JPS6363198B2 - - Google Patents

Description

[Detailed description of the invention] The present invention is directed to the field of recombinant DNA technology, i.e.
Methods used in engineered DNA technology and these
Concerning the product obtained by the method. More specifically, the present invention provides human leukocyte-derived
Mature human white that can be obtained by gene expression
It is a blood cell interferon with 165-166 amino acids.
partial amino acid sequence Cys-Ala-Trp
−Glu−Val−Val−Arg−Ala−Glu−Ile−Met
-Arg-Ser, and amino acid Asp at position 114,
This interferotype has one of Glu or Val
methionine at the N-terminus of the first amino acid of
If conjugated, it has 166-167 amino acids.
and the amino acid Asp, Glu or Val at position 115
Mature human leukocyte interferon with one
A DNA sequence characterized by consisting of a sequence that encodes
Regarding columns. A specific example of the above mature human leukocyte interferon and
Mature human leukocyte interferon (LeIF)
A, B, C, D, F, H, I and J are listed.
Ru. These mature human leukocyte interferon proteins
The amino acid sequences are shown in Table 3 on page 21 and Table 5 on page 24.
It is. That is, LeIF A in Table 3,
LeIF B, LeIF C, LeIF D, LeIF F and
LeIF H and each of I and J in Table 5
Signals indicated by numbers with S from the amino acid sequence
165 or 166 amino acids excluding peptides
polypeptides consisting of and these polypeptides
Add methio to the N-terminus of the first amino acid of
Nin is combined with 166 or 167 amino acids
The polypeptide is a mature human leukocyte protein of the present invention.
Tuffelon A, B, C, D, F, H, I and J
It is. First, the background of the present invention will be described. human leukemia
The ball interfaelon (LeIF) was initially
Isaacs and Lindenman
(Lindenmann), a very coarse precipitate and
was discovered and prepared [Proc. Ah
Le. Soshi. (Proc.R.Soc.) B147, 258−267
[1957]; United States Patent (U.S.P.) 3699222). do it carefully
Efforts to manufacture and characterize the
Comparatively derived from white blood cells obtained from leukemia donors
Leading to the preparation of homogeneous leukocyte interferon
(German Patent Publication No. 2947134). These interfaces
Elon makes their target cells virus resistant
Known to have the ability to inflict conditions
A group of proteins. In addition, interferon
inhibits cell proliferation and modulates immune responses.
It can act as if Due to these properties, viruses
leukocyte interface for the treatment of bacterial infections and malignant tumors.
The clinical use of Elon has been promoted. Leukocyte interferon is essentially homogeneous
(Rubinstein et al.,
Proc. Nattle. Acad. Sai. United States (Proc.
Natl.Acad.Sci.U.S.A.)76, 640-644 [1979];
Zoon et al., ibid.76, 5601-5605 [1979])
It is reported to have a molecular weight ranging from about 17,500 to about 21,000.
I was told. The specific activity of these preparations is very
high, 2×10⁸from 1×10⁹unit/mg protein
However, the yield from cell culture methods is extremely low. death
However, with advances in technology for determining the sequence of proteins,
The amino acid sequence of ZOON was determined.
etc., Science207, 527 [1980];
Levy et al., Proc. Nattle. Agad. sa
stomach. United States (Proc.Natl.Acad.Sci.U.S.A.)77,
5102-5104 [1980]). Various leukocyte interferotypes
The glycosylation of
However, the difference in glycosylation between types alone does not
It cannot explain the observed molecular weight distribution. So
Instead of leukocyte interferon, according to type
There are significant differences in amino acid composition and sequence.
amino acid homology may be lower than 80%.
stomach. Partial characterization by isolation from donor leukocytes
and sufficient quantities for limited clinical research.
Although it gave a uniform leukocyte interferon of
However, it does not require extensive clinical trials and widespread
necessary for preventive and/or therapeutic use.
insufficient to provide interferon
It is. Truly an interferon derived from human white blood cells
Current clinical use of antitumor and antiviral drugs
In the test, a crude (less than 1%) preparation was used.
and produce sufficient quantities even at impractical prices.
research in a wide range of areas has been delayed.
It's here. However, with the advent of recombinant DNA technology,
A huge variety of useful polypeptides can be found in minute quantities.
It has become possible to control production using living organisms. vinegar
In addition, modified by this technology, somatostasis
Chin, human insulin A and B chains and human insulin
It produces polypeptide chains such as growth hormone.
It was not until the existence of bacteria that were able to
Ru. (Itakura, etc., science
(Science)198, 1056-1063 [1977]; Godel
(Goeddel) et al., Nature281, 544
−548 [1979]). More recently, recombinant DNA
The technology is proinsulin and thymosin alf.
It made possible the production of A1, and furthermore, it
encodes human leukocyte interferon
Collection of DNA and resulting white blood cells
Regarding the production of proteins with interferon activity
(Nagata et al.,
Yah (Nature)284, 316-320 [1980]; Nantei
(Nantei) et al., GeneTen, 1-10
[1980]; Taniguchi et al.
(Nature)285, 547-549 [1980]). The recombinant DNA technology laboratory
present in microorganisms, often in numerous copies within cells
non-chromosomal double-stranded DNA that exists as
It's a loop, it's a plasmid. plasmid
The information encoded in DNA includes information that is stored in daughter cells.
Information necessary to reproduce lasmids (i.e.
“replicon”) and, usually, one type
or more than one selection characteristic, e.g. bacteria
In some cases, information on antibiotic resistance is available.
Ru. This information allows you to
host cell clones with
It becomes possible to preferentially grow underground. plastic
Sumids are useful because they
Recognize each part of the body as one type or another.
types of restriction endonucleases or “restriction acids”
The plasmid can be specifically cleaved by
That's what it means. After that, cut both ends of the cut area.
Or connect the reconstructed end to the cut site.
foreign genes or gene fragments.
can be inserted into a plasmid. DNA recombination is
“Recombination” can be carried out outside the cell, but it produces
Plasmids are transformed into cells by a method called transformation.
Introduced into cells and causing the transformed cells to develop
Recombinant plasmid containing a foreign gene
can be produced in large quantities. Furthermore, the foreign gene
transfer of the encoded DNA information within the plasmid.
Correctly inserted regarding parts governing copying and translation.
If inserted, the resulting expression vehicle
The actual polypeptide sequence encoded by the gene
actually used in the process called the production or expression of
can be used. Expression is recognized by RNA polymerase.
the region to which it binds, known as the promoter
Developed in In some cases, for example,
Tryptophar which is advantageous in carrying out the present invention
In some cases, such as the promoter or “trp” promoter,
The promoter region contains an “operator” region.
Overlapping, combined promoter-operator
- It's becoming. Operator may be eligible for certain promotions.
plays a role in regulating the likelihood of transcription initiation in
recognized by so-called repressor proteins.
This is the DNA sequence. RNA polymerase is
The information that travels along DNA and is contained in coding sequences.
information from the 5′ end to the 3′ end.
– transcribed into RNA, which then becomes the DNA code
translated into a polypeptide with an amino acid sequence that
be done. Each amino acid is a nucleotide
encoded by replets or “codons”
Ru. These are referred to as "structural remains" in connection with the purpose of the present invention.
The part called “gene”, that is, the gene that is expressed
It exists in the part that encodes the amino acid sequence of a substance.
After binding to the promoter, the RNA polymer
First, the DNA enzyme encodes the ribosome binding site.
transcribe the cleotide, then initiate translation or
“Start signal” (usually an ATG, the resulting signal
AUG in Tsenjiya RNA), then,
nucleotide codons present in the structural gene itself
Transcribe. A so-called stop at the end of a structural gene
Codons are transcribed. After that, the polymerase
forms additional sequences of messenger RNA.
Even if something happens, there is a stop signal, so
remains untranslated by ribosomes. ribo
The mRAN is usually forming
In bacteria, a special feature on messenger RNA
Binds to specific junction sites. Then start the translation
signal, and ends with the stopped signal mentioned above.
yielding the encoded polypeptide. Li
The sequence encoding the bosome binding site is
properly positioned with respect to the start codon, and the remaining
All codons are in phase
If the start codon is followed, the desired product will be produced.
It will be done. The resulting product lyses the host cells;
The product can be isolated from other bacterial proteins by appropriate purification methods.
It can be obtained by collecting it. We use recombinant DNA technology (i.e., interface
Inserting the Elon gene into a microbial expression vehicle
expressing them under the control of biological genetic regulatory elements
The use of large amounts of leukocyte interferon
We believe that this is a very effective way to provide this. that
glycosylation characteristics characteristic of human-derived materials.
Although it lacks sex, it is widespread and has viruses and viruses.
and may be used in the treatment of neoplastic diseases.
cormorant. The first method of collecting leukocyte genes of the present invention is
It consists of the following stages: (1) Human leukocyte interface purified to a homogeneous state
Using the partial amino acid sequence of Elon, partial amino acid
Possible nucleotides that can encode amino acid sequences
as a collection, representing all combinations of
Set of synthetic DNA probes containing codons
Configure. (2) Complementation from induced messenger RNA
Bacterial colony bank containing DNA (cDNA)
Prepare. Another radiolabeled induction
mRNA and plasmids from this bank
Hybridize with cDNA. hybrid
Elute mRNA and interact with oocyte analysis
Test the translation to Ron. Do it like this
has been shown to induce interferon activity.
