JPS5860998A - Production of interferon - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing interferon (FN). More specifically, the present invention relates to fragments of human genetic DNA.
This method relates to a method for producing human interferon in blast cells (βj type or leukocyte type) by introducing appropriate bacteria into cells. Human DNA taken directly from blood cells is cloned into an appropriate λ-phage. High interferon production is obtained in bacteria infected by the λ-phage, and the interferon can be harvested and produced from the bacterial lysate produced by this infection. Genetic DNA can also be introduced via plasmid expression vectors. BACKGROUND OF THE INVENTION Interferon and Knicht interferon are important in many fields, but their annual production is at a low level, and improvements in production methods have led to a rapid decline in production. Feeding and feeding in small areas such as the large intestine (E, coll).
■Category■-1-1--■-■--1-- Expression of animal genes is prevented by the presence of intervening sequences that interrupt the coding sequence of the skin gene. However, 9
There are also many genes that do not have introns. recently,
Nagata et al. 1■--111-■■--■M-- (1980) (Nature 287, 401-8
) showed that the human leukocyte interferon-a gene is one that lacks introns. They found low level expression of these genes in E. coli. Are human diploid fibroblasts induced by two M-stranded RNA K at least interferostatic? The two m that code
Contains RMA Y [Weisenbach et al.
enbach et al.), 19 f3 Q,
(PNA8)77.7152-7156). 0.
The 9 kilobase (xb) long (11s) mRNA is a live inter-elbow feron species produced by these cells.
-β□, but its amino acid sequence has been partially determined [Knight et al.
) 1980. 5science 207, 325-6). Using the Escherichia coli plasmid pBR322% as a vector, transfection of this engineered F to β□mRNA (D cDNA 'i' was achieved [Pl'y Dell et al.
l et al-) + 1980 rNucletc
Acta Ree, 8, 4057-4075)
. The nucleotide sequences of these DNA clones correspond to the determined protein sequences, and after generation of expression plasmids, IIFN
-β1 cDNA was demonstrated to direct the synthesis of human interferon in E. coli. The present inventors have developed a method for producing engineered FN-β□ smoke gene [Morley et al.
Ry athl, ), 1981, Kur, J.
Biochem. 120.197-202) and a human gene fragment carrying the engineered FN-ac gene. These genes lack introns and therefore can be expressed directly in E. coli. Summary of the Invention The present invention involves cloning a specific fragment of human genetic DNA extracted from human blood cells into a bacteriophage vector, directly using it to synthesize engineered FN in bacteria, and culturing the bacteria to produce interferon. Concerning a method of producing. Therefore, this method does not require long steps and does not require cloning full-length complementary DNA (ODNA) from mRNA. In accordance with the present invention, suitable NA fragments are taken directly from readily available human cells, such as leukocytes, and cloned into a λ-phage vector. The target protein 11IN gene is interrupted with an in)on and can direct the synthesis of protein FN in bacteria. By infecting a suitable bacterium such as Escherichia coli with a recombinant λ phage containing DNA Y into which the engineered FN-β□ gene or engineered FN-'o gene has been inserted, high I.
The engineered FN that produced FN activity was collected and purified from the bacterial lysate produced by infection. Phages attack bacteria) D
It was found that this vector is suitable for introducing an NA fragment. In particular, the λ (3h-aron 4 A phage is excellent and leads to high-efficiency FN production!) Furthermore, the present invention relates to the introduction of cloned human gene DNA fragments into plasmid expression vectors equipped with strong promoters. This method can be performed with engineered FN-α as well as IP'N-β1, provided that it lacks an intron. In one embodiment, DNA from an adult human human
It was fragmented by R digestion and cloned into 0haron 4A. The clone obtained in this way is 010n
e It was named O15. The clone had an approximately 17xb human DNA gene insertion, and it was identified as containing the gene corresponding to fibroblast interferon protein FN-β1. Restriction mapping showed that this gene, located on the EcoRI fragment of 1.8'b, was not interrupted by an intron. This human gene DNA fragment is capable of directing the synthesis of human IFN-β□, which is active in E. coli and other suitable microorganisms. Highly effective vN activity is recovered from the phage lysate. Under certain conditions, an activity of about 107 units/no was recovered from the phage lysate. The lysate is chromatographed, preferably by chromatography.
Del m-Sepha o −/< (C1bacron nu
sSepharos θ) or β1-like column. Does the thus obtained interfunilon have the same immunological properties and species specificity as engineered FN produced by human fibroblasts? have. FM-g, a gene identified in clone 18-3 containing a 13 Kb human gene fragment. obtained similar results. In other embodiments, the
gene? Contains all of the roe M prince DMA fragments.
was introduced into a cilasmid, such as cilasmid pBft 322, containing the A promoter. Cultures of E. coli transformed with these recombinant cilasmids were treated with roe by nalidixic acid.
When the A promoter was induced, a significant amount of IFN-β or IFN-1K was produced.4. IFN-β1
The gene fragment was cut at the codon corresponding to the first methionine of the mature engineered FN-β1 and ligated to the E. coli 1aC promoter at the bosome binding site. The lac promoter was modified to contain the RNA polymerase binding site of the tritphan promoter. This hybrid tryp-1
Driven by the ac promoter, the genetically engineered FN-β□DNA was transferred to E. coli minicells, strain p679-54pHl, at 108 sq/, j! We were able to produce FN-β1. -”
Like 111'N-α. By appropriately inheriting the gene to the tryp-1ac promoter, a sufficient amount of engineering 1'N-α can be obtained. I got it. On both platforms, FN activity was 3% with lysozyme.
