JPH02308791A - Polypeptide capable of bonding to immunoglobulin fc fragment and having luciferase activity - Google Patents

Abstract

PURPOSE:To obtain a fused protein for a labeled 2nd antibody having high sensitivity and short determination time by collecting a polypeptide capable of bonding to the Fc fragment of an immunoglobulin and having luciferase activity from a specific culture product. CONSTITUTION:The objective polypeptide can be produced by culturing a transformant containing a specific recombinant plasmid (M) and collecting a polypeptide capable of bonding to the Fc fragment of an immunoglobulin and having luciferase activity from the culture product. The recombinant plasmid (M) is produced by using a gene coding a polypeptide having a specific DNA sequence containing the fragments of formula I or formula II as a part thereof and integrating the gene into a plasmid of E.coli. The fused protein has short enzymatic reaction time compared with a peroxidase protein A commercially available as a labeled 2nd antibody.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a novel polypeptide having the ability to bind to the Fc region of immunoguprin and luciferase activity, a gene for the polypeptide, and a set containing the polypeptide gene. The present invention relates to a recombinant plasmid, a transformant transformed with the recombinant plasmid, and a method for producing a novel polypeptide using the transformant.

[Traditional technique (nei)]

Protein A is produced by the bacterium Staphylococcus aureus (
Staphylococcus aureus), especially Cowan 1 strain (SAC)
is known to have a particularly high content. On the other hand, protein A is produced in large quantities and commercially available by integrating the gene into an E. coli expression vector and culturing E. coli transformed with the expression vector. It is generally known that protein A specifically binds to the Fc region of immunoglobulin. Then, the structural gene of β-galactosidase is linked to the protein A gene to obtain a fusion protein that has the activity of protein A and β-galactosidase, and this is used as an enzyme to measure the amount of antigen or antibody by antigen-antibody reaction. Immunoassay (
In EIA), it is suggested to use it as a labeled second antibody instead of an enzyme-labeled antibody.

(Gene, 23369-378 (1983))
, and also protein A- produced by chemical methods.
Peroxidase conjugates are commercially available as labeled second antibodies in place of enzyme-labeled antibodies. However, these protein A-enzyme fusion proteins or protein-peroxidase complexes have the drawbacks of poor sensitivity and long measurement times.

[Problem to be solved by the invention]

The present inventors developed a fusion protein for a labeled second antibody that has higher sensitivity and a shorter measurement time in place of the conventional fusion protein for a labeled second antibody, and produced it in large quantities using genetic engineering techniques. The present invention was completed with the aim of

The purpose of the present invention is to provide a polypeptide having the ability to bind to the Fc31 region of immunoglobulin and luciferase activity.

Other objects of the present invention are a gene encoding the polypeptide, a recombinant plasmid containing this gene, a transformant transformed with the plasmid, and culturing the transformant to produce the polypeptide. The purpose is to provide methods for manufacturing each.

[Means to solve the problem]

The polypeptide of the present invention has the ability to bind to the Fc region of immunoglobulin and luciferase activity, and this polypeptide has Vibrio har
This is a fusion protein obtained by linking luciferase of I. veyi to a protein A-like substance. When used as a labeled second antibody, this fusion protein has higher sensitivity than conventional antibodies, and because the enzyme reaction time is short, the measurement time can be shortened.

Staphylococcus aureus cola 1f 1 strain (S
raphylococcus aureus Cowa
Protein A derived from n I 5train) 1 binds to the Fc portion of immunoglobulins and can be used as a second antibody and as an immunoadsorbent for purification/removal of immunoglobulins and Fc fragments.

The structure of protein A is shown in FIG. The characteristics of this structure are that it binds to the Fc part of immunoglobulin, and
There are four regions, peptide and D.
There is homology in NA base sequences.

On the other hand, luciferase of luminescent bacteria is known to consist of two subunits, α and β. Vibrio harvey is a type of luminescent bacterium.
luciferase has a protein with a molecular weight of 42,000 as a subunit α and a protein with a molecular weight of 37,000 as a subunit β. The proteins of these subunits α and β are encoded by genes named IuxA and IuxB, respectively. luxA gene and luxB
The gene constitutes a part of the luciferase operon, which consists of several gene groups, in chromosomal DNA. R, P. Legocki et al. (Proc.

Natl, Acad, Sci, USA, vol, 83
．． 9080-9084 (1986)) transformed the chromosomal DNA containing the Vibrio harveyi luciferase genes (1uxA and 1uxB) into Escherichia coli plasmid pB.
Insert into R322 to create recombinant plasmid pFITOO
This paper describes a method for preparing 1, introducing this plasmid into E. coli, transforming it, and expressing luciferase.