The plasmid DNA from the cloned colony was
Hybridization for the probe manufactured as in (1)
Test for deformation. (3) In parallel with the step (2) above, the inducer in the plasmid
Using cDNA derived from the derived mRNA,
Prepare another transformed colony bank. (One
Using the probe as a hybridization probe
to induce synthesis of radiolabeled single-stranded cDNA for
It was used to Synthetic probes can be used as templates.
Forms a hybrid with guide mRNA and is transferred by reverse transcription.
expanded, induced, radiolabeled cDNA
form. The radiolabel obtained in this way
Colonies hybridized to le cDNA
Find out more about clones from banks,
The presence of a full-length interferon-encoding gene
confirm. The estimation of the partial length obtained by (1) and (2)
The gene fragments that are generated are themselves full-length genes.
It can be used as a probe. (4) The full-length gene obtained above is a mature polypeptide.
may interfere with microbial expression of putido.
If there is a bad leader array, remove it.
and microbial promoter initiation signals.
expression vector with respect to the cell and ribosome binding site.
Allows you to position yourself appropriately in the vehicle.
Sea urchin was created using synthetic DNA.
The expressed interferon is purified and its properties are determined.
Confirm the activity and measure the activity. (5) Interferon gene prepared by the above method
The fragment itself can be isolated from other partially homologous white blood cells.
Produced by hybridization of Ntafaeron sp.
used for Applying the recombinant DNA technology outlined above
the corresponding preceding sequence or part thereof by
A mature polypeptide that does not essentially involve
A group of homologous leukocyte interferons (groups)
(not lycosylated), high yield, high purity
It became possible to produce it microbially. child
These interferons are found in animal and human viruses.
Suitable levels for use in cancer and malignant tumor diseases
can be directly expressed, harvested, and purified up to
I can do that. A group of insulins expressed in this way
Tafelon is effective in in vitro tests
was shown and the first microbially produced
In vivo testing as leukocyte interferon
But for the first time, it has been shown to be effective. “Mature leukocyte interface” as used herein
The term "elon" refers to a microorganism that lacks a glycosyl group.
Biologically (e.g., bacterially) produced interfaces
Meaning feron. The mature leukocyte interface of the present invention
Feron is the first amino acid codon of a natural product.
Directly from the immediately preceding translation initiation signal (ATG)
It will be expressed soon. Therefore, this “mature” polypep
Tide has methio as the first amino acid in its sequence.
(encoded by ATG), but the essence
Its properties are not affected. On the other hand, microbial hosts
processes the translation product to remove the first methionine.
I can hear it. Mature leukocyte interferon
can be expressed together with junctional proteins other than the
However, this conjugate does not interact with the extracellular or intracellular environment.
(British Patent Publication No.
(See 2007676A). Finally a mature interferon
is a microbial “sign” that transports the conjugate to the cell wall.
can be produced as a conjugate with a null peptide.
In the cell wall, the signal is processed and released.
Mature polypeptide is secreted. “Expression” of mature leukocyte interferon
For mRNA translation of leukocyte interferon genome
directly resulting in a glycosyl group or preceding sequence.
Does not contain interferon molecules of bacteria or other
Means production by microorganisms. The specific leukocyte interferon protein is here
is the determined DNA gene (Table 2 and Table 4)
and the deductively determined amino acid sequence (see Table 3).
and Table 5). These specific
of all the groups included here.
natural for leukocyte interferon protein
Allelic variation exists
However, it can also occur between individuals. These mutations
Amino acid differences throughout the sequence or in the sequence
Deletion, substitution, insertion, inversion or addition of amino acids
It appears as an addition. Each in-line targeted in this specification
Tuffelon, marked LeIF A to LeIF J
Therefore, such allelic variation is
included within its scope or definition and therefore included in this book.
shall be included within the scope of the invention. Next, the tables and figures will be explained.






Table 1 shows human
Separated from white blood cells and purified to homogeneity.
Two amino acids common to all types of interferons
The amino acid sequence is shown. encode these peptides
All possible mRNA sequences and corresponding DNA
Indicates an array. The letters A, T, G, C and U are
respectively, adenine, thymine, guanine, cytosi
Nucleotides containing the bases of ion and uracil
shows. The letter N represents the nucleotides A, G, C and
or U. The polynucleotide is
Written to read from 5′ (left side) to 3′ (right side)
has been done. and double-stranded (“d.s.”) DNA.
For the lower or non-coding strand,
The order is reversed. Figure 1 shows a possible LeIF
with lasmid³²p-label synthetic deoxyoligonuc
Autotradio showing hybridization with leotide
Gram. Table 2 shows the numbers from “A” to “H” respectively.
Leukocyte interferon production marked with the symbol
Eight gene fragments were isolated as candidates for the present study.
The nucleotide sequence (coding strand) of the fragment is shown. that
For each LeIF, the ATG translation start codon and
and the termination triplet are underlined. stop
A stop codon or termination codon followed by a 3′ untranslated region
area is shown. All remains of LeIF A included
The gene length is shown in the third column A line of Table 2.
In sea urchins, one codon present in other codons is missing.
I'm doing it. The 5′ untranslated region precedes the leader sequence.
ing. Fragment E, which remains separated, contains the leader's
There is no complete predecessor array. However, it is assumed that
Contains all genes for mature LeIF E. minutes
The fragment G that remains separated has an incomplete code sequence.
stomach. In Table 3, 8 nucleotides shown by the nucleotide sequence
We are comparing the LeIF protein sequences of . IUPAC−
LUB Commitment on Biochemicals
- Men Culture (Commission on
A sentence proposed by Biochemical Nomenclature
Letter abbreviations are used. In other words, alanine is A
So, cysteine is C, aspartic acid is D,
Glutamic acid is E, phenylalanine is F,
Glycine is G, histidine is H, isoleucine
ion is I, lysine is K, leucine is L, methane is
Onine is M, asparagine is N, and proline is
P, glutamine is Q, arginine is R, se
Phosphorus is S, threonine is T, valine is V, and T is
Lyptophan is represented by W, and tyrosine is represented by Y. number
indicates the position of the amino acid. S is signal peptide
shows. The LeIF A sequence is a 165 amino acid sequence.
The needlefish at position 44 in the row uses this LeIF A sequence as
Aligns with the 166 amino acid sequence of LeIF.
It's there for the purpose. The array of LeIF E is
extra nucleotide in the domain region (position 187 in Table 2)
It is determined by ignoring the Stars indicate infei
This shows the stop codon in the sequence. Common to all LeIFs
General amino acids (excluding pseudogene LeIF E)
is also shown. Underlined residue
is an amino acid also present in human fibroblast interferon.
It is a noic acid. Figure 2 shows eight types of LeIF cloned cDNA.
Restriction endonuclease map (from A to H)
shows. The hybrid plasmid has a dC:dG tag.
Ring method (Goeddel, D.V., etc.)
Yah (Natue)287, 411-416 [1980])
built. So the cDNA insert was cut using PstI.
sell. Line the ends of each cDNA insert on the sides.
Contiguous homopolymerized dC:dG tails are shown. Pvu,
Restriction sites for EcoR and Bgl are also shown.
The dark region indicates the coding sequence of mature LeIF. diagonal line
is the signal peptide coding sequence. White
The remaining parts are the 3' and 5' non-coding sequences. Figure 3 shows direct microbial synthesis of mature LeIF A.
The construction of the encoding gene is shown. restriction sites and
The remainder is indicated (“Pst” etc.). “b.p.” is salt
Indicates the base pair. Figures 4 and 5 show LeIF B mature leukocyte
Two genes used to express ntaferon
Restriction maps of child fragments are shown. The codon sequence shown is
Digested with restriction enzyme Sau3a for the two cases shown.
This is the end of the code chain generated by doing this. Tables 4 and 5 include types I and J.
In addition, the DNA and amino acid sequences of the five LeIF proteins were
No acids (the abbreviations are the same as in Table 3)
vinegar. In Table 5, asterisks indicate corresponding DNA sequences.
The hyphen indicates the stop codon in the sequence.
Indicates an omission or gap in the Next, advantageous specific examples for carrying out the present invention will be explained.
shows. A Microorganisms used In the described implementation, two types of microorganisms, one
Mari, E. described in U.S. Patent (U.S.P.
coli (E. coli) x 1776 and E. coli. Cori (E.
coli) K-12 294 strains (endo A, thi^-,hsr^-,
hsm⁺ _k, described in British Patent Publication No. 2055382)
ing. respectively, The American Type
Culture Collection (the American
Type Culture Collection)
ATCC No.31537 and 31446 (Budapest Convention)
Accession number 31446 based on the international deposit by
It is. All recombinant DNA experiments are
Regional Institute of Health
(National Institute of Health)
It was carried out in accordance with the guidelines. A most advantageous embodiment of the invention is defined above.
The strain of E. coli (E. coli) x 1776 and E. coli.
Not only E. coli K-12 strain 294, but also other
Known E. coli strain (hereinafter referred to as E.coli)
For example, E.coli and E.coli including E.coli B
This will be described in relation to other microbial strains.
Many of these are from The American Type Cal.
Cheer Collection (the American Type
Culture Collection) (ATCC)
The microorganisms deposited in the microorganism depository facility
hand is possible. German patent 2644432 was also found
stomach. These other microorganisms include Bacillus e.g.
Bacillus Subtilis and other intestinal
Bacteria such as Salmonella typhimurium
(Salmonella typhimurium) and Serratia
marcescens (Serratia marcescens)
capable of expressing and replicating foreign gene sequences
Use plasmids. Yeast such as Satsukaro
Saccharomyces cerevisiae
cerevisiae) also control yeast promoters.
Below, the gene encoding interferon is expressed.
