CN1387570A - Fibroblast growth factor-19 (FGF-19) nucleic acids and polypeptides and methods of use for treatment of obesity - Google Patents

Abstract

The present invention is directed to novel polypeptides belonging to the fibroblast growth factor family and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Furthermore, methods of treating obesity are provided.

Description

The nucleic acid and the polypeptide of fibroblast growth factor-19 (FGF-19) and the method that is used for the treatment of obesity

Invention field

The present invention relates generally to evaluation and separates new DNA, relating to reorganization and producing the novel polypeptide be called fibroblast growth factor-19 (FGF-19) polypeptide herein, relating to and use this polypeptide treatment fat and be used for method, composition and the assay method that preparation has the pharmaceutically active substance of treatment and pharmacological characteristics (comprise and treat relevant those of obesity).

Background of invention

Obesity is the very general chronic disease of modern society, and not only its aesthetic appearance is not relevant with society, and relevant with a large amount of medical problem with the lost of life, described a large amount of medical problem comprises bad mental development, dysgenesia such as polycystic ovarian disease, dermatology illness such as infection, varix, acanthosis nigricans and eczema, exercise tolerance is poor, diabetes, insulin resistance, hypertension, high blood cholesterol mass formed by blood stasis, chololithiasis, osteoarthritis, the type of rectifying damage, thrombotic diseases, cancer and coronary heart disease.Rissanen etc., British MedicalJournal, 301:835-837 (1990).

Existing bantingism comprises standard diet and exercise, very low calorie diet, behavior therapy relates to the pharmacotherapy of appetite-inhibiting agent, heat production medicine, food absorption inhibitor, medicine equipment such as jaw wire suture (iaw wiring), with a tight waist and air bag and operation.Jung and Chong, Clinical Endocrinology, 35:11-20 (1991); Bray, Am.J.Clin.Nutr, 55:538S-544S (1992).Existing report: it is effective aspect losing weight the teenager to control proteic modified form fasting method.Lee etc., Clin.Pediatr, 31:234-236 (April 1992).Heat restriction as bantingism causes the katabolism of body storage protein and produces negative nitrogen balance.Therefore, additional proteic diet is very popular as the measure that reduces nitrogen loss between the heat restricted period., reduces such diet the more effective ways that therefore need keep oneself slim health and albumen are stocked because only producing the nitrogen of appropriateness.In addition, if this class scheme also causes the body fat loss to be quickened, then can improve banting.The whole bag of tricks of this type of treatment comprises Weintraub and Bray at Med.Clinics N.Amer., 73:237 (1989); Bray, at Nutrition Reviews, those that discuss among the 49:33 (1991).

Consider fat extreme ubiquity and above-mentioned with fat relevant severe complication in society, any medicine that can be used for alleviating the obese individuals body weight potentially will be to their deep useful influence of health generation.This area need make the obese individuals whole body lose weight to its ideal body weight and help the body weight level after obese individuals keeps it to reduce and do not have the medicine of serious side effects.

Therefore, expectation provides and can be used for obese individuals and recover that it is normal, the treatment plan of ideal body weight.

Expectation further provides the bantingism that can keep under-weight over a long time.

Expect that also prevention of obesity and treatment Once you begin then stop the progress that is secondary to fat disease or prevent its outbreak, described secondary disease such as arteriosclerosis and polycystic ovarian disease.

The present invention also provides this class methods of treatment and compositions related.The present invention also provides new albumen and nucleic acid, and the method for screening the regulon of described new albumen and nucleic acid.Other method provided by the invention, treatment and composition will become apparent for those skilled in the art.

Summary of the invention

Identified the cDNA clone (this paper is called DNA49435-1219) of coding novel polypeptide, " fibroblast growth factor-19 " among member-the application of itself and fibroblast growth family (FGF-19) has some sequence similarity.

One aspect of the present invention provides the isolating nucleic acid molecule that comprises the nucleotide sequence of coding FGF-19 polypeptide.

On the one hand, isolated nucleic acid molecule contains with (a) coding and contains shown in Fig. 2 (SEQ ID NO:2) from the dna molecular of the PEACH polypeptide of about 1 or about 23 to about 216 aminoacid sequence (comprising the two ends residue), perhaps (b) (a) complementary strand of described dna molecular has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% nucleotide sequence homologous nucleotide sequence.

On the other hand, isolated nucleic acid molecule contains (a) coding and contains shown in Fig. 2 (SEQ ID NO:2) from the nucleotide sequence of the FGF-19 polypeptide of about 1 or about 23 to about 216 aminoacid sequence (comprising the two ends residue), perhaps (b) (a) complementary strand of described nucleotide sequence.

Again on the one hand, isolated nucleic acid molecule contain with (a) shown in Fig. 1 (SEQ ID NO:1) by the dna molecular of about 464 or about 530 to about 1111 nucleotide sequence (comprising the two ends residue), perhaps (b) (a) complementary strand of described dna molecular has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% nucleotide sequence homologous nucleotide sequence.

Also on the one hand, isolated nucleic acid molecule contains among (a) Fig. 1 (SEQ ID NO:1) from about 464 or about 530 to about 1111 nucleotide sequence, perhaps (b) (a) complementary strand of described nucleotide sequence.

In addition, the present invention relates to isolated nucleic acid molecule, it contains with (a) coding is the dna molecular of identical mature polypeptide of the people's albumen cDNA coding of 209480 (DNA49435-1219) at the ATCC of ATCC preservation preserving number by on November 21st, 1997, perhaps (b) (a) complementary strand of described dna molecular has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% nucleotide sequence homologous nucleotide sequence.In an embodiment preferred, it is the nucleotide sequence of people's albumen cDNA of 209480 (DNA49435-1219) identical mature polypeptide of encoding at the ATCC of ATCC preservation preserving number that isolated nucleic acid molecule contains (a) coding and on November 21st, 1997, perhaps (b) (a) complementary strand of described nucleotide sequence.

On the other hand, invention relates to isolated nucleic acid molecule, its contain with (a) on November 21st, 1997 be the full-length polypeptide encoding sequence of people's albumen cDNA of 209480 (DNA49435-1219) at the ATCC of ATCC preservation preserving number, perhaps (b) (a) complementary strand of described nucleotide sequence has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% nucleotide sequence homologous nucleotide sequence.In an embodiment preferred, it is the full-length polypeptide encoding sequence of people's albumen cDNA of 209480 (DNA49435-1219) at the ATCC of ATCC preservation preserving number that isolated nucleic acid molecule contains (a) on November 21st, 1997, perhaps (b) (a) complementary strand of described nucleotide sequence.

Also on the one hand, invention relates to the nucleic acid molecule of the active FGF-19 polypeptide that separated coding defines below, it comprise can with code pattern 2 (SEQ ID NO:2) in 1 or the nucleotide sequence of the complementary strand hybridization of the nucleotide sequence of about 23～about 216 amino acids (comprising two end points amino-acid residues).Preferably, hybridization takes place under stringent hybridization condition and wash conditions.

On the other hand, invention relates to the nucleic acid molecule of the active FGF-19 polypeptide that separated coding defines below, it comprises with Nucleotide about 464 or about 530 and about 1111 shown in Fig. 1 (SEQ ID NO:1) between the complementary strand of (comprising two end points residues) nucleotide sequence nucleotide sequence of hybridizing.Preferably, hybridization takes place under stringent hybridization condition and wash conditions.

Again on the one hand, invention relates to the isolating nucleic acid molecule that has at least about 22 Nucleotide, it is to make test dna molecule and (a) coding have the dna molecular of the FGF-19 polypeptide of the sequence of 1 or about 23 to about 216 (comprising the two ends residue) amino-acid residue among Fig. 2 (SEQ ID NO:2) under stringent condition, perhaps (b) (a) complementary strand of described dna molecular hybridize and produce, and, if this test dna molecule with (a) or (b) have at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% at least about 99% nucleotide sequence identity, then separate this test dna molecule.

On the other hand, invention relates to isolated nucleic acid molecule, it contains (a) coding compares with aminoacid sequence (comprising the two ends amino-acid residue) shown in Fig. 2 (SEQ ID NO:2) about 1 or about 23 to 216 residues, have at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about the nucleotide sequence of 99% scoring male polypeptide, perhaps (b) (a) complementary strand of described nucleotide sequence.

A particular aspects the invention provides isolated nucleic acid molecule, comprises that coding does not have the DNA of the FGF-19 polypeptide of N-terminus signal sequence and/or initial methionine(Met), the perhaps complementary strand of this coding nucleic acid molecule.Signal peptide tentatively has been defined as in sequence shown in Fig. 2 (SEQ ID NO:2) from about amino acid/11 position to 22 extensions of about amino acid (comprising the two ends residue).Yet, should be understood that, the C-end boundaries of signal peptide may be different, but most probable is no more than about 5 amino acid at signal peptide C-end boundaries two ends, the evaluation initial as this paper, normally used standard was determined (for example, Nielsen etc. when wherein signal peptide C-end boundaries may be used to identify this amino acid sequential element according to this area, Prot.Eng.10:1-6 (1997) and von Heinje etc., Nucl.Acids.Res.14:4683-4690 (1986)).And, it should be understood that also that in some cases the cracked signal sequence is incomplete same from the excretory polypeptide, thereby cause more than one secretion type.These polypeptide and polynucleotides encoding them are the contents that the present invention considers.The signal peptide of FGF-19 polypeptide shown in the application Fig. 2 (SEQ ID NO:2) extends to X from the amino acid/11 of Fig. 2 (SEQ ID NO:2), and wherein X is any amino acid of from 17 to 27 among Fig. 2 (SEQ ID NO:2).Therefore, the present invention includes the mature form of FGF-19 polypeptide, contain amino acid whose polypeptide of X to 216 among Fig. 2 (SEQ ID NO:2) and following their variant that will describe comprising those, wherein X is any amino acid of from 17 to 27 among Fig. 2 (SEQ ID NO:2).The nucleic acid molecule that also comprises these polypeptide of separated coding.

Another embodiment relates to the fragment of FGF-19 peptide sequence, comprises the encoding sequence that contains the encode fragment of peptide more than anti--FGF-19 antibody combining site as can randomly encoding in hybridization probe for example or the FGF-19 polypeptide.This type of nucleic acid fragment length is usually at least about 20 Nucleotide, perhaps at least about 30 Nucleotide, or at least about 40 Nucleotide, or at least about 50 Nucleotide or at least about 60 Nucleotide, perhaps at least about 70 Nucleotide, or at least about 80 Nucleotide, or at least about 90 Nucleotide, or at least about 100 Nucleotide, perhaps at least about 110 Nucleotide, or at least about 120 Nucleotide, or at least about 130 Nucleotide, or at least about 140 Nucleotide, perhaps at least about 150 Nucleotide, or at least about 160 Nucleotide, or at least about 170 Nucleotide, or at least about 180 Nucleotide, perhaps at least about 190 Nucleotide, or at least about 200 Nucleotide, or at least about 250 Nucleotide, or at least about 300 Nucleotide, or at least about 350 Nucleotide, or at least about 400 Nucleotide, or at least about 450 Nucleotide, or at least about 500 Nucleotide, or at least about 600 Nucleotide, or at least about 700 Nucleotide, or at least about 800 Nucleotide, or at least about 900 Nucleotide, or at least about 1000 Nucleotide, wherein this paper term " about " is meant that the nucleotide sequence associated length adds deduct 10%.In an embodiment preferred, nucleotide sequence fragment is derived from any coding region of nucleotide sequence shown in Fig. 1 (SEQ ID NO:1).Should be understood that, available ordinary method is measured the new segment of FGF-19 polypeptide-coding nucleotide sequence, any one is with the coding nucleotide sequence and the contrast of other known nucleotide sequence of FGF-19 polypeptide in numerous known sequence contrast programs by using for it, and determining wherein, which or which FGF-19 polypeptide-coding nucleotide sequence fragment is new.This paper has considered all this class FGF-19 polypeptide-coding nucleotide sequences, and can be measured with not loaded down with trivial details experiment.Also consider FGF-19 polypeptide fragment, preferably contain those FGF-19 polypeptide fragments of anti--FGF-19 antibody combining site by these nucleotide molecule fragment codings.

In another embodiment, invention provides the carrier of the nucleotide sequence that contains coding FGF-19 or its variant.This carrier can contain any isolated nucleic acid molecule of above-mentioned definition.

The host cell that contains examples of such carriers also is provided.For example, host cell can be Chinese hamster ovary celI, intestinal bacteria, infect insect cell or the yeast of baculovirus.The method of producing the FGF-19 polypeptide further is provided, is included under the condition of the suitable FGF-19 of expression and cultivates host cell and from cell culture, reclaim FGF-19.

In another embodiment, invention provides isolating by the coded FGF-19 polypeptide of arbitrary isolated nucleic acid sequences as defined above.

One particular aspects the invention provides isolating native sequences FGF-19 polypeptide, and in certain embodiments, it comprises shown in Fig. 2 (SEQ ID NO:2) by about 1 or about 23 aminoacid sequences to about 216 residues.

Another program, the present invention relates to isolating FGF-19 polypeptide, comprise with Fig. 2 (SEQ ID NO:2) in about 1 or about 23 to aminoacid sequence shown in about 216 residues (comprising the two ends residue) at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about the aminoacid sequence of 99% identity.

Again on the one hand, the present invention relates to isolating FGF-19 polypeptide, its contain with on November 21st, 1997 be that people's albumen cDNA amino acid sequence coded of 209480 (DNA49435-1219) has at least about 80% at the ATCC of ATCC preservation preserving number, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about the aminoacid sequence of 99% identity.In an embodiment preferred, isolating FGF-19 polypeptide contains by being people's albumen cDNA amino acid sequence coded of 209480 (DNA49435-1219) on November 21st, 1997 at the ATCC of ATCC preservation preserving number.

On the other hand, invention relates to isolating FGF-19 polypeptide, its contain with Fig. 2 (SEQ ID NO:2) in about 1 or about 23 to aminoacid sequence (comprising the two ends residue) shown in about 216 residues at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% scoring male aminoacid sequence.

One particular aspects the invention provides the FGF-19 polypeptide of the isolating N-of not having end sequence and/or initial methionine(Met), and this polypeptide is nucleotide sequence coded by the aforementioned aminoacid sequence of coding.This paper also discloses the method for preparing this polypeptide, and method comprises: under the condition of suitable expression FGF-19 polypeptide, cultivate the host cell of the carrier that contains the corresponding encoded nucleic acid molecule, and reclaim the FGF-19 polypeptide from culture.

Also on the one hand, the present invention relates to isolating FGF-19 polypeptide, comprise among Fig. 2 (SEQ ID NO:2) about 1 or about 23 to aminoacid sequence shown in about 216 residues (comprising the two ends residue) or its bioactive fragment, or be enough to provide fragment with anti--FGF-19 antibody combining site, wherein biologically active or the evaluation of the FGF-19 polypeptide fragment of anti--FGF-19 antibody combining site is provided can be finished with routine techniques well known in the art.Preferably, the FGF-19 fragment is keeping the biological activity of natural FGF-19 polypeptide in nature, comprises the ability that treatment is fat.

Again on the one hand, the invention provides the polypeptide that is prepared as follows: (i) under stringent condition, make test dna molecule and (a) coding have the dna molecular of the FGF-19 polypeptide of about 1 or about 23 sequences to about 216 amino-acid residues (comprising the two ends residue) among Fig. 2 (SEQ ID NO:2), perhaps (b) (a) complementary strand of described dna molecular hybridize, if and this test dna molecule with (a) or (b) have at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% nucleotide sequence identity, then (ii) under the condition of suitable this polypeptide of expression, cultivate and contain the host cell of this test dna molecule and (iii) from cell culture, reclaim this polypeptide.

In another embodiment, the invention provides the chimeric molecule that contains the FGF-19 polypeptide that merges with heterologous polypeptide or aminoacid sequence, wherein the FGF-19 polypeptide can contain aforementioned any FGF-19 polypeptide or its variant or fragment.An example of this chimeric molecule is the chimeric molecule that contains the FGF-19 polypeptide that merges with immunoglobulin (Ig) epi-position flag sequence or Fc district.

In another embodiment, invention provides following definitions and antibody aforementioned FGF-19 polypeptide specific combination.Randomly, antibody is monoclonal antibody, antibody fragment or single-chain antibody.

In another embodiment, the present invention relates to the agonist and the antagonist of the natural FGF-19 polypeptide of following definitions.In one embodiment, agonist or antagonist are anti--FGF-19 antibody or small molecules.

In another embodiment, invention relates to the agonist of evaluation FGF-19 polypeptide or the method for antagonist, and method comprises makes the FGF-19 polypeptide contact with candidate molecules, and detects the biological activity by described FGF-19 polypeptide mediation.Preferably, the FGF-19 polypeptide is natural FGF-19 polypeptide.

In the another one embodiment, the present invention relates to contain the agonist of FGF-19 polypeptide described herein, FGF-19 polypeptide or the composition of antagonist or anti--FGF-19 antibody and carrier.Randomly, carrier is pharmaceutically acceptable carrier.

Another embodiment of the present invention relates to FGF-19 polypeptide described herein or its agonist or antagonist or anti--FGF-19 antibody and is used for the treatment of the application in the medicine of the illness of FGF-19 polypeptide, its agonist or antagonist or anti--FGF-19 antibody response in preparation.

In one embodiment, provide the screening can be in conjunction with the method for the biologically active agent of FGF-19.On the one hand, method comprises in the FGF-19 sample and adds candidate bioactive agent, and measures combining of described candidate bioactive agent and described FGF-19, if combination wherein takes place, then show be can with FGF-19 bonded biologically active agent.

This paper provides screening can regulate the method for the active biologically active agent of FGF-19 in addition.In one embodiment, a method is provided, and this method comprises the steps: to add candidate bioactive agent in the FGF-19 sample, and detects the bioactive variation of FGF-19, if change, then show it is to regulate the active biologically active agent of FGF-19.In one embodiment, the FGF-19 activity is to reduce the picked-up of glucose in the cell.In another embodiment, the FGF-19 activity is to increase leptin to discharge from cell.In an embodiment preferred, the FGF-19 activity is to reduce glucose uptake and increase leptin to discharge from cell.Preferred this class cell is an adipocyte.In another embodiment, the FGF-19 activity is the oxidation that increases lipid and carbohydrate.Preferred this class cell is liver cell or muscle cell.

In another embodiment, invention provides the method for identifying the FGF-19 acceptor.In a preferred embodiment, this method comprises makes FGF-19 combine with the component that contains the cytolemma material, if the acceptor on described FGF-19 and the described cytolemma material forms mixture, identifies that then described acceptor is exactly the FGF-19 acceptor.In one embodiment, method comprises and makes described FGF-19 and the crosslinked step of acceptor.Cytolemma is taken from intact cell or cytolemma extract product (extract preparation).

Another aspect of the present invention provides the method for inducing leptin to discharge from cell (preferred fat cell), and in one embodiment, this method comprises the FGF-19 that uses the amount that can effectively induce leptin release to cell.

In method provided herein, FGF-19 can use with the nucleic acid form of expression FGF-19 or with the albumen form.As below will describing in detail, can use FGF-19 by infusion or extended release preparation.Preferably, FGF-19 is applied to individuality with pharmaceutically acceptable carrier.

The method of the glucose uptake minimizing of inducing cell (preferred fat cell) also is provided.In one embodiment, this method comprises the FGF-19 that uses the amount that can effectively induce the glucose uptake minimizing to cell.

The present invention provides treatment individual fat method on the other hand.In one embodiment, this method comprises composition from the FGF-19 that contains the fat amount of effective treatment to individuality that use.Also can treat by this way and fat relevant illness such as cardiovascular disorder.

