CA2262412A1 - Porcine leptin protein, nucleic acid sequences coding therefor and uses thereof - Google Patents

Abstract

A porcine adipocyte-specific polypeptide, termed leptin, is expressed in the fat tissue of pigs. Expression may be altered in over fat pigs, or expression may be in the form of a protein of lesser biological activity relative to that of leaner pigs. The porcing adipocyte polypeptide, DNA and RNA molecules coding therefor, methods for its preparation, and antibodies specific for the polypeptide are disclosed. Methods for determining the susceptibility of a pig to fat deposition are based on measuring the levels of the porcine adipocyte polypeptide in a biological fluid or tissue extract or by measuring mRNA encoding the porcine adipocyte polypeptide in cells of the subject. Methods of evaluating an agent related to the deposition of fat in swine comprise contacting the agent with an adipocyte in vitro and measuring the amount of the porcine adipocyte polypeptide or mRNA that is produced by the adipocyte. Methods of limiting fat deposition include administering porcine leptin or porcine leptin DNA, and methods of regulating intake include administering porcine leptin, porcine leptin DNA, or an antibody directed against porcine leptin.

Description

Wo 98104690 PcTIuss7ll2483 PORCINE LEPTIN PROTEIN, NUCLEIC ACID SEQUENCES CODING THEREFOR AND USES THEREOF

BACKGROUND OF THE INVENTION
1. Field of the Invention:
This invention relates to the regulation of energy intake and metabolism in growing, fini~hing, l~rtAting or nonlactating, and gest~ting swine. More specifically, it relates to a specific porcine polypeptide termed leptin which is secreted by adipocytes or other cell types and which influences energy intake and metabolism, fat deposition, and weight gain in swine. In addition, this invention relates to thenucleotide sequences encoding the porcine leptin polypeptide, the antibodies directed 0 against the porcine leptin polypeptide, and methods to determine susceptibility to fat deposition, alter energy intake, and minimi7e excessive fat deposition in swine.

2. Description of the Background Art:
Obesity has been declared a public health hazard by the National Tn~tihltes of Health and has prompted the food animal industry to seek methods of limiring fatdeposition in food animals. Additionally, the energetic cost of having food anim~l~
convert feed energy to fat rather than lean tissue provides considerable incentive to develop technology to facilitate the efficient production of leaner meat products and to accurately match the nutrient content of the diet to the nutrient needs of the animal.
2 o To combat these health and production problems, both prophylactic and therapeutic approaches are necessary. For prophylactic purposes, it would be useful to be able to predict and measure the propensity or susceptibility to excessive fat deposition. For Wo ,~ 1690 PCT/US97/12483 therapeutic purposes, it would be of great benefit to improve current methods ofminimi7ing the deposition of feed energy as fat in the adipocyte. Currently, neither of these desired objectives has been achieved completely.

Proteins from genes expressed only (or predomin~ntly) in adipose tissue and for which the level of expression can be related to fat deposition serve as prime targets for approaches directed toward prediction of fat accretion potential and the control of fat deposition. For example, a m~mm~ n adipocyte-specific polypeptide, termed plS4, was reported in USP 5,268,295 to Serrero, which is incorporated in its lo entirety herein by reference, as being expressed in high ql~ntities in adipogenic cell lines after cell differentiation and is abundant in the fat pads of normal and genetically obese mice. To date, however, there have been no reports of adipocyte-specific proteins expressed at dirrelclll levels in fat swine as compared with normal controls.

Leptin, the protein produced by the leptin (ob) gene, is possibly related to fatdeposition in swine because research has shown that mutations in genetically (oblob) obese mice resulting in excessive fat deposition are associated with altered expression of the leptin gene. Furthermore, at least one restriction fragment length polymorphism (RFLP) has been identified and related to the fat phenotype (Zhang et al., l9g4, Nature 371:425). The leptin gene is expressed specifically in the terminally differentiated adipocyte (Maffei et al., 1995, Proc. Natl. Acad. Sci. 92:6957; Leroy et al., 1996, J. Biol. Chem. 271(5):2365). Additionally, leptin is a regulator of feed intake (Pellymounter et al., 1995, Sci. 269:540; Halaas et al., 1995, Sci. 269:543;
Campfield et al., 1995, Sci. 269:546).

wo 98/04690 PCT/US97/12483 Although the murine leptin gene has been positionally cloned and a cDNA
sequence reported (Nature 371:425), neither the porcine leptin cDNA or genomic sequence is available. Thus, the insights obtained with respect to porcine metabolism is not accessible to porcine systems. Furthermore, the biologically active purified porcine protein (i.e., leptin) has not been obtained.

SUMMARY OF THE INVENTION
The present invention provides gene sequences, peptides, antibodies, and methods of using them which permit the prediction and modulation of fat deposition 0 and regulation of feed intake (i.e. appetite) in the porcine species.

In one aspect, this invention is directed to a porcine adipocyte polypeptide, the porcine leptin protein, subst~nti~lly free of other porcine proteins, or functional derivatives thereof. The present invention includes a porcine adipocyte polypeptide of at least about 8 amino acids of the amino acid sequence depicted in Figure l, preferably the amino acid sequence depicted in Figure 2, still more preferably, the amino acid sequence depicted in Figure 3, or functional derivatives thereof.