The present invention aims to synthesize the BRM region of protein A, which is one of the regions that binds the Fc portion of immunoglobulin (Fig. 2), by a DNA synthesis method. Polypeptide (MW
87,000), and using this gene, Escherichia coli is made to produce the polypeptide in large quantities. That is, the polypeptide of the present invention has immunoglobulin Fc.
This polypeptide has binding ability and luciferase activity, and when used as a labeled second antibody, it has higher sensitivity than conventional antibodies, and the enzyme reaction time is short, so the measurement time can be shortened. It has advantages.

The gene encoding the polypeptide of the present invention has the following formula D
What is represented by the NA sequence GCGACTGAACATGGCTACGA TGT
GA to TAA6 A'l'l'tiAL;L;QC:
1GTTTGTCTTA CATCACCTCCGT
CGATCATG ACTCAAATAGAfLi to L, l; TT to T TTAATC[;At; to A
GATTCT CAGTTTCAGTTGTT to AC
CAGTGA GTGTCACAAG ATCATC
AATG ATGCATTCACIt,ALiL;A
AfLiLi TL;ATT(iL;A6G ^A
CAAAAA to A GCTGACATCTCGCCT
TGTTA 48 GGGGCATA TCATA
CCAT TACCACAATAT or the following DNA sequence.

[1TTTGTA GA TAAAGATTTT CG
TGTCTTTG, GTACAGACATGGATA
ACAGCCGAGGCTTAA TGGACTGTT
G GTATGACTTGTTATGAATTCAAA
GCGTTCT TCTGAT to AAG TCAT
CGAAGATTTAAGTTGA AGACCCTA
TT AACACTTGACGAT The recombinant plasmid of the present invention is one in which the above gene is inserted into an E. coli plasmid. And as a plasmid of E. coli, p[1R
720, pKK233-3, etc. are used.

The transformant of the present invention is a host cell transformed with the recombinant plasmid. E. coli is used as this host cell.

The method of producing a polypeptide having the ability to bind to the FceN region of immunoglobulin and luciferase activity of the present invention includes the following steps: (a) chemically synthesizing DNA of the gene B region of protein A; constructing a plasmid vector for incorporating DNA (
C) constructing a plasmid vector for incorporating the luciferase gene; (d) constructing a recombinant plasmid by ligating the two plasmids; (e) transforming host cells using the recombinant plasmid. (f) culturing the resulting Form I transformant in a nutrient medium to produce a polypeptide having the ability to bind to the FcrJ region of immunoglobulin and luciferase activity. The step (f) is followed by the following step (□□□ collecting the produced polypeptide. The gene encoding the polypeptide has luciferase activity and the N-terminus is The host cell is characterized in that the host cell encodes a polypeptide containing E.co.
It is characterized by being li.

The present invention will be explained below in order according to each step.

A protein AOB ml region gene having the ability to bind to immunoglobulin Fc is synthesized using a DNA synthesizer. On the other hand, pDR72, an E. coli vector,
Digest 0 with restriction enzymes. This digested vector and the above synthetic gene are ligated using 74DNAIJ gauze to obtain recombinant DNA. This recombinant DNA ApS
PAPc-1 was transformed into Escherichia coli HBIOI to obtain a transformant into which the desired DNA fragment had been inserted.

Vibrio ))-bay ATCC14126 strain is cultured in LB medium, and the culture is centrifuged to obtain bacterial cells. The cells are suspended in physiological saline, lysozyme is added, and the suspension is incubated. Add an equal amount of phenol solution to this solution for extraction. Etaler is added to this extract to recover and separate the chromosomal DNA. This chromosomal DNA is digested with restriction enzyme 3^. On the other hand, E. coli plasmid pUc1B is digested with Bam old. These two digestive fluids are mixed, T4 ligase is added, and the mixture is reacted.

This reaction solution was brought into contact with E. coli JM109 strain to obtain an E. coli transformant. Among these E. coli transformants, those exhibiting luminescence were isolated to obtain a colony strain having luciferase activity. The plasmid contained in this strain was named pLUχ1801.

This plasmid ρLUX1801 was subcloned into plasmid ρLUX1802. pLUX1803.
pLUX1804 and pLtlX1805 were obtained.

Of these plasmids, pLUX1802 and pLUX1803 showed luciferase activity.

The recombinant plasmid pLUX1803 DNA-
Digest to completion with the restriction enzyme Hind m in f-buffer. Further, restriction enzyme 5alI was added to this DNA solution to completely digest it to obtain a 3.7 KbO 5ir-H4ndlI fragment. Machi 31 nuclease is added to this fragment and reacted, the reaction solution is extracted with phenol, and the DNA is recovered by ethanol precipitation. Dissolve this DNA in a buffer solution,
Bindl[[linker and T4 ligase are added and reacted to obtain a solution containing the luciferase gene with the N-terminus deleted.

On the other hand, the recombinant plasmid psPAFc-1 containing the protein A-like gene was dissolved in a buffer solution, and Htnd II
Add I, complete digestion, and collect DNA using a conventional method.