This paper also provides the method that reduces individual TBW, comprises the FGF-19 that uses significant quantity to described individuality.In an embodiment preferred, reduce individual fat (fat).

In addition, this paper provides and reduces the individual at least a triglyceride level and the method for free fatty acid levels, comprises the FGF-19 that uses significant quantity to described individuality.This paper also provides the method that increases individual metabolic rate, comprises to described individuality and uses significant quantity FGF-19.

This paper also is provided at the animal model that detects the effect of FGF-19 and instrumentality under various conditions and the state.In one embodiment, provide the genetically modified genomic animal that contains the FGF-19 that encodes, preferably rodent.

The accompanying drawing summary

Fig. 1 shows the nucleotide sequence (SEQ ID NO:1) of nucleotide sequence (Nucleotide 464-1111) cDNA that contains coding native sequences FGF-19, and wherein this nucleotide sequence (SEQ IDNO:1) is that this paper is called the clone of " DNA49435-1219 ".Also mark the position of initial sum terminator codon separately with boldface type and underscore.

Fig. 2 shows the native sequences FGF-19 amino acid sequence of polypeptide (SEQ ID NO:2) derived from encoding sequence among the SEQ ID NO:1.The apparent position that also shows various other important polypeptide structure territories.

Fig. 3 A and 3B show that proof MLC-FGF-19 transgenic mice body weight is than its not genetically modified brood mouse body weight light (Fig. 3 A) and histogram (Fig. 3 B) with lower circulation leptin level.Fig. 3 A show 6 week FGF-19 transgenic mouses in age (solid bar) and the brood mouse of transgenosis (wild-type) (stippling rod) weight of 24 hours (rightmost side) and after finishing fasting in 24 hours not (leftmost side) during the ad lib, fasting 6 hours and 24 hours.Fig. 3 B shows the serum (vertical bars) of mouse in leptin measures on the same group of phase shown in Fig. 3 A.

Fig. 4 A-4D shows that the FGF-19 transgenic mouse increases ingestion of food and urine amount but still has the histogram of normal plasma cell specific volume.Monitor during one group of mouse ad lib and finish after the fasting in 24 hours 24 hours ingestion of food (Fig. 4 A), water intake (Fig. 4 B), voided volume (Fig. 4 C) and hematocrit (Fig. 4 D) situation, wherein the result of FGF-19 transgenic mouse represents that with the solid black rod result of wild-type mice represents with the stippling rod in every group.

Fig. 5 is the histogram that expression FGF-19 transgenic mouse increases oxygen consumption rate.Shown that FGF-19 transgenic mouse (solid black rod) and wild-type (stippling rod) are in 24 hours oxygen consumption situation with night, fasting 24 hours and after finishing fasting in 24 hours in the daytime.

Fig. 6 A is to show with wild-type mice (stippling rod) to compare the histogram of FGF-19 transgenic mouse (solid black rod) triglyceride reducing (Fig. 6 A) and free fatty acids (Fig. 6 B) with 6B.

Fig. 7 A and 7B show with the placebo (stippling rod) that infusion does not contain FGF-19 to compare, giving not, transgenic mouse infusion FGF-19 (solid black rod) causes ingestion of food to increase the histogram of (Fig. 7 A) and oxygen consumption increase (Fig. 7 B), wherein " n " represents night, and " d " represents daytime.

Fig. 8 A and 8B show that FGF-19 increases leptin discharges (Fig. 8 A) and minimizing adipocyte ingestion of glucose (Fig. 8 B) from adipocyte histogram.

Fig. 9 shows FGF-19 transgenic mouse (shade rod) or wild-type (solid black rod) histogram of high fat diet (HFD) for some time posterior fat pad weight respectively, wherein along continuous straight runs is from left side beginning, be respectively 6 when week epididymises (HFD Ep) with peritonaeum after with the result of kidney week (HFD RP/PR), the result of epididymis when then being 10 weeks is behind the peritonaeum and the result in kidney week then.

Figure 10 shows FGF-19 transgenic mouse (shade rod) or wild-type mice (solid black rod) histogram (all 10 weeks of high fat diet) along with the glucose tolerance situation of time.

DESCRIPTION OF THE PREFERRED

I. definition

Term used herein " FGF-19 polypeptide ", " FGF-19 albumen " and " FGF-19 " comprise native sequences FGF-19 and FGF-19 polypeptide variants (will further define).The FGF-19 polypeptide can (as people's tissue or other source) separation obtain, also can make with reorganization and/or synthetic method from various sources.

" native sequences FGF-19 " comprises the polypeptide with aminoacid sequence identical with deriving from natural FGF-19.This native sequences FGF-19 can obtain from the nature separation, or make with reorganization and/or synthetic method.Term " native sequences FGF-19 " is particularly including natural truncation type or the secretor type (for example, the extracellular domain sequence) of FGF-19, natural variant (for example, replaceable shear-type) and natural allelic variant.In one embodiment of this invention, native sequences FGF-19 comprises 1 to 216 amino acid whose maturation or total length native sequences FGF-19 among Fig. 2 (SEQ IDNO:2).And, begin although the disclosed FGF-19 polypeptide of Fig. 2 (SEQID NO:2) shows the methionine residue that is defined as position 1 by this paper, among Fig. 2 (SEQ ID NO:2) another methionine residue in upstream, amino acid/11 position or downstream as the initial amino acid residue of FGF-19 polypeptide be can imagine with possible.

" FGF-19 variant polypeptide " refers to have active FGF-19 polypeptide at least about 80% amino acid sequence identity as what give a definition with following amino acid sequences: (a) aminoacid sequence of 1 or about 23 to 216 residues of FGF-19 polypeptide shown in Fig. 2 (SEQ ID NO:2), (b) aminoacid sequence of X to 216 residue of FGF-19 polypeptide shown in Fig. 2 (SEQ ID NO:2), wherein X is any amino-acid residue of 17 to 27 among Fig. 2 (SEQ ID NO:2), and perhaps (c) is derived from another specific fragment of aminoacid sequence shown in Fig. 2 (SEQ ID NO:2).This type of FGF-19 variant polypeptide comprises, for example, and in that sequence of N shown in Fig. 2 (SEQ ID NO:2)-and/or C-is terminal and the FGF-19 polypeptide that increases or delete one or more amino-acid residues in one or more internal structures territory.Usually, FGF-19 variant polypeptide and (a) aminoacid sequence of 1 or about 23 to 216 residues of FGF-19 polypeptide shown in Fig. 2 (SEQ IDNO:2), (b) aminoacid sequence of X to 216 residue of FGF-19 polypeptide shown in Fig. 2 (SEQID NO:2), wherein X is any amino-acid residue of 17 to 27 among Fig. 2 (SEQ ID NO:2), perhaps (c) has at least about 80% derived from another specific fragment of aminoacid sequence shown in Fig. 2 (SEQ ID NO:2), at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% amino acid sequence identity.The FGF-19 variant polypeptide does not comprise natural FGF-19 peptide sequence.Usually, the length of FGF-19 variant polypeptide is at least about 10 amino acid, or at least about 20 amino acid, perhaps at least about 30 amino acid, or at least about 40 amino acid, or at least about 50 amino acid, or at least about 60 amino acid, perhaps at least about 70 amino acid, or at least about 80 amino acid, or at least about 90 amino acid, or at least about 100 amino acid, perhaps at least about 150 amino acid, or at least about 200 amino acid, or at least about 300 or more a plurality of amino acid.

" amino acid sequence identity per-cent (%) " of the said relevant FGF-19 peptide sequence of this paper is meant the amino-acid residue of candidate sequence, carrying out the sequence contrast, and import the space where necessary obtaining the sequence identity of largest percentage, and the percentage ratio identical when any conservative replacement not being considered as sequence identity with the amino-acid residue of FGF-19 sequence.Can use this area the whole bag of tricks to carry out the sequence contrast, for example, use available computer software of the public such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software so that measure amino acid sequence identity per-cent.Those skilled in the art can determine to measure correlated suitable parameter, comprise at the sequence total length that is compared obtaining the required any algorithm of maximum contrast.Yet for this purpose, amino acid sequence identity % value is to use following sequence contrast computer program ALIGN-2 to obtain, and wherein whole source codes of ALIGN-2 program see Table 1.The author of ALIGN-2 sequence contrast computer program is Genentech, and source code shown in the Inc., table 1 and subscriber data have been submitted to together and be located in Washington D.C., 20559 U.S. Copyright Bureau, and its U.S.'s copyright registration registration number is TXU510087.The public passes through Genentech, Inc., and SouthSan Francisco, California can obtain the ALIGN-2 program, perhaps can work out from the source code that table 1 provides.The ALIGN2 program should be in UNIX operating system, preferably uses and works out at digital UNIXV4.0D.The ALIGN-2 program setting all sequences reduced parameter and constant.

For the object of the invention, given aminoacid sequence A is with respect to the following calculating of amino acid sequence identity % (perhaps say so: given aminoacid sequence A has or contain the % of given aminoacid sequence B same acid sequence) of given aminoacid sequence B:

X/Y ratio multiply by 100

Wherein X compares the same amino acid number that calculates behind the amino-acid residue of A and B with sequence contrast program ALIGN-2, and wherein Y is the amino-acid residue sum of B.Be appreciated that when the length of aminoacid sequence A and aminoacid sequence B is unequal A will be not equal to the amino acid sequence identity % of B with respect to A with respect to the amino acid sequence identity % of B.As an example that calculates amino acid sequence identity %, how table 2 and 3 explanations calculate the aminoacid sequence of being appointed as " reference protein " and the sequence identity % that is appointed as the aminoacid sequence of " PRO ".

Unless special declaration obtains otherwise all amino acid sequence identity % values used herein contrast computer program according to above-mentioned use ALIGN-2 sequence in addition.Yet amino acid sequence identity % also can use sequence contrast program NCBI-BLAST2 (Altschul etc., Nucleic acids Res.25:3389-3402 (1997)) to measure.NCBI-BLAST2 sequence contrast program can be downloaded from http://www.ncbi.nlm.nih.Gov, or from National Institute of Health, Bethesda, MD obtains.NCBI-BLAST2 uses several search arguments, wherein all these parameter settings are default value, for example comprise non-sheltering=yes, chain (strand)=all (all), appearance=10 of expection, minimum complicated length=15/5, many-journey e-value=0.01, many-journey constant=25, finally breachization is correlated falls (dropoff)=25 suddenly, and rating matrix=BLOSUM62.

When using NCBI-BLAST2 to carry out the aminoacid sequence comparison, given aminoacid sequence A is at the following calculating of amino acid sequence identity % (in other words, given aminoacid sequence A has or contain the % of the aminoacid sequence identical with given aminoacid sequence B) of given aminoacid sequence B:

X/Y ratio multiply by 100

Wherein X compares the same amino acid quantity that calculates behind A and the B amino-acid residue with sequence contrast program NCBI-BLAST2, and wherein Y is the amino-acid residue total quantity of B.Be appreciated that when the length of aminoacid sequence A and aminoacid sequence B is unequal A will be not equal to the amino acid sequence identity % of B at A at the amino acid sequence identity % of B.

" FGF-19 variant polynucleotide " or " FGF-19 variant nucleic acid sequences " be meant the coding following definitions active FGF-19 polypeptide nucleic acid molecule, it and (a) nucleotide sequence of the aminoacid sequence of 1 or about 23 to 216 residues of FGF-19 polypeptide shown in the code pattern 2 (SEQ ID NO:2), (b) nucleotide sequence of the aminoacid sequence of X to 216 residue of FGF-19 polypeptide shown in the code pattern 2 (SEQ ID NO:2), wherein X is any amino-acid residue of 17 to 27 among Fig. 2 (SEQ ID NO:2), perhaps (c) coding has the amino acid sequence identity at least about 80% derived from another specific segmental nucleotide sequence of aminoacid sequence shown in Fig. 2 (SEQ ID NO:2).Usually, FGF-19 variant polynucleotide and (a) nucleotide sequence of the aminoacid sequence of 1 or about 23 to 216 residues of FGF-19 polypeptide shown in the code pattern 2 (SEQ ID NO:2), (b) nucleotide sequence of the aminoacid sequence of X to 216 residue of FGF-19 polypeptide shown in the code pattern 2 (SEQ ID NO:2), wherein X is any amino-acid residue of 17 to 27 among Fig. 2 (SEQ ID NO:2), perhaps (c) coding has at least about 80% derived from another specific segmental nucleotide sequence of aminoacid sequence shown in Fig. 2 (SEQ ID NO:2), at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% nucleotide sequence identity.FGF-19 polynucleotide variant does not comprise natural FGF-19 nucleotide sequence.

Usually, the length of FGF-19 variant polynucleotide is at least about 30 Nucleotide, or at least about 60 Nucleotide, or at least about 90 Nucleotide, or at least about 120 Nucleotide, perhaps at least about 150 Nucleotide, or at least about 180 Nucleotide, or at least about 210 Nucleotide, or at least about 240 Nucleotide, perhaps at least about 270 Nucleotide, or at least about 300 Nucleotide, or at least about 450 Nucleotide, or at least 600 Nucleotide, or at least 900 or more a plurality of Nucleotide.

When " the nucleotide sequence identity per-cent (%) " of the nucleotide sequence about coding FGF-19 polypeptide described herein is meant in the candidate sequence and compares with the nucleotide sequence of coding FGF-19 polypeptide, percentage ratio with identical Nucleotide, this is in the contrast sequence, if necessary then introduce the space, with what obtain under the prerequisite that reaches maximal sequence identity per-cent.This area the whole bag of tricks be can use for the sequence contrast that mensuration nucleotide sequence homology per-cent carries out, for example, available computer software of the public such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software used.Those skilled in the art can determine to be used to measure correlated suitable parameter, comprise that the total length at institute's comparative sequences obtains the required any algorithm of maximum contrast.Yet for this purpose, nucleotide sequence homology % value such as following use sequence contrast computer program ALIGN-2 and obtain, and wherein whole source codes of ALIGN-2 program see Table 1.The author of ALIGN-2 sequence contrast computer program is Genentech, and source code shown in the Inc., table 1 and subscriber data have been submitted to together and be located in Washington D.C., 20559 U.S. Copyright Bureau, and its U.S.'s copyright registration registration number is TXU510087.The public passes through Genentech, Inc., and South San Francisco, California can obtain the ALIGN-2 program, and perhaps the source code that provides from table 1 is worked out.The ALIGN2 program should be in UNIX operating system, preferably uses and works out at digital UNIXV4.0D.The ALIGN-2 program setting all sequences reduced parameter and constant.

For the object of the invention, given nucleotide sequence C is with respect to the following calculating of nucleotide sequence identity % (perhaps say so: given nucleotide sequence C has or contain the % of the nucleotide sequence identical with given nucleotide sequence D) of given nucleotide sequence D:

W/Z ratio multiply by 100

Wherein W be the quantity of the identical Nucleotide that relatively calculates behind C and the D with sequence contrast program ALIGN-2 and wherein Z be the Nucleotide total quantity of D.Be appreciated that when the length of nucleotide sequence C and nucleotide sequence D is unequal C will be not equal to the nucleotide sequence identity % of D at C at the nucleotide sequence identity % of D.As an example that calculates nucleotide sequence identity %, how table 4 and 5 explanations calculate the nucleotide sequence and the identity % that is appointed as the nucleotide sequence of " PRO-DNA " of being appointed as " contrast DNA ".

Unless special declaration obtains otherwise all nucleotide sequence identity % values used herein contrast computer program according to above-mentioned use ALIGN-2 sequence in addition.Yet nucleotide sequence identity % also can use sequence contrast program NCBI-BLAST2 (Altschul etc., Nucleic Acids Res.25:3389-3402 (1997)) to measure.NCBI-BLAST2 sequence contrast program can be downloaded from http://www.ncbi.nlm.nih.Gov, or from National Institute of Health, Bethesda, MD obtains.NCBI-BLAST2 uses several search arguments, wherein all these parameter settings are default value, for example comprise, non-sheltering=yes, chain=all, appearance=10 of expection, minimum complicated length=15/5, many-journey e-value=0.01, many-journey constant=25, finally breachization is correlated falls suddenly=and 25, and rating matrix=BLOSUM62.

When using NCBI-BLAST2 to carry out the sequence comparison, the following calculating of the nucleotide sequence identity % of given nucleotide sequence C and given nucleotide sequence D (in other words, given nucleotide sequence C has or contain the % of the nucleotide sequence identical with given nucleotide sequence D):

W/Z ratio multiply by 100

Wherein W be the identical Nucleotide quantity that relatively calculates behind C and the D with sequence contrast program NCBI-BLAST2 and wherein Z be the Nucleotide total quantity of D.Be appreciated that when the length of nucleotide sequence C and nucleotide sequence D is unequal C will be not equal to the nucleotide sequence identity % of D at C at the nucleotide sequence identity % of D.

In another embodiment, FGF-19 variant polynucleotide be the coding active FGF-19 polypeptide nucleic acid molecule, it can with the nucleotide sequence hybridization (preferably under strictness hybridization and wash conditions, hybridizing) of total length FGF-19 polypeptide shown in the code pattern 2 (SEQ ID NO:2).The FGF-19 variant polypeptide can be by FGF-19 variant polynucleotide encoding.

As above-mentionedly carry out the term " positive " that amino acid sequence identity uses relatively the time, not only comprise amino-acid residue identical in the sequence that is compared, also comprise the amino-acid residue that wherein has similar characteristics.At the mark amino-acid residue of positive value of purpose amino-acid residue is those identical with the purpose amino-acid residue or the first-selection of purpose amino-acid residue replaces amino-acid residues.(see the following form 6 in definition).

For the object of the invention, given aminoacid sequence A is with respect to the following calculating of the positive value % (perhaps say so: given aminoacid sequence A has or contain the positive % of the aminoacid sequence identical with given aminoacid sequence B) of the aminoacid sequence of given aminoacid sequence B:

X/Y ratio multiply by 100

Wherein X compares the positive amino acid quantity of the scoring as defined above that draws behind A and the B with above-mentioned sequence contrast program ALIGN-2, and wherein Y is the amino-acid residue total quantity of B.Be appreciated that when the length of aminoacid sequence A and aminoacid sequence B is unequal A will be not equal to the positive % of B at A at the positive % of B.

When describing each peptide species of this paper, be meant and from the composition of natural surroundings, identify and the polypeptide that separates and/or reclaim with " isolating ".Preferably, isolated polypeptide and its natural bonded all components are without any combining.Pollutant component in the natural surroundings of polypeptide is that those will disturb the material that uses polypeptide to diagnose and treat, and can comprise enzyme, hormone and other albumen or non--albumen solute.In preferred embodiments, polypeptide is purified as: (1) uses rotary-cup type protein sequencing instrument, reach the degree that is enough to obtain N-end or at least 15 residues of internal amino acid sequence, perhaps (2) reach under non--reduction or reductive condition and dye the homogeneous degree that turns out to be through SDS-PAGE electrophoresis and Coomassie blue or preferred silver.Isolated polypeptide is included in the original position polypeptide in the reconstitution cell, because at least one component in the FGF-19 physical environment is non-existent.Yet, prepare isolated polypeptide with at least one purification step usually.

" isolating " nucleic acid molecule of coding FGF-19 polypeptide is to identify and isolated nucleic acid molecule usually and at least a impurity nucleic acid molecule of its bonded from the natural origin of FGF-19 coding nucleic acid.Preferably, described isolating nucleic acid and its natural bonded all components are without any combining.The nucleic acid molecule differ of one separated coding FGF-19 is in its natural form or combination (setting).Therefore the nucleic acid molecule of separated coding FGF-19 is different from its existence form in n cell.But the nucleic acid molecule of separated coding FGF-19 polypeptide comprises FGF-19-coding nucleic acid molecule contained in the cell of common expression FGF-19, and for example, described nucleic acid molecule is different with the chromosome position at n cell place.