The present invention is also directed to a single or double stranded DNA
molecule or an RNA molecule consisting essentially of a nucleotide sequence thatencodes the above polypeptide or functional derivatives thereof, the DNA or RNA
molecule being substantially free of other porcine DNA or RNA sequences. The DNA molecule is preferably a single or double stranded DNA molecule having a nucleotide sequence consisting essentially of at least about 20 nucleotides of the Wo 9~169~ PCT/US97/12483 nucleotide sequence depicted in Figure l, preferably, the nucleotide sequence depicted in Figure 2, still more preferably the nucleotide sequence depicted in Figure 3 or a sequence complementary to the nucleotide sequences depicted in Figures 1-3, substantially free of other porcine DNA sequences. The RNA molecule is preferably an mRNA sequence encoding the above porcine adipocyte polypeptide, or functionalderivatives thereof.

Included in the invention is a DNA molecule as described above which is cDNA or genomic DNA, preferably in the form of an expressible vehicle or plasmid.

The present invention is also directed to hosts transformed or transfected with the above DNA molecules, including a prokaryotic host, preferably a bacterium, aeukaryotic host such as a yeast cell, or a m~mm~ n cell.

The present invention also provides a process for preparing a porcine adipocyte polypeptide or functional derivatives as described above, the process comprising the steps of: (a) culturing a host capable of expressing the polypeptide under culture conditions; (b) expressing the polypeptide; and (c) recovering the polypeptide from the culture.

Also included in the present invention is a method for ~letectin~ the presence of a nucleic acid molecule having the sequence of the DNA molecule described above, or a complem~nt~ry sequence, in a nucleic acid-cont~ining sample, the method WO ~/01L9~ PCT/US97/12483 - -comprising: (a) contacting the sample with an oligonucleotide probe complementary to the sequence of interest under hybridizing conditions; and (b) measuring the hybridization of the probe to the nucleic acid molecule, thereby detecting the presence of the nucleic acid molecule. The above method may additionally comprise before step (a): (c) selectively amplifying the number of copies of the nucleic acid sequence.

Another embodiment of this invention is an antibody specific for an epitope of the porcine adipocyte polypeptide, or functional derivatives thereof, either polyclonal or monoclonal. Also intended is a method for detecting the presence or measuring the 0 ~uantity of the porcine adipocyte polypeptide leptin in a biological sample, comprising contacting the sample with the above antibody and detecting the binding of the antibody to an antigen in the sample, or measuring the quantity of antibody bound.

The present invention includes methods for determining the susceptibility of swine to fat deposition which comprises removing a biological sample from a pig and measuring therein the amount of the polypeptide or mRNA coding therefor, where the amount of the polypeptide or mRNA is related to susceptibility. The present invention also includes methods for determining the susceptibility of a subject to fat deposition which comprises removing a biological sample, extracting the DNA, digesting the 2 o DNA with restriction endonucleases, probing the sample with an oligonucleotide probe, separating the resulting fragments by gel electrophoresis, and relating the number of bands (banding pattern) generated by restriction enzyme digestion to fat ~ deposition (i.e., RFLP techniques).

Wo 98/04690 PCT~S97/12483 Another method provided herein is for evaluating the efficacy of a drug (or other agent) directed to the regulation of fat deposition and feed intake which comprises con~cting the drug being tested with an adipocyte culture in vitro andmeasuring the amount of the porcine adipocyte polypeptide or mRNA that is produced by the adipocyte, the efficacy of the drug or agent being related to changing the production of the polypeptide or mRNA.

BRIEF DESCRIPTION OF THE D~AWINGS
FIG. 1 depicts the nucleotide sequence of the porcine leptin gene and the 0 amino acid translation of the porcine leptin coding sequences.

FIG. 2 depicts the nucleotide sequence and the amino acid translation of the coding region of the entire porcine leptin cDNA (i.e., signal peptide and secreted protein).
FIG. 3 depicts the nucleotide sequence and the amino acid translation of the porcine leptin cDNA corresponding to the secreted porcine leptin protein.

FIG. 4 shows a comparison of the porcine leptin cDNA sequence 2 o corresponding to the entire porcine leptin protein with the murine and human sequences.

FIG. 5 depicts the Northern blot analysis of porcine leptin mRNA.

wo 98104690 PCT/US97tl2483 FIG. 6 depicts the isolation of a genomic DNA clone for porcine leptin.

FIG. 7 depicts a polyacrylamide gel electrophoresis of porcine leptin protein induction and purification in Escherichia coli.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention is directed to DNA and RNA molecules that encode a porcine adipocyte polypeptide, termed "leptin," or a functional derivative thereof, and the porcine leptin protein itself, or a functional derivative thereof. The porcine leptin 0 protein is useful for regulation of feed intake, energy metabolism, and fat deposition in swine. Such objectives can be achieved by ~mini~tering recombinant or purified porcine leptin, altering the expression of the porcine leptin gene or a~lministering an antibody directed against the porcine leptin protein to achieve neutralization, depending on the desired result. The porcine leptin DNA, RNA, and protein, or functional derivatives thereof, and antibodies specific for the protein are used in assays to predict the potential for fat deposition in swine. These molecules can also be utilized in the development of commercially valuable technology for altering feed intake and regulating fat deposition in swine, and for m~tching the nutrient content of the diet to the nutrient needs of the pig.
In its first aspect, the present invention provides a porcine adipocyte polypeptide termed "leptin". The term "polypeptide" as used herein is intended to include not only the porcine leptin protein, and functional derivatives thereof, but also amino acid sequences having additional components, e.g., amino acid sequences Wo 98/04690 PCT/US97/12483 having additional components such as a sugar, as in a glycopeptide, or other modified protein structures known in the art.

