JP2652534B2 - Recombinant inhibin - Google Patents

Description

Description: TECHNICAL FIELD The present invention particularly relates to the construction of cloning vectors containing a deoxyribonucleic acid (DNA) sequence encoding part or all of the hormone inhibin, host cells such as bacterial strains containing the vector, and It relates to a host cell, such as a bacterial strain that produces part or all of inhibin or a precursor thereof. The invention further relates to the production and use of the expression products of the vectors and strains, including natural and synthetic products, and the production and use of the expression products and fragments of the vectors.

BACKGROUND ART It was reported in 1932 that the gonads produce a nonsteroidal factor called inhibin, which is involved in the feedback regulation of pituitary function (Matsukla, DR
(1932) Science, 76 , 19-20 (McCullagh, DR (193
2) Scieuce 76 , 19-20)). Since that time, the anterior pituitary gland has been known to produce at least two gonadotropins, follicle-stimulating hormone or foritropin (FSH) and luteinizing hormone or lutropin (LH), which both regulate gonad development and function. ing. Sensitive radioimmunoassays make it possible to monitor each hormone accurately and individually, and also to clarify how the gonads regulate the feedback of these hormones. LH feedback control appears to be primarily through steroids, while FS
H's are mediated by proteins, glycoprotein factors and inhibin in addition to steroids.

Inhibin is currently defined as a protein or glycoprotein secreted from granulosa cells in the ovaries and Sertoli cells in the testis. It is secreted in the heart in response to FSH and acts on the pituitary as a feedback inhibitor of FSH synthesis and secretion, but has little involvement in basal synthesis and secretion of LH. It is not yet known whether inhibin, its mRNA or precursor is produced in other cells or tissues.

Since the early 1970s, various attempts have been made to purify inhibin from various testicular and ovarian gonads,
In various biological assays at that time, inhibition of FSH in pituitary cells in vivo and in vitro was presumed to be due to inhibin, and non-specific toxic effects of the subject were always checked. Instead of (Baker,
HWG et al. (1981) "Intragonadal Regulation of Repr
oduction (Franchimont, P and Channing, CP), Aca
demic Press, London, pp. 193-228; and Baker, HWG
(1982), Ann, New York Acad. Sci. 383 , 329-342),
The use of such a method was, to some extent, a contradictory result (de Jong, FH (1979) Mol.
Cell. Endocrinol, 13 , 1-10).

In recent years, inhibin from bovine follicular fluid (bFF) has been purified to homogeneity. (International patent application, PC
T / AU85 / 00119; and Robertson, DM et al. (1985) Bio
Chem. Biophys. Res. Commun. 126 , 220-226. ) This success has been attributed to a means of measuring the cytotoxic effects of analytes (Robertson, D.
M. et al. (1982) Mol, Cell, Endocrinol, 26 , 119-127).
(Endocrinsl, 107 , 1536-1542) of S. et al. (1980).
Nonspecific toxic effects that reduce the content were identified to be different from those of inhibin. The standard used was a bovine follicular fluid preparation having an inhibin activity of 20 U / ml with respect to the sheep testicular lymph standard giving any activity of 1 U / mg (Endo, Scott, RS et al. (1980)).
crinol, 107 , 1536-1542).

Inhibin from bFF was purified approximately 3,500 times and had a specific activity of 200,000 units / mg (protein), which was revealed by polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of sodium dodecylsulfonate. Allegedly, it is a 58 kD protein consisting of two disulfide-linked subunits A and B of about 43 kD and 15 kD, respectively. Each Sabuyunitsuto NH ₂ - amino acid sequence of terminal is determined. This purified substance was previously defined as a physiological property of inhibin, i.e., it inhibits the synthesis and release of endogenous FSH from rat pituitary cells, but
It has in vitro physiological properties that it is not involved in the synthesis and release of H, prolactin and thyroid stimulating hormone.

Since FSH is important in determining the incidence and rate of female ovulation and male spermatogenesis, administration of inhibin, its analogs or homologs, or antibodies to inhibin mainly suppresses or stimulates the gonads in humans and livestock animals. It could also be used as a diagnostic device for gonad function analysis. Many in vivo experiments have been performed using crude or partially fractionated gonad extracts or secretions to analyze the physiological effects of inhibin. In these experiments, the following were revealed as the effects of inhibin or antibodies to inhibin.

1. Suppression of gonad function. (Go of Moudgal, NR, etc. (1985)
nadal Proteins and Peptides and ttreir Biological
Signifieance (Sairam, MR and Atkinson, LE, ed.) Wo
rld Scientific Publishing Co., Singapore, pp. 21-37).

2. Increase in ovulation rate. (Proc, Au of O'Shea, T. et al. (1982)
St, Soc, Rep, Biol, 14 , 85; Proc, O'Shea, T, et al. (1983)
Aust, Soc, Rep, Biol, 15 , 22; and Henderson, KM et al. (19
84) J. Endocrinol. 102 , 305-309).

3. Promote the onset of puberty. (Al-Obaidi, FAR, etc. (1983
Proc, Aust, Soc, Rep, Biol, 15 , 80).

Developing these properties and / or other physiological studies in living animals on a market scale requires large amounts of purified natural or synthetic inhibin or its fractions or inhibin agonists and antagonists. . Obtaining such large quantities is not possible with current purification methods alone due to their limited sources (eg, from human follicular fluid); for example, extraction from 50 mm bFF requires only 5- Only 10 μg of purified product are obtained.

The invention is particularly directed to the use of E. coli (Escheric)
a host capable of synthesizing all or part or a precursor of this inhibin molecule, including its subunits, by identifying and identifying the gene encoding inhibin for introduction into a host such as H. coli. For example, by manipulating the cloned gene or a portion thereof to create a bacterial strain, one seeks to remove the aforementioned limitations.

DISCLOSURE OF THE INVENTION The present invention has the following constitution.

1. In the DNA encoding the bovine or human inhibin subunit, the DNA is the bovine prepro A subunit shown in FIG. 5 or the bovine pro B subunit shown in FIG. 20 or the human prepro A subunit shown in FIG. 7 or 21. A DNA comprising a DNA encoding each amino acid sequence of the human pro-B subunit shown in the figure.

2. The DNA of paragraph 1, which encodes a 43 kD subunit of inhibin.

3. The DNA of paragraph 1, which encodes a 15 kD subunit of inhibin.

4. Amino acid sequence of -60 to 166 of bovine prepro A subunit, amino acid sequence of bovine prepro A subunit -10 to 166, amino acid sequence of bovine prepro A subunit 1 to 166, -61 to 171 of human prepro A subunit
The amino acid sequence of -10 to 171 of the human prepro-A subunit, and the amino acid sequence of 1 to 171 of the human prepro-A subunit,
NA.

5. Bovine prepro A subunit described in Fig. 5 or 20
DNA encoding the bovine or human inhibin subunit, which encodes each amino acid sequence of the bovine pro-B subunit shown in the figure, the human prepro-A subunit shown in FIG. 7, or the human pro-B subunit shown in FIG. A recombinant DNA molecule comprising an insert DNA consisting of: and a vector DNA.

6. The recombinant DNA molecule according to the above item 5, wherein the vector DNA comprises a plasmid, virus or bacteriophage DNA.

7.pBR322, pUR290, pUR291, pUR292, pBTA286, pUC7, pUC8, pU
The recombinant D according to the above item 5, which comprises a plasmid selected from C9, pUC13, ptrpL1, pWT111, pWT121 or pWT131, and wherein the inserted DNA is inserted into the plasmid.
NA molecule.

8. The recombinant DNA molecule according to the above 5, 6, or 7, wherein the expression control sequence is effectively bound to the inserted DNA.

9. The recombinant DNA according to the above item 8, wherein the expression control sequence is selected from the E. coli β-galactosidase gene, the trp operon, the left promoter of bacteriophage λ or the long terminal repeat unit of Moloney leukemia virus.
molecule.

10. The recombinant DNA molecule according to the above item 8 or 9, wherein the expression control sequence comprises a promoter and a translation initiation signal.

11. The bovine prepro-A subunit described in FIG.
It encodes the bovine or human inhibin subunit characterized by encoding each amino acid sequence of the bovine pro-B subunit shown in FIG. 0, the human prepro-A subunit shown in FIG. 7, or the human pro-B subunit shown in FIG. A host transformed with a recombinant DNA molecule, which comprises an inserted DNA comprising DNA and a vector DNA.

12. The host of paragraph 11, wherein the host is selected from bacterial cells, bacteriophages, yeast, fungi, eukaryotic cells including vertebrates, and plant cells.

13. The bovine prepro A subunit or bovine pro B
13. The host according to the above item 11 or 12, wherein the host can express each amino acid sequence of the subunit, human prepro-A subunit or human proB subunit.

14. The host of paragraph 13, wherein the 43 kD or 15 kD subunit of inhibin can be expressed as a non-glycosylated form.

15. The host of clause 14, wherein the 43 kD or 15 kD subunit of the inhibin corresponds in sequence to a human or bovine inhibin.

16. Amino acid sequence of -60 to 166 of bovine prepro A subunit, amino acid sequence of bovine prepro A subunit -10 to 166, amino acid sequence of bovine prepro A subunit 1 to 166, -61 to 171 of human prepro A subunit
The amino acid sequence of human prepro A subunit -10 to 171; and the amino acid sequence of human prepro A subunit 1 to 171.
Or the host of clause 12.

The term "vertebrate" includes species of mammals including fish, amphibians, reptiles, birds and humans.

Inhibin Subunit Structure As described herein, inhibin secreted into follicular fluid is a 58 kD protein consisting of two subunits, a 43 kD and a 15 kD subunit (A and B, respectively). A, the 43 kD species, is a homologous protein between species that differs such that one inhibin species will act as an antigen when administered to another. Thus, it is believed that the heterologous inhibin can be used to raise anti-inhibin antibodies for the uses described below.

On the other hand, the B subunit, ie, the 15 kD subunit, is substantially identical in amino acid sequence between many species, for example, human and bovine species.

In some circumstances, the total protein in some cases be disconnected 31kD type consisting of two Sabuyunitsuto molecular weight 20kD and 15 kD (A _C and B, respectively). This 31 kD form has inhibin activity and is included within the scope of the present invention. Although polypeptide cleavage produces called A _N fragment by cutting the 58kD form to the 31kD form,
This itself is also included within the scope of the present invention since it has important effects on germline function.

BRIEF DESCRIPTION OF THE DRAWINGS As described above, the present invention covers a very wide range of fields, but preferred embodiments of the present invention will be described in more detail with reference to the following examples and the accompanying drawings.

FIG. 1 shows a method for synthesizing a recombinant plasmid containing a bovine granulosa cell cDNA.

FIG. 2 is a thin-layer chromatogram on polyethyleneimine cellulose of the product of the synthetic cDNA from bovine granulosa cell mensenger RNA.

FIG. 3 shows the sequences of the 43 kD (A) subunit and the 15 kD (B) subunit probe.

FIG. 4 shows a restriction enzyme (Pst I) map and a method for arranging seven inhibin cDNAs.

FIG. 5 shows the nucleotide sequence of bovine inhibin A subunit cDNA and its predicted amino acid sequence. The restriction sites chosen for this are shown above the DNA sequence.

FIG. 6 shows the nucleotide sequence of the bovine inhibin B subunit cDNA and its predicted amino acid sequence.

FIG. 7 shows the nucleotide sequence of human inhibin A subunit DNA and its predicted amino acid sequence.

FIG. 8 shows the nucleotide sequence of human inhibin B subunit DNA and its predicted amino acid sequence.

FIG. 9 shows the structure (A) of 58 kD inhibin (I) and 31 kD inhibin (II) as molecular weight standards (S is the size in kD) on SDS-PAGE gels; Results of conversion of native bovine inhibin of 58 kD to 31 kD form after overnight culture in castrated bovine serum (SS) and human postmenopausal serum (PMS) as determined by radioactivity measurements in sections (B) Is shown. In addition, PBS, bovine serum albumin, C
A represents carbonic acid dehydratase.

 FIG. 10 shows a method for obtaining a full-length A subunit cDNA.

FIG. 11 shows the linker used for expression of the A and B subunit cDNAs.

FIG. 12 shows a map of the plasmids pBTA302, pBTA303 and pBTA304.

FIG. 13 shows the expression of inhibin subunit fusion protein and β-galactosidase in E. coli strains.

FIG. 14 shows a method for constructing a full-length prepro-A subunit cDNA.

FIG. 15 shows serum FSH of rabbits immunized with a synthetic peptide.
Indicates a level. Arrows indicate the time of vaccine injection.

FIG. 16 shows the construction of a vector for expression of a bovine AN fragment.

FIG. 17 shows the structure of the synthetic prepro-B subunit gene.

FIG. 18 shows the structure of the synthetic prepro-B subunit gene of pBTA418.

FIG. 19 shows restriction enzymes of pBTA306 and pBTA307 (Pst I)
The map and base sequence method are shown.

Figure 20 shows the full-length beef Inhibin Pro B subunit cD
1 shows the nucleotide sequence of NA and its predicted amino acid sequence.

FIG. 21 shows the full-length human inhibin proB subunit coding DNA sequence and its predicted amino acid sequence.

FIG. 22 shows the sequence of the inhibin fusion protein encoded by plasmids pBTA303, pBTA308, pBTA309 and pBTA472. The bases produced by the synthetic linker are shown in lower case for each sequence.

BEST MODE FOR CARRYING OUT THE INVENTION The following abbreviations are used in the text.

ATP: Adenosine triphosphate bFF: Bovine follicular fluid bp: Base material cDNA: Complementary DNA CG: Villous gonadotropin Ci: Curie D: Dalton dATP: 2'-Deoxyadenosine triphosphate dCTP: 2'-Deoxycytidine trifos Phate dGTP: 2'-deoxyguanosine triphosphate DNA: deoxyribonucleic acid DTT: dithiothreitol dTTP: 2'-deoxythymidine triphosphate phosphate EDTA: ethylenediaminetetraacetic acid ELISA: ELISA (enzyme-linked immunosorbent assay) FSH: Foritropin or foam stimulating hormone XG: ~ twice the weight of the weight g: gram HPLC: high performance liquid chromatography k: (prefix) kg l: litre M: molarity m: (prefix) millimol: mol mRNA : Messenger RNA p: (prefix) pico n: (prefix) nano PAGE: polyacrylamide gel electrophoresis PCMB: parachloromercuribenzoic acid PMS: human menopause Post serum PMSF: phenylmethylsulfonyl fluoride PMSG: pregnant horse serum gonadotropin RIA: radioimmunoassay RNA: ribonucleic acid RP-HPLC: reverse phase HPLC SDS: sodium dodecyl sulfate SS: castrated bovine serum Tris: Tris (hydroxymethyl) Aminomethane μ: (prefix) micro In the text, the following terms are used interchangeably, regardless of their origin or state of glycosylation.

A subunit = 43 kD subunit = 58 kD large subunit of inhibin = amino acids His1 to Ile300 (Fig. 5)
= Amino acids His1 to Ile306 (FIG. 7).

From A _C Sabuyunitsuto = 20 kD Sabuyunitsuto = 31 kD inhibin large Sabuyunitsuto = amino acid Ser167 Ile300 (Fifth
(Figure) = amino acids Ser172 to Ile306 (Figure 7).

A _N fragment = NH ₂ -terminal part of A subunit = amino acid His
1 to Arg166 (FIG. 5) = amino acids His1 to Arg171 (FIG. 7).

B subunit = 15 kD subunit = 58 kD and 31 kD
Small subunit of inhibin = amino acids Gly1 to Ser116
(FIGS. 6 and 8).

"Having similar immunological activity" means a sufficiently homologous protein of inhibin or a part thereof,
(1) the immune system of the recipient bacterium has the same reactivity as the native protein, or (2) it can produce an antibody against the protein that can recognize endogenous inhibin when administered. It is meant to include proteins.

It should also be understood that the term "inhibin part" includes a subunit of inhibin.

The method and the product of the present invention will be described in detail below with reference to examples, but those skilled in the art can modify these, and the present invention naturally includes such modifications. Needless to say.

EXAMPLES Example 1 Isolation of Messenger RNA (mRNA) from Bovine Granulosa Cells Whole testis and whole ovaries or isolated Sertoli cells and granulosa cells can be used as a source of inhibin mRNA. In this case, isolated granulosa cells were used.

(A) Collection of Granulosa Cells Bovine follicular fluid (bFF) containing granulosa cells was collected from large follicles on the surface of fresh bovine ovaries at a local slaughterhouse using a needle and a syringe. The solution was stored on ice immediately after collection until transported to the laboratory. Cells from 70-100 ml of bFF were centrifuged at 500 × G for 5 minutes to remove the supernatant and purify the native inhibin. RNA was extracted from the cells as described below.

