JPS60248185A - Separation and expression of new interferon gene - 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 provides
The present invention relates to methods and compositions useful in rapidly filtering and isolating genes from.

The methods and compositions of the present invention utilize small NA probes (pro
use be). In one example of the present invention, novel human interferon genes are identified and isolated from genomic libraries by a method that utilizes small NA fragments containing complementary nucleotide sequences to all four known human interferon genes.

do. Also in accordance with the present invention, compositions and methods using recombinant DNA techniques allow for the replication and expression of novel human interferon genes in microorganisms. Furthermore, according to the present invention, the gene sequence of a novel mature human α-interferon gene has been obtained and can be expressed in bacteria.

Interferon is a protein of great clinical importance and potential. Although much research has been carried out to elucidate the molecular mechanisms of interferon induction and activity, progress has been slow primarily due to the low quantity of pure interferon proteins available for research. Recombinant DNA technology offers the opportunity to produce large amounts of interferon protein and interferon gene sequences.However, recombinant DNA technology has limited the ability to prepare recombinant DNA from the cellular messenger FtNA (mRNA). As such, it requires isolation of interferon DNA sequences, which is a difficult task.

mRNA is an interferon protein, or mRNA, that exists at extremely low levels and has an unknown heterogeneous structure.
The presence of the A molecule makes it even more complicated.

For example, at least six different classes of interferons have been identified in humans: fibroblast, leukocyte, and immune interferons. Each section contains a number of distinct interferon species that are known to be structurally related within a given network and structurally different in each interrogation.

Currently, the human interferon gene is a recombinant DNA
Two separate sources are isolated for use in the technique: m isolated from human leukocytes that synthesize interferon.
There are cDNA libraries prepared from RNA and genomic libraries prepared from human chromosomal DNA. These libraries are generated by inserting enzymatically synthesized DNA or native human DNA fragments without interferon gene sequences into a suitable vector such as a bacteriophage or plasmid. Insertion of the human DNA sequence into such a vector produces a recombinant or chimeric vector. Several general methods have been developed in recent years by which such recombinant vectors can be constructed. For example, Cohe
U.S. Pat. No. 4,257,224 to M. N. and Boyer describes a method for producing and ligating recombinant vectors using restriction endonuclease enzymes. This description uses U.S. Pat.
, No. 224.

The recombinant vector thus produced is then used to transform or infect cells with which the vector is compatible, allowing foreign DNA or genes to be introduced into the cell. Recombinant vectors must be capable of autonomous replication in the host cell and must contain at least one selection marker, a limited number of known restriction endonuclease cleavage sites, and a high copy number. . The resulting replication of the recombinant vector allows the production of multiple identical copies or the cloning of the recombinant, ie the heterologous gene inserted into this vector. Each clone or replicating recombinant vector contains a unique fragment of heterologous DNA or gene. The term "library" refers to the production of multiple distinct recombinant vectors from which a single unique gene sequence can be selected.

To date, the human interferon gene has been isolated using cDNA DNA libraries as well as cDNA libraries that use a heterogeneous mixture of cDNA probes containing interferon-specific DNA sequences.
It has been isolated from a genomic library using NA Proso. This is authored by Pestka19
“Record of Biochemistry and Biophysics J221” published in 1986
:1. The use of cDNA libraries for isolation of interferon genes has several advantages and disadvantages compared to genomic libraries containing chromosomal DNA.

c. The main advantage of DNA libraries is their specificity.

When prepared from mRNA that produces the desired gene product, the cDNA library must contain primarily only those DNA sequences that correspond to the desired gene product. However, a genomic library contains all the genes as well as the desired gene sequences and regulatory sequences necessary for the hardware from which the chromosomal DNA was isolated to function. But c D
The disadvantage of NA libraries arises from this significant specificity and mode of construction.

The cDNA library is composed of interferon-specific mRNA isolated from cells that produce interferon. This mFLNA represents only those interferon genes that are present at extremely low levels and actively expressed in these cells.

Interferon genes that are not actively expressed cannot be recovered by this method. Furthermore, interferon-
By normal cell processing that produces a specific message, the resulting mRNA molecule no longer contains a full complement of nucleotide sequences compared to the natural gene that produces this mFLNA. Among the eukaryotic genes that have been isolated so far, many of these genes contain sequences called "introns" within their respective gene sequences.
Removed during FtNA transcription and processing, the resulting mF
LNA molecules no longer contain introns. Another loss of genetic sequence occurs during the isolation of these highly unstable nuclease-sensitive mRNA molecules and their use in the synthesis of cDNA molecules for inclusion in cDNA libraries.

The need to transcribe cDNA molecules, such as mFlJA molecules, into double-stranded DNA that is then inserted into the library creates the risk of miscopying and thus altered structure and function of the final interferon gene product. Furthermore, the construction of cDNA and genomic libraries eliminates gene sequences during insertion into the vector used to hold the gene library.

Alternatively, the r-Tum library contains cloned DNA isolated from cellular r-Tum. If enough loans are given, 9
It is possible to generate a library containing some gene with a probability of 9%. Furthermore, these cloned genes are
In contrast to prokaryotic cloning systems, eukaryotes are advantageous in that they exist in their natural genetically intact form with regulatory signals. Since rTum libraries are generated from natural chromosomal DNA, no further replication is necessary prior to insertion of these libraries into the vector of choice. Furthermore, these inserted double-stranded DNA fragments are relatively unstable and nuclease resistant. Therefore, a preferred source for genes that are not actively expressed and are expressed in low copy numbers, or for which there is uncertainty regarding the site of synthesis, or for genes that are present in multiple copies or types, is the r-tum library. be.

The rTsum library was purified by restriction endonuclease to 1
Prepared by mechanically shearing chromosomal DNA into 6 to 20 kilobase (base) (k'b) fragments or by dividing this DNA and then inserting these DNA fragments into appropriate NA vectors. do. This is Maniatis (
Cell 15: 687, published in 1978, authored by Maniatis et al.
Isolation of structural genes from a library of NA.

One of the main tasks in isolating desired genes from r-tum libraries is to filter these libraries for the genes mentioned above.

A useful preliminary filtering method to identify and enrich these vectors containing foreign DNA sequences or genes uses "insertion inactivation." This method is based on Bocna
Journal of Bacteriology, published in 1980, whose authors include
Bacteriolog3') 145: 92
6, the heterologous DNA or gene is inserted into a plasmid vector at a preselected site. Such insertions render preselected plasmid genes inactive. Recombinant norasmid vectors are then identified by filtering the plasmid for loss of specific gene function, in this case loss of conventional antibiotic resistance. Another enrichment method for clones containing recombinant plasmid vectors is described in the 1977 book Nature by Seeburg et al.
) 220: 486 and Ulligh
5cie published in 1977 with authors such as
196:1313, the plasmid is treated with alkaline phosphatase prior to ligation with the heterologous DNA to be cloned. However, these methods are useful for targeting specific desired genes or D
Discriminative localization or separation of fragments of NA is not possible.

Identification and isolation of clones containing specific genes currently requires the use of gene sequence-specific prologues. In one example of such a method, Grunstei
n) and Hognes 1
Proceedings of the National Academy of Sciences, published in 1982.
ings of the Nation+al Aca
DemyofScience) 79: 1844
Bacterial clone or bentone (Bθ
In addition to filtration of bacteriophage plaques as described in Science 196:180, published in 1977 by David Davis and David Davis, natural DNA fragments and known proteins were extracted using differential hybridization. Synthetic NA fragments or genes based on sequence information
A labeled probe consisting of a specific mRNA molecule is used.

The use of gene-specific mRNA molecules includes m
I (NA isolation is problematic; likewise, it is not suitable to use natural DNA fragments as prologues in the absence of conventional cloning of natural gene sequences or DNA sequences.

The use of synthetic NA fragments based on known protein information requires isolation and sequencing of gene-specific protein products. When proteins are separated and arrayed, they become rice fields (
A region of given quality is selected and a gene sequence is generated. The gene sequence is as follows: Agarawl
Agn, ew, Ch, published in 1972 with authors such as
em, Eng, ma, Engl, 11:45
The phosphotriester method described in 1 or Itakura (It
:r, published in 1975 by authors such as Akura).
It is synthesized by the phosphodiester method described in Biol, Chem, 25024592. Combining solid-phase synthesis with the phosphotriester method results in the formation of gag
Nature 29, published in 1981, whose authors include
As described in 2nia56, whole genes such as alpha interferon can be synthesized.

Combining these gloves is an appropriate process. Furthermore, due to the degeneracy of the genetic code, protein genes with unknown sequences are isolated from cDNA libraries using oligodeoxynucleotide zolobes with mixed sequences.

This is a Pro book whose authors include Sugga et al.
c.

Nat'1. Acad, Sci, U, S, A,
78: It is described in 6613 and is in °C. Such a globe is a mixture of oligonucleotides representing each possible codon combination for a given range of amino acid sequences.

c These gloves, which are used only for filtration of DNA libraries, are described in Proc, Nat'1.
Acad.

Sci, U, S, A, 8 D: 11 to 17 nucleotides in length as described in 802, but the shortness and heterotypic admixture of these mixed groves means that there are Like the mammalian r-tums involved, they often lack specificity, characterized by complex probe sequences.

Traditionally, identification and isolation of mammalian genes from r-Tum libraries has required the use of synthetic NA prososins longer than 80 nucleotides in length. This is Anderson (An
Pr published in 1983 with authors such as aer80n)
oc, Nat'1. Acad, Sci, U, S
, A, 8026B5B. The generation of these relatively long DNA probes involves synthesizing multiple types of 12- to 20-nucleotide oligonucleotides, and then linking these oligonucleotides together to form a final probe of more than 80 nucleotides, which is used as a prologue. Obtain DNA molecules. The construction of these gloves is also a time consuming process. Therefore, it is desirable to develop a method that uses homogeneous short 10- to 20-nucleotide DNA sequences as groves to filter r-Tum libraries of mammalian genes.

Once the gene is identified in the rzum library, the bacteriophage or cilasmid recombinant vector containing the gene is isolated and replicated by standard methods to generate multiple copies of the recombinant vector.

However, expression of genes such as interferon contained within recombinant vectors requires several complex and specialized operations. These manipulations are determined by the host cell chosen to express the gene.

The expression of euphytobiotics such as interferons in prokaryotic hosts such as bacteria suggests that both transcription and translation of genes in prokaryotes have regulatory signals that are different from the required signals present in euphytogenes. , requiring the insertion of prokaryotic expression signals.