Production of interferon protein by expressing
It can be advantageous for use in construction. B LeIF mRNA raw material and purification LeIF mRNA can be collected from human leukocytes.
Ru. Usually, the method described in German patent no.
As in, Sendai virus or
Infected with Newcastle disease virus
Chronic bone induced to produce ntaferone
Uses material obtained from leukocytes of leukemia patients
do. Particularly advantageous are the
The raw material was derived from a patient with acute osteogenic leukemia.
This is a cell line called KG-1. this
The cell line is based on Koeffler, H.P.
Golde, D.W., Science
(Science)200, 1153 (1978).
As such, [Rosewall Park Memorial
Institute RPMI (Rosewall Park)
Memorial Institute)〕1640 plus 10% heat inactivation
FCS (fetal bovine serum), 25mM HEPES buffer
(N-2-hydroxyethyl-piperazine-
N'-2-entasulfonic acid) and 50μg/ml
Grows easily on gentamicin-containing medium of 1
Subculture twice a week (1 to 3 splits)
I can do it. Add 10% dimethyl sulfonate to the above growth medium.
By adding oxide, the cells can be frozen. KG
-1 is The American Type Culture
Collection (the American Type
Culture Collection) (ATCCNo.CRL8031)
It has been deposited. KG-1 cells contain Rubinstein
(Rubinstein) et al. Nattle.
Acad. Sai. United States (Proc.Natl.Acad.Sci.U.
S.A.)76, 640-644 [1979]), Sendai
(Sendai) or New Katsuru
(Newcastle) disease virus was used to induce leukocyte infusion.
Induced to produce Tahuelon mRNA
Ta. Cells were harvested 5 hours after induction and RNA was collected.
Guanidine thiocyanate-guanidine hydrochloride
Law (Chirgwin, etc.)
Mistry (Biochemistry)18, 5294−5299
[1979]). from uninduced cells
RNA was prepared similarly. oligodeoxythi
Midine (dT)-cellulose chromatography
and using sucrose gradient ultracentrifugation.
A 12S fraction of poly(A) mRNA was obtained. The method is
Green et al. (Ark. Biochem.
Arch.Biochem.Biophys.172,
74-89 [1976]) and Okuyuma
etc. (Arch. Biochem. Biofis.
(Arch.Biochem.Biophys.)188, 98−104
[1978]) was used. This mRNA is
Xenopus laevis oocyte analysis (Cavarilli)
(Cavalieri) et al., Proc. Nattle. Acad. sa
stomach. United States (Proc.Natl.Acad.Sci.U.S.A.)74,
3287-3291 [1977]) 8000-10000 units/my
Chromogram's interferon titer is shown. C Colony bank containing LeIF cDNA sequences
preparation of Standard method Wickens using 5 μg of mRNA
(Wickens) et al., J. Biol. Chemi. (J.
Biol.Chem.253, 2483-2495 [1978] and Go.
Goeddel et al., Nature
281, 544-548 [1979]), double-stranded cDNA
was prepared. cDNA was fractionated according to size by electrophoresis on a 6% polyacrylamide gel, and 230 ng of material ranging in size from 500 to 1500 b.p.
was obtained by electroelution. 100ng of this cDNA
470 ng tailed with a deoxycytidine (dC) residue (Chang et al., Nature 275 , 617-624 (1978); 470 ng tailed with a deoxyguanosine (dG) residue at the Pst site. plasmid of
annealed with pBR322 (Bolivar et al., Gene 2 , 95-113).
[1977]), and was used to transform E. coli x1776. About 130 tetracycline-resistant and ampicillin-sensitive transformants were obtained for cDNAng. In a second similar experiment, for cDNAng,
Approximately 1000 tetracycline-resistant and ampicillin-sensitive E. coli K-12 strain 294 transformants were obtained. In this case, ranging from 600 to 1300 b.p.
Size fractionated cDNA material was collected by electroelution and used for tailing with dC. D Preparation of Synthetic Oligonucleotides and Their Use Knowledge of the amino acid sequences of several tryptic fragments of human leukocyte interferon has allowed the design of synthetic deoxyoligonucleotides complementary to various regions of LeIF mRNA. two tryptic peptides
I chose T1 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 deoxyoligonucleotide probes were synthesized for each sequence. That is, each contains three (T-1A, B, C, D) or one (T-13A, B, C, D) oligonucleotides. The complementary deoxyoligonucleotide 11 base chain length shown was chemically synthesized by the phosphotriester method (Crea et al.
Proc. Nattle. Acad. Sai. United States (Proc.
Natl.Acad.Sci.USA) 75 , 5765−5769
[1978]). In the T-13 series, four individual probes were prepared. As shown in Table 1, 12 T-1 probes were prepared as 4 pools of 3 probes. 12 T-1 probes prepared as 4 pools of 4 individual probes of the T-13 series and 3 primers each were radiolabeled single heavy strands for use in hybridization probes.
Used to induce cDNA synthesis. template
mRNA was derived from 12S RNA from Sendai-induced KG-1 cells (8000 units IF activity/μg) or total polypeptides derived from uninduced leukocytes (<10 units/μg).
(A) mRNA. ³² p-labeled cDNA was subjected to known reaction conditions (e.g., Noyes) under known reaction conditions (Noyes, etc.).
Nattle. Acad. Sai. United States (Proc. Natl.
Acad.Sci.USA) 76 , 1770-1774 [1979]) from these primers. 20
mM Tris (hereinafter referred to as Tris) - HCl
(PH8.3), 20mM KCl, 8mM _MgCl2 , 30
Reactions were carried out in 60 μl volumes in mM β-mercaptoethanol. This reaction consisted of 1 μg of each primer (i.e., for the T-1 series,
12 μg total (4 μg total for T-13 series), 2 μg “induced” 12S fraction
10 μl of mRNA (or uninduced poly(A) mRNA)
g), 0.5mM dATP, dCTP, dTTP,
200μCi (α ^32p ) dCTP (Amersham, 2-3000Ci/mmole) and
60 units of reverse transcriptase (Bethesda Research Laboratory)
Laboratories)]. The product is 10ml Sephadex (hereinafter referred to as Sephadex).
Separate unbound labels by gel filtration on a G-50 column (indicated by ) and incubate at 70°C for 30 minutes.
Treat with 0.3N NaOH to degrade RNA and HCl
It was neutralized. Hybridization is carried out by Kafatos et al., NuCleic Acid Res. 7 , 1541-1552.
(1979). E Identification of clones pL1-pL30 500 E. coli K-12 strain 294 transformed strains (C
plasmid DNA
Birnboim et al., Nucleic Acids Res. 7 , 1513-1523.
(1979)'s rapid plasmid isolation procedure was used.
Each DNA sample was denatured and loaded on a nitrocellulose filter in triplicate according to the method cited above by Kafatos et al. Three sets of nitrocellulose filters containing 500 plasmid samples were tested for (a) induced cDNA primed with the T-1 set of primers, (b) induced cDNA primed with T-13, and (C) induced cDNA primed with the T-13 set of primers. was hybridized with uninduced cDNA prepared using both sets of cDNAs. A clone was considered positive if it hybridized more strongly to one or both of the induced cDNA probes than to all of the uninduced probes. Thirty "positive" clones pL1-pL30) were selected from the 500 clones and further analyzed. F Identification of clones pL31-pL39, isolation of plasmid containing LeIF gene fragment (No. 104) ³² p-label induced mRNA as a probe Lillenhaug et al., Biochemistry, 15 , 1858-1865
[1976]) using the colony hybridization method of Grunstein and Hogness (Proc. Natl. Acad. Sci. USA).
72, 3961-3965 [1975]) to screen for transformed strains of E. coli x1776. 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 labeled mRNA should occur selectively to colonies containing the induced sequences. Three groups of transformants were obtained. (1) 2 to 3% of colonies hybridized very strongly to ^32p -mRNA. (2) 10% had a significantly lower degree of hybridization than the above group (1). (3) The remainder showed no detectable hybridization signal. For positive colonies (groups (1) and (2)), interferon mRNA is
The presence of interferon-specific sequences was investigated by analysis by specifically hybridizing to DNA. First, tetracyclone (20 μg/ml), diaminopimelic acid (100 μg/ml),
ml), thymidine (20 μg/ml) and d-biotin (1 μg/ml) in 100 ml of M9 medium.
Each of the 60 strongly positive colonies (group 1) was grown individually. For 1 liter of M9 medium, Na ₂ HPO ₄ (6 g), KH ₂ PO ₄ (3 g),
Contains NaCl (0.5g) and _NH4Cl (1g),
After autoclaving, add 1 ml of sterile 1M _MgSO4 and 10ml of sterile 0.01M _CaCl2 . Ten cultures were pooled and plasmid DNA was isolated from six pools as described by Clewell et al., Biochemistry 9 , 4428-440 [1970]. 10 μg of each plasmid DNA pool
was cut with Hind, modified, and covalently bonded to DBM (diazobenzyloxymethyl) paper.
1 μg of purified mRNA from induced cells was hybridized to each filter paper. Unhybridized mRNA was removed by washing. Specifically hybridized mRNAs were eluted and translated in Xenopus oocytes. In this analysis,
All 6 pools were negative. Five pools of 10 colonies each and one pool of 9 colonies were prepared from 59 weakly positive colonies (group 2), and plasmids were prepared from these pools and examined as described above. Of the six pools tested,
One (K10) hybridized to interferon mRNA at levels significantly above background levels in each test. To identify specific interferon cDNA clones, plasmid DNA was prepared from 9 colonies of pool K10 and examined individually. Two of the nine plasmids (No. 101 and No. 104) bound interferon mRNA well above background levels. From plasmid No.104,
A unique Bgl restriction fragment containing 260 b.p. was isolated. This is described by Taylor et al. Acta (Biochim.
biophys.Acta) 442 , 324-330 (1976) and labeled with ^32p using the colony screening method (Grunstein et al.