(B) Extraction and purification of RNA Immediately after collection, 24 ml of 4.2 M guanidium isothiocyanate / 120 mM mercaptoethanol / 5% (v /
v) Sacrosil / 10 mM Tris-HCl (pH 7.4) (Chirgwin,
JM et al. (1979) Biochemistry 18 ; 5294-5299) was added and dissolved. The lysate was homogenized in a Sorvall Omnimix at speed 4 for 30 seconds and then centrifuged at 20,000 × G for 15 minutes to remove cell debris. To the supernatant was added 1 g of CsCl per 2.5 ml. The mixture was placed on a 9 ml dish of 5.1 M CsCl / 10 mM EDTA / 50 mM Tris-HCl (pH 7.8) and centrifuged at 122,000 × G at 15 ° C. for 65 hours.

Transfer the RNA pellet to 5 ml of 70% ethanol / 30% TE (1 mM EDT
(A / 10 mM Tris-HCl, pH 7.5) several times to remove CsCl. Finally, add 0.3 ml of 3M sodium acetate (pH 7.5)
And 6 ml of ethanol were added to precipitate from 3 ml of TE, then frozen at -70 ° C and centrifuged at 20,000 × G for 10 minutes. The RNA pellet was redissolved in 1 ml of TE and stored at -70 ° C until further use.

(C) Isolation of mRNA Total RNA by oligo dT cellulose chromatography
MRNA was extracted from. (Aviv, H and Leader, P (1972
Proc, Natl, Acad, Sci, USA, 69; 1408-1412).
First, RNA was converted to 1M NaCl / 1mM EDTA / 20mM Tris-HCl (pH7.5)
And heated to 70 ° C. for 2 minutes, quenched in ice water and a column of oligo dT cellulose (BRL) (dry bed weight 0.5
g). The fluid from the fractions was heated, cooled and the operation was repeated twice. The column was washed sequentially with 5 ml of loading buffer followed by 5 ml of the same buffer plus 0.5 M NaCl. mRNA eluted in TE at 60 ° C. Fractions (0.25 ml) were collected, the mRNA content was precipitated using sodium acetate and ethanol (see above), and the pellet was dried in vacuo. The mRNA was redissolved in 0.1 ml of TE and an aliquot (25 μl) was frozen at −70 ° C.

All of the RNA fractions from these extraction and purification steps were combined and 1-2 μg samples were examined by electrophoresis on an agarose-urea gel. That is, 5M urea and 0.01 each
% (W / v) of the tracking dye bromophenol blue and xylene cyanol FF in 10 μl of RN in TE.
Heating the A sample for 2 minutes 70 ° C., and 5.6M urea / 14 mM iodoacetate salt _{/ 1mM EDTA / 36mM NaH 2 PO} 4 / 40mM tris -NaOH (p
H7.4) on a 1.5% agarose gel. The electrophoresis was completed when the bromophenol blue dye reached the bottom of the gel, and the RNA was visualized under ultraviolet light after coloring by adding a solution of ethidium bromide.

The RNA concentration was measured by its absorbance at 260 nm and
The μg / ml RNA had an A ₂₆₀ of 1.0 using an optical path length of 1 cm. Specifically, 60-100 ml of bFF yielded 1-3 g wet weight of pelleted granulosa cells, and
900 μg of total RNA and 20-60 μg of mRNA were extracted.

Although this mRNA fraction contains mRNA specific for inhibin, its subunits or inhibin-like polypeptides, this mRNA fraction consists of a large number of different mRNA species, most of which are unnecessary. It should be understood that this is a mixture.

During the subsequent cDNA synthesis and tail formation steps, each of these functions similarly to mRNA specific for inhibin, its subunits or inhibin-like polypeptides, and their presence ultimately results in the production of large numbers of unused clones. Requires a screening step to identify the desired clone, ie, the clone containing the cDNA sequence encoding all, part or subunit of inhibin.

The step of producing cDNA from mRNA and using the vector pBR3
The steps introduced in FIG. 22 are shown in FIG. 1 and described in more detail in Examples 2 and 3, which are merely illustrative of one example of the present invention.

Providing a synthesis (a) the reaction mixture was a lack of synthesis of dATP first 1cDNA strand copy DNA from Example 2 mRNA (cDNA), the cDNA synthesis by adding out with ^alpha-32 P dATP takes its small portion The situation was monitored. Excess dATP was then added back to the main reaction mixture to complete the first strand synthesis. That is, a sample of the mRNA mixture (2 μg) was made up to 27 μl with water, heated to 70 ° C. for 2 minutes, and quenched in ice water. 500ng oligo dT
_10-17 primers (Boehringer), each 50 nmol dCTP, dTTP
And dGTP, 5 nmol dATP, 100 nmol DTT, 20 units AMV
Reverse transcriptase (Life Science), 2 μmol KCl, 400 nmol
The reaction was started by adding 23.5 μl of a mixture consisting of 2.5 μmol of MgCl ₂ and 2.5 μmol of Tris-HCl (pH 8.5).

Immediately after mixing, 2 μl of the sample was added to 0.5 μl of α- ³² P dATP.
The analytical reaction was performed in a separate tube containing (1800 Ci / nmol; 5 μCi / μl) and 0.5 μl of 60 mM dATP was added back to the main reaction and kept at 42 ° C. for 60 minutes. 0.5 μl was taken out from the analysis reaction system and rapidly frozen in an ethanol-dry ice bath to obtain a 0-hour control sample.

Keep the rest of the reaction at 42 ° C for 60 minutes, then add 0.5 μl
Was subjected to analysis.

Each radioactive samples were subjected to thin-layer chromatography of 0.75 M KH ₂ PO ₄ buffer (pH 3.5) medium polyethyleneimine (PEI) cellulose (Merck). The results of the autoradiography are shown in FIG. Unbound nucleotides were thus removed from the newly synthesized first strand (DNA-RNA hybrid) remaining in the starting material. After autoradiography, each spot was cut out and its bound radioactivity was measured to assess its binding capacity. 10% of the total binding (estimated maximum binding 33%) or more was sufficient for second strand cDNA synthesis. The remainder of the radioactive material was precipitated twice from 2M ammonium acetate / 67% ethanol, boiled in loading buffer (see above) for 2 minutes and then subjected to agarose-urea gel electrophoresis to separate the RNA and DNA strands. separated. This gel is then X
-Attached to the autoradiograph of line film (Fuji RX type). This step determines the size of the cDNA product, which is typically from more than 1000 bases to 20
The range was 0 bases or less.

First strand synthesis can also be performed by other methods known to those skilled in the art. In this example, oligo dT was used as a primer to generate poly (A)
One cDNA strand was synthesized. Any primer can be used to synthesize cDNA from the mRNA species. Alternatively, specific synthesis of inhibin cDNA using a primer having a sequence complementary to the inhibin mRNA portion is also possible. Oligo dT or another primer having a sequence complementary to the portion of the inhibin mRNA can also be used as a primer for the synthesis of the first strand.

(B) Second strand synthesis In the same manner as the first cDNA strand synthesis, the main reaction and the analytical reaction were performed from the starting reaction mixture deficient in dATP.

That is, the cDNA-RNA hybrid molecule of the first strand main reaction system was added to 50 μl of 3M ammonium acetate (pH 7.
Precipitated by adding 5) and 200 μl of ethanol, cooled to −70 ° C. and centrifuged at 15,000 × G for 5 minutes. Re-suspend this pellet in 50 μl TE,
Redeposited, it was dried and finally taken up in 50 μl of TE.

This was combined with 16 nmol of dCTP, dTTP and dGTP, 1 nmol of dA
TP, 3.5 units RNAse H (BRL), 92 units DNA polymerase I (Boehringer), 40 μg BSA, 6 nmol β-NA
D, 400 nmol (NH ₄ ) SO ₄ , 4 μmol KCl, 200 nmol MgC
l Solution consisting of ₂ and 800 nmol of Tris-HCl (pH 7.5)
(Gubler, V. and Hoffman, BJ (1983) Gene 25 ; 26)
350 μl of 3-269) was added.

Immediately after mixing, 2 μl of the sample was added to 0.5 μl of α ³² PdATP (18
The analytical reaction is performed in a separate tube containing (00 Ci / nmol; 5 μCi / μl) and 2 μl of 10 mM dATP is added back to the main reaction, which is kept at 15 ° C. for 60 minutes and then at 22 ° C. for 60 minutes. Was. At 0, 1 and 2 hours, 0.5 μl samples were removed from the analytical reaction, snap frozen in an ethanol-dry ice bath and thin layer chromatographed as in the first strand synthesis (FIG. 2). Analyzed by graph e. As a result of the second strand synthesis, at least 5% of the total number of bonds (estimated maximum binding of 8%) was obtained.

Second strand synthesis can also be performed by other methods known to those skilled in the art. Examples thereof include the following.

In this example, an RNAase (eg, RNAase H) specific for RNA in the DNA / RNA hybrid was used together with the DNA polymerase.

Alternatively, the RNA in the DNA / RNA hybrids generated during first strand synthesis may be chemically or enzymatically degraded or D
DNA isolated from NA may be used as a self-primer. Next, second strand synthesis is performed using DNA polymerase or reverse transcriptase, and the remaining hairpin loop is replaced with a single strand specific R
Digest with a factor such as S1 nuclease.

A third method is to add an oligonucleotide (eg, oligo dC) tail to the 3 'end of the first strand DNA product after removal or degradation of the mRNA. A complementary oligonucleotide (eg, oligo dG) is annealed to the tail and used as a primer in a second strand reaction with DNA polymerase or reverse transcriptase. Single-stranded or double-stranded DN having a sequence complementary or identical or similar to inhibin cDNA
A can also be made by chemical synthesis of clones.

Example 3 Construction of a Bovine Granulosa Cell cDNA Library in E. coli (a) Tailing and Annealing cDNA Obtained by second strand synthesis by adding 40 μl of 3 M sodium acetate (pH 7.5) and 800 μl of ethanol. The resulting double-stranded cDNA was precipitated, cooled to -70 ° C and
Centrifuged at 0,000 × G for 5 minutes. 50 μl of this pellet
redissolved in 1 l of TE, extracted twice with an equal volume of phenol (pre-equilibrated with TE), extracted three times with 100 μl of diethyl ether (pre-equilibrated with TE) and then extracted
50 μl of 4 M ammonium acetate and 200 μl of ethanol were added for precipitation and cooling to -70 ° C. Apart from this precipitation procedure, the cold mixture was returned to room temperature and centrifuged at 15,000 × G for 5 minutes. This operation is for removing unbound nucleotides. The pellet was dissolved in 50 μl of TE and precipitated using ammonium acetate and ethanol. The pellet was vacuum dried and resuspended in 30 μl of TE.

15 μl cDNA to 30 nmol CoCl ₂ , 3 nmol DTT, 1.5 μmo
An equal volume of buffer containing 1 l potassium cacodylate (pH 7.2), 5 μg bovine serum albumin, 30 nmol dCTP and 17 units terminal transferase (BRL) was added.
The mixture was incubated at 37 ° C for 3-5 minutes, frozen in an ethanol-dry ice bath and then heated at 65 ° C for 5 minutes. The tail forming DNA was stored at -20 ° C until further use. Pst I
A method known as annealing into a commercially available pBR322 plasmid molecule (Figure 1; Table 2) having about 24 dG residues on the 3 'overhang of the restriction enzyme site (BRL Cat, No. 5355 SA). Thus, cDNA species were ligated. By this annealing treatment, the dC tail of the cDNA forms a stable hybrid with the dG tail of the plasmid. Since the PstI site is in the pBR322B-lactamase gene, the resulting plasmid is ampicillin sensitive but still resistant to tetracycline.

In the annealing operation, 2.5 μl of dC tailed cDNA
And about 0.5 μg of dG tailed pBR322 with 0.1 M NaCl-containing TE
Heated to 65 ° C. for 5 minutes in μl and then incubated at 57 ° C. for 2 hours. The solution is then slowly cooled to room temperature over 1 hour and stored on ice until next use or at -20 ° C.
And stored frozen.

Insertion of the cDNA species into the vector molecule can also be performed by other methods known to those skilled in the art. For example, nucleotides can be added to the 3 'end of a DNA molecule using an end-transferase reaction known as tailing. These molecules can then be annealed with vector DNA molecules tailed with complementary sequences. Alternatively, the cDNA molecule may have a synthetic linker added by DNA ligase that, when cut with restriction enzymes, produces a sequence convenient for cloning. Alternatively, the cDNA may be directly digested with a restriction enzyme or DNAase and then directly subjected to cloning. Blunt-ended DNA
The molecule may be cloned directly into a vector, such vectors including plasmids, cosmids, bacteriophages and other viruses.

(B) Transformation of E. coli ED8654 with the recombinant plasmid E. coli ED8654 (Table 2) was transformed by the method of D. Hanahan (1983,
(J. Mol, Biol, 160 , 557-580) to confer reactivity, and 0.2 ml of the reactive cells were transformed with 10 μl of the annealed cDNA-pBR322 hybrid. After transformation, cells SOC (of 5 g / l of enzyme extract / 20 g / l of tryptone / 10 mM NaCl / 2.5 mM of KCl / 20 mM of MgCl _2/20 mM of 2 ml MgSO ₄
/ 20mM glucose) for 2 hours, then 2,000x
G for 5 minutes, resuspend in 0.5 ml of 0.85% NaCl, and add 10 μg / ml tetracycline-HCl to select the whole suspension and cells transformed with the pBR322 recombinant plasmid. 280mm LB containing (5g for each
Enzyme extract / 10g tryptone / 5g NaCl) agar (1
15 g of agar per plate). Thus 48 at 37 ° C
Cultured for hours. By this method, this transformation typically yields about 5 × 10 ^{3 to} 5 × 10 ⁴ colonies per μg of recombinant plasmid pBR322, or about 10 ⁴ to 10 ⁵ colonies per μg of cDNA. become. or
More than 78% of the transformants contained the recombinant plasmid as determined by random screening of plasmids from 14 tetracycline resistant colonies.

(C) Preservation of transformed cells Colonies from each culture plate were scraped into 10 ml of LB.
Cells resulting from an estimated 2000 independent transformations were collected, centrifuged at 1,500 x G for 5 minutes and resuspended in 5 ml LB (v / v) with 0.5 ml DMSO. Part of this (0.4m
l) was frozen in liquid nitrogen and stored at -70 ° C until use. Thus, five such pools were created.

Since each protransformant contains one recombinant DNA species from one complete or partial copy of the mRNA species in the mRNA fraction, the aggregate or pool of these transformants is a library. Called. In this case, bovine granulosa cells
A library of cDNAs has been formed in E. coli. Since eukaryotic cells are capable of producing about 1 × 10 ⁴ to 2 × 10 ⁴ different proteins, an aggregate of 10 ⁴ independent transformants will be able to produce all of the larger number of mRNA species. It will include those derived from it. In fact, the vast majority of cells in these libraries contain cDNAs unnecessary for the present invention, and only a few contain complete or partial copies of the inhibin mRNA species. The following steps are performed to identify and isolate these colonies.

Example 4 Screening of Inhibin Clone Library (a) Preparation of Library for Screening A frozen cell tube was thawed, diluted 5 × 10 ⁵ times, and
0.5 ml containing 10 μg / ml tetracycline-HCl 28
It was spread on the surface of a 0 mm LB agar plate and cultured overnight at 37 ° C.
In this way, about 10 ⁴ colonies are formed per culture plate,
Each transformant in the original library increased 1-5 fold. Transfer the replica to a nitrocellulose filter (Schl
eicher-Schuell) and a stereotaxic mark was made by piercing a blackened needle through a filter into agar. The mother culture plate was stored at 4 ° C. until further use, while replica filters were added at 10 μg / ml tetracycline-HC.
and cultured on fresh LB agar plates containing l for 8 hours. This was then transcribed onto LB agar plates containing 50 μg / ml chloramphenicol, and cells 1 of the recombinant plasmid
Cultured overnight to increase copy number per individual (Clewell, DE (1972) J. Bacteriol, 110 ; 667-676;
Hanahan, D and Meselson, M (1980) Gene 10 , 63-6
7). Grunstein, M and Hogness, DS
(1975, Proc, Natl, Acad, Sci, USA 72 , 3961-39)
65).

The filter was placed on Whatman 3MM paper saturated in 0.5 M NaOH / 1.5 M NaCl for 15 min to promote cell lysis and formation of single-stranded DNA, then 1.5 M NaCl / 0.5 M The paper saturated in Tris-HCl (pH 7.5) was changed twice and neutralized for 15 minutes. Air-dry these,
Placed on a Bwanner funnel, then saturated with 20 ml of CHCl ₃ and aspirated. The filters were then baked in a vacuum oven at 80 ° C. for 2 hours.

