DE3520404A1 - Porcine beta -FSH - Google Patents

Abstract

A cDNA sequence which encodes porcine beta -FSH.

Description

description

The invention relates to fertility hormones produced by recombinant DNA techniques.

Mammals, including humans, secrete various fertility hormones, e.g. B. luteinizing Hormone (LH) and the follut stimulating hormone (FSH), die heterodim.0: are and share a common alpha chain within a species, the ß-chain in each case confers a specificity. It is also known that FSH is sufficiently similar to a number of mammal species' so that the administration of an FSH obtained from the pituitary gland of one kind in the other is biologically effective. Pig FSH has been shown to induce superovulation in cattle, pigs, sheep and horses.

In general, in one aspect, the invention relates to a cDNA sequence encoding porcine β-FSH.

According to another embodiment, the invention relates to a vector, preferably a plasmid, containing a DNA sequence encoding porcine β-FSH.

The invention permits the production of highly purified porcine β-FSH without contamination with LH or other hormones.

The cDNA according to the invention is domestically genomic Porcine β-FSH DNA is superior because the cDNA does not contain nontransmitting introns and is therefore easier to move into

procaryotic host cells can be used.

Other features and advantages of the invention will appear from the following description of the preferred Embodiments and from the claims.

The accompanying drawings are explained first. Show it:

Fig. 1 shows the diagrammatic representation of the

Pig ß-FSH gene, including the flanking regions, the Show unique restriction sites and explain two regions of DNA that have been fused to form the complete one To form code sequence of porcine ß-FSH,

Fig. 2 ″ is a diagrammatic representation

a method of introducing the gene of porcine β-FSH into one Expression vector.

Pig β-FSH cDNA

The following shows the nucleotide sequence which has been determined for the porcine β-FSH cDNA according to the invention. The numbers indicate numbers of base pairs, the 53 flanking, non-coding base pairs shown at the beginning not being shown. The initial (5 ¹ ) base pairs shown are not translated. Around the ATG (bases 100 and 108-111), which leads the guide sequence, a box is attached · ^ w ^ split after the transfer. . The mature protein begins

with the amino acid Cys, which is encoded by the circled triplet TGT, bases 169-171. To get the base numbers 494 - 496, TAA, a conversion has taken place at the end of the coding area.

GTA CTT VaI Lau

TCB CTS Str Lau

CTQ ACC Leu Thr

ACC ACG Thr Thr

CCC AAC Per Asn

GGC TQT GIy Cys

GGC AAG

GIy Lys

TCC TTC Sar Ph

ATM GGA GIu GIy

AGA GAC Arg Asp

GGG CCT GIy Pro

CGO TCT OVq Sar

TCA CGG TCT CGT ACA CCA GCT CCT Sar Arq Sw Arq Thr Pro Ala Pro

CAG TTT TGC TTC CTA TTC TGT TGC Gin Pha Cys Ph Leu Ph Cys Cys

AAC ATC ACC ATC ACA STS GAG AAA Asn Ua Thr 11Dir VaI GIu Lvs

TGG TOT GCT 6GC TAT TGC TAC ACC Trp Cys Ala Bly Tyr Cys Tyr Thr

ATC CAG 11 · GIn

GCT CAC Ala His

TGT GAC Cys Asp

AGT GAA Sw GIu

CCA AGA Per arq

CCC ACT Per Thr

CAG TCC UIn Sw

AAA ACA Lys Thr

CAT GCA His Ala

AGT GAC

Sar Asp

ATB AAA Mat Lys

COT CCA Arq Pro

GAT CCC Asp Pro

CAT CAC His His

TTT TTT '

Ph · Ph · I

TGT ACC TTC AAG Cys Thr Ph · Lys

GAC TCC CTB TAT Asp Sat 'Lau Tyr

AOT ACT GAC TGC Sar Thr Asp Cys

,, 30Ϊ

GAA TAAIAGA GCA 61u | E £ dJArg AIa

AGA AGT TTG TGT Arq Sw Lau Cys

TGC TGT CCT GTO Cys Cys Pro VaI

CAC TCA ACC CTG His Sar Thr Lau

I TTA CTC AAT CAA> Lau Lau Asn älu TAA TTS TTT GGT TTC CAC CCC AAs IaTGI AAU ι End Lau Ph tilv Ph His Pro LvS riac Lvs'

TGG AAA GCC ATC TGC TGC AAT AGC (rÖj> SAG | Trp Lys AIa 11 · Cys Cys Asn Sar Cys GIu

232 |

GAG 'GAG TGT AAC TTC TGC ATA AGC ATC AAC I älu GIu Cys Asn rti * Cys Ua Sw II · Asn I

292!