This DNA is dissolved in a buffer solution, and a solution containing the N-terminally deleted luciferase gene and T4 ligase are added and reacted to produce a fusion protein gene containing both the protein A-like gene and the luciferase gene. Recombinant plasmids pt, uχ5PAFc-1 and pL
UXSPAFc-2 was obtained. The nucleotide sequence of this recombinant plasmid was determined and is shown in FIGS. 7 and 8.

From this result, pLUXsPAFc-1 and pLUXsP
It was confirmed that all AFc-2s contain a gene encoding a fusion protein in which a protein A-like gene and luciferase are fused.

The recombinant plasmids pLUXSPAFc-1 and pL
E. coli 11 using tlXsPAFc-2 according to the standard method.
8IOI is transformed to obtain a transformant. The transformant microorganism was then cultured by a conventional method to obtain a fusion protein consisting of a polypeptide having the ability to bind to the immunoglobulin Pc region and luciferase activity. The immunoglobulin Pc binding ability of the fusion protein produced by this bacterial cell was investigated, and the results are shown in FIG. 10, and all showed the binding ability.

〔Effect of the invention〕

The fusion protein obtained by this method, which has the ability to bind to the Fc region of immunoglobulin and has luciferase activity, can be used as a labeled second antibody. The enzymatic reaction time is shorter than that of peroxidase protein A, which is currently commercially available as a labeled second antibody. In addition, peroxidase protein A has peroxidase attached to protein A through a chemical bond.
Difficult to manufacture. This time luciferase protein A
is easy to manufacture because it is produced in the form of a fusion protein that is linked at the genetic level.

〔Example〕

The present invention will be explained below using examples. However, the present invention is not limited to these Examples.

Example 1 (1) Preparation of a recombinant plasmid and transformed microorganism containing a protein-like gene was obtained using the DNA synthesizer Genet (Fig. 2). pDR720DNA (
Pha, product purchased from rmacia company N [L27-49
30-01) 10XB10 with 1710 amount in 1100n
XBa buffer y-(100mM Tris-HCI(p
H8,0), 70n+M MgC1z, LM NaC
1, 20mM mercaptoethanol, 0.1% bovine blood serum albumin color, and 2 units of restriction enzyme BamHI were added for complete digestion at 37°C.

Mix the thus digested vector with g of the DNA digest encoding the above Fc-binding peptide, and add 1/10 of the amount
0XT4 ligase buffer - (660mM Tris
-t-ICI (pH 7,6), 66mM Mgch
, 100mM dithiothreitol, 1mM ATP
) and 10 units of T4 DNA ligase and incubate at 10°C.
The binding reaction was carried out overnight. The combined DNA was transformed into large intestine 111B101 competent cells (purchased from Takara Shuzo Co., Ltd.), and LB agar medium containing 50 dg/Id of ampicillin (Bactodributon ig,
Yeast extract 0.5g, NaCl 0.5g, agar 1
5%) was applied to 10 sheets and left overnight at 37°C. As a result, approximately 10,000 transformed colonies resistant to ampicillin were obtained. Plasmid DNA was extracted from the resulting colonies by the alkaline method, treated with restriction enzymes, and the insertion of the desired DNA fragment was confirmed by agarose electrophoresis (this recombinant plasmid was
PAFc-1) (Figure 3).

(2) Preparation of recombinant plasmid containing luciferase gene and transformed microorganism The luminescent bacterium Vibrio harveyi used in this example was purchased from American Type Culture Collection.
The strain is TCC14126. E. coli JM109 strain and plasmid pUc18 (Takara Shuzo product Nα3218) were purchased from Takara Shuzo.

Vibrio harvey ATCC14126 strain, 2% NaC
LB medium containing 1 (Bactodributon Ig, Yeast Esquidium 0.5 g, NaC 12 g/L001ni)
After 8 hours of incubation at 28°C for 100d, 8.00O
Centrifugation was performed for 10 minutes at rpm. The obtained bacterial cell precipitate was dissolved in physiological saline containing EDTA (NaClO, 88
g, EDTA-Nag 1.86g/100a+f.

pH 8,0) Suspended in 10d, washed, 8.00Orp
The precipitate was then suspended in 5 d of the above-mentioned physiological saline containing EDTA, lysozyme was added to a final concentration of 2 μ/d, and the precipitate was incubated at 37° C.
Incubation was performed at C for 20 minutes with shaking. Furthermore, 0.6 parts of 10% SDS was added to this solution,
Incubated at 60°C for 10 minutes. Then, an equal volume of phenol solution (phenol: chloroform: isoamyl alcohol: H2O 25:24:1:50) was added and gently stirred, followed by centrifugation to remove the supernatant (aqueous layer containing DNA). ) was obtained. Regarding this supernatant,
The above extraction operation using the phenol solution was repeated two more times.

