JPH03201994A - Fused polypeptide and its production - Google Patents

Abstract

NEW MATERIAL:A fused polypeptide obtained by inserting an amino acid sequence corresponding to a DNA having multicloning sites and also being integrated with an amino acid sequence from the fifth amino acid till N end of beta-galactosidase to an N terminal of a polypeptide derived from lck gene. USE:Diagnosis of adult T cell leukemia, etc. PREPARATION:A DNA having multiclonal sites is integrated at the desirable site with lck gene-containing DNA. Then, whole or a part of the integrated DNA is inserted to a manifestation vector of Escherichia coli to transform this bacilli with the recombinant vector. The transformed Escherichia coli is cultured and the objective fused polypeptide is collected.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a fusion polypeptide containing a novel fck gene-derived polypeptide and a method for producing the same.

[Prior art and problems of the present invention]

The lck gene is a polypeptide (p561ck; EC2-7-1-IL2) that exists specifically in human T cells and has tyrosine kinase activity that regulates the proliferation of the T cells.
) is a gene involved in the production of (Trevilla
nJ.

M,, Y, Lin, S, J, Chen+ C, ^,
Ph1llips, C, Canna and T, J.
Linna, Biochem Biophys, A
cta 888:286 (1986) + Yosuhi
ro Koga et al,, Journal o
f immunology vol 142. p, 4
493-4499. NQ12).

The lck gene is actively expressed in normal T cells, but in T cells that are activated and begin to proliferate.
It has been reported that in cells, the expression level decreases, and the production amount of the polypeptide having tyrosine kinase activity decreases (The EMBO Journal vo.
l 6. Na9. p, 2727-2734 (19B?
)).

On the other hand, in contrast to the expression of the fck gene, the amount of virus expression in T cells infected with the HTLV-I virus, which is the causative virus of T cell leukemia, increases with activation of the T cells. It is something.

Therefore, the expression levels of i and ck genes in T cells and HT
We confirmed that there is an inverse correlation between the expression levels of LV-r virus, and that T cells in living organisms are active.
Furthermore, by monitoring the production amount of the polypeptide derived from the fck gene in the cells using an antibody against the polypeptide, it is possible to diagnose adult T-cell leukemia, and the development of a diagnostic method using the antibody, etc. There is expected.

By the way, in order to obtain the antibody used for such diagnosis, it is necessary to immunize guinea pigs, mice, etc. with the polypeptide derived from the 1ck gene as an antigen.
This requires a large amount of the polypeptide.

However, in order to separate and purify the polypeptide derived from the natural lck gene present in T cells from T cells,
It is necessary to combine complicated operations such as gel filtration, and furthermore, it takes a huge amount of time to obtain the lck gene-derived polypeptide, which exists in extremely small amounts in T cells, in the amount necessary for immunizing the guinea pigs etc. However, it was not practical to separate and purify the polypeptide derived from the 1ck gene from T cells.

Against this background, the current situation is to synthesize a peptide with a molecular weight of about 3 kd, which is a part of the entire polypeptide chain encoded by the lck gene, and to use the synthetic peptide in guinea pigs.
Antibodies obtained by immunizing mice etc. are used (cel
l vol 55. p, 301-30B (1988)
), but this synthetic peptide is only a small part of the total polypeptide chain encoded by the ick gene, which has a molecular weight of 56 kd, and has a molecular weight of about 3 kd. When using the obtained antibody, not only immunoreaction with the LCK gene-derived polypeptide in T cells but also cross-reaction with other proteins occurs, making it difficult to accurately determine the expression level of the LCK gene in T cells. It was impossible.

Therefore, an object of the present invention is to provide a polypeptide capable of preparing an antibody that specifically causes an immune reaction only with a polypeptide derived from the lck gene in T cells, and a method for producing the polypeptide in large quantities by a simple means. The goal lies in trying to develop.

[Means to solve the problem]

As a result of extensive research to solve the above problems, the present inventors prepared a recombinant vector by incorporating the lck gene into the cloning site of the β-galactosidase gene in an Escherichia coli expression vector having the β-galactosidase gene. By culturing the transformant obtained by transforming E. coli using the recombinant vector, l
A fusion polypeptide in which a polypeptide derived from the ck gene and a polypeptide derived from an E. coli expression vector can be produced in large quantities, and the fusion polypeptide is also useful for the production of antibodies used in the diagnosis of T-cell leukemia, etc., as described above. We have found that this is extremely suitable and have completed the present invention.

That is, the present invention includes the following points (1) and (2) as its configuration.