Term " control sequence " refers to make in the specific host biology encoding sequence that can be operatively connected to express required dna sequence dna.Be suitable for procaryotic regulating and controlling sequence and can comprise, optional operon sequence, and ribosome bind site as promotor.Known eukaryotic cells utilizes promotor, polyadenylation signal and enhanser.

When a kind of nucleic acid and another kind of nucleotide sequence were in the relevant position of function, this nucleic acid " can be operated and link to each other " with this another kind nucleic acid.If for example a kind of expression of polypeptides is for participating in the excretory precursor protein of this polypeptide, then the DNA of presequence or secretion leader sequence can be operated with the DNA of this polypeptide and link to each other; Promotor or enhanser are when influencing transcribing this encoding sequence of Shi Keyu and can operating continuous of encoding sequence; Or ribosome bind site links to each other when its position can promote translation the time can operate with encoding sequence.Usually " can operate continuous " and refer to that connected dna sequence dna is adjacent, and, if the secretion leader sequence is then adjacent and be read state.Yet it is adjacent that enhanser needs not to be.Connection can realize by restriction site is continuous easily.Synthetic oligonucleotide adapter or joint can be used according to conventional practice in if there is no such site.

Term used herein " antibody " is meant broadest antibody, and particularly including strand for example anti--FGF-19 monoclonal antibody (comprising agonist, antagonist and neutralizing antibody), have multi-epitope specific anti--FGF-19 antibody compositions, strand be anti--fragment (stating as follows) of FGF-19 antibody and anti--FGF-19 antibody.Term " monoclonal antibody " herein is meant that promptly except the natural sudden change that may exist on a small quantity, each antibody in this antibody population is all identical from the antibody of the antibody population of homogeneity basically.

" the strict degree " of hybridization determined at an easy rate by those skilled in the art, rule of thumb calculates on the basis of considering probe length, wash temperature and salt concn usually.Generally speaking, long probe needs higher temperature so that correctly anneal, and short probe needs lesser temps.Reannealing ability when there is complementary strand in the DNA of sex change in being lower than the environment of melting temperature(Tm) is depended in hybridization usually.Required homology degree is high more between probe and the hybridization sequences, and then spendable relative temperature is high more.The result is, the higher reaction conditions that will make of relative temperature is tending towards strict more, and lesser temps then makes the rigorous degree of reaction less.About other details and the explanation of the strict degree of hybridization, see Ausubel etc., Current Protocols inMolecular Biologv, Wiley Interscience Publishers, (1995).

" stringent condition " of this paper or " height stringent condition ", can be defined as: (1) uses low ionic strength and high-temperature wash, for example, 50 ℃ of 0.015M sodium-chlor/0.0015M Trisodium Citrate/0.1% sodium lauryl sulphate; (2) in crossover process 42 ℃ use denaturing agent such as methane amide, for example, 50% (v/v) methane amide/0.1%Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer pH6.5 and the 750mM sodium-chlor that contains 0.1% bovine serum albumin, the 75mM Trisodium Citrate; Perhaps (3) 42 ℃ are used 50% methane amide, 5xSSC (0.75M NaCl, 0.075M Trisodium Citrate), salmon sperm DNA (50 μ g/ml), 0.1%SDS and 10% T 500 of 50mM sodium phosphate (pH 6.8), 0.1% trisodium phosphate, 5xDenhardt ' s solution, supersound process, neutralize at 0.2xSSC (sodium chloride/sodium citrate) in 42 ℃ and in 50% methane amide, to wash, then carry out highly strict washing in 55 ℃ of SSC that contain EDTA with 0.1x in 55 ℃.

" medium stringent condition " can be according to Sambrook etc., Molecular Cloning:A LaboratoryManual, New York:Cold Spring Harbor Press, 1989 definition, comprise and use strict degree (for example to be lower than aforesaid washing soln and hybridization conditions, temperature, ionic strength and SDS%).An example of medium stringent condition is, incubation spends the night in 37 ℃ of solution at following composition: 20% methane amide, 5x SSC (150mM NaCl, the 15mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5xDenhardt ' s solution, the salmon sperm DNA that 10% T 500 and 20mg/ml sex change are sheared then washs Hybond membrane in 37-50 ℃ in 1xSSC.Those skilled in the art understand and adjust conditions such as temperature, ionic strength how necessarily to adapt to such as factors such as probe length.

Term used herein " epi-position mark " is meant the chimeric polyeptides that contains the FGF-19 polypeptide that has merged with " labeling polypeptide ".This labeling polypeptide has abundant residue so that the epi-position at prepared antibody to be provided, and will enough lack so that the activity of the polypeptide of its nonintervention and its fusion.Labeling polypeptide also preferably is unique really, and cross reaction does not take place basically for antibody and other epi-position like this.Usually, suitable labeling polypeptide has 6 amino-acid residues at least, is generally about 8～50 amino-acid residues (being preferably 10～20 amino-acid residues).

Term " immunoadhesin " is defined as antibody molecule in this article, and it is with the binding domains and the constant region for immunoglobulin combination of allos " adhesin " albumen (for example acceptor, part or enzyme).On the structure, immunoadhesin comprises the adhesin amino-acid residue with required binding specificity and the fusion of constant region for immunoglobulin sequence, and described adhesin amino acid is different from the antigen recognition and the binding site (antigen binding site) (being " heterology ") of antibody.The adhesin of immunoadhesin molecule part normally comprise acceptor or part at least one binding site in abutting connection with aminoacid sequence.Constant region for immunoglobulin sequence in the immunoadhesin can derive from any immunoglobulin (Ig), as IgG-1, IgG-2, IgG-3 or IgG-4 hypotype, IgA (comprising IgA-1 and IgA-2), IgE, IgD or IgM.

Activity herein " " is meant and keeps complete or the biology of natural FGF-19 and/or the FGF-19 form of immunologic competence, wherein " biology " activity refers to the biological function (suppressing or excitement) that complete or natural FGF-19 causes, is not meant the ability of generation at the antibody of complete or the epi-position that natural FGF-19 had of inducing; " immunology " active then being meant induces generation at the ability complete or antibody that epi-position is arranged that natural FGF-19 gathered around.Preferred biological activity comprises following any one or a plurality of activity: increase individual metabolism (or metabolic rate), reduce whose body weight, alleviate individual fat, reduce glucose uptake and enter adipocyte, increase leptin and discharge, reduce individual triglyceride levels and reduce individual free fatty acids from adipocyte.Should understand that the part activity of FGF-19 is by the direct inductive of FGF-19, and some activity is an indirect induction, still, every kind of activity all is results that FGF-19 exists, if there is not FGF-19, will can not produce these results.

Term " antagonist " uses its broader sense herein, comprises partially or completely the bioactive any molecule of natural FGF-19 polypeptide described herein of blocking, suppress or neutralize.Similarly, term " agonist " also uses its broader sense, comprises the bioactive any molecule of simulation natural FGF-19 polypeptide described herein.Suitable agonist or antagonist molecules are particularly including the fragment of agonist or antagonist antibodies or antibody fragment, natural FGF-19 polypeptide or aminoacid sequence variant, peptide, little organic molecule etc.Identify that the agonist of FGF-19 polypeptide or the method for antagonist can be, the FGF-19 polypeptide is contacted with agonist to be measured or antagonist molecules, and measure one or more bioactive the record variation relevant under the normal circumstances with the FGF-19 polypeptide.

" treatment " is meant therapeutic treatment and preventive measure, its objective is stop or delay (alleviating) at pathological disorders.Those individualities that need treat comprise, have suffered from illness or ill tendency have been arranged or in order to prevent to suffer from the individuality of illness.

" chronic " administration refers to mode of administration is opposite fast reagent be used with the successive administration pattern, so that keep initial therapy effect (activity) in long-time." interruption " administration is meant that treatment is not carry out continuously off and on, but is feature with periodicity.

" Mammals " that is intended to treat is meant and is classified as mammiferous any animal, comprises people, domestic animal and farm-animals, and the animal in the zoological park, the animal that participates in sports events or pet such as dog, horse, cat, ox etc.Preferred described Mammals is human.

" individuality " is meant any main body, and preferably Mammals is more preferably the people.

" obesity " refers to that mammiferous weight index (BMI) is at least 25.9 situation, and this index is according to body weight (kg) square calculating divided by height (rice).Usually, the BMI with people of normal type be 19.9～less than 25.9.Obesity described herein can be any cause, no matter is due to heredity or the environmental factors.

Cause fat or the example of the illness of the fat reason of saying so comprises: excessive food consumption and Bulimia nerovsa, polycystic ovarian disease, craniopharyngioma, Prader Willi syndrome, Frohlich syndrome (Frohlich ' ssyndrome), type ii diabetes, GH-defective type individuality, normality variation of short and small stature, Turner syndrome (Turner ' s syndrome), show as with other that Metabolic activity lowers or REE accounts for the pathological disorders that the ratio of not fatty material total amount (fat-free mass) descends, for example, the children that suffer from acute lymphoblastic leukemia.

" with fat relevant illness " is meant the illness that causes obesity or obesity is increased the weight of, and such as but not limited to tetter such as infection, varix, acanthosis nigricans and eczema, utilization endurance is poor, diabetes, insulin resistant, hypertension, hypercholesterolemia disease, chololithiasis, osteoarthritis, anaplasty damage, thrombotic diseases, cancer and coronary heart disease (or cardiovascular diseases), particularly those cardiovascular disorders relevant with free fatty acids with individual high triglyceride.

Comprise administration simultaneously with one or more other therapeutical agents " associating " administration and with the coherent administration of any order.

" carrier " used herein is included under used dosage and the concentration pair cell or the avirulent pharmaceutically acceptable carrier of Mammals, vehicle, stablizer.Pharmaceutically acceptable carrier usually is aqueous pH solutions buffered.The example of pharmaceutically acceptable carrier comprises buffer reagent such as phosphoric acid salt, Citrate trianion and other organic acid; Antioxidant comprises xitix; Lower molecular weight (less than 10 residues) polypeptide; Albumen is as albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer such as polyvinylpyrrolidone; Amino acid such as glycine, glutamine, l-asparagine, arginine or Methionin; Monose, disaccharides and other carbohydrate comprise glucose, seminose or dextrin; Sequestrant such as EDTA; Sugar alcohol such as N.F,USP MANNITOL or sorbyl alcohol; Become salt ion such as sodium; And/or nonionogenic tenside such as TWEEN ^TM, polyoxyethylene glycol (PEG) and PLURONICSTM ^TM

" antibody fragment " comprises the part of complete antibody, preferably combination of the antigen of complete antibody or variable region.The example of antibody fragment comprises Fab, Fab ', F (ab ') ₂With the Fv fragment; Bivalent antibody; Linear antibody (Zapata etc., Protein Eng.8 (10): 1057-1062[1995]); The single-chain antibody molecule; With the multi-specific antibody that forms by antibody fragment.

Papain digestion antibody can produce two identical Fabs that respectively have single antigen binding site (being called " Fab " fragment) and remaining " Fc " fragment, and the title of Fc section has been reacted it and has been easy to the crystalline ability.Through pepsin can produce have two antigen binding sites and still can crosslinked antigenic F (ab ') ₂Fragment.

" Fv " contains the complete identification and the minimum antibody fragment of binding site.This district is made up of a variable region of heavy chain and the closely non-covalent dimer that is connected to form of variable region of light chain.Three of each variable region CDR interact in this conformation, at V _H-V _LThe dimer surface limits an antigen binding site.These six CDR give antibody jointly with antigen-binding specificity.Yet, even single variable region (or F _vHalf only contain three antigen-specific CDR) also have the identification and an ability of conjugated antigen, although to compare its avidity lower with complete binding site.

The Fab section also comprises first constant region (CH1) of constant region of light chain and heavy chain.Fab ' is that with the difference of Fab Fab ' has more several residues at the C-terminal of heavy chain CH1, comprises one or more halfcystines of antibody hinge region.Fab '-SH in this article refers to the Fab ' that has a free sulfhydryl groups in the constant region cysteine residues at least.F (ab ') ₂Antibody fragment has the hinge area halfcystine in that to be produced as Fab ' fragment at first right between them.Other chemical coupling of antibody fragment is well-known.

" light chain " of the antibody of any species of vertebrates (immunoglobulin (Ig)) can be classified as the type in the diverse amphitypy (being called k and λ) according to its constant region aminoacid sequence.

According to the aminoacid sequence of its CH, immunoglobulin (Ig) can be divided into inhomogeneity.Mainly contain 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them also can further be divided into " subclass " (isotype), for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

Strand Fv " or " scFv " antibody fragment, comprise the V of antibody _HAnd V _LStructural domain, these structural domains are present on the single polypeptide chain.Preferred Fv polypeptide is at V _HAnd V _LAlso comprise a peptide linker between the structural domain, it can make sFv form antigen in conjunction with required structure.See Pluckthun at " pharmacology of monoclonal antibody " about the summary of scFv, the 113rd volume, Rosenburg and Moore compile, Springer-Verlag, New York, 269-315 page or leaf (1994).

Term " bivalent antibody " is meant the small molecular antibody fragment with two antigen binding sites, and these fragments are at a polypeptide chain (V _H-V _L) on contain a continuous variable region of heavy chain (V _H) and a variable region of light chain (V _L).Utilize a kind of very short joint, it makes two structural domains on same the chain to match, have to another chain on the pairing of complementary structure territory, thereby form two antigen binding sites.At EP 404,097; WO93/11161; With Hollinger etc., institute of NAS periodical has the more detailed description of pair bivalent antibody among the 90:6444-6448 (1993).

" isolating " antibody is the antibody of identifying from the composition of its natural surroundings, separating and/or reclaim.The pollutant component of its natural surroundings is to disturb the diagnosis of antibody or the material that treatment is used, and can comprise enzyme, hormone and other albumen or non-albumen solute.In preferred embodiments, the purity of this antibody should reach: more than 95% of antibody weight that (1) is determined through the Lowery method, described weight more than 99% most preferably, (2) be enough to obtain N end or internal amino acid sequence with rotation cup-shaped (spinning cup) at least 15 residues that sequenator is surveyed, (3) are by the SDS-PAGE under reduction or the non-reduced condition and coomassie brilliant blue staining, the preferred silver-colored homogeneity that dyes and confirmed.Isolated antibody comprises the original position antibody in the reconstitution cell, because at least a component in the physical environment of this antibody does not exist.Isolated antibody can prepare by at least one purification step generally speaking.

Used herein " mark " speech, thus be meant detectable compound or the component that produces " mark " antibody directly or indirectly with antibody coupling.Mark itself can be recorded (for example labelled with radioisotope or fluorescent mark), perhaps if during enzyme labelling, but catalytic substrate compound or component generation chemical transformation, this variation is detectable.

" solid phase " is meant that antibody of the present invention can adherent non-aqueous matrix.The example of solid phase comprises those solid phases of partly or entirely being made by glass (as the glass in control aperture), polysaccharide (as agarose), polyacrylamide, polystyrene, polyvinyl alcohol and siloxanes herein.In some instances, content based on context, solid phase can comprise the hole of test panel; In other example, it can be purification column (as an affinity column).This term also comprises the discontinuous solid phase of disclosed discrete particles in the United States Patent (USP) 4275149.

" liposome " is by effectively transporting the small molecules vesica that each lipoids, phosphatide and/or the tensio-active agent of medicine (as anti-FGF-19 polypeptide disclosed herein and/or its antibody) are formed to Mammals.The component of liposome is arranged as double-deck form usually, with biomembranous lipid homotaxy.

" small molecules " is defined as molecular weight and is lower than about 500 daltonian molecules herein. Table 1(the computer command symbol of Insert Here original text 34-50 page or leaf)

Tablel

/* * C-C increased from 12 to 15 * Z is average of EQ * B is average of ND * match with stop is _M； stop-stop ＝ 0；J (joker)match ＝ 0 */#define _M     -8      /* value of a match with a stop */int    _day[26][26] ＝ {/*     A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*//* A */   { 2，0，-2，0，0，-4，1，-1，-1，0，-1，-2，-1，0，_M， 1，0，-2，1，1，0，0，-6，0，-3，0}，/* B */   { 0，3，-4，3，2，-5，0，1，-2，0，0，-3，-2，2，_M，-1， 1，0，0，0，0，-2，-5，0，-3，1}，/* C */   {-2，-4，15，-5，-5，-4，-3，-3，-2，0，-5，-6，-5，-4，_M，-3，-5，-4，0，-2，0，-2，-8，0，0，-5}，/* D */   { 0，3，-5，4，3，-6，1，1，-2，0，0，-4，-3，2，_M，-1，2，-1，0，0，0，-2，-7，0，-4，2}，/* E */   { 0，2，-5，3，4，-5，0，1，-2，0，0，-3，-2，1，_M，-1，2，-1，0，0，0，-2，-7，0，-4，3}，/* F */   {-4，-5，-4，-6，-5，9，-5，-2，1，0，-5，2，0，-4，_M，-5，-5，-4，-3，-3，0，-1，0，0，7，-5}，/* G */   { 1，0，-3，1，0，-5，5，-2，-3，0，-2，-4，-3， 0，_M，-1，-1，-3，1，0，0，-1，-7，0，-5，0}，/* H */   {-1，1，-3，1，1，-2，-2，6，-2，0，0，-2，-2，2，_M，0，3，2，-1，-1，0，-2，-3，0，0，2}，/* I */   {-1，-2，-2，-2，-2，1，-3，-2，5，0，-2，2，2，-2，_M，-2，-2，-2，-1，0，0，4，-5，0，-1，-2}，/* J */   { 0，0，0，0，0，0，0，0，0，0，0，0，0，0，_M，0，0，0，0，0，0，0，0，0，0，0}，/* K */   {-1，0，-5，0，0，-5，-2，0，-2，0，5，-3，0，1，_M，-1， 1， 3， 0， 0， 0，-2，-3，0，-4，0}，/* L */   {-2，-3，-6，4，-3，2，-4，-2，2，0，-3，6，4，-3，_M，-3，-2，-3，-3，-1，0，2，-2，0，-1，-2}，/* M */   {-1，-2，-5，-3，-2，0，-3，-2，2，0，0，4，6，-2，_M，-2，-1，0，-2，-1，0，2，-4，0，-2，-1}，/* N */   { 0，2，-4，2，1，-4，0，2，-2，0，1，-3，-2，2，_M，-1，1，0，1，0，0，-2，-4，0，-2，1}，/* O */{_M，_M，_M，_M，_M，_M，_M，_M，_M，_M，_M，_M，_M，_M，_0，_M，_M，_M，_M，_M，_M，_M，_M，_M，_M，_M}，/* P */   { 1，-1，-3，-1，-1，-5，-1， 0，-2，0，-1，-3，-2，-1，_M， 6， 0， 0， 1， 0， 0，-1，-6， 0，-5， 0}，/* Q */   { 0，1，-5，2，2，-5，-1，3，-2，0，1，-2，-1， 1，_M，0，4，1，-1，-1，0，-2，-5， 0，-4，3}，/* R */   {-2，0，-4，-1，-1，-4，-3，2，-2，0，3，-3，0，0，_M，0，1，6，0，-1，0，-2，2，0，-4，0}，/* S */   { 1，0， 0，0，0，-3，1，-1，-1，0，0，-3，-2，1，_M，1，-1，0，2， 1，0，-1，-2，0，-3，0}，/* T */   { 1，0，-2，0，0，-3，0，-1，0，0，0，-1，-1，0，_M，0，-1，-1，1，3，0，0，-5，0，-3，0}，/* U */   { 0，0，0，0，0，0，0，0，0，0，0，0，0，0，_M，0，0，0，0，0，0，0，0，0，0，0}，/* V */   { 0，-2，-2，-2，-2，-1，-1，-2，4，0，-2，2，2，-2，_M，-1，-2，-2，-1，0，0，4，-6，0，-2，-2}，/* W */   {-6，-5，-8，-7，-7，0，-7，-3，-5，0，-3，-2，-4，-4，_M，-6，-5， 2，-2，-5，0，-6，17， 0，0，-6}，/* X */   { 0，0，0，0，0，0，0，0，0，0，0，0，0，0，_M，0，0，0，0，0，0，0，0，0，0，0}，/* Y */   {-3，-3，0，-4，-4，7，-5，0，-1， 0，-4，-1，-2，-2，_M，-5，-4，-4，-3，-3，0，-2，0，0，10，-4}，/* Z */   { 0，1，-5，2，3，-5，0，2，-2，0，0，-2，-1，1，_M，0，3，0，0，0，0，-2，-6，0，-4，4}}；