Twice the amount of ethanol was added to the resulting supernatant, and the floating chromosomal DNA was collected by winding it up using a glass rod. 70 pieces of glass rod wrapped around this chromosomal DNA
% ethanol, 80% ethanol, 90% ethanol,
The DNA was dehydrated by sequentially immersing it in 100% ethanol. After further drying this at room temperature, the ld TEIO
-1 (10mM Tris-tlcl (pH 8,0),
1 mM EDTA4az).

Chromosome D of Vibrio harveyi obtained in this way
A solution containing 2 μg of NA (the amount of DNA is 0D260nr)
(obtained from the absorbance at a), 1/10 of the amount of IO
X5au3A buffer (100mM Tris-HCI
(pH 7,5), 70mM MgCIx, IM Na
C1) and 2 units of restriction enzyme 5au3A, 37
Partial digestion was carried out for 15 minutes at °C. Of this solution, D N
A Amount equivalent to 200 ng (1/1 of the above DNA digest)
1013) and E. coli plasmid DNA that had been completely digested with restriction enzymes. This "Escherichia coli plasmid DNAJ completely digested with restriction enzymes" is the DNA of the E. coli multicopy plasmid pUC18.
n, 1/101 of 10X Bam old buffer (100
mM Tris-11cI (pH 8,0), 70mM
MgC1z, IM NaCl.

20mM 2-mercaptoethanol, and 0.1%
bovine serum albumin], and 2 units of the restriction enzyme Bam
1lr was added and incubated at 37°C for 2 hours to completely digest. Add 171 to this mixture.
0 volume of 10XT4 ligase buffer (660mM Tris
-HCI (pH 7,6), 66mM MgC1z, 1
00mM dithiothreitol, and 1mM ATP
), and 10 units of T4 DNA ligase,
The reaction was carried out overnight at 10°C for ligation (see Figure 4).
. Prepare competent cells of Escherichia coli JM 109 strain,
This was contacted with the above ligation mixture to transform the E. coli. The cells were cultured on LB agar medium containing 50 dg/d ampicillin and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Bactodributon Ig per 100 d, yeast extract 0.5 g,
Contains 10.5g8 NaC and 1.5% Agar>i
Screening was performed by inoculating the plate onto 100 ml of paper and culturing the plate overnight at 37°C. To the 10,000 ampicillin-resistant transformed colonies obtained in this culture, n-decanal (
Hanui Chemical Co., Ltd.) was added in the form of a mist using a sprayer, and one colony that showed luminescence (having luciferase activity) was collected.
I got the stock. The recombinant plasmid containing luciferase activity contained in this colony-forming strain is pLUX.
The plasmid pLU was named 1801, and the plasmid pLU
X1801 was isolated and purified. This purified plasmid pLUX1801 contains restriction enzyme Hind III + 1. The following DNA fragments were obtained by digesting with PstI and ShiryoR1 in various combinations and electrophoresing on 0.8% agarose gel: (1) 4
．． 0 kbp (7) Hind m DNA fragment, (2
) 3.7 kbp (7) SalI - rule ndlI [DNA
Fragment, (3)3. OkbpO rulendI[I-匡[)NA
fragment, and (4) 1,5 kbp Htnd
m-EcoRI DNA fragment. (1) - (4)
20 ng of each DNA fragment and 10 ng of pUc1
8 DNA was digested with the same combination of restriction enzymes as in (1) to (4) and ligated with 10 units of T4 DNA ligase, and subcloned from plasmid pLUX1801 to obtain plasmid pLUX1802゜pLUX1803. pLUX1804, and pLU
X1805 was prepared (Fig. 5), that is, the above four recombinant plasmids each have the following Vibrio harveyi-derived (7) DNA fragment: pLU
X1802: 4.0kbp! find m D
NA fragment, pLUX1803: 3.7 kbp DNA fragment, pLUX1804
: 3.0 kbp HindI[-tl DNA fragment, and pLUX1805: 1.5 kbp Hindm
-t! coRI DNA fragment. Escherichia coli strain JM 109 was transformed with each of these recombinant plasmids in the same manner as described above. This transformant was cultured on LB agar medium, and luciferase activity was examined by the method using n-decanal described above. As a result, luciferase activity was detected in bacterial cells containing recombinant plasmids pLUX1802 and pL[JX1803.

The above plasmid pLU capable of expressing luciferase activity
X1802 and pLUX1803 (7), the above pLU
Details of the preparation from X1801 are as follows. To 10 μg of DNA of pLUX1801, add 1/101 of 10× ndI [[buffer (100mM Tris-HC
I (pH 7,5), 70mM MgCh,
600mM NaC1) and restriction enzyme 11indlI [
Add 20 units of
．． A reaction mixture solution containing a 0 kbp short ndlll DNA fragment is obtained. To the solution containing this HindlII DNA fragment was added 1710 volumes of 1150mM NaCl and 550ml of restriction enzyme.
After adding 20 units of ail and completely digesting at 37°C for 2 hours, 3 μg of the 3.7 kbp ndllI-riN) NA fragment was isolated and purified by electrophoresis on a 0.8% agarose gel. do. This 3.7kbp 11i
ndIII-5alI DNA fragment (20 ng) and DNA obtained by digesting pυC18DNAIOng with restriction enzymes 2 units of 5alI and 12 units of Hindll were combined with T4.
Ligation is performed using 10 units of DNA ligase to obtain recombinant plasmid pLUX1803.