(B) Method In order to identify a clone containing the inhibin cDNA gene or a part thereof, a radiolabeled oligodeoxyribonucleotide (oligonucleotide) probe was used. These were made to be complementary to the portion of the mRNA encoding the N-terminal amino acid sequence of each 43 kD and 15 kD subunit (probes 1 and 5, FIG. 3).
NH ₂ -terminal sequence of the 43kD and 15kD Sabuyunitsuto completely 58k
It is derived from the amino acids of the sequence of D-inhibin and its isolated subunits, the first 16 amino acids of each being described in international patent application PCT / AU85 / 00119. The amino acid sequence of the 43 kD subunit was then removed from the amino acid sequence of the complete 58 kD inhibin (see Example 6A, which was determined by the cDNA of the sequence in pBTA22 and pBTA23), as shown in Table 1. The original 15kD subunit analysis was improved and expanded. This improvement has revealed another area useful for making oligonucleotide probes, the area of amino acids, including 20-24. FIG. 3 shows the prepared oligonucleotide probe (probe 2). This is a 24-fold denatured 14-mer, pB
Used for isolation of TA293 and pBTA294. Since each amino acid (except methionine and tryptophan) has more than one DNA codon specific to it, it is preferred to include all possible combinations of codons in the probe. Thus, it encodes most of the amino acid sequence selected for the large subunit of inhibin
64 possible DNA sequences (probes 1, 3) and 24 possible DNA sequences (probes 2, 3) which encode the majority of the selected amino acid sequence for the small subunit of inhibin
Figure). To maximize the likelihood of obtaining a representative molecule of each of these sequences, have either dG and dA or dT and dA or dG or dG or dG or dT and dG at positions 2 and 6 from the 5 'end Collected from four independent composites. Probe 2 was synthesized in a single batch.

(C) Synthesis and purification of oligonucleotide The oligonucleotide probe was synthesized using an automatic DNA synthesizer (Model 380A, manufactured by Applied Biosystems Inc.). This device was first developed by Matteucci, MD and Caru
lhers, MH (1981, J. Amer, Che
m, Soc, 103, 3185-3191), an N, N'-diisopropylphosphoramidite deoxyribonucleotide derivative was input to the derivative-controlled porous glass support so as to be sequenced.

Applied Biosystems Inc. prescription method was used.
The modified oligonucleotide was purified by replacing the phosphoramidite deoxyribonucleotide with a single derived base. 20% (w / v) acrylamide / 1.0% (w / v) N, N'-
Gel containing bisacrylamide / 1 mM EDTA / 50 mM Tris-HCl (adjusted to pH 8.3 with solid boric acid) (20 cm × 20 cm ×
The oligonucleotide was purified from the oligonucleotide that stopped early by preparative electrophoresis in 1.5 mm). Electrophoresis was performed at 500 V for 1.5 hours, then the gel was visualized by placing it on Kieselgel F254 (Merek) thin layer chromatography so that it could be detected by UV light. The oligonucleotide shows a dark band. These were excised and eluted from the gel overnight in 0.8 ml of sterile water. Concentration of the eluted oligonucleotides were determined by measurement of absorbance at 260nm, which is because the A ₂₆₀ of using the optical path length of 1cm is 1.0, a single-stranded DNA of 35 [mu] g / ml concentration.

(D) 5 'end labeling reaction The purified oligonucleotide was made radioactive by adding ³² P-labeled phosphate to its 5' end.

That is, 40 pmol of oligonucleotide and 40 pmol of γ
^{- 32 P ATP (2000Ci / mmol} ; 5μCi / μl) Lyophilized was dissolved in the following and 4 units T ₄ polynucleotide kinase / 1
A buffer 20μl in containing 0mM of MgCl _2/10 mM of DTT / 1 mM spermidine / 50 mM Tris-HCl (pH 7.5) 90 min
The cells were cultured at 37 ° C.

The radioactive oligonucleotide was purified as described above and visualized by autoradiography on a Fuji RX type X-ray film for 5 minutes. Elution was overnight in 1.8 ml of sterile water. Each of the four oligonucleotide assemblies (see Example 1b), also including probe 1, was treated separately at this stage and then combined.

The 58KD inhibin was reduced, alkylated and the separated subunits were isolated by electroelution from a SDS-PAGE gel. 58 KD inhibin (80 pmol), A subunit (17 pmol) and B subunit (6 pmol) were subjected to Edman degradation in a continuous gas phase.

(Xaa) = amino acid determined from cDNA sequence.

Xaa ^* = amino acid identified by removing the A-subunit sequence from the double sequence of 58KD inhibin.

The regions used to make the oligonucleotide probe types are underlined.

(E) Use of probes for library screening Take each set of radioactive probes into 40 ml of 5 × SSC / 10 × Denhardt's solution and mix one representative filter of each library with gentle agitation. Dipped in this solution overnight. (Note that 20 × SSC is 3M NaCl / 0.3M trisodium citrate (pH 7.0), and the 50 × Denhardt solution is 1% (w
/ v) Ficoll / 1% (w / v) polyvinylpyrrolidone / 1%
(W / v) Bovine serum albumin. ) The filter was washed with 1xSSC / 0.1% SDS (sodium todecyl sulfate) several times at room temperature, and using Fuji RX type X-ray film and DuPont Cronex Hi Plus intensifying screen-
Autoradiography was performed at 70 ° C. for 3 to 5 hours. Develop the film and filter again with 1 × SSC / 0.1% SDS
Washed at 37 ° C. and autoradiographed overnight.

Areas that were stronger than the background after washing at room temperature or still visible after washing at 37 ° C. and corresponded to the location of the colonies on the mother culture plate were considered to be latent inhibin clones.

The above examples illustrate some of the means and methods for producing cells containing inhibin or inhibin-like DNA sequences and do not exclude modifications thereof. For example, in some cases, genomic DNA is used as the starting DNA and cDNA synthesis is not required (see Example 8). Also clone
It may be a cloning vector that expresses a DNA fragment and performs the screening procedure using anti-inhibin antiserum (see Example 10) or by directly detecting inhibin or inhibin-like biological activity.
Such methods are well-known to those skilled in the art.

Example 5 Identification of Putative Clones (a) Purification of Latent Inhibin Clones The area corresponding to the dark spot by autoradiogram was picked out, and a single colony streaked on an LB plate containing 10 μg / ml tetracycline-HCl. The cells were cultured at 37 ° C for 16 hours. Replicas of these culture plates are kept until next use.
Stored in ° C. The filter was placed on LB agar containing tetracycline-HCl for 3 to 6 hours, transferred to LB agar containing 50 μg / ml chloramphenicol for 16 hours, and then the colonies were decomposed as described above, and the radioactive oligonucleotide probe was again used. Was screened using From these filters, a single colony corresponding to the darkest spot on the autodiagram was picked and creased and analyzed.

(B) Restriction map of plasmid DNA The plasmid DNA content of at least one isolate of each putative inhibin was analyzed by extraction and digestion with the restriction enzyme PstI. This enzyme inserts cDNA from the plasmid
And further cleaves the cDNA at the internal Pst I site. Plasmid DNA extraction followed the method of Birnboim and Doly (1979, Nucl. Acids Res. 7; 1513-1523). Three
The cells cultured overnight in 1 ml of LB are centrifuged and 0.2 ml of digestion buffer (50 mM glucose / 10 mM EDTA / 0.2% lysozyme / 2
Resuspend in 5 mM Tris-HCl, pH 8.0) and add
Incubated for 0 minutes. 0.4 ml of alkaline SDS (0.2 M Na
OH / 1% (w / v) SDS) and after holding at 0 ° C. for 10 minutes, 0.3 ml of 3M sodium acetate (pH 4.8) was added. At 0 ° C
After 15 to 30 minutes, the precipitate formed was removed by centrifugation. Add 0.7 ml of isopropanol, cool to -70 ° C and centrifuge at 15,000 xG for 5 minutes.
Plasmid in 8 ml of the supernatant was precipitated. The pellet was redissolved in 0.4 ml of TE, then 40 μl of 3 M sodium acetate (pH 7.5) and 0.8 ml of ethanol were added, and the precipitate was precipitated by cooling and centrifuging as described above. . The finally obtained pellet was dried under vacuum and dissolved in 0.2 ml of TE. 40 μl (final volume) of TA buffer containing 10 μg / ml RNAase A (66 mM potassium acetate / 10 mM magnesium acetate / 0.5 mM DTT / 0.1 mg / ml BSA / 33 mM
Sample (20μ) in Tris-acetate (pH 7.9) at 37 ° C for 60 minutes
l) is digested with 20 units of Pst I (Boehringer) and then T
BE buffer (2.5 mM EDTA / 133 mM Tris-HCl / 89 mM boric acid)
In a polyacrylamide gel (acrylamide up to 10% / 0.27% bisacrylamide). Alu
A sample of pBR322 digested with I (Boehringer) was used as a standard. The clones initially identified are shown in Table 2.

(C) Sequencing method Suitable restriction enzyme fragments (one or more enzymes Pst
I, Pvu II, Sau 3AI or Sma I) from 0.5 M ammonium acetate / 10 mM magnesium acetate / 0.1 M EDTA / 0.1% S
Purify from polyacrylamide gels by eluting in DS at 37 ° C. overnight, then precipitate with ethanol, redissolve in TE, and sub-clone to obtain universal primers (17-mer; # 1211 or # 1211). 1212; New England B
dideoxy chain termination method (197, Proc, Natl, Acad, Sci, USA 74, 5463-546) using oligonucleotide primers complementary to the cDNA itself.
Bacteriophages M13mp8 and M13mp9 were used as replicating molecules for sequencing according to 7). The oligonucleotide primers are particularly useful for isolating overlapping cDNA species, for tensioning known sequences from overlapping cDNA species, or for sequencing from the dG / dC tail to the center of the cDNA.

Sequencing, subcloning and expression manipulations (Examples
Restriction enzymes were used in TA (see Example 5B) and T4 ligase was used according to its manufacturer's recipe for all of the above.

(A) Biotechnology Australia Pty. Ltd. Culture Coll
Section code name.

(B) “Size” (when noted) means Al
Represents the bp value estimated from electrophoresis on polyacrylamide gels against uI digested pBR322 standards.
This value differs slightly from the actual size obtained from the DNA sequence.

(C) Murray, NK et al. (1977), Mol. Gen. Genet. 150 ; 5.
3-61.

(D) Gene 2 ; 95-113 of Bolivar, F et al. (1977).

(E) BTA647 = E, coli K12, hsdR _k , supE44, SupF58, lacY
1, galT22, galK2, trpR55 / F'lacI ^q , Δ (lacZ) M15, lacY
⁺ A ⁺ , proA ⁺ B ⁺ , tra ⁺ .

(F) EMBO of Ruther, U and Muller-Hill, B, (1983)
J, 2 ; 1791-1794.

(G) BTA634 = E, coli K12Δ (lac, pro), supE (gln
V), thi, lon-1, zaj :: Tn5 / F′proA ⁺ B ⁺ , lacI ^q , Δ (lac
Z) M15, lacY ⁺ A ⁺ , traD36.

(H) pBTA286 is a derivative of pUR291 in which an HpaI-AvaI DNA fragment of 2355 base pairs has been removed from the coding sequence of the lacZ gene so that the coding sequence remains in frame.

(I) BTA652 = E, coli K12Δ (lac, pro), thi -, supE (g
lnV) 44, hsdPk17 endAl, gyrA96, relAl / F ', proA ⁺ B ⁺ , la
cI ^q , Δ (lacZ) M15, lacY ⁺ A ⁺ traD36.

(J) Gene 19 , 2 of Viera, J, and Messing, J, (1989)
59-268.

(K) BTA545 = E, coli ^{K12 thi -, Δ (lac,} argF), U169
Δ (lon) 100, hf1150, zje / ZJF :: Tn10, rpsL, hsdPk / F′la
cI ^q , Δ (lacZ) M15, lacY ⁺ A ⁺ , proA ⁺ B ⁺ , tra ⁺ .

(L) BTA637 = BTA652, lon-1, zaj :: Tn5.

[(M) Cepko et al (1984) Cell 37 , 1053-1062 Example 6 Inhibin clone (a) A (43 kD) subunit clone Two plasmids pBTA22 and pBTA23 encoding the 43-kD subunit were probed with 1 (Fig. 3). Sequence analysis of these cDNAs indicated that they were not long enough to isolate cDNAs extending from the 5 'position from pBTA22 (probe 3) and the 3' position from pBTA23 (probe 4). A sex oligonucleotide probe was made. pBTA290 was isolated using probe 3, while pBTA30 and pBTA295 were isolated using probe 4.

The method for sequencing the inserted cDNA from the PstI map and plasmid is shown in FIG. 4, and the composite nucleotide sequence encoding the A subunit is shown in FIG. This sequence is completely contained in the cDNA from plasmids pBTA290 and pBTA295. The 410 bp fragment of pBTA30 contains a stop codon followed by dG /
6 bases in the 3'-untranslated region before the dC tail. The CAT encoding the His1 of the A subunit is present in the 5'-PstI cDNA fragments of pBTA22, pBTA23 and pBTA290,
It is not present in either the 240 or 300 base pair Pst I cDNA fragment of A30. These two fragments, like the other clones, do not contain open reading frames or sequences and may be present in the unspliced portion of the intron.

The cDNA sequence contains an open reading frame from bases 1 to 1140. In this reading frame, the first ATG is at the 61st base (Met-60) and the amino acid sequence following Met-60 represents a signal peptide, but unlike many signal peptides, is hydrophobic leucine. No arginine or lysine before the string (Watson, MEE (1
Nucl, Ac. Res. 12 , 5145-5164). This signal peptide appears to terminate between Gly-44 and Gly-41, leaving a propeptide of about 40 amino acids before His-1. Thus, the A subunit of inhibin is first synthesized as a 38,810 dalton preproprotein extending from Met-60 to Ile-300.

The A-subunit is a arginine residue pair (-2, -1), which is a common signal for protein degradation of protein precursors.
Obtained from its precursor by cleavage at (Steine
Ann. NY Acad. Sci. 343 , 1-16, r, DF, etc. (1980). This A-subunit protein has -6, -5, 8, 9
And contains three additional pairs of arginine residues at positions 165 and 166. Cleavage at 165 and 166 residues of the A Sabuyunitsuto, of the same size, shown as in A _N and A _C Text 2
Pieces will be produced. A _C fragment consisting of 20-kDa Sabuyunitsuto of 31-kDa bovine inhibin (see Example 9). The A subunit appears to contain 11 cysteine residues, four of which are from His1 to Arg166.
It is in the A _N fragment. These four residues are internally bonded, are believed to allow the removal of A _N fragment from A _C Sabuyunitsuto. A _C Sabuyunitsuto is contained seven cysteine, one at least of which is believed to form a B Sabuyunitsuto and disulfide bonds. Subunit A is based on its recognition sequence Asn-X-Ser / Thr.
sn80 and Asn202 have two potential N-glycosylation sites (Wogh, PV and Bahl, OP (1981) CR).
C Crit, Rev, Biochem, 10 , 307-377), and Asn202 is A _C
Included in sub-units. The expected molecular weight of protein chains of Sabuyunitsuto A and A _C are 32,298 and 14,624,
This is about 25% less than that estimated by SDS-PAGE of native inhibin (43-kD and 20-kD, respectively). This appears to be due in part to the carbohydrate content in the original molecule whose molecular weight and molecular volume are both variable. These differences, both parts of A _N and A _C, but appear to be glycosylated at perhaps Asn80 and Asn202, is unknown whether there is O- linked glycosylation of serine and threonine residues . The carbohydrate contents of the glycoprotein hormones bovine LH, human CG and PMSG are said to be 16,29-31 and 45% by weight, respectively (Pierce, JG and Porson, T. et al.
F. (1981), Ann, Rev, Biochem, 50 ; 465-495). In the present invention, this is about 25% of the apparent molecular weight, so it is at the lower limit of the range.

The 3'-untranslated region in the mRNA of 42 bases can be predicted from the cDNA sequence. It contains a typical polyadenylation signal (AATAAA) 16 bases in front of the poly (A) tail (Nevins, J. et al.
R. (1983) Ann, Rev, Biochem, 52 , 441-466). The 3'-untranslated region differs from the coding sequence (66.3% G + C) and the 5'-untranslated region (71.6% G + C) in that A + T
(40.4% G + C).

(B) B (15 kD) subunit clones Five different clones were detected with probe 2 and two representative ones (cDNAs encoding mature B subunits from pBTA293 and pBTA294 are shown in FIG. 4. Three clones had the same restriction type as pBTA293, except that the 5'-PstI fragment contained a shorter cDNA.