(1) A polypeptide having an amino acid sequence corresponding to a DNA having a multiple cloning site at the N-terminus of a polypeptide derived from the fck gene, and having an amino acid sequence extending from the N-terminus of β-galactosidase to at least the fifth amino acid. fused polypeptide.

(2) DNA containing a multi-cloning site at the desired site of part or all of the β-galactosidase gene
In the E. coli expression system vector mediated by A, DNA having the l1ck gene is inserted into the desired cloning site of the multi-cloning site, a recombinant vector is prepared, and E. coli is transformed with the recombinant vector. By culturing the transformed transformant, the above (1)
A method for producing a fusion polypeptide, which comprises collecting the fusion polypeptide described in ).

The present invention will be explained in further detail below.

Among the fusion polypeptides of the present invention, the polypeptide derived from the LCK gene is a polypeptide derived from the LCK gene, which is a cancer-related gene, and is present specifically in human IT cells, and is a polypeptide derived from the amino acid tyrosine residue in the protein. It has tyrosine kinase activity that activates T cells by phosphorylating it.

The amino acid sequence of the polypeptide derived from this fck gene is shown in FIG.

The fusion polypeptide of the present invention has β-
A polypeptide having an amino acid sequence encoded by the gene from the 5' end of the β-galactosidase gene to an arbitrary cloning site is joined to an E. coli expression system vector using the galactosidase gene.

Therefore, the fusion polypeptide of the present invention can be used in an E. coli expression vector containing the β-galactosidase gene.
galactosidase l at the cloning site in the gene
It is produced by preparing a recombinant vector by incorporating DNA having the ck gene, transforming Escherichia coli using the recombinant vector, and culturing the obtained transformant, and used in the present invention. An example of the jack gene is a DNA having the base sequence shown in FIG. This lck gene may be synthesized using DNA synthesis based on the DNA sequence shown in Figure 2, but
It is preferably obtained by the following method.

That is, mRNA derived from the fck gene located in lymphocytes in the peripheral blood of a normal person is selected, and j!
Synthesis of ck cDNA, and even j! This is a method for synthesizing ck dsDNA. To explain this method specifically, first, lymphocytes are separated from normal human peripheral blood, RNA is extracted from the lymphocytes, and oligo(dT) is extracted from the obtained RNA.
) - Separate mRNA on a cellulose column 0 Then,
After fractionating the separated mRNA by sucrose density gradient centrifugation, the DNA encoding the lck gene product shown in FIG.
The fractionated mRNA was subjected to no-in blot hybridization using a DNA probe having a base sequence encoding any 20 or more amino acids synthesized based on the base sequence, and mRNA derived from DNA containing the fck gene was obtained.
Select the fraction where NA is located. Then, the selected m
cDNA and dsDNA are prepared from RNA by a known method using reverse transcriptase, RNase H1 Escherichia coli DNA polymerase I, and T4 DNA polymerase, and then EcoRI linkers are attached to both ends of the dsDNA.
After ligating to the EcoRI restriction enzyme cleavage site of any phage vector, the in vitro vector is used to create a recombinant phage solution. Finally, E. coli was infected with the phage and plaque hybridization was performed.
A DNA fragment containing the lck gene can be obtained by isolating a phage containing the lck gene, preparing a large amount of phage DNA from the isolated phage, and then cleaving it with EcoRI.

In addition, D containing the fck gene used in the present invention
The NA has a size that can be inserted into the cloning site of the β-galactosidase gene in an E. coli expression vector containing the β-galactosidase gene, and the 5' end of the NA is so large that it can be inserted into the cloning site of the β-galactosidase gene in an E. coli expression vector. There is no particular restriction as long as it is adjusted, and any DNA fragment can be used in a form connected to both ends of the lck gene.

However, usually j1! is created from the mRNA through cDNA! The DNA fragment containing the ck gene is lck
The start codon ATG of the gene is recognized by the Nco I restriction enzyme and about 3 oobp downstream of the stop codon TGA of the fck gene.
GCTT is the former ndln restriction enzyme recognition site.
Since it has the CGAA base sequence, the DNA containing the fck gene is approximately 1.8 kbp of Nco I-11 cut using Nco I and Hindnl restriction enzymes.
Preferably, the indllI fragment is used.

The method for producing the fusion polypeptide of the present invention begins with the above I! , ck genes are integrated into an E. coli expression system vector using the β-galactosidase gene to produce a recombinant vector.

FIG. 3 shows a typical base sequence of the β-galactosidase gene and an amino acid sequence of β-galactosidase.

