JPH08187100A - Deciding method of n-end sequence of secreted protein - Google Patents

Abstract

PURPOSE: To decide N-end sequence of a fused protein by inserting an N-end peptide gene containing a secreted signal sequence of a secreted protein at upstream of a marker protein gene, manifesting with introducing into an animal cell and using a marker protein as an indicator. CONSTITUTION: A DNA fragment coding an N-end peptide containing a secreted signal sequence of an arbitrary secreted protein is inserted into upstream of a gene coding a marker protein (e.g.; urokinase) and resultant fused gene is introduced into an animal cell (e.g.; CHO cell). The animal cell is cultured a fused protein of the protein coded with the DNA fragment and the marker protein is manifested, and the fused protein secreted into the cultured solution is purified using the marker protein as an indicator, then an N-end sequence of the purified fused protein is decided, thus the object N-end sequence after secretion of the secreted protein is decided.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for determining the secretory N-terminal sequence of a secretory protein. By this method,
After secreting a secretory protein useful as a medicine, it is possible to easily determine what kind of N-terminal sequence it has.

[0002]

2. Description of the Related Art Bioactive proteins produced by cells are
It is widely used in industry such as medicines, diagnostics, biosensors and bioreactors. Among them, secretory proteins are most promising as pharmaceuticals. For example, most of the protein drugs currently on the market such as interferon, erythropoietin, thrombolytic agents, etc. are secretory proteins.

The secretory protein has a hydrophobic sequence consisting of about 20 amino acid residues, called a signal sequence, at its N-terminus. After the protein moves into the parcel by the action of this signal sequence, the signal sequence is cleaved and removed by the action of the signal peptidase. Therefore, the protein actually secreted does not have a signal sequence. When using such a secretory protein as a medicine, it is necessary to know the N-terminal sequence of the secreted protein. Conventionally, in order to determine the N-terminal sequence of a secretory protein, the target protein is isolated from serum, or the gene encoding the target protein is expressed in cultured animal cells, and the secreted protein is isolated. After separation and purification, a method for determining the N-terminal sequence has been used. However, it was difficult to isolate and purify the secreted protein in a large amount and determine the N-terminal sequence.

[0004]

The object of the present invention is to provide a method for easily determining the secretory N-terminal sequence of a secretory protein.

[0005]

DISCLOSURE OF THE INVENTION As a result of earnest research, the present inventors have found that a DN encoding an arbitrary signal sequence peptide is used.
After fusing the A fragment with the gene for the marker protein,
It was found that when introduced into an animal cell, a fusion protein of a protein encoded by a DNA fragment and a marker protein is secreted into the culture medium, and by utilizing this, the fusion protein secreted in the culture medium has an affinity for the marker protein. The present invention was completed by constructing a method for determining the N-terminal sequence of the purified fusion protein after purification by utilizing sex.

That is, the present invention provides D encoding an N-terminal peptide containing a secretory signal sequence of any secretory protein.
NA fragment is inserted upstream of a gene encoding a marker protein, the fusion gene is introduced into an animal cell, and then the animal cell is cultured to fuse the protein encoded by the DNA fragment with the marker protein. A secreted N-secreted protein characterized by expressing the protein and purifying the fusion protein secreted in the culture medium using the marker protein as an index, and determining the N-terminal sequence of the purified fusion protein. A method for determining the terminal sequence is provided.

The present invention also provides a replication origin for Escherichia coli, a replication origin for animal cells and a promoter, and an N-terminal peptide encoding a secretory signal sequence of any secretory protein inserted at the cloning site located downstream thereof.
Provided is a NA fragment, and a plasmid vector including a marker protein coding region downstream of the NA fragment, which is in frame with the coding region of the gene inserted into the cloning site and can be purified using the gene as an index when secreted. To do.

Furthermore, the present invention introduces the above plasmid vector into an animal cell to express a fusion protein of a secretory protein and a marker protein, purifies the secreted fusion protein, and then the N-terminal sequence of the purified fusion protein. A method for determining the N-terminal sequence of a secretory protein after secretion is provided.

The present invention relates to a promoter for animal cells, DN encoding a signal sequence peptide of any secretory protein.
When a vector containing the gene encoding the A fragment and the marker protein (Fig. 1) is prepared and introduced into an animal cell, a fusion protein in which an arbitrary signal sequence peptide is added to the N terminus of the marker protein is synthesized in the cell. It was found that a fusion protein of the N-terminal peptide of the target secretory protein and the marker protein was secreted.