It was recovered from bacterial cells lysed with 0 thipropylene glycol and purified on a hydrophobic chromatographic carrier in the same manner as the phage lysate. A suitable carrier for engineered FN-β1 is Cibacron Blue Sepharose (01bacronBlu).
eSθpharose). The elution is ethylene glycol: I-#-? Preferably, propylene glycol is used. The production of F to βl or ao is
Further, immunoaffinity chromatography on monoclonal antibody columns or other conventional methods is performed. From the above it is clear that human gene DNA fragments can be used to produce human lPN in high yields in E. coli and other suitable host bacteria. As mentioned above, one method of the present invention involves introducing a human genetic DNA fragment into a suitable vector such as a phage, infecting a bacterial culture with the phage to lyse it, and collecting the interferon produced in the lysate. Consists of things. The present invention relates to a novel phage obtained by introducing human gene DNA, and a novel phage that is involved in the production of
one O15*EngFN-g. λ-type introducer phages such as clone 18.6 involved in the production of phage. Furthermore, in the present invention, human gene DNA 7 is introduced into a suitable phage, and a suitable NA fragment is extracted from the phage. It relates to a method for producing interferon, which comprises collecting the interferon, introducing it into an expression plasmid of a suitable bacterium, culturing the bacterium, and collecting the desired interferon. Other objects of the invention will become apparent from the detailed description below, which is not intended to limit the scope of the invention. An important advantage of the present invention is that by comparing the FN productivity of genes obtained from healthy or diseased individuals, genetic defects in FN production can be investigated. Preparation of: High molecular weight DNA 7β-thalassemia (β-thalas)
semia) was prepared from adult peripheral blood cells. DNA
A portion (40μg) of ``Fl:coR'' I
Oo, 20,0, 60 in 300 units
Digested for 1 minute to the side, heated at 65°C for 10 minutes, and subjected to a 10-40% sucrose gradient as previously described. 10-2
DNA fragment i between 0 K'b, Maniatis et al.
niatia at al,) (1978 se/L=(
λ0haron prepared by llf:coR digestion following the method of Cell) 15, 687-701).
4 In the A-DNA arm, Mory et al.
ah) (1980. L(ol, Biol, Reports t5, 203
The DNA was ligated with T and DNA ligase according to the description in 2008).In vitro packaging was
Hohn and Murray
) (1977. PNAS, 74.3259-5263), ligation reaction solution CD, 5 μg of vector DNA and 0.0 μg of vector DNA were added.
125 μg of human DNA) 8 μg of E. coli BI
(B, 2688 (N 205 recA-(21mm
434 c engineering t8b 2 red 31iam 4
Ham 7) /λ] and BHB 2690 (N 20
5 rech-(21mm 434 Q-ts b,'
2 red3 Dam 15 Saw 7) /λ]
This was done by mixing with 50 μm of the soybean extract. 3 x I Q5pfu per 1μg of recombinant, recombinant DNA
was gotten. - 108 for untouched λDNA
It was pfu/#g. Total amount of 1.8 x 1060 recombinant phages v 1 Q 111M Tris-
BOj (pH 7-5) r 1 0 mM MgS
The solution was diluted with 6d of solution containing O, and 0.01% seratin. Add 0.1 d of chloroform, remove the crushed material using a microfuge centrifuge, and add phage large & 1 l DP50 (Leder et al.
Planted at x 103 pfu/15 plates and increased by 105 times. 2X1 pfu for selection 1'
Benton et al.
a1. ) (1977, 5science 196.180
- 182)); continued copying. This operation was performed under P3 containment conditions. Engineering F'N-β, cDNA DNA arm: 100μg/l
E/poly(r): CTO) and 50 pg/d cycloheximide (containing zpg7w actinomycin DY for the last hour) Weissenbach et al. (1979)
, (Eur,+lr,Biochem,,) 98
The two peaks of interferon mRNA were fractionated by M-glucose gradient centrifugation described in detail by J. I., 1-8).
! J back'tttn'
ysloblaatosis Virus) (J. J3eard)
) as well as the earlier Weiäenb et al.
ach et ah) (1980, PNAS 77,
RNA-cDNA according to the method of 7152-7156)
A hybrid was prepared. This RNA-CDNA hybrid was used with the (10TP and (IG-strand-extended Pst-cut pBR322 vectors) for chain extension of the hybrid [Changat al., 1999].
78, Nature 275, 617-624]
using Ding et al. (1979, ae
directly cloned according to Ill 6, 851-861). Stenland et al.
Total number 12. as outlined by Tal, Ng (1980, Gena 1Q. 47-52).