The polypeptide of this invention has an amino acid sequence as depicted in Figures 1 and 2, and preferably as depicted in Figure 3. Also intended within the scope of the present invention is any peptide having at least about 8 amino acids present in the above-mentioned sequence. Sequences of this length are useful as antigens and for making immunogenic conjugates with carriers for the production of antibodies specific for various epitopes of the entire protein. Such peptides are also 0 useful in screening such antibodies and in the methods of the present invention directed to detection of the leptin protein in biological samples. It is well-known in the art that peptides of about 8 amino acids are useful in generation of antibodies to larger proteins of biological interest.

The polypeptide of this invention is sufficiently large to comprise an antigenically distinct determ~n~nt, or epitope, which can be used as an immunogen to produce antibodies against porcine leptin or a functional derivative thereof, and to test such antibodies. The polypeptide of this invention may also exist covalently or noncovalently bound to another molecule. For example it may be fused (i.e., a fusion 2 o protein) to one or more other polypeptides via one or more peptide bonds.

One embodiment includes the polypeptide substantially free of other porcine peptides. The polypeptide of the present invention may be biochemically or immunochemically purified from cells, tissues, or a biological fluid. Alternatively, Wo ~ 9~ PCTIUS97/12483 the polypeptide can be produced by recombinant means in a prokaryotic or eukaryotic host cell.

"Subst~nti~lly free of other porcine polypeptides" reflects the fact that because the gene for the porcine adipocyte polypeptide of interest can be cloned, the polypeptide can be expressed in a prokaryotic or eukaryotic organism, if desired.
Methods are also well known for the synthesis of polypeptides of a desired sequence on solid phase supports and their subsequent separation from the support.
Alternatively, the protein can be purified from tissue or fluids of the swine in which it 0 naturally occurs so that it is purified away from at least 90 percent (on a weight basis), and from even at least 99 percent if desired, of other porcine polypeptides and is therefore substantially free of them. That can be achieved by subjecting the tissue or fluids to standard protein purification techniques such as immunoadsorbent columns bearing monoclonal antibodies reactive against the protein. Alternatively, the purification from such tissue or fluids can be achieved by a combination of standard methods, such as ammonium sulfate precipitation, molecular sieve chromatography,and ion exchange chromatography.

As alternatives to a native purified or recombinant porcine adipocyte 2 0 polypeptide molecule, functional derivatives of the porcine adipocyte polypeptide may be used. As used herein, the term "functional derivative" refers to any "fragment", "variant", "analog", or "chP~nic~l derivative" of the porcine adipocyte polypeptide that retains at least a portion of the function of the porcine adipocyte polypeptide which permits its utility in accordance with the present invention.

Wo 98104690 PCT/US97/12483 A "fragment" of the porcine adipocyte polypeptide as used herein refers to any subset of the molecule, that is, a shorter peptide.

A "variant" of the porcine adipocyte polypeptide as used herein refers to a molecule substantially similar to either the entire peptide or a fragment thereof.
Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well-known in the art. Alternatively, amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synth~i7f~1 peptide (again using methods well-known in the art). Such 0 variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Obviously, the mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA
structures.

An "analog" of the porcine adipocyte polypeptide as used herein refers to a non-natural molecule substantially similar to either the entire molecule or a fragment 2 o thereof.

A "chemical derivative" of the porcine adipocyte polypeptide or peptide as used herein contains additional chemical moieties not normally a part of the polypeptide. Covalent modifications are included within the scope of this invention.

Wo ~8/01~ PcT/uss7tl2483 - -Such modifications may be introduced into the molecule by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

The polypeptide of the present invention is encoded by a nucleic acid molecule, one strand of which has the nucleotide sequence shown in Figure 1, preferably as shown in Figure 2, and still more preferably as shown in Figure 3. The present invention is directed to a DNA sequence encoding the polypeptide, or a functional derivative thereof, substantially free of other porcine DNA sequences.
Such DNA may be single stranded (i.e.~ sense strand, antisense strand or cDNA
sequence) or double stranded. The DNA sequence should preferably have about 20 or more nucleotides to allow hybridization to another polynucleotide. In order to achieve higher specificity of hybridization, characterized by the absence of hybridization to sequences other than those encoding the polypeptide or a functional derivative thereof~
a length of at least about 50 nucleotides is preferred.

The present invention is also directed to an RNA molecule comprising a mRNA sequence encoding the polypeptide of this invention, or a functional derivative thereof.

The present invention is further directed to the above DNA molecules which are functional in recombinant expression systems utili7.ing as hosts transfected or transformed with the vehicles and capable of e~ ressillg the polypeptide. Such hosts may be prokaryotic or eukaryotic. The DNA can be incorporated into the host W O 98/04690 PCTrUS97/12483 organism by transformation, tr~n~duction, transfection, or a related process known in the art.

