JPS6361960B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to the field of recombinant DNA technology.
That is, the methods used in recombinant DNA technology and
and the products obtained by those methods. More particularly, the present invention provides polypeptides, specifically
Specifically, mature human leukocyte interferons, those
It relates to a pharmaceutical composition containing. More specifically, the present invention provides human leukocyte-derived
A mature human that can be obtained by expressing the gene
Leukocyte interferon, which contains 165-166 alphas.
It has a amino acid and a partial amino acid sequence Cys-Ala-
Trp−Glu−Val−Val−Arg−Ala−Glu−Ile−
Contains Met-Arg-Ser and amino acid at position 114
This interface has one of Asp, Glu or Val.
Methio at the N-terminus of the first amino acid of feron
If nin is attached, 166−167 amino
acid and the amino acid Asp, Glu or at position 115
Mature human white characterized by having one of the Val.
Blood cell interferon and human leukocyte interferon
Antiviral composition containing Elon A (LeIF A)
relating to things. Identification of mature human leukocyte interferon of the present invention
For example, mature human leukocyte interferon
(LeIF) A, B, C, D, F, H, I and J are
can give. These mature human leukocyte interfaces
The amino acid sequence of Ron is shown in Table 3 on page 24 and Table 5 on page 27.
It is shown. That is, in Table 3
LeIF A, LeIF B, LeIF C, LeIF D, LeIF
F and LeIF H and I and in Table 5
Each amino acid sequence of J is indicated by a number with S.
165 or 166, excluding the signal peptide
Polypeptides consisting of amino acids and these polypeptides
At the N-terminus of the first amino acid of the repeptide
Methionine binds to 166 or 167 amino acids.
The mature human white polypeptide of the present invention
Blood cell interferon A, B, C, D, F, H, I
and J. First, the background of the present invention will be described. human leukemia
The sphere interfaelon (LeIF) was initially created by Isaacs and
and Lindenmann, a very coarse precipitate and
(Proc.R.Soc.
B147, 258-267 [1957]; U.S.P. 3699222). that
Efforts to refine and characterize the
or from white blood cells obtained from a leukemia donor.
Leads to the preparation of relatively homogeneous leukocyte interferon
(German Patent Publication No. 2947134). These in
Tahueron provides viral resistance to their target cells
Known to have the ability to confer sexual states
It is a group of proteins. In addition, interfero
inhibits cell proliferation and modulates immune response
It can act to Due to these characteristics, Wii
Leukocyte interaction for the treatment of infection and malignant tumors.
The clinical use of Feron was promoted. Leukocyte interferon is essentially homogeneous
(Rubinstein et al., Proc. Natl. Acad.
Sci.U.S.A.76, 640-644 [1979]; Zoon et al., ibid.
76, 5601-5605 [1979]) range from about 17,500 to about 21,000
It was reported to have a molecular weight of Preparation of these
The specific activity of the substance is extremely high, 2×10⁸From 1×
Ten⁹unit/mg protein, but from cell culture method
The yield is extremely low. However, the protein sequence
Advances in determination technology have led to the determination of partial amino acid sequences.
[Zoon et al., Science207, 527 [1980];
Levy et al., Proc. Natl. Acad. Sci. U.S.A.77,5102−
5104 [1980]). Various leukocyte interferon groups
Lycosylation is currently not completely clear, but species
Differences in glycosylation between species alone do not explain the observed
cannot explain the distribution of molecular weights. Instead
In general, leukocyte interferon levels vary depending on the type.
There are significant differences in amino acid composition and sequence;
The homology of amino acids is sometimes lower than 80%. 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 et al., Science198, 1056−1063
[1977]; Goeddel et al., Nature281, 544−548
[1979]). More recently, recombinant DNA technology
However, proinsulin and thymosin alpha
1, and in addition, some authors
encodes human leukocyte interferon
Collection of DNA and resulting white blood cells
Regarding the production of proteins with interferon activity
reported (Nagata et al., Nature284,316−
320 [1980]; Nantei et al., GeneTen,1-10
[1980]; Taniguchi et al., Nature285, 547−549
[1980]). The recombinant DNA technology laboratory
present in microorganisms, often in numerous copies within cells
It exists as a Non-chromosomal double-stranded DNA
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
It will be started 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.
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
Some are usually in the process of forming mRNA.
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 this is a very effective way to provide the following. 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 the 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. As a hybrid
The extracted mRNA was eluted and interfaced with oocyte analysis.
Test the translation to Elon. in this way
can induce interferon activity.
Plasmid DNA from the indicated colonies was
Hydraulic acid for the probe manufactured as described in (1) above
Test for brid formation. (3) In parallel with the step (2) above,
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. Estimation of partial length obtained by (1) or (2)
The generated gene fragments are themselves full-length genes.
Can be used as a child 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 properly in the car
It was created using sea urchin, synthesis, and 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 it. 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 microorganisms that lack glycosyl groups.
Physically (e.g., bacterially) produced interfaces
It means Elon. Mature leukocyte interface of the present invention
Elon is the first amino acid codon of natural products.
Immediately from the translation initiation signal (ATG) in front of the
It is expressed in Therefore, this “mature” polypeptide
has methionine as the first amino acid in its sequence.
(encoded by ATG), but essentially
Its properties are not affected by On the other hand, in the microbial host
In this case, the resulting translation product is converted into an aminopeptidase.
Methionine as the first amino acid
may be removed by cleavage. mature leukocyte inter
Ferrons interact with junctional proteins other than the normal leader.
Although expressed together, this conjugate is
or can be specifically removed in the intracellular environment.
(See UK Patent Publication No.2007676A). Finally,
Ripe interferon transports the conjugate to the cell wall.
as a conjugate with a microbial “signal” peptide.
can be produced. Signals are processed in the cell wall
The mature polypeptide is secreted.
“Expression” of mature leukocyte interferon
direct to mRNA translation of leukocyte interferon genome
containing glycosyl groups or preceding sequences, resulting in
Bacteria or other microorganisms that do not have interferon molecules
It means production by living things. 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
Read from 5' (left side) to 3' (right side)
Are listed. and double-stranded (“d.s”) DNA
When referring to the lower or non-coding strand
, the order is reversed. Figure 1 shows the possible
LeIF plasmid and 32p-label synthesis deoxyoli
Autotra showing hybridization with oligonucleotides
It is a geogram. 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 3' untranslated following a termination codon
The area is shown. All LeIF A included
The gene length is shown in the third column A line of Table 2.
, one codon present in other codons
is lacking. 5′ untranslated region becomes leader sequence
It's ahead. Fragment E, which remains separated, has a lead
There is no complete leading sequence of dars. However, temporary
Contains all the genes for mature LeIF E determined.
Ru. Fragment G, which remains separated, has a complete coding sequence.
Not all. In Table 3, 8 nucleotides shown by the nucleotide sequence
We are comparing the LeIF protein sequences of . IUPAC−
LUB Commission on Biochemistry
One letter omitted as proposed by Nomenclature is used.
It is. In other words, alanine is A and cysteine is
is C, aspartic acid is D, and glutamic acid is
E, phenylalanine is F, glycine is G
So, histidine is H, isoleucine is I, and
Gine is K, leucine is L, methionine is M
So, isparagine is N, proline is P, and glu
Tamine is Q, arginine is R, serine is S,
Threonine is T, valine is V, tryptophor
The molecule is represented by W and the tyrosine is represented by Y. number is amino acid
Indicates the location of S represents a signal peptide.
44 in the LeIF A sequence, which is a 165 amino acid sequence.
The next Datsushi uses this LeIF A array as another LeIF.
input to align with the 166 amino acid sequence.
It is. The sequence of LeIF E is of its coding region.
Nucleotides not found in other LeIF sequences (positions in Table 2)
187). Star (Table 3)
is a stop chord that correctly fits into the reading frame of the translation.
Indicates the Amino acids common to all LeIFs
soy genes (excluding LeIF E) are also shown.
Ru. Underlined residues are from human fibroblast protein.