CGS GAC CTG GTA TAC AAG GAC CCA GCC AGG I Arq Asp Lau VaI Tyr Lys Asp Pro AIa Arq

132!

GAS CTG GTG TAC GAS ACC GTG AAA GTA CCT GIu Lau VaI Tyr BIu Thr VaI Lys VaI Pro

ACB TAT CCA BTA SCC ACC GAA TGT CAC TGT Thr Tyr Pro VaI AIa Thr SIu Cys His Cys

472

ACC BTB AGA GGC CTG GGG CCC AGC TAC TGC Thr VaI Arq Bly Lau Bly per Sw Tyr Cys

GTS GAC ΛΤΤ TCA TGC TTC CTA CCC TTS TCT VaI Asp Ha Sar CyS Ph Lau Pro Lau Sar

BTA CAT STB CCC AGB CTG CAA ACC ACT ATG VaI His VaI Pro Arq Lau Bin Thr Thr Mat

»52 I.

GA6 6AS GAS CTC CAG BAA TGC AGA GTG CTA I GIu BIu SIu Lau Gin SIu Cys Arq VaI Lau

TAT TTT BGS TCT GOT TCC ATA AGT TTT ATT Tyr Ph · Sly Sar Bly Sar II · sw Ph · Ils

TTT TAT TAC ATT TAT AAT TQT AGC AAO GAT Ph Tyr Tyr II Tyr Asn CvS Sar Lys Asp

CAT CAC AA His His

The following is a reproduction of the above sequences, which are not broken up into triplets and show restriction sites. The beginning of the code area is boxed in with the ATG.

! .2 1-2 E2 ·. ·: Cc

^ TiCTTTCOC G3TCTCGTAC ACCAGCTCCT TSATTGTTTG GTTTCCACCC

I 1 1

1 = 2 t "2 112 IS3 1S2 <*;

rcGCTGCAGT TTTGCTTCCT ATTCTGTTOC TGBAA (HiCCA TCTGCTGCAA TAGCTGTGeG W 6 f. R.

BS BLL

vT υ u υ

u t t t

1B2 I «2 102 ~ 12 ZZZ ZZ? CTEACCAACA TCACCATCAC AGITJGAGAAA GASGAGTGTA ACTTCTGCAT AAGCATCaAC HM Ii

PN F

HL S

1 I L

242 ZZZ 2bZ ZTZ TB = Λ BCCACGTEGT GTGCTGGCTA TTGCTACACC CGSGACCTGG TATACAAGGA CCCAGCCAUG! = NAHS H ο S MC VSNV ä A AI ATAA Γ U 11 2 1 1 Z 11

jO2 -12 ;;: zzz --2 rs;

CCCAACATCC AGAAAACATG TACCTTCAAS GAGCTGSTGT ACGAGACCGT GAAAGTACCT P AR. ¹ AR r <B

0 F 3 LS 3 S K LA UA AT

1 3 1 Il 11

342 T.73 -32 --J2 5Π2 412 GSCTBTGCTC ACCATGCASA CTCCCTGTAT ACGTATCCaS TAGCCACCGA ATGTCACTGT HH H. S ■

BP I N

IH M A

Il 1 1

»22 4-52 44Ϊ 452 442 472 GGCAAQTQTS ACAQTGACAG TACTSACTGC ACCBTSAGAS GCCTGGGGCC CAGCTACTGC RR Π5Η a AS A

RS NTA S PA L

UA LUE T AU U

11 113 1 11 1

482 492 503 S12 S22 S3! TCCTTCASTG AAftTBAAASA ATAAAGAGCA BTGGACATTT CATGCTTCCT ACCCTTGTCT

= 42 552 = & 2 372 IB2 392 GAAS6ACCAA EACSTCCAAG AAGTTTGTGT GTACATGTGC CCAGGCTGCA AACCACTATG AA IRABB

VA * S F B B

ft T ft L T V

-22 13 11

6Ο2 il2 622 632 642 6S2 ASASACCCCA CTGATCCCTG CTGTCCTGTG BAGGAGSAGC TCCAGGAATS CASAGTGCTA B H H SA B

I N N AL S

N L L CU T

t I I 11 1

662 672 682 692 702 712

EEGCCTCAST CCCATCACCA CTCAACCCTS TATTTTGGGT CTGSTTCCAT AAGTTTTATT

SH no H

AA NO P UE LE H 13 11 1

722 732 742 752 762 773 'CGSTCTTTTT TTTTTAAATT ACTCAATBAA TTTTATTACA TTTATAATTB TAOCAASGAT

α it

H IA

A NU

3 »A

The code sequences given above encode porcine β-FSH, the amino acid sequence of which is in a number of factors differ from that reported in Closset et al, (1978) Eur. J. Biochem. 86, 115 is described. The differences can be summarized as follows.

