JPH10262673A - Production of b-cell differentiation factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently produce B-cell differentiation activating factor useful for the treatment of various infectious diseases and cancers as well as for the diagnosis and treatment of autoimmune diseases, immunodificiency diseases, etc., by culturing a specific transformed cell. SOLUTION: The objective B-cell differentiation activating factor containing a polypeptide as a polypeptide component and having BCG FII activity is produced by culturing a transformed cell containing a DNA coding for a B-cell differentiation activating factor having a base sequence expressed by the formula (underlined STP is a translation terminating codon) or a base sequence of the formula wherein the bases up to 54th C, 60th C or 63rd G of the 5' terminal are depleted and containing a plasmid such as pSP6K-mTRF23 introduced into the cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a B cell differentiation factor. More specifically, the present invention relates to a method for producing a B cell differentiation factor using a DNA sequence encoding a B cell differentiation factor produced by a mouse T cell line.

[0002]

2. Description of the Related Art B cell differentiation factor (TRF: T cell)
(Also referred to as Replacing Factor)
Is a factor consisting of a polypeptide that is produced from the T cell lineage in the blood and acts directly on B cells and differentiates and induces them into antibody-producing cells.

[0003] Antibodies have the function of reacting with xenobiotics 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 a B cell clone specific to a specific antigen (xenobiotic), that is, a B cell clone sensitized to a specific antigen, to an antibody-producing cell, and converts an antibody against the antigen into Because it is produced, TRF is a useful substance in treating various infectious diseases and cancer. That is, TRF
Is expected to be used not only for the diagnosis and treatment of autoimmune diseases and immunodeficiencies, etc., which are thought to be caused by the in vivo shortage of this factor, but also for the treatment of various infectious diseases and cancer.

Several studies have been made on TRF. For example, an attempt has been made to purify a substance having TRF activity from a culture of a T-cell hybridoma B151K12 strain known as a TRF-producing cell, and a monomer having a molecular weight of 18.0 or more, which is relatively stable at temperature and acidic pH.
It is estimated to be between 00 and 19,000 glycoproteins [Takatsu, K .; et. al. , J. et al. Imm
unol. 134 , 382, 1985, Harada,
N. et. al. , J. et al. Immunol. 134, 39
44, 1985 and JP-A-60-237022]. However, the TRF content in the culture is low,
Multi-stage purification process (ammonium sulfate precipitation, anion exchange chromatography, gel filtration, HPLC, slab electrophoresis, reverse phase H
Substances that can be collected via PLC, lectin affinity column chromatography, etc.)
Neither its structure (amino acid sequence) nor its physicochemical properties have been clarified. Further, genes (DNA sequences) involved in TRF production are not known. The above-mentioned purified TRF of the prior art is BCGF (B cell Gr).
owth Factor (B cell growth factor) II activity. The existence of multiple TRFs has also been suggested.

As described above, the structure of the factor (TRF) for differentiating and inducing B cells has not been elucidated so far, and it has not been possible to produce it in large quantities for practical use.

[0006]

That is, the present invention provides a TR
By isolating TRF mRNA from F-producing cells and elucidating the TRF gene (ie, DNA sequence) that governs TRF production and elucidating the structure (amino acid sequence) of the TRF molecule, recombinant DNA technology has It is intended to provide a possibility of mass production and application to medicine and the like.

[0007]

Means for Solving the Problems The present inventors have already developed Si
deras, P .; T cell lines are IgG ₁ induction factor BCDFγ producing cells that have been established by al 2.19
(Eur. J. Immunol., 15, 586-59.
3, 1985) found that TRF is produced about 100 times more than the conventionally used T cell hybridoma B151K12 strain. Therefore, this T cell line 2.1
As a result of diligent research to solve the above-mentioned problems using No. 9, the present invention has been completed.

To prepare the DNA of the present invention, first, an mRNA fraction is prepared from a T cell line 2.19 by an ordinary method, and a method already developed using poly (A) ⁺ RNA (Noma, Y. Et.al.Nature 3
19, 640-646 , 1986). Plasmid DNA of each cDNA clone obtained from this library was replaced with restriction enzyme Sal.
After linearization with I, SP6 in an in vitro system
Transcription is performed with RNA polymerase to obtain mRNA. The mRNA thus prepared is injected and cultured in Xenopus oocytes (oocyte), and TRF activity and BCGFII activity of a translation product (protein) from mRNA secreted into the culture supernatant are obtained. [Gronowicz, E. et al. et. a
l. , Eur. J. Immunol. 6, 588-59
0, 1976 and Takatau, K .; et. a
l. , J. et al. Immunol. 125, 2646-265
3, 1980]. The TRF activity and B
CGFII activity is defined by having each of the following activities.