It is an amino acid that is also present in tuffelon. 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 (Goedde I, D.V. et al., Nature287,411
Constructed by -416 [1980]. So cDNA
Inserts can be excised using Pst. each
The terminal line of the cDNA insert is
Polymerized dC:dG tail is shown. Pvu，EcoR
and Bgl restriction sites are also shown. dark area
shows the coding sequence of mature LeIF. The shaded part is
Signal peptide coding sequence. The white part
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 shown. (“Pst” etc.). “b.p.”
indicates a 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
For the two cases shown, the restriction enzyme Sau3a
This is the end of the coding strand produced by digestion with 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, viz.
E. coli x 1776 and E.
coliK-12 294 strains (end A, thi^-,hsr^-＋hsm
k⁺, described in British Patent Publication No. 2055382).
Ru. respectively, the American Type Culture
Deposited in the Collection, ATCC No. 31537 and
31446 (accepted under international deposit under the Budapest Treaty)
Consignment number 31446) is attached. Recombinant DNA fruit
The entire study was conducted by the National Institute of Health.
Performed in accordance with applicable guidelines. A most advantageous embodiment of the invention is defined above.
strain E. coli×1776 and E. coli K-12 strain 294.
as well as other known E. coli strains such as E. coli B
described in relation to E. coli and other microbial strains, including
Many of these are from the American
Like Type Culture Collection (ATCC),
It has been deposited in a known microbial deposit facility and is currently in use.
It's possible. I would also like to see German patent 2644432.
These other microorganisms include Bacillus e.g.
Bacillus Subtilis and other enteric bacteria e.g.
Salmonella typhimurium and Serratia
marcescens and may express foreign gene sequences.
Use a replicable plasmid. With yeast
For example, Saccharomyces cerevisiae is also a yeast protein.
Code the interferon under the control of the lomotor
interferolysis by expressing a gene that
may be advantageously used in the production of protein proteins. B LeIF mRNA raw material and purification LeIF mRNA can be collected from human leukocytes.
Usually, as described in German Patent No. 2947134,
with Sendai virus or Newcastle disease virus
Chronic bone induced to produce interferon
Uses those obtained from leukocytes of leukemia patients.
Ru. Particularly advantageous are the raw materials used in the work herein.
The sample was derived from a patient with acute osteogenic leukemia.
This is a cell line called KG-1. This cellular
In Koeffler, H.P. and Golde, D.W.
Science200, 1153 (1978).
RPMI (Rosewall Park Memorial)
Institute) 1640 plus 10% heat-inactivated FCS (fetal bovine
serum), 25mM HEPES buffer (N-2-hydrochloride), 25mM HEPES buffer
Roxyethyl-piperazine-N'-2-ethane
sulfonic acid) and 50 μg/ml geltamycin.
Grows easily on medium and can be subcultured twice a week
(1 to 3 splits) possible. Growth medium as above
By adding 10% dimethyl sulfoxide to the
Can be frozen. KG-1 is the American
Type Culture Collection (ATCCNo.CRL8031)
It has been deposited in KG-1 cells were prepared using the method described by Rubinstein et al.
(Proc.Natl.Acad.Sci.U.S.A.76, 640-644 [1979]
Causes Sendai or Newcastle disease virus
to produce leukocyte interferon mRNA.
was induced to do so. Cells were harvested 5 hours after induction.
The recovered RNA was treated with guanidine thiocyanate-guar.
Chirgwin hydrochloride method (Chirgwin et al., Biochemistry18，
5294-5299 [1979]. do not induce
RNA from cells was similarly prepared. oligodeo
xythymidine (dT) - cellulose chromatography
using E and sucrose gradient ultracentrifugation.
A 12S fraction of poly(A) mRNA was obtained. Method
Green et al. (Arch.Biochem.Biophys.172,74
−89 [1976]) and Okuyuma et al. (Arch.
Biochem. Biophys.188, 98-104 [1978]) method.
was used. This mRNA is found in Xenopus
oocyte analysis (Cavalieri et al., Proc. Natl. Acad.
Sci.U.S.A.74, 3287-3291 [1977]) and 8000-
10000 units/micrograms of interferonta
Show iter. C Colony bank containing LeIF cDNA sequences
preparation of Using 5 μg of mRNA, standard methods (Wickens et al.
J.Biol.Chem.253, 2483-2495 [1978] and
Goeddel et al., Nature281, 544-548 [1979]
A double-stranded cDNA was prepared. cDNA is 6% poly
By electrophoresis on an acrylamide gel, large
fractionated according to the size of 500 to 1500 b.p.
230 ng of material was obtained by electroelution. This cDNA
100 ng of
(Chang et al., Nature275, 617−624
(1978), deoxyguanosyl at the Pst site.
470ng tailed with dG residues
annealed with plasmid pBR322 of
(Bolivar et al., Gene2, 95-113 [1977]), this
E.coli×1776 was transformed using the vector. cDNAng
About 130 with tetracycline resistance,
A transformed strain sensitive to ampicillin was obtained. In a second similar experiment, for cDNAng,
Approximately 1000 tetracycline resistant and ampicillary
A convertible strain of 294 phosphorus-sensitive E. coli K-12 strains was obtained.
Ta. In this case, a size range of 600 to 1300 b.p.
The cDNA material fractionated by
It was collected and used for tailing with dC. D Preparation of synthetic oligonucleotides and their use
for Some triplets of human leukocyte interferon
The knowledge about the amino acid sequence of syn-degradable fragments is
Complementary synthesis for various regions of LeIF mRNA
Enables the design of synthetic deoxyoligonucleotides
And so. Two tryptic peptides T1 and
I chose T13. The reason is that their amino acid configuration
Columns describe all possible code sequences (Table 1)
Synthesize 12 and 4 undecamers to
This is because all you have to do is do it. For each array
and four sets of deoxyoligonucleotide probes.
synthesized. That is, three each (T-1A, B,
C, D) or one (T-13A, B, C, D)
Contains oligonucleotides. Complementary data shown
Oxyoligonucleotide 11 base chain length is phosphor
Chemically synthesized by the triester method (Crea et al.
Proc.NatlAcad.Sci.U.S.A.75, 5765−5769
[1978]). The T-13 series has four individual
Lobes were prepared. As shown in Table 1, the three
12 T-1 probes as 4 pools of lobes
was prepared. Each of the four probes of the T-13 series
and 4 pools of 3 primers each.
The 12 T-1 probes were combined with a hybridization probe.
Synthesis of radiolabeled single-stranded cDNA for use in
was used to induce The template mRNA is
Ndai-induced KG-1 cells (8000 units IF activity/μg)
12s RNA from or uninduced leukocytes (<10 monomers)
The total poly(A) mRNA derived from
Ru.³²p-labeled cDNA under known reaction conditions.
(Noyes et al., Proc. Natl. Acad. Sci. U.S.A.76，
1770−1774 [1979]), these primers were
Prepared from 20mM Tris-HCI (PH8.3),
20mM KCI, 8mM MgCI₂, 30mM β-merca
Perform the reaction in a volume of 60 μl in ethanol.
Ta. This reaction consisted of 1 μg of each primer (i.e.
For the T-1 series, 12 μg in total, T-
For the 13 series, 4 μg in total), 2 μg of
12S fraction mRNA (or uninduced mRNA)
(A) mRNA (10 μg), 0.5 mM dATP,
dCTP, dTTP, 200 μCi (α³²p)dCTP
(Amersham, 2-3000Ci/m mole) and 60
unit reverse transcriptase (Bethesda Research
Laboratories). The product is 10mlSehadex
Gel filtration on a G-50 column removes unbound
Separate from the removed label and apply 0.3N at 70℃ for 30 minutes.
Treat with NaOH to degrade RNA and neutralize with HCl.
Ta. Hybridization is performed using Nucleic Acid such as Kafatos etc.
Res.7, 1541-1552 (1979).
Ta. E. Identification of clones pL1-pL30 500 E. coli K-12 strain 294 transformed strains (section C
1 μg of plasmid DNA from each of the
Birnboim et al., Nucleic Acids
Res.7, 1513-1523 (1979)
A release operation was used. Each DNA sample is denatured,
Following the method cited above by Kafatos et al.