In addition to a DNA and mRNA se~uence encoding the porcine adipocyte polypeptide molecule, this invention provides methods for expression of the nucleic acid sequences. Further, the genetic sequences and oligonucleotides of the invention allow the identification and cloning of additional? yet undiscovered adipocyte polypeptides having sequence homology to the adipocyte polypeptide described herein.

0 The recombinant DNA molecules of the present invention can be produced through any of a variety of means, such as, for example, DNA or RNA synthesis, or more preferably, by application of recombinant DNA techniques. Techniques for synthesizing such molecules are disclosed by, for example, Wu, R., et al., Prog.Nucl. Acid. Res. Molec. Biol. 21:101-141 (1978), which is incorporated herein byreference. Procedures for constructing recombinant molecules in accordance with the above-described method are disclosed by Sambrook et al. Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), which is herein incorporated by reference.

2 o Oligonucleotides representing a portion of the porcine adipocyte polypeptide are useful for screening for the presence of genes encoding such proteins and for the cloning of porcine adipocyte polypeptide genes. Techniques for synthesizing sucholigonucleotides are disclosed by, for example, Wu, R., et al.. Prog. Nucl. Acid.
Res. Molec. Biol. 21:101-141 (1978).

Wo 3~10~6~t PCT/USg7/12483 A suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the porcine adipocyte polypeptide gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identified, synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells which are capable of expressing the porcine adipocyte polypeptide gene. Single stranded oligonucleotide molecules complementary to the "most probable" porcine adipocyte polypeptide-encoding sequences can be synthesized using procedures which are well known to those of ordinary skill in the art (See e.g., USP 5,268,295). Additionally, DNA synthesis may be achieved through the use of automated synthesizers. Techniques of nucleic acid hybridization are disclosed by Sambrook et al. (supra).

In an alternative way of cloning the porcine adipocyte polypeptide gene, a library of expression vectors is prepared by cloning DNA or, more preferably, cDNA
(from a cell capable of expressing the porcine adipocyte polypeptide) into an expression vector. The library is then screened for members capable of expressing a protein which binds to anti-porcine-adipocyte polypeptide antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as the porcine adipocyte polypeptide, or fragments thereof. In this 2 o embodiment, DNA, or more preferably cDNA, is extracted and purified from a cell which is capable of expressing the porcine adipocyte polypeptide protein. The purified cDNA is fragmentized (by shearing, endonuclease digestion, etc.) to produce ~ a pool of DNA or cDNA fragments. DNA or cDNA fragments from this pool are then cloned into an expression vector in order to produce a genomic library of Wo ~J'~ 1~h PCT/US97Jl2483 expression vectors whose members each contain a unique cloned DNA or cDNA

fr~gment, An "expression vector~ is a vector which (due to the presence of apl)lopliate transcriptional and/or translational control sequences) is capable of expressing a DNA
(or cDNA) molecule which has been cloned into the vector and of thereby producing a polypeptide or protein. Expression vectors of the present invention may be either prokaryotic or eukaryotic. Examples of suitable prokaryotic expression vectors include pASK75 (Biometra) or pET 21a-d (Novagen). Examples of suitable 0 eukaryotic expression vectors include pcDNA3 or pRc/RSV (In Vitrogen, Inc.).

A DNA sequence encoding the porcine adipocyte polypeptide of the present invention, or its functional derivative, may be recombined with vector DNA in accordance with conventional techniques such as those disclosed by Sambrook, et al.
(supra).

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to2 o nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.

The precise nature of the regu}atory regions needed for gene expression may W O 98/04690 PCTrUS97/12483 vary from organism to org~ni~m, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA
transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis. A promoter is a double-stranded DNA or RNA molecule which is capable of binding RNA polymerase and promoting the transcription of the "operably linked" nucleic acid sequence. The promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral. Strong promoters are, however, ple~llcd. Suitable promoters are repressible, or more preferably, constitutive. Examples of suitable prokaryotic promoters include the0 tetracycline (TetA) promoter for pASK75 and T71ac for pET21. Examples of suitable eurkaryotic promoters include alpha actin or beta actin. Examples of suitable viral promoters include Rous sarcoma or cyotmegala.

The present invention is also directed to an antibody specific for an epitope ofthe porcine adipocyte polypeptide, and the use of such antibody to detect the presence of, or measure the quantity or concentration of the polypeptide, a functional derivative thereof, in a cell, a cell or tissue extract, or a biological fluid. As used herein, the term "epitope" refers to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determin~nt~" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. An antibody is said to be ~ "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.

Wo 98 'û 1~9~ PcTluss7/l2483 The porcine adipocyte polypeptide of the present invention, or a functional derivative thereof, preferably having at least about 8 amino acids is used as an antigen for induction of a polyc}onal antibody or monoclonal antibody (mAb). As used herein, an "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the mlllti~lde of other antibodies which may be evoked by other 1 0 antigens.

The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), and chimeric antibodies. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of ~nim~l~ imm-lni7e~1 with an antigen. Monoclonal antibodies are a subst~nti~lly homogeneous population of antibodies to specific antigenic epitopes. MAbs may be obtained by methods known to those skilled in the art. (See, for example Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110; de St. Groth, S. F. et al.. J.
Imrnunol. Methods, 35:1-21 (1980); and Hartlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988).

Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having a variable region derived from a porcine mAb and a murine immllnoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad.
Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA
81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Neuberger et al., Nature 314:268-270 (1985); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Better et al., Science 240:1041-1043 (1988)). These l~rerellces are hereby incorporated by reference.

The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of 0 binding antigen. Fab and F(ab')2fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
The reaction of the antibodies and the polypeptides of the present invention aredet~cted by immlmnassay methods well known in the art (See, for example, Hartlowet al. supr~). The antibodies, or fragments of antibodies, useful in the presentinvention may be used to qll~ntitatively or qll~lit~tively detect the presence of cells 2 o which express the porcine adipocyte polypeptide protein. This can be accomplished by immlm(>fluorescence techniques employing a fluorescently labeled antibody coupled with microscopy, flow cytometric, or fluorimetric detection.

wos~J~l69o PCT/US97/12483 The antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in irnmunofluorescence or immunoelectron microscopy,for in situ detection of the porcine adipocyte polypeptide. In situ detection may be accomplished by removing a histological specimen from a pig, and providing a labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the porcine adipocyte polypeptide but also its distribution in the e~minP~l tissue. Using the present invention, those of ordinary skill will readily 0 perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Such assays for porcine adipocyte polypeptide typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested or cultured cells cont~ining adipogenic cells or adipocytes~ in the presence of a detectably labeled antibody capable of identifying the porcine adipocyte polypeptide, and cletçcting the antibody by any of a number of techniques well-known in the art, such as enzyme immlmt)assays (EIA or ELISA) or radioimm-lnoassays (RIA).

2 o The antibody molecules of the present invention may also be adapted for utilization in an immllnometric assay, also known as a "two-site" or "sandwich" assay.
In a typical immllnometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support (i.e., any support capable of binding antigen or antibodies) and a quantity of detectably labeled soluble antibody is added to permit Wo 98/04690 PCT/USg7/12483 detection and/or q~l~ntit~tion of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.

The binding activity of a given lot of antibody to the porcine adipocyte polypeptide may be determined according to well known methods. Those skilled in the art will be able to determine operative and optirnal assay conditions for each deterrnination by employing routine experimentation.

Antibodies can be used in an immunoaffinity column to purify the porcine 0 adipocyte polypeptide of the invention by a one step procedure, using methods known in the art.

According to the present invention, a pig that is susceptible to fat deposition is treated with the porcine adipocyte protein to limit such fat deposition. This treatment may be performed in conjunction with other anti-adipogenic therapies. A typical regimen for treating swine with a propensity for fat deposition comprises lmini~tration of an effective amount of the porcine adipocyte polypeptide ~(lmini~ered over a period of time.

2 0 The porcine adipocyte polypeptide of the present invention may be ~-lmini.~tered by any means that achieve its intended purpose, preferably to alter feed intake or limit fat deposition in a subject. For example, a-lmini~tration may be by ~ various parenteral routes including, but not limited to, subcutaneous, intravenous, intradermal, intr~mllsc~ r, and intraperitoneal routes. Alternatively, or concurrently, Wo 98,'~169 J PCTIUSg7/12483 - -~tlmini.ctration may be by the oral route which may be accomplished by the use of genetically-altered fee-lsnlffs, in which the porcine leptin gene has been inserted and expressed. Palenl~ldl a~mini.~tration can be by bolus injection or by gradual perfusion over time such as by implant of osmotic delivery device. Preparations for parenteral ~lministration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art. Ph~ ceutir~l compositions such as tablets and capsules can also be preparedaccording to routine methods.

0 It is understood that the dosage of porcine adipocyte polypeptide a-lmini~ered may be dependent upon the age, sex, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The most preferled dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art. The total dose required for each treatment may be atlministered by multiple doses or in a single dose. The porcine adipocyte polypeptide of the present invention may be a~lminict~red alone or in conjunction with other therapeutics directed toward the regulation of fat deposition.

In a preferred embodiment, the concentration of the porcine adipocyte 2 o polypeptide or mRNA of this invention is measured in a cell preparation, tissue extract or biological fluid of a subject as a means for determining the susceptibility or the plupellsily of the subject for fat deposition. The susceptibility of the subject to fat deposition is related to the level of the porcine adipocyte polypeptide or its mRNA.
Additionally, restriction fragment length polymorphisms in the porcine adipocyte gene Wo 98/04690 PcTruss7/l2483 will be used to predict fat deposition potential.

Another embodiment of the invention is evaluating the efficacy of a drug or other agent, directed to the increase or decrease of feed intake by measuring the ability of the drug or agent to stim~ te or suppress the production of the porcine adipocyte polypeptide, or mRNA of this invention by a cell or cell line capable of producing such polypeptides or mRNAs. Preferred cells are cells of an adipogeniccell line. The antibodies, cDNA probe or riboprobe of the present invention are useful in the method for evaluating these drugs or other agents in that they can be 0 employed to determine the amount of the porcine adipocyte polypeptide or mRNAs using one of the above-mentioned immllnnassays.

An additional embodiment of the present invention is directed to assays for measuring the susceptibility of a pig to fat deposition based on measuring in a tissue or fluid from the subject the amount of the mRNA sequences present that encode the porcine adipocyte polypeptide, or a functional derivative thereof, preferably using an RNA-DNA hybridization assay. The susceptibility to fat deposition is related to the amount of such mRNA sequences present. For such assays, the source of the mRNA
sequences is preferably a pig's adipogenic cells. The ple~lled technique for 2 o measuring the amount of mRNA is a hybridization assay using RNA (e.g., Ribonuclease Protection Assay) or DNA (e.g. Northern or Slot Blot Assays) of complementary base sequence.