500 E. coli MM'294 transformants of tatramp' were obtained. 118mRNA of induced ps11 cells (
6 μg) or total polyA''-RNA (3,
Colony hybridization (colony hybrid+1
cDNA synthesis was carried out using a synthetic complementary Zolimer [Narang et al.
al.), 1979 rMethods Knzym
ol, 68, 90-98 #teru]de 9, engineering FN-
5' GAGATOTT, which is the Bg-1 part of the βl sequence
OAGTTT (started with 33'~ml.jgp-5/end label [Houghton et al.
m,]. 1980, \Nuc1. Muc, Res 8, 1916
Using the -19614 ply-r-f, the expected 6
The 40 nucleotide long cDIJA was rarely seen only when the induced RNA Y-type was used. To iAd Zorope, 2Q p moles Nora (-?-' to 4μ) (7,4MKCI! p to 60
Anneal with M fllJA for 60 minutes at °C.
) L, 200 poi each “P-+ff4ATP and d
OTP (40001/mmolJ with double edge, each (J, 5
mM dGTP and dTTP, 5 Q mM Trim-
)(0)(pi-18,6), 4mM MgOnozs
The cells were incubated for 2 hours at 67°C in a 25μ mixture of 5mM zothiothreitol, 50μg/d Actinomycin D, 4mM pyrophosphate and 60% reverse transcription #. 2
After adding 5 μg of colon-1) NA, 0.3 M M
Treated with AOH for 7tJ”060 minutes.

[Neutralize the reaction, 5
0 mM TriB-HOJ (pt+s), 0, I
4m61P-t in M Na0J, 1Q mM EDTA, 0.5% Dodec! Fadex() -5Q(de
Filtered through phadex 1)-50). Each RN
5 × 10′ cpm cDNA was obtained from A. Colonies grown on nitrocellulose filters were fixed and 'DkIA'l Thayer (197
9, Anal, Biochem, 98, 60-63
). Each filter? 6 X
SET (8KT-150mM Na(!, i!, 2
mM EDTA. 3 Q zM Tria-HOJ (pH 8, )), 10
×Denhartz solution (Denhara s 5oluti
on ) (Denhardt) 1
966, Blochem, Biophys, Res
. Oomm, 25,641-646)), 0.1% sodium pyrophosphate, 0.1st dodecyl sulfate, 50ag/d denatured E. coli, 17NA mixed solution 10IIi/Y at 67°C.
Time pre-hybridized. filter? 0.5 each
x 10'cpm/filter of two "P-cDN"
A zolo-zo, 10 μg/wl polyA and 2.
5 pg/d poly-C? 67 °C in the same solution with
Hybridized for 14-18 hours. 67 °C in prehybridization solution for 1 h, then K 3
Q, 1 tipyrophosphoric acid, 0.1% dodecyl mimetic acid 67
°C, 3 times more for 30 minutes, then 25°C at 4 x SKT.
℃, 60 minutes - see 701]. The filters were dried and exposed to Agfa Ourix @-ray film with an intensifying screen at -70°C for 1-2 days. Of the 3,500 colonies screened, 14 hybridized preferentially to cDNA templated from induced 1-derived mRNA, and 130 also hybridized to hooked cDNA of non-stimulating mRNA. MA of cilasmids (0,6 μg) from the first group, 6xSSO110
× Filter with 5'-end 32P-labeled Zolaima (0,3 Q moleg, 4 x 1 Q' cpm) in Denharz solution [Kafatoe et al.
) 1979, NuolAc, Res, 7, 15
41-1552) for 21 h at 3-5 °C, then hybridized on 6 × 8eO (900 mM N
a0), 90 mM goue/sodium acid). Clone 1-5-5 was fold positive and DNA sequenced [Maxame'tal, 1980,
Methods]! inZ7mo1.65, 499
-560) K, the clone was found to contain approximately 370 nucleotides from the 3'-end of the F'1J-81 sequence. Sucrose gradient-purified derivative mRNA a
c chain extended double @ DNA (do-tailed da
-DNA) The other 50,00
0 clones were selected: The proportion of IFN-βl clones was 0.16% (80 clones), compared to
The percentage of total β2 clones was found to be 0.65%. The clone Fβ11-6-5 prepared from the F B-11 mRNA described above was used for the library selection. Mo♂, Biol, 113, 237-251)
According to
2 x 108 by Y nick translation
cpm/pg DNA was labeled and hybridized with the human gene library K I Q' cpm/filter. Filter preparation, DNA denaturation, and insolidization were performed as described above. Phage from noibrid positive plaques were added to 9 mock plates.
-2 x 102 pfu and 6 re-sorts were performed. This operation was repeated 13 times to hybridize more than 95 plaques on the plate with FN-β]cDNAK. Phage clone C15 was isolated from one of the plaques. The phages were prepared by Blattner et al.
l,)-1-■1--1)--1--h---(197
7, 5science 196.161-169). E. coli DP50Y1%
Tripton, 9.5 4 Mail, 0
．． 5% Fumu extract, 0.2% maltose, 0.25% M
gSO4,0,01 Thidiaminobimelic acid, 0.004
% thymidine overnight. Approximately 0.3 d of culture was preadsorbed with K 10 mM Mugso, 1Q mM
C! Ao-2 and 107 Phage mixture 0.3- and 3
Mixed for 20 minutes at 7°C. That culture? Z amine, 0.5 q6 yeast extract, Q in I-2 no 2 flask
, 5% Mail, 0.1% Catemi/Acid, 0.25
% Mg804. Preheated medium containing 0.01% diaminogimelic acid and 0.004% thymidine 50
Diluted at 0 d and heated at 37 °C for 15-18 min with good ventilation.
