JPS60222500A - Novel hybrid interferon - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel alpha interferon-type proteins that exhibit interesting antiviral and antitumor activities.

This protein can be produced by culturing an organism containing an expressible recombinant DNA encoding the novel alpha interferon type protein.

The antiviral and antitumor activities of various interferons are being widely explored. Recently, a hybrid interferon that does not naturally exist in nature has been created. See, eg, US Pat. No. 4,414,150, dated Nov. 8, 1983.

However, this hybrid interferon contains all parts of the native interferon sequence without deletions, such that the segments joined together are complete.

The present invention advances in that the novel alpha interferon-type proteins contain truncated or truncated segments or sequences (i.e., "pa subsegments" of native alpha interferon joined to segments or sequences from other alpha interferons). yet this new alpha interferon type protein surprisingly exhibits increased activity.

We have found that when this new hybrid is formed in specific conformance to the truncated alpha2 and alpha1 segments, the resulting interferon material exhibits a highly desirable biological profile.

As is well known in the art, the codon ATG has a unique meaning as a start codon, ie it codes for methionine as the first amino acid of a protein consisting of an amino acid chain. The host producing this protein (eg, by recombinant DNA technology) will sometimes cleave this amino terminal methionine from the protein and sometimes leave it intact. Two closely related substances are thus produced. According to the invention, a substance lacking methionine at the amine terminus would have 161 amino acids starting from glutamine. Also, a material whose amine terminus starts from methionine would have 162 amino acids.

The novel interferon substances of the present invention are CBa1TI) derived from the alpha-1 sequence, delta-4 alpha2CBryl ll-1) derived from the alpha-2 sequence, linked to a segment defined as alpha1
It is characterized by a “subsegment” defined as . It surprisingly exhibits enhanced antiviral and antitumor activity.

The protein of the invention was confirmed to exhibit an antiviral activity of at least 1X10' units/m9, using the NIH/WHO uncloned leukocyte interferon preparation 69/19 as a standard, essentially F"αm1llet
As described in the publication of Ti et al. (Reference 1)
It was determined by a cytopathic effect-inhibition assay using MC virus and human foreskin cells (1, S-71). Furthermore, the novel interferons of the present invention are generally non-toxic at therapeutic doses.

The gene encoding the novel alpha-interferon-type protein is a gene encoding the mature human alpha-2 interferon gene linked to the complete carboxy-terminal nucleotide coding sequence below a single Bgtu site in the mature human alpha-1 interferon gene. 1st Bgl
It consists of a portion of the amino-terminal nucleotide coding sequence of up to 11 sites (hence the terminology "Bgll-1"). That portion of the human alpha2 interferon gene used to generate the interferon-encoding gene of the invention lacks the first 12 nucleotides normally found in the sequence encoding mature human alpha2 interferon. and therefore the first four amino terminal amino acids (CY
Therefore, we use the technical term "delta-4" to reflect the characteristics of this subsegment.

The portion of the human alpha-1 interferon gene used to generate the interferon-encoding gene of the present invention consists of all nucleotides downstream from the unique BUln site found in the mature human alpha-1 interferon coding sequence; Therefore “CBg
l■) The technical term "Alpha 1" is used.

Thus, we have used the mature human alpha 2 gene encoding the interferon of the present invention.
To describe the nucleotide coding sequence from the interferon gene, use the term “delta-4alpha2CBql”.
We use the terminology "Ill-1)" and to describe the nucleotide coding sequence from the mature human alpha-1 interferon gene used to generate the interferon-encoding gene of the present invention (Bgjn
) Alpha 1” technical term is used.Furthermore,
We also use this terminology to define protein subsegments and protein segments.

Mature human alpha 1 interferon is defined as having the amino acid sequence shown below.

CYS ASP LEU PROGLU i'H1l
Hls 5ENLEU ASP ASN ARG AR
G THRLEUMETLEU LEU ALA GL
N MET SERARG ILESERPMOSl!
;RSERCYS LEU METASPARG HI
S ASP PHE GLY PHE PROGLNG
LU GLU PHE ASP GLY ASN GL
N PHEGLN LYS ALA PROALA I
LE SERVALLEU Hls GLU LEU
ILE GLN GLU ILEPHE ASN LE
U PHE THRTBRLYS ASPSERSER
ALA ALA TRP ASP GLU ASPLE
U LEU ASP LYS FEE CYS THR
GLULEU TYRGLN GLN LEU ASN
ASP LEUGLU ALA CYS VAL M
E'l' GLN GLU GLUARG VAL G
LY GLU THRPROLEUMh;TASN A
LA ASP SERILE LEU ALA VAL
LYS LYS 1'YRPHE ARG ARG I
LE 1'HRLEU TYRLEU Tl1lshi G
LU LYS LYS TYR8ERPHOCYS A
LA TRP GLU VAL VALARG ALA
GLU ILE MET ARG SERLEUSE
NotLEUSlShiR1'HRASNLEUGLNGL
UARG LEU ARG AEG LYS GLU Mature human alpha 2 interferon is defined as having the amino acid sequence shown below.

