JPS6229981A - Structural gene of sarcophaga lectin - Google Patents

Abstract

PURPOSE:A structural gene of sarcophaga lectin, consisting of a single-stranded DNA or double-stranded DNA coding a specific amino acid sequence of a specific protein and useful for producing a protein having physiological activity, e.g. antitumor effect. CONSTITUTION:The humor of larvae of Sarcophaga peregrina is collected and adsorbed and purified to give sarcophaga lectin (A). A mixture (B) of 8 kinds of 14 oligodeoxyribonucleotides, expressed by formula I and corresponding to part of cDNA sequence of the component (A) is then synthesized. RNA in a high mRNA concentration in the component (A) and the component (B) are hybridized to separate a clone and give the aimed structural gene having a signal sequence expressed by formula III linked to the 5'-terminal of sequence of about 780 nucleotides, e.g. nucleotides expressed by formula II (deoxyribonucleotide as follows; A is adenine; C is cytosine; G is guanine; T is thymine, and sequence for each codon corresponding to amino acids) and coding the amino acid sequence of alpha-subunit protein of the lectin protein.

Description

(a) Industrial application field
Sarcophaga peregrina) A protein with lectin activity (hereinafter referred to as Sarcophaga lectin) that can be produced in the body fluid of a larva when the body surface is injured, or that appears in the body fluid of a pupa in the early stages of pupa. of the α-subunit protein, which is a component of
It relates to DNA (cDNA) encoding an amino acid sequence.

(0) Conventional technology In general, in vertebrates such as insects and vertebrates such as wild birds, as well as medullary animals, changes in metabolism containing protein-4=platinic acid occur at the time of tissue injury, such as antibacterial release into body fluids. It is known that the appearance of active substances and thin 111 aggregating proteins occurs. This is thought to be the result of some kind of biological defense mechanism being activated in response to an external stimulus such as injury, but it is thought that vertebrates, which do not have the ability to produce antibodies, play a particularly important role in biological defense. Conceivable. When the body surface of the centipede fly larva is injured, proteins with remarkable antibacterial activity against Escherichia coli and Bacillus subtilis are released in the body fluid.
It has been revealed that both Norcophaga and lectin appear (for example, Mizuno et al., Mechanism of Biological Defense,
(See pages 41-54, published by Kawabankai, University of Tokyo, October 1980). Regarding the latter Sarcophaga lectin, its protein molecular weight, subunit structure, etc. have been clarified, and it has also been shown to be able to be used in tumor cells (C311/Hev mouse breast cancer-derived MM46 cells) in collaboration with macrophages in vitro. (e.g., Nakajima et al., Gann XVOI, 73.6
27-632. See August, 1082).

Furthermore, in stimulating macrophages derived from M1 mammals with Sarcophaga lectin, the molecular weight of approximately 30.0
The inventors have also found that 1 substance is produced between 0.00 and 70,000.

The molecular weight of this protein as determined by gel filtration is approximately 188,000.
Then, α with a molecular weight of about 32,000 was determined by 5DS-gel electrophoresis.
- Subunit l-4 molecules and β- with a molecular weight of about 30,000
The sub-knit is divided into two molecules.

(c) Structure of the invention The present inventors (C1 for α-subunit protein)
By successfully cloning NA and determining its nucleotide sequence, we were able to determine the amino acid sequence of α-1 nabuunit protein.

