JPS6312285A - B-cell differentiation factor - Google Patents

Abstract

PURPOSE:To produce TRF by selecting a clone having cDNA of TRF from a cDNA bank prepared from T-cell using TRF activity, etc., as indices and introducing the plasmid DNA of said clone into a host. CONSTITUTION:An mRNA fraction extracted from T-cell line 2.19 is inserted into an oocyte of Xenopus laevis, TRF mRNA of 16S is detected from TRF activity and a cDNA library is produced from said mRNA. A cDNA clone is selected from said library using a plasmid containing SPG promoter and digested with SalI. An mRNA is prepared from the obtained DNA fragment and a psp6k cDNA library is prepared therefrom. One clone having strong activity is selected from said library using TRF activity and BCGFII activity as indices and Escherichia coli is transformed with a plasmid psp6k-mTRF23 of said clone to produce TRF.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Application of E'R, Ming] The present invention relates to a Bmm follication factor. For more details,
This invention relates to a B cell differentiation factor produced by mouse T111 hybridoma and an ON^ sequence encoding the factor.

[Prior art] BIIIlla differentiated prisoner (TRI' factor T cel
1ReplacingFactor) is a factor consisting of a polypeptide that is produced from the 1 cell lineage in the blood, acts directly on B cells, and differentiates and induces them to become antibody-producing cells.

Antibodies have the function of reacting with foreign substances such as bacteria, viruses, or cancer cells that invade the living body, and inactivating or eliminating them.

B cell differentiation factor (hereinafter abbreviated as TRF) induces 8 cell clones specific to a specific antigen (bovine foreign body), that is, B cell clones sensitized to the specific antigen, into antibody-producing cells. TIIF is a useful substance in the treatment of various infectious diseases and cancers because it stimulates the production of antibodies against TIIF.

Therefore, it is expected that rRF can be used not only in the clinic and treatment of autoimmune diseases and immunodeficiency diseases, which are thought to be caused by an insufficient amount of this factor in the body, but also in the treatment of various infectious diseases and cancer. be done.

Several studies have been conducted regarding TRF. For example, [R "Ti1 known as a producing cell
Culture medium of 12 strains of L cell hybridoma 8151 Tn
Attempts have been made to purify the substance with F activity, and it has been determined that it is a monomeric glycoprotein with a molecular signature of 18,000 to 19,000, which is relatively stable at temperature and pH.
Takatsu, et, at, J, In
+muno1.134382, 1985.1larad
a, N, at, al,, J, I+++n+un
ol.

Lice 3944.1985 and JP-A-60-237022
Such〕.

However, there is little TRF content in the culture solution, and it is preparatively separated through a multi-step purification process (ammonium sulfate precipitation, anion exchange chromatography, Glu filtration, IIPLc1 slab electrophoresis, J1 reversed phase 11PLc, requin affinity column chromatography, etc.). Since the amount of the substance produced is extremely small, its structure (amino acid sequence) and physicochemical properties have not yet been clarified, and the genes (DNA sequence) involved in the production of TRI' are also unknown. Not yet. In addition, the purified TR of the above-mentioned prior art
r is BCGF (B cell crowdFac
tor: It has been shown that it also has the activity of B It cell proliferation captive factor (■). The existence of multiple TItFs has also been suggested.

In this way, conventional factors that differentiate and induce B cells (TR
)'s structure was not clear, and it was impossible to produce a ceiling-mounted telescope for practical use.

[Problems to be Solved by the Invention] That is, the present invention solves the problem of TRF m from TRr high-producing cells.
We isolated ItN8 and revealed the old gene (DNA sequence) that controls TIIF production, as well as 18
By clarifying the structure (amino acid sequence) of a single molecule, recombinant DNA technology can be used to improve human production and medicine of TRI.
This is intended to provide the possibility of application to, etc.