Incubated for hours. After completion of bacteriolysis, the cell lysate was transferred to an Elorvall GSA rotor (Elorvall GSA rotor).
The mixture was removed by centrifugation at 000 rpm for 15 minutes in a cold Em rotor. Phages were precipitated from this clear lysate with 7 polyethylene glycol 6000, followed by 2
Purification was carried out using multiple cesium chloride gradients. Phage O15
DNA Yphenol was purified and its structure was analyzed by restriction enzyme digestion and horizontal agarose del electrophoresis using 0.5 μg/d-ethidium zolomayrv in 20 mM) Lis base, 1Q mM sodium acetate, 1 mM KDTA, Nitrocellulose filter [Plaque (80uthern), 1975, J, M
o1. Biol, 9s, 1503-517) and hybridization to the nick-translated engineered PN-β cDNA described above. O
A HaoR fragment of 15 DNA was engineered into the EcoR site of pBR322 for accurate restriction mapping of lasmid DNA [Bolivar,
1979, hiethods Einzymo'
l. 68.245-267]. Cleared phage DingFN-β1015 prepared as above
Bathing solution 'k IJ acid buffered saline CPBS)
K dialyzed for 7 hours. Dialysate I Q wl V C1b
acron B'1ue-Elepharose CL
<5 B (Pharmacia Fine Ohe
m,) was placed on a 0.3 d column. Column'l 1
0-1.5++j 1M Mail, 0.02M
Washed with sodium phosphate buffer (pH 7.2) followed by 50% zoropyrene glycol 10d in wash solution. Fraction (0,51M1)Y was collected and stored at 4°C. 0 in some cases
．． 100% human blood albumin was added for stabilization. Weiesenbach et al.
! L1. ) (1979+1(!ur+J.Biochem, 98+'t-'8)'
Each fraction was serially diluted into 95-well microplates and assayed for interferon activity as described. Each well contained 2-3 x 10' IFS 11 human fibroblasts containing 0.1 sd of Minimum Essential Medium.
al Mediu, m, ()ibco), 5
Fetal calf serum (F'08) was added to a 0.541" tube. After 18 hours at 37°C, the medium was removed and vesicular stomatitis virus (vesicular stomatitis) was added.
tis virus. vsv) lk 1p in 2% FO13-containing medium
Added fu/cell. Cytopathic effect (cytopat)
hic effect) was inhibited by IFN-βN
4111La023-902-527, recorded 30-40 hours post-infection. In addition, the antiviral activity was determined as described above [Weissenpatzha et al.
Bach et al.), 1979. lur, J. Biochem, 98, 1-8, l.
, as measured by the decrease in 3H-uridine uptake following vsv infection. Antiserum for PH-β was obtained from rabbits and was purified as described above (We
igsenbabh et aley 1979,
Ffur,,r,Biocham. Sea i1. Sh. 98.1-8) Analyzed. For neutralization tests, 0.1
Antiserum (titer 104 units/dl) or non-immune serum was added to protein A-Sepharose beads (prote
in A-8epharoee beads) (P
harmacia-IPine Oh@m, )'s 50
The mixture was mixed with 0.2 ml of the suspension at 37° C. for 1 hour. Beads were washed twice with PB8 and 0.1-k of bacterial interferon (100 units/d) was added. After 2 hours at 37°C, the beads were centrifuged and the upper groove was analyzed to determine inhibition of 3H-lysine uptake in vsv g-stained cells. Using oligonucleotides, we directly screened the genetic library. The oligonucleotide is complementary to the consensus sequence of the FN-σ gene and is 5′ (!TOTGAOAAAOOTOO
O3' and 5'0CTTO'['()f)AAOT
G3' sequence, these oligonucleotides were <5' labeled as described above and then either directly or on Sendai virus-induced RNA from human leukemia cells.
32p-Olibinucleotyl or cast cDNA used as a paper mold (Zorai i-11-1pri) for synthesis If the total number is 500,000 as above
was hybridized to two recombinant phage. 9a - Approximately 5 X 10' cp per nitrocellulose filter
m used. Hybridization was carried out in minimal volume plastic sealed IP+P+ bags. Hybridization with a 15 base primer was performed as follows. First, pre-hybridization for 4 hours at 37°C in 6xSB 0.10x Denhardts solution, then hybridization for 18 hours at 67°C in 5xssc, 10x Denhardts solution (Denhardts), 6. 3 for 30 min each at 37°C in x ssa
Washed ~4 times until no radioactivity was detected. For hybridization with 13 base primers, the hybridization and washing temperature was lowered to 30°C. A total of 16 phages that showed positive hybridization were further analyzed by restriction mapping and I) NA sequencing. Does clone 18-3 contain three engineered FN-d genes? Carrying one of them. r Rubble et al. ((koeatlelet al,) [1
981. Nature, 290, 20-25) cDNA
It was proven that it had the same sequence as clone-8. Clone 18-6 was purified in the same manner as clone 015 above. The expression of clone 18-6 in the λ<i>f IJ , t7age and after insertion into the expression plasmid vector is described in the "Results" section below. Result complete - Zokuronga EcrR process part digested λ0har
isolated from a library of human DIJA cloned into the arm of on4A. The clone is human lfM
#4 = Two different engineered FN-β1 cDNA probes labeled separately from two different mRNA fractions of blast cells.