Multiple prokaryotic regulatory signals include Guaret.
Cell 20, published in 1980 by authors such as nte):
Site-specific insertion of these signals has been demonstrated and successfully used to obtain expression of bona fide plant genes in prokaryotic host cells, as described in 543. Each true plant gene is unique and requires at least a partial sequence analysis of the selected true plant genes. In particular, regulatory signals must be inserted at sites that allow for correct in-phase reading of authentic plant genes. Incorrect, out-of-phase insertion of prokaryotic regulatory signals results in premature termination, thus producing incomplete proteins, or producing nonsense proteins or failing to produce complete, recognizable gene products. Furthermore, the gene must be inserted in the correct 5' or 6' orientation within the expression system.Therefore, it is necessary to define a new expression system within each prokaryotic gene selected to be expressed in the prokaryotic host. There is.

The present invention provides methods and compositions for identifying, isolating, cloning, and expressing novel mature human interferon genes.

The present invention also provides methods and compositions that allow rapid filtration and separation from mammalian gene libraries. In the method of the present invention, a gene having a sequence complementary to the gene or genes to be identified and separated and having a length of about 1
A small 7 nucleotide DNA probe was synthesized and these DNA probes were then labeled.
In a method comprising hybridizing an NA probe to a recombinant vector contained within a host cell, the labeled D
An apparatus for filtering the NA probe prior to addition of the probe to the hybridization solution, using the labeled RDNA probe in the hybridization reaction, and simultaneously rotating and incubating the nitrocellulose filter during prehybridization and hybridization. using a prehybridization solution and an effective amount of dextran sulfate to the hybridization solution, and then identifying the gene to match the selected genomic library and label. In one example of the invention, at least two small labeled DNA probes of about 17 nucleotides in length are used to detect different complementary molecules present in at least four of the eight cDNA α-interferon genes described above. Synthesize to include the target sequence. These DNA probes are then used to filter the human genomic library for the human α-interferon gene.

Further, in accordance with the present invention, in a method of hybridizing a radiolabeled DNA globe to a recombinant vector contained within a host cell and affixed to a nitrocellulose filter, filtration of the labeled probe prior to addition to the hybridization solution is performed. Using at least one type of radiolabeled DNA globe of 17 bases in total, using an apparatus that simultaneously rotates and incubates nitrocellulose filters during prehybridization and hybridization, and adding 1 to 1 in the prehybridization solution and hybridization solution.
The novel method consists of adding 0% dextran sulfate.

The methods and compositions according to the invention are useful for isolating complement genes present in low copy numbers from genomic libraries and subsequently producing large amounts of said genes and gene products for clinical diagnosis and therapy. It is useful for and (.

The present invention relates to methods and compositions that allow for the rapid filtration and isolation of known and novel mammalian genes from genomic libraries. A method as described above for isolating genes from a genomic library that requires the synthesis and use of large DNA probes of more than 80 nucleotides or heterogeneous probe mixtures that identify a single site of the gene to be identified and isolated; In contrast, the method of the present invention uses small NA probes of about 17 nucleotides that contain sequences complementary to mutually different regions of the gene or genes that are to be identified and isolated.

The use of small NA probes as described above in the method of the present invention offers several advantages over the previously described methods of filtering and isolating mammalian genes from genomic libraries. Smaller DNA probes are easily and relatively quickly synthesized by conventional DNA synthesis methods, allowing selection of gene regions with high GiC content. In this way, a tighter and therefore more effective binding is obtained with the conventional hybridization filtration process, and individual identification of the desired genes can be made upon binding in a manner that mimics the Hess plating method. This discrimination eliminates the high rate of false positive gene discrimination in common hybridization filtration processes.

In one example of the present invention, two types of probes for human α-interferon are used, one of which is manufactured by Bio Logicals, Inc.
(Toronto, Canada) and one to Bachequent Incorporated (Toronto, Canada).
1 Inc., Torrance, Calif., USA).
It was synthesized using θnθMachine). Each probe of 17-base (17-mar) sequence contains eight cDNAs.
This is based on the sequence information of A interferon clone. This is Nature 290: 20, published in 1981 by Goθddel et al.
It is described in . Alternatively, sequence information useful in the generation of small NA probes can be obtained from gene-specific mRNAs.

In this case, the probe based on the mRNA sequence is
It is synthesized using the enzyme reverse transcription, which creates a cDNA copy from. This is a paper on molecular cloning (MolθClar
ning). For probe A,
Complementary sequences were present in at least four of the eight aforementioned cDNA α-interferon genes, but only one base mismatch existed for three of the remaining genes and two for the last gene. There were two base mismatches.

For probe B, there were complementary sequences in at least four of the eight cDNA α-interferon genes described above, whereas there was only one mismatch in the other four genes. The α-interferon sequences homologous to the two probes had G:C rich sequences separated by approximately 50 bases. The sequences of these probes were as follows.

A B 5'CAGCCAGGATGG4GTCC3'5'C
CTCCCAGGCACAAGGG3'prosoA is
J, Biol, published in 1975, authored by Tōyō et al.
It was synthesized and purified by the solid phase phosphotriester method as described in Chem. 250:4592. Probe B was manufactured by Bio Logicals, Inc. (Toronto, Canada). These probes were manufactured by BL Biochemicals (p,:[,.

T4-infected E. coli polynucleotide kinase from Biochθm1cals and New England Nuclear
) (Massachusetts, USA) 5000
It was labeled with 5' at both ends using (r62p)/mM of (r62p)-ATP (30' V C nanograms).
Probe A or Probe B was suspended in 10 μl of water. This water contains [1,5M) lysate with a pH of 7-6.
HC1,Q,"l MMgC12,5Q mM dithiothreitol, imM spermidine and imM Na
10 μl of 105× polynucleotide kinase (PNK) buffer consisting of 2 EDTA and 10 μl of [γ”'p]
-ATP (0,002tt mo17m) and 2
0 μl of water was added. Then 1 μ4 (5 units/μl) of PNK'i was added and the mixture was incubated for 20 minutes at 67°C. After this incubation, an additional 1 μl of PNK i was added. Alternatively, a non-isotopic label such as biotin coupled to thymine is typically added to the DNA during synthesis.
Let the probe work together. Biotin strongly binds to avidin, which is a 68,000 dalton protein.
g7) 126: 32 and Leary (Lθary
), published in 1986 by authors such as Proc, Nat.
'1. Acad, Sci, U, S, A, 8Q:
It can be detected by several biochemical means such as those described in 4045.

5 DN used to identify and locate a given gene sequence or its fragment or allele in a DNA library.
The minimum length of the A probe is determined by both the binding energy of the probe and the signal of the probe label used to detect the formation of stable hybrids.

At least 11 nucleotides are required to produce stable hybrids under the conditions conventionally used in hybridization procedures. In the method of the present invention, the length of the DNA probe that can be detected by a radioactive label associated with the probe is preferably small (at least about 17 nucleotides long).

The end-labeled RDNA probe was then stained with Sephadex (5e
phadex' ) G-50 column is used to separate it from uncooperative [γ~32p]L ATP by gel filtration techniques.

These towers are, for example, 3-OX 0-6cm and 2m13
into a disposable pipette. These towers are -7
Equilibrate with a buffer consisting of (trisoxymethylaminomethane)-HCt and 1mM Na2EDTA (sodium ethylenediaminetetraacetic acid) for 1 day. [Approximately 40 fragments of 1,057 d/fragment were collected, and these fragments containing the first peak of radioactive material eluted from the column were pooled and an appropriate amount of 2 Q
Final buffer concentration 6XNETiC by adding ET
, 4 verses. This buffer contains 9.9 M NaCl and t5
．． Q mM Na2EDTA and 0.09 M pH 7,
5 of Tris-HCt. End-labeled DNA probes were then manufactured by Gelman Sciences, Inc.
Filter with a 0.2μ ACROD Knee Filter Φ filter purchased from (Ann Arbor, Michigan, USA). These probes can be adjusted to pH 7 with this filter.
, 5 of 10 mM Tris-HCt, i-Q m
Pretreatment was performed by filtering 50 to 100 μ9 yeast transfer RNA purchased from Eoehringer Mannheim, West Germany in M Na2EDTA. The specific activity of the two probes is 1 to 5 x 108 counts/min (cpm
)/μll DNA, the preferred specific radioactivity is approximately 3
to about 5 x 10 days cpm/4 DNA.

Tom Maniatis (Tom Maniatis)
Harvard College, Boston, Massachusetts, USA (1978), authored by Lawn et al.
It is written in cell 15:1157 published in
The Star Destum library in Lambda Charon 4A was then filtered separately for each probe.

Escherichia coli DP, 50C Dr. Nidwin, M.D. Andersoff Hospital, Hyuston, Texas, USA
Dr. Win C, Mur
Phytolytes were pre-infected with λ Calon 4A' containing the library in NZ amine medium prepared as follows. 109 1iZ amine type A [Humco Cheffield, Memphis, Tennessee, USA (Hum, k.

5Mff1θla), 1 yeast extract (Difco Laboratories, Detroit, Michigan, USA) and 5I Na
C1 and 29 MgCl2.6H20 were dissolved in double distilled water to a final volume of -11. This medium is
Autoclaved at 21°Cz 15 pθ soil for 25 minutes. Thymidine and cyamopimelic acid (DAP) filter-sterilized with 0.049 thymidine and 0.19
It was added to a final concentration of DAP/1. Preinfection was generated by incubating the λ Charon 4A bacteriophage with E. coli for 15 minutes at 37°C. The infected E. coli was then incubated with NZ amine medium and 1.2
Fissure Bactoaga (Difco, Detroit, Michigan)
IX 104 plates per 80c1fL2 dish in NZ amine medium containing 0.7% Sea Kem 7Galose (FMC, Rockland, ME, USA) on prepared plates containing Plate at the appropriate concentration of pfu-forming units (pfu). These cultures were then incubated for 6 hours at 67°C.

All of the hybridization from about 1.8 X'103 to about 1.9
80 (1977)] using the plaque hybridization method. This can be done using the following modification. Namely, (1) the use of two sequentially different labeled probes, and the use of (H
) separation of said probe from contaminants and/or interfering substances prior to addition to the hybridization mixture; and (a) apparatus for rotating filters and controlling environmental temperature during prehybridization and hybridization. and ll) use of an effective amount of dextran sulfate in the hybridization solution. 1.8 x 105 pfu'a: When filtering, there is a 64% probability that a given DNA sequence will appear. This is described in A Laboratory Manual on Molecular Cloning, authored by Maniatis et al., published in 1982 by Cold Sphring Harbor Laboratories, New York City, New York.

In another example, the Benton and Davis plaque hybridization method includes the following variations. That is, (1) one type of D that is approximately 17 nucleotides in length and matches the site within the gene, gene fragment, or multiple genes or multiple gene fragments to be confirmed;
(It) separation of said probe from contaminants and/or interfering substances prior to addition to the hybridization mixture; and (Q) rotation of the filter during prehybridization and hybridization. (IV) the use of an effective amount of dextran sulfate in the prehybridization solution and the hybridization bath;
) Parallel hybridization by the probe on the filter of a pair of plaques to be filtered.