Table?1?(cont’)

/**/#include＜stdio.h＞#include＜ctype.h＞#define MAXJMP 16            /* max jumps in a diag */#define MAXGAP               24       /* don′t continue to penalize gaps larger than this */#define JMPS                 1024     /* max jmps in an path */#define MX                   4        /* save if there′s at least MX-1 bases since last jmp */#define DMAT                 3        /* value of matching bases */#define DMIS                 0        /* penalty for mismatched bases */#define DINS0                8        /* penalty for a gap */#define DINS1                1        /* penalty per base */#define PINS0                8        /* penalty for a gap */#define PINS1                4        /* penalty per residue */struct jmp {　　       short             n[MAXJMP]；      /* size of jmp (neg for dely) */　　       unsigned short    x[MAXJMP]；      /* base no. of jmp in seq x */}；                                           /* limits seq to 2″16 -1 */struct diag{　　       int               score；            /* score at last jmp */　　       long              offset；           /* offset of prev block */　　       short             ijmp；             /* current jmp index */　　       struct jmp        jp；               /* list of jmps */}；struct path{　　       int      spc；               /* number of leading spaces */　　       short    n[JMPS]；/* size of jmp(gap) */　　       int      x[JMPS]；/* loc of jmp(last elem before gap) */}；char                *ofile；                     /* output file name */char                *namex[2]；                  /* seq names：getseqs() */char                *prog；                      /* prog name for err msgs */char                *seqx[2]；                   /* seqs：getseqs() */int                 dmax；                       /* best diag：nw() */int                 dmax0；                      /* final diag */int                 dna；                        /* set if dna：main() */int                 endgaps；                    /* set if penalizing end gaps */int                 gapx，gapy；                 /* total gaps in seqs */int                 len0，len1；                 /* seq lens */int                 ngapx，ngapy；               /* total size of gaps */int                 smax；                       /* max score：nw() */tnt                 *xbm；                       /* bitmap for matching */long                offset；                     /* current offset in jmp file */struct    diag      *dx；                        /* holds diagonals */struct    path      pp[2]；                      /* holds path for seqs */char                *calloc()，*malloc()，*index()，*strcpy()；char                *getseq()，*g_calloc()；

Table?1?(cont’)

/* Needleman-Wunsch alignment program * * usage：progs file1 file2 * where file1 and file2 are two dna or two protein sequences. * The sequences can be in upper- or lower-case an may contain ambiguity * Any lines beginning with′；′，′＞′or′＜′are ignored * Max file length is 65535 (limited by unsigned short x in the jmp struct) * A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA * Output is in the file ″align.out″ * * The program may create a tmp file in/tmp to hold info about traceback. * Original version developed under BSD 4.3 on a vax 8650 */#include ″nw.h″#include ″day.h″static   _dbval[26] ＝ {　　     1，14，2，13，0，0，4，11，0，0，12，0，3，15，0，0，0，5，6，8，8，7，9，0，10，0}；static   _pbval[26] ＝ {　　     1，2|(1＜＜(′D′-′A′))|(1＜＜(′N′-′A′))，4，8，16，32，64，　　     128， 256，0xFFFFFFF，1＜＜10，1＜＜11，1＜＜12，1＜＜13，1＜＜14，　　     1＜＜15，1＜＜16，1＜＜17，1＜＜18，1＜＜19，1＜＜20，1＜＜21，1＜＜22，　　     1＜＜23，1＜＜24，1＜＜25|(1＜＜(′E′-′A′))|(1＜＜(′Q′-′A′))}；main(ac，av)                                                                                          main　　     int        ac；　　     char       *av[]；{　　     prog ＝ av[0]；　　     if (ac ！＝ 3) {　　                fprintf(stderr，″usage：％s file1 file2\n″，prog)；　　                fprintf(stderr，″where file1 and file2 are two dna or two protein sequences.\n″)；　　                fprintf(stderr，″The sequences can be in upper-or lower-case\n″)；　　                fprintf(stderr，″Any lines beginning with ′；′or ′＜′are ignored\n″)；　　                fprintf(stderr，″Output is in the file \″align.out\″\n″)；　　                exit(1)；　　     }　　     namex[0] ＝ av[1]；　　     namex[1] ＝ av[2]；　　     seqx[0] ＝ getseq(namex[0]，&len0)；　　     seqx[1] ＝ getseq(namex[1]，&len1)；　　     xbm ＝ (dna)？_dbval：_pbval； 　　     endgaps ＝ 0；                      /* I to penalize endgaps */　　     ofile ＝ ″align.out″；            /* output file */　　     nw()；           /* fill in the matrix，get the possible jmps */　　     readjmps()；     /* get the actual jmps */　　     print()；        /* print stats，alignment */　　     cleanup(0)；     /* unlink any tmp files */}

Table?1?(cont’)

/* do the alignment，return best score： main() * dna：values in Fitch and Smith，PNAS，80，1382-1386，1983 * pro：PAM 250 values * When scores are equal，we prefer mismatches to any gap，prefer * a new gap to extending an ongoing gap，and prefer a gap in seqx * to a gap in seq y. */nw()                                                                                                               nw{　　       char            *px， *py；                 /* seqs and ptrs */　　       int             *ndely，*dely；    /* keep track of dely */　　       int             ndelx，delx；      /* keep track of delx */　　       int             *tmp；             /* for swapping row0，row1 */　　       int             mis；              /* score for each type */　　       int             ins0，ins1；       /* insertion penalties */　　       register        id；               /* diagonal index */　　       register        ij；               /* jmp index */　　       register        *col0，*col1；     /* score for curr，last row */　　       register        xx，yy；           /* index into seqs */　　       dx ＝ (struct diag*)g_calloc(″to get diags″，len0+len1+1，sizeof(struct diag))；　　　     ndely ＝ (int*)g_calloc(″to get ndely″，len1+1，sizeof(int))；　　       dely ＝ (int*)g_calloc(″to get dely″，len1+1，sizeof(int))；　　       col0 ＝ (int*)g_calloc(″to get col0″，len1+1，sizeof(int))；　　       col1 ＝ (int*)g_calloc(″to get col1″，len1+1，sizeof(int))；　　       ins0 ＝ (dna)？DINS0：PINS0；　　       ins1 ＝ (dna)？DINS1：PINS1；　　       smax ＝ -10000；　　       if(endgaps){　　               for (col0[0] ＝ dely[0] ＝ -ins0，yy ＝ 1；yy ＜ ＝ len1；yy++){　　                              col0[yy] ＝ dely[yy] ＝ col0[yy-1] - ins1；　　                              ndely[yy] ＝ yy；　　               }　　　              col0[0] ＝ 0；     /* Waterman Bull Math Biol 84 */　　       }　　       else　　               for(yy ＝ 1；yy ＜＝ len1；yy++)　　                         dely[yy] ＝ -ins0；　　       /* fill in match matrix　　        */　　       for(px ＝ seqx[0]，xx ＝ 1；xx ＜ ＝ len0；px++，xx++){　　               /* initialize first entry in col　　                */　　　              if(endgaps){　　                        if(xx ＝＝ 1)　　                                 col1[0] ＝ delx ＝ -(ins0+ins1)；　　                        else　　                                 col1[0] ＝ delx ＝col0[0]-ins1；　　                                 ndelx ＝ xx；　　               }　　               else {　　　                               col1[0] ＝ 0；　　                                 delx ＝ -ins0；　　                                 ndelx ＝ 0；　　               }

Table?1?(cont’)

　　                                                                            ...nwfor(py ＝ seqx[1]，yy ＝ 1；yy ＜ ＝ len1； py++， yy++) {　　      mis ＝ col0[yy-1]；　　      if(dna)　　                mis +＝ (xbm[*px-′A′]&xbm[*py-′A′])？DMAT：DMIS；　　　    else　　                mis + ＝ _day[*px-′A′][*py-′A′]；　　      /* update penalty for del in x seq；　　       * favor new del over ongong del　　       * ignore MAXGAP if weighting endgaps　　       */　　      if(endgaps || ndely[yy] ＜ MAXGAP){　　              if(col0[yy] - ins0 ＞ ＝ dely[yy]){　　                        dely[yy] ＝ col0[yy] - (ins0+ins1)；　　                        ndely[yy] ＝ 1；　　              }else{　　                        dely[yy] -＝ ins1；　　                        ndely[yy]++；　　              }　　      }else{　　              if(col0[yy] - (ins0+ins1) ＞ ＝ dely[yy]){　　                        dely[yy] ＝ col0[yy] - (ins0+ins1)；　　                        ndely[yy] ＝ 1；　　              }else　　                        ndely[yy]++；　　      }　　      /* update penalty for del in y seq；　　       * favor new del over ongong del　　       */　　      if(endgaps || ndelx ＜ MAXGAP){　　              if(col1[yy-1] - ins0 ＞ ＝ delx){　　                        delx ＝ col1[yy-1] - (ins0+ins1)；　　                        ndelx ＝ 1；　　              }else{　　                        delx -＝ ins1；　　                        ndelx++；　　              }　　      } else {　　              if(col1[yy-1] - (ins0+ins1) ＞ ＝ delx){　　                        delx ＝ col1[yy-1] - (ins0+ins1)；　　                        ndelx ＝ 1；　　              }else　　                        ndelx++；　　      }　　      /* pick the maximum score；we’re favoring　　       * mis over any del and delx over dely　　       */

Table?1?(cont’)

　　                                                                                    ...nw　　                  id＝xx-yy+len1-1；　　                  if(mis ＞ ＝ delx && mis ＞ ＝ dely[yy])　　                              col1[yy] ＝ mis；　　                  else if(delx ＞ ＝ dely[yy]]){　　                              col1[yy] ＝ delx；　　                              ij ＝ dx[id].ijmp；　　                              if(dx[id].jp.n[0] && (！dna || (ndelx ＞ ＝ MAXJMP　　                              && xx ＞ dx[id].jp.x[ij]+MX) || mis ＞ dx[id].score+DINSO)){　　                                         dx[id].ijmp++；　　                                         if (++ij ＞ ＝ MAXJMP){　　                                                   writejmps(id)；　　                                                   ij ＝ dx[id].ijmp ＝ 0；　　                                                   dx[id].offset ＝ offset；　　                                                   offset+ ＝ sizeof(struct jmp) + sizeof(offset)；　　                                         }　　                              }　　                              dx[id].jp.n[ij] ＝ ndelx；　　                              dx[id].jp.x[ij] ＝ xx；　　                              dx[id].score ＝ delx；　　                 }　　                 else{　　                              col1[yy] ＝ dely[yy]；　　                              ij ＝ dx[id].ijmp；if(dx[id].jp.n[0]&&(！dna || (ndely[yy] ＞ ＝ MAXJMP　　                              && xx ＞ dx[id].jp.x[ij]+MX) || mis ＞ dx[id].score+DINSO)){　　                                       dx[id].ijmp++；　　                                       if(++ij ＞ ＝ MAXJMP){　　                                               writejmps(id)；　　                                               ij ＝ dx[id].ijmp ＝ 0；　　                                               dx[id].offset ＝ offset；　　                                               offset + ＝ sizeof(struct jmp) + sizeof(offset)；　　                                       }　　                              }　　                              dx[id].jp. n[ij] ＝ -ndely[yy]；　　                              dx[id].jp.x[ij] ＝ xx；　　                              dx[id].score ＝ dely[yy]；　　                 }　　                 if(xx ＝ ＝ len0 && yy ＜ len1){　　                              /* last col　　                               */　　                              if(endgaps)　　                                      col1[yy]-＝ ins0+ins1*(len1-yy)；　　                              if(col1[yy] ＞ smax){　　                                      smax ＝ col1[yy]；　　                                      dmax ＝ id；　　                              }　　                }　　    }　　   if(endgaps && xx ＜ len0)　　                col1[yy-1] -＝ ins0+ins1*(len0-xx)；　　   if(col1[yy-1] ＞ smax){　　                smax ＝ col1[yy-1]；　　                dmax ＝ id；　　   }　　   tmp ＝ col0；col0 ＝ col1；col1 ＝ tmp；}(void)free((char *)ndely)；(void)free((char *)dely)；(void)free((char *)col0)；(void)free((char *)col1)；}

Table?1?(cont’)

/* * * print() - only routine visible outside this module * * static： * getmat() - trace back best path，count matches：print() * pr_align() - print alignment of described in array p[]：print() * dumpblock() - dump a block of lines with numbers，stars：pr_align() * nums() -- put out a number line：dumpblock() * putline() - put out a line (name，[num]，seq，[num])：dumpblock() * stars() - - put a line of stars：dumpblock() * stripname() -- strip any path and prefix from a seqname */#include ″nw.h″#define SPC     3#define P_LINE  256     /* maximum output line */#define P_SPC   3       /* space between name or num and seq */extern  _day[26][26]；int     olen；          /* set output line length */FILE    *fx；           /* output file */print()                                                                                          prim{　　    int       lx，ly，firstgap，lastgap；     /* overlap */　　    if((fx ＝ fopen(ofile，″w″)) ＝＝ 0){　　              fprintf(stderr，″％s：can′t write ％s\n″，prog，ofile)；　　              cleanup(1)；　　    }　　    fprintf(fx，″＜ first sequence：％s (length ＝ ％d)\n″，namex[0]，len0)；　　    fprintf(fx，″＜ second sequence：％s (length ＝ ％d)\n″，namex[1]，len1)；　　    olen ＝ 60；　　    lx ＝ len0；　　    ly ＝ len1；　　    firstgap ＝ lastgap ＝ 0；　　    if(dmax ＜ len1 - 1) {     /* leading gap in x */　　            pp[0].spc ＝ firstgap ＝ len1 - dmax - 1；　　            ly -＝ pp[0].spc；　　    }　　    else if(dmax ＞ len1 - 1){ /* leading gap in y */　　            pp[1].spc ＝ firstgap ＝ dmax - (len1 - 1)；　　            lx -＝ pp[1].spc；　　    }　　    if(dmax0 ＜ len0 - 1){    /* trailing gap in x */　　            lastgap ＝ len0- dmax0-1；　　            lx-＝ lastgap；　　    }　　    else if(dmax0 ＞ len0 - 1){ /* trailing gap in y */　　            lastgap ＝ dmax0 - (len0 - 1)；　　            ly -＝ lastgap；　　    }　　    getmat(lx，ly，firstgap，lastgap)；　　    pr_align()；}

Table?1?(cont’)

/* * trace back the best path，count matches */staticgetmat(lx，ly，firstgap，lastgap)                                                            getmat　　       int     lx，ly；                   /* ″core″(minus endgaps) */　　       int     firstgap，lastgap；         /* leading trailing overlap */{　　       int             nm，i0，i1，siz0，siz1；　　       char            outx[32]；　　       double          pct；　　       register        n0，n1；　　       register char   *p0，*p1；　　       /* get total matches，score　　        */　　       i0 ＝ i1 ＝ siz0 ＝ siz1 ＝ 0；　　       p0 ＝ seqx[0] + pp[1].spc；　　       p1 ＝ seqx[1] + pp[0].spc；　　       n0 ＝ pp[1].spc + 1；　　       n1 ＝ pp[0].spc + 1；　　       nm＝0；　　       while(*p0 && *p1){　　              if(siz0){　　                       p1++；　　                       n1++；　　                       siz0--；　　              }　　              else if (sizl){　　                       P0++；　　                       n0++；　　                       siz1-；　　             }　　             else{　　                       if(xbm[*p0-′A′]&xbm[*p1-′A′])　　                               nm++；　　                       if(n0++ ＝ ＝ pp[0].x[i0])　　                               siz0 ＝ pp[0].n[i0++]；　　                       if(n1++ ＝ ＝ pp[1].x[i1])　　                               siz1 ＝ pp[1].n[i1++]；　　                       p0++；　　                       p1++；　　             }　　        }　　        /* pct homology：　　         * if penalizing endgaps，base is the shorter seq　　         * else，knock off overhangs and take shorter core　　         */　　        if(endgaps)　　                 lx ＝ (len0 ＜ len1)？len0 ：len1；　　        else　　                 lx ＝ (lx ＜ ly)？lx ：ly；　　        pct ＝ 100.*(double)nm/(double)lx；　　        fprintf(fx，″\n″)；　　        fprintf(fx，″＜％d match％s in an overlap of ％d：％.2f percent similarity\n″，　　                 nm，(nm ＝＝ 1)？″″ ：″es″，lx，pct)；

Table?1?(cont’)

　　      fprintf(fx，″＜ gaps in first sequence：％d″，gapx)；                                    ...getmat　　      if(gapx){　　              (void)sprintf(outx，″(％d％s％s)″，　　                       ngapx，(dna)？″base″：″residue″，(ngapx ＝＝ 1)？″″：″s″)；　　              fprintf(fx，″％s″，outx)；　　      fprintf(fx，″，gaps in second sequence：％d″，gapy)；　　      if(gapy){　　              (void)sprintf(outx，″(％d％s％s)″，　　                       ngapy，(dna)？″base″：″residue″，(ngapy ＝ ＝ 1)？″″：″s″)；　　              fprintf(fx，″％s″，outx)；　　      }　　      if(dna)　　              fprintf(fx，　　              ″\n ＜score：％d(match ＝ ％d，mismatch ＝ ％d，gap penalty ＝ ％d + ％d per base)\n″，　　              smax，DMAT，DMIS，DINS0，DINS1)；　　      else　　              fprintf(fx，　　              ″\n ＜ score：％d(Dayhoff PAM 250 matrix，gap penalty ＝ ％d + ％d per residue)\n″，　　              smax，PINS0，PINS1)；　　      if(endgaps)　　              fprintf(fx，　　              ″＜endgaps penalized. left endgap：％d％s％s，right endgap：％d％s％s\n″，　　              firstgap，(dna)？″base″：″residue″，(firstgap ＝＝ 1)？″″ ：″s″，　　              lastgap，(dna)？″base″：″residue″，(lastgap ＝＝ 1)？″″ ：″s″)；　　      else　　              fprintf(fx，″＜endgaps not penalized\n″)；}static            nm；             /* matches in core -- for checking */static            lmax；           /* lengths of stripped file names */static            ij[2]；          /* jmp index for a path */static            nc[2]；          /* number at start of current line */static            ni[2]；          /* current elem number -- for gapping */static            siz[2]；static char       *ps[2]；         /* ptr to current element */static char       *po[2]；         /* ptr to next output char slot */static char       out[2][P_LINE]； /* output line */static char       star[P_LINE]；   /* set by stars() *//* * print alignment of described in struct path pp[] */staticpr_align()                                                                            pr_align{　　       int              nn；   /* char count */　　       int              more；　　       register         i；　　       for(i ＝ 0， lmax ＝ 0；i＜ 2；i++){　　                nn ＝ stripname(namex[i])；　　                if(nn ＞ lmax)　　                         lmax ＝ nn；　　                nc[i] ＝ 1；　　                ni[i] ＝ 1；　　                siz[i] ＝ ij[i] ＝ 0；　　                ps[i] ＝ seqx[i]；　　                po[i] ＝ out[i]；　　       }