The nucleotide sequence of the B subunit cDNA (Fig. 6)
Primitively derived from the pBTA293 and pBTA294 sequences, and using Probe 6 (FIG. 3B), cDNAs overlapping those of pBTA306 and pBTA307 (FIG. 19) were obtained. The B subunit is synthesized as a proprotein consisting of 425 amino acids (FIG. 20), and is the C-terminal of each proprotein as in the Ac subunit. It is separated from its previous section by five consecutive arginine residues. It contains two pairs of lysine residues at positions B, 14 and 102,103, but is unlikely to have a reactive site (Steiner, D .;
(F. et al., 1980). There are 9 cysteine residues, and odd numbers indicate that at least one is crosslinkable with the Ac subunit. From this cDNA sequence, the protein chain of the B subunit is expected to have a molecular weight of 12,977 daltons, which is close to the apparent molecular weight of 14,900 expected from SDS-PAGE of the intact B subunit. No mature N-glycosylation sequence is present in the mature B subunit, but Asn-145 in its proprotein is capable of glycosylation.

The length of the signal peptide is not known for certain, but Gl
y-282 is believed to be the last amino acid of the signal peptide. There are several possible proteolytic cleavage sites in proproteins that exhibit the bioactive ability of these peptides. The 3'-untranslated region of the B subunit is 94 nucleotides long and lacks the AATAAA polyadenylation signal like the cDNA encoding the A subunit. The last 12 bases of the 3'-untranslated region before the poly (A) tail are also AA
It matches well with the last 12 bases of erythropoietin 3'-untranslated cDNA (ACTGAAACCACC) having no TAAA sequence (Jacobs, K. et al. (1985) Nature, 313 , 806-810). B
The G + C content of the 5'-untranslated region of the subunit is 50%, while the G + C content of the prepro-B subunit coding region is 56.8% and as high as that of the 3'-untranslated region (55.3%). And this differs from the differences seen in the A-subunit coding sequence and the 3'-untranslated region.

Example 7 Identification of Bovine Inhibin-Like Sequences in Genomic DNA Sequences similar or homologous to the genes encoding the large (43 kD) and small (15 kD) subunits of bovine inhibin
Identified in human, sheep, pig, fish and avian genomic DNA.

This is mainly due to the Southern Blot standard cross method (Maniatis
T. et al. (1982) Molecular Cloning, Cold Spring Harb
our). That is, genomic DNA is digested with restriction enzymes, and the obtained fragments are separated by size by electrophoresis on an agarose gel. DNA was transferred onto nitrocellulose (or other may be one) membrane and then it encodes a substantial portion of the 43kD or 15kD bovine inhibin sub Units - ^[32 P] - examining labeled cDNA. As a result, genomic DNA similar or identical to the nucleotide sequence
Fragments are detected.

Specifically, it is as follows. Genomic DNA was generated from the human microphage cell line U937 (ATCC CRL 1539), or from human, porcine and avian blood, or from bovine and sheep soft tissue. For the preparation from soft tissues and human cell lines, the method described in the above-mentioned literature such as maniatis, T. was used. DNA was prepared from blood as follows. 10 to 15 ml of blood (0.625% citrate added)
The cells were diluted to 40 ml with a cold solution of M EDTA / 10 mM Tris-HCl (pH 7.5) (TE10-10) and placed on ice for 5 minutes to break down the blood cells. After centrifugation at 4000 x G for 5 minutes, pellet
Resuspended in ml of cold TE10-10 solution, diluted to 40 ml with TE10-10 and centrifuged at 3000 × G for 5 minutes. The pellet was broken up in 10 mM Tris-HCl (pH 7.5) / 5 mM EDTA (same volume as starting blood). 10% SDS was added to 0.5%, proteinase K was added to 50 μg / ml, and the mixture was incubated overnight at 37 ° C. with gentle shaking. 0.1
0.1 mM Tris-HCl containing% 8-hydroxyquinoline
Was added and mixed gently for 5-10 minutes at room temperature. CHCl ₃ : isoacyl alcohol (2
4: 1), mix for 5-10 minutes, then add 1 ml at room temperature.
Centrifuged at 8000 × G for 0 minutes. The aqueous phase was re-extracted twice more with 15 ml of CHCH ₃ : isoacyl alcohol. 1/20 times the amount
5M NaCl and 2 volumes of ethanol were added at room temperature and mixed. The DNA was removed with a Pasteur pipet, air dried for 30-60 seconds, and dissolved in 4 ml of TE by gentle agitation at 37 ° C. for 3 hours. The DNA was reprecipitated as above and redissolved in 3 ml of TE.

Samples of this DNA (usually 10 μg) were cultured for 16 hours using up to 500 units / ml of enzyme and cleaved with various restriction enzymes at a concentration of 40-50 μg / ml according to the manufacturer's instructions. . The product is extracted with an equal volume of phenol and then extracted three times with chloroform to give 2.5 volumes of 0.2M ethanol.
Protein removal was performed by precipitation in NaCl. Dissolve the DNA pellet in water and 11x in TAE buffer
Size fractionation was performed by electrophoresis (75 volts / 4 hours) on a 14 cm 1% agarose gel. Run this gel with 1.5M NaCl / 0.5M
By immersing in a solution of NaOH for 45 minutes and then neutralizing for 2 x 45 minutes in 3.0 M NaCl / 0.5 M Tris-HCl (pH 7)
The DNA was denatured. This DNA was transferred to a nitrocellulose membrane (Schleicher-Schuell) by capillary action, and fixed by heating at 80 ° C. for 2 hours in a vacuum. Filters were precrossed for 2-4 hours with a solution consisting of 50% formamide, 5 × SSPE (Maniatis et al., Supra), 5 × Denhardt, and 25 μg / ml sonicated herring sperm DNA.

 Hybridization probes are as follows.

(A) 790 bp from the A subunit cDNA gene of pBTA23
Sph I-Sma I fragment (FIG. 5).

(B) 110 obtained from bovine B subunit clone pBTA293
9 bp Sph I-EcoRI fragment (FIG. 4). This corresponds to the B subunit cDNA of bases 361-718 (FIG. 6) and pBR322.
It contains a 751 bp fragment from Pst I to the EcoRI site.

These fragments were isolated from low gelling temperature agarose gels after electrophoresis and then Feinberg, AP and Vogel
Stein, B method (1984, Anal, Biochem, 137, 266-267)
Labeled with α- ³² PdATP.

Approximately 30 ng of DNA is labeled to have a specific activity of 2 × 10 ⁸ to 10 ⁹ counts / min (CPM) per μg of DNA;
After heating to 00 ° C., the probe at 3-4 × 10 ⁶ CPM was sonicated with 1 × Denhardt / 50% formamide / 5 × SSPE / 1 × Denhardt / 10% dextran sulfate / 25 μg / ml. Mixed with a solution consisting of purified DNA. The hybridization of the filters was carried out at 42 ° C. for 20 hours. Filter SS twice at room temperature
Rinse with C and 0.1% SDS, then double each SSC / 0.1%
SDS then 1x SSC / 0.1% SDS then 0.2x SSC / 0.1%
Were sequentially washed at 50 ° C. for 30 minutes. Crosses were made under conditions such that at least 70-75% of the hybrid sequence was homologous to the bovine cDNA sequence as a whole. After exposure to Fuji RX type X-ray film for at least 24 hours,
The size could be determined on the basis of the marker DNA fragment whose size was known.

The approximate sizes (in chlorine pairs) of human, bovine, ovine, porcine and avian genomic DNA fragments strongly crossed with the A or B subunit gene probes are as follows:

DNA from various animal species studied was restricted to a limited number (usually one and up to three exceptionally by digestion with PstI) of a large or small bovine inhibin subunit cDNA probe. Has fragments. Large sub-unit cD
An approximately 480 bp Pst I fragment common to all species examined by NA was characteristic. These hybrid fragments are found to be present in genes of non-bovine species that are similar or homologous to the bovine inhibin gene. Inhibin cDNA sequence not contained in the probe selected and used in this example is also
This does not preclude hybridization with genomic cDNAs other than those in the fragments shown in Table 3.

In another experiment, A and B in RNA from rat ovaries were detected using bovine subunit cDNA as a probe.
Subunit precursor mRNA species were examined. These mRNA species
They were found to be present in large amounts in the ovaries of rats treated with PMSG, indicating that inhibin DNA is useful as a diagnostic probe.

Thus, cDNAs encoding bovine A and B inhibin subunits can be used to identify DNA and RNA encoding inhibin molecules in the genomes and organs of other species. For this reason, their encoded inhibin molecules are homologous to bovine inhibin and can be used as well.

Example 8 Isolation of human inhibin-like sequence from genomic DNA A human DNA sequence similar or homologous to the cDNA encoding the A and B subunits of bovine inhibin was isolated. This involves constructing a human genomic library of cloned DNA in bacteriophage lambda and then encoding the cDNA encoding either the A or B subunit of bovine inhibin.
SakuTsuta from NA ^[32 P] - by the examining the bacteriophage off Urge plaques with labeled DNA fragments connexion KoTsuta. The phage DNA of the positive plaques was isolated, cut with restriction enzymes, and the inhibin-like DNA-containing fragment (determined by Southern Blot hybridization using the probe and method of Example 7) in M13. Closing was performed to determine the nucleotide sequence.

Specifically, it is as follows. A human genomic library was constructed in bacteriophage λEMBL3 by the method of Maniatis et al. (Supra), or a similar human genomic library was constructed in λ and constructed in 47 (Loenen, W. et al., Supra).
AM and Brammar, WJ (1980) Gene, 20, 249-25
9). Human genomic DNA (made as in Example 7)
Partial digestion with u3A Ito produced DNA ranging in size from about 5 to 30 kbp in length. This was performed using a gradient of TE and 1 M NaCl (Cmix = 15%, Cr = 31.5, Vm = 34.45, aK = 2.147) in a Beckman SW50.1 rotary machine at 12.5 μg of DN.
A is centrifuged at 50,000 RPM for 2 hours, and sucrose gradient centrifugation (McCarty, KS et al. (1974) Anal, Biochem, 61, 165-
183).

About 15-25 kbp of DNA was collected by this gradient separation, ethanol was precipitated with 0.5 μg of tRNA as carrier and EMB
Ligated to L3λarms. EMBL3 DNA (Promega Biote
c, Madison WI, USA) digested with the enzymes BamHI and Eco RI, deproteinized with phenol and chloroform, and
Precipitated with 0.6 volumes of isopropanol at 0-4 ° C in the presence of 0.3M sodium acetate. The 2.4 [mu] g, 10 mM Tris _{-HCl (pH7.5), 10mM MgCl 2} , 1mM ATP, T 4 DNA ligase 20mM dithiothreitol and 2 units (Boehri
nger Mannheim) in a total volume of 40 μl of a solution of 15-25 kb.
p was mixed with about 1 μg of human DNA and incubated at 15 ° C. for 20 hours. 95 μl of Puka Gene (Packagene, Promega Biot)
ec) and cultured at 22 ° C. for 2 hours. This mixture was given to the special host NM539 (see the formulation of Promega Biotec),
The concentration of approximately 50,000 plaques per plate diameter of 14 cm. Eight slabs were made from the reaction mixture.

A human library in bacteriophage L47 was also fed to NM539 to obtain similar plaque concentrations.

The plaques were transferred to nitrocellulose filters (two replicas per plate) by the method of Maniatis et al. (1982, supra) for examination with an inhibin cDNA probe. Labeled probes are as follows. The sequence corresponding to the A subunit of bovine inhibin contains 79
The 0 bp SphI-SmaI fragment was used for the λL47 library and used to contain pBTA29 containing the A-subunit cDNA (see Table 2).
The Bam HI-Hind III fragment from 7 was used for the EMBL3 library. The probe for the small subunit was the Sph I-Eco RI fragment described in Example 7. Preparation, labeling and hybridization conditions are the same as for the Southern Blot hybridization method described in Example 7. For screening of the first plaque, both probes were used in one hybridization solution. Latent positive plaques were eluted, re-plated and probed until a pure clone was obtained. Two clones are particularly important, one of which is a .lambda.A ₂ crossbreeding and large (A) Sabuyunitsuto obtained from L47 library and one was obtained from EMBL ₃ library small (B) Sabuyunitsuto ΛB ₁ that crosses with This λA ₂ is about 11-12 kbp
Human DNA was inserted. Digestion with Pst I produces a number of fragments, of which the [ ³² P]-
Three hybrids with the labeled Sph I-Sma I fragment (described above) were found. Digestion with Pvu II produces two more fragments that hybridize with the same probe (Table 4).

Table 4 Enzyme DNA size (bp) hybridization degree Pst I 2500 + 〃 1160 ++ 480 480 ++ ++ Pvu II 800 ++ 〃 420 ++ 1160 and 480 bp fragments (by Pst I digestion) and 800 bp fragments (by Pvu II digestion) Are similar to the Pst I and Pvu II fragments hybridized with the same probe in the Southern Blot analysis of human genomic DNA shown in Example 7.

Same λA ₂ Sazanburotsuto and (Southern blot), pBTA2
From 95 ^[32 P] - According to支雑with Pst I fragment labeled 500 bp, only 1160bp fragment of the resulting digestion with Pst I the .lambda.A ₂ were identified. Such a hybridization pattern is consistent with the hypothesis that the cloned human DNA fragment has a sequence that is similar or homologous to bovine inhibin DNA.
The .lambda.A ₂ was digested with Pst I, then cut 480,1160 and 2500bp fragments from low gelling temperature agarose gel, M13
The resulting sequence subcloned in and sequenced for its nucleotide sequence (see Example 5c) is shown in FIG. The DNA sequence of the 1.4-1.5 kbp intron between bases 275 and 276 is omitted. Since the sequence of the derived amino acid sequence and bovine inhibin play pro A Sabuyunitsuto are similar, it found to be homologous proteins, and which is a precursor of human inhibin A and A _C Sabuyunitsuto. A _N fragment of human inhibin are amino acids Arg171 from His1, A _C Sabuyunitsuto are amino acids Ile306 from Ser172.

.lambda.B ₁ clone has an insert DNA of about 21～26Kbp.
The DNA was similarly cut with the restriction enzyme PstI, size-fractionated by electrophoresis on a 1% agarose gel and transferred to a nitrocellulose filter. 136 bp purified from pBTA293
Alternatively, according to the hybridization with the 510 bp inhibin Pst I fragment ([ ³² P] -labeled) (FIG. 4), the smaller probe binds to a fragment of DNA of about 1300 bp (range 1100-1350 bp). The larger probe is approximately 800 bp in length (740-840
bp range). The nucleotide sequence of the portion of the fragment of 800bp was digested with .lambda.B ₁ with Pst I, about 800bp Ps
The tI fragment was purified on a low-gelation temperature agarose gel and
It was determined by cloning into the PstI site of mp9 and then determining the nucleotide sequence. The smaller fragment of the 800 bp fragment (PstI-Sau3AI; Sau3AI-Sau3AI) for sequencing and the nucleotide sequence resulting from subcloning into M13 are shown in FIG. According to the amino acid sequence determined by translation of the DNA sequence, this DNA encodes all of the B chain of inhibin and Gly1 to Ser1
Sixteen human B chains are identical to bovine chains. The active coding sequence of the human proB subunit determined from these lambda clones is shown in FIG. This coding sequence is 388
Disturbed by an intro between bases.

It can be seen that fragments of these lambda clones having the human inhibin DNA sequence, as well as similar synthetic fragments, can be inserted into eukaryotic or prokaryotic expression systems to produce human inhibin or polypeptides whose structure is related to inhibin. (See Example 10). The λ clone itself can also be transfected into an appropriate eukaryotic cell line and expressed.

Bovine cDNA was used as a probe to detect inhibin subunit genes in various vertebrate species (Example 7) and then to clone human inhibin subunit genes to reveal extensive similarities in amino acid sequences. The other vertebrate inhibin is homologous or otherwise identical to the bovine inhibin protein shown in International Patent Application PCT / AU85 / 00119 and FIGS. 5 and 6 It becomes clear.

We have detected inhibin activity in follicular fluid and ovarian extracts from a number of vertebrate species (eg, see Table 5),
Purification of native inhibin from human FF and sheep FF by the method of International Patent Application PCT / AU85 / 0019 and Example 9, and subunit structure similar in both species to bovine inhibin and functionally similar to bovine inhibin It was found to have 58 kD and 31 kD types.

Rabbit, bovine and human inhibin are also neutralized by rabbit antiserum to purified 58 kD bovine inhibin, described in International Patent Application PCT / AU85 / 00119, and cross-react in RIA to re-close tight structural homology. (Table 5).

However, the fact that native bovine inhibin is capable of eliciting an immune response in rabbits means that there are differences between rabbit and bovine inhibin that are recognized as foreign by the rabbit immune system. Thus, inhibin from one species can be used as an antigen in another species and antibodies are generated that recognize endogenous inhibin.

Even though all of the species shown in Table 5 are active in rat pituitary cell cultures that show structural homology to interact with the pituitary cell receptor, rabbit-generated antibodies will No significant cross-reactivity with sheep or rat-inhibin, which shows the specificity of the antiserum.