CYS ASP LLUU PIBI OGLN THRH
ls 5ERLEU GLY Sl! ；E ARG A
RG 'l'HRLEUMETLEU LEU ALA
GLN MET ARG ARG ILESERLE
U PHE SERCYS LEU LYS ASPA
RG Hls ASP PHE GLY PRE PR
OGLNGLU GLU PHE GLY ASN G
LN PHE GLNLYS' ALA GLU TH
RILE Pノ<(J VAL LEUHLs GLU
ME'l' ILE GLN GLN ILg PR
EASN LEU PHE SERI'HRLYS A
SP 5ER8EE ALA ALA i'RP AS
P GLU i'1i)1! LEULEU ASP
LYS PHE i″YRTHft GLU LEUT
YRGLN GLN LEU ASN ASP LEU
GLUALA CYS VAL ILE GLN G
LY VAL GLYVAL THRGLU THRP
ROLli, "U ME'l' LYSGLU ASP
5EII ILE LEU ALA VAL ARG
LYS TYRPRE GLN ARG'lLE TH
RLEUTYRLEU LYS GLU LYS LY
S i'YR5EliPROCYS ALA TRP
GLU VAL VAL ARGALA GLU IL
EMET ARG SEI PliE Sl! ;RL
EU SERTl1RASN LEU GLN GLU
SEIBILEU ARG SERLYS GLUThe present invention provides CBgl If) derived from alpha-1 sequence.
Delta-4 alpha 2 (B
The present invention relates to a new class of interferons or proteins that use a truncated segment or "subsegment" called gl[-1).

The term "segment" will be defined as a portion of an interferon amino acid chain that is joined at one end by a naturally occurring sequence found at one end of the mature molecule.

Also, "shortened segment"' and "subsegment"
The term is defined as a portion of an interferon amino acid chain that is not joined at either end by naturally occurring sequences found at both ends of the mature molecule;
It is the six segments of interferon that have been defined to emit light.”
is a shortened portion of the interferon amino acid chain in which the amino acids normally found at one end of the interferon are not adequately supplemented.

Furthermore, the present invention includes proteins having 161 or 162 amino acids as shown below.

(ME71) Engineering GLN '1'HRHIS SERLE
U GXYSERAEG ARG THRLEU ME
T LEU LEUALA GLN MET ARG
ARG ILE 5ERLEUPRE SERCYS
LEU LYS ASP ARG HlsASP PH
E GLY PRE PROGLN GLU GLUP
HE GLY ASN GLN PRE GLN LY
S ALAGLU THRILE PROVAL LE
U HIS GLUMET ILE GLN GLN
ILE PHE ASN LEUPHE THRi'H
RLYS ASP SER5ERALAALA TRP
ASP GLU ASP LEU LEU ASPL
YS PHE CYS THRGLU LEU TYR
GLNGLN LEU AS# ASP LEU GL
U ALA CYSVAL MET GLN GLU
GLU ARG VAL GLYGLU THRPRO
LEU MET ASN ALA ASPSl! ;RI
LE LEU ALA VAL LYS LYS '1
'YRPHE ARG ARG ILI! , “'I”
HRLEU TYRLEUTHRGLU LYS LY
S T”# SEI Bi pRo cysALA TRP
GLU VAL VAL ARG ALA GLUI
LE MET ARG 5EIi LEU SERLE
U 5ERTfiRASN LEU GLN GLU
ARG LEU ARGARG LYS GLU (wherein The second protein has the amino acid sequence of the first protein, but differs from the first protein in that methionine is placed in front of the entire amino acid sequence of the first protein.

The above protein or protein mixture is used as a standard by NIH/
'WHO Uncloned Leukocyte Interferon Preparation 6
Using 9/19, essentially pamill-etti
As described in the publication of et al. (Reference 1),
It was confirmed to have an antiviral activity of at least 1 x 107 units/m9 as determined by a cytopathic effect-inhibition assay using C virus and human foreskin cells CF'S-71).

The resulting protein or protein mixture is a novel interferon substance that is not alpha interferon that exists in nature.

The present invention further includes a DNA sequence encoding the above protein, preferably having the sequence shown below.