The E. coli expression system vector used in the present invention is β-
It contains part or all of the galactosidase gene, and the foreign gene can be introduced into E. coli by interposing the DNA with the above-mentioned multi-cloning site at the desired site of the gene. There are no particular restrictions on the plasmid vector as long as it has the conditions for a plasmid vector that can function in E. coli as a host, such as being able to replicate and express in E. coli and having useful markers such as auxotrophy, drug resistance, and temperature sensitivity. However, as the above-mentioned useful marker, it is preferable to use one having an ampicillin resistance gene for drug resistance.

Specific examples of Escherichia coli expression vectors that meet the above conditions include puc vector (manufactured by Takara Shuzo ■), p'rv vector (manufactured by Takara Shuzo ■), pUEX vector (manufactured by Amoosham ■), and the like. Among these vectors, pUc vector and pTV vector are EcoRI
, Kpn I , Bam1l 1 , χbat, 5a
il, Pstl.

5phl, a multi-cloning site such as the former ndI[I restriction enzyme cleavage site, was inserted into the 5′ of the β-galactosidase gene.
It has it near the end. Therefore, when the DNA containing the lck gene is integrated into the vector, transformants using this recombinant vector will no longer express a protein having β-galactosidase activity, and whether or not the recombination has occurred can be determined by, for example, Along with isopropyl-β-D-thiogalactoside (manufactured by Tsuba Biochem, hereinafter abbreviated as IPTG), X-gal (5-bromo-4-chloro-3
-indolyl-β-D-galactoside dinova (manufactured by Biochem ■)), and the presence or absence of β-galactosidase can be recognized by determining whether the color of the colonies that appear is white or green.

There are many more types of pUC vectors or pTv vectors, depending on the number of types of restriction enzyme cleavage sites present in the multi-cloning site and their directionality. However, in general, if pUC hectate, pHc118 and pUc119, and H-tet to pTV.
r) irL pTVl 18N and pTV119N are preferably used.

FIG. 4 shows the base sequence from the 5' end of the β-galactosidase gene of pUc119 to the multiple cloning site. In addition, pTV119N is obtained by changing A to G at the fourth base pair downstream of the 5' end in FIG. 4 above.

In the present invention, lck to the above E. coli expression system vector
Integration of the DNA containing the gene involves joining the 5' end of the DNA containing the lck gene and the 3' end of the cloning site cut with the restriction enzyme of the E. coli expression vector so that the reading frame of the fck gene is It is necessary to adjust it so that it does not move. No matter which cleavage site in the multi-cloning site of the vector used for integration is used, the resulting protein will be a fusion polypeptide containing the desired polypeptide derived from the lck gene.

However, the p[Ic119 and ρTV119N
The most preferably used cloning site is the old ncI [restriction enzyme cleavage site].

Next, the specific method of embedding is as follows. That is, - For boat fishing, first cut the E. coli expression vector with any restriction enzyme that corresponds to the cloning site used for integration, and then cut the linker of the same restriction enzyme with fc.
A common method is to connect both ends of the DNA containing the k gene, and finally connect the DNA ligated with the linker to the above-mentioned vector.

In addition, the integration of the DNA containing the fck gene into the E. coli expression system vector is performed using the DNA containing the lCk gene.
and a vector cut with any restriction enzyme and 4 dNTPs.
It is preferable to fill in the Klenow fragment by acting on it in the presence of , and then connect it.

Furthermore, the integration of the DNA containing the lCk gene into the E. coli expression vector is carried out in order to avoid self-ligation of the vector after cutting the E. coli expression vector with an arbitrary restriction enzyme. It is preferable to pre-treat with alkaline phosphatase.

DNA containing E. coli expression system vector and fck gene
The connection method can be performed by a known method using ligase. For example, the amount of DNA containing the lck gene to be integrated is 5 to 10 times the amount of DNA of the E. coli expression vector cut with a restriction enzyme. There is a method of adjusting to double the amount and connecting using T4 DNA ligase.

Isolation and confirmation of the recombinant vector incorporating the DNA containing the lck gene described above can be performed by the following method.

That is, for separation, first, the reaction solution obtained by performing the above-mentioned ligation reaction is added to E. coli (E, colt) competent cells, and transformation is performed by a known method. The E. coli used above is not particularly limited, but it is preferable to use strain JM109 (Na9052, manufactured by Takara Shuzo ■), strain MV 11B4 (manufactured by Takara Shuzo ■), and the like. The transformed E. coli can be obtained by culturing it in any selective medium, taking advantage of the fact that the introduced recombinant vector has a useful marker or DNA containing the fck gene incorporated into the structural gene.