Separation of the radiolabeled RDNA probe from contaminants and/or interfering substances prior to addition to the hybridization solution can be accomplished, for example, by filtration to remove particles having a solution diameter larger than the probe. This filtration ensures the removal of substances that would normally cause non-specific adhesion of these probes to the nitrocellulose filter and reduces the puncture ground observed when each probe is pre-filtered and unfiltered. . These substances are hereinafter collectively referred to as interferon substances. A preferred method of separating interferon substances from said glove comprises:
An inert filter with a pore size of approximately 0.2 μm or 2 μm skin is used. DN from such interferon substances
One example of such a filter useful for separating A-globes is the 0.2 μAC' manufactured by Shellman Sciences, Inc. (Mann Arper, Michigan).
This is ROD Engineering Sc @ Filter.

Dextran sulfate was added to the prehybridization solution or hybridization solution to reduce unspecific binding of DNA globules and remaining interfering substances to the nitrocellulose filter. Furthermore, the rate of cooperation of nucleic acids is accelerated in the presence of dextran sulfate. This is because the nucleic acid, in this case DNA globe, is excluded from the solution volume occupied by this polymer. The effective concentration of DNA globes is therefore increased. An effective amount of dextran sulfate is added to the hybridization solution to reduce the final solution volume and increase the solution concentration of DNA globes present in the hybridization solution. This increased DNA globe concentration allows the complementary D contained within the DNA of each o-tetonitrocellulose filter to
This will likely maximize the contact between the NA sequences and increase the rate of cooperation. Pro, whose authors include Wahl et al.
c, Nat'1. Acacl, Sci, 76:
3683 (published in 1979), this collaboration ratio is 10.
reported a 10-fold increase in the presence of % dextran sulfate. A preferred amount of dextran sulfate in the prehybridization solution and hybridization solution is about 10% (W/V).

Dextran sulfate has a marginal hybridization rate in detecting the sequences of interest, but its viscosity and difficulty in handling make it considered useless for many applications. This is described in the Molekiller Cloning (1982 publication) page 325 of the A Laboratory Manual, authored by Maniathes et al. of Cold Spring Harbor Laboratory. The use of constant rotation during prehybridization and hybridization reactions eliminates the viscosity problems associated with the use of dextran sulfate (and is thought to enhance the detection of the genetic sequences described in this invention). You can find out how to use

Paired nitrocellulose filter replicas were made from each plate according to the method described by Benton and Davis, Science 196:180 (published in 1977).

BA85SD nitrocellulose filter, Schreiker & Schnill Association Inc. (5ch
leicher and schuell, engineering nc,)
(Keene, Pushcher, USA). Next, copy the nitrocellulose filter, 3
MM watt? Whatman paper was denatured with alkali and treated with 0.5N NaOH and 1.5M NaCl.
Tris-HCj saturated for min, PI″17.51M
Neutralize with paper saturated for 5 minutes with 0.5M pH 7.5.
Neutralized again with paper saturated with Tris-HCI and 1.5 M NaCl for 5 minutes. Next, the nitrocellulose filter was heated at 80°C for 2 hours in a National Air
3 Noma ・NozrN口+(＾r+al Δηη1
4nnn^) Heavy ■6 - 20 to 25 inches in the city
Calcined under Hg vacuum. Next, the nitrocellulose filter is manufactured by Fisher Scientific (Fis.
Kabak/Scotch (KAP) manufactured by Her 5 Scientific (Pittsburgh, Pennsylvania, USA)
AK/5cotch) Placed in a PAK heat-sealable bag. This bag contains 6X NET and 0.1% weight/volume IR (
w/v) Sodium dotesyl sulfate) (EID
) and Sigma Chemical Company (SigmaCh
A prehybridization solution of 5a consisting of 10% (w/v) dextran sulfate (St. Louis, Mo., USA) was added. Next, this bag is made by Fisher Scientific (Bittsburg, Pennsylvania) and is made by Scotch P.
It was sealed with A-ream patch sealer (Pouch 5ealer). A 50% (w/v) dextran sulfate stock solution from Sigma Chemical Kannomani (St. Louis, MO) is prepared in deionized water and stored at -20°C.

Examples of methods and compositions according to the invention will now be described in detail with reference to the accompanying drawings.

The sealed bag was placed in the rotating apparatus shown in FIGS. 1, 2, and 3, which rotates the bag 360 degrees while creating a controlled temperature environment. A conventional electric motor 10 having a power on/off switch 11, a power cord 12 and a cover 13 as shown in FIG.・Oven (Stable-Therm Gravity 0ve
A suitable fastening in the middle of a common hook 20 such as n) is the attachment by means of a piece 14. Kama 20 is approximately 25 degrees below the outside temperature.
It can maintain a constant temperature in the temperature range down to 0°C.

The temperature inside the pot 20 is displayed by the temperature indicator 30. The front plate 22 located below the front hinged door 23 is
A main power on/off switch 22A for zero, a temperature indication on lamp 22B, and a temperature selection control device 22C are provided.

The kettle 20 is provided with its own power cord 28. As shown in FIG. 2, the shaft 40 is capable of continuous rotation about its central axis 72 and comprises, for example, a metal rod having a first end 41 and a second end 42 . The shaft 40 is
It extends horizontally through the center of the furnace 50, parallel to the floor 51 of the furnace 50, and perpendicular to the inner walls 21B, 24B of the furnace 50.

The position and rotational movement of the shaft 40 is controlled by the second end 4 by means of a bearing 45 mounted on the inner wall 24B by means of a piece 45A.
2 to the inner wall 24B, and proper tightening is done by attaching the bearing 4 to the inner wall 21B by the piece 48A.
Appropriate tightening of the inner wall 21B by 6A is achieved by the bearing 46B attached to the outer wall 21A by the piece 48B.
It is kept in the direction of arrow 74 in FIGS. 2 and 3 by mounting the first end 41 with a driven bevel gear 43. The rotation shown in FIG. 2 of shaft 40 is produced by meshing drive bevel gear 60, driven by electric motor 10, with driven bevel gear 43.

Axial movement of the shaft 40 along the axis 72 is achieved by a collar 47A and a set screw 47B located at the first end 41 of the shaft 40 and a set screw 47B located at the second end 42 of the shaft 40 as shown in FIG. This is prevented by the collar 44 and set screw 44A. A platform 70 for attaching the sealed hybridization bag 65 to the shaft 40 is shown in FIGS. 2 and 6. Platform 10 is formed from any suitable material, such as Styrofoam™ or wood. A sealed hybridization bag 65 is secured to the platform 70 by means of a fastening piece 66 such as a needle, bottle, nail, clasp or the like, without tearing the fluid-filled portion of the bag 65. The platform 10 is attached to the shaft 40 by a suitable locking piece 67, such as a locking piece 67, such as a locking bolt, bolt, and an attachment, as shown in FIG. 2, so that the platform 70 rotates as the shaft 40 rotates. In one method according to the invention, the bag was rotated at 10 rpm for about 12 to 24 hours at 67°C. Rotation of the filter in the apparatus of FIGS. 1, 2, and 6 optimizes movement of the prehybridization solution relative to the nitrocellulose filter. After incubation, the bag corner was cut open and the prehybridization solution was removed by suction. To the same 2 ml solution as the prehybridization solution, ten radiolabeled proso was added to a concentration of about 1 to about 10 ng/mJ. In this case, the preferred probe concentration is approximately 2 n
g/ml: solution.

In a modified hybridization method using a single DNA probe, in this case probe A or probe B, a single probe is added to the dual filtered hybridization solution. In this example of the invention, both Proso A, B were used, with only Probe A added to one filter hybridization reaction and Probe B added to the dual filter hybridization reaction.

Hybridization was carried out under constant rotation at 67° C. for about 20 to about 24 hours as described above in the prehybridization incubation. After incubation,
Remove the nitrocellulose filter from the bag and apply 6XSSC
into an appropriately sized Pyrex glass dish containing 250 ml of wash buffer consisting of: In this case 1X SSC
contains 0.15M sodium chloride and 0.015M<sodium citrate. Each filter is 250m at room temperature
Lightly wash in 1 wash buffer (2 washes with rocking)
Wash 4 times for 0 minutes each time, then wash the skin with 1% SDS g containing 250
Washed 4 times for approximately 1 hour in 6.times.ml of 6.times.SSC at a temperature of 45.degree. These nitrocellulose filters were then air dried at room temperature for approximately 60 minutes. Next TM T
M Kodak MAR film and two reinforced screens and Dupon Cronex Hi PlusTM
Autoradiography was performed using L1-M at 80° C. for about 64 to 72 hours. Filter and upper Kodak TMM
The AR film was sandwiched between two reinforced screens. D
Bacteriophage plaques hybridized to the NA probe were identified by a common autoradiographic signal with a dual filter, each hybridized to probe A1B.

Single DN during corresponding hybridization with double filter
By using and comparing the signals of the A probe or two sequentially different DNA probes, spurious positive hybridization signals that typically occur in a single hybridization reaction can be eliminated. Subsequently positive plaques were separated by multiple cycles of Norak spermatozoa as described by Benton and Davis, Science 196:180 (1977).

Initial filtration of Lambda Charon 4A genomic library 8
Isolated phages hybridized to both floes A and B by bacteriophage plating at high concentrations of approximately i x 1[]' pfu of 06IIL2 were
A second infection and plating with a lower concentration of E. coli DP, 50 at about 500 pfu per 80 CWL2 was performed as described for the initial filtration.

The bacteriophage plaques were then filtered at °C as described above. This second filtration yielded six bacteriophage plaques hybridized to both Proso A and B, designated λ/2, λWA and λ10-5, respectively. Multiple copies of λ/2, λWA, and λ105 are provided in the Laboratory Manual by Maniathes et al., published in 1982 by Cold Spring Nova La Horatories, New York City, New York. It was produced by a method described in Molecular Cloning.

Localization and Isolation of Specific Genes Contained in Recombinant Vectors Plaques hybridized to both Proso-AXE clones Japanese clone A F77, E-FI'wA, and E-F105 were then further analyzed to identify one or more specific genes. In this example, human α-interferon was localized and isolated.

Localization of a specific gene or gene fragment contained within a recombinant vector is performed, for example, by cleaving the recombinant vector with a restriction endonuclease enzyme. In this case, any restriction enzyme or its recombinant enzyme can be used. Ideally, the restriction enzyme used is the gene to be isolated or its -
8 must not be destroyed. Restriction enzymes used to localize and isolate genes therefore target the gene site, if known;
Selection is made according to the gene sequence and the cloning vector considered.