Since B-subunits are sufficiently present in most species and are likely to be identical, mutant B-subunit proteins are used as antigens to elicit antibodies that cross-react with endogenous inhibin B-subunits. It will be clear from the foregoing description that the use will be widely possible and effective in many species. Such antibodies may be used for in vitro mutagenesis of DNA, expression of new genes as described in Example 10, or chemical denaturation of the B subunit protein, or similar but not identical to those described in FIGS. Isolation of the B-subunit gene from a species having a different B-subunit, and Example 10
It can be produced by such means as comprising expression of this gene in the manner described above.

Example 9 Relationship between the 58 kD and 31 kD forms of inhibin Inhibin is produced by granulosa cells and secreted into follicular fluid. As already reported (international patent application PCT / AU / 85/00119), it initially exists as a 58 kD type.

Studies aimed at improving the efficiency of purifying inhibin from bovine follicular fluid have revealed another type of small inhibin. Robertson, DM, etc. (1985
Using the purification method of Biochem, Biophys, Research Commun, 126, 220-226; International Patent Application PCT / AU85 / 00119).
The precipitation step at 4.75 was performed during the first neutral and acidic buffer gel filtration chromatographic steps. As a result, it was clarified that a second biologically active species was produced, which was isolated from the first product of the acidic gel filtration step. After subjecting this second species to reverse phase HPLC and preparative polyacrylamide gel electrophoresis, the apparent molecular weights were 20,000 and 15,000.
Consists of two subunits with an apparent molecular weight of 31,000
It was found that a protein having the same biological activity as the original 58 kD type was produced. Since the apparent molecular weights of the smaller subunits of these two inhibin species are very similar (about 15 kD), the difference in apparent molecular weight between the two inhibin species is that the 43 kD subunit has been reduced to about 20 kD. It seems to be mainly due to

Also, pure 58 kD inhibin changes to the 31 kD form during culture in castrated bovine serum (SS) or human postmenopausal serum (PMS). This degradation does not occur during a similar culture on bFF (FIG. 9). Therefore, the smaller (31 kD) form of inhibin,
In particular, the larger (20 kD) subunit is described in International Patent Application PCT / AU85 / 00119 and Robertson, DM et al. (1985)
"Biochem. Biophys, Res, Commum, 126, 220-226", which is a direct derivative of the 58 kD form of inhibin, and is believed to be inducible from the subunit genes described herein.

Serum free from natural inhibin (SS and PM
It is clear that S) can be used to treat recombinant inhibin or its subunits, and that certain enzymes can be purified from serum or the cell type in which this reaction is performed and used for similar purposes. is there. This enzymatic reaction is characteristic for protease inhibitors. SS or PMS
The serum was incubated with iodized 58 kD inhibin for 17 hours at 30 ° C. in the presence of 3% parachloromercuribenzoate or 10 mM EDTA, changing the 58 kD to 31 kD inhibin by 24%. To 4.5%. Bacitracin and pepstatin A (0.3 mM each) had no effect. This pattern of protease inhibition is a feature of the acting enzyme (Lazure, C. et al. (1983) Cau. J. Biochem, Cell
Biol, 61 , 501-515), which is consistent with the finding that there is a standard reaction site (Arg-Arg) before Ser167 in the A-subunit used in this reaction (FIG. 5). .

Inhibin degradation upon release into serum results in two molecules,
That 31kD type and A _N fragment of inhibin (large Sabuyunitsuto acids 1-166) is generated. This 31 kD form is known to suppress the synthesis and release of FSH by pituitary cells, while one or both molecules can return to the gonad and directly regulate gonad function,
Or inhibin itself or inhibin fragments or their precursor molecules, for example, A _N may also to have different biological functions other than pituitary adjustment FSH. These are rabbits 69
The fact that FSH titer suddenly dropped to zero by immunizing 9 with peptide I is evidenced (see Example 15).

Both subunits of inhibin are produced as prepromolecules, and the propeptides from each subunit can have their own biological activities, including cell growth and suppression. Inhibin's own 31 kD form is defined by transforming growth factor-β (Derynck, R. et al. (1985) Nature 316, 701-70).
It shows high homology to 5), suggesting that it has an action of native molecules on cell regulation in addition to the already known action on FSH synthesis by pituitary cells.

Example 10 Expression of Inhibin cDNA (a) 43 kD and 20 kD subunits Clone BTA405 (Table 2) contains a cDNA encoding a portion of the large subunit of inhibin, which cannot be expected to produce an inhibin portion. This is because the cDNA has a dC tail (see Example 3a) and there is no ribose binding site or initiation ATG (f-Met) codon in its sequence. To express a portion of the inhibin gene, a fusion peptide was made by cloning the largest (about 480 bp) PstI fragment of the cDNA in pBTA23 into the PstI site of pUR292. Placing it in a stereotactic form produces a protein consisting of the majority of E, coli protein β-galactosidase followed by the 27-183 amino acids of the 43 kD subunit of inhibin. Plasmid construction was transformed into E. coli BTA647 (Table 2).

An immunological screening method was used to identify these clones. After transformation, cells were placed on LB agar plates containing 50 μg / ml sodium ampicillin at 37 ° C. for 16 hours to select for transformants. Colonies were transferred onto nitrocellulose membranes (Schleicher-Schuell) and stereotactically marked by penetrating a black ink needle from agar into the membrane. The mother culture plate was then stored at 4 ° C. until use, while the L-Plicafilter was cultured on a fresh LB agar plate containing 50 μg / ml sodium ampicillin for 3 hours. 0.5 mM isopropyl β-D-
The fusion protein was induced on LB agar containing thiogalactoside (IPTG) at 37 ° C. for 2 hours. 0.2M NaOH /
Watman 3MM saturated with 1% (w / v) SDS (Whatman 3M
M) Place on paper sheet for 5 minutes, then 0.5M Tris-HCl
Neutralized on paper saturated with (pH 7.5). The proteins released from the colonies bind to the nitrocellulose in situ. Filter with an additional 0.5% (v / v) Tween 20
TST (150 mM NaCl / 0.05) containing (Tween 20, Signa)
% (V / v) Tween 20/10 mM Tris-HCl (pH 8.0) for 1 hour with gentle agitation,
The protein binding sites remaining on the nitrocellulose were blocked. The filter is treated overnight at room temperature with a solution containing rabbit anti-bovine 58 kD inhibin antiserum (international patent application PCT / AU85 / 00019), diluted 1:50 in TST, and then the remaining antibodies are removed. For several times in TST. The filter was replaced with porcine anti-rabbit immunoglobulin-horseradish peroxidase conjugate compound (Dakopatts) by TST.
For 1 hour at room temperature, then washed in TST. Finally, these were added to 20 mM Tris-H.
Cl (pH 7.5) and washed in and 0.5 mg / ml of 4-chloro-naphthol /0.03Pasento(v/v) of H ₂ O ₂ / 20mM Tris-HCl (pH
7.5).

Colonies containing the fusion peptide were identified on the filter from areas that were more intensely colored than areas where the control strain BTA647 (pUR292) grew. A lawn of induced E. coli BTA647 (pUR292) was then grown up and the anti-E. Coli antibody titer of the antisera was reduced by pre-culturing a dilution of the rabbit anti-inhibin antiserum on a degraded filter. Despite attempts to reduce it, there were some backgrounds where another colony was colored.

Three such colonies were purified and their plasmid contents were analyzed. All contain pUR292 with the expected cDNA fragment in orientation and one of these strains was identified as BTA410.
And the plasmid was designated pBTA28 (Table 2). The fusion peptide was identified by the Western Blot method (Towbin, H. et al. (1979), in which the protein was denatured with sodium dodecyl sulfate, separated by size on polyacrylamide gel electrophoresis, and then transferred laterally onto a nitrocellulose sheet. Proc, Natl, Acad, Sci, USA, 76, 4350-435
4) went by. The nitrocellulose is then processed as described above to determine which protein bands contain the inhibin antigen determination group. A protein sample was prepared as follows.

BTA647 (pUR292) and BT cultured overnight in LB at 37 ° C
Dilute the A410 culture 1: 100 in fresh medium and adjust the turbidity to A.
Cultures were grown to ₆₀₀ = 0.4, then brought to 0.5 mM in IPTG and cultured for a further 2 hours before harvesting. The cell pellet was added to 0.1 volume of loading buffer (30% (w / v) sucrose / 1%
(W / v) electrophoresis buffer containing SDS / 0.1 M β-mercaptoethanol / 0.01% bromophenol blue) and then heated in a boiling water bath for 5 minutes. A sample (10-30 μl) was subjected to polyacrylamide gel (Laemmli, U.S.A.).
K. (1970) Nature, 227, 680-685; Mattick, JS et al. (1
980), Eur, J, Biochem, 114, 643-651) and electrophoresis was performed until the bromophenol blue dye reached the bottom of the gel. Lateral transfer was performed at 1 amp in 15.6 mM Tris-base / 120 mM glycine for 1 hour using a Hoeffer Transblot apparatus. Blocking and treatment of the filter with the antiserum is as described above.
To directly visualize proteins in polyacrylamide gels, gels were decolorized (10% (v / v) methanol / 5% (v
/ v) immersion in glacial acetic acid) for 30 minutes, then finished to the same solution
0.5% (w / v) Coomassie Brilliant Blue (Coomassi
e Brilliant Blue) R250 for 1 hour. The gel is decolorized by exchanging the decolorizing solution several times while gently stirring.
And the protein bands were compared with those detected by Western Blot and immunodetection. At least 2 strains BTA410
Contains about 5-10% of the total cell protein, these are induced by IPTG and detected by anti-inhibin antiserum (FIG. 13, track 10), and It became clear that these were not present in the strain BTA647 (pUR292) (FIG. 13, track 11). These are larger (about 130 and 117 kD) than native β-galactosidase (116 kD) and are, therefore, β-galactosidase-inhibin fusion proteins. The larger of this fusion protein is the one expected from DNA analysis (about 132kD)
Is comparable to

In a similar manner, the 410 bp PstI fragment of pBTA30 was expressed in pUR291. The resulting plasmid was designated as pBTA292 (Table 2), and again, the fusion protein was anti-inhibin antiserum (13th
It could be detected using the figure, track 9).

PBTA290 and pBT to express complete subunits
The cDNA from A30 was spliced and the method is shown in FIG. Briefly, Sau3AI fragments were first purified from each clone, cut at a unique RsaI site and ligated. The ligated product mixture is cut with Sau 3AI and the product is pUR29
Ligated during 0. Then, 5'-Sau 3AI- of pBTA290
To identify clones containing both the Rsa I fragment and the 3'-Rsa I-Sau 3AI fragment of pBTA30, clone and probe 3
And 4 (FIG. 3). pUR290 is Asp-10
Gives the correct reading frame for Ile-300, thus ensuring production of the β-galactosidase-inhibin fusion protein when the insertion is stereotactic. 480 and 410bp Pst I
Correct expression at the site can be separately checked by using specific antibodies isolated from a column containing the fusion product of pBTA28 and pBTA292 (see Example 9). The resulting plasmid was named pBTA296 (Table 2). This complete A subunit gene was also transferred to Sau 3A
It was cloned as an I-Sau 3AI fragment in the Bam HI site of pUC7, and the resulting plasmid (pBTA302: FIG.
BTA426 (ATCC67057) was obtained. The sequence Asp-Pro at the Sau 3AI site, because it is variable in formic acid,
It can be used to cleave the inhibin portion from upstream of the fusion protein (Nilsson, B, et al. (1985)
Nucl, Ac. Res, 13, 1151-1162). Expression of the full-length (amino acids Asp-10 to Ile-300) of the 43 kD subunit fused to β-galactosidase from strain BTA419 is shown in FIG.
It was shown to. In addition to the full-length β-galactosidase-inhibin protein, there are, in addition, at least three other bands of smaller size, which are approximately the same as the native β-galactosidase, probably the protein of the full-length product. It may be due to white matter degradation or premature termination of transcription or translation. The mass of the fusion protein is soluble. Inhibin DNA and flanking β-galactosidase DNA were excised from pBTA296 as EcoRI digestion fragments, and pBTA286
(Table 2, FIG. 13, track 4) to generate pBTA297 (Table 2) by insertion into pBTA286 and clones expressing inhibin DNA at the C-terminus of the β-galactosidase protein shortened from pBTA286 were cloned as described above. Selection was made by immunocrossing. Examination of such clones by phase contrast microscopy (eg, BTA420; Table 2) showed that they produced a fusion protein as an insoluble product called inclusion bodies. This inclusion body has a function of preventing protein degradation in cells and greatly simplifies purification for antigen production (Examples 12 and 13). Stock BTA4
20 was designated as ATCC67054, and the protein from the purified inclusion body of BTA420 is shown in track 3 of FIG. The expression of the A-subunit in BTA426 is shown in FIG. 13 and this protein is produced specifically as inclusion bodies. Unlike the fusion protein in BTA420, the longest fusion protein in BTA426 is Western
It is the most prominent and most reactive in Blot analysis and thus proves to be a particularly good host-vector system for the production of inhibin-like proteins.

From the foregoing, it is clear that other restriction enzyme sites are also useful for expression of the A-subunit. The first is a unique Sph I site linking codon His1, and an oligonucleotide linker, the A Sabuyunitsuto or A _N fragment of a variety of hosts and vectors as a fusion product, or a new f-Met codon CAG encoding His1
Or the signal peptide can be expressed so that it can be expressed as a non-fusion protein having a structure similar to that given before His1. Such a structure is well known to those skilled in the art, and one example is shown in FIG.

This expression linker uses a Cla I site to create a trp promoter plasmid such as ptrpLI (Edman, JC et al. (1981).
Year) after insertion into Nature 291 , 503-506)
Or to express a 43kD protein or A _N fragment via terminal methionine, or, beta-galactosidase by insertion of using Bam HI to pUC7, pUC13 or in pUR290 - as inhibin fusion protein, or, pWT121 (Talcon, W.
(1980) Molec, Gem, Genet, 177 , 427-438), which is made to be capable of expression as a trpE-inhibin fusion protein after insertion.

How to build a clone expressing A _N fragment shown in Figure 17. Briefly, pBTA297 at Bam HI and Sph I
Was digested, and then the linker described in 1. of FIG. 11A was inserted. As shown in FIG. 16, the full-length A subunit gene was isolated as a Sau 3AI fragment and partially digested with Hae II. Stopper linker containing a double-headed nonsense codon
The reaction product added to the Hde II site was cut with Bam HI, and the obtained fragment was inserted into pUC13.

The recombinant plasmid contains the oligonucleotide probe TCCTGG, which spans the Hae II site encoding amino acids 128/130.
Screen using CGCTCCTGC to confirm that the small Hae II fragment is in the correct orientation. Take a positive clone and purify it. The inserts were then sequenced to fix them. From each of the two clones, one contained the correct insert and the other contained a different source. This correct insert is purified, recloned into pUC13, and then introduced into E. coli, providing BTA1365 for primary expression. The three restriction sites at both ends of the molecule allow subcloning into the other vectors described above.

The recombinant protein produced by BTA1365 is excreted as inclusion bodies and has an apparent MW of 23.5 kDaltons and BTA42
6 and similar to the fusion protein from BTA427 (FIG. 13),
As expected, it is detected by Western blot with serum 474.

FIG. 14 shows a method for constructing a full-length prepro-A subunit gene. This method is basically based on pVTA295 from Pvu II-
Isolating the Pvu II fragment and then adding the linker (unphosphorylated) shown in FIG. 11 (Aii), whereby the first 4 '
Two amino acids (Met-60 to Gln-57) will be reconstructed at the first Pvu II site to provide a Bam HI site at the 5'-end. The linker binding molecule is cut with Sph I and ligated into pBTA297 cut with Bam HI and Sph I to give pBTA395.
This intermediate expresses the prepro-A subunit as a fusion protein. Inhibin DNA fragment was converted to Hind III-Hind III
Cut out as fragments or as EcoRI-EcoRI fragments and use in the next step. In particular, this EcoRI-EcoRI fragment is subcloned in pUC9. Its inhibin cDNA sequence is Ba
m HI-Bam-HI fragment, excised from the vector pZIP NeoS
V (X) 1 (Copko, CL et al. (1984) Cell, 37 , 1053-10)
Recloned in 62) to provide pBTA417.

Thus, the 5 'Pvu II, Sau 3AI and Sph I sites are all convenient linker sites for the expression of the 43 kD protein. Starting from base 734, the Hae II site is arginine 1
It is a convenient site for the expression of A _C portion of the starting A Sabuyunitsuto 65.