11! (Hybridized. Digestion of the phage DNA by the EcoR engineer produces two λ arms plus 12,
2.6 + 1.84 and 0.6 p O base (M I
The lr piece of A 1i41) is shown. Sadeen (5ou
thern) [197b. J, Mo1. Transfer to nitrocellulose and engineering F'R-βl by the method of B.Bol. 98, 503-517).
The 1.84 Kb fragment was found to contain the IFN-β1 gene by binding the 1-6-5 cDNA with the translated DNA. This EcaRI fragment? It was recloned into the EcoRI section of pBR322. Bg of this 1.841 subclone
:L IIPvu l, Pst I, Mine
The coding sequence of the FN-β, protein (H
lna 1 site) was ibed into the tAi box approximately 350 nucleotides from the BcoRI site (Figure 1A). Gene D
The distances between the various restriction enzyme sites in NA are the same as those in the c'DNA sequence in the four different parts of the collection (#1 East)
. Restriction site β1 cDNA sequence β, gene DNA
Nucleotide-nucleotide Taq r-Pvun 255
250H1ne I-Pvu H204210H
1nCI-Bgl I 570 57
0pvu l-Bgl m-366660Gy (G
ene J 10 (11-15). ') + 1.84 Kb, tears 11 from KcoR engineering fragment
Fixed. Considered Hlnc l and Taq Part 1 I arithmetic is 5 of genes
′ closest to the end. No unexpected restriction enzyme sites were found anywhere in the IFN-β1 coding region. The slippage is caused by a detectable intervening sequence (i
This shows that interveningsθqueneθ) does not exist. Ol 5 DNA RaoR partial oil digestion, '6
Kb 1coRI 醇I piece is 1.84 and 2. (5Kb 1
It was shown that it is located between the licoR fragments. Bam
The H engineering part is 11i in 2.6 Kb! 1lP9r was found, but not in other EcoR fragments of the inserted human DNA. Engineering? The KcoR fragment of N-β, cDNA Ic hybridizing 1,84' Kl) is O
It was found on a 9,6 Kb BamH fragment of l 5 DNA. As shown in Figure 1, this fragment is derived only from the BamH site of the λ right arm (Kcol'H to 5.1 K1:+) and corresponds to the KcoR knee BamM site of the inserted human DNA. Kb was released, indicating that the 1.84 Kb EicoR fragment must be adjacent to the λ right arm. Gene orientation (Figure 1A) was determined as follows. 015 DNA 4 Cut with Pvu I and make PN-
When the full-length or 3' half of the β1 cDNA was hybridized, a 0,66 Kl) fragment was found that hybridized only to the 5' end of FN-β1. However, the 1.84 Kb EcoR fragment Y Pvu
When cut with I, the 5' end of FN-β was found on a smaller 0.54 Kb fragment. inserted)
-0.6Kb Neon fragment of DNA contains Pvu
1 site is not found, so 0,66 xb Pvu
The lll1l'1 piece should end in the λ right arm, so the 5' end of FN-β, is closest to the λ right arm. The cooperage shown in Figure 1A is Hlnd I %
This is also confirmed from several other restriction mapping experiments using 8ma I and Bgl II digestion. The coding sequence of the engineered β1 gene in clone 015 is therefore controlled by the strong λPL promoter that regulates leftward transcription [Williams et al., 1980;
Genetic Finineering Vol.
l. pp201-281. Plenum Co.
It was inserted approximately 1,775 nucleotides away from rp)) in the proper left orientation. This, along with the absence of a detectable intron, prompted consideration of the possibility of producing interferon in E. coli infected with the al5 recombinant phage. The phage from clone a15 was
ds) Large & 1 DP50 culture solution 50 as described in K
Grown at 0 d. Dialyze 10d of clear bacteriolysis solution, 01b
The fractions were loaded onto acr'on Blue-8 epharoae small column (0,3d) and tested for inhibition of the cytopathic effect of vesicular vesicular inflammation, Rus (VSv).
-β was used as a control for analysis. Interferon activity in one experimental example conducted at a phage concentration in the lysate of 1.3 x 101'phage/d. It is shown in FIG. 5os zoropylene glycol-1 from the column
Total ffi 7,500 in both minutes eluted with M Na0J
Unit engineering FN activation? I collected it and took it. This corresponds to 0.75,10' units per lysate. However, measurable activity in the crude lysate was very low, suggesting that substances in the lysate were interfering with accurate analysis of FN activity. In reconstitution experiments, when standard human fly FN-β was added to the wild type λCharon 4 A lysate, the activity was one-tenth that of the input. This mixture was passed through a Blue-8 theta pharose column and 75 of the input activity was recovered. Wild type λ0haron 4 without 11M added as a control
The lysate from A phage was analyzed, but no FN activity was found (Figure 2). 11 from the lysate of clone 015! The chromatographic behavior of 'N is not constant. Phage concentration is 3 x 10”
In experiments with pfn 7 wt, pfn activity was high, but B
It was not retained on the lue-86pharose column. In this experiment, the total activity recovered from the column was 10-
of lysis solution (7-8 x 106 units/)) for 9 to 70
-80,000 units of engineering FN. The eluted fraction was readsorbed onto a second Blue-8 epharose column. At this time, the activity was retained on the column, but when the column was washed with PBB, it was eluted again. Human I
FN-βl is usually Blue-E only by hydrophobic solvents.