“Records of Biochemistry and Biophysics J2” published in 1986
A previous study of the α-interferon gene isolated from c DNA libraries according to the best paper of 21:1 °C showed that the α-interferon gene was approximately 4 in length.
00 nucleotides and the gene sequence contains BamH engineering or E
It is shown that it does not contain a coRI restriction endonuclease site. These restriction enzymes are therefore suitable for use in isolating the alpha interferon gene from plaque purified clones. Restriction endonuclease fragments of about 1 to 2 kilobases (kb) containing the gene are desirable in order to recover the entire gene from the purified bacteriophage and facilitate its subsequent insertion into cloning and expression vectors. . These enzymes can also be used to separate interferon gene fragments, tandem genes or alleles of this gene, or all of these. Alleles in this case consist of different gene sequences that are less than or equal to 1% of a given gene sequence.

In this example of the invention clone 1F 771. IFWA
and IF105 by restriction endonuclease cleavage with the restriction enzymes BamH, Ddel and EcoR.
, two end-labeled α-interferon probes A,
B was analyzed to identify restriction endonuclease fragments containing the α-interferon gene or portions thereof. The restriction enzyme mentioned above is Sadeene (5outhern).
Published in 1975 by J, Mol.

Biol, 98:505, Bethesda F (esearc
Purchased from H Laboratories, Inc. (Issersburg, MD, USA).

In particular, bacteriophage DNA from clones was obtained from Cold Spring, New York City, New York.
As described in Chapter 6 of the A Laboratory Manual by Maniathes et al. published by Harbor Publications in 1982, the following Restriction endonuclease BamH, EcoR or 1)
Each was digested separately by one of del. Restricted DNA was determined by Suzanne: J, Mo1. B111.98
: 503 (published in 1975), 9 Q mM Tris base, 9 Q mM l'l
One portion of agarose (Sθa Kem) from F'MC Corporation (Rockland, ME, USA) was added in a buffer consisting of oxalic acid and 2.5 mM NaBDTA.
% weight/volume (w/v) by agarose gel electrophoresis and transferred to nitrocellulose. Probes A and B were then hybridized separately to the °C-restricted DNA-containing nidocellulose in the same manner as described above for bacteriophage filtration to identify these fragments containing the α-interferon gene sequence. Appropriately labeled α
- Interferon-specific mRNA or cDNA is α
- Alternately used to separate the restriction endonuclease fragment fa0:R containing the interferon gene sequence. As shown in Example 1 below, clones λ77 and λWA were divided using the restriction enzyme HaoR, resulting in approximately i and 1 clones, respectively.
3kb and 2. A DNA fragment of Qkb was obtained. These EcoR-engineered restriction fragments are of appropriate size for efficient gene cloning and subsequent gene expression.

Cloning of Separated Genes Once a gene, gene fragment or allele has been isolated, the gene is inserted into a suitable cloning vector which provides a means for replicating the gene. Any suitable loning vector with a labeling function may be used.

This vector includes, for example, Versima field (He
Proc, Nat'1 by rshfield) etc.
．． Acad, Sci.

Col'E 1 and Bolivar as described in U, S, A71: 3455 (1974)
pBR622, described in Zone 2:95 (1977) by Oberon et al., and pB, described in Gene 4:121 (1978) by Oberon et al.
Scene with Rro25 and Rao etc. 7:79
(1979) and an E. coli plasmid vector containing pKc, 7, as described by Loene (1979).
Gene 10: 249 (1980) by Charon λL47.1 and Metssing (M
Gene 19: 269 (1
There is a colonic bacteriophage petator containing M 13 mp 8, which was described in 1982).

genes to generate recombinant cloning vectors,
Inserting a gene fragment or allele into a cloning vector involves cleaving the cloning vector with restriction enzymes or altering these ends to generate complementary sequences that produce cohesive ends complementary to the ends of the gene; This can be done by joining the two pieces next. If the gene or gene fragment does not have cohesive ends or following restriction yeast splitting, the gene is plant-end ligated into the split cloning vector. Such loning vectors can also be used to replicate entirely synthetic genes.

Once ligated, the recombinant cloning vector is used to transform or infect a suitable host cell. The exfiltrated host cell must be capable of replicating the recombinant clone, preferably at a high copy number.

In our example, Gene by Metzing et al. 19:269
(1982) was selected as an o-ning vector.

The M15mp8 vector can be separated into double-stranded (ds) DNA types and single-stranded m (ss) DNA types. Isolation of the DNA recombinant vector facilitates subsequent cloning of expression vectors as described below. Furthermore, the MpK of the 5sDNA recombinant vector facilitates isolation of a recombinant vector containing a gene in the appropriate 5' to 6' orientation for gene expression. Furthermore, M 15 mp 8 is compatible with genes or gene fragments ranging in length from 1 to 4 kb, ensuring cloning of the entire gene sequence.

In our example, each bacteriophage clone λ
1 kb and 1.3 kb EcoR from Shield A and λ/2
The I fragment was placed within the unique FCOR site of the M13mp8 vector, and gene 19:269 was generated by Messing et al.
('1982), a recombinant cloning vector was generated. Clones λWA and λ
/2 also separated Ec.

The R engineering fragments are α-interferon gene I.
Referred to as FWA and IF 77. M13f11p'8
I FWA or IF 77 into a cloning vector
The insertion was done as follows. Approximately 1.0μg M6m
p8 DNA in 50 mM Tris-H at -7,5
(4 and 0.01MMg('J2 and 5Q mM Na(
J, 1mM dithiothreitol, 100μg bovine serum albumin, etc., containing 0.5 to 1.0 units of EcoR.
, I (Bethesda Rizu Laboratories, Inc., Idesburg, Md.) with 100 μl of digestion buffer. The mixture was incubated for 60 minutes at 67°C and then treated with 1 unit of calf intracoal alkaline phosphatase (Boehringer Mannheim, West Germany) for 60 minutes at 37°C. The mixture was then subjected to two phenol extractions to remove the enzyme, followed by one ether extraction to remove residual phenol, and then precipitated with ethanol. 10μg
I ffA and IF 77 of M1 by EcoRI
Digested with EcoRI in the same manner as the mp3 and mp8 digests. Digested Mp 13 mp8, I FWA and I
Each F77 DNA was then electrophoresed separately in a 1% low melting temperature agarose del. Approximately 4 Q each
2 kb of EcoRI containing Ong DNA] Limited IF
The WA fragment and the 1.8 kb IF 77 fragment are defined as Molecular Cronin rK in A Laboratory Manual by Maniathes et al., published in 1982 by Cold Spring Harbor Publications, New York City, NY. It was separated from agarose and purified.

The M15mp8 DNA was digested with -7,6 [1,066M Tri, 5-H (4 and 5.Q mM
MgC4 and 5.0 mM cidaodoreitol and 1,0
Each EcoRJ restriction fragment was mixed in 20 μl of binding buffer consisting of mM ATP and 2 units of T4-induced DNA ligase (Bethesda Research Laboratories, Inc., Caithersburg, MD). The reaction mixture was incubated at 12°C for 16 minutes. M i 3 formed in this way
Recombinant cloning vectors containing mp 8-WA or M13mp 8-77 were then transfected with rM1o6 in canine boxes manufactured by Pethesda Research Laboratories, Inc. (Daithersburg, MD). It was used to generate

20 g of Bactodrypton, 10 g of Bacto yeast extract and 5 g of Na(4) dissolved in water to a final volume of 1 ml and sterilized by autoclaving as described above. Transfection of E. coli JM1D3 grown to a diameter was performed using Methods In Enzymology by meshing.
n Enzymolog'Y) 101:20(1
This was carried out by a method described in 1983). Briefly, rM103 cells were pelleted by centrifugation and cooled on ice with 5 Q mM Ca(J).

and then incubated on ice for 20 minutes. The JM 105 cells were then re-pelletized by centrifugation and resuspended in 5 [1 mM CaC] cooled on ice, including 0% of the original cell culture volume, and then placed on ice. Next, about 1 to 10 r1g of recombinant cloning vector DNA was added to Ca(J2-treated JM
i03 cells and this mixture was incubated on ice for 60 minutes. Transfected cells were heat-shocked at 42°C for 2 min and then treated with 0.7% Sea
2X YT medium containing Kem agarose and 10tt
2% (W/V) of 50 μt dissolved in dimethylformamide with l10100mM storage Innov0rovirthiogalactobiramoside CIPTG (Sigma Chemical Company, St. Louis, MO) and 2% (W/V) of 5-promo4-
Chloro-6 indoyl B-D-galactoside (x-ga
1) was added to 2'XYT top agarose consisting of 2,5 rnl.

The transfected cells were then 0.1-
of JM103 cells in logphase medium. This mixture was stirred and poured onto agar plates consisting of 2X YT medium containing 1.2% Bacto agar. These culture plates were incubated at 67°C for 12 to 24 hours.

M13 bacteriophage plaques of approximately 100 y and 500 y were then isolated. The bacteriophage DNA from each separated plate was then individually filtered in the presence of α-interferon-specific DNA by hybridization with one probe A of the end-labeled globe as described for the first bacteriophage filtration. did. M 13 mp 8 bacteriophage DNA hybridized to probe A was isolated as described above and subjected to restriction endonuclease digestion with EC0RI to ensure insertion into the M 13 mp 8 vector. Recombinant M13m
Engineer the α-interferon gene sequence at the correct site by positive hybridization of probe A to p8 vector DNA.
Only those recombinant vectors containing A or IF 77 are identified.

DNA sequence analysis of isolated whole genes or gene fragments was performed by Mayam and Gilbert to identify isolated interferon-specific gene sequences and facilitate subsequent insertion of these gene sequences into cloning expression vectors. Mesotsuzu by (()ilbert)
This was done by sequencing each separate restriction endonuclease fragment generated from isolated clones by the DNA sequencing method described in In Enzymology 36:271 (1980). All 5sD of recombinant cloning vector
Proc. by Sangerl et al. using interferon probes A, B or selected restriction fragments as primers for DNA sequencing reactions.

Nat'1. , Acad, Sci, U, S, A, 7
4:5463 (1977) using a dideoxy sequence system.

Approximately 80% of the gene sequence IF77 partial DNA sequence (IF77 was isolated from a human cDNA library as described in Annals of Biochemistry and Biophysics 221:1 (1983) by Plague Force) However, the complete DNA of I FWA shown in Figure 4 strongly suggests that it is the same as human α-interferon L.
The sequence is designed to represent a human α-interferon gene sequence not previously recorded in the available scientific literature, and thus represents a novel human α-interferon gene. The complete IFWA gene sequence shown in Figure 4 represents both the signal peptide (8) sequence and the mature protein (m) sequence. The signal pef0tide gene product guides the secretion of mature interferon proteins from interferon-producing cells. The isolation of the known human α-interferon gene IF 77 demonstrates the efficacy of the methods and compositions of the invention as described above in isolating such a gene as α-interferon from a denom library. Furthermore, the isolation of the novel human α-interferon gene I FWA, which has not been previously identified or isolated by conventional techniques, may indicate the sensitivity of the inventive methods and compositions.