CATG) CAA ACCCACAGCCTG GGT
AGCAGG ACCioTG A'l'G CTCC
TG GCACAG A1'G AGG AGA AT
C'l'ci'CTTTTCTCC1'GC'l'i
'G AAG GACAGA CAi'GAC1'T
T GGA TTT CCCCAG GAG GAG
i'TTGGCAAACCAG I°7'(,' CAA
AAG GCT GAAACCATCCCT Gi'
CC1'CCAT GAG A'l'GATCCAG
CAG Ai'Ci'Tc AACC1'Ci'TTA
CCACAAAAGAT7゛cA7゛cTGc71Gc
TTGG GAT GAG GACC1'CC1”A
GACAAATTCTGCACCGAA C1'CTA
CCAG CAGCTG AAT GACi'TG G
AA GCCTGT GTGATG CAG GAG
GAG AGG Gi'G GGA GAAAC1'
CCCCTG Ai'G AA1' GCG C;AC
i'ccATCT'l'G GCT GTG AAG
AAA TACi'TcCGA AGA Ai'CAC
7'CTC7'ATC1'G ACAGAG AA
G AAA 1'ACAGC(,:C1"'1'G1
'GCCTGG GAG Gi'T G'l'CAG
A GCA GAA ATCATG AGA TCCC
TCTCT Ti'A TCA ACAAAACT1'G
CAA GAA AGA TTA AGG AGGA
AG GAA [TAA] (wherein the start codon ATG is written in parentheses and the stop codon TAA is written in square brackets) The present invention further encodes the above protein, but the synonymy of the genetic code Accordingly, it also includes DNA sequences different from the above sequences.

The present invention also provides a cloning vector (
plasmids) and also hosts (eg bacteria, yeast or animal cells) transformed with this cloning vector.

Tests with the above-mentioned protein or protein mixture showed increased activity against viruses and cancers compared to the commercially available standard alpha 2 interfuron. Of particular interest, the protein or protein mixture has shown promising activity against adenoviruses (eg adenovirus-1) and cancers of the ovary and cervix.

The protein or protein mixture of the invention is engineered from two different interferon coding sequences by combining the sequence encoding the suff segment of mature alpha 2 interferon with the sequence encoding the segment of mature alpha 1 interferon. .

The 161 (or 162) amino acid chains of these proteins are theoretically thought to have a sulfur-sulfur bond between the cysteine residue at position 25 and the cysteine residue at position 134.

(In this numbering, when X is 1, the methionine residue present as the first amino acid was counted as position 0.) It is believed that the biological activity of the protein of the present invention is related to this disulfide bond. It is possible, and it is not possible, that this results from the characteristics of the specifically bound sequences provided by the segments and suff segments derived from the alpha=1 and alpha-2 mature coding sequences, respectively.

The general method for producing this new alpha interferon type protein was to prepare a hybrid interferon gene encoding this alpha interferon type protein. This involved first attaching a group of promoters with translation initiation signals to the beginning of the mature alpha-2 interferon coding sequence. A DNA fragment in which the promoter was linked to the alpha-2 amine terminal coding sequence (the first BglU restriction site was then identified, isolated, and immediately following the BQIR site found in the mature alpha-1 coding sequence) ligated to the carboxy-terminal coding sequence of the alpha1 interferon gene.

The experiments actually carried out can be divided into six broad areas of activity, and (1) alpha-1 interferon plasmid derivatives, in particular only one rather than two Eco
Plasmid Hi f-2 with R1 site (Reference 2)
(2) ATG translation initiation codon is attached to the end and “5hine-Da, Igarno”
a group (or genus) of promoter assemblies that differ in the number of nucleotides between the sequence and this start codon; (3)
(4) the binding of this group of promoters to the amine-terminal coding sequence of the alpha2 interferon gene; 1) comprising a promoter region linked to the amine-terminal coding sequence of the alpha2 interferon gene
Features of the invention include: dissociation of l''j fragments, (5) ligation of these promoter-containing fragments to the carboxy-terminal alpha-1 interferon coding sequence; and (6) screening of bacterial clones for active hybrid interferon production. and thus bacterial clones containing the appropriate recombinant molecules are isolated.

The method used to transform E. coli was essentially as described (13).

Restriction enzyme T4 DNA ligase and New England Biolab
s) Yohibethesda Research Institute CBethes
Other DNA modifying enzymes available from aaRegearch Laboratories were used generally according to the manufacturer's reports. How to recover DNA from gel.

Grosman and Mo1. dave
ed., Methods inEnzymology+ V
ol, 65, pcLrt1, 371-380
(1980), Academic Press, N.
Y, ``Recovery of DNA from ゛ge/L/''
, O, Smit7+J. Records regarding methods and operations not described in detail below (or essentially equivalent substitute methods) can be found in the numerous 'Experimental Handbooks' available, e.g. Each of these six areas of activity mentioned above is described in more detail below.