For example, when using a pUc vector as an E. coli expression system vector, since the vector contains an ampicillin resistance gene and the recombinant vector has integrated DNA containing the lCk gene into the β-galactosidase gene, The solution after transformation was treated with X-gal and IP
Inoculate on LB agar medium containing TG and ampicillin,
By culturing at 7°C for 14 to 20 hours, a single white colony that appears can be obtained as a transformed strain.

In addition, to confirm that the recombinant vector has correctly inserted the DNA containing the gene into lc, first perform mini-prevention of the recombinant vector present in the transformed strain using the boiling method or alkaline rinsing method. , obtain a recombinant vector DNA suspension. Next, the recombinant vector suspension is cut with any restriction enzyme that can confirm that the DNA containing the lck gene has been inserted in the correct direction.

In addition, if possible, cut with any restriction enzyme that can confirm whether the joint between the 3' end of the vector and the 5' end of the DNA containing the lck gene is properly ligated.

Then, the obtained cut product was subjected to agarose gel electrophoresis,
After staining with ethidium bromide, identification can be made based on whether a band exists at a predictable position and whether it can be cleaved with a restriction enzyme.

Specifically, for example, the DNA containing the lck gene is Ncol-Hind! When performing fill-in ligation using the If fragment and a vector cut at the old ncI [restriction enzyme site of p[Ic119] and confirming the connection, to check whether the cleavage is in the correct direction: Ncol-Hind■ containing the 3' end of the vector and the fck gene using BamHI restriction enzyme.
Nco I restriction enzyme can be used to check whether the ligation to the 5' end of the fragment is correct.

A fusion polypeptide containing a polypeptide derived from the fck gene is produced using a recombinant vector in which DNA containing the fck gene is integrated into the E. coli expression system vector described above.
It is produced by culturing transformed E. coli.

The culture is carried out by adding a β-galactosidase gene inducer of the E. coli expression vector used to the E. coli culture medium.

As the DNA containing the fck gene, the Nco 1
-Hindl [[DNA fragment, Escherichia coli When pUc vector is used as an expression system vector, the above culture will be specifically explained. First, the transformed strain is pre-cultured, and the pre-culture is transferred to H medium supplemented with IPTG and ampicillin (1 %
Tryptone, 0.5% NaCl 200-250
In this method, the cells are inoculated at a double dilution and cultured at 37°C for 115 to 20 hours.

The preculture medium is not particularly limited. For example, LB liquid medium to which ampicillin has been added may be used. Pre-culture time is preferably 12 to 20 hours at 37°C.

In addition, the final concentration of IPTG added to H medium was 0.
．． The range of use is 2-2mM. Note that the effect is the same whether IPTG is added before inoculating the preculture or after inoculating the preculture and culturing at 37° C. for about 5 hours.

After culturing, the culture obtained by centrifugation is used to purify a fusion polypeptide containing a polypeptide derived from the lck gene.

Note that the method for purifying the fusion polypeptide from the culture is not particularly limited, and can be performed by known means such as gel filtration. However, since the fusion polypeptide exhibits water-insoluble properties, it can be purified by a simple method as described below. That is, the culture is suspended in a neutral buffer such as Tris-HCl buffer, dispersed by sonication, centrifuged, and then RIPA (-3O3) buffer is added to the precipitate and suspended. After sonication again, centrifugation is performed to collect the precipitate.

Next, the precipitate obtained by the above method is dissolved in Laemmli buffer, the lysate is subjected to SDS polyacrylamide gel electrophoresis, and a band corresponding to the molecular weight of the fusion polypeptide is cut out from the SDS polyacrylamide gel. Purification to 90% or more can be achieved by elution from a cut out gel.

By the production method described above, a fusion polypeptide in which a polypeptide is joined to the N-terminus of the lck gene-derived polypeptide of the present invention can be produced.

The molecular weight of the produced fusion polypeptide is the molecular weight of the fck gene-derived polypeptide, 56 kd, plus the molecular weight of the conjugated polypeptide (usually about 1 kd).

Furthermore, the fact that the fusion polypeptide contains a polypeptide derived from the l1ck gene means that antibodies obtained by immunizing guinea pigs, mice, etc. with the obtained polypeptide are anti-1c.
As a K antibody, Jurka was added to cells (available from the Virus Division, National Cancer Center Research Institute) and MT-1 cells (Cancer Monograph Cancer Research Na2B (1
This can be confirmed by recognizing polypeptides derived from the natural fck gene expressed in T cell lines such as 982) P219 to P228).