It should be eluted from iepharose [Knight et al., 1980,
5science 2 Q 7. 525-526), therefore, the standard human fly Il'N-β was mixed with the same phage lysate and applied to the column. 60 active
% was not retained, and the active υ1 yield was less than 25%. This means that the components of the bacteriolytic solution are FN-β, especially FN-β produced by bacteria, and B111θ-8ep.
This suggests that the interaction between harosθ was destabilized. Interferon was not retained on the column, but a partial purification of the lysate in which more than 90 proteins of the lysate were removed from the active fraction with a specific activity of 4-5 Indispensable for removing chemical substances that hide their activity. In another experiment, the lysate was also subjected to carboxymethyl-Sepharose chromatography using clone 015. N activity was collected (not shown). Interferon activity recovered by chromatography reduced the uptake of 3H-uridine in vsv-g-ray human cells treated with actinomycin D [Weisaenbach 7 + 1979]
*Kur, T. Biochem, 98, 1-8
) The titer curve obtained with Nhk FN-β was the same as that obtained with the standard human fish FIJ-β (Figure 6).
. To investigate the immunological properties of bacterial engineered FN, the partially fertilized product was incubated with rabbit antibody PN-β serum (inactive against engineered FN-) or protein mucepha o-x (protein A).
-8epharose) and mixed with eel non-immune serum pre-bound. After centrifugation, FN activity analysis was performed. Anti-FN-βm was removed from all interferon activity, but is it a non-immune serum? When used, the activity remained (Fig. 6.) Substrate IFN (20 units 7 ml) was similarly neutralized by simply mixing it with Technique F to β (2θ units/-) on a cell culture, and ■5v11i11IJJll The species specificity of the bacterial interferon was analyzed by the comparison of the h version of the gene 1FN-β1c15 clone. Value unit/Mt Human Pull Bovine Mouse Mouseia, J-
7! o7 origin 'FSii BEC-i MDM
L A9 cells Cells Cells Cells 1, Human F1311 cells 1.500 32
32 <4 <42.15 Clone infected E. coli '1.000 32 32 <4
<4 Bacterial engineering FN is C1b of C15 lysate as shown in Figure 2.
It was used after chromatography on Acron Blue-8epha Oae. The activity of the clone 015-infected autologous product as human FN-β was less than 10% of that in human cells in pulled renal cell gsC-1 and bovine MDBK cells. There was no detectable antiviral activity on mouse L or A9 cells. The same results were obtained with 6,000 units/d of bacterial engineering FN produced by clone 015. Therefore, the present invention provides that the gene can be expressed in IP'N-βx3 isolated by direct cloning from the Eco'R engineered fragment of human genetic DNA into λ0haron 4A, and that the gene can be expressed in a significant amount in the lysate of the culture. It was shown that it is possible to accumulate IP'N activity of . The activity produced is clearly fibroblast interferon (β) due to its immunological properties and specificity. Active fly FN-β1cf5
It is produced only when infected bacteria are cultured by phage clones. The FN activity produced by clone 015 in E. coli is 01bacronBlue 5eph
The chromatographic properties on arosθ are similar to that of human F.
It is very similar to tβ. Highly active from bacterial lysate (7X 1
Chromatographic treatment is essential to recover the 0' units/)). Does interferon work? This is the first example of a biologically active protein produced by a bacterium under the direction of a DNA fragment (Human) obtained directly from the genes of a single individual. The clone is a chemically synthesized short oligonucleotide 1Ili! By hybridization, it was determined to be complementary to the sequence of IFN-α. Reassembly/0.5×10c'
Of the genes, 16 clones were positive for hybridization. RcoR hybridizes to oligonucleotides
As a result of examining the sequence of the engineering fragment, clone 18-3 was found to be the first clone.
As shown in Figure B, the 2 Kb HCOR fragment 18. where the j1 gene of FN-ac is located. -13 (1st BER
It was found that it contains the inserted part of I). This gene was clearly identified by Goedde: L et al.
Al, )' (1981, Nature 290. 2O-25) is the origin of the mRNA giving rise to the Ir N-' c clone. Clone 5-1 represents a duplicate DNA fragment. Clone 5-1 and clone 18-3 both contain a
-4 and two other engineering FN-f named a-64.
f gene? (1st BI&I). The recA promoter is considered one of the strongest bold promoters, usually when present on a multicopy plasmid such as pBR322.
The promoter is induced by cutting out its own suppressor in the presence of damaged DNA, and induction occurs to a very high degree. [5ancar and Ru
pp). 1979, PNA876, 6144-3148
). Substrates of recA are nalidixic acid, which prevents DNA synthesis, or mitomycin, which cross-links DNA. racA cloned to isolate the promoter on the approximately 1 kilobase Taq I-Ban)iI fragment.
A cilasmid containing pDR1453k was used. This is 01
It was ligated to pBR322 cleaved with a I and Ban I to construct cilasmid ftAP-1'r. In this case Ta
The q1-Ola1 linkage restored the Ola1 site, allowing grasmid to be cleaved uniquely in the third codon of the roem gene. RAP-2 is R
AP-1k was cut with 1aoRI and C1aI, filled in with sticky ends, and re-stretched with 31 rounds (31!) to create RAP.