Alpha 1 Interferon Code evening 11&! It is integrated into a plasmid called 11if-2hC Ref. 2) which has two EcoR1 sites. This plasmid DN
An aliquot of A was treated with the restriction endonuclease Ec under conditions expected to yield some partial digestion products.
Digested with oRI. The exposed end is nuclease S
1 followed by complete digestion with BamHI.

forward B in the non-coding region; the DNA of interest with the amino-terminal coding end of the alpha 1 interferon gene and the tetracycline resistance gene up to the coR1 site;
Fragments were identified and isolated by their mobility on agarose gels relative to genetic markers of size. In parallel, pBR322 (Reference 3) DNA was
, then treated with nuclease S1, and finally digested with flamH'l.

In this case, the isolated fragment contained the origin of replication for pBR322 and the carboxy-terminal coding sequence for the tetracycline resistance gene. The above two fragments were enzymatically linked with T4DNj4') gauze, and the recombinant DNA was used to transform E. coli strain 294 (Ref. 4). Plasmid DNA from the tetracytalline-resistant recombinant was subjected to restriction enzyme analysis to reveal that a single EcoR1 site within its structure was alpha-
1 was confirmed to be located upstream of the interferon coding sequence.

Array・・・CCTCGCCCTTTGCTTTAC
A plasmid with a unique Eco R1 restriction site approximately 30 base pairs upstream was constructed from TGATGGTCC... (obtained from the alpha 1 interferon gene 'leader' coding sequence in plasmid Hif-2h). This plasmid contains 7>BR322 Hind m
and Pvv, an approximately 40 base pair DNA fragment between the 11 sites (i.e., the Hae/pυull restriction fragment found within the region encoding the leader sequence of the human alpha 1 interferon gene of Hif-2h)'
was created by cloning in an orientation that reproduces the t11 site.

An aliquot of this plasmid DN'A was first linearized by enzymatic digestion at the unique EcoR1 restriction site. Different numbers of nucleotides between this point and the above (underlined) ATG codon were then removed through varying degrees of nuclease digestion using exonuclease G followed by nuclease S1 and other experiments. was removed using nuclease Earl 31, broadly following published methods (eg refs. 6 and 12) and/or manufacturer's recommendations. These truncated DNs
A molecule is 'Shine-D' for initiating translation.
Nomina IJ-12 with al-garno'' sequence
It was ligated to a 5 base pair 1acUV5 promoter (derived from pKB252 [Reference 5]) fragment. The sequence of this promoter fragment is as follows.

5'-GAATTCCAGTGAATCCGTAA'l
'CATGGTCATAGCTCAC1'CA1'1”
AGGCACCCCAGGCT'l'TACAC2'l
'TATGCTTCCGGCTCGTA'11AATG
'l'GTGGAATi'GTGAGCGGA1'AA
CAA'l"l'TcAcAcAGGAAAcAG-3
'The resulting population of recombinant DNA molecules was derived from E. coli strain 294.
was used for ampicillin-4 transformation. Plasmid DNA was isolated from a population of these ampicillin-resistant bacteria, and the promoter, operator and 5hine-Da, l garn.

was used to isolate a genus of lac promoters having the sequence and ending with the translation initiation codon ATG. Each promoter in this genus is 5hine-Dal
The number of nucleotides between the garno sequence and the ATG codon are different from each other and therefore expected to have different translation initiation efficiencies (References 4.6 and 7).

This DNA isolated from a non-methylating E. coli strain (lac
Aliquots (58 μg) of 2
Restriction endonuclease A in a 50 μl reaction volume
It was digested with υα■ (which recognizes CC and cleaves each DNA strand between G residues) with the nucleotide sequence GGCA/T. After this digestion, place the sample in ice and 10. ¥、S
'l salt (1,5M NaCl3.0.5M sodium acetate pH'4.0.0.06M Zn5 (h) 30μ
m3.5M NaCl320 til11 and nuclease SI C8igma catalog N-5255)2
μe (200 units) was added and the mixture was stirred at 11 °C for 10
Incubated for minutes. To this reaction mixture (0.3M
Sodium acetate, 0.01M EDTAlo, 2M
) Lys MCII pH 9,5) 0.5- was added and the DNA was precipitated by addition of ethanol. The centrifuged DNA was resuspended in an appropriate buffer and added to EcoE.
Digested with I. This reaction product was analyzed on an acrylamide gel, and a population of DNA fragments containing the promoter and translation initiation sequences was observed as a broad DNA band (approximately) 2125 base pairs in length.

Within this group was a collection of DNA fragments with a truncated EcoR1 site at one end and an ATG codon acting as a translation initiation signal at the other end (flush end). This entire collection of DNA fragments was isolated from acrylamide and ligated to the amine-terminal coding end of the alpha2 interferon gene in the next step as described below.