The present invention comprises the following three steps. Step 1 A DNA fragment encoding the N-terminal peptide of the target protein is recombined into an animal cell expression vector containing a gene region encoding a marker protein. Step 2 The obtained vector is introduced into animal cells to secrete and produce the fusion protein in the culture medium. Step 3 The fusion protein secreted in the culture medium is isolated and purified, and the N-terminal sequence of the isolated fusion protein is determined by amino acid sequencing.

Step 1 DNA encoding the N-terminal peptide of the target protein
A conventional method is used to recombine the fragment into an expression vector for animal cells. That is, a vector in which a promoter for animal cells, a cloning site (restriction enzyme cleavage site), and a gene encoding a marker protein are connected in series is used, and a target DNA fragment is inserted into the cloning site (FIG. 2, Method 1). When the cDNA encoding the target protein has already been recombined into an animal cell expression vector, the gene encoding the marker protein may be inserted into an appropriate restriction enzyme site located downstream of the signal sequence. Good (Figure 2, Method 2).

The expression vector for animal cells used in the present invention comprises a replication origin for E. coli, a replication origin for animal cells and a promoter, a cloning site existing downstream thereof,
It is a plasmid vector downstream of which is in frame with the coding region of the gene inserted into the cloning site, and which, in the case of secretion, is capable of isolating and purifying the marker protein.

The marker protein is preferably one that can be secreted by adding a signal sequence, and that can easily detect and isolate and purify the secreted marker protein. Examples include urokinase-type plasminogen activator (urokinase), tissue plasminogen activator, proteases such as blood coagulation factor, enzymes such as β-galactosidase, protein A, protein-binding factors such as antibody, etc. . When the marker protein is originally a secretory protein, those obtained by removing the signal sequence peptides derived from these proteins are used.

FIG. 3 shows the vector pSS used in the present invention.
The structure of D1 is shown. pSSD1 is a replication origin of SV40, an early promoter, a 16S splicing site,
Universal primer site for sequencing (U),
T7 promoter, BglII and EcoRV sites, urokinase translation region (including Pro from position 137 to Leu from position 411), poly (A) addition signal of SV40, origin of replication of pUC vector, β-lactamase gene, f1 phage Contains the origin of.
With this vector, single-stranded phage DNA (cDN
A) (including the antisense strand of A) can be easily prepared, and the base sequence of the cloned DNA fragment can be easily determined using universal primers.

Examples of secretory proteins include constituents of extracellular fluids such as blood and lymph, cytokines, extracellular matrix and the like. Since the membrane protein also has a secretory signal sequence, it falls within the category of the secretory protein of the present invention. Examples of membrane proteins include receptors, transporters, ion channels and the like.

The DNA fragment encoding the N-terminal of the secretory protein may be of genomic origin, cDNA origin, or synthetic DNA. In the case of genome or cDNA, a fragment cut with an appropriate restriction enzyme may be used, or a DNA fragment prepared by the polymerase chain reaction (PCR) method using an appropriate primer may be used. If an open reading frame starting from the initiation codon exists in the DNA fragment and the frame is in frame with the gene encoding the marker protein, a vector capable of expressing these fusion proteins can be prepared.

Step 2 Escherichia coli was transformed with the vector prepared in Step 1, and then colonies were formed into single colonies.
Animal cells are transfected with single-stranded DNA or single-stranded phage.

To introduce the expression vector into animal cells,
The calcium phosphate method, the DEAE-dextran method, the liposome method, the electroporation method and the like can be used (see, for example, Experimental Medicine Separate Volume “Gene Engineering Handbook”, Yodosha, 1991). Alternatively, if a single-stranded phage is prepared and then used for transfection, the gene can be more easily introduced into animal cells [M. Yokoyama-Kobayas
hi and S. Kato, Biochem. Bio
phys. Res. Commun. 192: 935-9
39 (1993)].

Any animal cell may be used as long as it functions as a replication origin or promoter incorporated in a vector. When the vector of the present invention is used, COS7, CHO cells and the like can be exemplified. It is also possible to use cells in which the secreted protein is actually secreted.