amino acid Closset et al. Pig ß-FSH, number encoded with cDNA (according to the invention) 2 GIx GIu 11th GIx GIu 12th VaI Lys 13th Lys GIu 14th Cys GIu 15th Leu Cys 16 Th r As η 31 Thr Tyr 33 Gly 35 ASX Asp 40 ASX Asp 45 Asx Asn 47 GIx gin 52 Tyr Phe 53 Arg Lys 54 Glx GIu 58 GIx GIu 70 Asx Asp 80 Glx GIu 87 ASX Asp 89 ASX Asp 92 Asx Asp 106 GIy Ser 108-110 . - Met-Lys-Gli

Production of cDNA

The first step in the production of porcine β-FSH cDNA was the removal of the pituitary glands Pigs 20 to 30 minutes after death. Thereafter, the total RNA was extracted as follows. The homogenization

of the tissue in a mixture of phenol: 100mM sodium acetate in the ratio 1: 1 (pH 5, S) ₇ 1 mmol EDTA, carried out the mixture was heated to 60 ⁰ C for 20 to 40 minutes. After cooling on ice for 10 min, the phases were separated by centrifugation. The hot phenol extraction was repeated twice, followed by two extractions with chloroform.

& NS was made from the last aqueous phase by adding precipitated from 2.5 parts by volume of ethanol.

In order to enrich the RNA in polyacids + mRNA, it was buffered over oligo- (dT) -cellulose in 0.5 M NaCl run with 10 mmol Tris-HCl of pH 7.5 ... left and washed with the same solution. Polyacid + mRNA was treated with 10 mmol Tris-HCl (pH 7.5), 1 mmol EDTA and 0.05% SDS eluted and precipitated twice with ethanol.

A library of porcine β-FSH cDNA was constructed by reverse transcription from pituitary mRNA according to the synthesis on the second phase, using E. coli DNA polymerase I (large fragment), Treatment with SI nuclease and appending a homopolymer isats (de) terminated with deoxynucleotidyltransferase, all of these procedures according to usual techniques were carried out.

The cDNA molecules were then attached to DNA fragments of the Plasmids pBR322 "tempered", which were digested with PstI and to which dG "tails" were attached. These recombined plasmids were then used to transform E. coli cells and generate a cDNA library (using transformed cells selected on the basis of tetracycline resistance became).

The cDNA library was then sieved, using of "guessed" long probes, the basic concept of such probes in Jaye et al. (1983) Nucleic Acids Research 11 (8), page 2325, and is that when the amino acid sequence of one desired protein is at least partially known, a long probe can be constructed in which thoughtful questions regarding triplet coding of each amino acid are made '... by which each amino acid is encoded which is encoded by more than one and no more than four different triplets can be. All of the right questions increase the amount of homology and increase the specificity of the results. To obtain desired regions from cDNA, two labeled 45-mer probes are used: TB36, the one with Amino acids 56 to 70 are homologous to porcine β-FSH, and TB21, which is homologous to amino acids 72-87. These Probes have the following nucleotide composition (corresponding amino acids are also shown):

TB36: Val-Tyr-Glu-Thr-Val-Lys-Val- (AA56-70) 3 'CAC ATG CTC TGG CAC TCT CAC

Pro-Gly-Cys-Ala-His-His-Ala-Asp GGT CCG ACG CGG GTG GTG CGA CTG

TB21: Tyr-Thr-Tyr-Pro-Val-Ala-Thr- (AA72-87) 3 'ATG TGC ATG GGT CAC CGA TGT

GIu-Cys-His-Cys-Gly-Lys-Cys-Asp CTC ACA GTG ACG CCG TTT ACG CTG

The named probes were used to scan the cDNA library as follows. The probes were with

P marked and used for scanning the cDNA library

the colony hybridization method of Grunstein et al. (1975) PNAS USA 72, page 3961 used. The for

Prahybridisierung solution used was maintained at 55 ⁰ C and had the following composition 0.75 M NaCl; 0.15 M Tris-HCl, pH 8.0; 10 mmoles EDTA; 5 χ Denhardt's solution; 0.1% sodium pyrophosphate; 0.1% SDS; 100 g / ml E. coli t-RNA. The hybridization solution had the same composition except that it was kept at 42 ⁰ C and a probe in a concentration of about 0.4 χ 10 cpm / ml.