Binding of this group of promoters to the amino-terminal coding sequence The starting material used in this step has a Hind m restriction site with the nucleotide sequence...AAGCT'7'qT...
The alpha-2 interferon gene was located at the beginning of the mature alpha-2 coding sequence with... The truncated codon (TGT) codes for cysteine, the first amino acid of mature alpha2 interferon. The preparation of this mold is described in Reference 8 (FIG. 9, Structures 1-3). This plasmid containing the alpha2 interferon gene also has a unique EcoR1 site upstream of this unique Hind■ site.
It had parts.

Therefore, stepwise digestion of this DNA with Hindm, nuclease S1 and EcoRI yields a vector into which the lac promoter (prepared in step 2 above) can be ligated in a predetermined orientation.

More specifically, 50 μl of this plasmid carrying the alpha2 interferon gene was completely digested with Hind Ill in a reaction volume of 350 μβ. This sample was transferred to ice, IOX, S'l salt 50μly,
5 M NaC 1135 μl J and nuclease S1
2μ7 (200 units) was added. The mixture was incubated at 11° C. for 10 min, then 41 rnl of a solution containing 0.3 M sodium acetate, 0.01 M EDTA, J: and 0.2 M Tris-HCII pH 9,5 was added, and 2.5 volumes of DNA by addition of ethanol
was precipitated. After resuspending this DNA, it was
After digestion with oRl, the enzyme was inactivated by heating to 65°C. The DNA was again precipitated with ethanol and finally resuspended and enzymatically linked by 1'4DNA') gauze to a collection of previously isolated sita promoters. The recombinant DNA molecule was then transformed into E. coli strain D1
210 (References 9 and 13) to ampicillin resistance.

Two promoters -l-α- linked to the amine-terminal coding sequence of the mature alpha 2 interferon gene!
- Raw dirt)! - Isolation of DNA @ Pieces Plasmid DNA was isolated from the entire population of ampicillin-resistant transformants of D1210 obtained in step 3 and aliquoted into 60μ
s is the restriction endonuclease EcoR1 and Bglu
was digested. The reaction product was subjected to electrophoresis on an 856 polyacrylamide gel. As expected, a blurred broad band of fragments approximately 330±20 base pairs in length was observed. These fragments therefore represent the amine-terminal coding region (first Bg
It was expected to contain the lac transcription and translation initiation signals fused to the ln site). A broad band surrounding this group of fragments was isolated from the gel.

Formation of alpha2/alpha1 hybrid interferon gene by binding to plasmid DNA containing the alpha1 interferon gene (step 1
The Nariquat (prepared in Isolated after gel electrophoresis. This larger DNA fragment was then ligated to the population of lac promoter fragments isolated in step 4.

G. Isolation and Characterization of Clones Expressing Hybrid Interferon with Antiviral Activity by Screening Bacterial Clones for Active Vibration Interferon Production Bound DNA Formed in Step 5
The molecule, which should have the lac regulatory element fused to the alpha2/alpha1 high frit interferon gene, was used to transform an E. coli strain to tetracycline resistance. Individual colonies were allowed to grow and extracts were prepared. These extracts were assayed for the presence of interferon activity using methods essentially described in references 1 and 10.

Plasmid IpNA was monocyclized from the clone that produced the highest level of antiviral activity. The junction between the lac promoter and the amino-terminal coding end of this hybrid interferon coding sequence was then subjected to DNA sequence analysis. This unexpectedly indicates that the coding sequence for the first four amino acids was removed during the above-described construction, possibly indicating that the single-stranded -
This may be due to the "fraying" activity that is sometimes known to be associated with nuclease Sl.

(Reference 6). The partial nucleotide sequence from this region is expressed as follows.

(fmet) Gin Thr His Ser Le
14...AGGAAACAGACTG A7″G C
AA ACCCACAGCCTG... Other features of the present invention include a method for preparing a plasmid containing a gene encoding and capable of expressing the novel alpha interferon defined herein, as well as a method for preparing a host transformed with the plasmid of this building. α) From a plasmid containing the alpha2 interferon gene, a fragment (approximately 300
b) hair-annealing the coding strand of this fragment with a synthetic oligonucleotide (which may be about 15 base pairs or less in length), which synthetic oligonucleotide sequence is the mature alpha -2 Starting from the first nucleotide residue of the fifth codon of the interferon coat sequence; C) Filling in the non-coding strand in the 5'→3' direction 1llin
), and consume the code strand in the 3'→5' direction ea
taway), digesting the double-stranded DNA fragment thus obtained with BglU; d) converting the alpha-1 interferon plasmid, which has only one EcoR1 site upstream of the coding sequence of alpha-1 interferon, into Ec.

Digesting with R1, filling it in, and digesting it with BglH: e) Add the thick fragment from this second plasmid to step C.
(thereby binding to the amino-terminal coding region of alpha2 interferon DNA derived from E
f) transforming a suitable host; and g) modifying the plasmid to enable expression of the gene.

A suitable host for step f is E. coli, which can be cultured to propagate the plasmid, making its modification step in step g easier.