It was placed on a trocellulose filter. Nitrocell containing 500 plasmid samples
The three sets of loin filters are a Primed with the T-1 set of primers.
derived cDNA, b T-13 primed induced cDNA and c prepared using both sets of primers.
hybridized with uninduced cDNA. Non-invitation
than for the entirety of the probe. for one or both of the cDNA probes.
If it hybridizes strongly, the clone is positive.
Thought. 30 “positive” clones out of 500
(pL1−pL30) was selected for further analysis. F. Ku
Identification of the loan pL31-pL39, containing the LeIF gene fragment.
Isolation of plasmid (No. 104) containing KG-1 cells were infected with Sendai virus or
Leukocyte interface induced by Katusuru disease virus
lon mRNA is produced, the same cells are collected,
Prepare mRNA and label it using standard methods.
³²p label induced mRNA was obtained. as a probe
³²p-label induced mRNA (Lillenhaug et al.
Biochemistry,15, 1858-1865 [1976])
Grunstein and Hogness colony hives
Lydization method (Proc.Natl.Acad.Sci.U.S.A.72,3961
−3965 [1975]) transformed strain of E. coli×1776
was screened. Non-labeled from non-induced cells
Le mRNA³²present in p-label preparations
probe to compete with uninduced mRNA.
It was mixed at a ratio of 200:1. labeled
Hybridization of mRNA includes the derived sequence.
This should occur selectively in colonies that have this. three
A group of transformed strains were obtained. (1) 2 to 3% of the colonies³²p-mRNA
Always strongly hybridized. (2) 10% have a higher degree of hybridization than group (1) above.
was significantly lower. (3) The remainder is a detectable hybridization pattern.
He showed me Gunar. Regarding positive colonies (groups (1) and (2))
The interferon mRNA is plasmid DNA
Analysis by specifically hybridizing to
We investigated the existence of interferon-specific sequences.
Beta. First, tetracycline (20 μg/ml),
Aminopimelic acid (100μg/ml), thymidine
(20 μg/ml) and d-biotin (1 μg/ml).
60 strongly positive colonies in 100ml of added M9 medium
(Group 1) were grown individually. M9
For 1 liter of medium, Na₂HPO_Four(6
g), KH₂P.O._Four(3g), NaCl (0.5g) and
N.H._FourContaining Cl (1 g), autoclaved
and sterile 1M MgSO_Four1ml and sterile 0.01M
CaCl₂Add 10ml of 10 cultures pool
Clewell et al., Biochemistry9,4428−440
[1970] As described, more than 6 pools
DNA was isolated. Each plasmid DNA pool
10 μg of Hindlll-cleaved, denatured DBM
(diazobenzyloxymethyl) covalently bonded to paper.
1 μg of purified mRNA from induced cells each
hybridized to filter paper. not hybrid
mRNA was removed by washing. specifically hybrid
The extracted mRNA was eluted and extracted from Xenopus laevis.
was translated in oocytes. In this analysis, six
All pools tested negative. weakly positive colonies
5 pools of 10 colonies each from 59 (2nd group)
Prepare one pool of 9 colonies with
Prepare the plasmid from the sample and test as above.
Ta. One of the six pools tested (K10)
The background level is significantly improved each time the exam is taken.
interferon mRNA at levels that exceed
Hybridized. Specific interferon cDNA
9 columns of pool K10 to identify clones.
Prepare plasmid DNA from each knee and test individually.
Beta. Two of the nine plasmids (No.101 and
and No. 104) are interferon mRNA and
bound well above background level. P
Cut lasmid No. 104 with Bgl restriction enzyme to obtain
The 260 b.p. fragment was separated by gel electrophoresis.
A Bgl restriction fragment containing 260 b.p. was obtained. this,
Taylor et al., Biochim. Biophys. Acta442,324−
330 (1976)³²Label with p and place
Colony screening method (Grunstein and
and Hogness, Proc. Natl. Sci. U.S.A.72,3961−
3965 [1975]), 400 E. coli 294 convertible stocks,
Used for probes for individual screening
there was. Hybridized to varying degrees with this probe
Nine colonies (pL31-pL39) were identified.
Ta. Furthermore, using a labeled 260 b.p.
In the same way, 4000 E.coli294 convertible stocks were individually
was screened. This probe and species
Fifty colonies that hybridized at each time were identified.
One is LeIF G fragment, one is LeIF H
One of the fragments is called LeIF Hl (apparently
Top, allele of LeIF H).
The resulting hybrid plasmid is “pLeIF H” etc.
It was called. G. Isolation and sequence of the first full-length LeIF gene
decision In total, 39 possible LeIF c DNA clusters
Plasmid DNA was prepared from the loan. and,
The hybridization procedure of Kafatos et al. (cited above)
and rescreened using the same 260 b.p. DNA probe.
I did a ning. This probe and three plasmids
(PL4, pL31, pL34) is a very strong hybrid
give a signal, and 4 (pL13, pL30.pL32,
pL36) was moderately hybridized, and three (pL6,
pL8, pL14) were weakly hybridized. 39 possible LeIF cDNA recombination plus
Mido also³²p-label synthetic undecamer
T-1 primer each consisting of 3 types of primers
each of the pools or that of the T-13 primer.
each) directly as a hybridization probe.
searched. detectable hybridization signal
It seems that a complete base-base pair is required to give
We selected the hybridization conditions (Wallace et al.
Nucleic Acid Res.6, 3543–3557 [1979]). Two
plasmid DNA from 39 clones.
Standard supernatant method (interface cells with plasmids)
After lysing the bacteria using an activator, etc., and centrifuging,
Collect the supernatant containing Mido, and use the plastic from this supernatant.
This is a method for isolating Sumid. Isolation from supernatant
The ethanol precipitation method is used. )(cleared
lysate procedure, Clewell, etc.),
Biorad Agarose A-50 (gel manufactured by Bio-Rad)
Agarose gel for filtration) Column chromatography
It was purified by A sample (3 μg) of each preparation was
Treated with EcoR to open the ring, denatured with alkali,
Spoon onto two separate nitrocellulose filters.
I got it. 1.5 μg per spot. Up
See the cited reference by Kafatos et al. synthetic deoxy
Oligonucleotide T-13 primer and T-
1 primer pool, each of (γ³²P) ATP
It was phosphorylated as follows. 50pmole oligo
Nucleotides and 100pmole (γ³²P) ATP
(New England Nuclear, 2500Ci/m mole)
, 50mM Tris-HCl, 10mM MgCl₂and
of a mixture of 15mM β-mercaptoethanol
Combined in 30 μl. 2 units of T4 polynucleotide
Add dokinase and after 30 min at 37°C.³²p−label
Primer is 10ml Sephadex On G-50 column
It was purified by chromatography. Ten⁶cpm
Primer T-13C or 3x16⁶cpm primer
-Pool T-1C was used for hybridization. C
Hybridization follows the method cited above by Wallace et al.
Then, 6×SSC [1×SSC=0.15 MNaCl,
0.015M Sodium Citrate, PH7.2), 10x
Denhardts [0.2% bovine serum albumin, 0.2% polyvinyl
Nilpyrrolidone, 0.2% Ficoll] solution at 15 °C.
Hybridized for 14 hours. Filter is 6xSSC
Washed three times for 5 minutes at medium 0°C. Dry and X-ray
exposed to ilm. The results are shown in Figure 1.³²p-pu
Limer T-13C and primer pool T-1C
The case of . Plasmid DNA from clone 104 was
Markpool T-1C and primer T-13C
hybridized to the author. However, other undecomas
- indicates no detectable hybridization
Ivy. As shown in Figure 1, there are 39 possible
Some of the LeIF plasmids (pL2, 4,
13, 17, 20, 30, 31, 34) of these probes.
Hybridized with both. However, restriction analysis?
et al., only one of these plasmids,
pL31 also contained a 260 b.p. internal Bgl fragment.
Digestion of pL31 with Pst resulted in cDNA insert
The size was approximately 1000 b.p. The entire Pst insert of pL31 was sequenced using Maxam
-Gilbert Chemical Methods (Methods Enzymol.65,499−
560 [1980]) and dideoxy chain termination method
(Smith, Methods Enzymol.65,560−580
[1980]). At that time, the Sau3 a fragment was
13 vector.