That is, the present invention provides a single strand of approximately 780 nucleotides that encodes the amino acid sequence of α-subunit l-protein of Lilcophaga lectin. double-stranded DNA consisting of
It is. The single-stranded nucleotide sequence of the present invention is more specifically represented by the following formula (1) or a biologically equivalent sequence thereof. 5'...GTG CC
CCAA TTA CAA△AG GCT
TTA CAT GCCACA CAA
TΔTCDingT ΔTTGAA ACA CAA
CTT ΔAGTACAAT TGG
CAT CAAGCCTGG CAT GAA
TG DingGCCCGCCAT GAT C△
ΔCAΔ CTT GTA ACA AT
CGΔ△ AGT GCT GAT AAGA
AT AAT GCCATT ΔTCGA
T CTG GTG AAA CGGG
TCGTT GCA AAA TCTCAT
AAT TTA TGG TTGGGC
GGCAAT GAT GAGTAT A
GT TCA AGT CGTGACTA
T GGCAGA CCCTTT TTC
TGG TCA CCCACCGGT CA
A GCA TTCTCCTTT GCCTA
CTGG TCG CAA AACAAT CCC...(
1) GAT AAT TAT AAG
CATCAA G, AA CAT TG
T GTCCAT ATA TGG
GAT ΔCΔAΔG CCCTTA T
AI” CΔATGG AACGAT A
AT GATTGT AAT GTT
AAA ΔTGGGT TACATA T
GT GΔACCCAAT CAT TT
CCGGGΔA ACA TAT GAT
CAAGCA CTCAAG CAA
ΔAAATGCGΔA GCA ATT
AAGATA ACA AAT TCA
AAAATT TCA ACA GAA
Lower TTGAT CAA TTG CAT
GCCAAA CAA TCAT
TG GAATTT GAT ACT
ATA ACGCAA AAT GTA
GCA AAAGTG 8AT GAA
GAT TGGAAA ATT GAA
ATCCA△AΔA CTA CAG
AAT GCCACA CAA ATT
GCCΔTACAA CAG ATT ATG
GAAAAT CAT GAG AAG
AAGATA AGA GAT TTA ΔG
TGAT AAT CTA CTT AAGCA
G CTA CAA GAT TCCAAT
GAA CAA CTG AAACAG T
CCACT GACCATATG AAT GC
A TCCI TTTGGT GAG AAA
TTG AAAGGCCAAA CAA GC
A GAAAAT AAT GAA ATT
TGT...3' (In formula (1), A, C, G, and T mean deoxyribonucleotides having adenine, cytosine, guanine, and dimine bases, respectively, and are shown as sequences for each codon corresponding to an amino acid. ) A biologically equivalent nucleotide sequence is one that has the same amino acid sequence as the nucleotide sequence of formula (1), even though it differs from the sequence of formula (1) in one or more types of nucleotides. ]-means the nucleotide sequence 911 as shown in FIG. In addition, when performing operations such as changing the -terminal strand DNA, the DNA complementary to it, i.e., the Δ9 complementary to the
= JT, DNA in which the de'oxyribonucleotide sequence was transferred in G versus C.

However, such double-stranded DNA is also included within the scope of the present invention.

Particularly preferred in the present invention is a nucleotide sequence in which a signal sequence of the following formula (2) or (3) is bound to the 5' end of the nucleotide sequence of formula (1).

5'...ATG AAG AACGTA GA
AGGCTTCGTT ATA TTTTTA
GTA ATT TTT'ACGTCT ACA
GCG GCA...3'...(2
) or 5'...ATG AGT TTA AC
A ΔTGAAG AACGTA GAA
GGCTTCGTT ATA TT'l-TTA
GTA ATT TTT ACG T
CTACA GCG GCA...3'
(3) In addition to a signal sequence, the 5' end of the nucleotide sequence of the present invention contains a known translation initiation codon and a Goldberg-Hogness box (promoter).

It is said that there is a 1q nucleotide sequence including the transcription start point, and 11 nucleotide sequences including a stop codon, poly(A) addition signal, and poly(A) are present on the 3' end. Not even.

Sarcophaga lectin in the present invention can be used in nature by impairing the body surface of the fly larva, for example, by penetrating the larvae with a single injection.
produced in and from this body fluid, e.g.
Extract using the following method.

Purified and isolated. Body fluids, e.g. Sepharose 4B
Most of the proteins contained in body fluids are collected in the unadsorbed fraction when the column is applied to
．． Elute the adsorbed protein by flowing 2M galactose solution-1
Sarcophaga lectin is then eluted. from now,
If galactose is removed by dialysis or the like, a pure aqueous solution of Sarcophaga lectin can be obtained.