-2 cleaved at the R-engineering site, foreign DNA vreaA I
M white can enter the third codon K J4. The RAP-1 vector was used for indirect detection of interferon activity using gene fragments from the human gene library. The IFN-β1 gene and several isolated a subspecies IFN-α genes produced strong φ interferon 1 activity when inserted into the plasmid of the human plasmid, according to a semi-antiviral assay. was recognized. RI fragment of DNA in which these genes are present (Figures 1A and B) V
Secondary cloned into the RI site of RAP-1 cilasmid. The plasmid is introduced into a reCA+ strain, the bacteria are cultured, induced with nalidixic acid, the cells are harvested,
The cells were lysed with lysozyme and 30% progylene glycol, and the extract 0 was analyzed for antiviral activity. The extent of activity clearly depends on induction by nalidixic acid (at high 50μ
g/d (Table 6). 1, nAp-1 (1853)-EngF'N-Direct. +Nalizoxic acid 500u4p-1 (1833)-
FN-40-nalidixic acid <622, RA
P-1 (1833)-Eng 7N-ao Right direction 10
00RAP-1 (6!+1)-Eng FN-81 right direction
3000RAP-1 (631) - Engineering F to β1 Reverse direction
750 clone RAP-1 (1833) is engineered FN-α
. Contains an ICcoR fragment carrying the gene (Fig. 1B, 1)
Clone RAP-1 (631) contains a KcoR fragment carrying the FN-βl gene (Fig. 1A). The fragment was introduced at the beginning of the recA gene. The direction is rec
This is related to the orientation of the promoter. Interesting: Is the gene fragment active in two possible orientations relative to the recA promoter? It also says that it will be produced. 1. The active FN-β1 and PN-a genes were located on a 1.8-2 kilobase DNA BcoR fragment. The distance from the promoter to the ATG start codon of the interferon gene is approximately several hundred nucleotides. It is clear from these experiments that the promoter functions as expected, and it is possible to rapidly determine the presence or absence of activity of interferon-like genes from gene libraries. Cilasmid pb, r 3 (:John
srud). pNAs, 1978.75.5314-5318) contains two 1ac promoters, each 95 base pairs long, separated by 95 unrelated nucleotides. This 285 nucleotide fragment is inserted into the Eco11 site of cilasmid PMB9. The 95 base pair long lac sequence contains operator and promoter sequences and stops just before the ATG of β-galactosidase. Encoding D with an ATG initiation codon inserted at an appropriate distance from the ribosome binding site of β-galactosidase
The NA sequence should be correctly expressed in E. coli cells. The 285 base pair BCoR fragment was converted into 1) BR322 E
Re-cloned into the coRI site. 404 to recombinant 1
Cells were grown and sorted on plates containing g/d X-ga'l and 15 μg/d tetracycline. Only positive blue colonies were collected and purified by DNA 7 cesium chloride gradient centrifugation. Two directions can be expected for the laa promoter: the ampicillin gene direction of pBR322 and the tetracycline gene direction. The authors are interested in lac! Plasmid DNA K
RNA polymerase binds to this site and preserves this site, while the peripheral IcoR enzyme cleaves it and cleaves the DNA.
A/Klenow 7 fragment of limerase (Klenow
fragment) and recombined. After transformation of MM294 large and parapet cells, zolamid was isolated and the distance between the EcoRI site and the BamH site of pBR322 was measured. The plasmid containing the 375 base pair fragment has the 1aC promoter in the tetracycline gene direction and the 670#A base pair fragment (675±295 b, p,
) A plasmid containing ψ was derived from an EcoRI1-84
The gll fragment was excised. The mature IFN-β□ protein contains a methionine at its amino terminal end, which can be used as the η initiation codon in the large intestine. Two Alu 1 adjacent to this jATG codon are separated by 16 nucleotides.
There are two sites, one is 8 nucleotides before ATG, the other is A
There are two nucleotides after TG. Partial A'lu T digestion was performed on a piece of Y Hlncl-Bg1ml and a fragment of approximately 510 base pairs was isolated on a keagarose sprue. 18. Tetracycline gene direction. Restricted with the vector f7EcoR containing the C promoter, restriction site 7 DNA yje
It was embedded with IJ merase and recut with BamH. E embedded with Alul-Bg1m fragment
Clone 5 was obtained to allow differentiation between clones containing the cATG codon (16 nucleotides long) in which the WQOR engineering site was restored by ligation of the coRI-BamHI vector. Restricted with EcoRI and Pstl, the expected 15
A clone containing a 1 base pair fragment was sequenced. Then, the KcoR engineering site Y M lm14 was cleaved, and the km between the ribosome binding site and ATG was shortened by Bal 3' 1 digestion and religation. E. coli transformed with these plasmids were selected by IFN-β production. Clone L-11 produced approximately 3 x 10' units/unit of F to β. The lac promoter region in this clone was cut with HpaH, ie, 17 nucleotides before the start of the mRNA. To cut out the engineering FN-β, regressor,
Enzyme 8au 3 av and its DNA 7a-■FN
- Cut 3 nucleotides beyond the stop r:/'Jgt of β1. This HpatoSau3a1ac-FN-β1 fragment was equally sized between the 01&1 site and the Hlnd1 site of the tryp promoter plasmid prepared as follows. 18 of -21 to -201 before the start of try NA
Try a 0 nucleotide V-length Taq I-Taq I fragment.