A) TRF activity: 1) activity to differentiate mouse chronic B leukemia cells (BCL ₁ ) into IgM antibody-producing cells; 2) antigen (DNP-KLH) -sensitized mouse spleen B cells into antigen (DNP) Ovalbumin) to stimulate differentiation into specific antibody (anti-DNP-IgG) producing cells, or 3) I of B cell blasts activated in vivo
gM synthesis inducing activity.

[0010] B) BCGFII activity: ₁₎ BCL 1 cells of mitogenic, or 2) mitogenic of dextran sulfate stimulate resting B cells.

[0011] Among the above methods for measuring the activity, in particular, using the activities of A) -1) and B) -1) as indices,
It is convenient to measure the TRF activity and the BCGFII activity of the mRNA transcribed in the above step, and to select a clone having the TRF cDNA from the cDNA library. Next, the selected clone (pSP6K-mTRF2
The nucleotide sequence of the cDNA fragment inserted into the plasmid DNA in 3) can be determined by a known method (dideoxy method and unidirectional deletion method described in Examples described later).
(See FIG. 1), meaningful open reading frame (base sequence encoding polypeptide)
And the amino acid sequence of TRF can be determined. At this time, in the inserted DNA region of the selected clone pSP6K-mTRF23, two types of open reading frames encoding a polypeptide consisting of 133 and 62 amino acids are found, but the latter (from position 544 in FIG. 1). The polypeptide corresponding to the 729th nucleotide sequence)
The molecular weight is about 6,500, which is the monomer molecular weight of TRF produced from 2.19 cells (about 18,00
Compared to 0), even if a sugar chain is provided, it is too small, and mRNA prepared from a clone prepared by cleaving the ACC I cleavage site present in this open reading frame with the restriction enzyme is: It is not a TRF polypeptide because it does not lose its ability to produce TRF in Xenopus oocytes. Therefore, 2.
The TRF that 19 cells first produce in the body is 133
It is determined to be a polypeptide consisting of two amino acids (see FIG. 1). In addition, M at the N-terminus of this polypeptide
et (methionine) is involved in the post-translational modification process (post t
translational modification
process), it may be formylated or acetylated, and Met may be removed.

The thus determined N-terminal region of the polypeptide has a leader sequence specific to a secreted protein (FIG. 1).
And a highly hydrophobic peptide region corresponding to the amino acid sequence from the N-terminus to the 20th position). This is consistent with the fact that TRF is secreted into the 2.19 cell culture supernatant. Usually, it is known that a leader sequence undergoes processing (eg, cleavage with a signal peptidase) in a living body and becomes a mature polypeptide from which the region has been removed. Cleavage sites of leader sequences known so far [generally alanine (Ala) or glycine (Gly)]
C-terminal side of an amino acid such as Watson, M .; E. FIG.
E. FIG. , Nucl. Acid. Res. 12, 5145-
5164, 1984] from 2.1 used in the present invention.
TRF activity (or BCGFII activity) secreted by 9 cells
Approximately 18,000 molecular weight protein (glycoprotein) with
It is considered to be a molecule containing the 19th, 21st or 22nd to 133rd polypeptides in the amino acid sequence shown in FIG. This is considered from the fact that TRF is a glycoprotein, and is appropriate from the viewpoint of the molecular weight (about 12,300 to 12,700) of these polypeptides. That is, the molecular weight (about 12,300) calculated from this polypeptide is determined by the three N-glycosyl groups present in the amino acid sequence corresponding to the open reading frame (the nucleotide sequence from the 44th to the 442th in FIG. 1). Possible site (underlined amino acid sequence in Fig. 1)
There is no contradiction. In addition, the N-terminal amino acid residue of the polypeptide generated by the above-described processing is:
Further, it may be acetylated or, if it is Glu (glutamic acid), pyrrolated.