■The unique EcoR1 site can now be utilized in step G. That is, the plasmid is digested with EcoRI, both single-stranded ends are removed, a flash end is generated, and the flash end contains in its sequence the promoter, liposome binding site, and translation initiation codon (at its end). The fragment is ligated in front of and between the hybrid alpha2/alpha1 interferon genes to control them, and the plasmid is circularized again.

This method is more flexible than the first method described here, as it allows the incorporation of different genetic regulatory sequences. Preferred embodiments are described below.

The starting material for this work was previously described and reference 8
There is a Hind restriction endonuclease site in the first part of the mature alpha-2 interferon coding sequence, as described in 7 derivatives of the alpha-2 interferon gene and the previously described CBglU) alpha-
Preferably, it contains one fragment. The method seems to cover the region of interest (nominally) 276 base pairs Hind
It consists of isolating the Ill/Pvun fragment from this plasmid containing the alpha2 interferon gene. This fragment is then annealed with a synthetic oligonucleotide (which can be custom synthesized commercially), such as 15 nucleotides with the following sequence: 5'-CAAACCCACAGCC2''G-3'. The kinased oligonucleotide is used as a template. (Heat-denatured version of the above Hindm/PvrbII DNA fragment containing a portion of the nucleotide coding sequence of the mature alpha 2 interferon gene) should be hair-annealed.

The synthetic oligonucleotide will form a double-stranded DNA structure starting from the first nucleotide of the codon for the fifth amino acid of the mature alpha-2 interferon. Klenow of E. coli DNA polymerase I in the presence of four deoxynucleoside triphosphates.
The combined action of the polymerase and 3'-5' exonuclease activities of the fragment, followed by digestion with Il, of the alpha 2 interferon (the first Bg
up to the IU site), but this would lose the codons for the first four amino acids. The fragment of interest is easily visualized by autoradiography, for example if the oligonucleotide is kinased with gamma-''P-ATP (Ref. 11).

The alpha-1 fragment recipient plasmid is digested with EcoRI and then digested with D in the presence of e.g.
6 filled in with the Klenow fragment of NA polymerase 1
lleti in)”, then digested with Bg#.

5′ to reduce background in the next process.
- Removal of terminal phosphates is recommended. The larger fragment generated by these manipulations is then conjugated to the amine-terminated coding region of alpha 2 isolated in the steps described above. Therefore, the recombinant DNA isolated after transformation of a suitable E. coli host and selection of tet clones is a delta-4 alpha 2 CBgl ll
-1/Bgl II) One EcoR1 immediately in front of the first part of alpha 1 interferon structural gene
It has parts.

This gene can be expressed in a number of ways, for example by digesting this molecule with oRI and then with any of a number of single-strand-specific nucleases (ref. 12) to generate flash ends. to which a flash-terminated fragment containing the promoter and liposome binding site and ending with the translation initiation codon can be ligated.Producing clones will have the regulatory elements positioned in the correct orientation with respect to the coding sequence of the novel interferon. This is probably what has been done.

The present invention further includes a pharmaceutical composition comprising the above-defined novel protein or protein mixture as an active ingredient together with a pharmaceutical carrier or excipient.

The novel interferons are preferably administered to patients parenterally (eg, intravenously, subcutaneously, intramuscularly), similar to the methods and doses used for human alpha 2 interferon. The new interferons may be useful when applied topically (eg, to the cornea to treat infectious viral infections of the eye).

A range of 105 to 108 U/M'' surface level for 7 days is suitable for topical administration, but the correct dose will be determined by the clinician depending on the particular patient and the exact condition being treated. European Patent Application No. 824 dated June 29, 1983
No. 81.

Lyophilization solution (1000 vials, 1-/vial)
Combination interferon 7.5X1o10IU glycine, U
SP 20. O9/13 Human Albumin, USP 1.09/1 Distilled Water for Injection,
USP Total amount 1. The above interferon is a hybrid interferon or a mixture of these interferons according to the present invention.

Appendix: Reference 1, Familletti, Ph1li'p C,+
5ara Rubens-teinr and 5icl
ney Pe5tka :Methods in Enz
ymolog IB 78: 387-394 (198
1), Academic Press Jnc2, Ma
nte'i,N,,M',Schwarzstein,
M, 5tre-uli, S, Panem+ S, N
a, gatar and C, We-i 88mnnn
:Cloned human leukocyte interferon cDNA
Nucleotide sequence of A.

Gene 10:1-10 (1980) a Boliv
ar+F, rR,L, Rodriguez+P,J.

Greety M, C, Betlach, 11, L
, Heyneker + H, W., Boyer IJ, H.
Crosa+ and S.