α-interferon gene IFWA & HiIF 7
As shown in Figs. 4 and 5, sequence analysis of I
a single βuI restriction endonuclease site in the signal peptide site of the FWA and IF 77 trough genes;
The presence of a single EcoR1 site of about 500 base pairs downstream from the terminal code/cod of each of the genes in Ml3mp8 DNA was identified. To locate the restriction sites of AluT and ECOR, fully mature IFWA and IF 77 were isolated from the M16mp 8-tuplet cloning vector by double digestion of these vectors with AluI and EcoR,T. I can do it. 1 kb generated by the 2M digestion described above: [FWA, D
The NA fragment is then inserted into an appropriate expression vector.

Insertion of the α-Interferon Gene into an Expression Vector As described above, a suitable expression vector carries the necessary transcription and translation signals for the different gene product in the selected ID cell. , along with a labeling function for identification of these expression vectors into which the gene sequence has been inserted.

Several expression vectors have been described for heterologous gene sequences in prokaryotic host cells.

In the example of the present invention, Slosocom (5locoI11be)
Proc, Nat'l Acad, Sci.
The expression vector M13mp11 was selected, as described in , U.S.A., 79:5455 (1982). In the M13mp11 expression thread, a heterologous gene in the LacZ gene fragment contained within the expression vector, in this case IFW
Insert A. Insertion of the gene into the LacZ gene fragment is carried out by combining the Lac Z gene fragment contained in the expression vector with the Lac Z gene fragment contained in the sacchromatin DNA of the large intestine taJM103 (4 main fibers 11'fl). Breaking normal complementarity, the host cell is no longer able to metabolize the lactose present in the bacterial growth medium.
E. coli JM103 containing the cZ gene fragment without insertion into M16mp11 can metabolize the lactose present in the bacterial growth medium, and E. coli JM103
VC produces unique blue plaques when grown on agar containing 2X YT medium. The large intestine infected with M13mP11 containing the gene inserted into the LacZ gene fragment [the plump eyebrow color of JM103 is due to the fact that the large intestine is 2X
When grown on YT medium, it becomes dry. Therefore M16
Positive intrusion of the heterologous gene into the recombinant M1
Infection of E. coli JM103 with the 3mp11 expression vector was then identified by colorless 7° Lark formation on 2x YT agar.

In the example of the present invention, the IFWA gene fragment is extracted from a 1v15mp8 cloning vector containing this gene using the restriction enzyme Alu
It was separated by double digestion with E. coli and EcoRI. A of recombinant M13mp8 cloning vector
Digestion with lu■ leaves a plant end at one end of the 1 kb α-interferon gene, but Ec
Digestion with oRI produces a staggered end at the other end of the i k,b gene fragment. By creating mutually different ends on the 1 kb I) NA fragment, insertion of the overpressure gene orientation strip into the expression vector is greatly facilitated. The digested DNA was then analyzed by Suzuin J, Mo], Bi
Preliminary agarose gel electromobilization in 1% agarose (Vl/V) was performed as described in J.D. ol, 98:5D3 (197,5).
bDNA fragments were isolated.

The presence of the 1 kb IFWA gene fragment was determined by the presence of the 1 kb IFWA gene fragment in J, Mo1. Biol.

98+505 (1975). Next, the 1 kb IFWA gene fragment was produced by Fuke and Tho.
J, Mo1. Biol, 52:
395 (1970), and from this agarose gel the expression vector can be inserted into the M15mp11 vector. In the first variant, I-Ichua 292 by Etsuji et al.
: 756 (1981) are similarly inserted into the expression vector.

As mentioned above, the heterologous gene contained in the expression vector must maintain the overpressure reading frame of the heterologous gene sequence by inserting the front 4-self heterologous gene into the vector. In an example of the present invention, sequence analysis of the Mlomp11 expression vector revealed that the expression vector contained a single restriction endonuclease Hi in the LacZ gene fragment as shown in FIG.
Single-KcoRI is shown downstream from the nc 11 site. Restriction enzyme Hin of M13mp11 expression vector
M13m by digestion with c II and Eco RI
The p11 expression vector - has the added advantage of producing plant ends at the site of Hincl digestion and staggered ends at the site of vector DNA K Eco Rl digestion.

In this case, both ends of the i kb IFWA fragment are ligated into the expression vector [L, as shown in FIG.
kb, an in-phase matchmaker for TFW A DNA M pieces is created.

Therefore, approximately 100 ng of M15mp11 as DNA obtained from BL Biochemicals (Milwaukee, Wis., USA) was obtained from Y.
Double digestion was performed with 1 to 5 units of Hinc II and 1 to 5 units of EcoRI in whitening buffer. The digested DNA was then precipitated with ethanol and plated in 1% (w/v) agarose as described above. Divide by X shift, smaller Hinc 1
- Separate the desired larger linearized M15mp 1i Hacte IJ phage DNA fragment from the Eco'RI restriction endonuclease cleavage fragment.

The larger linear M1. '+ ml) 11 The part of the gel containing the DNA is
Mol.

Biol., 52:395 (1970). The 1kb IFWA DNA fragment is a 2 year old T4
Mix 10 ng of each DNA in 100 μl of binding buffer in the presence of inducing ligase, incubate this mixture for 17 h at 12 °C, and then remove the DNA with ethanol.
Linear M 13 mplI DNA by precipitating
inserted inside.

The precipitated DNA was then purified by the method described by Messing in Methods in Enzymology 101: 20 (1983), manufactured by Pethesda Research Laboratories, Inc., Ithersburg, Maryland. Recombinant M13 by transfecting E. coli, TM I
Filtered for generation and presence of mp11 expression vector. The transfected E. coli JM103 was 2
Grow in agar containing X XT medium for 20 hours at 37°C.

Six unrelated clear plaques were identified and phages were isolated from these plaques and then replicated to generate six separate IFWA-expressing clones.

DNA samples from six buildings were collected at the sites shown in Figures 6 and 7.
It was divided separately by coRI and Hindl. It was confirmed that the 1 kb IFWA gene fragment was present in all six clones.

Mature human α-interferon WA (ff FW
A-M) to minimize antigenic disturbances that may occur during isolated clinical urine test training.
To optimize the clinically functional use of gene products,
Removal of the portion of the Lac Z gene originally isolated along with the human α-interferon WA gene sequence in the expression vector and the signal peptide coding region of the gene that normally encodes for secretion of interferon from interferon-producing cells. is desirable. These areas are shown in FIG. Removal of the LacZ and signal peptide regions can be achieved by dividing the protein-negative portion of the gene from the resulting fusion peptide following expression of genes containing these sequences, or by removing the LacZ and signal peptide regions from each gene prior to expression. This is done by removing the sequences or by dividing the undesirable gene or protein sequences by the bacteria expressing the fusion gene.

Removal of these peptide or gene sequences produces mature interferon protein or mature interferon protein, respectively.

In our example, the Lac Z and signal peptide gene sequences are linked to the final mature gene (IFWA,
-M) was removed prior to cloning.

For removal of each gene sequence of LaCZ and signal peptide,
Oligonucleotide-directed mutagenesis was used. Science 209 by Wallace et al.:
1396-1400 (1980) and Schiller (Zol
ler) and Mentazo by Smith (Sm1shiL)
In Ench Seven Logi-100: 468-501 (19
A similar method was described in 1983).

A 25 base oligonucleotide spanning the region upstream from the initiation codon ATG and downstream from the N-terminus of mature interferon was obtained from Bachem-Gentec, Inc. (Torrance, California, USA). I bought it. Or 2
The 5-mer may be synthesized by the phosphotriester method or the phosphoramicite method. This oligonucleotide is 2
It consists of a 5-mer sequence.

5'AGGCAGATCACACATAFCTGTT
TCC3' This 25-mer formed a #: box shadow against MI3mp11 phage DNA containing the human α-interferon WA gene. The above recombinant DNA phage is hereinafter referred to as interferon WA Lac Z-conjugated phage DNA, as described below. Single chain interferon WA
25-ma bound to LacZ fusion phage DNA
r is for this combination, Bethesda Research Labora) IJ's.

DNA polymerase Flenow (purchased from Inc., Gaithersburg, MD)
A total interferon WALac Z fusion phage with the LacZ and signal pezotide DNA fragments excluded using a 3-deoxynucleoside triphosphate purchased from BL Biochemicals (Milwaukee, Wis.). D
Serves as a zolimer in the synthesis of the second DNA strand complementary to NA. Add T 4 DNA I to the DNA synthesis reaction mixture
By adding J gauze and ATP, a covalently closed two-stranded circular DNA molecule with a single-stranded 57 base loop can be formed as shown in FIG. This newly synthesized D
NA is then used to generate two distinct bacteriophage populations. One population still contains the 57 base sequence, and the second bacteriophage population does not contain this sequence. This ninth group must contain the IffA-M gene.

As shown in Figure 9, DNA containing a 57 base sequence is Hin.
A bacteriophage containing the IfA-M gene, which contains the IffA-M gene and the IffA-M gene, contains the restriction endonuclease sites H.
It must contain no indl or Pstl restriction enzyme sites and only one Ava■ site.

However, confirmation of the presence or absence of these restriction endonuclease sites in the DNA of the resulting bacteriophage population requires
These enzymes only cut double-stranded DNA, so 2
Requires isolation of full-stranded replicating (RF) bacteriophage DNA. That is, removal of the Lac Z gene portion and signal peptide gene sequence and concomitant isolation of the bacteriophage vector containing the IFWA-M gene were performed as follows. 25-mar as interferon WA La
A double-stranded covalently closed circling DNA containing a single-stranded 57-base loop was synthesized by hybridization to the cZ fusion DNA.

This newly synthesized DNA was used to generate a transfection of E. coli to generate two populations of bacteriophage. One population contains interferon WATJaCZ fusion DNA and the second population contains I
Contains the FWA-M gene. The RF” DNA of each of these bacteriophage populations was then isolated by HindllPs.
tl and Aval to determine which isolated bacteriophage contained the IffA-M gene. The bacteriophage containing the IFWA-M gene was then used to generate multiple copies of the IfA-M gene by infecting E. coli with the bacteriophage. Bacteriophages that are isolated after E. coli infection are isolated in the first step, and the DNA of these bacteriophages is
A isolates the sequence shown in FIG. 5 and confirms the presence of the TFWA-M gene.

25- as a primer for the synthesis of the DNA strand of at
A typical reaction used to generate a double-stranded circular DNA molecule with a 57-base single-stranded loop using mer is performed as follows. Approximately 5/1 g of one interferon WA La-c Z fusion phage DNA is 200
mM Na(J) and 15 mM Tris at pH 7,4.
H.C., I.

and i [approximately
It was boiled for a minute and immediately cooled to 06G. After cooling to 0°C, 0.5μ/17) 100mM ATP and 5mM each of the four deoxynucleotide triphosphates
5 μl (5 units) of Flenow fragment of DNA polymerase I and 5 μl (10 units) of T from Pethesda Research Laboratories (Gaithersburg, MD).
4. Generate 5 μl of solution containing DNA ligase.