Wo 981046gO PCT/US97/12483 Nucleic acid detection assays, especially hybridization assays, can be pre~ ated on any characteristic of the nucleic acid molecule, such as its size, sequence, susceptibility to digestion by restriction endonucleases, etc. The sensitivity of such assays may be increased by altering the manner in which detection is reported s or signaled to the observer. Thus, for example, assay sensitivity can be increased through the use of detectably labeled reagents. A wide variety of such labels have been used for this purpose. Kourilsky et al. (U.S. Pat. No. 4,581,333) describe the use of enzyme labels to increase sensitivity in a detection assay. Radioisotopic labels are disclosed by Falkow et al. (U.S. Pat. No. 4,358,~35), and by Berninger (U.S.0 Pat. No. 4,446,237). Fluorescent labels (Albarella et al., EP 144914), chemical labels (Sheldon III et al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No.
4,563,417), modified bases (Miyoshi et al., EP 119448), etc. have also been used in an effort to improve the efficiency with which detection can be observed.

One method for overcoming the sensitivity limitation of nucleic acid concentration is to selectively amplify the nucleic acid whose detection is desired prior to performing the assay. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or 2 0 RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al.
(U.S. Pat. No. 4,237,224), Maniatis, T., et al., etc.

W O 98/04690 PCTrUS97/12483 Recently, an in vitro enzymatic method has been described which is capable of increasing the concentration of such desired nucleic acid molecules. This method has been referred to as the "Polymerase Chain Reaction" or "PCR" (Mullis, K. et al.,Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP 50, 424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194). The polymerase chain reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequenre has not been previously purified and is present only in a single 0 copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

2 o EXAMPLE I
ISOLATION OF PORCINE LEPTIN cDNA
The putative secreted portion of porcine leptin gene product was amplified - from adipose tissue mRNA using reverse transcriptase-polymerase chain reaction.
Four separate cDNA synthesis reactions were carried out using 1-2 ~g of porcine W O 98104690 PCT~US97/12483 -adipose tissue total RNA or 1-2 ,ug of poly A+ mRNA, 150 pmol of random hexamer oligonucleotides, 500 nM dNTP, 200 U of MM~V RNase H- reverse transcriptase (Life Technologies, Inc.) in 20 ,ul of the supplied buffer. The reactions were in~uh~ted for 1 h at 37 C and termin~ted by heating to 70 ~C for 10 min. The leptin cI)NA product was amplified by PCR using the following degenerate oligonucleotide primers with restriction site linkers for R~m~lBsa I and EcoRII~co47 III, respectively:
Sense strand:
5'-GGATCCGGTCTCAGGCC GTGCC(C/T)ATCCA(A/G)AAAGTCC-3' Antisense strand:
5'-GAATTCAGCGCT GCA(C/T)(C~T)CAGGGCT(G/A)A(G/C)(G/A)TC-3' These oligonucleotide primers were designed from a multiple sequence alignment of the mouse and human cDNA sequences. Approximately 100 ng of adipose tissue cDNA was added as template to 50 ,ul PCR reactions made in the m~nllf~turers buffer with 100 pmol of each primer and 2.5 U of Taq DNA polymerase (Life Technologies, Inc.). Athree stage amplification was carried out under the following conditions; Stage 1- 95 ~C, 3 min; 52 ~C, 1 min, 72 C 1 min, 1 cycle; Stage 2- 94 C, 45s; 52 DC, 45s, 72 ~C, 1 min, 4 cycles; Stage 3- 94 C, 45 s; 55 C, 30 s, 72 C 1 min, 28 cycles. Template 2 0 cDNA from three out of four cDNA reactions produced a 466 bp product.

The PCR products were prepared for ligation into the protein expression vector pASK75 (Biometra Inc.) by complete digestion with ~co47III and partial digestion with Bsa I. The restriction enzyme digested PCR products were purified by electrophoresis in ,, , . . , . , ~

Wo 98/04690 PCT/USg7/12483 low melting point agarose and a 437 bp product was excised from the gel and ligated into the vector. The ligations were transformed in ~. coli XL1-Blue (Stratagene Inc.) and plated on LB plates cont~inin~ 50,ug/ml ampicillin for plasmid selection. Twelve E. coli colonies were isolated that contained the porcine leptin cDNA, and plasmid DNA was isolated for DNA sequencing.

The nucleotide sequence of the porcine leptin gene comprising 5917 base pairs, and the amino acid translation of the leptin coding sequences are depicted in Fi~ure 1.
The nucleotide sequence and the amino acid sequence of the entire porcine leptin cDNA
0 (i.e., signal peptide and secreted proteins) comprising 501 base pairs and 167 amino acids are depicted in Figure 2. The nucleotide sequence and the amino acid sequence of the porcine leptin cDNA corresponding to the secreted protein alone and comprising 435 base pairs and 145 amino acids are depicted in Figure 3.

There was an 83 % identity between the pig and human cDNA sequence and a 76% identity between the pig and mouse cDNA sequence as depicted in Figure 4.