Falkow: Generation and characterization of a new cloning vehicle. ■Multipurpose cloning system. Gen
e2: 95 (1977) 4 13ackman
, K., and ,M., ptashne: Inhuman.”
Maximizing gene expression of recombinant plasmids in this plant. C
e1l 13:65-65-71 (1978) FLBa
C.K., K., M., Ptashne and W.G.
11-bert: Construction of a plasmid containing the CI gene of bacteriophage lambda. Proc, Nat.

Acad, Sci., 73:4174 4178 (19
76) G Neuoberts, I'homas M, and Ga, il D.

Lauer: Maximizing gene expression of recombinant plasmids in vitro. Methods in Enzy
mology+ 68: 473-482, Acad
emicpr (3ss Jnc, C1979).

? Roberts+1'homasM,+Raytn
ondKacic.

and Mαγk Ptα5hne: General methods for maximizing expression of cloned genes. Proc.

Hat, Acad, Sci, + Vol.
76,/162, pp.

760-764 (1979) EL Weissmnn, C. Cloning of ninterferon and other mystics: Interfe.
υ011+1981, Voi3, pp, 101, Jon
Edited by Gre-sser, Academic Press
+ N, Y, (1979) 9, 5adler+J
,R9+M,i'ecklenburg and J.L.
Betz: Plasmid Gene 8:279- Containing Many Tandem Copies of the Synthetic Lactose Operator
300 (1980) IQ Nagatar S, + H
,1'aira+ A, Hal l, L, Jo
hn-srud, M, 5trerblir J,
Ecsodi, W., Hall.

K, Can t e l l + and C, We i
s smann: Synthesis in E. coli of a polypeptide with human leukocyte interferon activity. Nαtur
e284:316-320 (1980) 11, Goeddel, David V, rH, Mi
chael 5hepa-rtL Elizabeth
Yelvertonr David Leung,
and Roberto Crea: Synthesis of human fibroblast interferon by Escherichia coli.

NrLcleic Ac1ds Res,, Vol.
8, /I6.18, pp, 4057-4074 (19
80) 12. Documentation regarding methods and operations not specifically described above or essentially equivalent substitute methods can be found in numerous reusable "laboratory manuals." For example, 7", Maniα-tis, E, F, F
r1tsch and J. Sambrook+6 Molecular Cloning - Experiment x Ma Yual # (1982)
, edited by the Co1d Spring Harbor Institute, Co1d Spring Harbor.

N.Y.

la Dagert, M., and S-D., Ehrl.
ich: Long-term culture in calcium chloride improves the capacity of E. coli cells. Gene fL: 23-28(
Continuing from page 1 ■Int, C1,' Identification symbol Internal reorganization procedure amendment (
Format) May 17, 1985 Manabu Shiga, Commissioner of the Patent Office 1, Indication of the case - μ 1988 Patent Application No. 268225 3, Person making the amendment - Relationship to the case Applicant Address Name Schling Corporation 4 Agent 5, date of amendment order April 60, 1985 (shipment date)
6. Correction target lyophilization solution (1000 vials: 1ml/) < vial) 1 button interferon 7.5X 10” I U disodium hydrogen phosphate anhydrous, 2.27g/(IUSP (United States Pharmacopeia), reagent hydrogen phosphate Disodium USP O,55!?#!Glycine, USP 20.Oy#!H) Albumin,
USP 1. Og//l distilled water for injection, USP Total amount 1.01 The above interferon is according to the present invention)
interferon or a mixture of these interferons.

1,7 Amiretschi Philip Sea
Ti.

Ph+lip C,)+Sarah Rubenstein (S a
raRubens te in) and Sitney
SidneyPestka: “Methods in Enzymolog”
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H, L, Heyneker) + Work Dub')
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F, 'Fritscl+) and George Sambruch (J.

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2328

Claims (1)