Further, it is known that the C-terminus of a polypeptide produced in a living body may be deleted in the course of processing in the living body, and the C-terminal of the polypeptide shown in FIG. It is thought that the terminal amino acid or a part of the peptide may be deleted.

These polypeptides have the DN shown in FIG.
A, in particular, a DNA having a nucleotide sequence corresponding to the amino acid sequence from 1 to 133 including a translation termination codon (TGA) or a nucleotide sequence substantially equivalent thereto is inserted into an appropriate expression vector, and this is inserted into an appropriate expression vector. By introducing (transforming) into a microorganism or an animal host cell and culturing the transformant, it can be produced more efficiently and in a larger amount than in the conventional cell culture.

Hereinafter, the present invention will be described in more detail by way of examples.

[0016]

【Example】

(1) Preparation of TRF mRNA As a TRF producing cell, T cell line 2.19 was used. This cell line is available from Sideras, P.L. (Sideras, Pe).
t. al. Eur. J. Immunol. , 15, 58
6-593, 1985).

First, Noma, Y. et al. Were obtained from 2.19 T cells activated with ConA (concanavalin A). (Nature, 319, 640-646 , 1986)
Poly (A) ⁺ RNA (mRNA fraction) was extracted according to the procedure described above, and poly (A) ⁺ RNA was extracted by a sucrose density gradient centrifugation (36,000 rpm, 15 hours) using a 5 to 22% concentration gradient.
(A) The ⁺ RNA extract was fractionated into 16 fractions. Next, an appropriate amount of each fraction was added to Xenopus (Xenop).
us) oocytes, and cultured at 20 ° C. for 36 hours.
CGFII activity was measured.

The measurement of the TRF activity was carried out by using a sample of the oocyte culture supernatant containing 1.5 × 10 ⁵ BCL ₁ cells.
After 2 days, the cells were cultured in 2 ml of RITC medium (serum free containing 5 mg / ml bovine serum albumin, 50 μM of 2-mercaptoethanol, and antibiotics).
Gronowics, E .; Et al., Protein A plaque assay [Eur. J. Immunol. 6, 588-59
0, 1976] to determine the number of IgM secretory producing cells. On the other hand, measurement of BCGFII activity
At the end of 6 hours 2 days of culture the proliferation degree of the BCL ₁ cells
³ H thymidine is added and taken up by the above BCL ₁ cells
^The measurement was performed by measuring the amount of ³ H thymidine.

As a result, as shown in FIG. 2, BCGFII activity (black bars in the figure) was observed in the mRNA fractions of 9S and 16S, and TRF activity (white bars in the figure) was observed in the mRNA fraction of 16S.
It was found only in the NA fraction. In the figure, P is TRF
T cell hybridoma B151K1, one of the producer strains
Culture supernatants of two strains showed a positive control was added to the BCL ₁ culture medium at a concentration of 50%, N is the, mRNA
Negative controls, in which a phosphate buffer was injected into oocytes instead of the A fraction and cultured, are shown.

The molecular size of 9S mRNA corresponds to that of interleukin (IL) -4 [Noma. Y. et. al. Nature, 319,
640～646,1986], and the 9S mRNA fraction is the BCL ₁ growth promotion activity, since the differentiation activity of the IgM secreting producing cells BCL ₁ cells is not observed, the present inventors have found that 2. 16S mRNA of 19T cells
It was determined that TRF mRNA was present in the fraction,
An attempt was made to create an NA library.

(2) Preparation of cDNA library and TR
Selection of FcDNA clones Total poly of 2.19 T cells activated with ConA
(A) The cDNA library of ⁺ RNA was prepared using the plasmid Noma, Y. [Nature, 319, 640-649 , 198].
6] to obtain about 5 × 10 ⁴ cDNA clones (pSP6K cDNA library).

Next, plasmid DN of these clones was used.
A mixture was digested with SacI and SalI, respectively, to linearize the plasmid, and then in vitro (in vitro).
In o), mRNA was synthesized using SP6 RNA polymerase. Xenopus oocytes (oocyt) were prepared from the mRNA solution thus prepared.
e) was injected and cultured at 20 ° C. for 36 hours, and the secreted product was collected in the culture solution, and TRF activity and BCGFII activity were measured by the measurement method described in Example (1). As a result, mR prepared from the DNA fragment digested with SalI
Both activities were observed in NA, but no activity was observed in mRNA from a DNA fragment digested with SacI.