(d) Effects of the invention The Sarcophaga lectin according to the present invention is
In an in vitro test, it not only destroyed C3l-(/l-1e mouse mammary carcinoma-derived MM46 tumor cells together with macrophages), but also in an in vivo test, it inhibited the proliferation of ascitic carcinoma 8180 cells transplanted into ICR mice. It has been confirmed that sulfaga lectin can be used as an anti-tumor agent, as it has been confirmed to suppress cancer and prolong and cure the survival of mice.It has also been confirmed that it induces the production of interferon in human white blood cells. Therefore, it is expected that the α-nab unit protein of Sarcophaga lectin obtained by genetic manipulation using the DNA of the present invention will also have physiological activities such as antitumor effects.

1T, from another study by the present inventors, the Sarcophaga lectin α-1)-buunitsu1~ and β-zabuconite1~
Since it is known that the primary structures of [alpha]-subknits 1~ and [beta]-sub]nits are derived from one common structural gene, it is assumed that [alpha]-subknits 1~ and [beta]-subknits are derived from one common structural gene.

It is thought that one of them will be modified after translation, and as a result, the two will exhibit different behavior in 5DS-polyacrylamide gel electrophoresis. In order to investigate this possibility 1, p.
mRNA with poly(A) (poly(A)”
RNA) and translated it in VitrO,
The translation products were analyzed by immunoprecipitation. In other words,
poly(A)” RNA was in v using a cell-free extract of rabbit reticulocytes containing S-methionine.
It'ro translation was performed, the translation products were immunoprecipitated with antibodies against Sarcophaga lectin, and the immunoprecipitates were analyzed by 5DS-polyacrylamide gel electrophoresis followed by indirect projection.

As a result, the protein corresponding to the α-subunit was precipitated by the antibody against S. faga lectin, whereas the protein corresponding to the α-subunit
- No hunt for 4-knob unit was detected. From this, α-→Nobu] to β-1 to Nobukonitsu 1 are derived from the same structural gene, and a3 is derived from the same structural gene.
ly (A)” RNA.

(e) Reference Example (1) Collection of body fluids from lenticin fly larvae The lentic fly larva wets its body surface with water to prevent the formation of a peristaltic shell and synchronize with the third instar. I used something. To cause damage to the body surface, the third instar larvae are placed on ice to slow their movement, and they are lined up on a glass plate, and a Note 04♀I is passed through the lower part of the first body cylinder. Using. Two days after the injury, the heads of the larvae were cut off with surgical scissors, and cooled on ice.
, 9 cm) and squeezed out the body fluid. With this operation, approximately 20,000 larvae to approximately 120
body fluids of me were obtained. Collect this in a Spitz tube, 3.0
OOrr+m (5 minutes, 4℃, KOKSAN MOD
The body fluid cells were removed by centrifugation in E+ 103R), and the supernatant was stored at 180°C as a body fluid preparation. If this preparation is to be used as a purification material, the stored material should be thawed in a 37°C water bath and then io, oooz (10 minutes, 4°C) to further remove any contaminating fat bodies. The centrifuged supernatant was collected.

(2) When the body fluid obtained as described above was passed through a 12-pharose 4B column, most of the proteins were recovered as an unadsorbed fraction. This is a protein that has antibacterial properties, unlike C. faga lectin. After thoroughly washing the column with physiological saline, a 0.2M galactose solution was passed to elute the adsorbate, ie, Sarlophaga lectin. From this, galactose was removed by dialysis to obtain approximately 1 mg of purified Sarcophaga lectin.

(3) Properties of Sarcophaga lectin When both protein-containing fractions obtained as described above were analyzed by native gel electrophoresis, a single protein band was observed, and the portion of the gel corresponding to this band was observed. When the protein was extracted and the activity was measured, sphere agglutination activity was detected.