Hodness), Proc. Nattle. Sai. USA (Proc.Natl.Sci.USA) 72 , 3961−3965
[1975]), 400 E. coli 294 transformants were used as probes for individual screening. Nine colonies (pL31-pL39) were identified that hybridized to varying degrees with this probe. Furthermore, using a labeled 260 b.p.
Similarly, 4000 E. coli 294 transformants were individually screened. Fifty colonies were identified that hybridized to a species-specific degree with this probe. One is LeIF G fragment, one is
One of the LeIF H fragments is called LeIF Hl (apparently it is an allele of LeIF H)
Contained. The resulting hybrid plasmid is
It was called "pLeIF H" etc. G Isolation and Sequencing of the First Full-Length LeIF Gene Plasmid DNA was prepared from a total of 39 potential LeIF cDNA clones. and,
The same 260 b.p. DNA probe was rescreened using the hybridization procedure of Kafatos et al. (cited above). Three plasmids (pL4, pL31, pL34) gave very strong hybridization signals with this probe, four (pL13, pL30, pL32, pL36) moderately hybridized, and three (pL6, pL8, pL14) ) were weakly hybridized. The 39 potential LeIF cDNA recombinant plasmids were also directly hybridized with ^32p -labeled synthetic undecamers (each of the T-1 primer pool or each of the T-13 primers, each consisting of 3 primers). I searched using it as a probe. Hybridization conditions were chosen such that complete base-base pairing was required to give a detectable hybridization signal (Wallace et al., Nucleic Acid Res. 6 ). , 3543
−3557 [1979]). In other words, plasmid DNA was extracted from 39 clones using the standard supernatant solution method (cells containing the plasmid were lysed using a surfactant, etc., the supernatant containing the plasmid was collected after centrifugation, and the plasmid was isolated from this supernatant. Ethanol precipitation is used for isolation from the supernatant.) (cleared lysate
procedure; Clewell, etc., above)
prepared in Biorad agarose (Biorad
Agarose) A-50 (agarose gel for gel filtration manufactured by Bio-Rad) column chromatography. A sample (3 μg) of each preparation was
to open the ring, denature with alkali, and 2
The samples were spotted onto two separate nitrocellulose filters. 1.5 μg per spot.
See the literature by Kafatos et al. cited above. Synthetic deoxyoligonucleotide T-13
Each of the primers and the T-1 primer pool was phosphorylated with (γ ³² P)ATP as follows. 50 pmole of oligonucleotide and 100 pmole of (γ ³² P) ATP (New England
Nuclear), 2500Ci/mmole), 50mM
Tris-HCl, 10mM _MgCl2 and 15mM β
- Combined in 30 μl of a mixture of mercaptoethanol. After adding 2 units of T4 polynucleotide kinase and 30 minutes at 37°C, ^{the 32} p-labeled primer was purified by chromatography on a 10 ml Sephadex G-50 column at 10 ⁶ cpm. Hybridization was performed using primer T-13C or 3 x ¹⁶⁶ cpm of primer pool T-1C. Hybridization was carried out according to the method cited above by Wallace et al.
6 x SSC [1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate, PH7.2], 10 x Denhardts [0.2
% bovine serum albumin, 0.2% polyvinylpyrrolidone, 0.2% Ficoll] solution for 14 hours at 15°C. Filter at 0℃ in 6×SSC
I washed it three times for 5 minutes. It was dried and exposed to X-ray film. The results are shown in Figure 1 for ^32p -primer T-13C and primer pool T-1C.
The case is shown below. Plasmid DNA from clone 104 was combined with primer pool T-1C and primer T-13C.
It was noticeably hybridized. However, it showed no detectable hybridization with other undecamers. As shown in Figure 1, some of the 39 potential LeIF plasmids (pL2, 4, 13, 17, 20, 30, 31, 34) hybridized with both of these probes. However, restriction analysis showed that only one of these plasmids, pL31, also contained a 260 b.p. internal Bgl fragment. As a result of digesting pL31 with Pst,
The size of the cDNA insert was approximately 1000 b.p. The entire Pst insert of pL31 was sequenced using Maxam-Gilbert chemistry (Methods Enzymol.).
65, 499-560 [1980]) and dideoxy chain termination methods (Smith, Methods Enzymol. 65 , 560-580).
[1980]). At that time, the Sau3a fragment
It had been subcloned into the M13 vector.
The DNA sequence is shown in Table 2, "A". Information about available protein sequences, known
The range of LeIF molecular weights and the relative presence of stop triplets in the three possible reading frames suggested a suitable translational reading frame. Then, the entire LeIF amino acid sequence was shown, including the prey or signal peptide. The first ATG translation initiation codon is located at the 60th nucleotide from the 5' end of the sequence, and 188 codons later, there is a TGA termination triplet. The 3' end contains 342 untranslated nucleotides followed by a poly(A) sequence. The postulated signal peptide (presumably involved in secretion of mature LeIF from leukocytes) is 23 amino acids long. mature
The calculated molecular weight of the 165 amino acids that make up LeIF is 19,390. We designated this LeIF encoded by pL31 as "LeIF A." Table 3
From the sequence code (“A”) of LeIF A, tryptic peptides T1 and T13 are 146-150 and
It turns out that they correspond to 58-62, respectively. The actual DNA coding sequences found in these two regions are those represented in primer pool T1-C and primer T-13-C (Table 4).
). H. Direct Expression of Mature Leukocyte Interferon A (LeIF A) 1 General Description Manipulations for directly expressing LeIF A as a mature interferon polypeptide include the combination of synthetic (N-terminal) and complementary DNA. This is a variation of the method used for human growth hormone (Goeddel et al., Nature 281 , 544-548 [1979]). As shown in Figure 3, the Sau3a restriction endonuclease site is Conveniently, LeIF A
It is located between codons 1 and 3 of
Contains the ATG translation initiation codon and contains 1 amino acid
Repairs the codon for (cysteine),
Involves creating new EcoR sticky ends. Two synthetic deoxyoligonucleotides were designed. These oligomers were ligated to the 34b.p.Sau3a-Ava fragment of pL31. The resulting 45 b.p. product has two additional
It was ligated to a DNA fragment to create an 865 b.p. synthetic-natural hybrid gene. It encodes LeIF A and is flanked by EcoR and Pst restriction sites. This gene contains EcoR and
It was inserted into pBR322 between the Pst sites to create plasmid pLeIF Al. 2 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 pGMI (ATCC31622) was constructed from E. coli containing the deletion ΔLE1413. coli tryptophan operon (Miozzari, etc.)
J.A. Bacteriology (J.
Bacteriology) 133 , 1457-1466 [1978]).
Therefore, a fusion protein is expressed that contains the first six amino acids of the trp leader and approximately the last third of the trp E polypeptide (hereinafter collectively referred to as LE') as well as the entire trp D polypeptide. This is all under the control of the trp promoter-operator system. Plasmid pGM1 can be replaced by other readily available equivalent trp promoters (e.g. Bennett, J. Mol. Biol (1978)
121, 113-137, p. 135, Fig. 10) may be used. Add 20μg of this plasmid to restriction enzyme
Digested with Pvu. This converts the plasmid into 5
Cut at different parts. This gene fragment is then ligated with an EcoR linker (made up of the self-complementary oligonucleotide of pCATGAATTCATG) to provide an EcoR cleavage site for later cloning into an EcoR site-containing plasmid.
20μg of DNA fragment obtained from pGMl,
In the presence of 200 pmoles of the 5′-phosphorylated synthetic oligonucleotide pCATGAATTCATG, 20μ
_T4 DNA ligase buffer (20mM TrisPH
7.6, 0.5mM ATP, 10mM _MgCl2 , 5m
10 units of _T4 DNA in M dithiothreitol)
Treated with ligase overnight at 4°C. solution next
Ligation was stopped by heating at 70°C for 10 minutes. The linker was cleaved by EcoR digestion, and the fragments with EcoR ends were separated using 5% polyacrylamide gel electrophoresis (hereinafter referred to as PAGE). The fragment was located and separated by cutting out the target part from the gel. Place each gel fragment (300μ0.1×TBE) into a dialysis bag.
And 0.1× TBE buffer (TBE buffer is
10.8g Tris base, 5.5g boric acid, 0.09g
Electrophoresis was performed at 100V for 1 hour in Na ₂ EDTA in 1 liter of H ₂ O). An aqueous solution was collected from a dialysis bag, extracted with phenol, extracted with chloroform, and adjusted to a concentration of 0.2M sodium chloride.
DNA was collected in water after ethanol precipitation.
trp promoter with EcoR sticky end
The operator-containing gene was identified by the procedure described below. That is, inserting a fragment into a tetracycline-sensitive plasmid, which
Tetracycline resistance was achieved by inserting a promoter-operator. Plasmid pBRHI (Rodriguez et al., nuclear acid
Research (Nucleic Acids Res.) 6 , 3267
-3287 [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 EcoR site, the plasmid can be made resistant to tetracycline. pBRHI was digested with EcoR, the enzyme was removed by phenol extraction, chloroform extraction, and ethanol precipitated into water. The resulting DNA molecules present in separate reaction mixtures merge with each of the three DNA fragments obtained above;
They were then ligated with _T4 DNA ligase as described above. DNA present in the reaction mixture
using standard methods (Hershfield et al., Proc. Natl. Acad.