[Interim means for solving the problem] The present inventors
One-rm spore Labiline 2.19, which is a DIG1-induced convict 8C leaf γ-producing cell, which has already been established by 5idOraS, P. et al.
15586-593.1985) is more TI than the conventionally used Tl cell hybridoma 8151 strain 12).
It was found that approximately 100 times more M was produced. So this T
As a result of extensive research to solve the above problems using cell line 2.19, the present invention has been completed.

To prepare the DNA of the present invention, first, an mRNA fraction is prepared from TI[I cystin 2.19 by a conventional method, and 1) O
IV(A)' Methods already developed using RNA (Noma, 'f, at, al, Natur
cDNA according to e 319 640-646゜198G)
^Create a DNA rally. After linearization of the plasmid DNA of each cDNA clone obtained from this library with restriction thread Sal.

mRNA is obtained by transcription using SF3118A polymerase in an in vitro system. The mRNA thus prepared was injected into Xenopus oocytes and cultured, and the Ti1l' activity of the translation product (protein) from the mRNA secreted into the culture supernatant was detected. and measuring the presence or absence of BCGF I activity (Gronowicz, E., et.
[ur, J, In+muno1.6588-590
．． 1976J3 and TakatSu, at, a
T.

J, Iamunol, 125 2646-2653
．． 1980), and the TIIF activity and BCGF If live bamboo described herein are each defined by having the following activities.

8) TRF activity: 1) Mouse chronic B leukemia cells (BCLl) were infected with IfJ14.
2) Antigen (GNP-L) 1) Stimulate the sensitized mouse intrapancreatic B cells with the antigen (DNP-ovalbumin) and generate a specific antibody (anti-DNP-1 (IG)). ) activity to differentiate into producing cells, or 3) activity in vivo''?

B) BCGr [Activity: 1) Mitogenesis of BCL1 cells, or 2) Mitogenesis of dextran sulfate stimulated resting B cells.

Among the above activity measurement methods, especially 8)-1) and B)-1
) activity was used as an indicator to measure the TRF activity and BCGt'' activity on mRNA transcribed in VitrO.
It is convenient to select from DN^DNA rally. Next, the base sequence of the cDNA1gi piece inserted into the plasmid DNA of the selected clone (psP6K-mrR "23)" was determined by known methods (the dideoxy method and the unidirectional bleed region method described in the Examples below). The amino acid sequence of Tlt[ can be determined by searching for a flexible open reading frame (1!! base sequence that -1-codes the polypeptide) as shown in Figure 1. At this time, the selected clone psP6K Some Tl1
In the tp human DNA region of r23, two open reading frames encoding polypeptides consisting of 133 and 62 amino acids are found; The polypeptide (equivalent) has a molecular weight of approximately 6.500, and this value is smaller than the molecular weight of a single chain (approximately 18,000) of Til produced from 2.1'111 cells, even if the sugar chain is 1. Even if 0.0 mRNA is present, it is too small; TR with oocytes of
Since it does not lose its F-producing ability, it is not an old polypeptide. Therefore, it has been determined that rRF, which is first produced by 2.19all cells in vivo, is a polypeptide consisting of 133 amino acids (see Figure 1). Note that Net (methionine) at the N-terminus of this polypeptide is caused by a post-translational modification process (post-translational modification process).
odification process), it may undergo fumylation or acetylation, and Net may also be removed.

In the N-terminal region of the polypeptide determined in this way,
A leader sequence (a highly hydrophobic peptide region corresponding to the amino acid sequence from the N-terminus to about the 20th amino acid sequence in FIG. 1) unique to secreted proteins is present. This is 2.19
@I! This is consistent with the fact that TRI is secreted into the culture supernatant. It is known that the leader sequence is usually processed in vivo (cleavage by signal bevudaase, etc.) and becomes a mature polypeptide from which that region has been removed.

Cleavage sites for leader sequences known so far [generally the C-terminal side of amino acids such as alanine (^la) and glycine (Gly); Watson, H, E, [, Nu
cl.