Recombinant plasmid pLtlX1802 was prepared by similarly digesting the DNA of pLUX1801 with the restriction enzyme ndllI, a 4.0 kbp ndl DNA fragment, and pUc18 DNA (previously digested with HindI[I
(digested with ) and ligated using T4 DNA ligase.

(3) Creation of fusion protein with immunoglobulin Fc binding ability and luciferase activity Recombinant plasmid pLUX1803 DNA 10
μg contains 1710 amounts of 1OXHindI [[buffer y −
(100n+M Tris-11CI (pH 7,5),
Add 70mM MgCl (1g, 600mM NaCl) and 1120 units of restriction enzyme HtndI, and incubate at 37°C for 2 hours.
The time was completely consumed. Then, add 1/10 to this DNA solution.
1150mM NaCl and 120 units of restriction enzyme 5al were added and completely digested at 37°C for 2 hours.
3.7 by electrophoresis on an 8% agarose gel.
3 μg of the 5alI HindII fragment of kbp was purified.

To 3 μg of this purified 3.7 kbp 5all-form ndlu fragment was added 173 amounts of 3 x Ba131 buffer y (
60mM Tris-HCI (pH 8,0), 300m
M NaCl, 15mM CaCIz, 15mM M
gCIz.

3mM EDTA/2Na) and 1.35 units of Ba131 nuclease were added and reacted at 30°C for 1 minute and 10 seconds. Then, an equal amount of phenol was added, slowly stirred, centrifuged (10,000 rpm, 5 minutes, 4°C), and the supernatant was collected. DNA was recovered by ethanol precipitation by adding 0) and twice the amount of ethanol.The obtained DNAa:ii was washed with 70% ethanol and dried.This DNA was mixed with 20 μ2 of Tf!10-1 (
lhM Tris-HCI (pH 8,0), 1mME
Dissolve in DTA/2Na). Add 500p to this DNA solution.
aao 1 t-1indllI linker (C-A-A
-G-G-T-T-G) and 1710 amount of 10XT4
Ligase/" (y 7 y- and 10 units of T4 DNA IJ gauze were added, and the ligation reaction was carried out for 10'C1-night. To this ligated DNA, 1710 volumes of 10X Hindl
1 buffer and 2 units of the restriction enzyme Hindll were added to complete digestion at 37°C, and the restriction enzyme Hindll was inactivated by heat treatment at 65°C for 5 minutes. The solution thus obtained will be referred to as "rBa131 nuclease-treated DNA solution." This solution contains luciferase genes with various N-terminal deletions.

Next, recombinant plasmid pSPAFc-I DNA
A 1/10 amount of 10x ndII [buffer and 2 units of restriction enzyme Hind] were added to 1100n, and the mixture was completely digested at 37°C. Add an equal amount of phenol to this DNA solution, stir slowly, and centrifuge (10,000 rpm+,
5 minutes at 4°C), and the supernatant was collected. After that, 1,710 volumes of 3M sodium acetate (pH 5,0) and 2 volumes of ethanol were added to the supernatant, and D was purified by ethanol precipitation.
NA was collected. The recovered DNA was incubated with 20μ2 TEI.
Dissolve to O-1.

Add 1710 amounts of IM Tris-11c to this DNA solution.
I (pH 8,0) and 2 units of alkaline phosphatase were added and treated at 37°C for 2 hours. This DNA solution was extracted with phenol and then subjected to ethanol precipitation to recover DNA. The recovered DNA is dissolved in 20μ2 of TEIO-1.

To this DNA solution (of this solution, use an amount equivalent to 100 nH of DNA), add the rBa131 nuclease-treated DNA solution obtained as described above (of this solution, use an amount equivalent to 1100 n of DNA).
and 1710 amounts of l0XT4 ligase buffer and T4
10 units of DNA ligase was added and the ligation reaction was carried out at 10°C for 5 nights. This combined DNA is transferred to E. coli)! B.I.
Transform into OI competent cells,
0μs/ad! of ampicillin and 40μg/1ttfl
The mixture was spread on 10 plates of LB agar medium containing indole acetic acid (IAA) and left overnight at 37°C. N-decanal was sprayed onto 1000 ampicillin-resistant transformed colonies that had grown using a sprayer to obtain 22 colonies that emitted light (having luciferase activity).
(named PAFC-1-22).