The reaction mixture thus produced is 12 to 2
Incubate for 4 hours at 12°C.

The double-stranded covalently closed circular DNA molecule containing the single-stranded 57-base loop produced by the above synthesis reaction is then converted into the mature interferon gene by first generating multiple copies of the above gene sequence in the large intestine. (rFWA, -M) was used as a source of sequences. E. coli JM103 was transformed with the newly synthesized covalently stranded DNA using the same calcium chloride method as described above in the cloning of isolated genes.

M1 containing the interferon gene sequence as described above
The 3mp11 vector is a single-stranded DNA bacteriophage. Transformation of Escherichia coli with the double-stranded DNA form of the interferon-containing vector, in which the single strand contains a 57-base single-stranded loop and the complementary strand does not contain the loop, thus produced two mutually different M13mpH phage populations. do. One population contains 57 base sequences consisting of LaCZ and single penotide gene sequences. The second M15 phage population has lost the 57 base sequence. Therefore, it is necessary to separate the replication form (RF), which is a double-stranded DNA molecule of M13 bacteriophage, and filter the bacteriophage population that contains or does not contain the 57 base gene sequence.

RF DNA from both bacteriophage clusters was isolated and identified as follows. Separate E. coli-infected bacteriophage plaques of M13mp11-infected foci were isolated by transformation by removing the plaques from the plates and growing the bacteriophage-infected E. coli in 2×YT medium overnight at 67°C with agitation. did. The double-stranded FtF DNA of the bacteriophage is described in Chapter 6 of A Laboratory Manual on Molecular Cloning by Maniathes et al., published in 1982 by Cold Spring 7%-Barr Publications, New York City, NY. isolated from infected E. coli.

Next, RF DNA is treated with each restriction endonuclease Hi
, nd I, Pst I and Ava I, and both bacteriophage populations were digested with IFWA-M.
We determined whether it contains the gene. Restriction endonuclease fission reactions were generated as follows. 0.1 MNa
(4 and PH7, 4[], 11MTris-HCJ and 1
Q mM or 5H
bacteriophage DNA was added. This mixture is 3
Incubated for an hour at 7°C. This incubation is followed by the addition of an additional 1 to 5 units of restriction enzyme,
This mixture was incubated again for 1 hour at 67°C. Digestion was then stopped by phenol and ether extraction, and the digested DNA was then precipitated with ethanol. The digested DNA was then analyzed by agarose scale resolution as described in the Examples below.

RF resistant to Hincl I, Pst I digestion.
These bacteriophage plaques containing DNA and having only one Ava II restriction endonuclease site, in this case the mature human α-
It was planned to contain the interferon WA gene.

Bacteriophage containing the IFWA-M gene insert were then propagated by reinfecting an overnight culture of colonic [JM103] grown by agitation at 37°0 in 2XYT medium to contain a large number of IffA-M genes. A replica was generated. Assume that each Prague 1 contains approximately 1011 bacteriophage particles. Each plaque is 1 2 x
Dissolve in YT medium and then add 103 per fnl medium.
The bacteriophages were diluted to a final bacteriophage concentration of 7. Next, approximately 0.1 diluted bacteriophages were collected from the large intestine [JM1060, collected from guests at the wake, Cold Spring, New York City, NY - 1984 from Barr Publications.
Plate culture was performed using the soft top-mounting method described in Molekilla Cloning, page 64 of the A Laboratory Manual published in 2010.

The bacteriophage plaque was isolated again. [FWA-M in the separated M 13 mp 11 DNA
Confirmation of the presence of the gene sequence is performed using the DNA ICHind
11, restriction endonuclease cleavage with Pstl and Ava II, followed by agarose del cleavage as described above, and Proc, Nat'1 by Sanger et al.
．． Acad, Sci, U, S, A.

74:5463 (1977) by determining the nucleotide sequence around the 25-mar junction. The complete sequence of the coding strand of the rFWA-H gene is shown in FIG.

Expression of isolated genes Heterologous or functional genes in host cells In this case: [F
Positive expression of WA or engineered FWA-M is gene-specific mR
This can be determined by analyzing the presence of NA or gene-specific protein products or both. Gene-specific mRNA is mRNA produced and present within the host cell.
or by first isolating the mRNA present in the host cell and subsequently hybridizing said isolated mRNA to a gene DNA probe. Positive identification of gene-specific protein production by host cells can be achieved by physical identification and/or purification of the gene-specific protein, or by analysis of the known activity or function of the desired protein product. is performed by all of these.

In the present example, positive expression of the IFWA and IF'WA7M genes was determined by analyzing virus propagation interferon for gene-specific protein (interferon) activity. However, interferons exhibit multiple additional biological activities that are analyzed separately, such as antiproliferative activity, natural killer cell activity, stimulation of muscle cell differentiation, and suppression of cloning efficiency of human bone marrow cells. (Refer to p. 24 of Annals of Biochemistry and Biophysics 221:1-37, 1983)
.

Co-purification of anti-viral activity or anti-viral activity alone with one or more of these additional biological activities is a feature and is referred to as "interferon-like" activity.

In this example: [The representations of FWA and IFWA-M were obtained as follows. Large intestine [as follows in JM103 I
Plaque-purified recombinant bacteriophage M 13 mp 11 containing FWA was infected. 1. #Q)2XY
Colon muscle JM103 grown in T medium to an external diameter of 600 was mixed with recombinant bacteriophage containing IFWA or TFWA-M at a multiplicity of infection of 100. This mixture was incubated for 5 minutes at room temperature, then distributed 1:100 in 2X YT medium and incubated at 67°C for approximately 2 hours. After this incubation, IFWA or: [FWA-M gene expression was performed using isopropylthiogalactoside (pTo) from Bethesda Research Laboratories, Inc. (K9, Isserpurg, MD) at a final concentration of 5mM. This terminal culture was incubated for 2 hours by adding up to 6
After incubating at 7°C, E. coli was incubated at 6.00° C. in a Beckman JA 140-meter.
] The bacteria were separated and pelleted by centrifugation at ~pm. This medium is then removed and pF (
8,0 of 5 [1mM Tris4 ((J and 50mM
It was suspended in a 100-ml solution consisting of NaCJ.

Lysozyme was added to this solution to give a final concentration of 1 to 11t. The resuspended bacteria were then cooled to 09C and kept at 06C for 60 minutes. The bacteria were then subjected to six freeze-thaw treatments consisting of freezing the bacteria to -80°C and thawing the bacteria at room temperature. Bacterial cell debris was removed by centrifugation at 100,000×1 for 1 hour in a Beckman ultracentrifuge to pellet the debris.

Next, the excreta were removed and interferon-like activity was analyzed as described below.

Analysis of Interferon In one example of the present invention, the top of the Bacteria #Is extract prepared as described in the previous section was prepared using Stewart.
t) II interferon system (
Tb+e Interferon System) page 17 and 1 from New York City's Beete (■ 工ena)
Published in 1979, Sujiringa Farrock (Spri
5cand J Immunol by GerFerlock and Barb (Eerg)
, Immunol) 6 Near 7 (1977)
is obtained from Flow Laboratories (Fl) by the method described.
ow Laboratories) U, S, A.

WISH cells from vesicular stomatitis virus (vsV)
The enzymes are analyzed for antiviral activity, as measured by their ability to protect against cytopathic effects (CPE). A protective effect is said to occur when 50% of WISH cells are protected from CPE resulting from ■S■ infection. Alternatively, since interferon exerts its effect by rendering cells unable to support virus multiplication, interferon can be measured by the degree of inhibition of new virus production.

In this example, when performing CPE N-based analysis, the CPE was suspended in 100 μl of RPMI medium manufactured by Flow Laboratories U, S, A! 20,000 WISH cells from Flow Laboratories U, S, A, containing 10% serum from a newborn calf of R were placed on 96 micrograsp plates (Nunc, Denmark). The plates are then incubated for 24 hours at 37°C in a humidified atmosphere containing 95% air and 5% CO2. This incubation is followed by filtration for the formation of a complete cell monolayer by microscopic examination. Only the micrografts containing the complete cell monolayer are used for virus interference titration analysis performed as follows.

100 μt of bacterial cell extract supernatant is added to the first 11 microclamps of the first row of microclamps on the plate. Titer Tack 'U
, S.A., Inc., makes 6 serial dilutions and adds the next trough dilution to the next series of 11 minute dilutions. A total of 8 such serial dilutions were made. The plates are then incubated at 67°C for approximately 24 hours in a humidified atmosphere containing 95% air and 5% CO2. Following this incubation, the medium was removed by gentle suction from all microcapsules except the first four microcapsules in the last row, which served as controls for analysis, and the medium was removed by gentle suction from Dr.
.

Immuno Modulators Laboratories Limited (Immuno Mo
dulat-ors Laboratories, Lt.
d.) (Heuston, Texas) v3v manufactured by W
Replace the medium with 100 pg2 fbRPMI medium containing 50-000 PE at the desired concentration for the SH cells. V
No SV is added to the four control microdroplets. Each plate is then incubated at 67° C. for 24 hours in a humidified atmosphere containing 95 inches of air and 5% 002. Following this incubation, each separate micrograsp is microscopically examined for CPE. 100% protection from CPE is observed for all four micrograps to which no VSV was added.

National Institutes of Health (Na)
tional In5titude of Heali
h) The International White Plate Interferon Reference Standard is obtained from Bacterial cell extracts in separate but identical 96 micrograsp plates treated in the same manner as the cutillary cell extract top plate test plates. It is included in place of skim. WI St (The ability of bacterial cell extract supernatants to protect cells is determined against international reference standards. At least 10 per 11 bacterial culture extracts) international white interferon units were obtained from VF3V inbound CPE.

The methods and compositions of the present invention allow rapid filtration and isolation of mammalian genes that are present in low copy numbers in genomic libraries. In an example of the present invention, a known and novel human α-interferon gene was isolated from the Human Detum library. In addition, the gene expression of the regular genes I FWA and IFWA-M in bacteria is indicated by the interferon activity that can be isolated from bacterial cells.
This method can be used to express . Further, although preferred embodiments of the present invention have been described in detail, various modifications thereof will be apparent to those skilled in the art after reading this specification, but such modifications and variations do not fall within the scope of the present invention. Of course it's inside.

EXAMPLE The following example displays the filtration and isolation of two conventional human α-interferon genes from a human library. This example is presented for a better understanding of the present invention. It is not intended to limit the scope of the invention.