Ac1d, Res, 12 5145-5164.1
984], the protein (glycoprotein) is considered to be a molecule containing polypeptides from the 19th, 21st, or 22nd to the 133rd, respectively, in the amino acid sequence shown in FIG. Considering that 11 (") is a glycoprotein, the molecular weight of these polypeptides (approximately 1
2.300 to 12,700), b is reasonable. That is, the molecular weight derived from this polypeptide (approximately 12
．． 300) has three N-glycosylation possible sites (underlined in Figure 1) present in the amino acid sequence corresponding to the open reading frame (nucleotide sequence from 44th to 442nd in Figure 1). There is no contradiction considering the amino acid sequence). In addition, the N-terminal amino acid residue of the polypeptide produced through the above processing may be further alletylized, or may be converted to /ll? In the case of GIU (glutamic acid), it may be pyrrolated.

Generally, it is known that the C-terminus of polypeptides produced in vivo may be deleted during the process of 71 lusing in vivo, and the C-terminus of the polypeptide shown in Figure 1 is It is thought that a portion of the terminal amino acid or peptide may be deleted.

These polypeptides are the DNA shown in FIG.

In particular, the base sequence corresponding to the amino acid sequence from 1 to 133 including the translation stop codon (IOA), or 1! which is substantially equivalent to this! Insert the DNA having the i-base sequence into an appropriate expression vector, introduce it (transformation) into an appropriate microorganism or animal host cell, and culture the transformant.
- By doing so, it is possible to produce a large amount of cells more efficiently than conventional cell culture.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

[Example] (1) Preparation of TItFmRN^ Cell line 2.19 was used as TRF-producing cells. This cell line has already been established by 5idOraS, P. et al. (Sidaras, P.
.

at, al, Eur, J, ll1illJnO
1,,15,586=593°1985).

First, 2 activated with Con^ (concoverin A)
, 19TIil cells to the method of N0IIla, Y. et al. (
NatLIrQ.

319 640-646.1986) Poly(A)'' RNA (IIIRHA fraction) was extracted and subjected to sucrose density gradient centrifugation using a 5-22% concentration gradient (36,000 rpm, 15 hours). ) by poly(^
)” The RNA extract was fractionated into 16 fractions. Next, an appropriate amount of each fraction was added to the African frog (Xcnopu
T
RI' activity and BCGFII live bamboo were measured.

To measure TflF live bamboo, oocyte culture supernatant thimble,
0.2m containing 1.5X 10 BC111B cells
(2 days later, G
Protein Δ plaque assay method of Ronowicz, E. et al. (Eur, J., ll1lnlunO1,658
8-590.1976) to measure the number of Ig+4 secreting cells. On the other hand, BCGFII
To measure the activity, the proliferation rate of the BCL1ill cells was determined by adding 3 to 1 demidine for the last 6 hours of the 2-day culture.
This was carried out by measuring the l-1 demidine prefecture taken up into 1111 cells.

As a result, as shown in Figure 2, BCGF II activity (black frame in the figure) was observed in 9S and 163 mRNA^RNA, and In the figure, P stands for Tl1l',
¥; Til+ cell hybridoma 815, one of the live strains
1 indicates a positive control in which the culture supernatant of 12 strains was added to the BCL1 differential medium at a concentration of 50%, and N is
Negative controls in which oocytes were injected with phosphate buffer instead of mRNA and cultured are shown.

The fraction size of each 93 mRNA is interleukin (IL
) - corresponds to an mRNA size of 4 (NOla,
Y.

at, al, Nature, 319 640-6
46.1986) and the 95mRNA fraction have activity to promote the proliferation of BC11HA cells, but no differentiation activity into IN secretion producing cells was observed in the BC11HA vesicles. It was determined that DingRr mRNA was present in the 163 ml NA fraction of the cells, and then cDN^D
An attempt was made to create NA rally.