Next, the mR of the 2.19 T cell line
The pSP6K cDNA library prepared from NA was divided into 18 pools (about 3,000 clones per pool), and the TRF activity and BCGFII activity of each were measured according to the method described above. As a result, only one pool (No. 17) showed both activities. Therefore, this pool was further divided into subgroups, and TRF was
And BCGFII activity were measured to obtain one group having positive activity consisting of 60 clones. Subsequently, it was confirmed that the DNA fragment (cDNA) inserted into the plasmid DNA of these clones had a SacI cleavage site and no SalI cleavage site in the nucleotide sequence of 1 Kb or more. Clone Nos. 16, 18, 23, 27, 42, 51 and 53) were obtained. The TRF activity of these 7 clones (Ig
It is shown in Table 1 for M plaque forming cell number) and BCGFII activity ⁽³ H-thymidine uptake). These 7
Among the clones, No. Since 23 clones showed the strongest activity, the plasmid of this clone was called pSP6K-m
Analysis after naming TRF23 (see FIG. 3) was performed.
E. coli HB transformed with this plasmid
101 / pSP6K-mTRF23 was obtained from Escheri
Chia coli SBM 285, which has been deposited with NIKKEI under the accession number of FERM P-8828.

[0024]

Table 1 Table 1 TRF and BCGFII activities of cell culture supernatant or mRNA-injected oocyte culture supernatant -------------------------- ------------------------------------ Cell culture supernatant or sample IgM plaque ³ H-thymidine oocyte Amount added to cells * Forming cells mRNA template to be taken up (%) / culture (cpm / culture) --------------------------- ----------------------------------- 1) Cell culture supernatant: B51K12 hybridoma 25 1834 7069 10 903 4222 2.19 T cell line 1.0 3134 13726 0.25 1716 6847 Control 0 211 1289 2) mRNA template: Sac I digested library 5 500 8396 Sal I digested library 5 799 14342 Phosphate buffer 5 563 6732 cDNA clone No. 16 5 199 1728 〃 18 5 233 1969 〃 23 5 3132 17970 〃 27 5 366 2001 〃 42 5 215 1650 〃 51 5 201 1862 〃 53 5 180 1883 Control (distilled water) 5 150 1654 pSP6K-mTRF23 1 3229 10824 〃 0.25 1662 6615 〃 0.06 772 3037 〃 0.015 232 1625 〃 0.008 213 149 7 ------------------------------------------------- ------------- * Indicates the ratio of the sample amount to the medium amount for activity measurement.

(3) Analysis of cDNA base sequence and TRF
The cDNA inserted into the polypeptide pSP6K-mTRF23 was once subcloned into the BamHI site of the pUC18 plasmid, and was subjected to the dideoxy method [Sanger, F. et al. et.
al. Proc. Natl. Acad. Sci. US
A, 74, 5463-5469 , 1977] and the unidirectional deletion method [unidire].
Cational deletion method, Yanisc
h-Perron, C.I. et al. , Gene 3
3, 103-119 , 1985]. FIG. 4 shows the strategy for base sequence determination. As a result, a sequence consisting of 1,533 base pairs excluding the polyA tail region was obtained as shown in FIG.

As a result of detailed analysis of this nucleotide sequence, 13
Three amino acid residues (base sequence from position 44 to position 442 in FIG. 1) and 62 amino acid residues (5 in FIG. 1)
(Opening frames 44 to 729), and (i) T
RF polypeptide (glycoprotein) is secreted and produced from the 2.19T cell line and the oocytes injected with the mRNA, and its monomer (analyzed by SDS polyacrylamide gel electrophoresis) has about 18,000 (Ii) a DNA obtained by cleaving the AccI cleavage site (the nucleotide sequence of 624 to 629 in FIG. 1) present in the open reading frame consisting of 62 amino acid residues with the enzyme and linearizing it; Is capable of producing TRF and BCGF in oocytes; (iii) the molecular weight of a polypeptide consisting of 62 amino acid residues (about 6,
500) is the estimated molecular weight of the TRF monomer of about 18,000.
However, even if a sugar chain is added (there is one N-glycosylation signal sequence in the sequence), the TRF or BCGFII activity initially produced in the 2.19T cell line is too small, etc. The resulting polypeptide (TRF polypeptide) was determined to consist of the 133 amino acid sequence shown in FIG. However, Met (methionine) at the N-terminus of this polypeptide is subjected to post-translational modification (post-translational modification).
Modification process)
Formylated, acetylated or Met
The removal of residues may be considered from the in vivo modification process of polypeptides reported so far.