This example was carried out to locate and begin to isolate the interferon-specific gene sequence from a bacteriophage clone that was shown by positive hybridization with both probes A, B to contain said gene sequence. DNA isolated from bacteriophage clones λ77, λWA and λ105 was purified by Bethesda Research under the following digestion conditions:
The following six valley restriction enzymes Ba manufactured by Laboratories, Inc. (Daithersburg, Maryland)
mHI.

Digested separately with EcoRI and DdeI.

(a) Bam HI digested 2'OITIM Tris H (J and 10
1 to 5 μg of bacteriophage clone DNA was dissolved in 100 μl of a buffer consisting of 0 mM Na(J), 2 mM 2-merkanoteethanol, and 1001 n9/lnt of bovine serum albumin (BSA). Between 1 and 5 units of BamHI were added and the mixture was incubated for 1 hour at 67°C. After this incubation, an additional 1 to 5 units of BamHI was added.
was added and the mixture was incubated again for 1 hour at 67°C. Digestion was then stopped by phenol extraction and ether extraction, and the digested DNA was then precipitated with ethanol.

(b) EcoRff digestion p) 47.5 of 5 Q mM Tris-H (J and 10
mM MgCl4. and 50mM Na1J and 1mM dithiothreitol and BA at a final concentration of 100μg/-
1 to 5 μg of bacteriophage clone DNA was dissolved in 100 μl of buffer consisting of A. Next 1
Add 5 to 5 units of ECoRI and heat the mixture to 67°
Incubate for 1 hour at C. After this incubation, an additional 1 to 5 units of EcoRI was added and the mixture was incubated again at 67°C for 1 hour.

Digestion was then stopped by phenol and ether extraction, and the digested DNA was then precipitated with ethanol.

(c) DdeI digestion p)I of 7.5 1Q Q mM Tris-HCJ and 5 mM MgCl2 and 100 mM Na (J and
1 to 5 μg of bacteriophage clone DNA was dissolved in 100 μl of buffer U consisting of M 2-mercazotoethanol and BSA at a final concentration of 100-/-. Then 1 to 5 units of DdeI were added and the mixture was incubated at 37°C for 1 hour. After this incubation, an additional 1 to 5 units of Dde I was added and the mixture was incubated again for 1 hour at 67°C. Digestion was then stopped by phenol and ether extraction, and the digested DNA was then precipitated with ethanol.

(d) Agaro-scale split approximately 0.2 μg of digested bacteriophage clone D
NA was dissolved in 20 pl of buffer consisting of 10% sucrose and 0.03% bromophenol blue;
90 mM Tris base and 90 mM oxalic acid and 2.5
1% in a buffer consisting of mM Na2 EDTA (
w/v) of agarose (Sea Kem) 1? Resolution was performed by neutral agarose del electrophoresis in a mX 15 m slab del. The DNA was electrophoresed at 200v for 1.5 hours at room temperature.

The digested DNA was split in an agarose del and then
J, Mo1. Biol, 98:50
3 (1975) to nitrocellulose filter paper.

Separate hybridizations with valley end-labeled probes A and B were performed as described for the initial bacteriophage filtration. It was fixed whether the DNA fragments generated by l1lJ151j endonuclease digestion contained the α-interferon gene sequence and the size of these α-interferons, including the DNA restriction fragments.

The results of restriction endonuclease digestion of the λ/2 and λWA DNAs formed in Globe A, BKmPIi are shown in Table 1 below. As shown in this table, restriction enzyme Dd
DNs of λ/2 and λWA by e X and EcoRr
Digestion of A yielded DNA fragments of appropriate size for cloning and expression of the α-interferon gene. However, λ105 with the three indicated restriction enzymes
Cleavage of the DNA did not yield fragments within the 1-2 kb range desired for gene cloning or expression.

Table 1 Identification of interferon gene sequences in clones of λ/2, λWA and human λ105 Restriction Endonuclease A λ/2 >20.000 1,080 1,820
A λWA >20', 000 1,02o 2. Roa.

A λ105 na na 7,000B λ/2 >
20.000 1,08OL820B λWA, >2
0.000 1,020 2,070B λ105 n
ana 7,000 yellow end-represents the size of the restriction fragment hybridized with the labeled zorobe (base pair size). na-
not available

[Brief explanation of drawings]

FIG. 1 is a partially cutaway perspective view of an incubation rotating device used in one embodiment of the method of the present invention. FIG. 2 is an enlarged sectional view taken along line 2-2 in FIG. 1. FIG. 6 is an enlarged perspective view of the stand on which the reactor for miscellaneous food forming reaction mixture is fixed in the apparatus of FIG. 2; FIG. 4 shows the entire base sequence of human α-interferon carried in vector M13mp8 along with the corresponding amino acid sequence of the encoded protein. In this table, S□ indicates the first of the 23 amino acids of the interferon signal peptide, and 1 indicates the onset of mature interferon protein production. FIG. 5 shows the entire gene sequence of the mature human 1-α-interferon wA gene. Figure 6 shows vector M1 used for insertion of human α-interferon genes IFWA and IF77 into vector M13mp8 and the restriction endonuclease cleavage site.
3 shows the restriction endonuclease cleavage site for the VcM present in mp8 and determining the insertion of the gene into this vector. FIG. 7 shows that the human α-interferon gene WA is present in the vector M13mplll, which is used to insert the Hincl restriction endonuclease site and the Hndl restriction endonuclease site of the vector M13mp11 into the vector M13mp11 of the human α-interferon gene WA. Restriction endonuclease cleavage sites useful for KM intrusion into the cell are shown. Figure 8 shows Hinc II with better M13mp11 expression.
1 restriction endonuclease site M13 containing the human α-interferon WA gene inserted into the expression vector.
The mp11 expression vector is shown. Figure 9 shows the 25-me reaction against M13mp11 phage DNA containing the human α-interferon WAM gene.
Figure 3 shows hybridization of r oligonucleotides. 10...Electric @ machine, 20...Kettle, 22c...Temperature selection control device, 40...Shaft, 65...Mongrel type, storage, 7L
l...One unit. Engraving of the drawing (no changes to the content) FIGURE 5 CYS LED LYS TGT GAT CTG Continuation of the first page ■Int, CI,' Identification symbol Internal serial number Priority claim @ July 3, 1988 [phase] United States (US) 662
65910 Inventor Motohiro, Fuke United States T., Tulsa, DE Street 0 Inventor Richard, Michael, United States Torczynski Farmers Buohkurahouma 74137, Tulsa Curran East One Hundred and Sure South 4522 Dallas, Thixus State 7523/ Lunch County, Lunch,
Chaudil 24247#r Procedure supplement IR, yen (method) % formula % 3, Person making the amendment Relationship to the case Patent applicant 7, Contents of the amendment As shown in the attached sheet

Claims (1)