The hybridization scanning procedure revealed two cDNA sequences, namely PP55 and PF434, which are shown in FIG which also shows its relationship to the entire sequence of porcine β-FSH. The two Sequences were cut with Aval, these cuts being made at the single Smal site and the Both sequences were fused to form the complete porcine β-FSH clone with untranslated en flanking areas. The cDNA sequence introduced into E. coli plasmid pBR322 was laid down in the Agricultural Research Culture Collection (NRRL), Peoria, IL, with NRRL No. B-15793. J-Es in agreement found that after a Patents all restrictions subject to availability of the laid down P] »asfrrjr9sunwiderruf lent away be and ^ jäarer ^ as plasmid over the life of the but

Insertion into an expression vector

The porcine β-FSH cDNA sequence according to the invention can be converted into any one using conventional techniques A variety of expression vectors for the production of porcine ß-FSH can be inserted using common practice.

Referring to Fig. 2, it should be noted that the porcine β-FSH cDNA sequence, for example, in a Bovine papilloma virus (BPV) expression vector can be inserted in which the cDNA sequence is under control of the promoter for mouse metallothionein (MMT) is. The first step in this procedure is to cut the cDNA from the pBR322, below Use of Pstl and Sacl, which are immediately at the 5 'end and cuts below the ATG and below at the 3 'end the end of the coding region (see the restriction map above). A synthetic sequence will be attached at the 5 'end, which completes the gene up to the ATG and contains an overhang BamHI. At the 3 'end the overhang of Sacl is blunted and a BamHI connector attached. The modified one Sequence is inserted into pBR322 at the Barn site, forming the plasmid pFSH 55/434 Bam (FIG. 2).

The plasmid CL28 (which is identical to the plasmid JYMMT (E); cf. Hamer et al. (1983) J. Mol. Applied Gen. 1, pages 273-288) and the mouse metallothionein promoter as well as SV40 DNA and the pBR322 sequences with the restriction endonuclease BgIII cut. At this point the FSH cDNA is inserted, which has an untranslated region at the 3 'end having.

The plasmid obtained is digested with the restriction enzymes BamHI and Sail to reveal the SV40 DNA sequences to release.

The plasmid pB2-2, which contains the entire genome BPV and some pBR322 sequences, is digested with BamHI and Sail and gives the BPV genome with BamHI / SalI ends; this fragment is transferred to the abovementioned plasmid, which contains the metallothionein FSH sequences.

constricts to form the expression plasmid ppFSH, as stated.

The plasmid ppFSH can then be used to transform mammalian host cells and porcine β-FSH can be isolated and purified according to standard procedures.

use

The porcine ß-FSH can be used for production of anti-ß-FSH antibodies, using conventional methods, which are labeled and in radioimmune samples from biological fluids from pigs and other animals can be used, as well as in humans, at the ß-FSH. Measuring ß-FSH levels can be important for diagnosis, recording and characterization of pituitary tumors and other pituitary dysfunction.

The porcine β-FSH according to the invention can also be used alone or in combination with alpha-FSH for Induction of superovulation in pets, e.g. B. pigs, cattle, horses, sheep and goats. Alpha-FSH (from cattle) has been cloned and sequenced and described by Erwin et al. (1983) Biochemistry 22, page 4356. FSH is also available commercially, e.g. B. Reheis division, Armor Pharmaceutical Company, Kankakee, IL, USA, and the alpha subunit could be obtained from the commercially available dimer, ß- and alpha-FSH can spontaneously combine to form the dimeric form without the need for special reaction conditions. It also needs the species, 'on from which the two subunits are derived may not be the same to form a functional dimer.

Other uses of porcine FSH are the treatment of genital infantilism, gonadal regression, incomplete follicular development, persistent luteal cysts, aspermia, and infertility Animals.

The porcine β-FSH according to the invention can be administered to animals according to conventional procedures and dosages for FSH administration administered, either alone or in the form of the dimer with alpha-FSH. ß-FSH alone can be effective because the pituitary gland normally overproduces the alpha subunit necessary for dimer formation with ß-FSH is available in vivo.

Other embodiments are within the scope of the claims possible.

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Claims (3)

APPROVED BY THE EUROPEAN PATENT OFFICE EUROPEAN PATENT ATTORNEYS MANDATAIRES EN BREVETS EUROPEENS DR. DIETJ-R: vf ^ iP DIPL. ING. PETE * R SCHÜTZ DIPL. ING. WOLFGANG HEUSLER PATENTANWÄLTE MARlA-THEREStA-STRASSE 23 POST BOX 86 02 60 D-eOOO MUENCHEN TELEPHONE (0691 4706006 TELEX 522 638 TELEGRAM SOMBEZ FAX CR II + III (0891 2 7160 Massachusetts 6, INTEGRATED GENETICS V St. A. 818 Dr.Be/nc pig-ß-FSH patent claims 1. A cDNA sequence encoding porcine β-FSH. 2. Vector containing a DNA sequence which encodes porcine ß-FSH. 3. The vector of claim 2, wherein the vector is a plasmid is.