At the N-terminus of the polypeptide comprising 133 amino acid residues shown in FIG. 1, a highly hydrophobic peptide comprising about 20 amino acid residues is present. This sequence (leader sequence) is unique to a secreted protein, and it is known that this sequence is removed when the protein is secreted from the cell. Generally, alanine (Al
a) and C-terminal of amino acid residues such as glycine (Gly) are often cleaved (Watson, ME et al.
E. FIG. , Nucl. Acid. Res. 12, 5145-
5164, 1984), and the leader sequence is usually about 20 peptides.
The polypeptide having RF or BCGFII activity is considered to be a molecule containing the polypeptide at the 19th, 21st, or 22nd to 133rd positions in the amino acid sequence shown in FIG. The molecular weight (approximately 12,300-12,700) of these polypeptides is almost consistent with the fact that the TRF monomer is a glycoprotein with a molecular weight of about 18,000.

The N-terminal amino acid of the polypeptide produced by removing the leader sequence as described above is further acetylated in vivo or, if it is glutamic acid (Glu), pyrrolated. This may be considered in view of the peptide structure that has been found in vivo.

(4) TRF activity of pSP6K-mTRF23 plasmid
MRNA transcribed by RNA polymerase is injected, ³ H to cultured TRF activity (BCL ₁ differentiation activity of the cells into IgM secreting producing cells) of culture supernatant of oocytes and BCGFII activity (BCL ₁ cells Thymidine incorporation) is shown in Table 1.

Further, Takatsu, K .; [J. I
mmunol. 125, 2646-2653 , 198
0], the effect of the above oocyte product (abbreviated as γTRF) on the anti-DNP-IgG antibody production response reaction was examined. First, BALB / c mice were transformed with DNP-keyhole limpet hemocyanin [Keyhole li
mpet hemocyanin; KLH]
Six to eight weeks later, spleen cells were collected, B cells were enriched by anti-Thy1.2 antibody and complement treatment, and γTRF anti-DN
The ability to promote P-IgG plaque forming cells was examined. As a result, γTRF can be used for antigen (DNP-ovalbumin: DNP
In the presence of -OA), DNP-sensitized B cells were stimulated and clearly showed an activity of differentiating into anti-DNP-IgG producing cells (Table 2). Although no further data is shown, γTRF
Is the I of B cell blasts activated in vivo.
It also showed gM synthesis-inducing activity. In addition, IL-1, IL-
2, No activity of IL-3 and BSF-1 was shown. From these results, pSP6K in the present invention
-It was confirmed that the cDNA cloned into mTRF23 (see FIG. 1) controls the production of TRF polypeptide.

[0031] Incidentally, pSP6K-mTRF23 clone IgM plaque forming cells promoting activity and BC of BCL ₁ cells
L ₁ that is the growth promoting activity always have (see Table 1)
This suggested that TRF and BCGFII were the same molecule.

[0032]

Table 2 Effect of γTRF on anti-DNP-IgG plaque formation --------------------------------- -------------------------- Anti-Thy1.2 antibody + complement γTRF amount Anti-DNPIgG plaque Antigen * Pretreatment (%) / Culture ------------------------------------------------ ------------None 0 127-DNP-KLH 0 1792 + None 0 127 + DNP-KLH 0 199 + DNP-OA 0 92 + DNP-OA 1695 + DNP-OA 0.5 884 + DNP-OA 0.25 555 + DNP-OA 0.125 382 ------------------------------------- ---------------------- * Indicates the ratio of γTRF added to the culture volume.

[Brief description of the drawings]

FIG. 1. cDN inserted into pSP6K-mTRF23
BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the base sequence of A and the amino acid sequence of a TRF polypeptide area | region.

FIG. 2 is a graph showing TRF activity (plaque-forming cells: PFC) and BCGFII activity (incorporation of ³ H thymidine) in the mRNA fraction.