[Scope of Claims] (1) A 161 amino acid sequence consisting of an amino acid chain that does not exist in nature as alpha interferon,
or a protein having a 162 amino acid sequence due to the optional addition of methionine to the first amino acid of the sequence, wherein said sequence is CBQln) a delta-4 alpha 2 CB preceding an alpha 1 segment.
QI II-1) It is characterized by containing different portions of the two types of alpha interferon described above, which are defined as subsegments and have the respective subsequences of two different alpha interferons existing in nature combined. protein. (2) The following 161 or 162 amino acid sequence: C
M'ET), GLN THRH'IS SERLEU
GLYSEfl ARG ARG THRLEU M
ET LEU LEUALA GLN MET ARG
ARG ILE 5ERLl! ;UPHE SERC
YS LEU LYS ASP ARG HISASP
PH'E GLY 'PHE PROGLN GLN
GLUPHE GLY ASN GLN PHE G
LN LYS ALAGLU THRILE PROV
AL LEU HIS GLUMET ILE GLN
GLN ILE PHE ASN LEUPRE T
HRTHRLYS ASP SERSERALAALA
TRP ASP GLU ASP LEU LEU
ASPLYS PHE CYS THRGLU LEU
i'YRGLNGLN LEU ASM ASP L
EU GLU ALA CYSVAL M'ET Gl
,N'GLU GLU AIIG VAL GLYG
LU THRPROLEU MET ASN ALA
ASPSERILE L, l! ；U ALA VAL
LYS LYS i'YRPHE ARG ARG I
LE THRLEU TYRLEUTHEGLULYS
LYSTYR8l'RPNON0CYSALA TRP
GLU VAL VAL AItG ALA GLU
ILE Ml! ;TARG 5hHI LEU SE
RL12: USI! J'RTHRASN LEU G
LN GLU ARG LEU ARGARG LYS
GLU (wherein X is O or 1) and has an antiviral activity of at least about 1 x 107 units/ln9 as determined by '/f4++ in a cytopathic-inhibition assay. (3) A mixture of proteins according to claim 1 or 2. (4) A protein or protein mixture according to any one of claims 1 to 3 as an active ingredient for use in pharmaceutical preparations. (5) A pharmaceutical composition comprising a carrier or an excipient containing the protein according to claim 1 or 2 and having the following sequence: CATG) CAA ACCCACAGCCTG GGT
AGCAGG AGG ACC1'TG Ai”G C
TCCTGGCA CAG ATG AGG AGA
ATCTCTCTTTTTCTCCTGC1'TG AA
G GACAGACATGACTTT GGA TTT
CCCCAG GAGGAGTTTT GGCAACC
AG T1'CCAA AAGGCTGAA ACCA
TCCCT G7'CCTCCAT'GAGATG A
TCCAG CAG ATCT'l”CAACCTCT
TT ACCACA AAA GAT TCA TCT
GCT 'GC1' TGG GAI' GAG GA
CC1'CC1'A GACAAA 'l'Tc TG
CACCGAA CTCTACCAG CTG
AAT GACTTG GAA GCCTGTGTG
ATG CAG GAG GAG AGG Gi'G
GGAGAA ACT CCCCTG ATG AAT
GCG GACTCCATCTTG GCT GTG
AAG AAA TAC1'TCCGA AGA A
TCACT CTCTAT CTGACA GAG A
AG AAA TACAGCCCT1'GTGCCTG
G GAG GTT GTCAGA GCA GAAA
TCATG AGA TCCCTC'I'CT TI'
A TCAACA AACT1”G CAA GAA
AGA Ti'A AGGAGG AAG GAA [
7° AAI (where the start codon ATG is written in parentheses and the stop codon TAA is written in square brackets)
and a DNA sequence encoding a protein according to claim 1 or 2, but differing from the above sequence due to synonymous genetic code. (6) A vector containing the DNA sequence according to claim 5 and capable of expressing the DNA sequence. (7) A host transformed with the vector according to claim 6. (8) A method for preparing a vector according to claim 6 or a host according to claim 7, comprising: (CL) producing a derivative of alpha 1 interferon plasmid having only one EcoR1 site; What to do: <b> Create a group of promoters that have an ATG translation initiation codon at their end and differ in the number of nucleotides between the 'Shine-Dalgarno' sequence and this initiation codon; (C) Produce this group of promoters with alpha Cd) I comprising a promoter region linked to the amino-terminal coding sequence of the alpha 2 interferon gene;
) isolating the NA fragments; (C) ligating these promoter-containing fragments to the carboxy-terminal alpha-1 interferon coat sequence; transforming a host with the resulting plasmids to produce active hybrid interferon; The above-mentioned method for preparation comprises: screening a host clone for use in the preparation of the host. (9) A plasmid containing the gene encoding the novel alpha interferon according to claim 1 or 2 and capable of expressing the interferon, or a method for preparing a host transformed with such a plasmid. (G) isolating from a plasmid harboring the alpha2 interferon gene a fragment extending from the restriction site preceding the gene to the Pvu II restriction site within the gene; (b) the fragment; synthetic oligonucleotide hair-annealing of the coding strand of (where the synthetic oligonucleotide sequence starts from the first nucleotide residue of the fifth codon of the mature alpha-2 interferon coding sequence). (C) Filling in the non-coding @ 5'→3' direction, consuming the coding strand in the 3'→5' direction, and digesting the double-stranded DNA fragment thus obtained with Bgln. (d) alpha-1 interferon plasmid fEc with only one EcoR1 site upstream of the alpha-1 interferon coding sequence. Digest with R1, fill it in, and fill it with B (digest with Ilm; Ce). The above method for preparation comprises: (a) transforming a suitable host; and (σ) modifying the plasmid to enable expression of the gene. (g) A method for producing the novel interferon-type protein' according to any one of claims 1 to 3, which comprises culturing the host according to claim 7.