The DNA sequence is shown in Table 2, "A". Obtained
Information about LeIF protein sequences, known LeIF components
range of quantities, stopping points in three possible reading frames.
A more appropriate translation reading frame than the relative existence of replets
was suggested. Then the play or signal
The entire LeIF amino acid sequence, including the peptide, is shown.
Ta. The first ATG translation initiation codon is at the 5' end of the sequence.
Located at the 60th nucleotide from the end, 188 cods
After the turn, there is a TGA termination triplet.
There are 342 untranslated nucleotides at the 3′ end;
This is followed by a poly(A) array. Assumed signal
peptide (probably more mature LeIF than leukocytes)
(which probably participates in secretion) is 23 amino acids long.
Ru. The molecular weight of the 165 amino acids that constitute mature LeIF
The calculated value is 19,390. We have this pL31
The LeIF encoded by LeIF was designated "LeIF A".
From the sequence data (“A”) in Table 3, tryptic peptide
T1 and T13 are 146-150 and 58 of LeIF A
It can be seen that each corresponds to −62. These two
The actual DNA coding sequences found in the two regions are
Primer pool T1-C and primer T13
-C (see Table 4). H Direct release of mature leukocyte interferon A (LeIFA)
contact expression 1 General description LeIF A as mature interferon polypeptide
The procedure for direct expression is synthetic (N-
end) and complementary DNA combinations
The method used for human growth hormone in
etc., Nature281, 544-548 [1979].
Ru. As shown in Figure 3, the Sau 3a restriction endonucleus
The lyase site is conveniently located at the codome of LeIF A.
It exists between lines 1 and 3. ATG translation
Contains the start codon and amino acid 1 (cysteine)
Repair the codon for EcoR and create a new EcoR sticky end.
Two synthetic deoxygenates, including the production of
designed thioligonucleotides. these
The oligomer is 34b.p.Sau 3a−Ava of pL31
The fragments were concatenated. The resulting 45 b.p. product has two additional
865 b.p. synthesis - natural miscellaneous
It was used as a seed gene. This codes LeIF A,
Flanked by EcoR and Pst restriction sites
There is. This gene contains EcoR and Pst sites
Insert into PBR322 and insert the plasmid between
De-PLeIF Al was used. 2 Tryptophan regulatory element (E.coli trp promoter)
operator, operator and trp reader revolver
Contains the some binding site but for translation initiation
Construction of (lacking ATG sequence) Plasmid pGMI (ATCC 31622) deleted
E. coli tryptophan op containing ΔLE 1413
(Miozzari et al., J.Bacteriology133，
1457-1466 [1978]). Therefore, the TRP leader
the first six amino acids and the TRP E polypeptide
Approximately the last 1/3 of the
) as well as the entire trp D polypeptide.
Express the fusion protein. This is all trp pro
Under the control of the motor-operator system
Ru. Readily available alternative to plasmid PGM1
Other equivalent trp promoters (e.g.
Bennett, J. Mol. Biol. (1978) 121, 113-137.
(See Fig. 10 on page 135) may also be used. This plastic
20 μg of Sumid was digested with restriction enzyme Pvu. this
cuts the plasmid at five sites.
This gene fragment is then attached to the EcoR linker.
(self-complementary oligonucleotide of pCATGAATTCATG)
formed from cleotide), and later,
Clone into a plasmid containing an EcoR site
EcoR cleavage site for Obtained from pGM1
20 μg of DNA fragments were added to 200 Pmol of 5′-phosphate.
synthetic oligonucleotide pCATGAATCATG
In the presence of 20μlT_FourDNA ligase buffer (20mM
Tris PH7.6, 0.5mM ATP, 10mM MgCl₂，
10 units T in 5mM dithiothreitol)_FourDNA
Treated with ligase overnight at 4°C. The solution is then heated to 70℃
The mixture was heated for 10 minutes to stop the ligation. The linker is
Fragment cut with EcoR digestion and having EcoR ends
5% polyacrylamide gel electrophoresis
(hereinafter referred to as PAGE), and the larger
The three fragments were first stained with ethidium bromide.
Color and position the fragments with UV light and target them from the gel.
It was separated by cutting out the part. Each gel cut
Place the pieces (300 μl 0.1 x TBE) in a dialysis bag and
0.1× TBE buffer (TBE buffer is 10.8 g
Tris base, 5.5g boric acid, 0.09g Na₂EDTA
1 liter of H₂100V electricity for 1 hour in (contained in O)
It migrated. Collect the aqueous solution from the dialysis bag and
Nol extraction, chloroform extraction, 0.2M sodium chloride
DNA in water after ethanol precipitation.
was collected. trp promo with EcoR sticky ends
The operator-operator-containing genes are listed below.
It was identified by the following procedure. In other words, tetracycline
Insert the fragment into a susceptible plasmid, which
By inserting a motor-operator, the tetracycle
It was made phosphorus resistant. Plasmid pBRHI (Rodriguez et al., Nucleic
Acids Res.6, 3267-3287 [1979])
develops resistance to cillin and tetracycline
Contains resistance genes. But combined with that
Since there is no promoted promoter, its resistance
Does not express sex. This plasmid is therefore tetra
Cyclin sensitive. This EcoR site
Introducing a motor-operator system
makes the plasmid tetracycline resistant
It can be done. pBRHI was digested with EcoR, and the enzyme was phenol.
Extraction Removed with chloroform extraction and ethanol precipitation
Let it go into the water. present in separate reaction mixtures
The generated DNA molecules are the three DNA molecules obtained above.
merge with each of the fragments, and as above
To, T_FourIt was ligated with DNA ligase. reaction mixture
Standard methods using DNA present in
(Hershfield et al., Proc. Natl. Acad. Sci. U.S.A.71，
3455-3459 [1974]) showed that the DNA uptake ability was
This bacterium was transformed into E. coli K-12 strain 294.
with 20 μg/ml ampicillin and 5 μg/ml te
LB containing tracycline (Luria-Bertani)
plate was inoculated. some tetracyclides
Select resistant colonies and isolate plasmid DNA.
and confirm the presence of the desired fragment by restriction enzyme analysis.
I chewed it. The resulting plasmid is called pBRH trp.
Ru. EcoR and hepatitis B viral genomes
BamH digestion products were collected in a conventional manner and
EcoR and Sumid pGH6 (ATCC 31539)
cloned into the BamH site (this plasmid
pGH6 was described in Nature by Goeddel et al.281, 544 (1979)
from plasmid pKB268 as shown in
The isolated 285 base pair EcoR fragment (the fragment is
Two 95 base pair heterologous DNA fragments
UV-5 Consists of lac promoter and how to construct it
Law Johnsrud Proc.Natl.Acad.Sci.USA,75
(11) 5314-5318 (1978)).
Inserting into the EcoR site of lasmid pBR322
Constructed by In addition, UV-5 lac promo
The nucleotide sequence of the data was published in Science by Dickson et al.187,27
−35 (1975), Simpson et al.18, 277-285
(1979), Siebenlist et al.20, 269-281
(1980), etc., and is publicly known. ],
Lasmid pHS32. Plasmid pHS32
Cleavage with Xba, phenol extraction, and chlorophor
rum was extracted and ethanol precipitated. The precipitation is
Contains 0.1mM dTTP and 0.1mM dCTP
30μl polymerase buffer (50mM potassium phosphate
PH7.4, 7mM MgCl₂, 1mM β-mercaptoeta
1 μl E.coli DNA polymerase,
Klenow fragment (Boehringer-Mannheim), 0
℃ for 30 minutes and then at 37℃ for 2 hours. This place
4 complementary to the 5′ overhang of the Xba cleavage site.
2 out of 3 nucleotides are filled. 5′CTAGA− 5′CTAGA− 3'T- TCT- Two nucleotides dC and dT are incorporated
give an end with two overhanging 5' nucleotides.