[Scope of Claims] (1) A method for identifying genes, gene fragments, or alleles contained in an r-Tsum library, comprising: (a) at least one type of DNA contained in the gene, gene fragment, or allele thereof; (b) synthesizing at least one unique sequence DNA probe having a DNA sequence homologous to the sequence and of an effective size to stably hybridize to said gene, gene fragment or allele; labeling the DNA probe; (c) hybridizing with the labeled DNA probe against the r-Tsum library; and (cl) then identifying the gene or gene fragment to match the selected label. A method for identifying genes, gene fragments, or alleles. (2) The method according to the preceding item (1), wherein human chromosomal DNA contained in a bacteriophage or plasmid vector is used as the rTsum library. (3) The method according to the preceding item (1), wherein interferon is encoded by a gene, gene fragment, or allele. (4) The method according to the preceding item (3), wherein human interferon is encoded by a gene, gene fragment, or allele. (5) Human α by gene, gene fragment or allele
- The method described in the preceding section (4), which encodes interferon. (The method described in the preceding paragraph (1), in which the 61 DNA probe is synthesized using an enzyme or chemically. The method described in the preceding paragraph (7), in which the probe contains a DNA base sequence rich in guanine and cytosine. (9) The DNA probe is composed of a 17-mar having a base sequence of 5'CAGCCAG()ATGGAGTCC5'. , previous paragraph (7)
The method described in. QO) The DNA probe is composed of a 17-mer having the base sequence 5'CCTCCCAO()CACAAGGG6' (7) above.
The method described in. 01) Using an improved method of hybridizing labeled DNA probes to recombinant vectors contained within host cells and fixed to nitrocellulose filters, the labeled DNA probes are hybridized to the r-Tum library, as described in the previous section (1).
) The method described in K, wherein the improvement includes (a) adding 710 the labeled DNA probe to the hybridization solution;
(b) separating the labeled DNA probe from particulate matter having a solution diameter larger than the 7° rope; and (C) using an apparatus that simultaneously rotates and incubates the nitrocellulose filter during prehybridization and hybridization. 02) The method according to item (11) above, wherein the nitrocellulose filter can be continuously rotated 3600 times by the device. (13) The method described in the previous item Ql), in which the labeled DNA probe is separated from the particulate matter by forming a carrier that retains particles larger than about 0.2 μm or passing the labeled DNA probe through an active filter. . 04) Effective amount of dextran sulfate about 10%
(w/v), the method described in the preceding paragraph 0υ. (]51 The prehybridization solution was 3.9 M NaCl
and 6. QmNa, 2 EDTA and pH 7,5 Q, 9
M Trj,s (trisoxymethylaminemethane) -HCI and 0.1% (w/v) sr+s and 10%
(w/v) dextran sulfate-1-,! The method described in the preceding paragraph Ql), consisting of J. C6) The hybridization solution was mixed with 0.9 M NaCl and 75. Q
0.09 M T of mM Na-2EDTA and pH 7.5
ris-HCI and o, i% (w/v) SDS and 10
% (W/V) dextran sulfate and a filtered labeled DNA probe. 071 s'CAGCCAOG, A, TGGAGTCC
A 17 base DNA fragment with a 6' sequence. (]-8j 5' CCTCCCAG()CACAAG
A 17 base DNA fragment with a GG3' sequence. Q.9) A method for identifying an interferon gene, a gene fragment, or an allele thereof in an r-zum library, which includes: 0-) a DNA sequence homologous to at least one type of DNA sequence contained within the interferon gene or a fragment thereof; (b) synthesizing at least one uniquely sequenced DNA probe having a DNA sequence of an effective size to stably hybridize to the gene fragment; (b) labeling the DNA probe; (c) hybridizing this labeled DNA probe to the rTsum library; and (d) identifying said interferon gene or fragment thereof to match the selected label. How to identify. (2(1) The method described in the preceding paragraph (19), in which the rTsum library is contained in a bacteriophage or plasmid vector. (21) The method described in the preceding paragraph, Cod, in which the length of the DNA probe is approximately 17 nucleotides. (2 匈 DNN artificial lobe 5' CAGCCA, () GATGGA () TCC 3'
The method according to the preceding item 09), consisting of the sequence. (23) The method according to the preceding item Q9), wherein the DNA probe consists of the sequence 5'CCTCCCAG()CACAA()GG3'. (2) Using an improved method of hybridizing a labeled DNA probe to a recombinant vector contained within a host cell and fixed to a nitrocellulose filter, the labeled DNA probe was hybridized to a recombinant vector contained in a host cell and fixed to a nitrocellulose filter. The method described in the preceding paragraph (19) for hybridization is improved by: (a) prior to addition of the labeled DNA probe to the hybridization solution, the labeled DNA is removed from the particles having a solution diameter larger than these labeled globes. separating the labeled DNA probe; (b) adding the labeled DNA probe to a hybridization solution containing an effective amount of dextran sulfate capable of increasing the concentration of the DNA probe in the hybridization solution; and (C) prehybridization. and a device that simultaneously rotates and incubates the nitrocellulose filter during hybridization. The method described in the preceding paragraph (241), in which the labeled DNA probe is passed through the filter to separate the labeled DNA probe from the particulate matter. (26) The nitrocellulose filter can be continuously rotated 360° by the device. The method described in the previous section (2IO), in which an effective amount of dextran sulfate was added to
(W/V), the method described in the preceding item Ca. (28) The prehybridization solution was 3.9 M NaC] and 6.1] mM Na2EDTA and 9 M skin at pH 7.5.
TrjS-HCI and 0.1% (w/v) SDS and 1
0% (W/v) dextran sulfate,
The method described in the previous section (241). (29) The hybridization solution contains 0.9 M NaCl and 6.Q.
mMNa 2EDTA and pH 7.5 Q, 9 M Tr
is-HCI and 0.1% (w/v) SDS and 10% (
W/,) The method described in the preceding paragraph (241) consisting of dextran sulfate and a labeled DNA probe. (a) using at least one radiolabeled DNA, Azoroso, with a DNA sequence homologous to the recombinant vector; and (b) introducing the radiolabeled DNA probe into a hybridization solution of the probe. (c) using a device that simultaneously rotates and incubates each filter during prehybridization and hybridization; (d) in the hybridization solution. An effective amount of dextran sulfate capable of increasing the concentration of the DNA probe was added to the prehybridization solution and the hybridization solution, and (,) the radiolabeled DNA probe hybridized to the recombinant vector was selected. A method of hybridizing a radiolabeled DNA probe comprising identifying a radiolabeled DNA probe to match a radiolabel. The previous item (7) is composed of DNA with a length of
The method described in. G21 The method according to the preceding paragraph 0D, wherein the length of the radiolabeled DNA probe is approximately 17 nucleotides. C33) The method of paragraph 30, wherein the radiolabeled DNA probe is separated from the particulate matter by passing the radiolabeled DNA probe through an inert filter that forms a retainer that retains particles larger than about 0.2μ. (Foundation) Effective amount of dextran sulfate approximately 10%
(w/r), the method described in the preceding paragraph (30). t351 prehybridization solution was 3.9 M NaCl
and 75. QmMNa 2EDTA and pH 7.5 0.9
M Tris-HCI and 0.1% (w/v) SDS
and 10% (w/v) dextran sulfate. (36) The hybridization solution was mixed with 3.9 M NaC] and 6.
0mM Na2EDTA and pH 7.5 Q, 9M
Tris-HCI and 0.1% (w/v) SDS and 1D
% (W/■) dextran sulfate and a filtered radiolabeled DNA probe. 0η The method according to the preceding item (to), wherein the device allows the nitrocellulose filter to be continuously rotated by 360°. (To) A human α-interferon WA gene having a coding strand consisting of the base sequence shown in FIG. (39) The fragment or allele of the gene described in the preceding paragraph (38). (40) The human α-interferon gene, its gene fragment, or its allele isolated by the method described in the preceding paragraph (1) or (l Kama). ( 4I) Human α-interferon WA gene isolated by the method described in the preceding paragraph (1) or (19) and having a coding strand consisting of the base sequence shown in Figure 4. (42) In the preceding paragraph (1) or θ9). A fragment or allele of the human α-interferon WA gene isolated by the method described above and having a coding sequence consisting of a partial base sequence of the base sequence shown in Figure 4. (4) From the base sequence shown in Figure 5. (44) A fragment or allele of the gene described in (49) above, isolated by the method described in (1) or (19); The human σ-interferon WA-M gene has a coding sequence consisting of the base sequence shown in Figure 5. (46) A partial base of the base sequence shown in Figure 5 isolated by the method described in (1) or ■ above. A fragment or allele of the human α-interferon WA-M gene having a coated chain consisting of the sequence. (47) Human α-interferon WA or human α-
In a method for producing an expression vector containing the gene for interferon WA-M, (a) human α-interferon WA or HA-
(b) cloning the gene of M into the unique restriction endonuclease site of the cloning vector to create a recombinant cloning vector; (b) introducing the recombinant into a host cell capable of replicating the recombinant cloning vector; (c) replicating the recombinant cloning vector in said host to create a replicative recombinant cloning vector; (d) isolating these replicative recombinant cloning vectors; (e) These replicating recombinant cloning vectors are cleaved with a restriction enzyme capable of cleaving at a single site not within the human α-interferon WA or WA-M gene to generate restriction fragments. (f) the human α-interferon WA or WA
- identifying said restriction fragment comprising the gene of M or a fragment thereof; (g) said human α-interferon WA or WA;
- isolating said restriction fragment containing the gene of M or a fragment thereof, and (h) separating said human α-interferon W A or 4
'j-WA, -M gene or fragment thereof is expressed in r)NA of the vector, in which the human α-interferon WA or WA, -1vi gene or fragment thereof can be in-phase transcribed. A method for producing an expression vector comprising inserting the DNA of said expression vector at a unique restriction endonuclease site. (Tsukuda) The production method described in the previous section (47), in which bacteriophage M13mp8 is used as a cloning vector. (49) The production method described in the previous section (47), in which the unique restriction endonuclease site of the cloning vector is an EcoRI site.
Production method described in. Replicated recombinant cloning vector can be cleaved at a site that is not within human α-interferon WA or WA-M or a fragment thereof, and A]υ and EcoRI are used as restriction enzymes, as described in the previous section (47). Production method. 511 Human α-interferon Wj” (Also HaWA-
At least one labeled DNA probe having a sequence homologous to the DNA sequence contained in M or a fragment thereof is hybridized to the restriction fragment, and then the human α-interferon WA or WA-M or The production method according to the preceding item (47), wherein the restriction fragment is identified by identifying the restriction fragment containing the fragment so that it matches a selected label. The production method according to the preceding item (47), wherein a restriction fragment containing the human α-interferon WA or WA-M gene or a fragment thereof is separated by r-cleavage. (53) Bacteriophage M13'mp1 into which the human α-interferon WA or WA, -M gene or a fragment thereof is inserted by the production method described in item (47)
A recombinant vector consisting of 1. l54) At least one type of human α-interferon W
DNA inserted at an insertion site consisting of the A gene or a fragment thereof
A recombinant DNA vector consisting of a vector. 6 at least one type of human α-interferon WA-
DNA inserted at an insertion site consisting of the M gene or a fragment thereof
A recombinant DNA vector consisting of a vector (〇Hiro)) by inserting the human α-interferon WA or WA-M gene or a fragment thereof, and in-phase transcription of mRNA corresponding to the inserted gene or gene fragment in a bacterial host. and the recombinant DNA vector described in the preceding item 64) or the gloss that enables translation. I5η Using bacteriophage or plasmid DNA as a DNA vector, 54) or I! 5!5)
The recombinant vector described in . 68) The recombinant DNA vector described in the preceding paragraph (') 4), which uses M13mplI DNA as the DNA vector. 59 Ml3mpF3D as a DNA vector
The recombinant DNA vector described in the preceding section 6, which uses NA. (60) A method for replicating a human α-interferon WA gene or a human α-interferon WA-M gene or a fragment thereof in a bacterial host, comprising: (a) a human α-interferon WA or WA-M gene or a fragment thereof; (b) transfecting the recombinant DNA vector with the recombinant DNA vector into a bacterial host capable of replicating the recombinant DNA vector and replicating the recombinant DNA vector; Generating a recombinant DNA vector and (C) Selecting the recombinant DNA vector for replication (61) The replication method described in the previous section, using Escherichia coli as a bacterial host. (521 The replication method described in the previous section (60) using bacteriophage or plasmid DNA as a DNA vector. (63) Mlplompl or M as a DNA vector.
13mpF3 DNA is used, the replication method described in the preceding paragraph. (61g Recombinant DNA vector produced by the replication method described in the previous section (60). (65) Human α-interferon WA in bacteria
or a method for expressing the human α-interferon WA-M gene or a fragment thereof, in which: (a) at least one human α-interferon WA or WA-M gene or fragment thereof is placed adjacent to a bacterial promoter; The at least one human α-interferon WA or WA-M gene or (b) producing a recombinant vector comprising a DNA vector into which said fragment has been inserted at an insertion site comprising said recombinant vector; (c) growing said infected bacteria by incubating said infected bacteria in a bacterial growth medium; and (d) growing said infected bacteria in a bacterial growth medium located adjacent to said gene or fragment thereof. (e) incubating said induced infected bacteria with said human α-interferon WA or WA-
An expression method consisting of enabling the transcription and translation of the M gene or its fragment. (66) The expression method described in the previous line, using E. coli as the bacterium. (6η The expression method described in the previous section (65) using bacteriophage or plasmid DNA as a DNA vector. (68) M 13 mp 11 as a DNA vector
The expression method described in the previous section (67) using DNA. (69 The expression method described in the previous section (64), in which β-lactosidase is used as the bacterial promoter. (70) The expression method described in the previous section (69), in which inpropylthiogalactoside is used as the inducing factor. 6υ Interferon activity protein produced by the method described in the preceding paragraph (641). (72) Human α-interferon WA0 produced by the method described in the preceding paragraph (Foundation) (73) Human protein produced by the method described in the preceding paragraph (64) The sequence of the NA70 lobe is useful in identifying the α-interferon WA-M0 (74) gene or its gene fragment when it is one of the genes of the family consisting of the cDNA.
A method determined by hybridization of said DNA probe to said gene or gene fragment thereof contained in an A library or a genomic library or a recombinant vector;
at least one oligonucleotide sequence of sufficient length to compare NA sequences and stably hybridize to genes or gene fragments thereof that are occupied by at least half of said genes or gene fragments in said substrate; A method for determining a DNA Proso sequence, which comprises determining. (76) The method described in the previous section (75), in which the DNA probe has the sequence 5'CAGCCAGGAT()GAGTCC'. (77) The method according to the preceding item (75), wherein the DNA probe has the sequence 5'CCTCCCAGGCACAAG()G'.