Sai. USA (Proc.Natl.Acad.Sci.USA)
71, 3455-3459 [1974]), and the bacteria were transformed with LB (Luria -Bertani) 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 called pBRH trp. EcoR and hepatitis B viral genomes
BamH digestion products were collected by conventional methods,
EcoR of plasmid pGH6 (ATCC31539)
[This plasmid pGH6 was cloned into the BamH site of Goeddel et al.
281, 544 (1979), a 285 base pair EcoR fragment isolated from plasmid pKB268 (the same fragment is a 95 base pair heterologous fragment)
It consists of two UV-5lac promoters interposed with DNA fragments, and its construction method is as follows:
Johnsrud's Proc.Natl.Acad.Sci.USA, 75 (11)
5314−5318 (1978)),
It is constructed by inserting into the EcoR site of plasmid pBR322. In addition, UV-5lac
The nucleotide sequence of the promoter was obtained from Dickson et al.
Science 187 , 27−35 (1975), Simpson et al.
Cell 18 , 277-285 (1979), Siebenlist et al.
It is described in Cell 20 , 269-281 (1980), etc., and is well known. ], plasmid pHS32. Plasmid pHS32 was cut with Xba, extracted with phenol, extracted with chloroform, and precipitated with ethanol. Precipitation is 0.1mM
30μl containing dTTP and 0.1mM dCTP
Polymerase buffer (50mM potassium phosphate
1 μl E. coli DNA polymerase, Klenow fragment (Boehringer-Mannheim) in pH 7.4, 7 mM MgCl ₂ , 1 mM β-mercaptoethanol).
Mannheim) for 30 minutes at 0°C, then at 37°C.
It was treated for 2 hours. This treatment fills in two of the four nucleotides complementary to the 5' overhang of the Xba cleavage site. 5'CTAGA-5'CTAGA-3'T-TCT- Two nucleotides dC and dT are incorporated to give an end with two overhanging 5' nucleotides. This linear residue of plasmid pHS32 (phenol and chloroform extraction followed by ethanol precipitation and taken up in water) was
I cut it with. large plasmid fragment,
The smaller EcoR-Xba fragments were separated by PAGE and separated by electroelution.
This DNA fragment from pHS32 (0.2 μg) was used to transform the EcoR
- ligated to the Taq fragment. In this method of ligating the PHS32 fragment described above to the EcoR-Taq fragment, although not fully Watson-Crick base-paired,
The overhanging end of Taq is ligated to the remaining overhanging end of Xba. -T+CTAGA-→-TCTAGA--AGC+TCT-→-AGCTCT- A portion of this ligation mixture was used for transformation of E. coli 294 cells, heat treated and plated in LB plates containing ampicillin. 24 colonies were selected and grown in 3 ml LB (Luria-Bertani) and the plasmids were isolated. Six of these colonies were caused by E.coli.
Xba regenerated by DNA repair and replication
It was found that there are parts of the body. −TCTAGA−→−TCTAGA− −AGCTCT−→−AGATCT− These plasmids contain EcoR and
It was also found that both Hpa were cut to give the expected restriction fragments. One plasmid, designated pTrp14, was used to express foreign polypeptides as discussed below. Plasmid pHGH107 (ATCC31538) [This plasmid pHGH107 was derived from Goeddel et al.
The method described in Nature 281 , 544 (1979) (see, for example, Fig. 4 in the same document)
It is constructed by inserting a DNA fragment encoding human growth hormone downstream of the lac promoter of pGH6. ] is a gene for human growth hormone consisting of 23 amino acid codons generated from a synthetic DNA fragment and 163 amino acid codons obtained from complementary DNA generated by reverse transcription of human growth hormone messenger RNA. Contains. This gene lacks the codons of the human growth hormone "preceding" sequence, but contains the ATG translation initiation codon. This gene is from 10μpHGH107,
EcoR treatment, followed by E.
coli DNA polymerase Klenow (hereinafter referred to as Klenow) fragment and was isolated through dTTP and dATP treatment.
The ethanol-precipitated plasmid after phenol and chloroform extraction was treated with Bam HI. Human growth hormone (HGH) gene-containing fragments were separated by PAGE followed 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, so that when subsequently cloned into an expression plasmid, it contains the insert. plasmids can be identified by the restoration of tetracycline resistance. The EcoR end of the fragment is
As filled in with the Klenow polymerase process, this fragment has one blunt end and one sticky end. This ensures correct orientation when later inserted into an expression plasmid. Expression plasmid pTrp14 was then prepared to accept the HGH gene-containing fragment prepared above. That is, pTrp14 is
Digest and remove the resulting sticky ends with dATP
Klenow with dTTP dGTP and dCTP
It was measured using the polymerase 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 pTrp14 is 1
having five blunt ends and one sticky end;
This made it possible to recombine with the aforementioned HGH gene-containing fragment in the correct orientation. HGH gene fragment and pTrp14ΔXba−Bam
The HI fragment was merged and interconnected under conditions similar to those described above. Filled XBA and
The EcoR end is ligated with a blunt end,
Both the Xba and EcoR sites were rearranged. [Table] This construction also creates a tetracycline resistance gene. Plasmid pHGH107 is
This construct, designated pHGH207, allows expression of the tetracycline resistance gene under the control of the tryptophan promoter-operator, since the tetracycline resistance is expressed from a promoter located upstream of the HGH gene (lac promoter). Then, in the concatenated mixture
E. coli 294 was transformed and colonies were selected on LB plates containing 5 μg/ml tetracycline. Plasmid pHGH207 was EcoR digested and trp
A 300b.P. fragment containing the promoter-operator and trp leader ribosome binding site but lacking the ATG sequence for translation initiation,
PAGE followed by electroelution. this
The DNA fragment was cloned into the EcoR site of pLeIFA. The above-mentioned modified trp regulon (E.
The expression plasmid containing the E. coli trp operon can be grown to a predetermined level in medium supplemented with tryptophan in an amount sufficient to suppress the promoter-operator system. The desired product can then be expressed by removing the tryptophan and deinhibiting the system. To be more specific, with reference to Figure 3,
Digest 250 μg of plasmid pL31 with Pst,
The 1000 b.P. insert was separated by gel electrophoresis on a 6% polyacrylamide gel. Approximately 40 μg of insert was electroeluted from the gel, divided into three portions, and further digested as follows. (a) 16μ of this fragment
g samples were partially digested with 40 units of Bgl for 45 min at 37°C, and the reaction mixture was purified on a 6% polyacrylamide gel. Fragment of approximately 670 b.p. desired
2 μg was collected. (b) Another sample (8 μg) of the 1000 b.p.Pst insert was digested with Ava and Bgl. After gel electrophoresis, μg of the 150 b.p. fragment described above was obtained. (c) 16μg of 1000b.p.
and treated with Ava. After electrophoresis on a 10% polyacrylamide gel, approximately 0.25 μg
A 34 b.p. fragment of (10 pmole) was obtained. The two indicated deoxyoligonucleotides, 5'-
dAATTCATGTG (fragment 1) and 5′-
dGATCACACATG (fragment 2) was synthesized by the phosphotriester method. Fragment 2 was phosphorylated as follows. 200μl (approx. 40pmole) of ( ^γ32P )
ATP (Amersham), 5000Ci/
60mM Tris-HCl (PH
8), resuspended in 30μ of a solution containing 100pmole of DNA fragment and 2 units of T4 polynucleotide kinase, consisting of 10mM MgCl ₂ , 15mM β-mercaptoethanol. After 15 minutes at 37℃, 1μ
10mM ATP was added and the reaction was continued for an additional 15
It progressed by a minute. The mixture was then heated at 70°C for 15 minutes to remove 100 pmol of the 5′-OH fragment 1 and 10 pmol
The 34b.p.Sau3aAva fragments were merged. 20mM
Tris-HCl (PH7.5), 10mM _MgCl2 , 10m
M dithiothreitol, 0.5mM ATP and
4 for 5 hours in 50μ of 10 units T4 DNA ligase
Ligate at °C. The mixture was electrophoresed on a 6% polyacrylamide gel and the 45 b.p. product was collected by electroelution. 30ng of 45b.p. product
(1pmole) of the 150b.p.Ava−Bgl fragment
0.5μg (5pmole) and 670b.p.Bal−Pst
1 μg (2 pmole) of the fragment was combined. 20 units
Ligation was performed at 20°C for 16 hours using T4 DNA ligase. The ligase was inactivated by heating at 65°C for 10 minutes. The mixture was digested with EcoR and Pst to remove gene polymers. The mixture is 6%
Purified by PAGE. Approximately 20ng (0.04pmole)
865 b.p. product was isolated. Half of this (10ng)
was inserted between the EcoR and Pst sites of pBR322 (0.3 μg). Transformation of E. coli294 resulted in 70 tetracycline-resistant, ampicillin-sensitive transformants. Plasmid DNA was isolated from 18 of these and EcoR
and digested with Pst. Of the 18 plasmids, 16 had an 865 b.p. long EcoR-Pst fragment. One of these pLeIF Al
1 μg was digested with EcoR and ligated to a 300 b.p. EcoR fragment (0.1 μg) as prepared above, containing the E. coli trp promoter and trp leader ribosome binding site. Transformants containing the trp promoter were identified using a ³² P-trp probe in conjunction with the Grunstein-Hogness colony screening method. The asymmetrically located Xba site in the trp fragment indicates that the trp promoter
This allowed the identification of recombinant products oriented in the direction of the A gene. In vitro and in vivo activity of I LeIF A Extracts for IF analysis were prepared as follows. 1 ml of culture was grown in L broth containing 5 μg/ml tetracycline and A ₅₅₀
The value (absorbance at a wavelength of 550 nm) is approximately 1.0,
It was then diluted into 25 ml of M9 medium containing 5 μg/ml of tetracycline. When A ₅₅₀ reached 1.0, centrifuge a 10 ml sample and collect the cell mass.