Table?1?(cont’)

　　        for(nn ＝ nm ＝ 0，more ＝ 1；more；){                                           ...pr_align　　                  for(i ＝ more ＝ 0；i＜2；i++){　　                             /*　　                              * do we have more of this sequence？　　                              */　　                             if(！*ps[i])　　                                       continue；　　                             more++；　　                             if(pp[i].spc){    /* leading space */　　                                       *po[i]++ ＝′′；　　                                       pp[i].spc--；　　                             }　　                             else if(siz[i]){    /* in a gap */　　                                       *po[i]++ ＝ ′- ′；　　                                       siz[i]--；　　                             }　　                             else{              /* we′re putting a seq element　　                                                */　　                                       *po[i] ＝ *ps[i]；　　                                       if(islower(*ps[i]))　　                                               *ps[i] ＝ toupper(*ps[i])；　　                                       po[i]++；　　                                       ps[i]++；　　                                       /*　　                                        * are we at next gap for this seq？　　                                        */　　                                       if(ni[i] ＝ pp[i].x[ij[i]]){　　                                                /*　　                                                 * we need to merge all gaps　　                                                 * at this localion　　                                                 */　　                                                 siz[i] ＝ pp[i].n[ij[i]++]；　　                                                 while(ni[i] ＝ pp[i].x[ij[i]])　　                                                          siz[i] +＝ pp[i].n[ij[i]++]；　　                                       }　　                                       ni[i]++；　　                             }　　                   }　　                   if(++nn ＝ olen || ！more && nn){　　                              dumpblock()；　　                              for(i ＝ 0；i＜2；i++)　　                                       po[i]＝out[i]；　　                              nn ＝ 0；　　                  }　　        }}/* * dump a block of lines，including numbers，stars：pr_align() */staticdumpblock()                                                                           dumpblock{　　        register i；　　        for(i＝0；i＜2；i++)　　                   *po[i]-＝′\0 ′；

Table?1?(cont’)

　　                                                                                                   ...dumpblock　　      (void)putc(′\n′，fx)；　　      for(i＝0；i＜2；i++){　　                if(*out[i] && (*out[i]！＝″|| *(po[i])！＝″)){　　                          if(i＝0)　　                                  nums(i)；　　                          if(i＝0 && *out[1])　　                                  stars()；　　　                          putline(i)；　　                          if(i＝0 && *out[1])　　                                  fprintf(fx，star)；　　                          if(i＝1)　　                                  nums(i)；　　                 }　　       }}/* * put out a number line：dumpblock() */staticnums(ix)                                                                                 nums　　       int       ix；     /* index in out[] holding seq line */{　　       char               nline[P_LINE]；　　       register           i，j；　　       register char      *pn，*px，*py；　　       for(pn＝nline，i＝0；i＜lmax+P_SPC；i++，pn++)　　                 *pn＝′′；　　       for(i＝nc[ix]，py＝out[ix]；*py；py++，pn++){　　                 if(*py＝″|| *Py＝′- ′)　　                          *pn＝′′；　　                 else{　　                          if(i％10＝0||(i＝1 && nc[ix]！＝1)){　　                                   j＝(i＜0)？-i：i；　　                                   for(px＝pn；j；j/＝10，px--)　　                                           *px＝j％10 + ′0′；　　                                   if(i＜0)　　                                           *px＝′-′；　　                          }　　                          else　　                                   *pn＝′′；　　                          i++；　　                 }　　      }　　      *pn＝′\0′；　　      nc[ix]＝i；　　      for(pn＝nline；*pn；pn++)　　              (void)putc(*pn，fx)；　　      (void)putc(′\n′，fx)；} /*  * put out a line (name，[num]，seq，[num])：dumpblock()  */staticputline(ix)                                                                              putline　　           int        ix；{

Table?1?(cont’)

　　                                                                                                       ...putline　　      int               i；　　      register char     *px；　　      for(px＝namex[ix]，i＝0；*px && *px！＝′：′；px++，i++)　　              (void)putc(*px，fx)；　　      for(；i＜lmax+P_SPC；i++)　　              (void)putc(′′，fx)；　　      /* these count from l：　　       * ni[]is current element(from 1)　　       * nc[]is number at start of current line　　       */　　      for(px＝out[ix]；*px；px++)　　              (void)putc(*px&0x7F，fx)；　　      (void)putc(′\n′，fx)；}/* * put a line of stars (seqs always in out[0]，out[1])：dumpblock() */staticstars()                                                                               stars{　　      int               i；　　      register char     *p0，*p1，cx，*px；　　      if(！*out[0]||(*out[0]＝″ && *(po[0])＝ ″)||　　         ！*out[1]||(*out[1]＝″ && *(po[1])＝″))　　                return；　　      px＝star；　　      for(i＝lmax+P_SPC；i；i-)　　                *px++＝′′　　      for(p0＝out[0]，p1＝out[1]；*p0 && *p1；p0++，p1++){　　                if(isalpha(*p0) && isalpha(*p1)){　　                          if(xbm[*p0-′A′]&xbm[*p1-′A′]){　　                                  cx＝′*′；　　                                  nm++；　　                          }　　                          else if(！dna && _day[*p0-′A′][*p1-′A′]＞0)　　                                  cx＝′.′；　　                          else　　                                  cx＝′′；　　                }　　                else　　                          cx＝′′；　　                *px++＝cx；　　      }　　      *px++＝′\n′；　　      *px＝′\0′；}

Table?1?(cont’)

/* * strip path or prefix from pn，return len：pr_align() */staticstripname(pn)　　     char      *pn；    /* file name (may be path) */{　　     register char      *px，*py；　　     py＝0；　　     for(px＝pn；*px；px++)　　               if(*px＝′/′)　　                        py＝px+1；　　     if(py)　　               (void)strcpy(pn，py)；　　     return(strlen(pn))；}

Table?1?(cont’)

/* * cleanup()--cleanup any tmp file * getseq()--read in seq，set dna，len，maxlen * g_calloc()--calloc()with error checkin * readjmps()--get the good jmps，from tmp file if necessary * writejmps()--write a filled array of jmps to a tmp file：nw() */#include ″nw.h″#include＜sys/file.h＞char    *jname＝″/tmp/homgXXXXXX″；               /* tmp file for jmps */FILE    *fj；int     cleanup()；                                 /* cleanup tmp file */long    lseek()；/** remove any tmp file if we blow*/cleanup(i)                                                                              cleanup　　       int        i；{　　       if(fj)　　                  (void)unlink(jname)；　　       exit(i)；}/* * read，return ptr to seq，set dna，len，maxlen * skip lines starting with ′；′，′＜，or′＞′ * seq in upper or lower case */char       *getseq(file，len)                                                                       getseq　　       char    *file；   /* file name */　　       int     *len；    /* seq len */{　　       char              line[1024]，*pseq；　　       register char     *px，*py；　　       int               natgc，tlen；　　       FILE              *fp；　　       if((fp＝fopen(file，″r″))＝0){　　                fprintf(stderr，″％s：can′t read％s\n″，prog，file)；　　                exit(1)；　　       }　　       tlen＝natgc＝0；　　       while(fgets(line，1024，fp)){　　                if(*line＝′；′||*line＝′＜′||*line＝′＞′)　　                         continue；　　                for(px＝line；*px！＝′\n′；px++)　　                         if(isupper(*px)||islower(*px))　　                                   tlen++；　　       }　　       if((pseq＝malloc((unsigned)(tlen+6)))＝0){　　                fprintf(stderr，″％s：malloc()failed to gct％d bytes for％s\n″，prog，tlen+6，file)；　　                exit(1)；　　       }　　       pseq[0]＝pseq[1]＝pseq[2]＝pseq[3]＝′\0′；

Table?1?(cont’)

　　                                                                                                          ...getseq　　      py＝pseq+4；　　      *len＝tlen；　　      rewind(fp)；　　      while(fgets(line，1024，fp)){　　               if(*line＝′；′||*line＝′＜′||*line＝′＞′)　　                         continue；　　               for(px＝line；*px ！＝′\n′；px++){　　                         if(isupper(*px))　　                                    *py++＝*px；　　                         else if(islower(*px))　　                                    *py++＝toupper(*px)；　　                         if(index(″ATGCU″，*(py-1)))　　                                  natgc++；　　               }　　      }　　      *py++＝′\0′；　　      *py＝′\0′；　　      (void)fclose(fp)；　　      dna＝natgc＞(tlen/3)；　　      return(pseq+4)；}char      *g_calloc(msg，nx，sz)                                                                             g_calloc　　     char    *msg；               /* program，calling routine */　　     int     nx，sz；             /* number and size of elements */{　　     char            *px，*calloc()；　　     if((px＝calloc((unsigned)nx，(unsigned)sz))＝0){　　              if(*msg){　　                      fprintf(stderr，″％s： g_calloc() failed％s(n＝％d，sz＝％d)\n″，prog，msg，nx，sz)；　　                      exit(1)；　　              }　　     }　　     return(px)；}/* * get final jmps from dx[]or tmp file，set pp[]，reset dmax：main() */readjmps()                                                                                       readjmps{　　      int              fd＝-1；　　      iht              siz，i0，i1；　　      register i，j，xx；　　      if(fjj){　　               (void)fclose(fj)；　　               if((fd＝open(jname，O_RDONLY，0))＜0){　　                        fprintf(stderr，″％s：can′t open()％s\n″，prog，jname)；　　                        cleanup(l)；　　               }　　      }　　      for(i＝i0＝i1＝0，dmax0＝dmax，xx＝len0；；i++){　　               while(1){　　                        for(j＝dx[dmax].ijmp；j＞＝0 && dx[dmax].jp.x[j]＞＝xx；j-)　　                                 ；

Table?1?(cont’)

　　                                                                                                                 ...readjmps　　                      if(j＜0 && dx[dmax].offset && fj){　　                              (void)lseek(fd，dx[dmax].offset，0)；　　                              (void)read(fd，(char *)&dx[dmax].jp，sizeof(struct jmp))；　　                              (void)read(fd，(char *)&dx[dmax].offset，sizcof(dx[dmax].offset))；　　                               dx[dmax].ijmp＝MAXJMP-1；　　                      }　　                      else　　                               break；　　           }　　           if(i＞＝JMPS){　　                     fprintf(stderr，″％s：too many gaps in alignment\n″，prog)；　　                     cleanup(1)；　　           }　　           if(j＞＝0){　　                     siz＝dx[dmax].jp.n[j]；　　                     xx＝dx[dmax].jp.x[j]；　　                     dmax +＝siz；　　                     if(siz＜0){              /* gap in second seq */　　                               pp[1].n[i1]＝-siz；　　                               xx +＝siz；　　                               /* id＝xx-yy+len1-1　　                               */　　                               pp[1].x[i1]＝xx-dmax+len1-1；　　                               gapy++；　　                               ngapy -＝siz；/* ignore MAXGAP when doing endgaps */　　                               siz＝(-siz＜MAXGAP||endgaps)？-siz：MAXGAP；　　                               i1++；　　                     }　　                     else if(siz＞0){  /* gap in first seq */　　                               pp[0].n[i0]＝siz；　　                               pp[0].x[i0]＝xx；　　                               gapx++；　　                               ngapx +＝siz；/* ignore MAXGAP when doing endgaps */　　                               siz＝(siz＜MAXGAP||endgaps)？siz：MAXGAP；　　                               i0++；　　                     }　　           }　　           else　　                     break；　　       }　　       /* reverse the order of jmps　　        */　　       for(j＝0，i0--；j＜i0；j++，i0--){　　                   i＝pp[0].n[j]；pp[0].n[j]＝pp[0].n[i0]；pp[0].n[i0]＝i；　　                   i＝pp[0].x[j]；pp[0].x[j]＝pp[0].x[i0]；pp[0].x[i0]＝i；　　       }　　       for(j＝0，i1-；j＜i1；j++，i1-){　　                   i＝pp[1].n[j]；pp[1].n[j]＝pp[1].n[i1]；pp[1].n[i1]＝i；　　                   i＝pp[1].x[j]；pp[1].x[j]＝pp[1].x[i1]；pp[1].x[i1]＝i；　　       }　　       if(fd＞＝0)　　                   (void)close(fd)；　　       if(fj){　　                   (void)unlink(jname)；　　                   fj＝0；　　                   offset＝0；　　       }}

Table?1?(cont’)

/* * write a filled jmp struct offset of the prev one(if any)：nw() */writejmps(ix)                                                                        writejmps　　        int        ix；{　　        char       *mktemp()；　　        if(！fj){　　                   if(mktemp(jname)＜0){　　                            fprintf(stderr，″％s：can′t mktemp()％s\n″，prog，jname)；　　                            cleanup(1)；　　                   }　　                   if((fj＝fopen(jname，″w″))＝0){　　                            fprintf(stderr，″％s：can′t write％s\n″，prog，jname)；　　                            exit(1)；　　                   }　　        }　　        (void)fwrite((char*)&dx[ix].jp，sizeof(struct jmP)，1，fj)；　　        (void)fwrite((char*)&dx[ix].offset，sizeof(dx[ix].offset)，1，fj)；}

Table 2

PRO XXXXXXXXXXXXXXX (length=15 amino acid)

Reference protein XXXXXYYYYYYY (length=12 amino acid)

Amino acid sequence identity %=(quantity of the identical match amino-acid residue of measuring with ALIGN-2 between two peptide sequences) divided by (sum of amino-acid residue in the PRO polypeptide)=5 divided by 15=33.3%

Table 3

PRO XXXXXXXXXX (length=10 amino acid)

Reference protein XXXXXYYYYYYZZYZ (length=15 amino acid)

Amino acid sequence identity %=(quantity of the identical match amino-acid residue of measuring with ALIGN-2 between two peptide sequences) divided by (sum of amino-acid residue in the PRO polypeptide)=5 divided by 10=50%

Table 4

PRO-DNA NNNNNNNNNNNNNN (length=14 Nucleotide)

Contrast DNA NNNNNNLLLLLLLLLL (length=16 Nucleotide)

Nucleotide sequence identity %=(quantity of the identical match Nucleotide of measuring with ALIGN-2 between two nucleotide sequences) divided by (sum of Nucleotide in the PRO-DNA nucleotide sequence)=6 divided by 14=42.9%

Table 5

PRO-DNA NNNNNNNNNNNN (length=12 Nucleotide)

Contrast DNA NNNNLLLVV (length=9 Nucleotide)

Nucleotide sequence identity %=(quantity of the identical match Nucleotide of measuring with ALIGN-2 between two nucleotide sequences) divided by (sum of Nucleotide in the PRO-DNA nucleotide sequence)=4 divided by 12=33.3%

II. the compositions and methods of the invention

A. total length FGF-19 polypeptide

The invention provides new evaluation and isolating nucleotide sequence (perhaps being also referred to as UNQ334) that the code book application is called the polypeptide of FGF-19.More specifically, as the following examples are further disclosed in detail, identified and isolated the cDNA of coding FGF-19 polypeptide.It should be noted that expressing the albumen that produces in the circulation in difference may have different PRO numbers, still, the UNQ number of any specific DNA and its encoded protein is unique, and will can not change.Yet, for brevity, all other natural homologues and variant with the FGF-19 (also claiming PR0533 sometimes) of DNA49435-1219 encoded protein and aforementioned definitions all is called " FGF-19 " in this manual, no matter and its source or preparation mode how.

As the following examples were disclosed, the DNA49435-1219 cDNA of this paper cloned in the ATCC preservation.This clone's actual nucleotide sequence can use this area ordinary method by the preservation clone being carried out sequential analysis and recording at an easy rate by those skilled in the art.Use this area routine techniques can predict the aminoacid sequence that this is nucleotide sequence coded.About FGF-19 polypeptide described herein and its coding nucleic acid, the applicant has determined what is being read on the frame and to have sequence information now the most consistent this moment.

Use above-mentioned ALIGN-2 sequence contrast computer program, have found that: the natural FGF-19 sequence of total length (seeing Fig. 2 and SEQ ID NO:2) has certain amino acid sequence identity with AF007268_1.Therefore, it is believed that the newcomer that the disclosed FGF-19 polypeptide of the application is the fibroblast growth factor protein family at present, and may have one or more biology and/or immunologic competence or characteristic that this protein family has usually.

B. FGF-19 variant

Except the FGF-19 polypeptide of total length native sequences described herein, can also prepare the FGF-19 variant.Suitable Nucleotide changes and/or synthetic required FGF-19 polypeptide by introducing in FGF-19DNA, can prepare the FGF-19 variant.Those skilled in the art will recognize that amino acid changes process after the translation will change FGF-19, for example changes the quantity and the position of glycosylation site or changes the film anchor and characteristic.

For example use the arbitrary technology and the guideline of the conservative and non--conservative sudden change of in No. 5364934, United States Patent (USP), setting forth, can in natural full length sequence FGF-19 or various structural domains, make variation at FGF-19 described herein.Variation can be the one or more codons that replace, lack or insert coding FGF-19, and they cause changing with respect to the FGF-19 aminoacid sequence of native sequences FGF-19.Randomly, variation is that at least one amino acid in the one or more structural domains of FGF-19 is by any other aminoacid replacement.FGF-19 sequence and homologous known protein molecule are compared, and make height homologous region aminoacid sequence number change minimum, can set up which amino-acid residue of decision and can be inserted into, replace or lack and the governing principle of the no negative impact of required activity.Aminoacid replacement can be that an amino acid is had the displaced result of amino acid of similar structures and/or similar chemical property by another, as Serine displacement leucine, i.e. conservative amino acid replacement.Inserting or lack can be randomly in about 1～5 amino acid whose scope.By systematically in aminoacid sequence, inserting, lack or replace and detect the active situation that the gained variant shows total length or ripe native sequences, can determine the variation that can allow.

This paper provides FGF-19 polypeptide fragment.Such fragment can be for example, to compare with the total length native protein, in N-end or terminal fragment brachymemma or that lack inner residue of C-.Some fragment lacks those for the non-essential amino-acid residue of FGF-19 polypeptide expection biological activity.

The FGF-19 fragment can prepare according in the wide variety of conventional technology any one.Can the required peptide fragment of chemosynthesis.Other method relates to by enzymic digestion and produces the FGF-19 fragment, for example, handles albumen with known enzyme at the site crack protein that particular amino acid residue limited, or with the digestion with restriction enzyme DNA that suits and separate required fragment.Another suitable technology comprises separates and with the dna fragmentation of the required polypeptide fragment of polymerase chain reaction (PCR) amplification coding.The oligonucleotide that limits the required end of dna fragmentation is used for 5 of PCR primer ' and 3 ' end.Preferably, the FGF-19 polypeptide fragment has at least a biology and/or the immunologic competence of natural FGF-19 polypeptide shown in Fig. 2 (SEQ ID NO:2).

In specific embodiments, the conservative title that replaces titled with preferred replacement of target is shown in the following table 6.Cause biological activity to change if this class replaces, then can introduce more material alterationses and screen product, the exemplary replacement of described change called after in table 6 is perhaps as hereinafter about the detailed description of amino acid kind.

Table 6

The exemplary replacement of original residue preferably replaces

Ala(A) val；leu；ile val

Arg(R) lys；gln；asn lys

Asn(N) gln；his；lys；arg gln

Asp(D) glu glu

Cys(C) ser ser

Gln(Q) asn asn

Glu(E) asp asp

Gly(G) pro；ala ala

His(H) asn；gln；lys；arg arg

Ile(I) leu；val；met；ala；phe；

norleucine leu

Leu(L) norleucine；ile；val；

met；ala；phe ile

lys(K) arg；gln；asn arg

Met(M) leu；phe；ile leu

Phe(F) leu；val；ile；ala；tyr leu

Pro(P) ala ala

Ser(S) thr thr

Thr(T) ser ser

Trp(W) tyr；phe tyr

Tyr(Y) trp；phe；thr；ser phe

Val(V) ile；leu；met；phe；

ala；norleucine leu

Can replace by selectivity the substantial modifications of the biological characteristics of FGF-19 polypeptide and to finish, the effect of described replacement is being kept the structure that (a) replaces district's polypeptide backbone, for example laminated structure or helical conformation, (b) electric charge of this molecule target site or hydrophobicity, (c) there were significant differences for these several respects of the size of side chain.Natural residue can be divided into according to total side chain characteristic:

(1) hydrophobicity: nor-leucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidity: asp, glu;

(4) alkalescence: ash, gln, his, lys, arg;

(5) influence the residue of chain orientation: gly, pro; With

(6) aromatic series: trp, tyr, phe.

Non-conservative replacement will limit the member of above-mentioned a certain class by another kind of replacement.Also the residue that replaces can be incorporated into conservative replacement site, perhaps more preferably introduce all the other (non--conservative) sites.

Can use (fixed point) mutagenesis, L-Ala scanning and the PCR induced-mutation technique of means known in the art such as oligonucleotide-mediation to produce variation.Can carry out site-directed mutagenesis [Carter etc., Nucl.Acids Res., 13:4331 (1986) to clone's DNA; Zoller etc., Nucl.Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells etc., Gene, 34:315 (1985)], restriction selects mutagenesis [Wells etc., Philos. change .R.Soc.London SerA, 317:415 (1986)] or other known technology to produce FGF-19 modification D NA.

Scanning amino acid analysis also can be determined one or more amino acid along contiguous sequence.Preferred scanning amino acid is relatively little neutral amino acids.This amino acid comprises L-Ala, glycine, Serine and halfcystine.Usually, L-Ala is a preferred scanning amino acid in this group, because unprotected side chain and unlikely change the main chain conformation [Cunningham and Wells, Science, 244:1081-1085 (1989)] of variant on its β-carbon.Another reason of preferred L-Ala is because it is the most frequently used amino acid.And it had usually both appeared at shelters the position and also appears at exposure position [Creighton, The Proteins, (W.H.Freeman ﹠amp; Co., N.Y); Chothia, J.Mol.Biol., 150:1 (1976)].If L-Ala replaces the variant that can not produce q.s, then can use isoteric amino acid.

The modification of C.FGF-19

The present invention includes the covalent modification of FGF-19.A kind of covalent modification comprises the target amino acid residue that makes the FGF-19 polypeptide and can react with organic derivatize agent that the selected side chain of FGF-19 or N-or C-terminal residue react.Derivatize with difunctionality agent is very useful, for example, FGF-19 and purifying be anti--the FGF-19 antibody method in used water-soluble carrier matrix or surface-crosslinked, vice versa.Normally used linking agent for example comprises; 1; two (two azo the ethanoyl)-2-phenylethanes of 1-; glutaraldehyde; N-hydroxyl succinimide ester as with the salicylic ester of 4-azido-, high difunctionality imido grpup ester comprises that two succinimide esters are as 3; 3 '-dithionic acid two (succinyl phosphorons amino propyl acid esters), difunctionality maleimide ester such as two-N-maleimide-1,8-monooctyl ester and chemical reagent such as methyl-3-[(be right-azidophenyl) two sulphur] propine imines ester.

Other modification comprises that glutamine and asparagus fern acyl residue deacylated tRNA amine become corresponding glutamy and aspartyl residue respectively, proline(Pro) and Methionin hydroxylation, the hydroxyl phosphorylation of seryl or threonyl residue, the alpha-amino group of Methionin, arginine and the Histidine side chain [T.E.Creighton that methylates, Proteins:Structure and Molecular Properties, W.H.Freeman ﹠amp; Co., San Francisco, pp.79-86 (1983)], the amidation of the terminated acetylated and any C-terminal carboxyl(group) of the N-of amine.

The present invention also comprises the another kind of covalent modification of FGF-19 polypeptide, and described covalent modification comprises the Natively glycosylated pattern that changes polypeptide." change Natively glycosylated pattern ", the purpose here is that one or more carbohydrate partly (perhaps glycosylated site take place or delete glycosylation by chemistry and/or enzyme means by removing) among the native sequences FGF-19 in order to delete, and/or adds one or more non-existent glycosylation sites in native sequences FGF-19.In addition, this phrase comprises the glycosylated change of properties of native protein, comprises the variation of different carbohydrate part on character and ratio.

Can be implemented in the FGF-19 polypeptide and add glycosylation site by changing aminoacid sequence.This change can be for example, by adding in native sequences FGF-19 (glycosylation site that connects for O-) or replace one or more Serines or threonine residues is finished.Randomly, can will translate into the amino acid whose codon of expectation thereby particularly undergo mutation to produce, change the FGF-19 aminoacid sequence by the variation of dna level by previously selected base among the DNA that makes coding FGF-19 polypeptide.

The other method that increases the carbohydrate partial amt on the FGF-19 polypeptide is, with glycoside chemical or enzyme ground and polypeptide coupling.Prior art has the description of these class methods, the WO 87/05330 that announced on September 11st, 1987 for example, and Aplin and Wriston, CRC Crit.Rev.Biochem. 259-306 page or leaf (1981).

With chemistry or enzyme method, perhaps replace the codon of coding as the amino-acid residue of glycosylation target spot by mutagenicity, realize removing the carbohydrate part in the FGF-19 polypeptide.Chemical de-glycosylation technology is well known in the art, and is described in for example following document: Hakimuddin etc., Arch.Biochem.Biophys., 259:52 (1987) and Edge etc., Anal.Biochem., 118:131 (1981).Use Thotakura etc. at Meth.Enzvmol., describe among the 138:350 (1987) various in-and outer-Glycosylase, can finish the enzymatic lysis of carbohydrate part in the polypeptide.

The another kind of covalent modification of FGF-19, comprise in mode described in United States Patent (USP) 4640835,4496689,4301144,4670417,4791192 or 4179337, with one of FGF-19 polypeptide chain and various non-protein polymkeric substance, for example polyoxyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.

FGF-19 of the present invention also can be modified to chimeric molecule, comprises FGF-19 and another heterologous polypeptide or aminoacid sequence fusion.

In one embodiment, chimeric molecule contains the syzygy of FGF-19 and labeling polypeptide, the alternative bonded epi-position of anti--traget antibody that labeling polypeptide wherein has.The epi-position mark generally is positioned at the amino of FGF-19-or carboxyl-end.Use the antibody of anti-labeling polypeptide, can detect the existence of this type of epi-position-marking type FGF-19.In addition, the providing of epi-position mark makes and utilizes anti--traget antibody or another kind of and epi-position mark bonded affinity matrix to come affinity purification FGF-19 to become very easy.Various labeling polypeptides and separately antibody be well-known in the art.Example comprises poly--Histidine (poly-his) or poly--HIS-GLY (poly-his-gly) mark; Flu HA labeling polypeptide and antibody 12CA5[Field etc. thereof, Mol.Cell.Biol., 8:2159-2165 (1988)]; C-myc mark and its 8F9,3C7,6E10, G4, B7 and 9E10 antibody [Evan etc., Molecular and Cellular Biology, 5:3610-3616 (1985)]; With herpes simplex virus glycoprotein D (gD) mark and antibody [Paborsky etc., Protein Engineering, 3 (6): 547-553 (1990)] thereof.

Other labeling polypeptide comprises Flag-peptide [Hopp etc., BioTechnology, 6:12041210 (1988)]; KT3 epitope peptide [Martin etc., Science, 255:192-194 (1992)]; Alpha-tubulin epitope peptide [Skinner etc., J.Biol.Chem., 266:15163-15166 (1991)]; With T7 gene 10 protein peptide marks [Lutz-Freyermuth etc., Proc.Natl.Acad.Sci.USA, 87:6393-6397 (1990)].

In another embodiment, chimeric molecule can comprise the fusion of FGF-19 and immunoglobulin (Ig) or immunoglobulin (Ig) specific region.For the chimeric molecule (also referring to " immunoadhesin ") of bivalent form, fusion can be the fusion with IgG molecule Fc district.Ig merges the soluble form (membrane spaning domain disappearance or inactivation) that preferably includes the FGF-19 polypeptide and replaces at least one variable region of Ig intramolecularly.In a particularly preferred embodiment, immunoglobulin (Ig) merges hinge area, CH2 and the CH3 district that comprises the IgG1 molecule, or hinge area, CH1, CH2 and CH3 district.The United States Patent (USP) 5428130 that the preparation of immunoglobulin (Ig) syzygy is also issued referring to June 27 nineteen ninety-five.

D. prepare FGF-19

Below description relate generally to by cultivate to transform or transfection contain the carrier of FGF-19 nucleic acid cell prepare FGF-19.Therefore, consider that certainly selective method well known in the art can be used for preparing FGF-19.For example, the method for the direct peptide synthesis by utilizing solid phase synthesis technique prepare FGF-19 sequence or its part [referring to for example, Stewart etc., Solid-Phase Peptide Synthesis, W.H.Freeman Co., San Francisco, CA (1969); Merrifield, J.Am.Chem.Soc.,, 85:2149-2154 (1963)].Make by hand or operative technique automatically, it is synthetic outward to carry out proteoplast.For example, according to the indication of manufacturers, (Foster City CA), can finish synthetic automatically to use practical biosystem peptide synthesizer.Can distinguish the each several part of chemosynthesis FGF-19, utilize chemistry or Enzymology method to make total length FGF-19 then.

1. the separation of the DNA of coding FGF-19

Can obtain the DNA of coding FGF-19 from the cDNA library, described cDNA library is to have FGF-19mRNA and with the tissue preparation that can record horizontal expression FGF-19 from it is believed that.Correspondingly, as described in the embodiment, from the cDNA library of people's tissue preparation, can obtain people's FGF-19DNA easily.Also can obtain the FGF-19-encoding gene from genomic library or by known synthetic method (for example nucleic acid is synthetic automatically).

Use to recognition objective gene or its encoded protein designed probe (as FGF-19 antibody or at least about the oligonucleotide of 20-80 base), can screen the library.Use standard operation, as Sambrook etc. at Molecular Cloning:A Laboratory Manual (New York:Cold Spring HarborLaboratory, 1989 publication) method described in is carried out the screening of cDNA or genomic library with the probe of selecting.Another selected for use method of separating the FGF-19 encoding gene be to use PCR method [Sambrook etc., ibid; Dieffenbach etc., PCR Primer:A LaboratorvManual (Cold Spring Harbor Laboratory, 1995 publish)].

Following embodiment describes cDNA library screening technology.The oligonucleotide sequence that is elected to be probe should have enough length and enough clear, so that make false positive the probability minimum occur.Oligonucleotide is mark preferably, like this by with the library of screening in DNA hybridization and can be detected.Marking method is well known in the art, comprise use radioactively labelled substance as ³²The ATP of P-mark, vitamin H or enzyme labelling.Comprise medium strictness and highly strict hybridization conditions, ibid to see Sambrook etc.

The sequence identified in screening library method and the sequence in other known preservation sequence or public database such as GenBank or other the privately owned sequence library can be compared.Use method known in the art and described herein, can measure the sequence identity (no matter at amino acid levels still at nucleotide level) of localized area in the molecule or full length sequence.

Use the disclosed first putative amino acid sequence of this paper and if necessary, use Sambrook etc., the conventional primer extension program that ibid describes, be not reversed precursor and the processing intermediate that record is the mRNA of cDNA to detect, can obtain to have the nucleic acid of albumen coded sequence from selected cDNA or genomic library.

2. the selection of host cell and conversion

With expression or cloning vector transfection or the transformed host cell of producing FGF-19 described herein, and in conventional nutritional medium, cultivate host cell, described substratum is improved the gene that makes it to be suitable for inducing enhanser, selective conversion body or the required sequence of amplification coding.Need not loaded down with trivial details test, those skilled in the art just can select culture condition such as substratum, temperature, pH etc.Usually, make principle, scheme and the practical technique of cell culture maximum production see Mammalian CellBiotechnology:a Practical Approach, M.Butler writes (IRL publishes, 1991) and Sambrook etc., and ibid.

The method of transfecting eukaryotic cells and conversion prokaryotic cell prokaryocyte is that those skilled in the art are known, for example, and CaCl ₂, CaPO ₄, liposome-mediation method and electroporation.According to used host cell, use the standard technique that is suitable for this host cell to transform.Calcium as ibid described use calcium chloride such as Sambrook is handled, or electroporation generally is used for prokaryotic organism.At Gene, as described in the WO 89/05859 that 23:315 (1983) and on June 29th, 1989 announce, the infection of agrobacterium tumefaciens is used to transform the certain plants cell as Shaw etc.For the Mammals that does not have cell walls, can use Graham and van derEb at Virology, the calcium phosphate precipitation method described in the 52:456-457 (1978).Total situation that the mammalian cell host system transforms is existing the description in United States Patent (USP) 4399216.According to Van Solingen etc., J.Bact., 130:946 (1977) and Hsiao etc., Proe.Natl.Acad.Sci. (USA), the described method of 76:3829 (1979) can finish to zymic transforming usually.Yet, also can use other method of DNA being introduced cell, for example pass through the fusion of microinjection, electroporation, bacterium protoplastis and the intact cell of nuclear, perhaps polycation such as polybrene, poly ornithine.The whole bag of tricks of transformed mammalian cell is seen Keown etc., Methods in Enzymology, 185:527-537 (1990) and Mansour etc., Nature, 336:348-352 (1988).

The suitable host cells of cloning or express DNA in the carrier described herein comprises prokaryotic organism, yeast or higher eucaryotic cells.Suitable prokaryotic organism include but not limited to eubacteriums such as Gram-negative or gram positive bacterium, such as enterobacteriaceae (Enterobacteriaceae) is as intestinal bacteria.Various e. coli strains all are that the public is available, as e. coli k12 strain MM294 (ATCC 31446); Intestinal bacteria X1776 (ATCC 31537); E. coli strains W3110 (ATCC 27325) and K5772 (ATCC53635).Other suitable prokaryotic host cell comprises enterobacteriaceae such as Escherichia, for example, intestinal bacteria, enterobacter, Erwinia, Klebsiella, the mycetozoan bar belongs to, Salmonella (as Salmonella typhimurtum), Serratia (as serratia marcesens) and Shigella etc., and bacillus such as subtilis and Bacillus licheniformis (for example Bacillus licheniformis 41P described in the DD 266710 that published on April 12nd, 1989) etc., the pseudomonas bacteria pseudomonas of Rhodopseudomonas, and streptomycete.These examples be used for the explanation and be not to be limited to this.The W3110 strain is a particularly preferred host or female host, because it is host's strain commonly used of recombinant DNA product fermentation.Preferably, a small amount of proteolytic ferment of secretory host cell.For example, modify the W3110 strain so that the gene generation genetic mutation of coding host endogenous protein, this type of host's example comprises intestinal bacteria W3110 strain 1A2, and this strain has complete genotype tonA; Intestinal bacteria W3110 strain 9E4, this strain has complete genotype tonA ptr3; Intestinal bacteria W3110 strain 27C7 (ATCC 55244), this strain has complete genotype tonA ptr3 phoAE15 (argF-lac) 169 degP omp T kanr; Intestinal bacteria W3110 strain 37D6, this strain has complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kant; Intestinal bacteria W3110 strain 40B4, it is the strain that non--kantlex resistance degP deletion mutantion takes place 37D6; E. coli strains with the mutant periplasm protein enzyme that discloses in the United States Patent (USP) 4946783 with issue on August 7 nineteen ninety.Perhaps, clone's in vitro method, for example PCR or other nucleic acid polymerase reaction suit.

Except prokaryotic organism, eukaryotic microorganisms such as filamentous fungus or yeast also are to be suitable for making the carrier cloning of coding FGF-19 or the host of expression.Yeast saccharomyces cerevisiae, or bread yeast commonly used are the most commonly used in low microorganism such as eucaryon host such as grade.Other eukaryotic microorganisms comprises: schizosaccharomyces pombe (Schizosaccharomyces pombe) (Beach and Nurse, Nature, 290:140[1981]; The EP 139383 that on May 2nd, 1985 announced); Genus kluyveromyces (Kluyveromyces) host (United States Patent (USP) 4943529; Fleer etc., Bio/Technologv, 9:968-975 (1991)), Kluyveromyces lactis (Klactis) (MW98-8C, CBS683, CBS4574 for example; Louvencourt etc., J.Bacteriol., 154 (2): 737-742[1983]), Kluyveromyces fragilis (K.fragilis) (ATCC 12424), Bulgarian kluyveromyces (K.bulgaricus) (ATCC 16045), Brunswick Man kluyveromyces (K.wickeramll) (ATCC24178), K.waltii (ATCC 56500), fruit bat kluyveromyces (K.drosophilarum) (ATCC36906; Van den Berg etc., Bio/Technology, 8:135 (1990)), heat-resisting kluyveromyces (K.thermotolerans) and Crewe Vickers yeast belong (K.marxianus) etc.; Yarrowia (EP 402226); (EP 183070 for pichia pastoris phaff (pichia pastoris); Sreekrishna etc., J.Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244234); Neurospora crassa (Case etc., Proc.Natl.Acad.Sci.USA, 76:5259-5263[1979]); Permitted Wang Shi yeast belong (schwanniomyces) and permitted Wang Shi yeast (schwanniomyces occidentalis) (EP 394538 that announce October 31 nineteen ninety) etc. as the west; And filamentous fungus, for example neurospora, Penicillium, Tolypocladium (WO 91/00357 that on January 10th, 1991 announced) and Aspergillus host such as Aspergillus nidulans (Ballance etc., Biochem.Biophys.Res.Commun., 112:284289[1983]; Tilburn etc., Gene, 26:205-221[1983]; Yelton etc., Proc.Natl.Acad.Sci.USA, 81:1470-1474[1984]) and aspergillus niger etc. (Kelly and Hynes, EMBOJ., 4:475-479[1985]).Having a liking for methyl (Methylotropic) yeast in this article suits, include but not limited to be selected from the yeast that can grow in methyl alcohol of following genus: Hansenula anomala belongs to (Hansenula), Candida (Candida), Kloeckera (Kloeckera), Pichia (Pichia), yeast belong, torulopsis and Rhodotorula.The inventory of the illustrative specific yeast belong of this type of zymic is seen C.Anthony, The Biochemistrvof Methylotrophs, 269 (1982).

Be used to express the suitable host cell of glycosylation FGF-19 from multicellular organism.The example of invertebral zooblast comprises insect cell (for example Drosophila S2 and fall army worm Sf9) and vegetable cell.Useful mammalian host cell line comprises Chinese hamster ovary (CHO) cell and COS cell.More special example comprises the monkey kidney CV1 clone that has transformed SV40 (COS-7, ATCC CRL 1651); Human embryonic kidney cell line's (293 cells or subclone cultivate 293 cells of growing in suspending nutrient solution, Graham etc., J.Gen Virol.36:59 (1977)); Chinese hamster ovary cell/-DHFR (CHO, Urlaub and Chasin, Proc.Natl.Acad.Sci.USA, 77:4216 (1980)); Mouse Sai Ertuoli cell (TM4, Mather, Biol.Reprod., 23:243-251 (1980)); Human pneumonocyte (W138, ATCC CCL 75); People's liver cell (Hep G2, HB 8065); (MMT 060562, ATCCCCL51) with the mouse breast cancer cell.Selecting the suitable host cell is this area general knowledge.