To explain this confirmation method specifically, first, a 1:1 mixture of the fusion polypeptide and the completed ash band was applied to one guinea pig, and the fusion polypeptide was mixed with 100 to 2
Inject the amount to be 00 μg. The injection procedure is repeated 3 to 4 times at one week intervals. Next, blood is collected from the guinea pigs subjected to the above procedure, and antibodies are obtained by obtaining serum.

On the other hand, a T cell line expressing the fck gene product, such as Jurkat cells or MT-2 cells, labeled with [3SS] is suspended in RIPA buffer or the like to obtain a cell lysate. Next, this cell lysate is reacted with the antibody obtained above, and the reaction product is immunoprecipitated with protein A Sepharose, and further subjected to 5OS-polyacrylamide gel electrophoresis. After gel drying, bands are detected by autoradiography. The molecular weight positions of the detected bands and the same experiment as above were determined using MT-2 cells (Cancer Monograph on Cancer Research Na28).
19B2) P219-P228; National Institutes of Health (J
The fact that no band was detected when using a T cell line that does not express the lck gene product, such as CRB 0063)), indicates that the above antibody is an anti-fck antibody.
From the above, it is confirmed that the fusion polypeptide of the present invention contains a polypeptide derived from the lck gene.

[Effects of the Invention] The fusion polypeptide of the present invention has 56
It has a polypeptide with a molecular weight of kd, and by immunizing guinea pigs, mice, etc. with this,
Compared to the conventional polypeptide derived from the LCK gene, which has a molecular weight of only about 3 kd, it is possible to obtain an extremely specific antibody that can cause an immune reaction only with the polypeptide derived from the LCK gene in T cells. be. Moreover, the fusion polypeptide of the present invention can be efficiently produced in large quantities by a simple method of transforming and culturing E. coli, and is also water-insoluble, which makes separation and purification extremely easy. Thus, the above-mentioned antibody can also be obtained in large quantities.

Therefore, it becomes possible to accurately diagnose adult T-cell leukemia and the like using the antibody, and this will greatly contribute to the development of medical technology.

〔Example〕

The present invention will be explained in detail below using examples.

However, the present invention is not limited to these Examples.

(Obtaining DNA containing the lck gene) Lymphocytes are separated from the peripheral blood of a normal person according to a known method, and R
Approximately 10+ag of NA was extracted.

The RNA was fractionated by chromatography using a column packed with 1-oligo(dT) cellulose (Nα27-5649-Of, manufactured by Pharmacia ■).
00 μg of poly(A)” RNA was obtained.

The obtained poly(A)” RNA solution (1μg/μ7ri1
5-20 in tube volume 12I11 for 00μl
% siam density gradient centrifugation (40,00 Orpm, 15 m
1np 15g).

After centrifugation, 0.4 fractions of each of the 30 fractions were precipitated with ethanol. The obtained RNA sample was labeled with a 60mer (:l!p) that can hybridize to the 4th to 63rd bases downstream of the 5' end of the mRNA derived from the fck gene. No-in blot hybridization was performed as a probe. The synthetic nucleotides used were as follows.

5' CTCACA CACATCGAT GT
T TTCCAT CCA GTCTTCTTC
CGG GTG TGA GCT GCA
As a result, a fraction enriched in poly(A)'' RNA derived from fckDNA was selected.

5 μg of the selected poly(A)" RNA was denatured, and using the poly(A)" RNA as a template, a random primer (dP(N)i, (Takara Shuzo Co., Ltd., Nα38) was used.
01) 5 ttg, 4 dNTP each 40 g a
+oles), [a-”P] dCTP10μCi
3000C4/msgol), reverse transcriptase 50
Using Unit (manufactured by Takara Shuzo Co., Ltd. NO. 2610^),
An RNA-cDNA hybrid was created.

RN for the created RNA-cDNA hybrid.
Using 2 units of ase H (Na215OA, manufactured by Takara Shuzo Co., Ltd.), the mRNA was nicked to fragment the mRNA into eight-part fragments.

Furthermore, E. coli DNA polymerase I
(Nct2130A manufactured by Takara Shuzo) was reacted with 100 units, and the fragmented RNA was used as a primer for the second
A strand of DNA was created. After heat inactivation, 10 units of T4 DNA polymerase (Takara Shuzo Co., Ltd. No. 2040^) are added and reacted. Phenol extraction and two ethanol precipitations were performed to obtain 5 μg of dsDNA1.