15% sucrose, 50mM Tris-HCl (PH8.0),
Suspended in 1 ml of 50mM EDTA. 1mg
of lysozyme was added, and after 5 minutes at 0°C, the cells were disrupted by sonication. Samples were centrifuged for 10 minutes (15000 rpm) and interferon activity in the supernatant was determined by inhibition of cytopathic effect (CPE) assay compared to standard LeIF. To measure the number of IF molecules per cell, 4×
A LeIF specific activity of 10 ⁸ units/mg was used. As shown in Table 6, the clone pLeIF A trp25 in which the trp promoter was inserted in the desired orientation showed high activity (2.5×10 ⁸ units/). As shown in Table 7, E. coli K-12 strain 294/pLeIF A
The trp25-generated IF behaved similarly to the human LeIF preparation. Stable to treatment with PH2 and neutralized by rabbit anti-human leukocyte antibodies. The apparent molecular weight of this interferon is approximately 20,000.
Clone pLeIF A trp25 has been deposited with the American Type Culture Collection (ATCC). In vivo effects of interferon include macrophages and natural killers (NK).
cells are required. And in vivo effects appear to involve stimulation of these cells.
Thus, interferon produced by E. coli294/pLeIF A25 has antiviral activity in cell culture assays but may be ineffective in infected animals. Furthermore, in vivo studies of bacterially produced non-glycosylated LeIF A
Antiviral activity may be different from glycosylated LeIF, which is derived from human "buffy coat" leukocytes. Therefore, we investigated the biological activity of LeIF A (2% purity) synthesized by bacteria in squirrel monkeys.
A comparison was made with Buffy Coat LeIF (8% purity) in lethal doses of encephalomyocarditis virus (EMC) infection of humans (Table 8). [Table] [Table] *E.coli294/pLeIF A trp25 listed in Table 6
250,000 units per ml of extract was diluted 500 times with minimum essential medium to give a specific activity of 500 units/ml.
Leukocyte interferon standard (Wadley Institute) was prepared in advance by NI.
Titers were determined against leukocyte interferon standards, also diluted to a final concentration of 500 U/ml. Adjust the pH of 1 ml to 2 with 1NHCl and adjust to 52 at 4℃.
Incubate for an hour and neutralize by adding NaOH.
IF activity was measured with a standard CPE inhibition assay. A 25μ aliquot of the 500 units/ml sample (untreated) was incubated with 25μ rabbit anti-human leukocyte interferon for 60 min at 37°C.
Incubate for 5 minutes, centrifuge at 12,000 x g for 5 minutes,
The supernatant was analyzed. [Table] All monkeys were male (average weight 713 g) and did not have EMC virus antibodies before infection. Monkeys were infected intramuscularly with 100×LD ₅₀ EMC virus (measured in mice). Control infected monkeys died at 134, 158, and 164 hours postinfection. 10 ⁶ units interferon treatment was administered intravenously 4 hours before infection and 2, 23, 29, 48, 72, 168 and 240 hours after infection. Bacterial leukocyte interferon is a column chromatography fraction from the melt of E.coli294/pLeIF A25, and has a specific activity of 7.4×10 ⁶ units/mg.
It was protein. The control bacterial protein was E.
The total protein amount was doubled in the corresponding column fraction from the coli294/pBR322 melt. Leukocyte interferon standard, chromatographically, 32
It was a Sendai virus-induced interferon from normal human "batty coat" cells purified to a specific activity of x10 ⁶ units/mg protein. Control monkeys exhibited progressive coma, loss of balance, flaccid hind limbs, paralysis, and lachrymal drainage starting approximately 8 hours before death. Interferon-treated monkeys did not exhibit any of these abnormalities, were always active, and did not exhibit symptoms of viremia. One control monkey, which did not show viremia until day 4, died late (164 hours after infection), but high titers of virus were detected in the heart and brain postmortem. Interferon-treated monkeys did not produce antibodies against the EMC virus when tested 14 and 21 days after infection. These results indicate that the antiviral effect of LeIF preparations in infected animals can be attributed solely to interferons. The reason is that the proteins that contaminate bacteria and buffer coat preparations are very different. Furthermore, these results indicate that glycosylation is not required for the in vivo antiviral activity of LeIF A. J Against other leukocyte interferons
Isolation of cDNA DNA from a fully characterized LeIF A cDNA-containing plasmid was cut with Pst, separated by electrophoresis, and labeled with ^32p . Using the resulting radiolabeled DNA as a probe, screen additional E. coli 294 transformants obtained as in Section C above using the Grunstein and Hogness in situ colony screening method described above. did. 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 were isolated by Pst cleavage and characterized in three ways. First, these Pst fragments were characterized by restriction endonuclease digestion patterns using the enzymes Bgl, Pvu and EcoR. This analysis was performed on at least eight different types (LeIF A, LeIF B, LeIF
C, LeIF D, LeIF E, LeIF F, LeIF G and LeIF H). this is,
The approximate positions of various restriction breakpoints are shown for the previously known leading and coding sequences of LeIF A. One of these, LeIF D, is believed to be the same as reported by Nagata et al. in Nature 284 , 316-320 (1980). Second, regarding some of those DNAs,
From poly-A-containing KG-1 cell RNA, LeIF
Regarding the ability to selectively remove mRNA, Cleveland et al.
20, 95-105 (1980). LeIF A, B, C and F were positive in this assay. Third, these Pst fragments were inserted into expression plasmids and E.
coli294 was transformed with the plasmid and the fragment was expressed. The expression product believed to be preintaferon showed no CPE for interfaeron activity, except for a marginally positive LeIF F fragment.
All analyzes were positive. In addition to the above, all of the LeIF types listed above have been sequenced. K second mature leukocyte interferon (LeIF
Direct expression of B) The sequence of the isolated fragment containing the gene for mature LeIF B shows that the first 14 nucleotides of types A and B are the same. So,
A fragment containing the trp-promoter-operator, the ribosome binding site and the first codon of the LeIF A (=B) gene was isolated from pLeIF A25 and ligated with the rest of the B sequence to construct the expression plasmid of LeIF B. . in this case,
The remainder of the B sequence begins at the second codon of LeIF B. pL4 (LeIF B shown in Figure 2)
Pst (plasmid containing Pst terminal gene)
The prominent restriction maps for the fragment and part of pLeIF A25 are shown in Figures 4 and 5, respectively. To obtain a Sau3a-Pst fragment of approximately 950 b.p. from the Pst fragment using the sequence shown in Figure 4,
Since there is one or more intervening Sau3a restriction sites, the simple method of cutting with Pst and Sau3a is not applicable and requires several steps. In other words, it becomes as follows. The LeIF B expression vector and a method for producing a transformant containing the same are described below. 1 The following fragments were isolated. (a) 110 b.p. from Sau3a to EcoR; (b) 132 b.p. from EcoR to Xba. (c) >700 b.p. from Xba to Pst. Two fragments (1a) and (1b) were ligated. Xba
and cut with Bgl, Sau3a and Xba
Prevented self-polymerization via the terminal (related
The Sau3a site is within the Bgl site; Bgl cleavage left Sau3a sticky ends). A 242 b.p. fragment was isolated. 3. Pst and
Cut with Bgl. An approximately 950 b.p. fragment from Sau3a to Pst (Figure 4) was isolated. This fragment is not common to LeIF A, LeIF B
Contained part of the gene. 4 LeIF A's trp promoter - approximately containing the operator, ribosome binding site, ATG initiation signal and cysteine codon.
300b.p. fragment (from Hind to Sau3a),
It was isolated from pLeIF 25. 5 Approximately 3600 b.p. fragment from Pst to Hind
Isolated from pBR322. replicon and encodes tetracycline resistance, but does not contain ampicillin resistance. 6 Triple ligate the fragments obtained in steps 3, 4, and 5 and use the resulting plasmid to infect E. coli K.
−12 shares converted 294. Convertible strains were screened using mini-screening (Birnboim et al., Nucleic Acids Res. 7 , 1513-1523).
[1979]) and the plasmid samples were digested with EcoR. The digest yielded three fragments, which were characterized by: (1) EcoR-EcoRtrp promoter fragment; (2) internal EcoR-EcoR fragment of pL4; and (3) protein translation initiation signal of pL4.
EcoR fragment. CPE assay (The interferon) is a conventional analytical method based on the cytopathic effects of viruses.
System, Springer-Verlag, Wien/Austria,
1979, pp. 17 and 18), bacterial extracts from clones prepared as described above typically gave an interferon activity of about 10×10 ⁶ units/A ₅₅₀ =1. One representative clone prepared in this way was E.coli294/pLeIF
B trp7. This clone has been deposited at ATCC. Direct expression of further mature leukocyte interferons (LeIF C, D, F, H, I and J) Additional full-length gene fragments containing other LeIF types, similar to those in LeIF A, can be used to tailor expression. can be placed in an expression vehicle for. For the expression of mature LeIF A, an approach as described in Section H above, i.e., removing the leading sequence by restriction cleavage and replacing the N-terminal amino acid codon lost by removing the leading sequence by ligation of a synthetic DNA fragment. Complete sequencing by conventional methods will determine whether a restriction site exists sufficiently close to the first amino acid codon of the mature interferon type to determine whether substitution can be conveniently performed. Probably. 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 area surrounding that point, the double chain
Convert the DNA into single-stranded 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 single-stranded region generated in step (1). Hybridize complementary primers to the stranded DNA. 3. The portion of the second strand removed in step 1 in the 3' direction from the primer is treated in the presence of adenine-, thymine-, guanine-, and cytosine-containing deoxynucleotide triphosphates.