FIG. 3 (A) is a schematic diagram of the plasmid of the pSP6K-mTRF23 clone, and FIG. 3 (B) is a schematic diagram of the periphery of the insert cDNA of (A).

FIG. 4. cDN inserted into pSP6K-mTRF23
FIG. 2 is a diagram showing a strategy for determining the nucleotide sequence of A.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 21/02 A61K 37/02 ADS // C07K 14/52 ADU (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (6)

[Claims] [Claim 1] Formula I: 1 ATG AGA AGG ATG CTT CTG CAC TTG AGT GTT 31 CTG ACT CTC AGC TGT GTC TGG GCC ACT GCC 61 ATG GAG ATT CCC ATG AGC ACA GTG GTG AAA 91 GAG ACC TTG ACA CAG CTG TCC GCT CAC CGA 121 GCT CTG TTG ACA AGC AAT GAG ACG ATG AGG 151 CTT CCT GTC CCT ACT CAT AAA AAT CAC CAG 181 CTA TGC ATT GGA GAA ATC TTT CAG GGG CTA 211 GAC ATA CTG AAG AAT CAA ACT GTC CGT GGG 241 GGT ACT GTG GAA ATG CTA TTC CAA AAC CTG 271 TCA TTA ATA AAG AAA TAC ATT GAC CGC CAA 301 AAA GAG AAG TGT GGC GAG GAG AGA CGG AGG 331 ACG AGG CAG TTC CTG GAT TAC CTG CAA GAG 361 TTC CTT GGT GTG ATG AGT ACA GAG TGG GCA 391 ATG GAA GGC STP (I) wherein STP represents a translation stop codon. Or a plasmid containing a DNA encoding a B cell differentiation activating factor having a base sequence represented by the following sequence or a base sequence in which the 5 'end thereof has been deleted up to C at position 54, C at position 60 or G at position 63: A method for producing a B cell differentiation activating factor having a polypeptide as a polypeptide component by culturing the introduced transformed cell. 2. The method of claim 1, wherein the factor produced also has BCGFII activity. 3. The method according to claim 1, wherein the plasmid is pSP6K-mTRF23.
the method of. 4. The following formula II: [In the formula, X represents H (hydrogen atom), fMet (formylmethionine), AcMet (acetylmethionine) or Met (methionine). From the N-terminal to the 18th Ala, up to the 20th Ala,
Alternatively, a method for producing a factor having B cell differentiation activity by culturing a transformed cell into which a plasmid containing a DNA encoding a polypeptide in which the amino acid residue up to Met No. 21 has been deleted has been introduced. 5. The method of claim 4, wherein the factor produced also has BCGF II activity. 6. The polypeptide of formula II wherein [In the formula, X represents H (hydrogen atom), fMet (formylmethionine), AcMet (acetylmethionine) or Met (methionine). From the N-terminal to the 18th Ala, up to the 20th Ala,
Or a polypeptide in which the amino acid residues up to the 21st Met have been deleted, or Formula III: Y-Glu-Ile-Pro-Met-Ser-Thr-Val-Val-Lys-Glu-Thr-Leu -Thr-Gln-Leu-Ser-Ala-His-Arg- Ala-Leu-Leu-Thr-Ser-Asn-Glu-Thr-Met-Arg- Leu-Pro-Val-Pro-Thr-His-Lys-Asn -His-Gln- Leu-Cys-Ile-Gly-Glu-Ile-Phe-Gln-Gly-Leu- Asp-Ile-Leu-Lys-Asn-Gln-Thr-Val-Arg-Gly-Gly-Thr-Val -Glu-Met-Leu-Phe-Gln-Asn-Leu- Ser-Leu-Ile-Lys-Lys-Tyr-Ile-Asp-Arg-Gln-Lys-Glu-Lys-Cys-Gly-Glu-Glu-Arg -Arg-Arg- Thr-Arg-Gln-Phe-Leu-Asp-Tyr-Leu-Gln-Glu-Phe-Leu-Gly-Val-Met-Ser-Thr-Glu-Trp-Ala-Met-Glu-Gly (III) wherein Y is H (hydrogen atom), Met or Thr-Ala-Met
With the proviso that the N-terminal amino acid may be acetylated, or the N-terminal Glu when Y is H may be pyrrolated. The method according to claim 4, which is a polypeptide represented by the formula: