Porcine Beta FSH

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

Mammals, including humans, secrete several fertility hormones, e.g., leuteinizing hormone (LH) and follicle stimulating hormone (FSH), which are heterodimeric and, within a species, share a common alpha chain, the beta chain of each conferring specificity. It is also known that the FSH of a number of mammalian species is similar enough so that administration of FSH obtained from the pituitary gland of one species is biologically effective in another. Porcine FSH has been shown to induce superovulation in cattle, pigs, sheep, and horses.

In general, the invention features, in one aspect, a cDNA sequence encoding porcine beta FSH.

In another aspect, the invention features a vector, preferably a plasmid, containing a DNA sequence encoding porcine beta FSH.

The invention can permit the production of highly purified porcine beta FSH, without contamination from LH or other hormones.

The cDNA of the invention is superior to genomic porcine beta FSH DNA because the cDNA does not include untranslated introns, and therefore can be more easily used in prokaryotic host cells.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

The drawings will first be described.

FIG. 1 is a diagrammatic representation of the porcine beta FSH gene, including flanking regions, showing unique restriction sites, and illustrating two DNA regions which were fused to form the complete porcine beta FSH coding sequence.

FIG. 2 is a diagrammatic representation of a procedure for inserting the porcine beta FSH gene into an expression vector.

Table 1 shows the nucleotide sequence which has been determined for the porcine beta FSH cDNA of the invention. The numbers indicate numbers of base pairs; the initial 53 flanking, non-coding base pairs are not given. The initial (5') base pairs shown in Table 1 are untranslated. There is a box around the ATG (bases 108-111) which begins the leader sequence, which is cleaved post-translationally. The mature protein begins with the amino acid Cys encoded by the circled triplet TGT, bases 169-171. There is a box around base numbers 494-496, TAA, the end of the coding region.

Table 2 is the sequence of Table 1, not broken into triplets, showing restriction sites; the ATG beginning the coding region is boxed.

The coding sequence of Tables 1 and 2 encodes porcine beta FSH whose amino acid sequence differs in a number of respects from that which has been published in Closset et al. (1978) Eur. J. Biochem. 86: 115. The differences are summarized in Table 3.

The first step in the production of porcine beta FSH cDNA was to remove the pituitary glands from pigs 20-30 minutes after death. Next, total RNA was extracted, as follows. Homogenization of the tissue was carried out in a 1:1 mixture of phenol:100 mM Na-acetate (pH 5.5) containing 1 mM EDTA, warmed to 60.degree., for 20 min. After cooling on ice for 10 min., the phases were separated by centrifugation. The hot phenol extraction was repeated twice more followed by two extractions with chloroform.

RNA was precipitated from the final aqueous phase by the addition of 2.5 volumes of ethanol.

In order to enrich for poly A+ mRNA, RNA was passed over oligo (dT)-cellulose in 0.5M NaCl buffered with 10 mM Tris-HCl, pH 7.5, and washed with the same solution. Poly A+ mRNA was eluted with 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.05% SDS, and precipitated twice with ethanol.

A porcine beta FSH cDNA library was constructed by reverse transcription of pituitary mRNA, second strand synthesis using E. coli DNA polymerase I (large fragment), treatment with SI nuclease, and homopolymer tailing (dC) with terminal deoxynucleotidyl transferase; all such procedures were by conventional techniques.

The cDNA molecules were then annealed to DNA fragments of the plasmid pBR322, which had been digested with PstI, and to which dG "tails" have been added. These recombinant plasmids were then used to transform E. coil cells to generate a cDNA library (transformed cells were selected on the basis of tetracycline resistance).

The cDNA library was then screened using "guessed" long probes; the basic concept of such probes, set forth in Jaye et al. (1983) Nucleic Acids Research 11 (8), 2325, is that, if the amino acid sequence of a desired protein is at least partially known, a long probe can be constructed in which educated guesses are made as to the triplet encoding any amino acid which can be encoded by more than one and not more than four different triplets. Any correct guesses increase the amount of homology, and improve the specificity, of the results. To obtain desired regions of cDNA, two labeled 45-mer probes were used: TB36, homologous with amino acids 56-70 of porcine beta FSH, and TB21, homologous with amino acids 72-87. These probes have the following nucleotide compositions (corresponding amino acids are also given):

The above probes were used to screen the cDNA library as follows. The probes were labeled with .sup.32 P and used to screen the cDNA library by the colony hybridization procedure of Grunstein et al. (1975) PNAS USA 72, 3961. The prehybridization solution was maintained at 55.degree. C. and had the composition: 0.75M NaCl; 0.15M Tris HCl, pH 8.0; 10mM EDTA; 5.times. 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 maintained at 42.degree. C., and contained probe in a concentration of about 0.5.times.10.sup.6 cpm/ml.