The above is shown in Figure 6. After each colony was grown in an LB liquid medium containing 50 μg/mi of ampicillin, plasmid DNA was extracted from the resulting bacterial cells by the alkaline method, and the restriction enzyme 11indl11. P! A fish! Processed with
Insertion of the desired DNA fragment was checked by agarose electrophoresis. Among these, recombinant plasmid pLIJXsPAFc-1, which seems to have protein A and luciferase fused at the protein level,
The DNA base sequence of pLUXsPAFc-2(7) was determined. The DNA base sequence was determined according to the Sanger method. The resulting sequences are shown in FIGS. 7 and 8. Results of DNA base sequence determination pLUXSPAF
It was found that c-1 and pLIIXsPAFc-2 encode a single protein in which Protia A and luciferase are fused. In Fig. 7, bases 16 to 1275 surrounded by squares are α of protein A and luciferase.
This is a base sequence that encodes a fusion protein with the subunit, and bases 1302 to 2276 are the base sequence that encodes the β subunit of luciferase.

In Figure 8, bases 16 to 1266 surrounded by squares are:
This is a base sequence that encodes a fusion protein between protein A and the alpha subunit of luciferase, and is located at base 1292.
2267 is a base sequence encoding the β subunit of luciferase.

Also, pLUXSPAFc-1 and pLLIXsP, respectively.
50 μg 7 ml of E. coli 118IOI containing AFc-2
The cells were cultured in 1.5 Ill LB medium containing ampicillin, and when the cell number reached 5 x 10'' cells/ml, indole acetic acid (IAA) was added at a rate of 40 μs/d. ρLUXSPAFc-1 and pLUXSP
Since AFc-2 has a tryptophan promoter (P trp) derived from pDR720, the fusion protein can be induced by addition of IAA. By continuing the culture for an additional 2 hours, the transformant was allowed to produce the fusion protein. This culture was centrifuged at 800 rpm for 5 minutes, and the resulting precipitate was added to 500 μl of TE10-1 buffer (
pH 8.0). 20μ2 were taken from this suspension and centrifuged at 8000 rpm for 5 minutes. The resulting precipitate was suspended in 5 μ2 of sterile water, and 5 μ2 of 2×
Sample buffer (0.13M Tris-HCI (pH 6°8), 4% SOS, 20% glycerol, 10% mercaptoethanol, and 0.002% bromophenol blue) was added and heat treated at 90°C for 3 minutes. E. coli was denatured by heat, and 5O5-PAGE of intracellular proteins was performed.The gel after electrophoresis was stained and analyzed using a densitometer, and it was found that overproduction of molecules with molecular weights of 49,000 and 37,000 was present (the molecular weight of 49,000 was protein A+ The luciferase α subunit, molecule 3137000, is thought to correspond to the luciferase β subunit).5
The results of O5-PAGIE are shown in FIG.

Next, the Fc binding activity of immunoglobulin, which is an intracellular protein, was examined by the following method. pLUXsPAFc-
E. coli HB 101 containing 1 and pLUXsPAPc-2
to produce the fusion protein in the same manner as above, centrifuging the resulting culture, suspending the resulting precipitate in TEIO-1 buffer, and taking a portion of the suspension, followed by centrifugation. I did it. In addition, the recombinant plasmid pLUX1803 containing the luciferase gene obtained in section (2) above was added to JM1.
Exactly the same operation was performed on the transformed 09 and HB101.

100μ of the precipitate obtained by centrifugation! (expressed as dilution factor x 1) and 10001! f (dilution factor x 1
0) in 8M urea. Using this suspension as a sample, the binding of immunoglobulin to Fc was examined by enzyme immunoassay. Enzyme immunoassay is BIO-D
OT (immun-blot system: Bi
o-RAD) according to the instructions for use.

The sample solution was adsorbed onto a nitrocellulose (NC) filter set in an 810-DOT device. Next, the peroxidase-labeled anti-human TgG antibody was brought into contact with the above-mentioned adsorbent and washed.Next, the nitrocellulose filter was removed from the device, and the coloring solution [Solution A: 30% 11oz
12μIt+TBS (20M Tris-HCI.

pH7,5,500+++M NaC1) 20d, B
Liquid: 4-chloro-1-naphthol 1211Ig + methanol 4m, Mix A and B solutions. ). If there is something that binds to the Fc of immunoglobulin in the sample solution, a yellow color develops.

The results are shown in FIG. FIG. 10 shows that the fusion protein has the ability to bind to immunoglobulin Fc.

Example 2 Large-scale preparation of fusion protein Recombinant plasmid pLt obtained in Example 1 (3)
E. coli strain 88101 (transformant) containing 1XsPAFc-1 was transformed into 11 strain containing 50 Mg/uti ampicillin. The cells were cultured in large quantities in LB medium. When the cultured cells reached 5 x 10" cells/ml, add 40 Mg of IAA.
oal to induce the above fusion protein, and culture was continued for an additional 2 hours. 800 ml of this culture
Centrifuge at 0 rpm for 10 minutes, and the resulting precipitate is
d Buffer A (50μItM Tris-HCI
(pH 7,0), 5mM BDTA-Na
z.