EXAMPLE II
ISOLATION OF mRNA CORRESPONDING TO PORCINE LEPTIN cDNA
2 o The porcine leptin cDNA was used as a probe for detection of the full length mRNA. A northern blot cont~inin~ porcine adipose and bovine adipose poly A+ mRNAas well as ob~ob mouse adipose total RNA was provided by Dr. M. Spurlock of Purina Mills Inc. The blot was hybridized with an [32p] dCTP labeled porcine leptin cDNA in hybridization solution (HY; 0.9 M NaCl, 0.09 M sodium citrate, 0.05% ficoll, 0.05%

.

W O 98J~1~5~ PCT~US97/12483 . .-polyvinylpyrolidone, 0.0~% BSA, 0.5% SDS, 0.1% sodium pyrophosphate, 10 mM
EDTA and 100 mg/ml sonicated salmon sperm DNA at 60 ~C for 15 h. The blot was washed to a final stringency of 0.2X SSC (0.03M NaCl, 0.003 M sodium citrate), 0.1%
SDS at 60 ~C and exposed to X-ray film. A 3,090 bp leptin mRNA was detected in porcine and bovine adipose tissue and a 3,240 bp leptin mRNA was detected on oblob mouse adipose tissue. As shown in Figure 5, lanes 1 and 2 contain the porcine adipose poly A+mRNA, lane 3 contains the adipose total RNA from a control mouse and lanes 4 and S contain the adipose total RNA from an ob/ob mouse, and lane 6 contains the bovine adipose poly A+mRNA.

EXAMPLE III
ISOLATION OF GENOMIC DNA CLONE
CORRESPONDING TO PORCINE LEPIIN
The porcine leptin cDNA was also used to screen a porcine genomic DNA library.
Specifically, a porcine genomic library cont~ining 4.64 X 105 recombinants was previously constructed in SuperCos 1 (Stratagene, Inc.) and screened for porcine leptin.
Specifically, two sets of replica filters were prehybridized for 2 h at 60~C. Filters were hybridized overnight with [-32p] dCTP labeled probe at 5 X 105 cpm per ml of hybridization solution at 65 C. Filters were sequentially washed in 2X SSC (0.3 M
NaCl, 0.03 M sodium citrate), 0.5% SDS; lX SSC, 0.5% SDS; and 0.2X SSC 0.5%
SDS with each wash at 60' C for 30 min. Positive clones that showed signals on both replica filters were recovered from the agar plates and individual colonies were isolated by a second low density replica plating and hybridization step. A cosmid designated Obg-361 was isolated that hybridized to the porcine ob cDNA probe and had essentially the Wo 98/04690 PCT/USg7/12483 same restriction enzyme digestion pattern as found in porcine genomic DNA.

Figure 6 illustrates the isolation of the cosmid Obg-361. Specifically, lanes 1-4 are an agarose gel cont~ining Kb ladder molecular mass markers (lane 1), cosmid Obg-361 digested with Eco RI (lane 2) and Hind III (lane 3) and biotinylated lambda/Hind III
molecular mass markers (lane 4).

Southern blot analysis of the gel in lanes 2-4 were probed with the porcine leptin cDNA in~lic~t~ that the EcoRI fragments (lane 5) and the Hind III fragments (lane 6) contain leptin sequences. Lane 7 is lambdalHind III molecular mass markers.

Porcine genomic DNA digested with BAM HI (lane 8), EcoRI (lane 9) and Hind III (lane 10) and hybridized with a Bsa I fragment (300 bp) of the porcine leptin cDNA
showed equivalent bands that contain leptin sequences indicating that the porcine leptin gene was isolated in cosmid Obg-361.

The 5917 bp Hind III fragment was subcloned into Bluescript II SK+ (Stratagene, Inc.). Both strands of the sequence was deterrnined using progressive nested deletions using Exonuclease III and Mung Bean nll~!P~e. Sequencing reactions were carried out with Sequenase V2Ø This sequence was 5917 bp in length and contains the entirecoding region in two exons (Figure 1). There was 78.6% nucleotide identity between the pig and human as well as 71.2% nucleotide identity between pig and mouse coding sequences. The splice junctions for the two exons were confirrned by the cDNA
sequence. The cDNA sequence of the protein coding region is shown in Figure 2. The Wo 98J ~ 1690 PcTtuss7/l2483 ~01 bp sequences encodes 166 amino acid residue leptin polypeptide with a predicted molecular mass of 18,334 Da.

A clone was obtained using the process described above, Obg H3-15, was 5 deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852-1776, on July 11, 1996, and has been deci~n~t~l ATCC No.
97653. This microorganism was deposited under the conditions of the Budapest Treaty on the International Recognition of Deposit of Microor~ani~m~ for the purpose of Patent Procedure. All restrictions on the availability to the public of the material so deposited 0 will be irrevocably removed upon the granting of a patent. This deposit will be m~int~ined for a time period of 30 years from the date of deposit or 5 years after the last request for the material, whichever is longer.

EXAMPLE IV
PURIFICATION OF THE PORCINE LEPrIN GENE PRODUCT
The polypeptide sequence encoded by the porcine leptin cDNA was synthesized and purified using the Strep-Tag system (Biometra, Inc.). The pASK plasmid contains the ompA leader sequence for secretion of the protein into the periplasmic space of E.
coli as well as a ten amino acid carboxyl terminus that binds to strepavidin for affinity 20 chromatography. Synthesis of the porcine leptin protein by E. coli strain X~1-Blue was in~ ced with 200 ~g/l of anhydrotetracycline and the cells harvested after 3 h. The proteins in the periplasmic space were isolated by osmotic shock by suspending the cells in 100 mM Tris-HCl pH 8.0, 500 mM sucrose, 1 mM EDTA and 0.02% NaN3 for 30 m at 4 ~C. The cells were removed by centrifugation and the porcine leptin protein was W 0 9~ 9~ PCT~US97/12483 purified from the periplasmic proteins by strepavidin affinity chromatography as depicted in Figure 5.

Specifically, Figure 7 shows the polyacrylamide gel electrophoresis of porcine 5 leptin protein induction and purification in E. coli. Molecular mass markers are located in lane 1. Lane 2 contains total protein from XL-1 Blue and an pASK/Ob cell line before (lane 3) and after (lane 4) anhydrotetracycline induction. Affinity purified porcine leptin protein is located in lane 6.

ANTIBODIES TO PORCINE LEPIIN PROTEIN
AND l~l~;l~ USE TO DETECT PORCINE LEPIIN IN ADIPOGENIC CELLS
Polyclonal and/or monoclonal antibodies are produced with the recombinant porcine leptin protein. The techniques used for producing, screening, ~letecting~ and/or ~uantifying antibodies for porcine leptin are ~liscllcsed extensively in "Antibodies: a Laboratory Manual" (Harlow et al., 1988, Cold Spring Harbor laboratory). All media or medium components, mouse or cell strains (e.g. BALB/C mouse, sp2/0 myeloma cells, JA744A.1 macrophages etc.) are commercially available.
A. lm mlni7qt:on of ~nimql.
l. Rabbits:
Purified porcine leptin protein is iniected into rabbits for production of polyclonal antibodies. Specifically, each rabbit receives repeated subcutaneous injections with antigen in Freund's complete adjuvant followed by at least 1 booster injection of about 200 ~g to l mg. When the serum titer of the immunized rabbits is sufficiently high when tested using the porcine leptin as antigen, rabbit serum is harvested as the polyclonal W O ~8101690 PCTrUS97112483 - -antiserum for porcine leptin.

2. BALB/C mice (4-week old):
Purified porcine leptin protein is injected into BALBIC mice for production of 5 monoclonal antibodies. Specifica}ly, each mouse is injected with about 50 ~Lg porcine leptin protein with Ribi's S-TDCM adjuvants (RIBI ImmunoChem Research, Inc., Hamilton, Montana). The number of injections depends on the titer of the antibody in the serum of immllni7e-1 mice as determined by ELISA using porcine leptin as the antigen. In the course of producing monoclonal antibodies against porcine leptin protein, the spleens 0 of immllni7~d mice are used to prepare spleenocytes. ~ybridoma cells are made by fusing the spleenocytes with sp2/0 myeloma cells (treated with 8-Azaguanine cont~ining medium) in the p,~sellce of 50% PEG-1500. Hybridoma cells are incubated in selection HAT (hypox~nthin~, aminopterine, and thymidine) medium. Subsequent screening for posltive clones uses the recombinant porcine leptin as antigen in ELISA methodology.
5 Positive clones that produce strong anti-porcine-leptin antibody are characterized for specificity, subtype, affinity, binding sites, etc.

When large quantities of purified antibody are needed, the positive clones are cultured in large scale and antibody purified from the culture supernatant, or injected into 20 the intraperitoneal cavity of BALBIC mice for production of ascites. The latter procedure requires about 1-2x106 hybridoma cells per mouse, and usually takes about 7-14 days.
Large quantities of antibody is then purified from ascites by ammonium sulphate precipitation and ion exchange chromatography (e.g. DEAE-Trisacryl M).

, . .. .... . .

wo 98/04690 PcTruss7/l2483 Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While this invention has been described in connection 5 with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential 0 features hereinbefore set forth as follows in the scope of the appended claims.

Claims (12)

What is claimed is:

1. An isolated single or double-stranded DNA molecule consisting of a nucleotide sequence which encodes a porcine adipocyte polypeptide leptin having the amino acid sequence of SEQ ID NO:4, or the complement to the DNA molecule.

2. An expression vector comprising the DNA molecule of Claim 1.

3. The vector of Claim 2 in which the vector is a plasmid.

4. A host cell transformed or transfected with the plasmid of Claim 3.

5. An isolated mRNA molecule encoding a porcine adipocyte polypeptide leptin, the mRNA molecule encoded by the nucleotide sequence of SEQ ID NO:3.

6. The DNA molecule of Claim 1 consisting of the nucleotide sequence of SEQ ID NO:3.

7. A host cell transformed or transfected with the vector of Claim 2.

8. An isolated mRNA molecule consisting of a nucleotide sequence that encodes a porcine adipocyte polypeptide leptin having the amino acid sequence of SEQ
ID NO:4.

9. An expression vector comprising the DNA molecule of Claim 6.

10. The vector of Claim 7 in which the vector is a plasmid.

11. A host cell transformed or transfected with the vector of Claim 9.

12. A host cell transformed or transfected with the plasmid of Claim 11.