Furthermore, when this protein was subjected to 5DS-polyacrylamide gel electrophoresis, it was separated into two subunits 1- with molecular weights of 32,000 (α-subunits 1~) and 30,000 (β-subunits 1~). The temperature ratio of the stained image was 2:1.

Furthermore, by gel filtration using Bio-Get P-300, the molecular weight of this protein was found to be approximately 188,000.

In addition, Requin-like live bamboo (hemagglutination activity ↑ (I), affinity chroma 1-graphie with Sepharose IB, native gel electrophoresis, SDS polyacrylamide gel electrophoresis, and gel filtration with Bip-Get p-300) Law is The Journal ofB io
Logical Chemistry, Vol. 2
55, No. 7.

P 2919-2924 (1980).

Example (1) Partial degradation of Sarcophaga lectin and determination of amino acid sequence In order to synthesize a DNA probe for elevating mRNA, it is first necessary to know the partial amino acid sequence of Sarcophaga lectin. Therefore, 100 μ9 of Sarcophaga lectin obtained in Reference Example was digested with TPCK-treated trypsin at an enzyme-protein weight ratio of 1:30 in a triethylamine/bicarbonate buffer at 37° C. for 12 hours. The resulting peptide was dissolved in 100 μΩ distilled water and attached to a G11sonl-IPLC system for 3 ynchr.

pak RP-P (C18) (250X 4.1
It was passed through a column of reversed phase high performance liquid chromatography of 1.5 mm, Synchrp lnc, . Then, it was added to solution A (0.05% (V/
V) Solution 3 for TFA in 1-120)
Elution was performed with a linear gradient of 5-60% (0.05% (V/v) TFA in acetonyl-lyl). As a result, the J: beat chromato pattern shown in FIG. 1 was obtained with good reproducibility. Among the various peptides, Beak ■ and Beak ■ were easy to isolate and collect, so the amino acid sequences of the peptides corresponding to these were determined.

The 18 peptide is l-l 11nkapil
Applied @ iosystem 470A using the program described by L.
Botein 5equencer was used for automatic sequence analysis (J, Immunol, 115,
1617-1624 (1975)). And PTI~1 derivative of amino acid is Unicil pack
3 htmazu equipped with Q C18 column
It was identified uniformly using an HPLC system. As a result, in the amino acid sequences of these peptides, the peak ■ is Val-A 5n-G 1u-ASll-Tr
In l'l-L VS, the peak ■ is Glu-Thr-Ty
r-ΔSn-GIn-Ala-Leu-
It turned out to be L vs. S.

Next, a mixture of 8 types of 14 homogeneous oligodeoxyribonucleotides represented by the following formula (4), which corresponds to the CD N A sequence of a part of the peptide of peak T, was synthesized.

......(4) These oligodeoxyribonucleotides are △sn
-Glu-Asp-Trll-LyS, including all possibilities of complementary sequences, provided that
The third nucleotide residue of lysine has been removed).

In addition, the synthesis of oligodeoxyribonucleotides is improved 1.
~Performed according to the Riester method (Tetrahedr
on Lett, 28.2449-2452゜(1
978)).

(2) Preparation of polV(A)'' RNA containing Sarcophaga lectin mRNA A This Sarcophaga lectin is expressed in the fat body as III RNA (polV(A)) with poly(A>).
y(A>'' RNA) was isolated from the fat body collected from a larva of the centinic fly that had been wounded with a syringe needle 6 hours earlier. In order to obtain RNA, it is preferable to inject, for example, 1×10 G of sheep red blood cells into the larvae of the centinic fly. This method is a very effective way to increase Sarcophaga lectin production. Three hours after the injection of the sheep red blood cells, fat bodies were removed from the peritoneal cavity of the centinic fly larva under a microscope. Total RNA was extracted from 60 fat bodies and
Δ)+RNΔ is determined by oligo(dT) cellulose column.
Prepared.

(3) Monkey] Cloning of the cDNA of Faga lectin - Part 1 The method of Kayama and Berg (MOI, Ce
11. Biol.