Suspended in 10mM MgCh, 0.1mM dithiothreitol, 10% glycerol, 2111M phenylmethylsulfonyl fluoride, 6011M NH4C1),
Ultrasonic treatment was performed.

This was centrifuged at 10.00 rpm for 10 minutes, and the resulting supernatant was fractionated with 70% saturated ammonium sulfate. The obtained precipitate was added to 2d buffer B C10ttrMNat11POa-
Na11zPC10ttr, 8>.

100 μh phenylmethylsulfonyl fluoride, 100
Dissolved in μM BDTA・Naz), 10.00Orp
Centrifugation was performed for 10 minutes at 1.5 ml, and the resulting supernatant was equilibrated with buffer B. It was added to a Sephacryl 5200 column (manufactured by Pharmacia LKB) with a height of 90 cm, and gel chromatography was performed at 30 d/h using buffer B as an eluent. Molecular weight with luciferase activity 8
6,000 fractions were subjected to protein 5DS-polyacrylamide gel electrophoresis (SOS-PAGE), and the gel after electrophoresis was stained with Comasi-Brilliant Blue.
Bands were observed at molecular weights of 49,000 and 37,000. These two bands correspond to the fusion peptide of the Fc-binding peptide of immunoglobulin and the luciferase subunit α of Vibrio harveyi and the luciferase β subunit, respectively, and their purity was determined by desyntomization.
When analyzed using a micrometer, it was confirmed that it was 90% or more. Furthermore, molecule 18 obtained by gel chromatography
6.000 fractions were dialyzed against 12 buffer A overnight at 4°C. This dialyzed sample was equilibrated with buffer A and had an inner diameter of 1.61 mm.
, 17cm high Q Sepharose (Pharmacia IJ
B) and fractionated with a 20mM to IM NaCl linear concentration gradient of buffer A containing NaCl at a flow rate of 30y1/h, and the activity of the fractionated product was measured using the "luciferase activity measurement method" described below. Then, the buffer fraction containing 0.3M to 0.35M NaCl, which has luciferase activity, was subjected to protein 5DS-polyacrylamide gel electrophoresis, and when the gel after electrophoresis was stained with Comasi-Brilliant Blue, molecules 1F49.000 and 37 A band was observed at .000, and its purity was confirmed to be 95% or more when analyzed using a densitometer. When the amount of protein in this solution was measured by the Lowry method using bovine serum albumin (BSA) as a standard substance, it was found that
It was found that it contains proteins.

“Measurement method of luciferase activity” Luciferase activity can be measured using NAD(P)ll measurement kit (Boehringer Mannheim Yamanouchi), NADH(
Herringer Mannheim Yamanouchi) and NADH oxidase were used.

2 in bottle 1 that comes with the NAD(P)ll measurement kit
．． Add 0 d of deionized water (FMN final concentration 62.5
μmol/l) (Solution 1) 0 Next, add 5 x 10'mol/l of potassium phosphate buffer (pH solution) to final concentrations of 31.25mM and 0.125mM, respectively.
50 ul of NADH solution was added. Add this solution to 20
Dispense 0μ2 into small test tubes and add 10 units of N to each.
AD) Add l oxidase and add 10 μ2 to 20 μ
2 of the sample for measuring luciferase activity was added, then 5 μ2 of n-decanal was added, and the amount of photons was measured using a Beckman liquid scintillation counter LS3801 with a diameter of 1 minute.

Example 3 In Example 2, I) the recombinant plasmid pLIJX obtained in (3) of Example 1 was used instead of E. coli HB 101 strain (transformant) containing LUXSPAFc-1.
The fusion protein was produced in the same manner as in Example 2, except that E. coli HB 101 strain (transformant) containing sPAFc-2 was used, and almost the same results as in Example 2 were obtained. It was done.

Example 4 Quantification of alpha-fetoprotein using fusion protein BIO-DOT (immun-blot system)
) device (Figure 11). Nitrocellulose (NC) filter (Bio-RAD, 9X14cm
) in TBS (20mM Tris-1c1 (pH 7,
5), soak in 500mM NaCl) for about 5 minutes. Thereafter, the water was gently drained on filter paper and mounted as shown in FIG. 11.

Wash twice with 500μ2 TBS per well,
After suction drying for a minute, the pressure was returned to normal. Then 10-'.

7 Xl0--10-@, 6.8X10-' and I
Q-'mol/j! 20 μl of concentrated alpha-fetoprotein was dotted into each well. After 5 minutes, Co.
After opening, the mixture was naturally filtered, and after 2 hours, it was filtered with suction and returned to normal pressure.