I can do it. Plasmid pHS32 (phenol and chloride)
After roform extraction, precipitate with ethanol and take in water)
This linear residue of was cleaved with EcoR. Large size
PAGE the lasmid fragment into a smaller EcoR
−Xba fragments and separated by electroelution.
Ta. This DNA fragment from pHS32 (0.2 μg)
Tripp derived from pBRH trp was added under conditions similar to those described above.
EcoR-Taq of Tojuan operon (approximately 0.01μg)
I fragment. The above pHS32 fragment was converted into an EcoR-Taq fragment.
In this way, the complete Watson-
Although it is not a click base pair, the Taq
Connect the emerging end to the remaining overhanging end of Xba
do. -T −AGC+CTAGA− TCT−→−TCTAGA −AGCTCT− − A portion of this ligation mixture was added to E.coli294 cells.
used for conversion, heat treatment, and ampicillin-containing LB plastic.
It was applied during the rate. Select 24 colonies,
Grow in 3 ml LB (Luria-Bertani) and plate
Sumid was isolated. 6 of these colonies
due to DNA repair and replication by E. coli.
It was found to have a regenerated Xba site. −TCTAGA −AGCTCT− −→−TCTAGA −AGATCT− − These plasmids contain EcoR and Hpa
to give the expected restriction fragment.
I also found out that it can be done. One protein, called pTrp14,
lasmids, as discussed next,
It was used to express peptides. Plasmid pHGH107 (ATCC 31538) (this plasmid
Lasmid pHGH107 was published in Nature by Goeddel et al.
281, 544 (1979) (for example, the method described in the same document)
(see Fig. 4), the DNA encoding human growth hormone is located downstream of the lac promoter of pGH6 mentioned above.
Constructed by inserting fragments. ] 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 messager RNA. Contains. This gene lacks the codons of the human growth hormone "preceding" sequence, but contains the ATG translation initiation codon. This gene was extracted from 10μpHGH107 with EcoR treatment followed by E. coli DNA polymerase as described above.
Klenow fragment and isolated via dTTP and dATP treatment. Plasmids were extracted with phenol and chloroform and then precipitated with ethanol.
Treated with BamH. 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 when subsequently cloned into an expression plasmid, it contains the insert. plasmids can be identified by the restoration of tetracycline resistance. The EcoR ends of the fragment have been filled in using the Klenow polymerase method, so the fragment has only one blunt end.
end) and one sticky end. This ensures correct orientation when later inserted into an expression plasmid. The expression plasmid pTrp14 was then adjusted to accept the HGH gene-containing fragment prepared above. Briefly, pTrp14 was digested with Xba and the resulting sticky ends were filled in using the Klenow polymerase method using dATP, dTTP, dGTP and dCTP. After phenol and chloroform extraction and ethanol precipitation, the resulting DNA is
The resulting large plasmid fragment was
Separated by PAGE and electroelution. The pTrp14-derived fragment had one blunt end and one sticky end, allowing it to recombine in the correct orientation with the HGH gene-containing fragment described above. HGH gene fragment and pTrp14ΔXba−Bam H
The fragments were merged and interconnected under conditions similar to those described above. The filled Xba and EcoR ends are blunt-ended ligated to the Xba site and
Both EcoR sites were reconstituted. [Table] This construction also creates a tetracycline resistance gene. Plasmid pHGH107 HGH
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 (lac promoter) located upstream of the gene. E. coli294 was then transformed with the ligation mixture and transformed into LB containing 5 μg/ml tetracycline.
Colonies were selected on the plate. Plasmid pHGH207 is EcoR-digested and contains the trp promoter-operator and trp leader ribosome binding site but not for translation initiation.
A 300b.P. fragment lacking the ATG sequence was collected by PAGE followed by electroelution. This DNA fragment
It was cloned into the EcoR site of pLeIFA. An expression plasmid containing the modified trp regulon described above (an E. coli trp operon in which the attenuator sequence has been deleted for regulation to increase expression levels) is prepared with a sufficient amount of trp regulon to suppress the promoter-operator system. They can be grown to predetermined levels on tryptophan-supplemented media. The desired product can then be expressed by removing the tryptophan and deinhibiting the system. To be more specific, with reference to Figure 3,
250 μg of plasmid pL31 was digested 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 A 16 μg sample of this fragment was partially digested with 40 units of Bgl for 45 min at 37°C and the reaction mixture was purified on a 6% polyacrylamide gel. Approximately 2 μg of the desired 670b.P. fragment was collected. b1000b.p, another sample of Pst insert (8μ
g) was digested with Ava and Bgl. After gel electrophoresis, 1 μg of the 150 b.p. fragment described above was obtained. 16μg of c1000b.p. fragment was added to Sau3a and Ava.
Processed with. After electrophoresis on a 10% polyacrylamide gel, approximately 0.25 μg (10 pmole) of 34b.
p. fragment was obtained. The two deoxyoligonucleotides shown, 5'-dAATTCATGTGT (fragment 1) and 5'-dGATCACACATG (fragment 2), were synthesized by the phosphotriester method. Fragment 2 was phosphorylated as follows. 200μl (approximately 40pmole) of (γ
^32P ) ATP (Amersham, 5000Ci/mmole) was dried, 60mM Tris-HCl (PH8), 10mM
100 pmol of DNA fragment and 2 units of T4 from MgCl ₂ , 15mM β-mercaptoethanol
Resuspend in 30 μl of polynucleotide kinase containing solution. After 15 minutes at 37°C, 1 μl of 10 mM ATP was added and the reaction was allowed to proceed for an additional 15 minutes. The mixture was then heated at 70°C for 15 minutes and 100 pmol of the 5'-OH fragment 1
and merged with 10pmole of 34b.p.Sau 3a Ava fragment. 20mM Tris-HCl (PH7.5),
_10mMgCl2 , 10mM dithiothreitol,
0.5mMATP and 10 units of T4 DNA ligase
Ligation was carried out in 50 μl for 5 hours at 4°C. The mixture is 6%
Electrophoresed on polyacrylamide gel, 45 b.p.
The product was collected by electroelution. 45b.p. product
30 ng (1 pmole), 0.5 μg (5 pmole) of the 150 b.p.Ava −Bgl fragment and 670 b.p.Bgl −
It was merged with 1 μg (2 pmole) of Pst fragment. 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 was digested with EcoR and Pst to remove gene polymers. The mixture is 6% PAGE
It was purified with Approximately 20 ng (0.04 pmole) of 865 b.p. product was isolated. Half of this (10ng) was added to pBR322
(0.3 μg) was inserted between the EcoR and Pst sites. Transformation of E. coli294 resulted in 70 tetracycline-resistant, ampicillin-sensitive transformants. Plasmid DNA was isolated from 18 of these and digested with EcoR and Pst. Of the 18 plasmids, 16 were 865b.
p. length EcoR-Pst fragment. One of these, 1 μg of PLeIF Al, was digested with EcoR, containing the E. coli trp promoter and trp leader ribosome binding site, as prepared above.
It was ligated to a 300 b.p. EcoR fragment (0.1 μg). trp
Transformants containing promoters are Grunstein-
Identification was performed using a ³² P-labeled DNA fragment containing the trp promoter sequence as a probe in combination with the Hogness colony screening method.
The asymmetrically located Xba site in the trp fragment allowed the identification of recombinant products in which the trp promoter was oriented in the direction of the LeIF A gene. In vitro and in vivo activity of LeIF A Extracts for IF analysis were prepared as follows. 1 ml of the culture was grown in L broth containing 5 μg/ml tetracycline to give an A ₅₅₀ value (absorbance at a wavelength of 550 mm) of approximately 1.0, and then
M9 medium containing 5 μg/ml tetracycline
Diluted in 25ml. 10ml when A ₅₅₀ reaches 1.0
A sample was collected by centrifugation, and the cell mass was suspended in 1 ml of 15% sucrose, 50mM Tris-HCl (PH8.0), and 50mM EDTA. After adding 1 mg of lysozyme and incubating at 0°C for 5 minutes, 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. A LeIF specific activity of 4×10 ⁸ units/mg was used to measure the number of IF molecules per cell. 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/l). As shown in Table 7, the IF produced by E. coli K-12 strain 294/pLeIF A trp25 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). The in vivo effects of interferons require macrophages and natural killer (NK) cells. And in vivo effects appear to involve stimulation of these cells. So, E.