2, 161-170 (1982)).
DNA containing A and 3μq of vector/primer structure (
A cDNA library was created using Ok-ayama-Ber (I vector). In this case, Mo
Methods in En
zymol, 68. 326-331 (1979)).

coliHB 101 was developed by Hanehan and Mes.
Selected for ampicillin resistance as reported by Elson (Gene 10.63-6
7 (1980)). Then, at 36°C (1)
Screening was performed by hybridization with a mixture of eight synthetic oligodeoxyribonucleotides obtained in .

That is, using these synthetic oligonucleotides as probes, cD
By examining approximately 10,000 transformed strains of E. coli derived from the NA library, five hybridipesion-positive clones were isolated.

When the DNA nucleotide sequence of the positive recombinant plasmid was analyzed, it was found that only one clone contained the nucleotide sequence corresponding to the amino acid sequence of Bebutide in Peak I (this was named pIF2). . N
The amino acid residue following the terminal Val could be determined to be I-ys.

This agrees well with the specificity of trypsin digestion.

Thus, it is possible that this clone contains the cDNA of L. lectin, but I'1LE2 does not contain poly(dA)poly.
(dT) sequence, and its molecular weight revealed that it was approximately half the size of the DNA segment corresponding to III RNA of Monkey Faga lectin.

(4) Cloning of cDNA of Sarcophaga lectin - Part 2 Next, the following experiment was conducted to extract the complete mRNA. As a probe, the EcoRI-3au3AI fraction obtained from the 5' end of ρ1-E2 (see Figure 2)
A newly created c in the same manner as in (3) above using
Approximately 50,000 transformed strains of the DNΔDNA library were screened. And 24 hybridization positive clones were identified as 19.

Among these, the 10 clones with the same restriction enzyme map appeared to contain considerable coverage of the complete length of the NlRNA of S. faga lectin. One of these clones, riL E 10, was further analyzed for sequencing. This clone was pOIV(dA) pol
y(dT) and ll0IV<dG) I) OIV(dC
cDNA of approximately 1,3 Kb containing the tail part of )
It contained an insertion sequence.

(5) Assignment of base sequence and amino acid sequence of ril-Flo Figure 2 shows the restriction enzyme map of rlLElo and the method used to determine its complete base sequence.

Horizontal arrows indicate the direction and range of base sequencing of each restriction enzyme digest. The label portion at the 5' end is indicated by a slightly vertical line at the end of the arrow. In addition, the region encoding the protein is indicated by a white frame, and poly(dA)I)oly(dT)
The poly(dG) poly(dC) tail portion is not shown in the restriction map. The base sequence of nl Elo (complementary sequence to CDNA) revealed as a result of these tests is shown in FIG. 3 together with the amino acid sequence for α-subunit of Sarcophaga lectin.

In addition, the restriction endonuclease digest and the 5' restriction enzyme digested fraction of synthetic oligodeoxyribonucleotides
Terminal labeling was performed using the method of Kakidani et al. (N
ature 29, 245-249 (1982)
). Nick translation by [α-””P]-dCTP was performed using the method of Wc+n5tark et al. (Proc, Natl, A cad, S c
i, USA 75.1299-1303 (19
78) ). The DNA sequence is M a
xam and G11bert method (M methods
nE nZVmof, 65. 499-560 (1
980) J: was decided.

Plot hybridization of RNA and DNA
50% (V/V) Borumamide 15XSCC (1x3
cc= 0.15 MNa C9,0,015Mri I
:/acid sodium) / 5 X D enbarcl
t's solution (1X Denllardt'S solution - 0,02% (w/v) F 1co11-400.
The test was carried out at 42°C for 18 hours in a solution of 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone-40/50°C and 1 g/day of sonicated denatured salmon sperm DNA. And the plot is 10 at room temperature in 2XSSC.
and 0.1×SSC at 42° C. for 30 minutes.

(6) Base sequence of the cloned cDNA encoding Sarcophaga lectin (Monkey) In Figure 3, the deduced amino acid sequence of Sarcophaga lectin is superimposed on the base sequence (sequence complementary to the CDNA). Indicated.