1.5 μg of the dsDNA was methylated at the EcoRr recognition site using 20 units of EcoRI methylase (Takara Shuzo Co., Ltd. Na1540A), and after the reaction, phenol-chloroform extraction and ethanol precipitation were performed. A ligation reaction was performed on the ethanol-precipitated dsDNA using 500 mg of phosphorylated 10a+er-EcoRr linker (Na4740, manufactured by Takara Shuzo Co., Ltd.) and 200 units of T4 DNA ligase (Na2011A, manufactured by Takara Shuzo Co., Ltd.). Then, after heat inactivation, Eco
RI restriction enzyme (Takara Shuzo Co., Ltd. Na1540A) 20
ds EcoRI linker by cutting at 0 unit.
Connected to DNA.

A column filled with Sepharose CL-4B (manufactured by Pharmacia ■) was equilibrated, and the dsDNA digested with EcoRI was loaded onto the column and fractionated in 100 μl portions.

Each fraction was poured onto agarose, dried, and autoradiographed to select fractions containing 1 to 3 kbp dsDNA.

Next, using 1.0 #g of the dsDNA fragment, 2 μg of λgtlo vector (manufactured by Amsham Company), and T4 DNA ligase (Na2011A, manufactured by Takara Shuzo Company),
ds to the EcoRI cleavage site present in the tlo vector.
A DNA fragment was inserted.

Next, after performing in vitro packaging, the treated solution was centrifuged, and the supernatant was used as a recombinant phage solution for hybridization to Brachts.

After infecting E. coli c600hf1 strain (manufactured by Takara Shuzo ■) with the recombinant phage solution to form plaques,
Plaque hybridization was performed using the above probe to isolate phage containing lck dsDNA.

A large amount of phage DNA is prepared from the isolated phage, and the phage DNA is digested with EcoRI to create fck
400 μg of a 2.0 kbp DNA fragment (both 5' end and 3' end are EcoRI ends) containing dsDNA was obtained.

200 μg of the 2.0 kbp DNA fragment was added to l1indll! in a cleavage buffer with a NaCl concentration of 100 mM. Intermittent cleavage was performed using restriction enzymes. After cutting, add N at the final concentration.
NaCl was added so that the aC11 degree was 150 mM, and cleavage with Nco I restriction enzyme was performed. the result,
The 5' end containing the lck gene is the Nco I end and 3
1.8 kbp DNA fragment whose end is the old ndll end (hereinafter abbreviated as Nco I-H1nd■ fragment) 10
0 μg was obtained.

Creation of recombinant vector Escherichia coli expression vector pUc119 (manufactured by Takara Shuzo)
IOμg was digested with old ncll restriction enzyme, and the resulting reaction solution was digested with alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.).
k2120), phenol extraction and ethanol precipitation.

pUc cleaved with old ncII thus obtained
119 0.2 μg and Nco 1 obtained by the above method
-Hindl [Fragment 2ttg is shown in Table 1, Man-''-
Part 0.05M) Lis-○CI (pH 7,4) 0,
OIM Mg01g 0.01M Dithiothreitol 1mM Spermidine 1mM ATP 0.1+mg/I11 Mix in a solution of bovine serum albumin and add 4 dNTPs to a final concentration of 50-M, then add 10 units of Klenow fragment. A fill-in reaction was performed using

Further, hexamine cobalt solution was added to a final concentration of 1-, and 300 units of T4 DNA ligase was added, followed by reaction at 16°C for 15 minutes.

Finally, MCI was added to a final concentration of 30mM,
The connection reaction was further carried out for 3 hours or more.

E. coli JM
Add to 200 μl of 109 strain competent cell suspension,
It was left on ice for 30 minutes. Thereafter, it was heat-shocked at 42°C for 2 minutes and further left on ice for 5 minutes.

After taking it out from ice, 800 μl of LB liquid medium was added and cultured with gentle shaking at 37°C for 1 hour.

1OOμl of the culture was treated with ampicillin 100μs/l1d.
After inoculating a 151 dl plate of LB agar medium containing 3
The cells were cultured at 7°C for 15 hours.

After culturing, 24 white single colonies that appeared were taken out and each was treated with L containing 100 u of ampicillin.
It was inoculated into 1.5 d of B liquid medium and cultured at 37°C for 9 hours.