Recover by reaction using DNA polymerase. 4. Digest any remaining single-stranded DNA that protrudes beyond the point to be cut. A short synthetic DNA having a translation initiation signal ATG at the 3' end of the coding strand is then ligated to the resulting gene tailored for the mature interferon, for example by blunt-end ligation,
Then insert this gene into an expression plasmid,
It is under the control of a promoter and its associated ribosome binding site. In the same way as used in the K term above, LeIF
The gene fragments encoding C and LeIF D were
Suitably sequenced for direct bacterial expression. As a strategy for the expression of these additional leukocyte interferons, in each case the trp promoter-operator of LeIF A from pLeIF A25, the ribosome binding site.
A fragment of approximately 300 b.p. (from Hind) containing the ATG initiation signal and cysteine codon.
Sau3a) is used. In contrast, gene fragments from other interferon genes encoding the respective amino acid sequences beyond the first cysteine, which is common to all, are ligated. Using each of the obtained plasmids, E.
294 coli K-12 strains were transformed. The connections to form each gene are as follows. LeIF C pIsolate the following fragments from LeIF C: (a) 35 b.p. from Sau3a to Sau96. (b) >900 b.p. from Sau96 to Pst. (c) As in section K(4) above. pLeIF A-
Fragment of about 300 b.p. from 25 (Hind-Sau3a)
Separate. (d) Separate the approximately 3600 b.p. fragment of section K(5) above. Construction (1) Connect (a) and (c). Cut with Bgl, Hind and isolate a product of approximately 335 b.p. (2) Triple ligate (1) + (b) + (d) and transform E. coli with the resulting plasmid. A representative clone prepared in this way is E.
coli K-12 strain 294/pLeIF ctrp35. This clone has been deposited at ATCC. LeIF D pLeIF Separate from D as follows: (a) 35 b.p. from Sau3a to Ava. (b) 150 b.p. from Ava to Bgl. (c) Approximately 700 b.p.p. from Bgl to Pst. pLeIF A25
Separate the following fragment from: (d) 300 b.p. from Hind to Sau3a. Separate the following fragment from pBR322: (e) Approximately 3600 b.p. from Hind to Pst. Construction (1) (a) + Concatenate (b) and cut with Bgl, 185b.p.
Purify the product (1). (2) Ligate (1) + (d), cleave with Hind, Bgl, and purify the approximately 500 b.p. product (2). (3) Connect (2) + (c) + (e), and the resulting plasmid
Transform E. coli. A representative clone thus prepared is E. coli K-12 strain 294/pLeIF D trp11. This clone has been deposited at ATCC. LeIF F Fragment containing LeIF F contains LeIF B and
Direct expression can be achieved by rearrangement that takes advantage of the complete homology of amino acids 1 to 13 of LeIF F. above
From pHGH207, the trp promoter-containing fragment (a) with suitable sequence ends is obtained via Pst and Xba digestion and then separation of a fragment of approximately 1050 b.p. The second fragment (b) is obtained as the larger of the fragments resulting from Pst and Bgl digestion of plasmid pHKY10. (This Pst
- The Bgl fragment contains part of the ampicillin resistance gene and all of the tetracycline resistance gene except the combined promoter. Therefore, without using plasmid pHKY10, this fragment can also be prepared from other available plasmids, such as plasmid pBR322 (ATCC31344). ) Fragment (a) contains approximately half of the gene encoding ampicillin resistance; fragment (b) contains the remainder of the gene and also contains all of the tetracycline resistance gene except the combined promoter. do. Fragments (a) and (b) are joined by T4 ligase and the product is treated with Xba and Bgl to prevent dimerization and contains the trp promoter-operator and the genes for tetracycline and ampicillin resistance. Let it be fragment (c). Fragment (d) of approximately 580 b.p. is obtained by digestion of pLeIF F with Ava and Bgl. This is LeIF F
contains codons for amino acids 14 to 166. Fragment (e) (49 b.p.) is obtained by Xba and Ava digestion of pLeIF B. Fragment (e) encodes amino acids 1 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 LeIF F gene, was under the control of the trp promoter-operator, allowing for transformation with the desired plasmid. Bacteria can be selected on tetracycline-containing plates. Representative clones prepared in this way are E. coli
K-12 strain 294/pLeIF F trp1. This clone has been deposited at ATCC. LeIF H The complete LeIF H gene can be arranged as follows for expression as a mature leukocyte interferon. 1 Plasmid pLeIF H into Hae and Rsa
Upon digestion, the signal peptide amino acid 10
An 816 b.p. fragment extending from the 3′ non-coding region was isolated. 2 This fragment was denatured and the Klenow fragment of DNA polymerase (Klenow et al., Proc. Natl. Acad. Sci. USA)
Synthetic deoxyribo-oligonucleotide primer 5'-dATG TGT AAT CTG
Subject to repair synthesis using TCT. 3 The obtained product was cleaved with Sau3a, and from 1
A 452 b.p. fragment representing 150 amino acids is isolated. 4 Digest pLeIF H with Sau3a and Pst,
The resulting 500 b.p. fragment is separated to obtain a gene encoding the sequence from the 150th amino acid to the end of the coding sequence. 5 The fragments separated in steps (3) and (4) are ligated to obtain fragment 1 166 met cys asp-stopped ATG TGT...-|...GAT TGA-...-Pst Sau3a. This encodes 166 amino acids of LeIF H. 6 Digest pLeIF A trp25 with Xba,
blunt ends using DNA polymerase I,
Digest the product with Pst. The resulting large fragment can be separated and ligated with the product of step (5) to form an expression plasmid. This is E.coli K
-12 strain 294 or other host bacteria can be used to express mature LeIF H. A human genome phage λCharon4A recombinant library (Cell, 15 , 1157 (1978)) constructed by LeIF I Lawn et al. , Cell 15 , 687
(1978) for leukocyte interferon. cDNA clone
Approximately 500,000 plaques were screened using a radioactive LeIF probe derived from LeIF A. Six LeIF genomic clones were selected.
Following rescreening and plaque purification, one of these clones, λHLeIF2, was selected for further analysis. Using the methods described above, other probes may also be used advantageously to isolate other LeIF clones from the human genome. These can then be used to produce other leukocyte interferon proteins according to the invention. 1. The 2000 b.p. EcoR fragment of clone λHLeIF2 was subcloned into pBR325 at the EcoR site. Obtained plasmid
LeIF I was cut with EcoR and a 2000 b.p. fragment was isolated. Deoxyoligonucleotide dAATTCTGCAG to this 2000b.p.EcoR fragment
(EcoR-Pst converter) was connected.
The resulting product is cleaved with Pst to have a Pst end. It was made into a 2000b.p. fragment. this
Cut with Sau96. A 1100 b.p. fragment with one Pst and one Sau96 end was isolated. 2 Plasmid pLeIF C trp35 was digested with Pst and Xba. Large pieces were separated. 3 Smaller Xba-Pst than pLeIF C trp35
The fragment was digested with Xba and Sau96.
A 40b.p.Xba-Sau96 fragment was isolated. 4. The fragments separated from steps (1), (2) and (3) were ligated to form a pLeIF I trp1 expression plasmid. This clone has been deposited at ATCC. LeIF J 1 Plasmid pLeIF J contains a 3.8 kilobase Hind fragment of human genomic DNA containing the LeIF J gene sequence. A 760 b.p.Dde-Rsa fragment was isolated from this plasmid. 2. Cut plasmid pLeIFB trp7 with Hind and Dde to create 340b.p.Hind-Dde.
The fragments were separated. 3 Cut plasmid pBR322 with Pst,
incubate with DNA polymerase;
(Klenow fragment), then Hind
I digested it. A large (approximately 3600 b.p.) fragment was isolated. 4. The fragments separated in steps (1), (2), and (3) were ligated to form the expression plasmid pLeIF J trp1. This clone has been deposited at ATCC. M Purification The leukocyte interferon content in bacterial extracts can be increased as follows. 1 Polyethylene-imine precipitation. Here, most of the cellular proteins, including interferons, remain in the supernatant. 2 Ammonium sulfate fraction. Here, interferon is precipitated from a solution of 55% saturated ammonium sulfate. 3. Suspend the ammonium sulfate pellet in 0.06M potassium phosphate, 10mM Tris-HCl, PH7.2 and dialyze against 25mM Tris-HCl, PH7.9. Interferon activity remains in solution. 4 Adjust the above supernatant to pH 8.5 and perform chromatography on a DEAE-cellulose column (0 to 0.2M in 25mM Tris-HCl, pH 8.5).