The amino acid residues are numbered starting with the first methionine. Nucleotide numbers are also placed to the right of each line. In FIG. 1, the amino acid sequences corresponding to the peaks ■ and ■ of the trypsin-digested fraction are surrounded by solid lines. Furthermore, the amino acid sequence obtained by analysis of Sarcophaga lectin is indicated by a dotted line. The predicted signal sequence is indicated by a solid line below it.

The nine amino acid residues from the N-terminus of the complete Sarcophaga lectin are Vat-Pro-G1n-
1eu-Gln-LVS-Ala-Leu-Asp, which is 24-32 in Figure 3.
It corresponded to the second part. Therefore, the N-terminus of this protein is assigned to the 24th valine. Upstream of this position there are two candidate start codons giving two signal peptides of 19 and 23 amino acid residues, respectively. At the same time, it is difficult to determine from which initiation codon translation begins. From the genetic code corresponding to the N-terminal valine, 2 before the first stop codon (TAA).
There is an open reading frame (a DNA base sequence that can be translated as an amino acid sequence) for 60 amino acid residues.

The polyA sequence is about 100J from the stop codon.
The sequence AATAAA for the 1101 yA addition signal is located 17 bases upstream of the DolVA addition site.

The amino acid composition predicted from this base sequence is shown in Table 1, and this is consistent with that obtained by analysis of the complete Monkey Faga lectin.

(Left below) Table 1 Lysine 22 (8,5>
(8,7) Histidine
11 (4,2)
(4,4) Arginine 7 (
2,7) (2,8>Aspartic acid 18 (6,9) △S
p+ASn (1!i, 6) asparagine
22 (8,5)Threonine
10 (3,8) (3
,7) Serine 16 (6,1
) (5,7) Glutamic acid
21 (8,1) Glu+Gl
n (17,7)glutamine 2
4 (9,2>proline
6 (2,3) (2,0)
Glycine 9 (3,5)
(3,7) Alanine
15 (5,8) (
6,0) cysteine 6 (2
,3) (1,9)valine
9 (3,5> (
3,4) Methionine 3 (1
,2) (1,2) Isoleucine 17 (6,5)
(6,3) Leucine 1
9 (7,3> (7,6) Dirosine 9 (3,5)
(3,7) Phenylalanine
8 (3,1> (3
,5)1~Lyptophan 8 (3
, 1) (2,4)*: A: Number of residues B: Mol%

[Brief explanation of the drawing]

Figure 1 shows the partially degraded product of Sarcophaga lectin.
The flow pattern for reverse phase high performance liquid chromatography is shown. Figure 2 shows the restriction enzyme map of clone IILEIO containing the mRNA of Sarcophaga lectin. Figure 3 shows the amino acid sequence of the α-subunit protein of Sarcophaga lectin and the corresponding W gene. The base sequence of Patent Applicant: Teijin Co., Ltd. Company Announcement Tomi Seiyaku Co., Ltd. Day

Claims (1)

[Scope of Claims] 1. A single-stranded DNA consisting of approximately 780 nucleotides encoding the amino acid sequence of the α-subunit protein of Sarcophaga lectin, or two strands consisting of this DNA and its complementary DNA. Stranded DNA. 2. The single-stranded or double-stranded DNA according to claim 1, which has a nucleotide sequence of the following formula (1) or a nucleotide sequence biologically equivalent thereto. [There is a gene sequence]...(1) (In formula (1), A, C, G, and T mean deoxyribonucleotides having adenine, cytosine, guanine, and thymine bases, respectively, and each codon corresponding to an amino acid 3. A signal sequence of the following formula (2) or (3) or a nucleotide sequence biologically equivalent thereto is linked to the 5' end of the approximately 780 nucleotide sequence. Single-stranded or double-stranded DNA according to claim 1 or 2. [There is a gene sequence]...(2) or [There is a gene sequence]...(3)