Next, 1IR1 was collected from each liquid medium,
The recombinant vector present in E. coli in each collection medium was purified by the alkalinosis method to obtain a recombinant vector suspension. Next, a portion of each obtained recombinant vector suspension was subjected to a cleavage reaction using BamHI restriction enzyme, and each reaction solution was subjected to agarose electrophoresis.
A recombinant vector was selected in which the Nco I-Hlndl [I fragment containing the ck gene was inserted into pUc119 in the correct orientation.

In addition, a part of the selected 11-change vector suspension was subjected to a cleavage reaction with Nco I restriction enzyme, and the cleavage was subjected to electrophoresis, and by confirming that the cleavage with Nco I resulted in one band, A suspension containing a recombinant vector for fck gene expression was found.

The obtained i, ck gene expression vector was transformed into pLcK11.
9(a), and the recombinant vector pLcK119
(a) owns 109 shares of coli JM!
, coli-1ck 1, was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology. (FERM P-11129) The method for preparing the above recombinant vector pLCK119 (a) is shown in FIG.

(Production of fusion polypeptide) E, coli 1 ck 1 was added to LB liquid medium 1 containing ampicillin to a concentration of loOμg/Id.
It was inoculated at 0d and cultured at 37°C for 12 hours. The culture 2
00uj2 was inoculated into H medium containing ampicillin (100 μg#d) to which IPTG had been added to a final concentration of 2 mM, and cultured for 115 hours at 37°C.

After culturing, the culture was collected and centrifuged (3000 rp
H+4'C+15m1n), the precipitate was added to Laemmli buffer 7d, suspended, sonicated, boiled, and centrifuged. The supernatant was used as a sample for SOS polyacrylic acid gel electrophoresis.

SDS polyacrylamide gel electrophoresis was performed using 20 μl of the sample, and the gel was coated with Coomassie Brilliant.
It was stained with Blue (manufactured by Σ) staining solution.

As a result, a band was detected in this sample at around 56 kd, which corresponds to the molecular weight of the fusion polypeptide to be obtained. (Illustrated in FIG. 6) In addition, FIG. 6 shows confirmation of polypeptide expression in E. coli. In Figure 4, Q anel is lc
E possessing pUc119 in which the k gene has not been inserted
, 1a is a cell lysate when E. coli containing pUc119 is cultured in a medium without IPTG, 1a
ne3 is B, a cell lysate obtained when cal i-1ck 1 was cultured in a medium without IPTG, 1ane 4 is E, a cell lysate obtained when colt-lck 1 was cultured in a medium supplemented with IPTG, and polyacrylamide Gel electrophoresis and Coomassie brilliant blue staining.

(Purification of fusion polypeptide) ε, colif obtained in the production of the fusion polypeptide
Tris-HCl (pH 7,4
), 25% shi = 11! , 0.01%NP-40, 1mM
Dithiothreitol, 1mM PMSF, l
μg/ad! After adding, suspending and sonicating a solution with the composition of lysozyme,
Centrifuged for minutes. Next, for the obtained precipitate,
RIPA(-3O3) buffer 25- was added and suspended. After sonicating this suspension, 4°C 18000r
After centrifugation at p-, the precipitate was subjected to SOS polyacrylamide gel electrophoresis. After electrophoresis, fill the gel with 2M KC
Immersion in I solution, cut out the white band that appeared at a position around the molecular weight of 56 kd, and add 0.1M Tris, 0.1M
) Elution using t volume buffer of lysine, 0.1% SDS yielded 3 fusion polypeptides. still,
FIG. 7 is an SOS polyacrylamide gel electrophoresis chart showing the purification status at each purification step in the above purification process. In Figure 7, 1ane 1 is the supernatant of the Tris-HCl treatment, 1ane 2 is the supernatant of the RIPA (-5[lS) buffer treatment, 1ane 3 is the precipitate of the RIPA (-3DS) buffer treatment, and j2ane 4 is the precipitate of the RIPA (-3DS) buffer treatment. This is the gel eluate after electrophoresis. Scanning the fane 4 gel revealed that the fusion polypeptide was purified with a purity of over 90%.

(RA recognition of fusion polypeptide) The fusion polypeptide obtained by the above purification was
After dilution with the elution buffer to a concentration of g/d,
0.5 d of Band Adjuvant Complete Friend (manufactured by Deaf Collaborative Laboratories) was added to 0.5 d of the diluted solution and suspended.

The suspension was then injected transdermally into guinea pigs. Similar injections were performed one week and two weeks after the first injection, and blood was drawn from the guinea pigs three weeks after the first injection. The collected blood was stored at 4"C overnight, then centrifuged (3000 rpm, 4°C, 20 m
1n) By removing the supernatant, the anti-I! ,ck
10 d of serum containing antibodies was obtained.