Excise from p plasmid pBP 121-221, pB
Cloned into the 01a T site of p322. We carried the tryp promoter fragment in a clockwise orientation. This operation inserts an Ola 1 site at position -21 of the ftryp promoter. After ligation to the 1ac-FN-βl fragment, a hybrid promoter tryp-1ac is formed in front of the PN-β□ gene. This plasmid TL-11 was used to transform E. coli MM294 or colon Ciminicell strain p679-54. These bacteria have an OD of 6. . 10 shots #kimarui; and 108
Unit/j! The company will produce FNJl. The results show that this engineered PN-βl gene DNA fragment has high activity (Fig. 4).

[Brief explanation of drawings]

Circle (at λC! Schematic diagram of Haron 4A. The two arms will be shown in the second part. (bl) Emotion of human D'i axis inserted into clone 015. Blacked part is engineered FN-β] Hybridized into cDNA The EcoR fragment is shown. lc) An enlargement of the blacked-out fragment from b is shown as the -a line. The single line is the part of the λ right arm. PL is the λ left promoter. (tl) position, the upper scale corresponds to (al and (bl), the lower scale corresponds to (Ql
and (cl). See text for details. Restriction map Y of two λ0haron 4A clone inserts is shown. The yy-ac gene is indicated by arrow alpha c4. The other two engineered FN-ff genes are engineered
. exists near. The insert contains fragment 18-, which contains the gene gene in FN-a83 and 1coR-2, which was cloned into the recA plasmid as described in the text.
33 is shown. The symbol corresponding to one restriction enzyme site is shown. Analysis using 5epharose The crude lysate of E. coli DP5Q infected with phage 015 was fractionated into 1 fraction per "Materials and Methods", and the EFN activity was analyzed in both fractions (To 1).The protein concentration was determined in the same fraction. Figure 6 1: Gene clone FN-β1c15 interferon titer 2 Fraction of phage 0151FN from the column in the figure (approximately position 103j1/*/)? 25 units/ml of standard human fibroblast interferon was serially diluted on a microplate for phase separation of engineered FH by dilution of VSV RNA synthesis by diluting /1:40. Analyzes were conducted in parallel (1-------.). Control without VEIV (0) and control with VSV and without F'N (-)
The two columns on the right indicate 0, as shown in “Method”.
Residual activity of phage 015-FN after adsorption to immobilized FN-β or non-immunogenic gG (final dilution '1:40)
) is shown. The engineered FN-producing TLl 1 plasmid was hybridized as shown in the text.
p~], contains the sequence encoding the aC zolomotor and the human gene fragment-derived mature human FN-β1. Hoso'dMk11t New Delnswick 4@
(lieu Brunawick fermento
At the indicated times, bacteria were incubated with lysozyme-propylene glycol and challenged with VEJVK human cells to measure EFN activity. l'N activity is calculated per 1 bacterial culture. Agent: Asamura Akira, 4 people, 21 sides, 31 people 4. Continued from page 1 of i Inventor Louise Cheno 52 Matt Aviv Tagore Street, State of Israel 0 Inventor Jelton Israel Feinstein 33 Farnham Avenue, New Haven, Connecticut, United States of America Procedural amendment Letter (method) January 1948 - Mr. Commissioner of the Japan Patent Office 1, Indication of the case Showa -! Near Year Patent Application No. θz29 No. 3, Relationship with the person making the amendment Patent applicant 4, Agent 5, Date of amendment order: December 4, 1950 6, Number of inventions increased by amendment 7, Amendment (No change in content) 8. Contents of amendments as attached.

Claims (1)

[Claims] (1) Human gene DNA is introduced into a suitable phage. Either a suitable bacterium is infected with the recombinant phage, or an appropriate NA fragment is collected from the phage. Production of fibroblast (β) type and leukocyte (-1 type human interferon) is carried out by introducing this into an appropriate bacterial expression plasmid, culturing the resulting cells, and collecting interferon. (2) The method according to claim 1, wherein the interferon is the (β) type and the phage used is the λ type. haron 4 A. (4) The method according to claim 1, wherein the interferon produced is the (-) type and the phage used is the λ type. The method according to claim 4. (5) The method according to claim 4, wherein the phage is λ-0haron 4 A involved in the production of Cl0n018-6. (6) The phage used for infection is the λ type, and The method according to any of claims 1 to 5, wherein the bacterium is Escherichia coli (F!, coli). -5Q. (8) A human gene DNA fraction is collected from a phage, introduced into a plasmid expression vector of a suitable bacterium, and cultured in the bacterium to produce interferon. The method according to claim 1, characterized in that: (9) the method according to claim 8, wherein the DNA is introduced into a plasmid biased for a strong Zodiac motor; The method according to claim 8, wherein the gene to be introduced is the gene F'N-β1 or the gene IFN-α. Claim 10
The method described in section. α2 A human gene DNA fragment containing the FN-g or FN-β gene, taken from a phage or plasmid. The E. coli 1ac promoter, which has been modified to contain the RNA polymerase binding site of the tritphan promoter, is cleaved next to the codon corresponding to the first methionine of the mature engineer FH linked to the bosomal binding site to generate a hybrid try.
The method described in item 8 or 10 of the patent scope, characterized in that the plasmid is reintroduced into a bacterium and the bacterium is cultured to produce interferon.