixed with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described in Remington's Pharmaceutical Sciences by Margin ([.14.8artin)].
Pharmaceutical Sciences). This is cited as a reference in this specification. In these compositions, an effective amount of inkferon protein is combined with a suitable amount of a throat vehicle to form a pharmaceutically acceptable composition suitable for effective administration to a host. One advantageous mode of administration is parenteral administration.

Claims (1)

[Scope of Claims] (1) A polypeptide comprising the amino acid sequence of human mature leukocyte inkferon, comprising the partial amino acid sequence Cys
-toIa -Trp -Glu -Val -Val
-＾rg -Ala -Glu -11e -Net
-Arg-3er, and without a preceding sequence, having 165-166 amino acids, or a methionine attached to the N-terminus of the first amino acid of said interferon. If so, 1
A replicable expression vehicle characterized in that it contains an NA sequence consisting of a sequence encoding a polypeptide having 66-167 amino acids. (2) If it is not accompanied by a preceding sequence, it has amino acid 11fAsp, Glu or Val at position 114, and if Aruhamedionine is bound to the N-terminus of the first amino acid of interferon, it has amino acid As at position 115.
Expression vehicle according to claim 1, characterized in that it contains a DNA sequence consisting of a sequence encoding a polypeptide having p, Glu or Vat. (3) Human leukocyte interferon A (LelF A
2. A launch vehicle according to claim 1, characterized in that it contains a DNA sequence encoding the following. (4) Human leukocyte interferon B (LelF B
2. Expression vehicle according to claim 1, characterized in that it contains a DNA sequence encoding .). (5) Human leukocyte interferon C (LeIF C)
Expression vehicle according to claim 1, characterized in that it contains a DNA sequence encoding. (6) Human leukocyte ink Feron D (LOIF D
2. Expression vehicle according to claim 1, characterized in that it contains a DNA sequence encoding .). (7) Human leukocyte ink Feron F (LelF F
An expression vehicle according to claim 1, characterized in that it contains a DNA sequence encoding >. (8) Human leukocyte interferon H (LelF If
2. Expression vehicle according to claim 1, characterized in that it contains a DNA sequence encoding .). (9) Human leukocyte ink Feron I (LelF I
2. An expression vehicle according to claim 1, characterized in that the expression vehicle contains a DNA sequence encoding the following. (10) Human leukocyte interferon J (LelF
Expression vehicle according to claim 1, characterized in that it contains a DNA sequence encoding J). (11) Claim 1, characterized in that it contains a DNA sequence encoding a polypeptide obtained by allelic mutation of the polypeptide of any one of Claims 3 to 10. expression vehicle. (12) The expression vehicle of claim 1, which contains a DNA sequence encoding a polypeptide resulting from an allelic variation of LelFA. (13) The expression vehicle of claim 1, which contains a DNA sequence encoding a polypeptide that differs from LelFA in only one amino acid. (14) The expression vehicle of claim 1, which contains a DNA sequence encoding a polypeptide that differs from LelFA in the amino acid at position 23. (15)' LelFA in that it contains Arg at position 23
An expression vehicle according to claim 1, characterized in that it contains a DNA sequence encoding a polypeptide different from the expression vehicle of claim 1. (16) An expression vehicle according to any one of claims 1 to 15, wherein the expression vehicle is a plasmid. (17) Plasmid pLelF A trp 25,
pLelF B trp 7, and pLelF F
17. The expression vehicle of claim 16 selected from the group consisting of trp1. (18) The expression vehicle of claim 16 selected from the group consisting of plasmids pLelF Ctrp 35 and pLelF D trp 11. (19) Plasmids pLelF G and pLeIF
Claim 16 selected from the group consisting of If
Expression vehicle for terms. (20) The expression vehicle of claim 16 selected from the group consisting of plasmids pLclF I trp 1 and 3゜pLelF J trp 1 . (21) A polypeptide comprising the amino acid sequence of human mature leukocyte inkferon, the partial amino acid sequence Cy
s-hela -Trp -Glu -Val -Val
-Arg-8la -Glu -11e-Net-^
rg-3ep and without preceding sequence, having 165-166 amino acids, or with methionine attached to the N-terminus of the first amino acid of said interferon. is 166-
A microorganism with bopeptide No. 21 having 167 amino acids. (23) The microorganism according to claim 22, wherein the microorganism is an E, CO1+ strain. (24) The microorganism is E. coli K-12 strain 294
The microorganism according to claim 23, which is