Interferon produced by coli294/pLeIF A25 has antiviral activity in cell culture assays, but may be ineffective in infected animals. Furthermore, the in vivo antiviral activity of non-glycosylated LeIF A produced by bacteria is
It may be different from glycosylated LeIF, which is derived from human "buffy coat" leukocytes. So LeIF synthesized by bacteria
The biological activity of Buffy Coat LeIF (8% purity) was demonstrated in a lethal dose of encephalomyocarditis virus (EMC) infection of squirrel monkeys.
% purity) (Table 8). [Table] [Table] *250,000 units per ml of E.coli294/pLeIF A trp25 extract listed in Table 6 in the minimum essential medium.
It was diluted 500 times to give a specific activity of 500 units/ml. The leukocyte interferon standard (Wadley Institute) was previously titered against the NIH leukocyte interferon standard, also diluted to a final concentration of 500 U/ml. Adjust 1ml to PH2 with 1NHCl,
Incubate for 52 hours at 4°C, neutralize by addition of NaOH, and measure IF activity using a standard CPE inhibition assay.
A 25 μl aliquot of the 500 units/ml sample (untreated) was incubated with 25 μl of rabbit anti-human leukocyte interferon for 60 minutes at 37° C., centrifuged at 12,000×g for 5 minutes, and the supernatant was analyzed. [Table] All monkeys were male (average weight 713 g) and did not have EMC virus antibodies before infection. For the monkey,
100×LD ₅₀ EMC virus (measured in mice) was infected intramuscularly. Control infected monkeys were infected at 134 p.i.
He died at 158 and 164 hours. 10 ^6- unit interferon treatment was performed 4 hours before infection;
It was administered intravenously at 2, 23, 29, 48, 72, 168, and 240 hours after infection. Bacterial leukocyte interferon was column chromatographically fractionated from the lysate of E.coli294/pLeIF A25 and had a specific activity of 7.4×10 ⁶ units/mg protein. The control bacterial protein was E.coli294/
In the corresponding column fraction from pBR322 melt, 2
The total protein amount was doubled. Leukocyte interferon standard is chromatographically 32 x ¹⁰⁶ units/mg
It was a Sendai virus-induced interferon from normal human "buffy coat" cells purified to specific protein activity. 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 monkey in the control group, which did not show viremia until day 4, died late (due to infection).
(164 hours later), 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, the Grunstein method described above
Another colony bank of transformants containing the leukocyte interferon cDNA obtained in Section C above was screened using E. coli 294 as the host microorganism using the in situ colony screening method of Hogness and Hogness. As a result, colonies that hybridized to the probe to various degrees were isolated. Plasmid DNA from these colonies and from the 10 hybridized colonies shown in Section G above were transformed into Pst
It was separated by cutting and characterized in three ways. First, these Pst fragments were characterized by restriction endonuclease digestion patterns using the enzymes Bgl, Pvu and EcoR. Analysis of Pst fragments obtained from the colonies described above revealed that at least eight different types (LeIF A, LeIF B,
LeIF C, LeIF D, LeIF E, LeIF F, LeIF
G and LeIF H). This indicates the approximate location of the various restriction breakpoints with respect to the previously known leading and coding sequences of LeIF A. One of these, LeIF D, was reported by Nagata et al. in Nature 284 , 316.
This is believed to be the same as reported in -320 (1980). Second, some of these DNAs were extracted from polyA-containing KG-1 cell RNA by LeIF
Regarding the ability to selectively remove mRNA,
The hybridization selection assay described by Cleveland et al., Cell 20 , 95-105 (1980) was used. LeIF
Pst fragments A, B, C and F were positive in this assay. Third, these Pst fragments were inserted into expression plasmids and E. coli 294 was transformed with the plasmids to express the fragments. All expression products believed to be preintaferon were positive in the CPE assay for intertaferon activity, except for the LeIF F fragment, which was marginally 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, trp−
A fragment containing the promoter-operator, ribosome binding site and the first codon of the LeIF A (=B) gene was isolated from pLeIF A25, and LeIF B
was combined with the remainder of the B sequence to construct an expression plasmid. In this case, the remaining part of the B sequence starts from the second codon of LeIF B.
Pst fragment of pL4 (plasmid containing the LeIF B Pst terminal gene shown in Figure 2) and pLeIF
Significant restriction maps for parts of the A25 are shown in Figures 4 and 5 respectively. To obtain a Sau 3 a-Pst fragment of approximately 950 b.p. for the Pst fragment with the sequence of FIG. 4, one or more intervening Sau 3
Since the a restriction site is present, simply Pst and Sau
The method of cutting using 3a cannot be applied 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 Sau3 a to EcoR; b 132 b.p. from EcoR to Xba. c >700 b.p. from Xba to Pst. 2 Fragments 1a and 1b were ligated to create Xba and
Cleavage with Bgl prevented self-polymerization via the Sau3a and Xba termini (associated Sau
3 The a site is within the Bgl site; Bgl
Cleavage left Sau3a sticky ends).
A 242 b.p. fragment was isolated. 3 To link the products of 2 and 1c and prevent self-polymerization again, Pst and Bgl
I cut it with. An approximately 950 b.p. fragment from Sau3a to Pst (Figure 4) was isolated. This fragment contained a portion of the LeIF B gene that is not common to LeIF A. 4 LeIF A trp promoter-operator,
Contains a ribosome binding site, an ATG initiation signal and a cysteine codon. An approximately 300 b.p. fragment (Hind to Sau3a) was isolated from pLeIF25. 5. An approximately 3600 b.p. fragment from Pst to Hind was isolated from pBR322. replicon and tetracycline resistance encoded;
Does not contain ampicillin resistance. 6. Triple ligate the fragments obtained in steps 3, 4, and 5 and incubate E. coli K-12 with the resulting plasmid.
294 shares were converted. Convertible stocks are screened by mini-screening (Birnboim et al.
Nucleic Acids Rcs. 7 , 1513-1523 [1979]) and the plasmid samples were digested with EcoR. The digest gave three fragments, their characteristics were as follows:
1 EcoR-EcoR trp promoter fragment; 2) internal EcoR-EcoR fragment of pL4; and 3) protein translation initiation signal of pL4-EcoR
piece. CPE analysis (The Interferon System, a conventional analytical method based on the cytopathic effects of viruses)
Springer-Verlag, Wien/Austria, 1979,
(see pp. 17 and 18), bacterial extracts from clones prepared as described above typically have A ₅₅₀ =1
gave an interferon activity of approximately 10×10 ⁶ units/l. One representative clone thus prepared is 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. mature
For the expression of 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 a convenient substitution can be made. Dew. If that doesn't work, you can use the following operation. In short, the first stage of the mature polypeptide
The fragment containing the preceding sequence is cut exactly before the start of the codon for the amino acid. In other words, 1 double strands within the area surrounding that point.
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, and convert the single-stranded DNA into single-stranded DNA. hybridize complementary primers. 3 Add adenine, thymine,
Reconstitution is achieved by a reaction using DNA polymerase in the presence of guanine and cytosine containing deoxynucleotide triphosphates. 4 Projects beyond the point to be cut,
Digest the remaining single-stranded DNA. A short synthetic DNA carrying the 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, and this gene is It is inserted into an expression plasmid and placed under the control of a promoter and associated ribosome binding site. In the same way as used in the K term above, LeIF C
and LeIF D encoding gene fragments were suitably sequenced for direct bacterial expression. In each case, pLeIF as a strategy for the expression of these additional leukocyte interferons.
Approximately 300 b.p. containing the LeIF A trp promoter-operator, ribosome binding site, ATG initiation signal and cysteine codon from A25.
fragments (from Hind to Sau 3a) are 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. E. coli K-12 strain 294 was transformed using each of the obtained plasmids.