On the other hand, after Jurkat, MT-1, and MT-2 cells were labeled (35S) by a known method, each 1×
10' cells were dissolved in I'l IPA buffer 1d shown in Table 2 to obtain a cell lysate.

Difference-j Heat Table RIPA Buffer 110 l 11 Tris-HCl (pH 7,4) 1% N
P-40 0.1% Sodium deoxycholate 0.1% 5DS O, 15M NaCl 1a+M εDTA 2 sM PMSF 1% Triton-X 1mM Dithiothreitol To each of these, 450 μl of RIPA buffer was added and mixed, and then the above Add 10 μl of anti-1ck antibody and incubate at 4°
The cells were allowed to stand overnight at C for an antigen-antibody reaction.

After standing, each reaction solution obtained was heated at 15,000 rpm for 10 minutes.
After centrifugation for a minute, 30 ul of protein A Sepharose was added to the supernatant, followed by shaking at 4°C for 1 hour.

Next, after the above treatment, centrifugation was performed at 15,000 rpm + 4"C for 10 minutes, and the precipitate was washed twice with 1 ml of high salt wash buffer having the composition shown in Table 3. Furthermore, low salt wash buffer 1
Washed twice with ttrl. Then, each precipitate was dissolved in 20 μl of Laemmli buffer, boiled and centrifuged, and the supernatant was subjected to SOS polyacrylamide gel electrophoresis.

High 20 Suri- 0.5M 1mM 065% 1% Yi-:L-Omote Salt Wash Buffer Tris-HCl (pH 7,4) aCI EDTA NP-40 DOC (Margin below Honshin) Daiichi Niri Ichimugi Law Salt Wash Buffer 10■H Tris-11cI (pH 7,4) 10+++M NaCl 1s+M EDTA O85% NP-40 1% DOC Next, staining was performed, and after decolorization, the amplified gel was dried.
autoradiography. The results are shown in FIG.

As controls for the anti-1ck antibody, non-immune guinea pig serum and anti-1ck antibody pre-adsorbed with the fusion polypeptide of the present invention to render it inactive were also used for comparison. Table 5 shows the types of antigens and antibodies for each 1 ane in FIG. 8.

(Blank below the text) As can be seen from Fig. 8, the anti-fck antibody immunized with the fusion polypeptide of the present invention is Jurkat, MT-
It recognizes the lck gene product expressed in one cell and forms a hand at each 56 kd position.

This band was detected when non-immune guinea pig serum and preadsorbed anti-FCK antibody were used as antibodies (fanel,
3.4.6) Or, even if an anti-1ck antibody is used as the antibody, if MT-2 cells that do not express the fck gene product are used as antigen-side cells <1ane7, 8.
9) cannot be confirmed. From this, it was confirmed that the fusion polypeptide obtained by the present invention contained the fck gene product.

[Brief explanation of drawings]

Figure 1 shows the amino acid sequence of the polypeptide derived from the fck gene, Figure 2 shows the DNA sequence of the lck gene, and Figure 3 shows the β-
DNA sequence of galactosidase, Figure 4 is pUc119
The DNA sequence of the multi-cloning site of the vector, Figure 5 is a diagram of the construction of the recombinant vector of the present invention, Figure 6 is an SDS polyacrylamide gel electrophoresis diagram of the fusion polypeptide of the present invention, and Figure 7 is the diagram of the fusion polypeptide of the present invention. SDS polyacrylamide gel electropherograms at each purification step of the peptide, and FIG. 8 are immunograms showing that the antibody obtained using the fusion polypeptide of the present invention can recognize the polypeptide derived from the lck gene in each cell. Autoradiography of the precipitate.

Claims (1)

[Claims] 1. An amino acid sequence corresponding to a DNA having a multiple cloning site is interposed at the N-terminus of a polypeptide derived from the lck gene, and an amino acid sequence from the N-terminus of β-galactosidase to at least the fifth amino acid A fusion polypeptide in which polypeptides having the sequence are joined together. 2. In an Escherichia coli expression vector containing a DNA containing a multi-cloning site at a desired site of part or all of the β-galactosidase gene, lc is inserted into a desired cloning site of the multi-cloning site.
The fusion polypeptide according to claim 1 is produced by integrating a DNA having the k gene, preparing a recombinant vector, transforming E. coli with the recombinant vector, and culturing the obtained transformant, A method for producing a fusion polypeptide, which comprises collecting the polypeptide.