Animal cells into which the expression vector has been introduced are cultured under appropriate conditions, and then a culture supernatant is prepared. Whether or not the marker protein is secreted therein is examined using the method for detecting the marker protein used. In the case of enzymes,
Use the corresponding enzyme activity detection method. When there is an antibody against the marker protein, enzyme immunoassay may be performed using this antibody. Urokinase secreted when used in the examples can be detected by a method using an artificial substrate peptide or a fibrin plate method. In the point that a large amount of sample can be easily detected with a small amount of sample,
The fibrin plate method illustrated in the examples is suitable.

Step 3 If the marker protein is detected in the culture medium,
The fusion protein of the N-terminal peptide of the target protein and the marker protein is secreted. Therefore, after culturing on a large scale, affinity chromatography,
That is, the fusion protein can be easily isolated and purified by passing the culture solution through a column packed with a gel carrier on which a substance that specifically binds to the marker protein is immobilized. The N-terminal of the isolated and purified fusion protein can be easily determined using an amino acid sequencer.

[0022]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

The basic manipulations and enzymatic reactions for DNA recombination are described in the literature ("Molecular Clon").
ing. A Laboratory Manual
l ", Cold Spring Harbor Lab
Oratory, 1989). Unless otherwise specified, the restriction enzymes and various modifying enzymes used were those manufactured by Takara Shuzo. The buffer composition of each enzyme reaction and the reaction conditions were in accordance with the attached instructions.

Preparation of vector for cloning signal sequence 1 μg of 10 μg of plasmid pUC19 (Pharmacia)
After digestion with 00 units of ScaI followed by 100 units of PstI, it was subjected to 0.8% agarose gel electrophoresis to isolate and purify the large fragment containing the origin from the gel. Plasmid pKA1 (described in JP-A-4-117292) 10
μg is 100 units of ScaI, then 100 units of Ps
After digestion with tI, it was subjected to 0.8% agarose gel electrophoresis to isolate and purify a small fragment containing f1 origin from the gel. After ligation of both fragments, E. coli HB1
01 transformation was performed. 10 μg of the plasmid isolated from the transformant was treated with 100 units of PvuII and then 1
After digestion with 00 units of PstI, it was subjected to 0.8% agarose gel electrophoresis to isolate and purify the large fragment containing the origin from the gel.

Plasmid pKA1 (JP-A-4-1721)
92) (10 μg) was digested with 100 units of AccI, blunt-ended by treatment with Klenow fragment, then digested with 100 units of PstI, and subjected to 0.8% agarose gel electrophoresis, and the gel containing small SV40 origin was digested. The fragment was isolated and purified. After ligation of both fragments, Escherichia coli HB101 was transformed. A plasmid was isolated from the transformant, and it was confirmed from the restriction enzyme map that it was the desired plasmid pKA1U. That is, pKA1U is the pBR3 that pKA1 had.
The origin of 22 is replaced with the origin of pUC.

After digesting 1 μg of pKA1U with 10 units of BstXI, its ends were blunted with T4 DNA polymerase and phosphorylated BglII with T4 DNA ligase.
The linker was connected. This was digested with 20 units of BglII and cyclized with T4 ligase to construct pKA1UB. pKA1UB1μg, 20 units EcoR
After digestion with 20 units of Vpn KpnI, the terminal phosphate group was removed by alkaline phosphatase derived from Escherichia coli C75.

Human fibrosarcoma cell line HT-10
80 cDNA libraries (JP-A-4-117292)
As a result of the nucleotide sequence determination of the clone arbitrarily selected from the above, the clone p containing the full-length cDNA of urokinase
KA1-UPA was obtained.

[0028] The following two kinds of synthetic DNA oligomers having a cDNA sequence corresponding to the N terminus of the human urokinase activity domain (Pro to 137th to Glu to 142th) and a sequence of urokinase 3'untranslated region were DN:
It was synthesized using an A synthesizer (Applied Biosystems). SSD1 5'-GGCGATATCCCTCCTCTCCTCCAGAAG-3 'SSD2 5'-CGCGGTACCAGCAAGAAAGCGGGTGG-3'

PCR was carried out using these synthetic oligomers as primers, and the urokinase activity domain cD
NA was amplified. That is, Ps of pKA1-UPA
PCR was carried out for 30 cycles using 1 ng of the 1.2 kbp fragment isolated from the tI digest as a template and 1.8 nmoles of SSD1 and SSD2 as primers. PCR product with 40 units of EcoRV and 30 units
After digestion with units of KpnI, 0.7% agarose gel electrophoresis was performed to isolate a fragment of approximately 860 bp. These fragments were ligated to the vector fragment described above using 4DNA ligase to construct pSSD1.