By using the Hae II site linker described in FIG. 11 (Aiii), a fusion product of β-galactosidase was expressed in pUR290, pBTA286 and pUC13. Purify the desired Hae II-Sau 3AI fragment from pBTA30 to achieve this,
A linker was added. The resulting construct was cut with Sau 3AI and the DNA was then precipitated twice with isopropanol, which efficiently precipitates large DNA fragments but does not sufficiently precipitate small DNA fragments such as linkers. The precipitated DNA was redissolved in TE and ligated into pUR290. BT
Transformed into A634. The resulting clones were copied onto a copy filter, then one filter of each pair was replaced with [ ³²
P] -labeled Hae II linker (DNA shown in FIG. 11 (Aiii)
Using the paired tip strands, the other was screened using probe 4 (FIG. 3) as in Example 4. Clones detected from both probes were picked and Western Bl.
Tested for expression by ot analysis. One of these clones is shown in FIG. 13, track 7, and was named BTA421 (Table 2). Inhibin A _C Sabuyunitsuto and its side chain β-
The DNA fragment encoding the galactosidase DNA sequence is also EcoR
Excised as an I fragment and p for expression as a fusion protein
Ligated during BTA286. This fusion protein is again insoluble and is therefore convenient for purification and prevention of degradation.
This strain was named BTA422 (ATCC67059, Table 2; FIG. 13,
Track 2).

The new Bam HI-Sau AI fragment encoding the A _C Sabuyunitsuto also cut from PBTA218, is subcloned into pUC13 for short chain fusion protein product. Share BTA427
(ATCC67056; Table 2) produced this protein as an inclusion body and analyzed it by gel analysis (Fig. 13, track 15). When it was combined with BTA426 again, the longest fusion protein became West.
It turns out again to be the most prominent and most conservative in the ern Blot analysis. This indicates that BTA427 is an excellent host-vector binding system for the production of inhibin-like proteins. Human A _C in pUC13
Subunit gene expression is 436 bp of human genomic DNA.
This is done by isolating the Pvu II-Bam HI fragment,
This fragment is derived from λA ₂ (section 8),
Those containing Corde viewing area of the A _C Sabuyunitsuto.

After HgaI digestion, (Linker 1) was added and the product was cleaved with Bam HI followed by pUC13 B
Ligation to am HI site.

Plasmid preparations of E. coli BTA652 transformed clones expressing hA _C Sabuyunitsuto as a fusion product was isolated. This clone is BTA1367 (= BTA652 <pBTA47
2>), and the sequence of the fusion vector is shown in FIG. Equivalents to this fusion protein are detected by antisera 474 on Western blots, with the largest band being the most prominent.

The striking feature of each linker shown in FIG. 11 and linker 1 above is that when the DNA is correctly expressed from the Bam HI site, its amino acid sequence Asp-Pro has been introduced into the fusion protein. As described above, this sequence is easily denatured in formic acid and is upstream of a protein without an Asp-Pro sequence in the A-subunit protein (Nilsson, B. et al. (1985)).
Nucl, Ac, Res, 13; 1151-1162) can be used to cleave inhibin protein. Other methods of cleaving the desired sequence from the fusion protein can also be used in combination. Examples include protease recognition sequences such as the Xa-factor recognition sequence (Nagai, K and Thogerson, HC
(1984) Nature 309 , 810-812) or the collagenase recognition sequence (Promin. N of Sermino, J. and Bastia, D (1984)).
atl.Acad.Sci.USA 81, a method of introducing 4692-4696). The possibility of using enzymes that recognize arginine pair residues has been described above (Example 9). By introducing methionine thereon prior to the inhibin sequence, the fusion product can be cleaved with cyanogen bromide to obtain a β-calactosidase-free inhibin fragment. Since the methionine residue in the inhibin sequence can be changed to another amino acid by in vitro mutation of the cDNA, and therefore the full-length inhibin-like protein will not contain the fusion protein precursor, This increases the utility of this cyanogen bromide cutting method. By changing the internal methionine residue, a molecule having more favorable antigenicity or inhibin-like agonism or antagonism can be obtained.

(B) Expression of 15 kD subunit A 510 bp PstI cDNA fragment of pBTA293 (FIG. 4) was ligated to the PstI site, and this plasmid was then ligated to E. coli BTA6.
34. The resulting clone is first used for pBTA
293 and pBTA294 (probe 2, FIG. 3) were used for isolation. Screening was performed using a 24-denatured 14-mer oligonucleotide to confirm the presence of the transforming plasmid. Then, several plasmids were digested with the restriction enzyme Hind II.
Map using and insert with stereotaxic
One plasmid was analyzed for expression on a polyacrylamide gel. This single strain was named BTA423 (Table 2).

The 510 bp Pst I cDNA fragment of pBTA213 was directly
Cloned into the I site and B for fusion protein expression
Transformed into TA634. pBTA286 is derived from pUR291,
This direct cloning is therefore possible because of the reading frame across its Pst I site. The obtained strain was transformed into BTA424 (ATCC67).
058; Table 2), which produces the fusion protein as inclusion bodies.

The 510 bp Pst I cDNA fragment of pBTA293 was also
And cloned into BTA637 for expression of the insoluble short fusion protein into strain BTA1360 (ATCC6
7055; Table 2).

A and A _C Sabuyunitsuto is Despite respectively, BTA423, BTA424 and fusion tan white matter by the both rabbit anti-inhibin antiserum connexion from BTA1360 is never to have been detected (Figure 13, track 1, 6 and 16 ). Thus, notice that level of antibodies to 15kD protein is very low or zero biological effects associated with immunity accordance connexion inhibin achieved only by connexion to the use of A or A _C Sabuyunitsuto It seems possible (see also Examples 17-21).

Also, expression of the 15 kD sequence was accomplished by isolating the entire 15 kD sequence and arginine-5 to -1 and with the HaeII fragment, and adding the HaeII site linker shown in FIG. 11 (Aiii). Achieved. The resulting fusion code is useful for expression of the β-galactosidase fusion in pUR290 / pUC7 / pUC13 using the Bam HI site, as shown in FIG. 11 (Bi) and described above, and in the Asp-Pro sequence. It also has the function of cleaving the fusion with formic acid. Cla I
The site is p, where ATG functions as a new translation initiation site.
It can also be used for expression in trpL ₁ (Edman, JC et al. (1981) supra).

A third means possible for expression uses a unique Hind II site and encodes amino acids 1-8 with the desired linker and Met-1 sequence, as shown in FIG. 11 (Bii). Is to synthesize a DNA sequence.

Thus, the Pst I, Hae II and Hind II sites are all convenient sites for adding an expression linker. Also, 510bp Ps
After partial digestion of the tI fragment with HaeIII, a HaeIII site joining amino acids 1 and 2 (GGCC) can also be used. Incidentally, it is well known to those of ordinary skill in the art to alter the expression systems various, any possibility described above with respect to A and A _C Sabuyunitsuto applies to B Sabuyunitsuto.

Pre-pro A subunit and pro B subunit cD
FIG. 17 shows the structure of a synthetic prepro-B subunit based on NA. The B subunit of inhibin was 510 bp Pst I
It was isolated from pBTA293 as a fragment (FIG. 4). After digestion of the Pst I fragment with Hae II, the separated 365 bp Hae II fragment is converted to a Bam HI fragment by coupling to a Hea II site linker (FIG. 11, B1). The obtained Bam HI fragment was pZip
Ligation was carried out at the BamHI site of Zeo SV (X) 1, and then E. coli was transformed to obtain BTA1419 (= ED8654 pBTA416). To construct a synthetic preprogene for pZipNeo, A
Subunit signal sequence was changed from pBTA to 160bp Bam HI-S
Isolated as the au 3AI fragment. This was linked to the Bam HI site of the pBTA416 derivative from which the downstream Bam HI site had been deleted, resulting in pBTA4
A synthetic prepro B vector of 18 was obtained. This construction
See Figure 18. The sequence of amino acids -61 to -1 contains one cysteine residue added to the signal peptide. Thus, during expression in eukaryotic cells, folding of the B-subunit is expected to occur due to the formation of the correct disulfide, and processing of the B-subunit from its synthetic precursor is 5% prior to Gly1. It is still expected to occur at arginine residues.

Reliable and complete prepro-B subunit genes are available for expression by eukaryotic cells in pBTA306, pBTA294 and pBTA294.
Reconstructed by splicing segments from 308, and that portion of the Sabuyunitsuto precursor expressed in prokaryotic or eukaryotic cells independently inhibin A _C and B Sabuyunitsuto sequences to study their respective functions Is obvious.

Since the amino acid sequences of bovine and human B-subunits are identical, when the cDNA encoding the bovine B-subunit is expressed, the product is identical to the product obtained by the same expression using the human DNA sequence. is there. Therefore, the stock BTA423,
It should be considered that BTA424 and BTA1360 also produce the B subunit of human inhibin. DNA encoding full-length pro-B subunit mRNA contains a special Sac I site (767
~ 770 residues, FIG. 20), plasmids pBTA294 and pBTA30
Created by splicing the cDNA from 6.

c. Expression in eukaryotic cells The expression of inhibin subunits in eukaryotic cells was independently or simultaneously carried out by introducing the vectors pBTA417 and pBTA418 into NIH3T3 cells by the following procedure.

The day before the Transfection, about 2 × 10 ⁵ NIH3T3
Cells were plated in a 5 ml T25 flask. DNA (retrovirus or bacteriophage lambda DNA) 100ng-1μ
g to Parker, BA and Stark, G, R. (1979, J. Virol, 31 , 360-3
69) Graham, FL and Van der Eb, AJ modified according to
(1973, Virol, 52 , 456-467). Four hours later, the cells were stimulated with glycerol, and after 72 hours, the cells were collected in 1/3 aliquots into T75 flasks, which contained G4 / ml.
A complete medium containing 200 μg of 18 is placed in advance. Wash cells every 3-4 days with G418 containing medium and divide for 14-21 days until confluent growth is completed.

NIH3T3 cells contain 10% fetal calf serum (v / v), glutamine,
Dulb supplemented with penicillin and streptomycin
It was maintained in ecco modified Eagle medium (complete medium).

The transformed cells and supernatant were identified for inhibin expression when the cells reached confluent growth, ie, about 3 days after sorting. The cell lysate was prepared by trypsinizing about 2 × 10 ⁷ cells, washing with 10 ml of PBS, and adding 1 mM of 5 mM sodium phosphate buffer.
Prepared from those resuspended in l and frozen at -20 ° C.

To prepare RNA, cells (2 × 10 ⁷ ) are collected by centrifugation, and 2 ml of 2 × NETS / phenol (2 × NETS) at 95 ° C.
= 200mM NaCl, 2mM EDTA, 20mM Tris-HCl pH7.5,1.0% SD
Resuspended immediately in S). After vigorous stirring, the mixture was cooled to room temperature before the phases were separated by centrifugation.

The aqueous phase was re-extracted with phenol and the RNA precipitated from the final aqueous phase, at which time 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were added. 10 on dry ice
After one minute, the RNA was pelleted at 11,000 g for 10 minutes at 4 ° C. Dissolve RNA in water and convert DNA and tRNA from total nucleic acid to LiCl
Was added by adding up to 2.5M. The aqueous solution was allowed to stand at 4 ° C. overnight, and LiCl-precipitated RNAs were 11,000 g for 10 minutes,
Separation was performed by centrifugation at 4 ° C through an equal volume of 3MLiCl cushion. The final RNA pellet was reprecipitated from water and
Washed with 0% ethanol, dried and dissolved in sterile distilled water. After measuring the concentration by absorbance at 260 nm, the RNA was stored at -80 ° C until ready for use.

Northern blot and dot blot analyzes were used to estimate the amount and size of the mRNA transcript resulting from the vector. RNA (5 μg) was added to Thomas, PS, 1980, Proc . Natl . Aca
After glyoxylation as described in d . Sci . USA 77 , 5201-5205, the gel was separated on a 1% agarose gel. RN
A was directly transferred to nitrocellulose in the presence of 20 × SSC and left overnight at room temperature. Then, it was heated at 80 ° C. under reduced pressure for 2 hours.
Filter at 42 ° C. for 4 hours with 50% formamide, 5 ×
SSC, 10mM sodium acetate pH6.5,250μgml ^-1 DNA0.02% PVP
Prehybridization in 25,0.02% Ficoll 400, followed by hybridization overnight at 42 ° C., with the prehybridization buffer containing up to 10% dextran sulfate and 1 μg DNA 2 x 1 per probe
Running was performed in a buffer containing 10 ⁶ to 10 ⁷ cpm of ⁰⁹ cpm. 365 probe A _C Sabuyunitsuto is comprising 788 to 1149 bases (Figure 5)
bp cDNA fragment, B subunit probe is 504 ~
This is a 369 bp cDNA fragment containing 872 bases (FIG. 6). The filter is washed at 65 ° C. with 0.1 × SSC, 0.1% SDS and then −8
Autoradiography was performed at 0 ° C.

For the preparation of genomic DNA, the collected cells (2 × 10 ⁷ ) were
E buffer (10 mM Tris HCl, 400 mM NaCl, 2 mM EDTA, pH
8.2) and resuspended in SDS added to a final concentration of 0.5%. After mixing, 20 μg ml ^{−1 of} proteinase K was added, and the mixture was incubated overnight at 37 ° C. The DNA was extracted twice with an equal volume of phenol for 15 minutes each and then once with chloroform: isoamyl alcohol (24: 1).

DNA was recovered from the final aqueous phase by ethanol precipitation, rolled up, and then redissolved in TE (10 mM Tris HCl, 1 mM EDTA pH 7.5). Add 400μgml- ¹ heat-treated RNase, 37
After incubating at 30 ° C. for 30 minutes, SDS was added to 0.5%. Proteinase K was added to 20 μg ml ⁻¹ and the incubation was continued for a further 2 hours. The DNA was separated twice by phenol treatment and once by chloroform: isoamyl alcohol treatment, followed by ethanol precipitation. The rolled-up DNA was dissolved in TE until ready for use, and stored at 4 ° C.

Southern blot analysis was used to detect integration of the vector into the genome, but it also has the ability to estimate the number of integrated copies. DNA (10μg) to DNA 1μ
Digest with 8-10 units of enzyme per gram, usually at 37 ° C overnight under conditions recommended by the supplier.
The sample was phenol extracted and precipitated with ethanol, 40 mM
Electrophoresis is performed on a 0.7% agarose gel containing Tris acetate, 1 mM EDTA, pH 8.2. After visualization under UV, denaturation and neutralization, the DNA is transferred from the gel to nitrocellulose, usually in the presence of 20 × SSC, as described by Southern (1975). After overnight transfer, filter 5x
Wash lightly with SSC and heat under reduced pressure at 80 ° C for 2 hours.

Prehybridization was performed for at least 4 hours with 50% formamide, 1 × Denhardts, 25 μgml ⁻¹ DNA, 5 × SSPE (SS
PE; 180 mM NaCl, 10 mM NaH ₂ PO ₄ , 1 mM EDTA)
The hybridization Chillon 42 ° C., carried out overnight, at a pre-hybridization Chillon in cross Hua containing 10% sulfuric acid dextrin and 10 ⁶ to 10 ⁷ cpm probe (specific activity 2 × 10 ⁹ cpm per 1 [mu] g DNA) . The probe is as described above. The filter was washed with 65 ° C., 0.1 × SSC, 0.1% SDS, and autoradiographed at −80 ° C.

RIA is mostly performed by McLachlan, RIetal (1986; Mol. Cell.E.
ndocrinol, 46 , 175-185), but using I ¹²⁵ -labeled 31KD bovine inhibin as a tracer. This assay detects morphologically accurate inhibins or subunits rather than denatured subunits. The standard used is HPLC purified 31KD inhibin,
It has a B / I ratio of 0.4, compared to bFF with a B / I ratio of 1.0.

The pituitary cell culture of the dispersed rats used sheep reticulated fluid as a standard, as described in International Patent Publication WO86 / 00078.

In all cases, the correct DNA was observed in the genome, indicating integration of the vector to be transformed. MRNA was also produced in these cells. However, there was no clear relationship between integrated copy number, mRNA levels and expressed protein.

On the other hand, new cell culture media are bioassay and RI
In A it was impossible to detect inhibin,
Progress medium from cells transformed with the vector pBTA419 confers low levels of FSH suppression in bioassays, which appears to be a non-specific suppression effect (Table 6).

In contrast, the A subunit gene of pBTA417 is expressed and secreted to provide a detectable substance for both bioassays and RIA. This suggests that one or more proteins from the prepro-A subunit gene alone may have inhibin-like biological activity, but previously only AB
Or a combination of Sabuyunitsuto the A _C B in which had been classified as inhibin.

The amount of immunoreactive substance produced is proportional to the number of cells in the culture medium [B subunit blot]. However,
It cannot still be ruled out that the host cells themselves make low levels of the B subunit. Since the B / I ratio of the sample from Transfection 3 is close to that of natural inhibin, this recombinant material can be used anywhere natural activity is required, such as in the development of contraceptives. This indicates that it can be substituted for inhibin. Naturally active substances are also found in supernatants from cotransfected cells with pBTA417 and 418 or supernatants from mixed aggregates after transfection of each vector separately, but reconstitution of inhibin. Is represented.