3. the selection of replicating vector and application

The nucleic acid (for example cDNA or genomic dna) of coding FGF-19 can be inserted in the replicating vector to clone (DNA cloning) or to express.Various carriers are that the public can obtain.Carrier can be the form of plasmid, clay, virion or phage for example.Making ins all sorts of ways can insert carrier with suitable nucleotide sequence.Usually, utilize technology known in the art that DNA is inserted into suitable restriction endonuclease site.The carrier integral part generally comprises but is not limited to: one or more signal sequences, replication orgin, one or more marker gene, enhancer element, promotor and transcription termination sequence.The standard interconnection technique of using those skilled in the art to know makes up the appropriate carrier that comprises one or more elements in these integral parts.

The FGF-19 preparation of not only can directly recombinating, but also can be prepared into fusion polypeptide with heterologous polypeptide, described heterologous polypeptide is signal sequence or other polypeptide that has the specificity cracking site on the N-terminal of maturation protein or polypeptide.Usually, signal sequence is a component of carrier or a part that is inserted into the FGF-19-coding DNA of carrier.Signal sequence can be to be selected from for example prokaryotic signal sequence of alkaline phosphatase, penicillinase, 1pp or thermally-stabilised enterotoxin 1 I leader.For yeast secretary, can be for example yeast invertase leader, alpha factor leader (the alpha factor leader that comprises saccharomyces and genus kluyveromyces, the latter states in United States Patent (USP) 5010182), or acid phosphatase leader, albicans glucoamylase leader (EP 362179 that announce April 4 nineteen ninety), perhaps signal sequence described in the WO90/13646 that announces November 15 nineteen ninety.When mammalian cell expression, can use the mammalian signal sequence with direct secretion albumen, as derive from the signal sequence and the viral secretory leader of same species or relevant species secreted polypeptides.

Expression vector and cloning vector all contain the nucleotide sequence that carrier is duplicated in one or more selected host cells.In various bacteria, yeast and virus, such sequence is well-known.Replication orgin from plasmid pBR322 is suitable for most of gram negative bacteriums, and 2 μ plasmid replication starting points are suitable for yeast, and various virus replication starting points (SV40, polyomavirus, adenovirus, VSV or BPV) are suitable for the cloning vector in the mammalian cell.

Expression vector and cloning vector comprise screening-gene usually, but are also referred to as selection markers.Typical screening-gene proteins encoded, this albumen (a) provides microbiotic or other toxin, resistance as penbritin, Xin Meisu, methotrexate or tsiklomitsin etc., (b) remedy auxotrophy, or (c) provide the crucial nutritive substance that from complex medium, can not obtain, the gene of the bacillus D-alanine racemase of for example encoding.

The selection markers example that is suitable for mammalian cell is those mark that enables to admit the cell of FGF-19 nucleic acid to obtain identifying, for example DHFR or thymidine kinases.When using wild-type DHFR, appropriate host cell is active defective type Chinese hamster ovary (CHO) clone of DHFR according to the method preparation of descriptions in Pric.Natl.Acad.Sci.USA 77:4216 (1980) such as Urlaub and breeding.Be applicable to that the suitable screening-gene of zymic is trp1 gene [Stinchcomb etc., Nature, the 282:39 (1979) that is present among the yeast plasmid YRp7; Kingsman etc., Gene, 7:141 (1979); Tschemper etc., Gene, 10:157 (1980)].The trp1 gene provides selection markers [Jones, Genetics, 85:12 (1977)] for the yeast mutant (for example ATCC 44076 or PEP4-1) that can not grow in tryptophane.

Expression and cloning vector contain the promotor that is operably connected to the FGF-19-nucleic acid sequence encoding usually, and be synthetic to instruct mRNA.Promotor by various potential host cell identifications is well-known.Be applicable to the promotor of prokaryotic hosts, comprise β-Nei Xiananmei and lactose promoter systems [Chang etc., Nature, 275:615 (1978); Goeddel etc., Nature, 281:544 (1979)], alkaline phosphatase, tryptophane (trp) promoter systems [Goeddel, Nucleic acids Res., 8:4057 (1980); EP36776] and hydridization promotor such as tac promotor [deBoer etc., Proc.Natl.Acad.Sci.USA, 80:21-25 (1983)].Be applicable to the promotor of bacterial system, also contain Shine-Dalgarno (S.D.) sequence of the DNA that is operably connected to coding FGF-19.

The example that is applicable to the initiating sequence of yeast host comprises glycerol 3-phosphate acid kinase [Hitzeman etc., J.Biol.Chem., 255:2073 (1980)] or other glycolytic ferment [Hess etc., J.Adv.EnzymeReg., 7:149 (1968); Holl and Biochemistrv, 17:4900 (1978)] promotor, described other glycolytic ferment such as Hydratase, phosphoenolpyruvate, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, glucose-6 phosphoric acid isomerase, the glycerol 3-phosphate mutase, pyruvate kinase, triosephosphate isomerase, glucose phosphate isomerase and glucokinase.

Other Yeast promoter, be those inducible promoters with the advantage of transcribing by growth conditions control, be the promoter region of following gene, i.e. the enzyme of alcohol dehydrogenase 2, different cell pigment C, acid phosphatase, degrading enzyme, metallothionein(MT), glyceraldehyde-3-phosphate dehydrogenase and responsible maltose and the galactose utilization relevant with nitrogen metabolism.The suitable carrier and the promotor that are used for yeast expression system in EP 73657, have been further described.

In mammalian host cell, transcribe FGF-19 by carrier and can be subjected to following promoter regulation, described promotor is from viral genome such as polyomavirus, bird pox virus (UK2211504 that on July 5th, 1989 announced), adenovirus (as adenovirus 2), bovine papilloma virus, avian sarcomata virus, cytomegalovirus, retrovirus, the promotor of hepatitis B virus and simian virus 40 (SV40), perhaps from the mammiferous promotor of allos, as actin promoter or immunoglobulin promoter etc., with from the heat-shocked promotor, prerequisite is that these promotors are compatible with host cell systems.

In carrier, insert enhancer sequence, can increase DNA the transcribing in higher eucaryote of coding FGF-19.Enhanser is the cis-acting elements that acts on the DNA that promotor transcribes with increase, general about 10～300bp.Known the enhancer sequence of a lot of mammalian genes (globin, elastoser, albumin, alpha fetal protein and Regular Insulin) at present.Yet people use the enhanser of eukaryotic cell virus usually.Example is included in the SV40 enhanser (bp100-270) of its replication origin side in late period, and the sub-enhanser of cytomegalovirus early promoter is at the polyoma enhanser and the adenovirus enhanser of its replication origin side in late period.Described enhanser can montage be gone into 5 ' or 3 ' end of FGF-19 encoding sequence, but is preferably placed at 5 ' end of promotor.

Be used for the expression vector of eukaryotic host cell (yeast, fungi, insect, plant, animal, people or from the karyocyte of other multicellular organism), also comprise Transcription Termination and the necessary sequence of stable mRNA structure.These sequences are usually from 5 ' (being 3 ' once in a while) non-translational region of eucaryon or viral DNA or cDNA.These zones comprise the segmental nucleotide fragments of polyadenylation in the non-translational region of mRNA that is transcribed into coding FGF-19.

In the recombinant vertebrate cell cultures, adapt to other proper method of FGF-19 synthetic, carrier and host cell, see Gething etc., Nature, 293:620-625 (1981); Mantei etc., Nature, 281:40-46 (1979); EP 117060; With the description among the EP 117058.

4. detect gene amplification/expression

Use is based on the probe of the appropriate flags of sequence provided herein, utilize the Northern trace [Thomas of routine techniques such as Southern trace, the mensuration mRNA amount of transcribing, Proc.Natl.Acad.Sci.USA, 77:5201-5205 (1980)], dot blotting (DNA analysis) or in situ hybridization, can directly in sample, measure gene amplification and/or expression.Perhaps, use can be discerned the antibody of special duplex, and described duplex comprises DNA duplex, RNA duplex and DNA-RNA heteroduplex body or DNA-albumen duplex.Traget antibody successively combines with which position, surface duplex and to analyze, thereby based on the formation of duplex on the surface, detects and duplex bonded antibody.

Perhaps, measure the gene product of expressing, use determination of immunological methods genetic expression, immunohistochemical staining and the cell culture or the body fluid analysis of described method such as cell or tissue section for direct quantitative.Be applicable to the antibody that immunohistochemical staining and/or sample liquid are analyzed, can be monoclonal antibody or polyclonal antibody, and can in any Mammals, prepare antibody.The antibody that can prepare anti-native sequences FGF-19 polypeptide easily, or the antibody of anti-synthetic peptide based on dna sequence dna provided herein, perhaps anti-the fusion with FGF-19DNA and the antibody of the exogenous array of coding specific antibody epi-position.

5. peptide purification

Can from substratum or host cell lysate, reclaim FGF-19.If FGF-19 combines with film, use suitable washing agent solution (for example Triton-X 100) so or make it to discharge from cytolemma by enzymatic lysis.Use various physics or chemical means,, make and express the used cell rupture of FGF-19 as freeze-thaw cycle, ultrasonic, mechanical disruption or cell lytic agent.

Can expect purifying FGF from recombinant cell protein or polypeptide-19.Following program is illustrative suitable purification step: fractional separation on ion-exchange column; Ethanol sedimentation; Reversed-phase HPLC; Chromatography on silica or positively charged ion-exchange resin such as DEAE; Chromatographic focusing; SDS-PAGE; Ammonium sulfate precipitation; For example using, sephadex (Sephadex) G-75 carries out gel-filtration; Cross the albumin A agarose column to remove pollutent such as IgG; With metal chelator post in conjunction with epi-position-mark pattern FGF-19.Can use the whole bag of tricks of protein purification, these methods are well known in the art, and at for example Deutscher, Methodsin Enzymology, 182 (1990); Scopes, Protein Purification:Principes and Practice, Springer-Verlag, New York is described in (1982).The selection of purification step is for example depended on, the character of production method and the specific FGF-19 that is produced.

The application of E.FGF-19

The nucleotide sequence (or its complementary strand) of coding FGF-19 serves many purposes in biology field, comprises as hybridization probe, in karyomit(e) and gene mapping and the purposes in preparation sense-rna and DNA.FGF19 nucleic acid also is very useful for preparing the FGF-19 polypeptide by recombinant technology described herein.

Total length native sequences FGF-19 gene (SEQ ID NO:1) or its part, the hybridization probe that can be used as the cDNA library is to isolate total length FGF-19Cdna, or isolate and Fig. 1 (SEQ ID NO:1) in disclosed FGF-19 sequence have expectation sequence identity other cDNA (for example, those encode natural FGF-19 variant or from the cDNA of the FGF-19 of other kind).Randomly, probe length is about 20～about 50 bases.Hybridization probe can stem from SEQ ID NO:1 nucleotide sequence to the small part newly developed area, wherein said newly developed area is that those do not need very loaded down with trivial details experiment with regard to confirmable zone, or the zone of determining from the native sequences FGF19 genome sequence that comprises promotor, enhancer element and intron.For instance, screening method comprises: use the selected probe of synthetic about 40 bases of known dna sequence, the coding region of separating the FGF-19 gene.But various signs on the hybridization probe mark, comprise the radioactive nuleus thuja acid as ³²P or ³⁵S or enzyme labelling, described enzyme labelling is as being coupled alkaline phosphatase and probe by the avidin/biotin coupling system.Have the label probe with FGF-19 gene complementation sequence of the present invention, can be used for screening human cDNA library, genomic dna or mRNA, hybridize to measure probe and which library.The following examples will describe in further detail hybridization technique.

The method of using this paper to disclose, the disclosed est sequence of the application can be used as probe similarly.

The useful fragment of other of FGF-19 nucleic acid, comprise contain can with the antisense of target FGF-19mRNA (justice) or FGF-19DNA (antisense) sequence bonded single-chain nucleic acid sequence (RNA or DNA) or positive MODN.According to the present invention, antisense or positive MODN comprise the fragment of FGF-19DNA coding region.This fragment generally contains at least about 14 Nucleotide, preferably by about 14～30 Nucleotide.Obtain the ability of antisense or positive MODN based on coding one given proteic cDNA sequence, at for example Stein and Cohen (Cancer Res.48:2659,1988) and in the article of van der Krol etc. (BioTechniques 6:958,1988) description is arranged.

Antisense or positive MODN combine with target nucleic acid sequence, cause blocking the duplex formation that target sequence is transcribed or translated ripe preceding termination or alternate manner that described blocking way comprises to be increased the duplex degraded, transcribe or translate by one or more modes.Therefore, antisense oligonucleotide can be used for blocking the proteic expression of FGF-19.Antisense or positive MODN further comprise the oligonucleotide (or other sugar chain connects those as describing among the WO 91/06629) of the sugared phosphodiester backbone with modified, and wherein to connect be the nuclease of anti-the endogenous to this type of sugar chain.Has oligonucleotide that the resistance sugar chain connects and is in vivo stable (that is, can antienzyme degraded), simultaneously reservation queue and target nucleotide sequences binding specificity.

Other example of justice or antisense oligonucleotide comprises the oligonucleotide of those and organic molecule covalent bonding, as those oligonucleotide of describing among the WO 90/10048, increase the molecule of oligonucleotide and target nucleic acid sequence avidity with other, as poly--(L-Methionin).In addition, intercalator (as ellipticine) and alkylating agent or metal complex can be articulated on justice or the antisense oligonucleotide, to modify antisense or the positive MODN binding specificity to target nucleotide sequences.

Use any gene transfer method antisense or just oligonucleotide can be introduced in the cell that contains target nucleic acid sequence, described method for example comprises, the DNA transfection of CaPO4-mediation, electroporation or use gene transfer vector such as Epstein-Barr virus.In a preferable methods, antisense or positive MODN are inserted in the suitable retroviral vector, in vivo or the external cell that contains target nucleic acid sequence that makes contact with recombinant retroviral vector.Suitable retroviral vector includes but not limited to, derives from Murine retroviral M-MuLV, N2 (deriving from the retrovirus of M-MuLV), or is called the double rendition virus of DCT5A, DCT5B and DCT5C (seeing WO 90/13641).

Also can form the form of conjugate, and justice or antisense oligonucleotide are incorporated in the cell that contains target nucleotide sequences, see the description of WO91/04753 by making justice or antisense oligonucleotide and ligand binding molecules.Suitable ligand binding molecules includes but not limited to: cell surface receptor, somatomedin, other cytokine or can with other part of cell surface receptor bonded.Preferably, the conjugation of ligand binding molecules is not disturbed the ability of ligand binding molecules molecule corresponding with it or receptors bind basically, or blocking-up justice or antisense oligonucleotide or its conjugated type enter cell.

Perhaps, the method according to WO 90/10448 describes by formation oligonucleotide-fat complexes, and is incorporated into justice or antisense oligonucleotide in the cell that contains target nucleic acid sequence.Preferably, justice or antisense oligonucleotide-fat complexes be to use endogenous lipase and with the mixture of cellular segregation.

Also can in round pcr, use probe, be used to identify one group of sequence near the FGF-19 encoding sequence with generation.

The nucleotide sequence of coding FGF-19 also can be used for being structured in describes to encode the gene mapping of FGF-19 and carry out the hybridization probe that uses in the genetic analysis to suffering from the inherited disease individuality.Use known technology, as the linkage analysis of in situ hybridization, anti-known chromosomal marker thing and with the screening by hybridization technology in library, nucleotide sequence provided herein can be put in order into to karyomit(e) and chromosomal specific region.

When the encoding sequence of FGF-19 coding can be with another protein bound albumen (for example, when FGF-19 is acceptor), FGF-19 can be used in the analysis of identifying other albumen that participates in association reaction or molecule.Use this class methods, can identify the inhibitor of receptor/ligand association reaction.Also the albumen that participates in this association reaction can be used to screen the micromolecular inhibitor or the agonist of peptide or association reaction.And acceptor FGF-19 can be used for separating associated ligands.Screening is analyzed and be can be used for designing the main compound of seeking natural FGF-19 biological activity of simulation or FGF19 acceptor.This type of screening is analyzed and is comprised that using chemistry to carry out height-throughput screening with database analyzes, and makes it to be suitable for especially identifying small molecules candidate medicine.Small molecules comprises synthetic organic or inorganic compound.Can use various forms of analytical methods, comprise the protein-protein binding analysis, the biochemical screening analysis, immunoassay and based on the analysis of cell, these analytical methods all are well known in the art.

The nucleic acid of coding FGF-19 or its modified forms also can be used for producing transgenic animal or " rejecting " animal, and the gained animal is very useful for exploitation and the suitable therapeutical agent of screening conversely.Transgenic animal (for example mouse or rat) are the animals that contains transgenic cell, and described transgenosis is introduced in animal or pregnancy period for generations in the animal, for example embryonic stage.Transgenosis is the DNA that is integrated into cellular genome, develops into transgenic animal by described cell.In one embodiment, use the technology of having set up and be used to produce the genome sequence of the transgenic animal that contain the cell of expressing coding FGF-19DNA, the cDNA of coding FGF-19 can be used for the genomic dna of clones coding FGF-19.Produce the method for transgenic animal, particularly, become this area routine techniques, and for example in the United States Patent (USP) 4736866 and 4870009 description has been arranged such as mouse or the such animal of rat.Usually, the FGF-19 transgenosis that has tissue-special enhanser is integrated in the specific cells.Be included in and be integrated into genetically modified transgenic animal of copy coding FGF-19 of stem cell animal embryonic stage, can be used for detecting the effect that increases coding FGF-19DNA expression.This type of animal can be used as to detect thinks the experimental animal that might have the medicament of provide protection to the pathologic conditions relevant with overexpression.According to an aspect of the present invention, carry genetically modified animal and compare with untreated, the animal of with medicament treatment, the incidence of its pathologic conditions reduces, and this shows that medicament has potential treatment intervention effect for pathologic conditions.

Perhaps, non--humanized FGF-19 is used to make up " rejecting " the animal of FGF19, this animal has FGF-19 genes encoding defective or that change, and this is the result who the homologous recombination thing between the coding FGF-19 genomic dna of endogenous FGF-19 genes encoding and change is introduced animal embryonic stem cell.For example, according to the technology of having set up, the cDNA of coding FGF-19 can be used for the genomic dna of clones coding FGF-19.The part of coding FGF-19 genomic dna can be deleted or replace with other gene, as be used to monitor the genes encoding replacement of the selective key of integration.Usually, the unaltered side DNA of thousands of base pairs (5 ' or 3 ' end) is incorporated in the carrier [referring to for example Thomas and Capecchi, Cell, 51:503 (1987) is to the description of homologous recombination vector].Carrier is introduced embryonic stem cell line (for example passing through electroporation), and select and introduced and the cell of the DNA of endogenous dna homologous recombination [referring to for example, Li etc., Cell, 69:915 (1992)].Then, the cell of selecting is injected the blastular of animal (for example mouse or rat), to form to form aggregation chimera [referring to for example, Bradley, in Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, E.J.Robertson writes (IRL, Oxford, 1987), 113-152 page or leaf].Chimeric embryo is implanted in the suitable female raising animal body of pseudopregnancy, and embryo growth promptly becomes " rejecting " animal to mature.Use standard technique, identify the filial generation animal that in its stem cell, carries homologous recombination DNA, go out the animal that in all zooblasts, all contains homologous recombination DNA with these filial generation animal reproductions.Reject animal and be characterised in that, for example, resist the ability of some pathologic conditions and pathologic conditions takes place owing to animal lacks the FGF-19 polypeptide.