Tumor necrosis factor-derived signal sequence peptide /
Construction of Expression Vector of Urokinase Fusion Protein 1 μg of pSSD1 was completely digested with 10 units of EcoRV, and then partially digested with 2 units of EcoRI at 37 ° C. for 45 seconds, and the terminal phosphate group was removed with Escherichia coli C75 alkaline phosphatase.

Human tumor necrosis factor (TNF-α) full length c
Plasmid pKA1-TNF containing DNA (Patent Document 4)
117292) 1 μg BalI and EcoR
Subject the I digest to 1.5% agarose gel electrophoresis,
A cDNA fragment of approximately 500 bp was isolated from the gel. Escherichia coli JM109 was transformed with the T4 DNA ligase reaction product of this fragment and the vector fragment. The plasmid contained in the obtained transformant was analyzed, and urokinase cDNA was analyzed.
Two EcoRI sites in which the TNFcDNA fragment was inserted upstream of the urokinase cDNA 5'side were selected as pSSD1-TNF (Fig. 4).

The transformation sample was prepared as follows. E. coli JM carrying the plasmid pSSD1-TNF
A small amount was collected from a glycerol stock of 109 transformants with a sterile toothpick and inoculated into 2 ml of 2xYT containing 100 µg / ml ampicillin. After incubation for 1 hour and 30 minutes at 37 ° C., 50 μl of a helper phage M13KO7 suspension
Was added and the cells were further cultured for 16 hours. This culture solution was centrifuged twice at 15,000 rpm for 5 minutes twice to obtain a supernatant from which Escherichia coli was completely removed. 1.2 ml of this supernatant
To 20% polyethylene glycol, 2.5M Na
Add 0.3 ml of Cl solution and mix well, then at room temperature 1
After standing for 0 minutes, centrifugation was performed at 15,000 rpm for 10 minutes, and the precipitate was collected. The obtained precipitate was suspended in 120 μl of Tris-EDTA (pH 8.0), and this suspension was used for transformation.

Transfection of COS7 cells and preparation of culture supernatant COS7, a monkey kidney-derived cultured cell, was cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum for 5 days.
Culture was performed at 37 ° C. in the presence of% CO ₂ . 2x10 ⁶ CO
S7 cells were seeded in a culture bottle (Nunc, culture area 175 cm 2) and cultured at 37 ° C. for 20 to 22 hours. After removing the medium, the cell surface was washed with phosphate buffer, and
DMEM containing mM Tris-HCl (pH 7.5) (TDM
It was washed again with EM). Phage suspension 2 described in the previous section
0 μl of T containing 0.4 mg / ml DEAE dextran
After mixing with 15 ml of DMEM, this solution was added to COS7 cells and cultured at 37 ° C. in the presence of 5% CO ₂ . After 3 hours
150 μl of 10 mM chloroquine was added, and the cells were further cultured for 2 hours. After removing this solution and washing the cell surface with TDMEM, 40 ml of DMEM containing 10% fetal bovine serum was added, and the mixture was cultured at 37 ° C. for 2 days in the presence of 5% CO ₂ . The culture solution was removed, and the cell surface was washed with 10 ml of phosphate buffer. This washing was repeated twice. After that, 40 ml of DMEM without serum was added, and the cells were further cultured for 3 days. The medium was recovered, and floating cells and the like were removed by centrifugation (5,000 rpm, 10 minutes, 4 ° C.) and filtration (3MM filter paper). Sodium chloride was added to this culture supernatant to a final concentration of 0.5M.
Was added.

Assay of culture supernatant 10 ml of human thrombin (Mochida Pharmaceutical Co., Ltd.) was added to 10 ml of 50 mM phosphate buffer (pH 7.4) containing 2% bovine fibrinogen (Miles), 0.5% agarose and 1 mM calcium chloride. In addition, a fibrin plate was prepared by solidifying in a plate having a diameter of 9 cm. 10 μl of the culture supernatant of the transfected COS7 cells was placed on a fibrin plate and incubated at 37 ° C. for 15 hours. As a result, formation of a dissolution circle was observed and secretion of the urokinase fusion protein was observed.

Preparation of urokinase antibody-immobilized column Cyanogen bromide activated Sepharose 4B (Pharmacia)
1 mM hydrochloric acid was added to 0.5 g and swollen at room temperature for 15 minutes to obtain a gel having a volume of 1.5 ml. The gel was washed with 1 mM hydrochloric acid on a glass filter G4, and further coupled with coupling buffer A (0.1 M sodium hydrogen carbonate, pH).
8.5, 0.5 M sodium chloride). On this gel, human low molecular weight urokinase antibody (goat origin, biopool) 5 dissolved in 5 ml of coupling buffer A was added.
mg was added and reacted at 4 ° C. for 16 hours. After removing the supernatant, the gel was filled with blocking buffer (1M glycine,
4 ml of 0.1 M sodium hydrogen carbonate, pH 8.0) was added and reacted at room temperature for 4 hours. After removing the supernatant,
The gel was washed with Coupling Buffer A and Coupling Buffer B (0.1 M sodium acetate, pH 4.5, 0.5 M sodium chloride), alternating 4 times. 0.5 more
20 mM phosphate buffer, pH 7, containing M sodium chloride.
Washed with column 5, column (Econo column, diameter 1 cm, Bi
oRad) and stored at 4 ° C.

Isolation and purification of the fusion protein The low molecular weight urokinase antibody-immobilized column described in the above paragraph was
Equilibration was performed by flowing a 20 mM phosphate buffer (pH 7.5) containing 0.5 M sodium chloride in an amount 10 times the column volume or more. COS7 for 4 culture bottles in this column
The cell culture supernatant was applied and the fusion protein in the culture supernatant was adsorbed on the column. After this, 0.5M sodium chloride containing 2
Run 0 mM phosphate buffer (pH 7.5) and
It was washed until the absorption at 80 nm was reduced to the background level. Next, 2 containing 2.5M potassium thiocyanate
A 0 mM phosphate buffer (pH 7.5) was flown, and the effluent was fractionated by 0.5 ml. The urokinase activity of each fraction was measured by the fibrin plate method.

N-terminal sequencing of the fusion protein The fractions in which the urokinase activity was detected were combined into one,
This was applied to a NAP10 column (Pharmacia) equilibrated with 0.2 M ammonium carbonate, 1 ml at a time, to give 1.5
Elute with ml 0.2 M ammonium carbonate. The obtained eluates were combined and concentrated to about several tens μl by speed back. This concentrate is used as an amino acid sequencer (AB
I, model 477A) to determine its N-terminal sequence. As a result, the N-terminal sequence of the obtained fusion protein was
Val-Arg-Ser-Ser-Ser-Arg-T
Decoded as hr-Pro-Ser-Asp-Lys.
This sequence represents phorbol ester stimulated HL-60.
N-terminal sequence of TNF-α purified from cell culture supernatant [B. B. Aggarwal et al. J. Bi
ol. Chem. 260: 2345-2354 (19
85)].

N-terminal sequencing of plasminogen activator inhibitor 1 mature protein (1) Construction of fusion protein expression vector Plasminogen activator inhibitor 1 (PA
Plasmid pKA1-P containing the full length cDNA of I1)
AI1 [Kato et al. , Gene 150: 2
43-250 (1994)] 1 μg of HindIII
The StuI digested product was subjected to 1.5% agarose gel electrophoresis, and a cDNA fragment of about 900 bp was isolated from the gel. This fragment was designated as HindIII-Ec of pSSD1.
After ligation with the oRV fragment, E. coli JM109 was transformed with the T4 DNA ligase reaction product. The plasmid contained in the obtained transformant was analyzed, and PAI1cDN was added upstream of the urokinase protease domain cDNA 5 ′ side.
The one in which the A fragment is inserted is selected, and pSSD1-
It was PAI1.

(2) Expression of fusion protein The f1 phage particles having pSSD1-PAI1 were introduced into COS7 cells according to the above method to express the fusion protein. After culturing for 3 days, the medium is collected and 5000 rp
The culture supernatant was obtained by centrifugation for 5 minutes.

(3) Partial Purification of Fusion Protein Into the above-mentioned column for immobilizing low molecular weight urokinase antibody, 1M sodium chloride, 0.01 times or more of the column volume was added.
20 mM phosphate buffer (pH 8.
0) was flowed to perform equilibration. COS7 cell culture supernatant for 4 culture bottles was applied to this column, and the fusion protein in the culture supernatant was adsorbed to the column. After that, 20 mM phosphate buffer (pH 8.0) containing 1 M sodium chloride and 0.01% Tween 80 was flown, and the effluent was washed until the absorption at 280 nm was reduced to the background level.
Next, 1M sodium chloride and 0.01% Tween80-containing 0.3% ammonia water (pH 11.5) were run, and the effluent was fractionated in 2 ml portions. Adjusted to. The urokinase activity of each fraction was measured by the fibrin plate method.

(4) N-terminal amino acid sequence determination of fusion protein Fractions with detected activity were combined and concentrated to about 0.1 ml with a centrifugal concentrator (Centricon-10). Trichloroacetic acid was added to this concentrated solution at a final concentration of 10
% To obtain a protein precipitate. This precipitate was used as a sample buffer for electrophoresis (60 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol,
It was dissolved in 0.025% bromphenol blue, 14 mM mercaptoethanol) and subjected to SDS-polyacrylamide gel electrophoresis. After completion of the electrophoresis, the gel was immersed in a transfer buffer (25 mM Tris, 192 mM glycine, 15% methanol) for 20 minutes. Using a semi-dry blotting apparatus (manufactured by Sartorius), three pieces of filter paper dipped in cathode buffer (25 mM Tris, 40 mM ε-aminocaproic acid, 20% methanol, pH 9.4), PVDF membrane dipped in gel, methanol and transfer buffer (Bio Rad), two pieces of filter paper dipped in anode buffer # 2 (25 mM Tris, 10% methanol, pH 10.4), anode buffer # 1 (0.3 M Tris, 10% methanol, pH)
10.4) Soak one filter paper in this order, 80mA, 2
It energized for a time and blotting was performed. The PVDF membrane after blotting was washed with distilled water, and a staining solution (0.2%
It was stained with Coomassie Brilliant Blue, 45% methanol for 5 minutes, decolorized with 90% methanol, and dried.
A band corresponding to the desired fusion protein was cut out from this membrane and subjected to analysis by an amino acid sequencer. As a result, Val-His-His was added to the N-terminus of the fusion protein.
-Pro-Pro sequence confirmed. This sequence is PA
It matched the sequence from the hypothetical signal cleavage site of I1.

INDUSTRIAL APPLICABILITY According to the present invention, the secretory protein useful as a medicine is encoded by the secretory protein without any isolation and purification of the secretory protein without isolation and purification. It can be easily determined using the existing cDNA.

[Brief description of drawings]

FIG. 1 is a diagram showing the structure of a fusion gene.

FIG. 2 is a diagram showing a step of producing a fusion gene.

FIG. 3 is a diagram showing the structure of pSSD1.

FIG. 4 shows the structure of TNF-α signal sequence peptide / urokinase fusion protein cDNA.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location G01N 33/68

Claims (4)

[Claims] 1. A DNA fragment encoding an N-terminal peptide containing a secretory signal sequence of an arbitrary secretory protein is inserted upstream of a gene encoding a marker protein, and then the fusion gene is introduced into animal cells, Culturing an animal cell to express a fusion protein of the protein encoded by the DNA fragment and the marker protein, purifying the fusion protein secreted in the culture medium using the marker protein as an index, and then purifying the fusion A method for determining the secretory N-terminal sequence of a secreted protein, which comprises determining the N-terminal sequence of the protein. 2. The method according to claim 1, wherein the marker protein is urokinase. 3. A replication origin for Escherichia coli, a replication origin for animal cells and a promoter, a DNA fragment encoding an N-terminal peptide containing a secretory signal sequence of an arbitrary secretory protein inserted in a cloning site existing downstream thereof, and its downstream. And a plasmid vector containing a region encoding the marker protein, which is in frame with the coding region of the gene inserted into the cloning site and can be purified using it as an index when secreted. 4. The plasmid vector according to claim 3 is introduced into an animal cell to express a fusion protein of an N-terminal peptide containing a secretory signal sequence of a secretory protein and a marker protein, and the secreted fusion protein is purified. A method for determining the N-terminal sequence of a secreted protein after secretion, which comprises determining the N-terminal sequence of the purified fusion protein.