Data were determined in Bioassay (B) or RIA (I) and are given as units / ml (culture supernatant).

ND: Level below detection.

*: Data obtained from purified cell colonies are shown. The remainder was obtained from the transformation supernatant containing the aggregate of the mixed cells.

+: Indicates a cultured cell obtained by independently transforming each vector and then mixing them.

/: Indicates cells in which each vector was cotransfected simultaneously.

Due to the similarities between humans, cattle and other animal species (Example 7), similar constructs as described above can be used to express biologically active molecules from any species, including humans. However, expression of the inhibin protein or inhibin-like protein or peptide is not limited to the examples described above.

The expression of the sequences in the present invention is applicable to many different expressions used in conventionally known prokaryotic and eukaryotic cells.

Example 11 Isolation of Anti-Inhibin Antibody The protein fraction containing the β-galactosidase-inhibin fusion protein from BTA410 and BTA415 was subjected to the carbonyldiimidazole method (Bethell, GS et al. (1979)).
J. Biol, Chem, 254 ; 2572-2574) using Sepharose C
Antibodies to each fusion protein that bound to L-6B were isolated from 1 ml of rabbit anti-inhibin antiserum by affinity purification. The adsorbed antibody was eluted from the column with 3M MgCl ₂ and dialyzed against 10 mM Tris-HCl / 150 mM NaCl (pH 7.5).

The eluted antibodies were used for Western Blot analysis of each fusion protein, which revealed that they only bound the isolated fusion protein. That is, there was no cross-linking, and the amount of the anti-β-galactosidase antibody was negligible compared to the anti-inhibin antibody. Similar experiments were performed on fusion proteins from strains BTA425, 420, 422 and 424.

As a result of these experiments, recombinant inhibin or inhibin-like protein was purified anti-inhibin antibody or screening of monoclonal or polyclonal antibody preparations for anti-inhibin antibody and inhibin analysis system, such as EL
It can be used as a standard in ISA and RIA.

Example 12 Synthesis of synthetic peptides An alternative to the production of inhibin-like proteins in eukaryotic and prokaryotic cells is to chemically synthesize some or all of the inhibin. Such synthetic peptides can be used as analogs, agonists and antagonists of inhibin, and have uses as described in Examples 10 and 11.

The synthesis, purification and characterization of the synthetic peptide were performed in the following manner. 430A peptide synthesizer (Applied Bios
ystems Inc.) was used. The 1.00 version software was used according to the manufacturer's prescription without any changes. All drugs used were supplied by Applied Biosystems. The C-terminal amino acid (0.5 mmol) supplied covalently to the PAM resin was charged to the reaction vessel. The state of the synthesis was monitored by a ninhydrin reaction after coupling of each resin. The total yield of resin bound to the peptide was about 75%.

Peptide-bound resin was treated with scavenger cresol (0.
The peptide was cleaved and deprotected from the resin by treatment with hydrofluoric acid (10 ml) at 0 ° C. for 1 hour in the presence of 5 ml) and thiocresol (0.5 ml). This cleavage and deprotection is performed on a Teflon HF-type reactor (Protein Research Fou
ndation, Osaka). The reaction product was converted to ether (3
(× 50 ml), filtered, then the peptide was dissolved in acetic acid (10%), filtered, and the filtrate was lyophilized.

The residue was dissolved in trifluoroacetic acid (0.1%) containing acetonitrile (10%). The peptide was purified to homogeneity by reversed-phase high-pressure liquid chromatography. Gradient elution was carried out using trifluoroacetic acid (0.1%) and acetonitrile increasing linearly from 10 to 80%. The eluate was continuously monitored at 220 nm at a flow rate of 1 ml / min using an Altex C8 column for analytical chromatography. Preparative RP-HPLC for Vydac Protein and Pept
2-4 ml using ides C ₁₈ column (Cat, No.218 TP1010)
Manipulated in minutes. The eluent gradient was adjusted for optimal purification of each peptide. The purified peptide was recovered by evaporating the acetonitrile and the aqueous phase was lyophilized.

Amino acid analysis of synthetic peptides was performed using the Waters Pico-tag System. Perform according to the manufacturer's prescription and set the system to PT
Scaled with C amino acids. The amino acid composition of each peptide was consistent with the expected ratio of each amino acid.

Two regions of the A-subunit amino acid sequence from the cDNA sequence were first used to generate synthetic peptides. The first is Hi
from s1 to Ala26, the second from Ser167 to Asp195, which is 43 kD (A) and 20 kD of inhibin
(A _C ) contains the N-terminal of the subunit. The entire sequence of peptide 1 is shown below. Here, each number is shown according to the amino acid sequence of FIG.

This sequence can be abbreviated as (H ₁ -A ₂₆ ) K using the last one amino acid code, where the amino acids in the sequence are the inhibin sequences listed in FIG.

Peptide 2 is Y (H ₁ -A ₂₆ ) K. This is obtained by adding an NH ₂ terminal tyrosine to peptide 1. The synthesis was performed starting from lysine. A portion of the resin (75%) was removed (peptide 1) and then tyrosine residues were added to the remaining 25% of the peptide to produce peptide 2.
Lysine residues were added to facilitate hapten production and tyrosine was added to create sites for radioiodination.

Example 13 Production and purification of inclusion bodies Strains BTA420, BTA422 and BTA424 produce inhibin-like protein as a fusion product. All of the protein from these three strains is found in vivo as insoluble masses called inclusion bodies, which are produced and purified by the following steps.

Overnight cultures are grown in 2 liter flasks
× 1 fresh LB (10g tryptone / 5g yeast extract / 5
g / NaCl; 1), and this was shaken at 37 ° C. until the culture concentration was OD = 0.3-0.4. Isopropyl-BD-thiogalactosidase (IPTG: final concentration 0.1 mM) was added, and the culture was continued for 2 to 7 hours. The inclusion bodies first became visible as light-phase granules after 2 hours and then reached their maximum size and quantity experimentally after 7 hours.

Collect cells and place this pellet at -80 ° C overnight for convenience.
Frozen. 20 ml of H ₂ O per cell of original culture
During resuspension, it was crushed using a French Press. The suspension was made 0.1 mM in phenylmethylsulfonyl bromide (PMSF) and 5% Triton X-100 and then centrifuged at 1200 xG for 10 minutes.

Discard the supernatant and sonicate the pellet to 50 ml.
In 1 M NaCl / 5% Triton X-100 and centrifuged. This washing step was repeated and the pellets were finally sonicated with 2.5 ml of 1 per original culture.
Re-suspended in M NaCl / 5% Triton X-100.

The suspension was layered on a 60% (w / v) sucrose density gradient in a Beckman SW28 rotator,
Centrifuged at 0 × G for 60 minutes. Inclusion bodies were pelleted through sucrose cushions and isolated from cell debris. They can be washed with H ₂ O and stored frozen at -80 ° C. for at least 3 months without any change as proteinaceous material found on polyacrylamide gels.

Example 14 Production of Antigen Purified inclusion bodies were adjusted to a concentration of 2 mg / ml with 8 M urea / 0.1 M DTT / 0.
It was dissolved in 1M Tris-HCl (pH 8.0) at 37 ° C. for 2 hours under N ₂ atmosphere. The solution was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was changed at least 2 × 5 l overnight at 4 ° C. to 50 mM NaH ₂ PO ₄
Dialysis was performed against / 150 mM NaCl (pH 7.5) (PBS). The protein in the dialysis bag became a white floc, which was used without further purification. When 1 mg of the substance was injected intraperitoneally into mice, the substance was found to be free of pyrogens and lipopolysaccharides. The floc suspension is combined with an equal amount of an oily adjuvant, namely Marco152: Montanide88.
Emulsify with 8 (9: 1) and finally 100-250μg antigen per 1ml
Concentration. If there were antigens from two or more starting materials, the concentration of each antigen was 100-250 μg / ml.

Keyhole limpet haemocynin (Keyhole limpet haemocynin) was synthesized according to the glutaraldehyde method.
anine, KLH) (Briad, JP et al. (1985) JI
mmunol, Methods, 78 , 56-59) and the conjugate was emulsified as described above.

It should be noted that there are many other means for producing antigens and many other methods and methods for immunization. Alternative methods of antigen production include, for example, emulsification of Freund's in complete or incomplete adjuvant, emulsification in anhydrogel or saponin, or digestion of antigen-containing polyacrylamide gel sections. Alternatively, the antigen may be administered without the use of adjuvant. Examples of the administration form include oral administration, administration through mucosa, intramuscular administration, intraperitoneal administration, and subcutaneous administration. There are many methods for enhancing the antigenicity of a peptide or protein, such as cross-linking with itself, cross-linking with other proteins, denaturation of residues, or change in sequence.

Example 15 Immunization Method The following animals were first injected with the injection shown in FIG. 1 (week 0), and then injected with the enhancer 4 weeks later. The dose of each Flock antigen is as follows.

Rabbit 100 μg / time Sheep 250 ブ タ Pig 200-250 〃 Rat 100 〃 In addition, synthetic peptide (peptide 1, Example 10) conjugated to keyhole limpet hemocyanin (KLH) was conjugated with 100 μg of conjugate per rabbit Was used.

Example 16 Antiserum Analysis Blood samples are collected regularly. A typical exsanguination method is as follows. One exsanguination prior to the first injection, one or more exsanguinations prior to the booster administration, and a weekly exsanguination during and after the booster administration. The blood is allowed to clot overnight at 4 ° C. and the serum is removed after centrifugation. The serum was split into several aliquots and −2 during antibody and FSH titer analysis.
Stored frozen at 0 ° C.

 Serum was run under three different experimental conditions.

(A) Tracer binding The antibody binding of the iodinated 58 kD and 31 kD inhibins was measured to determine whether the antibodies could recognize native usinhibin. The tracer antibody complex is precipitated using a second antibody and the radioactivity in the pellet is
It was expressed as a percentage of the total count in the reaction system.

The iodination step is as follows. Purified 58kD or 31
kD inhibin (1-2 μl in 25 μl electroelution buffer)
g) (International patent application PCT / AU85 / 00119) was added to 25 μl of 0.5 M phosphate buffer (pH 7.2). Na ¹²⁵ I (0.5 mCi, 5 μl,
Amersham, Bucks, UK). Chloramine T (40μ
l) was added at a ratio of chloramine T: hormone = 8: 1. The reaction was carried out at room temperature for 60 seconds with stirring, and the reaction was stopped by adding 20 μl of sodium metabisulfite (3 mg / ml). The reaction mixture was added to 20 mM phosphate buffer / 0.1% BSA or 0.5% Polypep (p
H6.0) to 500 μl, and Sephadex G25 column (PD1
0, Pharmacia, Uppsala, Sweden)
¹²⁵ I was excluded. Collect the inactive fractions to 20 ml and add 200 μl of M
Supply to atrex Red A (Amicon, Danvers, Mass., USA)
The eluate was then removed by washing with a phosphate buffer containing 400 mM KCl. ¹²⁵ I-inhibin in phosphate buffer
Eluted with 1, M KCl / 4M urea. The ligated inhibin was further gel filtered on a Sephadex G25 column with an appropriate RIA buffer (see below) to remove KCl / urea.

Following iodination of the 58 kD and 31 kD inhibins, 60 μCi and 25 μCi, respectively, were collected in the inactive volume fraction after gel chromatography on Sephadex G25.
About 30% was eluted with 1M KCl / 4M urea buffer. SDS-PAGE
The ¹²⁵ I-inhibin determined from the molecular weight in was found in this fraction.

The specific activity of an iodine preparation is determined by the self-replacement method using the hormone used for iodination of the reference body (Marana et al. (1979)
Acta Endocrinol (Kbh.) 92,585-598). The specific activity of ¹²⁵ I-58 kD inhibin was 50-60 μCi / μg, that of ¹²⁵ I-31 kD inhibin was 24 μCi / μg, and the recovery of each was 5-25%.

Anti-inhibin antiserum from rabbit 474 immunized with native 58 kD inhibin has lost the biological activity of native inhibin and binds strongly to iodinated 58 and 31 kD inhibin, but little to a separate subunit of each inhibin. Do not combine. This indicates that iodination has not significantly changed the molecule. Therefore, the ability of a test antiserum to bind to iodized inhibin is considered to be the ability of the antiserum to recognize native inhibin.

(B) In vitro biological quantification (bioassay).

The bioassay described above (international patent application PCT / AU85 / 001
In 19), since the antiserum has the ability to suppress the biological activity of native inhibin, it is recognized that a neutralizing antibody is present.

(C) FSH.

Serum levels of sheep FSH were determined by radioimmunoassay using rabbit anti-sheep FSH serum and iodinated sheep FSH as tracer. The serum level of rabbit FSH was determined by a radioimmunoassay method using guinea pig anti-rabbit FSH serum and iodinated rabbit FSH as a tracer. An increase in FSH titers would be seen upon removal of endogenous inhibin from antibody recruitment that occurred against the recombinant.

Example 17 Sheep experiment A group of 9 animals (white sheep, chosen not to be in estrus during the experiment) were immunized with one or a pair of Flock antibodies shown in Table 7 and the serum was run. Analyzed as in Example 16.

(A) Tracer binding experiment.

Serum (2 weeks after booster administration) having remarkable tracer binding ability (Table 7) was collected from 19 mice. All of these animals were from groups other than the placebo control group (Group A) and the group immunized with the 15 kD subunit fusion protein from pBTA301 (Group C). The highest response was seen in animal # 673 (Group D), which showed 10% binding to 31 kD tracer and 3.1% binding to 58 kD tracer under 1: 1000 dilution.

All animals not listed in the table had less than 1% binding.

Thus, the recombinant inhibin subunit has the ability, when used as an antigen, to raise antiserum in sheep that can recognize native inhibin. still,
(I) these experiments detect free antibody in serum, and the antibody with the highest affinity for sheep inhibin is bound to sheep inhibin in the experiment and not detected in the analysis of this experiment; and (Ii) It should be noted that the screening was performed at a dilution of 1: 1000 which probably exceeded the dilution titer of the weakly positive sera.

(B) In Vitro Bioassay Data Under the conditions of the experiment (1 μl of antiserum per 2 V inhibin), none of the sera showed neutralization of in vitro inhibin activity in pituitary cell culture bioassays.

(C) FSH titer The serum FSH level of each animal group is shown in Table 8. Although no remarkable difference was observed from the control group (A), a remarkable increase was observed in all test groups (except one group) even at one time.

Thus, recombinant inhibin can be used in livestock animals for FSH
It can be used as a titer increasing antigen. FSH
Is known to increase ovulation, so immunization against recombinant inhibin can be expected to increase ovulation, and this can be used as a fertility enhancer to improve fertility. There is expected. Furthermore, since FSH has the effect of regulating spermatogenesis in males and oogenesis in males, the administration of biologically active recombinant inhibin lowers the FSH level, and therefore reduces the use of both males and females. It can also be used as a contraceptive.

The data are group mean ± standard deviation. The test groups are the same as in Table 4. Statistical comparisons between the data for the fourth week and the data for the following weeks were calculated using a pair of t
-Made in the test.

(*) P <0.05 (**) p <0.01 Example 18 Rabbit experiment With native 58 kD or 31 kD inhibin or plasmid pBTA
Antisera from uncastrated male rabbits immunized with fusion protein from cells having 297, 299 or 301, or with conjugated protein containing synthetic peptide 1 (Example 10),
The analysis was performed using a tracer binding system. The results are shown in Table 9.

The serum of animal number 474 was used at a dilution of 1: 2000, and all others were diluted at a dilution of 1: 1000.

These data also indicate that recombinant inhibin subunits and synthetic peptides can elicit antibody responses, including those that recognize and bind native inhibin. Response varies from animal to animal, but recombines 15kD
Since the antigen (from BTA424) does not elicit a significant response in all tested rabbits, all antibodies in serum from rabbit # 474 (which were raised against the native 58kD inhibin) It appears to be an antibody to 43 kD inhibin as far as this experiment is concerned, thus the 15 kD subunit is found to have little antigenicity in rabbits. However, this does not mean that this subunit will not be useful in other species. These results also suggest that in order to achieve a sufficient antigenic response to the 15 kD subunit, it would be necessary to make some changes to its structure, sequence or configuration. Similarly, the 20 kD subunit is also a poor antigen in rabbits.

The serum level of rabbit 699 suddenly decreased to 0 after boost immunization with peptide 1-KLH conjugated antigen (FIG. 15), and
Rabbit 698 shows a gradual decrease. Each animal's response correlates with its antiserum titer to the 58 kD tracer. Also, the synthetic peptides themselves or their antisera have no effect on rat pituitary cell bioassays in vitro and they have no direct effect on inhibin receptors. Thus, since FSH is involved in spermatogenesis and oogenesis (see Example 19), this peptide and its derivatives can be used as contraceptives for both males and females. Furthermore, this is NH ₂ the A _N fragment - because it is end, A _N fragment itself or bovine, other synthetic peptides derived from human or other vertebrate species A _N fragment was also observed here If the response is to be mediated by antibodies, it is deemed to have a similar application.
Also, when the biologically active _AN fragment or a part thereof is administered not as an antigen, the FSH level is expected to increase,
Therefore, they are expected to act as reproductive enhancers for improving fertility (see also Example 19).