After that, add TTBS (TBS containing 0.5% Dryde:/X100) and dissolve BSA to a concentration of 3%.
100 μ2 dots were placed per well and blocked. After 5 minutes, open the stopcock and filter the water by gravity.
The cells were washed 4 to 5 times with 00 μl of TTBS, dried by suction for 5 minutes, and returned to normal pressure. 100IIJl per well! The first antibody [anti-alphafetoprotein monoclonal antibody (purchased from Toyo Jozo Co., Ltd.) at 1 μg/d: once a time,
% BSA-containing TTBS] was poured into the solution, and the stopcock was opened to allow natural filtration. After 2 hours at room temperature, it was filtered with suction. The wells were washed 4 to 5 times with 500 μ2 of TTBS per well, dried under suction for 5 minutes, and returned to normal pressure.

(with erase activity) to a concentration of 1ag/ml, 1%
[dissolved in BSA-containing TTBS]] was added, and the stopper was opened for natural filtration. After 1 hour, it was filtered with suction. Wash 4 to 5 times with 5001 Jf of TTBS per well,
Thereafter, each well was washed 2 to 3 times with 500.1 TBS, and then dried by suction for 5 minutes. Remove the nitrocellulose filter from the apparatus, cut the filter along the well, and place it in a luminescence counter Biolumat LB9500 (
The cut filter was placed in a measuring tube (Berthold). Then, 200 uA of reaction mixture (
25mM sodium phosphate buffer (pH 7,0), 10
01! M dithiothreitol, 2,5! MFMNH,
,40mg/j! ) Rydon X-100, 20u mo
l/i myristeic aldehyde], 1 behind B
Measured with iolumat LB9500. (The results are shown in Figure 12.)

[Brief explanation of drawings]

Figure 1 shows the structure of protein, Figure 2 shows the protein A-like gene sequence, Figure 3 shows the construction of psPAFc-1, Figure 4 shows the construction of pLUX1801, and Figure 5 shows the construction of pL.
IJX1801.1802.1803.1804 and 1
Figure 6 shows the DNA fragment of pLUXsI'
Construction diagram of AFc-1, pLIJXsPAFc-2, No. 7
The figure shows the DNA base sequence of pLUXsPAFc-1, Figure 8 shows the DNA base sequence of pLLIXsPAFc-2, Figure 9 shows HBIOI (pLLIXsPAFc-1), IIB
805-PAG of IOI (pLUXsPAPc-2)
E, Figure 10 is a diagram showing the Pc-binding ability of immunoglobulin, a bacterial cell-produced protein, and Figure 11 is a diagram showing the Pc binding ability of immunoglobulin, a bacterial cell-produced protein.
FIG. 12 is a diagram showing the measurement results of alpha-fetoprotein. Figure 2: Protein, 1! The genetic sequence of ysLeuC:ysMe is shown in Fig. 3. Construction of psPAFc-1 Fig. 4 T4 DNA ligase ligation ↓ Inji H1/Kanho Fig. 7 (Part 1) DNA base sequence of pLUXsPAFc-1 Figure 7 (Part 2) Figure 7 (Part 3) Figure 7 (Part 4) ALIIulAIIl 1lAAl+'l”1LiA
A ljA Nishishi■^T-f^ ACA to TTGA to GT Figure 8 (Part 1) DNA base sequence of pLUXsPAFc-2 Figure 8 (Part 2) Figure 8 (Part 3) Figure 8 (Part 4) GCCGATAGCG CTAACTAATA G
AGGCATTτA TA Ding GGACGTAG Ding CA
TTGCAG GAACAAAAAACAGCTGAC
ATCTCGCCTTGTTGGAAGATCTCAA
CGATATCCCCCGTTTTCCCCTACTTCT
ATTDING TTAAGTTGA AGACCCTATT
AACAC Ding TGACGAT Figure 9 1, Molecular weight marker 2, HBIQI 3, JM109 (p LUX1803) 4, HBI
OI (pLUXsPAFc-D5, HBIOI (pLU
XSPAFc-2)HBIOI(pLUXsPAFc-
1). HBIOI (pLUXSPAFc-Rino5DS-PAG
E Figure 10 Measurement of Fc binding ability of immunoglobulin, z + ++, no color development/yellow color development 4: H8101 (pLUXsPAFc-2) 1st
1 Figure 2: Sample template 8: Suction cock

Claims (1)

[Scope of Claims] 1. A polypeptide having the ability to bind to the Fc region of immunoglobulin and luciferase activity. 2. A gene encoding the polypeptide according to claim 1, having a DNA sequence of the following formula. [There is a gene sequence] 3. A gene encoding the polypeptide according to claim 1, which has a DNA sequence of the following formula. [Gene sequence is available] 4. A gene that encodes a polypeptide with luciferase activity and an N-terminus. 5. A recombinant plasmid in which a DNA sequence containing the gene according to claim 2 or 3 is integrated into an E. coli plasmid. 6. A transformant obtained by transforming a host cell with the recombinant plasmid according to claim 5. 7. A transformant containing the recombinant plasmid according to claim 5 is cultured in a medium, and a polypeptide having the ability to bind to the Fc region of immunoglobulin and luciferase activity is collected from the culture. Method for producing peptides.