(2) CDN^DNA rally preparation and TRF cDNA
Clone Selection Total Po1y of 2,19Tll cells activated with Con^ (
A) “COMA library to RN is SPG Pro [
N0IIla, Y, using a plasmid containing -
(Nature, 319 640-649.1)
Approximately 5 x 10' cDNAs
A clone (1lsP6KcON^DNA rally) 4
! Also.

Next, the mixture of these clones into plasmid DN was digested with Sac I and Sal II respectively to avoid linearization of the plasmid, and then 5P was added in vitro (in VitrO).
1ltN^ was synthesized using 6RNA polymerase.

The IRN^RNA thus prepared was injected into African frog (Xcnopus) oocytes (oocytc).
DAt, after culturing at 20°C for 36 hours, the products secreted into the culture solution were collected and determined by the measurement method shown in Example (1).
Rr activity and BCGF It activity were measured. As a result, 1R prepared from pieces of ON8 digested with Sat.
Both active bamboos were observed in the NA, but no activity was observed in the DNA fragments digested with Sac I.

The pSP6K cDNA library prepared from the mRNA of the 2.19 T cell line described above was then divided into 18 pools (approximately 3,000 genes per boule), each of which was analyzed for TIIF activity and BCGr I according to the method described above.
I activity was measured. As a result, one pool [,,17
) showed both activities. Therefore, this pool was further divided into subgroups and TRF and BCG
``II activity was measured and a group of 60 clones with positive activity was identified.

Next, we confirmed that the DNA fragment (cDNA) inserted into the plasmid DNA of these clones had IKI1 or higher, had a Sac I cleavage site in its base sequence, and had no Sal I cleavage site, and selected 7 clones from the 60 clones mentioned above. (clones positive 16.18.23.27.42.51 and 53) were obtained. rllll' activity of these 7 O-ons (
1 (1M plaque-forming cell number) and BCG “U activity (
3H boumidine uptake) are shown in Table 1.

Among these 7 clones, 23 and 23 [1-] clones showed the strongest activity, so the plasmid of this clone was used as pSP.
The protein was named P6K-mTRF23 (see Figure 3) and was used for subsequent analysis. The Escherichia coli 118101/psP6K-mTRF23 transformed with this plasmid was named [5cherichia coltSBH285, and the accession number of rLRHP-8828 was given to FEIKEN as 19.
It has been deposited in

(3) COMA! ! t, sequence analysis and TR,F polypeptide + 1sP6K-ITRF23Nell/, = CDN8H once pUcI8I lasmid B
Am1l I site was rib-cloned using the NDE Axi method (Sanger, F., et al., Pro.
c.

atl, ACad, Sci, us^, 74 5
463-5469.1') 77) 3 and unidirectional direction method 2 unidirection
al deletion method, YaniSCh
-erron, c, Otal,, Gen833
103-119.1985) were combined and the nucleotide sequence was determined. The strategy for determining the base sequence J is shown in FIG. As a result, as shown in FIG. 1, a sequence consisting of 533 base pairs excluding the poly^tail region was obtained.

As a result of detailed analysis of this base sequence, we found 133 -mino acid residues (base sequence from 44th to 442nd in Figure 1) and 62 amino acid residues (from 44th to 442nd in Figure 1). Two open reading frames were found consisting of the 729th base sequence), but i) TRF polypeptide (glycoprotein) was found in 2.19T follicles and oocytes injected with the mRN^. It is secreted and produced, and its monomer (SOS polyacrylamide gel electrophoresis analysis) has a molecular weight of approximately 18,000, (i
i) ON8 which was linearized by cleaving the photopic site of ^cc I (salt chain sequence 624-62'l in Figure 1) existing in the oven reading frame consisting of 62 amino acid residues with the enzyme. 018N8 from T in oocytes
Ability to produce RF and BCGr (iii) 62
The molecular weight of a polypeptide consisting of 6 amino acid residues (approximately 6
．． 500) is the molecular suspension of the TRF biom approximately 18,00
Compared to 0, TRr or BCGF II, which is first produced in the 2.19T cell line, is too small even if a sugar chain is added (there is one N-glycosylated Sigril sequence in the sequence). It was determined that an active polypeptide (abbreviated as TRI' polypeptide) was based on the 1133 amino acid sequence shown in FIG. However, Hat (methionine) at the N-terminus of this polypeptide is involved in the translation 11 modification process (post trans
tional modification processes
It is thought from the previously reported hyperplasia of in vivo polypeptides that s) results in formylation, acedelylation, or removal of Met residues.