The hybridization screening procedure yielded two cDNA sequences, PF55 and PF434, shown in FIG. 1, which also illustrates their relationship with the entire porcine beta FSH sequence. The two sequences were cut with AvaI, which cuts at the unique SmaI site, and fused to form the complete porcine beta FSH clone, with untranslated flanking regions. The cDNA sequence, inserted into the E. coli plasmid pBR322, has been deposited in the Agricultural Research Culture Collection (NRRL), Peoria, IL. and given NRRL Accession Number B-15793. It is agreed that, upon issuance of a patent on this application, all restrictions on availability of the deposited plasmid will be irrevocably removed, and that the plasmid will be maintained throughout the life of the patent.

The porcine beta FSH cDNA sequence of the invention can be inserted, using conventional techniques, into any of a variety of expression vectors, for the production of porcine beta FSH.

Referring to FIG. 2, the porcine beta FSH cDNA sequence can, for example, be inserted into a bovine papilloma virus (BPV) expression vector in which the cDNA sequence is under the control of the mouse metallothionein (MMT) promoter. The first step in this procedure will be to cut the cDNA out of pBR322 using PstI and SacI, which will cut at the 5' end just downstream from the ATG, and at the 3' end, downstream from the end of the coding region (see restriction map, above). A synthetic sequence will be added to the 5' end, which will complete the gene up to ATG and contain a BamHI overhang. At the 3' end, the SacI overhang will be made blunt and a BamHI linker will be attached. The modified sequence will be inserted into pBR322 at the Bam site to form plasmid pFSH 55/434 Bam (FIG. 2).

Next, the plasmid CL28 (identical to plasmid JYMMT(E); Hamer et al. (1983) J. Mol. Applied Gen. 1, 273-288), containing the murine metallothionein promoter, SV40 DNA, and the pBR322 sequences, is cut with the restriction endonuclease BglII. At this site is inserted the FSH cDNA, containing an untranslated region at the 3' end.

The resulting plasmid is digested with the restriction enzymes BamHI and SalI to release the SV40 DNA sequences.

Plasmid pB2-2, which contains the entire BPV genome, and some pBR322 sequences, is digested with BamHI and SalI to yield the BPV genome with BamHI/SalI ends; this fragment is ligated into the above plasmid containing the metallothionein-FSH sequences, to form expression plasmid ppFSH, as shown.

Plasmid ppFSH can then be used to transform mammalian host cells, and porcine beta FSH can be isolated and purified, according to conventional methods.

The porcine beta FSH can be used to produce anti-beta FSH antibodies, using conventional methods, which can be labeled and used in radioimmunoassays of biological fluids of pigs and other animals, as well as humans, for beta FSH. Measuring beta FSH levels can be important in the diagnosis, monitoring, and characterization of pituitary tumors and other pituitary malfunctions.

The porcine beta FSH of the invention can also be used, alone or in combination with alpha FSH, to induce superovulation in farm animals, e.g., pigs, cattle, horses, sheep, and goats. Alpha FSH (bovine) has been cloned and sequenced and described in Erwin et al. (1983) Biochemistry 22, 4856. FSH is also available commercially, e.g. from the Reheis division of Armour Pharmaceutical Company, Kankakee, IL., and the alpha subunit could be obtained from the commercial dimer. Beta and alpha FSH can associate into the dimeric form spontaneously, with no special reaction conditions being required. Also, the species from which the two subunits are derived may not need to be the same for the formation of a functional dimer.

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

The porcine beta FSH of the invention can be administered to animals according to established FSH administration techniques and dosages, whether administered alone or in the form of a dimer with alpha FSH. Beta FSh alone can be effective because the pituitary normally overproduces the alpha subunit, which is available for dimer formation with beta FSH in vivo.

Other embodiments are with the following claims.