Example 19 Pig Experiment The method of this experiment is different from that of the previous example.

Endogenous sows (22-23 weeks old) were treated with 750 μg of BTA425-derived β-galactosidase Floc antigen (16 mice, control group) or 250 μg of each of BTA422, BTA424 and BTA426-derived subunit Flot antigens. (16 animals, test group) were immunized respectively. After the first immunization
A similar booster was administered on day 25 and further day 47. Serum was collected 60 days after the first immunization and analyzed for anti-inhibin antibodies by the tracer binding method. These sows exhibited estrus and were mated during the first estrus 60 days later.

When sera from 15 test animals were analyzed for tracer binding ability under 1: 100 dilution conditions, 10 of these sera showed a range of 1.7-9.0% (= 4.9 ± 2.5) of ¹²⁵ I-58k
The ability to bind to D-inhibin was shown. Further, ¹²⁵ one example capable of binding 2.0% with respect to I-58 kD inhibin ¹²⁵ I-31 kD
It also showed 6.4% binding ability to inhibin. None of the six control sera showed intact binding to both tracers, indicating that the recombinant inhibin subunit is capable of eliciting an antigenic response in pigs and thus recognizes native inhibin It became clear that it could be an antibody.

The estrus period of the pigs is a 21-27 day cycle after the blood collection on day 60, and all the test animals in this experiment were expected to be eruptive and to mate. Table 10 shows the mating data for the control and test groups. However, the results of the following analysis indicate that the animals in the test group did not mate by this experimental treatment.

Having a p <0.02 (applying Yates correction) anti 58kD inhibin antibodies; - Table 10 Mating data X ² 16 analysis unit mating success mating unsuccessful meter control group 14 2 16 Test group 6 10 total 20 12 32 X ² = 6.5 9 out of 10 sera
1: Since has failed to detect the 31kD inhibin in 100 dilution conditions, the above data are those showing antibody activity against A _N fragment of 58kD inhibin, most of the antibody
It is believed to be A _N specific. This hypothesis is that in rabbit experiments, ie, when peptide 1 was administered as an antigen, the FSH level was reduced (Experimental Example 18) and as a result, the estrous cycle and follicle formation were arrested in female vertebrates. Is supported by what it will do. One case that produced sera with higher antibody titers to 31 kD inhibin than 58 kD inhibin mated normally during the course of this experiment. It can also be explained as follows. For example, the 43 kD antigen has the amino acids -10 to -1 of the prepro-A subunit, and this sequence provides this effect. However, whatever the functioning mechanism in which this paradoxical result was achieved in vivo, the data underscores the action of FSH and thus of inhibin as a regulator of reproductive function and its processes, and This demonstrates the action of recombinant inhibin as a male and female contraceptive and as a molecule that suppresses female estrus.

Example 20 Rat Experiment A test group of rats was immunized in the same manner as in sheep. The sera was 1:50 by iodine tracer binding assay.
The analysis was performed under a dilution condition of 0, and the binding data (%) of each group was shown in Table 11 as a mean value ± standard deviation. The antigens used in groups (A-F) are the same as in Table 7.

In these data, comparing the tracer binding ability of group (A) with groups (B) and (E) or (D),
It can be seen that the A (43 kD) subunit is a better antigen than the A _C (20 kD) subunit. From the results of the tracer binding test, the B subunit had no effect as an antigen. These results indicate that 58 kD native inhibin produced excellent antibody responsiveness and neutralizing antibodies in rabbit 474, while neutralizing antibodies were produced in rabbit 461 and two other animals immunized with 31 kD native inhibin. It emphasizes and emphasizes the observation that no antigen responsiveness was observed.

For other species (sheep, rabbit, pig), neutralizing antibodies were not detected in any of the animals immunized with the recombinant inhibin subunit. Also, in most species, a significant immune response to the β-galactosidase portion of the antigen was seen. These observations indicate that the conformational epitope is important and that BTA426, BTA427 and BTA13
It appears that antigens such as those from 60 or from the trp vector (Example 10) are of high value. Because the β-galactosidase portion of the fusion is substantially reduced (FIG. 12) or deleted, experiments were performed to generate conformational epitopes and to improve antigen production. It is because it can be performed.

However, animal experiments in the present invention have revealed that recombinant inhibins, subunits, fragments and synthetic analogs or homologs thereof can be used as follows.

(A) Production of antibodies that recognize native inhibin.

(B) Change of FSH level.

(C) Use as a reproductive enhancer or contraceptive.

The production and use of pUC- derived fusion protein as Example 21 immunogen pBTA303, pBTA308, pBTA309 and excellent immunogenicity pUC- derived short chain fusion protein from pBTA472 cattle A _C from inclusion bodies from BTA427 like Illustrated by what is obtained by neutralizing the antibodies of sheep immunized with the product.

This protein converts inclusion bodies into Tris-HCl pH 8.0 (8M urea and
It is prepared by dissolving at 37 ° C for 2 hours. The solution was brought to pH 3 by adding glacial acetic acid, then centrifuged, then concentrated and chromatographed on a Sephadex x G50 or G75 column with 0.1 M acetic acid. The fraction containing the recombinant protein was fixed by SDS-pAGE,
Pooled, reversed-phase chromatographic columns (Vydac C4
Or Dy namax C18). In each case, the protein was eluted from the column using an acetonitrile gradient containing 0.1% TFA. The eluted protein was fixed again with SDS-pAGE, and the required fractions were pooled and concentrated to 1-10 mg / ml,
Dialyzed with BS. Identification of protein NH ₂ - was confirmed by end sequencing.

4 sheep were immunized with 300MyugKLH, sheep etc. The number A _C fusion protein 30 [mu] g, and 300MyugKLH hybrid comprising a binding A _C (1
07: 1 molar ratio) using the glutaraldehyde method (Briand et al.,
1985 ibid ).

The primary immunizer was Freund's complete adjuvant on day 0, and the booster was Marcoll on days 21 and 50.
l) 52: Performed on Montanide 888 (9: 1). The 26-day serum was tested for its ability to recognize the native bovine 31KD tracer by RIA and the result was% (count)
Are shown in Table 12.

Significant binding to native 31KD inhibin indicates that antibodies have been generated that recognize morphologically correct inhibin. In addition, antibodies from sheep 6, 7, 8, and 10 were tested in in vivo bioassays, which neutralized inhibin bioactivity. Sheep 5-12 were immunized at 93 days with an unmixed immunogen (Montanide 888: 100 μg in Markol 52), and then co-treated with a progestogen estrous cycle to examine the ovulatory response. These groups receiving recombinant inhibin have significantly higher ovulation rates than KLH-immune controls or untreated controls (C1-C5: Table 13), alone or hybridized with carrier molecules. The superior properties of the short-chain fusion protein as an immunogen were recognized when using E. coli, demonstrating that antibodies against a single subunit of inhibin can neutralize the biological activity of inhibin in vivo and in vitro. . One (9) was laparoscopic and ovarectmisted (ovarectmist)
d) I know what I'm doing.

These methods are used in humans and livestock, such as pigs, dogs,
It is clear that the protein white matter from pBTA303, pBTA308, pBTA309, and pBTA472 can be used alone or in combination in horses, cattle, sheep, goats and the like.

Example 22 Method for Producing Inhibin Host cells with a recombinant plasmid carrying the genetic information for inhibin production are stored as freeze-dried vials in a production culture assembly. The cells from the storage vial are regenerated, planted on a particular medium, and the cells from this medium are used to prepare the inoculum for fermentation. This inoculum is used to make a fermentation material containing a suitable growth medium, and the fermentation is performed under conditions suitable for inhibin protein production. After completion of the fermentation, the cells are harvested, and the product is isolated from the cells and purified. The product is analyzed and conditioned and stored under storage conditions sufficient for its stability. Under severe hygiene conditions, the product is formulated for the application in combination with other components.

INDUSTRIAL APPLICABILITY Use of inhibin Inhibin or a part thereof produced according to the present invention can be used as an antigen or as a bioactive substance. The effect of one use may be opposite to the effect of another.

In one form, inhibin, or a portion thereof, produced according to the present invention and / or subsequently modified, can be used as an antigen and can thus be used to regulate fertility. Increased ovulation rates in vertebrates, including humans, improve fertility and thus increase fertility, ie, improve reproductive efficiency.
The products of the present invention can be used for increasing ovulation rates and can therefore be used for in vivo fertilization programming, for example for livestock and humans. It can also be used for animals other than domestic animals, for example, to facilitate crossing in zoos. The products of the present invention may also be used to accelerate the onset of sexual maturity and puberty to extend the reproductive period, and / or to shorten the period between copulation and convergence in estrus in livestock animals. It can be used to lengthen and / or shorten seasonal or postpartum nonestrus. In the case of males, the compounds of the invention can be used to stimulate spermatogenesis.

Thus, administration of inhibin is beneficial in raising livestock species such as cattle, sheep, pigs, goats, deer, horses, fish and birds.

Inhibin can also be used as an active substance to suppress ovulation in females or to reduce spermatogenesis in males, and can therefore be used as a contraceptive or as a means to have the same ovulation. it can.

Portions of inhibin, such as synthetic peptides or _AN fragments
It can be used as an antigen to lower the FSH level and acts as a contraceptive or as an agent to suppress estrus.

The product of the invention can be used for antibody production,
This antibody is also included in a part of the present invention. Further, anti-inhibin antibodies can be used as diagnostic agents. For example, such applications include means for monitoring the state of the reproductive cycle in vertebrates, including humans. In this way, it is possible to predict the time of ovulation and the number of ovulated eggs, and thus determine the treatment used to regulate the reproductive cycle.

This antibody can detect female granulosa cell function, or be used as a marker for male Sertoli cell function, or perhaps as a marker for genetic selection of highly fertile livestock.

Thus, the above-described recombinant proteins and their derivatives, homologs and analogs have many different uses, for example:

(A) Use as an antigen or an inhibin antagonist for suppressing or inhibiting a part or all of the physiological action caused by native inhibin or its precursor in vivo.

(B) Use as an antigen for producing monoclonal or polyclonal antibodies.

(C) Use as precursor for the production of physiologically active recombinant inhibin in vivo or in vitro.

(D) Use as a standard in inhibin radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) or in other detection systems such as chemiluminescent or fluorescent assays.

(E) Use in screening monoclonal or polyclonal antibody preparations.

(F) Use in purification of an antibody that recognizes inhibin or an inhibin-like amino acid sequence (see Example 9).

(G) Use as an inhibin agonist to enable part or all of the physiological action caused by native inhibin or its precursor in vivo. Biologically active recombinant inhibin is produced by simultaneously expressing both subunits or their precursors in the same cell, or by correctly reconstructing individual subunits or their precursors, analogs, homologs or derivatives in vitro. be able to.

The inhibin cDNA sequence has various uses, examples of which are listed below.

(1) Use as a cDNA template for protein synthesis in prokaryotic or eukaryotic cells (see Example 8).

(2) DN for production of radiolabeled inhibin cDNA probe
AUse as a mold. These probes can be used to detect and isolate inhibin or inhibin-like sequences in genomic DNA of various species or in the mRNA of cells that make inhibin or inhibin-like peptides (see Examples 7 and 8). ). Thus, they have the ability to diagnostically detect inhibin synthesis and regulation in humans and domestic animals, or have potential use in the cloning and processing of inhibin genes.

(3) Use as a DNA template for production of a radiolabeled RNA probe for the same use as in (2) above.

(4) Use as a DNA template for production of radiolabeled inhibin.

Continued on the front page (31) Priority claim number PH3157 (32) Priority date October 29, 1985 (33) Priority claiming country Australia (AU) (31) Priority claim number PH3960 (32) Priority date 1985 December 19, 1988 (33) Priority claiming country Australia (AU) (31) Priority claim number PH3961 (32) Priority date December 20, 1985 (33) Priority claiming country Australia (AU) Referee number Hei 6-13851 (73) Patent holder 999999999 Monash Medical Center Victoria 3004, Australia, St Kilda Road, Melbourne (no address) (73) Patent holder 999999999 St. Vincents Institut Unit・ Medical Research Victoria 3065, Australia, Fitzroy, Victoria Park 41 (72) Inventor Forage, Robert Gregory New Sau, Australia Su-Wales 2088, Mossman, Awaba Street 80 (72) Inventor Stewart, Andreu Gyoji, Australia New South Wales 2073, Pimble, Robin Head Road 26 ( 72) Inventor Robertson, Devitt Mark Victoria, Australia 3150, Glen Waverley, Florite Court 6 (72) Inventor De Cretztour, Devited Mori Tutu Victoria 3127, Australia, Surrey Hills, Liura Street 1 (56) Reference FEBS LETTERS, 175 [2] (1984) 349-355

Claims (16)

(57) [Claims] 1. A DNA encoding a subunit of bovine or human inhibin, wherein said DNA is a DNA encoding each amino acid sequence of the following bovine prepro-A subunit, bovine proB subunit, human preproA subunit or human proB subunit: DNA comprising: Bovine prepro A subunit: Bovine pro B subunit: Human prepro A subunit: Human pro-B subunit: 2. The DNA of claim 1 which encodes a 43 kD subunit of inhibin. 3. The DNA of claim 1 which encodes a 15 kD subunit of inhibin. 4. The bovine prepro-A subunit from -60 to 166.
Amino acid sequence of bovine prepro-A subunit-10 to 16
6 amino acid sequences, bovine prepro A subunit 1 to 1
66 amino acid sequence, -61 of human prepro A subunit
2. The DNA of claim 1 which encodes the amino acid sequence of -1 to 171 of the human prepro A subunit and the amino acid sequence of 1 to 171 of the human prepro A subunit. 5. A DNA encoding a bovine or human inhibin subunit, which encodes the amino acid sequence of the following bovine prepro A subunit, bovine pro B subunit, human prepro A subunit or human pro B subunit. A recombinant DNA molecule comprising an inserted DNA and a vector DNA. Bovine prepro A subunit: Bovine pro B subunit: Human prepro A subunit: Human pro-B subunit: 6. The recombinant DNA molecule according to claim 5, wherein said vector DNA comprises a plasmid, virus or bacteriophage DNA. (7) pBR322, pUR290, pUR291, pUR292, pBTA286, pU
C7, pUC8, pUC9, pUC13, ptrpL1, pWT111, pWT121 or pWT131
6. The recombinant DNA molecule according to claim 5, comprising a plasmid selected from the group consisting of: (a) and said plasmid wherein said inserted DNA is inserted. 8. The recombinant DNA molecule according to claim 5, wherein the expression control sequence is effectively bound to the inserted DNA. 9. The expression control sequence according to claim 1, wherein said expression control sequence is selected from the β-galactosidase gene of E. coli, trp operon, left promoter of bacteriophage λ or long terminal repeat unit of Moloney leukemia virus. Item 8. The recombinant DNA molecule according to item 8. 10. The recombinant DNA molecule according to claim 8 or 9, wherein said expression control sequence comprises a promoter and a translation initiation signal. 11. A DNA encoding a bovine or human inhibin subunit, which encodes the amino acid sequence of each of the following bovine prepro-A subunit, bovine pro-B subunit, human prepro-A subunit or human pro-B subunit: A host transformed with a recombinant DNA molecule, which comprises an inserted DNA and a vector DNA. Bovine prepro A subunit: Bovine pro B subunit: Human prepro A subunit: Human pro-B subunit: 12. The host according to claim 11, wherein said host is selected from bacterial cells, bacteriophages, yeast, fungi, eukaryotic cells including vertebrates, and plant cells. 13. The host according to claim 11, wherein said host is capable of expressing each amino acid sequence of said bovine prepro A subunit, bovine pro B subunit, human prepro A subunit or human pro B subunit. 14. The host of claim 13, wherein the 43 kD or 15 kD subunit of inhibin can be expressed as a non-glycosylated form. 15. The host of claim 14, wherein the 43 kD or 15 kD subunit of said inhibin corresponds in sequence to a human or bovine inhibin. 16. The bovine prepro-A subunit from -60 to 16
Amino acid sequence of 6, bovine prepro A subunit -10 to 1
66 amino acid sequences, from bovine prepro A subunit 1
166 amino acid sequences, human prepro A subunit -6
Capable of expressing the amino acid sequence of 1 to 171; the amino acid sequence of -10 to 171 of the human prepro-A subunit; and the amino acid sequence of 1 to 171 of the human prepro-A subunit.
13. The host according to claim 11 or 12.