At the N-terminus of the polypeptide shown in FIG. 1, which consists of 1133 amino acid residues, there is a highly hydrophobic peptide consisting of about 20 amino acid residues.

This sequence (leader sequence) is unique to secretory proteins, and it is known that this sequence is removed when the protein is secreted from inside the cell.

In general, the C-terminus of amino acid residues such as alanine (Ala) and glycine (Gly) is often cleaved (1).
4atFiOn, H, E, E,, NIJC+, ^
cid, Rcs, 125145~5164.198
4), and since the leader sequence is usually a peptide around 20Il1, TRr or 8CGrI in the present invention [the polypeptides that determine the activity are the 19th and 21st peptides in the amino acid sequence shown in Figure 1]. Or 22
It is thought that the molecule contains the polypeptide from position 1 to 133. The molecule ff1 of these polypeptides (
to approximately 12,300, 12. γ00) is almost consistent with the fact that the single m form of TRF is a glycoprotein with a molecular length of about 18,000.

In addition, the N-terminal amino acid of the polypeptide resulting from removal of the leader sequence as described above is further acetylated in vivo, or if it is glutamine 1 (Glu), it is biolylated. It is natural to think that this may be the case, considering the structure of peptides that have been discovered in living organisms.

(4) TR of pSP6K-mlRF23 plasmid
mlt transcribed by 5P611N^ polymerase from one 5TRF23 plasmid ON8 to F-active psPG
The TRF activity of the culture supernatant of 111 oocytes injected and cultured (differentiation activity of BCL1ill follicles into rgH secretion producing cells) and 8CGF7I activity46 (3-day thymidine uptake suspension into BCL1 cells) ) are shown in Table 1.

Additionally, Takatsu, K. et al. (J, Immun.
The above oocyte product (abbreviated as γTRF) was purified according to the method of
The effect of promoting the NP-IQG antibody production response was investigated. First, BALB/C mice were treated with DNP-keyhole limpet hemocyanin (eyeholc Iimpct).
sensitized with helocyanin; 111), 6-8
After a week, pancreatic cells were collected and enriched with B111 cells by anti-'rhy1.2 antibody and complement treatment.
NP-1 (IG) was examined for its ability to promote plaque-forming cells.

As a result, γTRI' stimulates ONP-sensitized B cells in the presence of antigen (DNP-ovalbumin 20NP-0^) and clearly exhibits an activity that causes them to differentiate into anti-DNP-1 (IG-producing cells). Table 2). Although further data are not shown, 71RI is associated with activated B111 in vivo.
It also showed the I (JH synthesis
-1, It-2, 11-3 and BSF-1 activities were not increased. From these results, in the present invention, pSP6K-mTRF23 was
It was confirmed that psP6K-mTRF23Rio->1 (1M plaque-forming cell-promoting activity and BCL1 proliferation-promoting active bamboo (see Table 1), T
RF! : It was suggested that BCGFII is the same molecule.