Elute with a linear gradient of NaCl). 5. Adsorb to Cibachrome Blue-Agarose or hydroxylapatite and elute with 1.5M KCl or 0.2M phosphate (conducted as necessary). 6 Size the molecules using a Sephadex G-75 column. 7 Perform cation exchange chromatography on CM-cellulose in 25 mM ammonium acetate at pH 5.0. Develop with an ammonium acetate gradient (up to 0.2M ammonium acetate). The above method 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 can be mixed with Milstin, Scientific
Scientific American 243 , 66
(1980) and eluted with 0.2M acetic acid, 0.1% Triton and 0.15M NaCl. In another advantageous embodiment, the leukocyte interferon produced in the above procedure can be purified in the following step. 1. Grind the frozen cell mass containing the expressed leukocyte interferon manually or with a suitable grinding device. Partially melted cells are heated to PH7.5−
0.1M Tris adjusted to 8.0, 10% (w/v)
Sucrose, 0.2M NaCl, 5mM EDTA,
0.1mM PMSF and 10−100mM _MgCl2
Suspend in 4 volumes of containing buffer A. The suspension is kept at approximately 4°C. The suspension is passed through a homogenizer at about 6000 psi and then a second time at less than 1000 psi.
The effluent from the homogenizer from both passes is cooled in an ice melt. 2. Slowly add polyethylene-imine to the homogenate to make a concentration of about 0.35%, and add about 30%
Leave it for a minute. Solids are removed by centrifugation or filtration. This step is carried out quickly enough to control the temperature or keep the supernatant (filtrate) below 10°C. Concentrate the supernatant (filtrate) by ultrafiltration to approximately 1/10 of the original volume. If particulate matter or cloudiness is observed, it may be removed with a suitable filter, such as a micropore membrane. 3. The clarification solution is flowed directly onto the monoclonal antibody column at 5-8 cm/hour (eg, 25-40 m ^<1> /hour on a 2.6 cm diameter column).
This column was then diluted with approximately 10 column volumes of 20mM.
Wash with Tris-HCl, PH 7.5-8.5 (containing NaCl (0.5M) and Triton X-100 (0.2%) or equivalent surfactant). After washing, the column is passed through approximately 10 column volumes of a solution containing 0.15 M NaCl and Triton X-100 (0.1%) or equivalent surfactant. Use this column as Triton.
Elute with 0.2M acetic acid containing X-100 (0.1%) or equivalent surfactant. Collect the protein peak (measured by UV absorption or other methods) from the monoclonal antibody column and
Adjust the PH to approximately 4.5 with NaOH or 1.0M Tris base. 4. Transfer the pooled interferon peaks to a suitable buffer such as ammonium acetate PH4.5.
Whatman CM52 cellulose or equivalent cation exchanger equilibrated with (50 mM). After loading, wash the column with equilibration buffer and drain the effluent.
The UV absorption is brought to a point where almost no protein is eluted from the column. The column was then eluted with 25mM ammonium acetate/0.12M sodium chloride or a combination that maximized recovery of interferon.
The lyophilized cake has satisfactory appearance and solubility. The monoclonal antibodies in this preferred embodiment described above are described by Staehelin et al. in Proc. Nattle. Acad. Sai. United States (Proc.
Natl.Acad.Sci.USA) 78 , 1848−52 (1981)
It can be prepared by the method described in . Monoclonal antibodies are purified and covalently coupled to Affigel-10 as described below. Preparation and Purification of Monoclonal Antibodies from Ascites Fluid Five female Balb/c mice were each inoculated with 5-10×10 ⁶ hybridoma cells harvested at mid-log phase. Approximately 5× obtained from ascites fluid produced by mice
10 ⁶ viable cells were inoculated intraperitoneally into each of 10 or more mice. This ascites fluid was collected repeatedly (2 to 4 times) from each mouse. 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 (500-1000 x g) for 15 minutes,
Cells and debris were removed from the ascites fluid. Then
Centrifugation was performed at 18,000 rpm for 90 minutes. The supernatant was frozen and stored at -20°C. After melting,
Centrifugation was performed at 35,000 rpm for 90 minutes to further remove fibrin and particulate matter. A batch of ascitic fluid from each transfer was tested in a solid phase antibody binding assay (Staehelin et al., cited above).
Specific antibody activity was tested and pooled if satisfactory. The protein concentration in the pooled solution is 1 mg protein at 280 nm in a 1.0 cm thick cuvette.
It was measured using an approximation method that showed an absorbance value of 1.2.
Ascites fluid with high concentrations of antibodies contains 30-35 mg protein/ml. This corresponds to 4-7 mg specific antibody/ml. This liquid was diluted with PBS (0.01M sodium phosphate, PH7.3, 0.15M NaCl) to a protein concentration of 10 to 12 mg/ml. To each 100 ml of the diluted solution, 90 ml of a saturated ammonium sulfate solution at room temperature was added at 0° C. with vigorous stirring. The suspension was kept on ice for 40 to 60 minutes. Then, it was centrifuged at 4°C and 10,000 rpm for 15 minutes. The supernatant was removed by straining, and the liquid was thoroughly drained. The protein mass was dissolved in 0.02M Tris Hcl (PH
7.9)/0.04M NaCl (buffer).
Add the protein solution to 100 volumes of buffer.
Dialysis was performed for 16 to 18 hours at room temperature. During this time, the buffer was exchanged at least once. dialysis solution
Centrifugation was performed at 15,000 rpm for 10 minutes to remove insoluble matter.
Approximately 30 to 35% of the initial amount of total protein in ascites
was estimated and collected based on the absorbance value at 280 nm. A solution containing 30-40 mg of protein per ml was then applied to a DEAE-cellulose column equilibrated with Buffer. At least 100% per gram of protein added
The column bed volume was ml. 0.02M Tris HClPH
The antibody was eluted from the column by linearly changing the NaCl concentration from 0.04M to 0.5M NaCl in 7.9. Peak fractions from 0.06 to 0.1 M NaCl were pooled, added with an equal volume of saturated ammonium sulfate solution (room temperature), precipitated and centrifuged, and concentrated. Protein mass in 0.2M NaHCO ₃ (PH approx. 8.0) / 0.3M
Dissolved in NaCl (buffer) and dialyzed against the same buffer (3 changes) at room temperature. The dialyzed solution was centrifuged at 20,000 xg for 15 minutes to remove insoluble matter.
Protein concentration was adjusted to 20 to 25 mg/ml with buffer. Preparation of immunoadsorbent Affigel-10 [Bio-Rad
Bio Rad Laboratories, Richmond, California
California) was washed three times with ice-cold isopropanol on a glass filter and then three times with ice-cold distilled water. The gel slurry (approximately 50% in cold water) 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 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 washed twice with buffer (0.1M NaHCO ₃ /0.15M NaCl).
Unbound antibody was removed. When the washing solution was combined and protein was measured, more than 90% of the antibody was bound to the gel. In order to block unreacted areas, the gel was mixed with an equal volume of 0.1 M ethanolamine HCl (PH 8) and treated at room temperature for 60 minutes by rotating the long axis in a circular motion. The gel slurry was washed with PBS to remove the reactants and stored in PBS at 4°C in the presence of 0.02% (w/v) sodium azide. N Parenteral Administration LeIF may be administered parenterally to patients in need of anti-tumor or anti-viral therapy. It may also be administered to patients exhibiting immunosuppressed conditions. The dosage and rate of administration will be similar to that currently practiced clinically for substances of human origin. for example,
For a purity of greater than 1% at about (1-10) x 10 ⁶ units/day, it can be up to, for example, 5 x 10 ⁷ units/day. A suitable dosage form of essentially homogeneous bacterial LeIF in a form for parenteral administration includes, for example:
25 ml of 3 mg of LeIF with a specific activity of 2 x 10 ⁸ units/mg
Dissolved in 5N human serum albumin, pass this solution through a biological filter, and aseptically divide the filtered solution into 100 bottles, each containing 6 x 10 ⁶ units of purity, suitable for parenteral administration. Contains interferon. It is desirable to keep these bottles in a cool place (-20°C) before use. The compounds of the invention can be formulated according to known methods for preparing pharmaceutically useful compositions. The polypeptide is then mixed with a pharmaceutically acceptable carrier vehicle. For suitable vehicles and their formulation, see
As described in Remington's Pharmaceutical Sciences by EW Martin. This is cited as a reference in this specification. In these compositions, an effective amount of interferon protein and a suitable amount of vehicle are combined into a pharmaceutically acceptable composition suitable for effective administration to a host. One advantageous mode of administration is parenteral administration.

[Brief explanation of drawings]

Figure 1 shows a possible LeIF plasmid and ^32p
1 is an autoradiogram showing hybridization with a labeled synthetic deoxyoligonucleotide. Second
The figure shows restriction endonuclease maps of eight types (A to H) of LeIF cloned cDNAs. Figure 3 shows the construction of a gene encoding direct microbial synthesis of mature LeIF A. Figures 4 and 5 show restriction maps of the two gene fragments used to express the LeIF B mature leukocyte interferon.

Claims (1)

[Scope of Claims] 1. A mature human leukocyte interferon that can be obtained by expressing a gene derived from human leukocytes, which has 165-166 amino acids and has a partial amino acid sequence Cys-Ala-Trp-Glu-Val- Val−Arg−
Contains Ala−Glu−Ile−Met−Arg−Ser, and
If the interferon has one of the amino acids Asp, Glu, or Val at position 114, and methionine is attached to the N-terminus of the first amino acid of this interferon, 166
−167 amino acids and amino acid at position 115
A DNA sequence characterized in that it consists of a sequence encoding a mature human leukocyte interferon having one of Asp, Glu or Val. 2. The DNA sequence of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon A (LeIF A). 3. The DNA sequence of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon B (LeIF B). 4. The DNA sequence of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon C (LeIF C). 5. The DNA sequence of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon D (LeIF D). 6. The DNA sequence of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon F (LeIF F). 7. The DNA sequence of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon H (LeIF H). 8. The DNA sequence of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon I (LeIF I). 9. The DNA sequence of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon J (LeIF J).