The connections to form each gene are as follows. LeIF C pIsolate the following fragments from LeIF C: (a) 35 b.p. from Sau 3 a to Sau 96. (b) >900 b.p. from Sau 96 to Pst. (c) As in section K4 above. , a fragment of about 300 b.p. (Hind-San 3a) is isolated from pLeIF A-25. (d) Isolate the approximately 3600 b.p. fragment of section K5 above. Construction (1) Connect (a) and (c). Cut with Bgl and Hind to separate 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 From D, the following is separated: a Sau 3 35b.p from a to Ava. b 150b.p from Ava to Bgl. c About 700b.p from Bgl to Pst. pLeIF From A25, the following Separate the fragment: d From Hind to Sau 3 a 300 b.p. Separate the following fragment from pBR322: e From Hind to Pst about 600 b.p. Construction (1) Connect (a) + (b). , cut at Bgl, 185b.p.
Purify product 1. (2) Ligate (1)+(d), cleave with Hind, Bgl, and purify approximately 500 b.p. product 2. (3) Connect (2) + (c) + (e) and use the resulting plasmid to create E.
convert coli. Representative clones prepared in this way are
E. coli K-12 strain 294/pLeIF D trp 11. 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 fact that amino acids 1 to 13 of LeIF F are completely homologous. From the above pHGH207, the trp promoter-containing fragment a with appropriate sequence ends was obtained via Pst and Xba digestion and then isolating a fragment of approximately 1050 b.p.
get it. The second fragment b is the plasmid pHKY10
The larger of the fragments resulting from Pst and Bgl digestion of (This Pst-Bgl fragment contains part of the ampicillin resistance gene and all of the tetracycline resistance gene except the combined promoter. Therefore, the plasmid
Even if pHKY10 is not used, other available plasmids, such as plasmid pBR322
This fragment can also be prepared from (ATCC 31344). ) 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 resistance gene except the associated promoter. Fragments a and b are joined by T4 ligase, converting the products into Xba and Bgl
to prevent dimerization,
Fragment c contains the trp promoter-operator and the genes for tetracycline and ampicillin resistance. 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 pLeIFB. Fragment e encodes amino acids 1 to 13 of LeIF F. Fragments c, d and e, in the presence of T4 ligase,
Triple concatenate. The adhesive ends of each fragment are
The composite plasmid is properly circularized so that the tetracycline resistance gene, along with the mature LeIF F gene, is under the control of the trp promoter-operator, so that bacteria transformed with the desired plasmid can be placed on tetracycline-containing plates. You can choose from. A representative clone thus prepared was 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, an 816 b.p. fragment spanning from signal peptide amino acid 10 to the 3' non-coding region is isolated. 2 This fragment is denatured and treated with DNA polymerase.
Klenow fragment (Klenow et al., ProC, Natl. Acad.
Sci. USA 65 , 168 [1970]), a synthetic deoxyribo-oligonucleotide primer 5′-
dATG TGT AAT CTG TCT is used for repair synthesis. 3 The resulting product was cleaved with Sau 3 a and 1
A 452 b.p. fragment representing 150 amino acids is isolated from. Digest pLeIF H with 4 Sau 3 a and Pst, isolate the resulting 500 b.p. fragment, and obtain a gene encoding from the 150th amino acid to the end of the coding sequence. 5. Connect the fragments separated in steps 3 and 4,
Fragment 1 166 met cys asp stop ATG TGT…｜…GAT TGA−…−Pst
Get Sau 3 a. This encodes 166 amino acids of LeIF H. 6 Digest pLeIF A trp25 with Xba,
Make blunt ends using DNA polymerase,
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
For use in transforming strain 294 or other host bacteria,
capable of expressing mature LeIF H. Human genome phage λ Charon 4A recombinant library (Cell,
15, 1157 (1978)) were screened for leukocyte interferon by the method of Lawn et al., cited above, and the method of Maniatis et al., Cell 15 , 687 (1978). Approximately 500,000 plaques were screened using a radioactive LeIF probe derived from the cDNA clone 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 2000b.p.EcoR fragment of clone λHLeIF2 was
Subcloned into pBR325 at the EcoR site. The resulting plasmid LeIF I
It was cut with EcoR and a 2000 b.p. fragment was isolated.
This 2000b.p.EcoR fragment was combined with deoxyoligonucleotide dAATTCTGCAG (EcoR-Pst
converter) was connected. The obtained product
2000b cut with Pst and with a Pst end.
p. Fragmented. This was 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 create a pLeIFtrp1 expression plasmid. This clone has been deposited at ATCC. LeIF J 1 Plasmid pLeIF J is the previously mentioned pLeIF I
3.8 kilobases of human genomic DNA containing the LeIF J gene sequence obtained from the human genomic Phage λ Charon4A recombinant library (total human DNA digested and inserted into the Phage λ vector) constructed by Lawn et al.
Contains the Hind fragment. from this plasmid
A 760b.p.Dde-Rsa fragment was isolated. 2. Plasmid pLeIF B trp7 as Hind and
It was cut with Dde and the 340b.p.Hind-Dde fragment was isolated. 3 Cut plasmid pBR322 with Pst,
Incubated with DNA polymerase to blunt ends (Klenow fragment) and then digested with Hind. 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 (25mM Tris-HCl, 0 to 0.2M in PH8.5).
Elute with a linear gradient of NaCl). 5 Cibachrome B lue−Agarose or
Adsorb to hydroxylapatite and add 1.5MKCl or
Elute with 0.2M phosphate (if 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 substances from step 4 above
Milstein, Scientific American 243 , 66 (1980)
Monoclonal antibody column prepared as described plus 0.2M acetic acid, 0.1% Triton and 0.15M NaCl
It can be eluted with 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 thawed cells were treated with 0.1M Tris, 10% (w/v) sucrose, 0.2M NaCl, 5mM EDTA, 0.1mM PMSF adjusted to PH7.5-8.0.
and suspended in 4 volumes of buffer A containing 10-100 mM _MgCl2 . 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 a permanent bath. 2. Slowly add polyethylene-imine to the homogenate to a concentration of approximately 0.35% and leave it for approximately 30 minutes. Remove solids 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 applied at a rate of 5-8 cm/hour (e.g.
Flow directly onto the monoclonal antibody column using a 2.6 cm diameter column at a rate of 25-40 ml/hour). This column was then treated with approximately 10 column volumes of 20mM Tris-HCl,
PH1.5−8.5 (NaCl (0.5M) and Triton X−
100 (0.2%) or equivalent (containing surfactant). After washing, add 0.15M NaCl to this column.
Approximately 10 column volumes of solution containing Triton X-100 (0.1%) or equivalent surfactant are passed through. The column is eluted with 0.2M acetic acid containing Triton X-100 (0.1%) or equivalent surfactant. Protein peaks from monoclonal antibody columns (measured by UV absorption or other methods)
Collect and PH with 1N NaOH or 1.0M Tris base
Adjust to approximately 4.5. 4. Transfer the pooled interferon peaks to a suitable buffer such as ammonium acetate PH4.5.
(50 mM) equilibrated with Whatman CM52 cellulose or equivalent cation exchanger.
After addition, the column is washed with an equilibration buffer to reach a point where the UV absorption of the effluent becomes irritating and almost no protein is eluted from the column. The column is then eluted with a combination of 25mM ammonium acetate/0.12M sodium chloride or interferon for best recovery resulting in a lyophilized cake with satisfactory appearance and solubility. Monoclonal antibodies in this advantageous embodiment described above have been described by Staehelin et al. in Proc. Natl. Acad.
Sci. USA 78 , 1848-52 (1981). Monoclonal antibodies are purified and covalently coupled to Affigel-10 as described below. Preparation and Purification of Monoclonal Antibodies from Ascites Fluid Five female Balb/c mice were each inoculated with 5-10×10 ⁶ hybridoma cells harvested at mid-log phase.