Table 2 Anti-DNP-1 (IG plaque formation promoting effect-DNP-KLll 0
17920 None 0127 + DNP-KLII O1990
Go to Open P-〇〇92-4-
[INP-08 1
695〇NP-OA
0.5 884+ DNP-O
A 0.25 5! i5

[Brief explanation of drawings]

Figure 1 is a diagram showing the base sequence and amino acid sequence of the TRI polypeptide region of the cDNA inserted into I) Sr1-111TRF23. Figure 2 shows the TRF activity (plaque formation) of the a+ RNA fraction. Figure 3 (8) is a schematic diagram of the plasmid of the psP6K-mTIIF23 clone, and Figure 3 (B) is a graph showing the plasmid of psP6K-mTIIF23 clone. (
Figure 4 is a schematic diagram of the surroundings of the in-lead cDNA of psI) 6K-mTRF23. Patent applicant
Representative Yu Patent attorney 4 Yoe J'-]21 (5 others)

Claims (18)

[Claims] (1) The following formula I: [There is a base sequence] (I) [In the formula, \STP\ represents a translation stop codon. A DNA encoding a B cell differentiation activating factor having a base sequence represented by the following or a base sequence in which part of its 5' end is partially deleted up to G at position 63. (2) The following formula I: [There is a base sequence] (I) [In the formula, \STP\ represents a translation stop codon. A plasmid containing a DNA encoding a B cell differentiation activating factor having a base sequence represented by the following or a base sequence in which part of the 5' end of the base sequence is deleted up to the 63rd G. (3) The plasmid according to claim 2, which is represented by pSP6K-mTRF23. (4) The following formula I: [There is a base sequence] (I) [In the formula, \STP\ represents a translation stop codon. ] or a part of the 5' end of the nucleotide sequence up to the 63rd G is deleted. Or a factor having B cell differentiation activity, which has a polypeptide produced by translation within a prokaryotic cell as a polypeptide component. (5) The factor according to claim 4, which also has GCGFII activity. (6) The amino acid sequence of the polypeptide is formula II: [There is an amino acid sequence] (II) [where X is H (hydrogen atom), fMct (formylmethionine), AcMet (acetylmethionine), or Mct (methionine) represents. ] The factor according to claim 4, which is represented by: (7) Following formula III: [There is an amino acid sequence] (III) [In the formula, Y is H (hydrogen atom), Met or Thr-
represents Ala-Met, provided that the N-terminal amino acid may be acetylated, or
The terminal Glu may be pyrrolated. ] It has an amino acid sequence represented by
A patent claim having B cell differentiation activity, which has as a polypeptide component a polypeptide produced by processing the N-terminal peptide of the polypeptide represented by Formula II given in Section 2 in a eukaryotic or prokaryotic cell. The factor described in range item 4. (8) The factor according to claim 7, which also has BCDFII activity. (9) The following formula I: [There is a base sequence] (I) [In the formula, \STP\ represents a translation stop codon. ] Transformation in which a plasmid containing a DNA encoding a B cell differentiation activating factor having a base sequence represented by the following or a base sequence in which part of the 5' end of the base sequence is deleted up to the 63rd G is introduced. cell. (10) The transformed cell according to claim 9, wherein the plasmid is pSP6K-mTRF23. (11) The following formula I: [There is a base sequence] (I) [In the formula, \SIP\ represents a translation stop codon. ] Transformation in which a plasmid containing a DNA encoding a B cell differentiation activating factor having a base sequence represented by the following or a base sequence in which part of the 5' end of the base sequence is deleted up to the 63rd G is introduced. A method for producing a B cell differentiation activating factor having a polypeptide as a polypeptide component by culturing cells. (12) The method according to claim 11, wherein the produced factor also has BCGFII activity. (13) The method according to claim 11, wherein the plasmid is pSP6K-mTRF23. (14) A DNA having a biologically equivalent function to the base sequence represented by the above formula I which encodes the polypeptide represented by the above formula II. (15) A plasmid containing all or part of the above-mentioned substantially equivalent DNA. (16) A method for producing a factor having B cell differentiation activity by culturing transformed cells into which the plasmid according to claim 15 has been introduced. (17) The method according to claim 16, wherein the produced factor also has BCGFII activity. (18) The method according to claim 16, which is a polypeptide represented by formula II or formula III.