Approximately 5 x 10 ⁶ viable cells obtained from the ascites fluid produced by the mice were inoculated intraperitoneally into each of 10 or more mice. This ascites fluid was 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. Batches of ascites fluid from each transfer were tested for specific antibody activity by solid phase antibody binding assays (Staehelin et al., supra) and pooled if satisfactory. The protein concentration in the pooled solution was determined using the approximation that 1 mg of protein has an extinction value of 1.2 at 280 nm in a 1.0 cm thick cuvette. 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 mixed with PBS (0.01M sodium phosphate,
PH7.3, 0.15M NaCl), 10 to 12 mg/ml
The protein concentration was set to To each 100 ml of 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. 0.02M Tris HCl (PH7.9)/
Dissolved in 0.04M NaCl (buffer). The protein solution was dialyzed against 100 volumes of buffer for 16 to 18 hours at room temperature. During this time, the buffer was exchanged at least once. The dialysis solution was centrifuged at 15,000 rpm for 10 minutes to remove insoluble matter. Approximately 30 to 35% of the initial amount of total protein in the ascites was collected as estimated from absorbance values at 280 nm. A solution containing 30-40 mg of protein per ml was then applied to a DEAE-cellulose column equilibrated with Buffer. A column bed volume of at least 100 ml was used for each gram of protein added.
NaCl from 0.04M in 0.02M Tris HCl PH7.9
The antibody was eluted from the column by changing the NaCl concentration linearly to 0.5M NaCl concentration. 0.06 to 0.1M
Pool the peak fractions of NaCl, add an equal volume of saturated ammonium sulfate solution (room temperature), and centrifuge for precipitation.
Concentrated. Protein mass was dissolved in 0.2M NaHCO ₃ (pH approx.
8.0)/0.3M NaCl (buffer) and dialyzed against the same buffer (three changes) at room temperature.
The dialyzed solution was centrifuged at 20,000 xg for 15 minutes to remove insoluble matter. Adjust the protein concentration to 20 to 25 mg/
ml. Preparation of immunoadsorbent Affigel−10 (Bio Rad Laboratories,
Richmond, California) was washed three times with ice-cold isopropanol and then three times with ice-cold distilled water on a glass filter. Gel slurry (approximately 50% in cold water)
%) was transferred to a plastic tube and briefly centrifuged to sediment. Remove the supernatant with an aspirator,
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. Add an equal volume of gel to fill up any unreacted parts.
Mix with 0.1M ethanolamine HCl (PH8),
The long axis was rotated in a circular shape and treated at room temperature for 60 minutes.
Wash the gel slurry with PBS to remove the reactant.
Stored in PBS in the presence of 0.02% (w/v) sodium azide at 4°C. 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. Dosages and rates of administration will be similar to those currently practiced clinically for substances of human origin. For example, about (1−
10) x 10 ⁶ units/day with a purity of greater than 1%, which can amount 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, 2×
Dissolve 3 mg of LeIF with a specific activity of 10 ⁸ units/mg in 25 ml of 5N human serum albumin, pass this solution through a biological filter, and aseptically divide the filtered solution into 100 bottles suitable for parenteral administration. Thus, one bottle contains 6×10 ⁶ units of pure interferon. Store these bottles in a cool place (-20℃) before use.
It is desirable to keep it at 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 EW
Remington's Pharmaceutical by Martin
As described in Sciences. This is incorporated herein by reference. 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. Leukocyte interferon A (LeIF A) of the present invention
Acute toxicity experiment (A) Intramuscular administration acute toxicity in mice, rats, ferrets and rabbits (1) Formulation No. 1 (Lyophilizate) [Table] LeIF A lyophilized preparation (Formulation No. 1) 30 x ¹⁰⁶ units/Kg for mice (10/each sex), rats (5/each sex), and white weasels (3/each sex)
The dose volume was 5 ml/Kg for mice, 2.0 ml/Kg for rats and 1.67 ml/Kg for white ferrets. Control mice (10/each sex), rats (5/each sex) and white weasels (3
animals/sex) were given equal amounts and doses of vehicle. Treated animals were observed for 31 days post-dose for abnormal signs and mortality. Body weights were recorded on the day of administration and on days 7, 14, 21 and 28 after treatment. All animals in this experiment survived the 31-day observation period, except for one LeIF A-treated female rat that was found dead in the cage on day 7 post-treatment. . The death of this female rat
It appears to be unrelated to treatment with LeIF A. No abnormal symptoms were observed in any of the animals during the period of this experiment. Normal weight gain was observed in all groups of animals, except for the female white weasel. The average body weight of female albino ferrets 28 days after treatment decreased slightly in the LeIF A-treated group (-6%) and the vehicle-treated group (-12%). (2) Formulation No. 2 (Liquid) [Table] Liquid parenteral formulation of LeIF A (Formulation No. 2) was administered to mice (10 animals/each sex), rats (5 animals/each sex), and rabbits (2 animals). /each sex) at doses of 500 x 10 ⁶ units/Kg and 100 x 10 ⁶ units/Kg, respectively; the dose volumes were 10 ml/Kg and 2 ml/Kg for mice, rats and rabbits, respectively. Ta. Separate groups of mice (10/each sex), rats (5
rabbits/each sex) and rabbits (2 animals/each sex) were used as controls and received equal and equal doses of vehicle. 14 Regarding lethality and abnormal symptoms in treated animals
Observed for days. Body weights were recorded on the day of administration and on days 7 and 14 after treatment. No deaths occurred in any of the treatment groups. There were no signs of abnormality and weight gain was observed throughout the study. (B) Intravenous Acute Toxicity in Mice and Rats A lyophilized preparation of LeIF A (Formulation No. 1) was administered to mice (10/sex) and rats (5/sex) at 3 x 10 ⁶ units. /Kg; dosage volumes of 5.0 ml/Kg and 2.0 ml/Kg were used for mice and rats, respectively. Control mice (10/each sex) and rats (5/each sex) received vehicle at equal doses and dose volumes. Treated animals were observed for abnormal signs, venous irritation, and mortality for 14 days after administration. Body weights were recorded on the day of administration and on days 7 and 14 after treatment. All animals survived the 14 day observation period. No abnormal symptoms were observed in any of the animals, and weight gain was observed in all groups of mice and rats. No venous irritation was observed in rats. In mice, an area of darkening around the injection site was seen 4 hours to 3 days after injection of either LeIF A or vehicle. Signs of venous irritation subsided 6-7 days after administration. (C) Acute toxicity of subcutaneous administration in mice, rats, and albino ferrets A lyophilized preparation of LeIF A (Formulation No. 1) was administered to mice (10/each sex), rats (5/each sex), and albino ferrets ( 3 animals/each sex) 30×10 ⁶ units/Kg
The dose was 5.0 ml/Kg for mice, rats, and ferrets, respectively.
2.0ml/Kg and 2.0ml/Kg. Control mice (10/each sex), rats (5/each sex) and albino ferrets (3/each sex) received vehicle at equal and equal dose volumes. Treated animals are subject to abnormal signs and mortality14
Observed for days. Body weights were recorded on the day of administration and on days 7 and 14 after treatment. No mortality or treatment-related abnormalities were observed in any of the animals. Normal weight gain was seen in all groups of animals. (D) Acute toxicity of oral, intravenous and intramuscular administration in mice (3) Formulation No. 3 (Liquid) [Table] [Table] Liquid parenteral administration formulation of LeIF A (Formulation No. 3)
was administered orally, intravenously and intramuscularly to mice (10 mice/each sex/dose level). Control group mice (10
Animals/each sex) received 0.5% human serum albumin in sterile saline at equal doses and doses by each route of administration. No mouse mortality occurred after administration by any of the three routes of administration. There were no abnormal signs, weight gain, or necropsy findings that could distinguish treated mice from control mice. 【table】

[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 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 mature human leukocyte interferon characterized by having one of Asp, Glu or Val. 2. The interferon of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon A (LeIF A). 3. The interferon of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon B (LeIF B). 4. The interferon of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon C (LeIF C). 5. The interferon of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon D (LeIF D). 6. The interferon of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon F (LeIF F). 7. The interferon of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon H (LeIF H). 8. The interferon of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon I (LeIF I). 9. The interferon of claim 1, wherein the mature human leukocyte interferon is mature human leukocyte interferon J (LeIF J). 10. An antiviral composition containing mature human leukocyte interferon A ((LeIF A)) as an active ingredient.