Also the nucleic acid of coding FGF-19 polypeptide can be used for gene therapy.In gene therapy is used, gene is introduced cell so that reach synthetic in vivo treatment efficient gene product, for example replace dcc gene." gene therapy " both comprised the conventional gene therapy that just can obtain permanent effect through single therapy, also comprised the administration of gene therapeutic agents, and the latter relates to once or repeat repeatedly the DNA or the mRNA of administering therapeutic significant quantity.Sense-rna and DNA can be used as the therapeutical agent that specific gene is expressed in the blocking-up body.Proved already that the short chain antisense oligonucleotide can be integrated in the cell, in described cell, the short chain antisense oligonucleotide plays inhibitor, no matter and because how the restricted picked-up of cytolemma causes its low IC.(Zamecnik etc., Proc.Natl.Acad.Sci.USA83:4143-4146[1986]).But modified oligonucleotide is to strengthen its picked-up, and for example never charged group replaces the phosphodiester with negative electric charge.

There are various available technology that nucleic acid is incorporated in the viable cell.Technology according to whether nucleic acid shifted cultured cell in vitro into or the host inner cell that is intended in and different.The appropriate technology that nucleic acid is transferred to the external cell of Mammals comprises liposome, electroporation, microinjection, cytogamy, DEAE-dextran, the calcium phosphate precipitation method etc. used.At present popular vivo gene transfer technology comprises with viral (being generally retrovirus) carrier transfection with the transfection of viral dressing albumen-liposome-mediated (Dzau etc., Trends inBiotechnology 11,205-210[1993]).In some cases, be desirable to provide the nucleic acid source that has at the material of target cell, the membranin on described material such as pair cell surface has specific antibody, or the part etc. of acceptor on the target cell.When using liposome, will with the protein bound albumen of cell surface membrane that participates in endocytosis as target and/or promote the material of picked-up, for example the ad hoc type cell is had tropism's viral capsid protein or its fragment, proteic antibody, target that internalization takes place are located (localization) and increased the transformation period in the cell in cell albumen in circulation.The technology of the endocytosis of relevant acceptor-mediation is at for example Wu etc., J.Biol.Chem.262,4429-4432 (1987); With Wagner etc., Proc.Natl.Acad.Sci.USA 87, and 3410-3414 has description in (1990).Summary about genetic marker and gene therapy scheme sees also Anderson etc., and Science 256,808-813 (1992).

FGF-19 polypeptide described herein also can be used as proteic molecular weight sign in the electrophoresis.

Coding FGF-19 polypeptide described herein or its segmental nucleic acid molecule are very useful in karyomit(e) is identified.In this regard, owing to have only a few available chromosome marker agent relatively based on the actual sequence data, thereby exist for the demand of identifying new chromosome marker always.

FGF-19 nucleic acid molecule of the present invention can be used as chromosome marker.

FGF-19 polypeptide of the present invention and nucleic acid molecule also can be used for tissue typing, and FGF-19 polypeptide wherein of the present invention is differentially expressed between different tissues.The FGF-19 nucleic acid molecule also can be used for preparing PCR, Northern analysis, Southern analyzes and the probe of Western analysis usefulness.

FGF-19 polypeptide described herein and its conditioning agent be useful as therapeutics also.According to known preparation beneficial drugs method for compositions, can make FGF-19 polypeptide of the present invention and its conditioning agent, prepared FGF-19 product becomes to be the mixture combination with pharmaceutically acceptable carrier.Convenient in order to treat preparation stored, the active ingredient that will have required purity is mixed (Reminaton ' sPharmaceutical Sciences with pharmaceutically acceptable carrier, vehicle or the stablizer chosen wantonly, the 16th edition, Osol, A. write (1980)), be prepared into freeze-dried preparation or aqueous solution form.Acceptable carrier, vehicle or stable do not have toxicity at used dosage and concentration range to the recipient, and it comprises buffer reagent such as phosphoric acid salt, Citrate trianion and other organic acid; Antioxidant comprises xitix; Lower molecular weight (less than about 10 residues) polypeptide; Albumen is as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer such as polyvinylpyrrolidone, amino acid such as glycine, glutamine, l-asparagine, arginine or Methionin; Monose, disaccharides and other carbohydrate such as glucose seminose or dextrin; Chelating such as EDTA; Sugar alcohol such as N.F,USP MANNITOL or sorbyl alcohol; Become salt ion such as sodium; And/or nonionogenic tenside such as TWEEN ^TM, PLURONICS ^TMOr PEG.

The preparation that is used for vivo medicine-feeding must be aseptic.This can be in lyophilize and realizes easily by the degerming membrane filtration before or after the preparation again.

The therapeutic composition of this paper generally is contained in the container with aseptic access port, and for example this container is IV bag or the bottle that has stopper that can be by the subcutaneous injection needle penetration.

Route of administration is consistent with currently known methods, administration in for example injection or venoclysis, intraperitoneal, encephalic, intramuscular, intraocular, intra-arterial or the damage, local application or the administration of use sustained release system.

The dosage of pharmaceutical composition of the present invention and required drug level will be different according to the specific end use of anticipation.Decision optimal dose or route of administration are that the general medicine doctor fully can fine competent thing.Animal experiment provides reliable guidance for the used dosage of decision treatment human patients.Toxicokinetics and New Drug Development (the Pergamon Press that can write according to Yacobi etc., New York 1989) in the book, Mordenti, the ratio of J. and Chappell effective dose between the given method conversion kind of W. " The use of interspecies scaling intoxicokinetics " 42-96 page or leaf.

When using FGF-19 polypeptide or its agonist or antagonist in the body, depend on route of administration, every day the standard dose scope for about 10ng/kg extremely up to 100mg/kg weight of mammal or more, preferably about 1 μ g/kg/ day～10mg/kg/ day.Document provides the guidance and the method for releasing of relevant given dose, referring to for example United States Patent (USP) 4657760; 5206344; Or 5225212.For difference treatment compound and different syndromes, can expect that different preparations can prove effective, for example, those preparations that are applied to organ or tissue make be different from the mode that is applied to other organ or tissue discharge become essential.

When expectation FGF-19 polypeptide or conditioning agent have in the preparation of the release characteristics that is suitable for treating any disease that need use FGF-19 polypeptide or conditioning agent or illness sustained release administration, consider to use microencapsulation.The recombinant protein microencapsulation that continue to discharge usefulness has been successfully used to the lasting release of human growth hormone (rhGH), Interferon, rabbit-(rhIFN-), interleukin-2 and MN rgp120.Johnson etc., Nat.Med., 2:795-799 (1996); Yasuda, Biomed.Ther., 27:1221-1223 (1993); Hora etc., Bio/Technologv8:755-758 (1990); Write at Powell and Newman Vaccine Design: The Subunit and Adjuvant Approach(Plenum Press:New York, 1995) 439-462 page or leaf in the book, Cleland, " Design and Production of Single Immunization VaccinesUsing Polylactide Polyglycolide Microsphere Systems "; WO 97/03692, WO96/40072, and WO 96/07399; With United States Patent (USP) 5654010.

Because poly-lactic-coglycolic acid (PLGA) polymkeric substance has the biodegradation character of biocompatibility wide range, so use this polymkeric substance to prepare proteic extended release preparation.The degraded product of PLGA, promptly lactic acid and oxyacetic acid are removed in vivo fast.And, can regulate its degradation characteristic according to the molecular weight and the formation of this polymkeric substance, can be not wait several months to the several years.At M.Chasin and R.Langer (writing), Biodegradable Polymers as Drug Delivery Systems1-41 page or leaf in (MarcelDekker:New York, 1990) book, Lewis, " Controlled release of activeagents from lactide/glycolide polymer ".

FGF-19 therapeutical agent and the composition of containing provided herein can serve many purposes.These purposes comprise treatment obese individuals or the illness relevant with obesity.On the one hand, significant quantity FGF-19 is applied to the individuality that needs FGF-19, thus the treatment illness.Preferably, illness is at least a following illness that need treat: metabolism increase, weight loss, body fat minimizing, triglyceride level reduce, free fatty acids reduces, glucose increases from adipocyte release from adipocyte release increase and/or leptin.Use standard method, can measure each parameter wherein, for example, measure metabolic rate, use scale to measure body weight, and measure fatty situation by measurement size by measuring oxygen-consumption.

In addition, use standard method, can measure the content of triglyceride level, free fatty acids, glucose and leptin.In the specific embodiment below, the illustrative mensuration situation that has provided each parameter.

FGF-19 and contain the FGF-19 composition preferably uses in vivo.But, as described below, also can be at treated in vitro, when for example in the method for following screening FGF-19 conditioning agent, using.In addition, should understand, also can identify the conditioning agent of FGF-19 by using animal model and the sample that picks up from the patient.

Invention comprises the method for SCREENED COMPOUND, to identify those simulations or to strengthen FGF-19 polypeptide (agonist) or the compound of prevention or inhibition FGF-19 polypeptide (antagonist) effect.In this article, agonist and antagonist are called conditioning agent.The drug screening test of antagonist candidate, purpose are to identify to combine or the compound compound with the FGF-19 polypeptide of the genes encoding of being determined by this paper, and be perhaps opposite, identifies that those interference bases are because of encoded polypeptides and the interactional compound of other cell protein.This type of shaker test comprises uses the screening of high throughput chemline to analyze, and makes it to be particularly suitable for identifying small molecules candidate medicine.

Test can be carried out according to any way, comprises protein-protein binding analysis, biochemical screening analysis, immunoassay and based on the analysis of cell, these analytical procedures in this area by fine description.

Antagonist test common aspect is that they make the candidate medicine contact the enough time with the FGF-19 polypeptide of the nucleic acid encoding of being determined by this paper so that these two kinds of component interactions.

In in conjunction with test, interaction is combination, and the mixture of formation can be separated or detected in reaction mixture.In one particular,, and the FGF-19 polypeptide or the candidate medicine of the genes encoding of being determined by this paper are fixed on the solid phase, for example, are fixed on the microtiter plates by covalency or non--covalently adhere to.Non--covalent attachment, generally by with FGF-19 polypeptide solution encapsulated solid surface and carry out drying and realize.Perhaps, sessile antibody, for example, fixed has specific monoclonal antibody to the FGF-19 polypeptide, can be used for making the FGF-19 polypeptide to be fixed to solid surface.Test is carried out like this: adding on-fixed component in fixing component, described on-fixed component can mark on detectable marker, for example, the dressing surface is contained makes FGF-19 polypeptide fixed component.When reaction is finished, remove unreacted component (for example, passing through mode of washing), and detect the mixture that is fixed on solid surface.When original on-fixed component has detectable, detect the marker that is fixed on the surface, just show that mixture forms.If non-originally-fixedly component is not carried marker, then can detect compound, for example, by using the antibody with the fixed complex specific combination.

If but the specific FGF-19 polypeptide of the genes encoding that candidate compound and this paper determine interacts do not combine with it again, so, use the method for known detection protein-protein interaction to analyze interaction with that polypeptide.This type of analytical procedure comprises usual method, for example, crosslinked, altogether-immunoprecipitation and by gradient or chromatography column altogether-purifying.In addition, according to Fields and its colleague (Fields and Song, Nature (340:245-246 (1989); Chien etc., Proc.Natl.Acad.Sci.USA, 88:9578-9582 (1991)) and Chevray and Nathans, Proc.Natl.Acad.Sci.USA, 89:5789-5793 (1991) disclosed method uses yeast-Ji genetic system can detect protein-protein interaction.Many transcriptional activation agent, as yeast GAL4, it is made of two fully decentralized molecular structure territories, and one is as the DNA-land, and another is as the district of activated transcription.The yeast expression system of describing in the aforementioned publication (referring generally to " two-factor heterological system ") utilizes this characteristic, and uses two hybridization albumen, and merge the DNA-land of the target protein of one and GAL4, and merge another candidate's activator and active region.The expression of GAL1-lacZ reporter gene depends on the GAL4 activity of rebuilding by protein-protein interaction under the control of GAL4-activated enhanser.Chromogenic substrate with right-tilactase detects the flora that contains the interaction polypeptide.Can be purchased a complete set of test kit (MATCHMAKER) that utilizes protein-protein interaction between two differential proteins of two-factor heterozygosis technical evaluation from Clontech.This system also can be extended to describes to participate in the interactional protein structure domain of differential protein, and the accurate amino-acid residue that plays a crucial role for these interactions.

The FGF-19 polypeptide of intervening this paper genes identified coding with other cell the interior or interactional compound of extracellular component can followingly measure: usually under controlled conditions, preparation contains in product gene and the cell or the reaction mixture of extracellular component product, two kinds of products is interacted and in conjunction with certain hour.Suppress the bonded ability in order to test candidate compound, under the condition that contains and do not contain test-compound, react.In addition, can join placebo in the 3rd reaction mixture, as positive control.According to the method described above, combining between the interior or extracellular component of test-compound and cell (mixture formation) in the monitoring mixture.In control reaction, there is mixture to form, and in containing the reaction mixture of test-compound, do not have mixture to form, show the interaction that is tried between mixture intervention test-compound and its compatibility compound.

For analyzing antagonist, the FGF-19 polypeptide can be joined in the cell with screened compound, the compound of certain activity and ability suppresses the relevant activity of the FGF-19 polypeptide of coexistence, shows that then compound is the antagonist of FGF-19 polypeptide.Perhaps, FGF-19 polypeptide receptor or recombinant receptor on making FGF-19 polypeptide and potential antagonist under proper condition and being combined in film combine, and test by competitive inhibition and detect antagonist.But flag F GF-19 polypeptide as radio-labeling, like this, is combined in the FGF-19 peptide molecule quantity on the acceptor, just can be used for measuring the effectiveness of potential antagonist.Use numerous method known in the art, for example, part panning and FACS ordering, gene that can the identification code acceptor.Coligan etc., Current Protocols in Immun., 1 (2): Chapter 5 (1991).The preferred cloning by expression that uses, wherein polyadenylic acid RNA is to preparing the FGF-19 polypeptide permissive cell, and the cDNA library of being set up by this RNA is divided into several storehouses, is used for rotaring redyeing COS cell or other to the FGF-19 polypeptide cell of susceptible not.The transfectional cell of growing on the glass wave carrier piece is contacted with the FGF-19 polypeptide of mark.The flag F that can in all sorts of ways GF-19 polypeptide comprises the embedding of the recognition site of iodate or site-specific protein kinase.Behind the fixing and incubation, slide glass is accepted radioautographic analysis.Identify positive storehouse, preparation Ya-Ku, and use interactive Ya-Ku to carry out transfection again, and carry out screening process again, infer the single clone of acceptor until obtaining encoding.

As another approach that acceptor is identified, can make affine connection of extraction goods light of FGF-19 polypeptide with the cytolemma or the expressed receptor molecule of mark.Remove cross-linked material with PAGE, be exposed to X-line film.Can excise the labeled complex that contains acceptor, be decomposed into peptide fragment, accept albumen micrometering preface.From the aminoacid sequence that the micrometering preface obtains, be used to design the degenerate oligonucleotide probe in a cover screening cDNA library, infer the gene of acceptor with identification code.

In another analytical procedure of antagonist, under the condition that the candidate compound exists, the FGF-19 polypeptide incubation of the membrane product of mammalian cell or expressed receptor and mark.Measure the compound enhancing then or block interactional ability.

The more special example of potential antagonist comprises the fusions bonded oligonucleotide with immunoglobulin (Ig) and FGF-19 polypeptide, particularly antibody includes but not limited to polyclone and monoclonal antibody and antibody fragment, single-chain antibody, resists-idiotype antibody, with chimeric antibody or humanized antibody or antibody fragment, and people's antibody and antibody fragment.Perhaps, potential antagonist is closely-related albumen, for example, and the mutation of FGF-19 polypeptide, latter's identification receptor but do not cause effect, thereby the effect of competitive inhibition FGF-19 polypeptide.

In the embodiment of this paper, done competitive binding experiment, FGF acceptor 4 or FGF-19 antibody are as competitor.

Another potential FGF-19 polypeptide antagonist is to use the sense-rna or the DNA construct of antisense technology preparation, and wherein for example, sense-rna or dna molecular play a part direct blocking-up mRNA translation by hybridizing with said target mrna and stoping protein translation.Antisense technology can be used for controlling the genetic expression by triple helix structure or antisense DNA or RNA, and two kinds of methods all are based upon on polynucleotide and DNA or the RNA bonded basis.For example, 5 ' encoding part of polymerized nucleoside acid sequence, this part ripe FGF-19 polypeptide described herein of encoding is used to design the antisense rna oligonucleotide of about 10～40 base pair length.The DNA oligonucleotide is designed to participate in the complementary strand in open gene district, and (triple helix is referring to Lee etc., Nucl.Acids Res., 6:3073 (1979); Cooney etc., Science, 241:456 (1988); Dervan etc., Science, 251:1360 (1991)), stop to transcribe thus to produce with the FGF-19 polypeptide.Antisense rna oligonucleotide is hybridized with mRNA in vivo, and blocking-up mRNA molecule is translated into the FGF-19 polypeptide, and (antisense is seen Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotide is as the antisense inhibitor (CRC Press:Boca Raton, FL, 1988) of genetic expression.Also can be released into above-mentioned oligonucleotide in the cell, sense-rna or DNA can express in vivo like this, and suppress the generation of FGF-19 polypeptide.When using antisense DNA, preferably derived from the translation initiation site oligodeoxynucleotide, for example, the pact of target gene nucleotide sequence-10 and+10.

The potential antagonist comprises and avtive spot bonded small molecules, receptor binding site or somatomedin or other relevant binding site of FGF-19 polypeptide, thus the normal biological activity of blocking-up FGF-19 polypeptide.

Micromolecular example includes but not limited to, little peptide or class-peptide molecule, and the peptide and the synthetic of preferred dissolution be non--peptide organic or inorganic compound.

Ribozyme is the RNA molecule that can catalysis RNA specificity cracked has enzymatic property.Ribozyme, then plays a role by cracking in the nucleic acid in target RNA complementary strand by sequence-specific hybrid.Use known technology, can identify the special hammerhead ribozyme cleavage site point that is positioned at the potential rna target.Further details sees also for example Rossi, Current Biology, 4:469-471 (1994) and PCT publication WO97/33551 (announcement on September 18th, 1997).

The nucleic acid molecule that is used to the triple helix configuration that suppresses to transcribe should be a strand and be made of deoxynucleotide.These oligonucleotide based compositions design, and it promotes the formation of triple helix by Hoogsteen base pair principle like this, and this generally needs in the duplex purine or the sizable extension of pyrimidine on the chain.Further details for example see also, PCT publication WO97/33551, and ibid.

With any one or a plurality of analytical procedure of the shaker test of above-mentioned discussion, and/or use any other triage techniques well known to those skilled in the art, can identify these small molecules.

Thankfully, analytical procedure provided herein can be used for screening multiple candidate biologically active agent.Term used herein " candidate biologically active agent ", " candidate agent " or " candidate medicine " or phraseological Equivalent, be meant the biologically active agent that can directly or indirectly change cytoactive phenotype or FGF-19 sequence expression (both having comprised that nucleotide sequence also comprised protein sequence) in order to screen, and tested any molecule, for example albumen, oligopeptides, little organic molecule, polysaccharide, polynucleotide, purine class etc..

Although representational candidate agent is an organic molecule, the candidate agent can comprise numerous chemical species, preferred molecular weight greater than 100 less than about 2500 dalton's (d) little organic compound.This paper can further limit the small molecules that molecular weight is 50d～2000d.In another embodiment, micromolecular molecular weight is less than 1500 or less than 1200 or less than 1000 or less than 750 or less than 500d.In one embodiment, small molecules molecular weight used herein is about 100～200d.The candidate agent comprises with protein-interacting necessary structural functional group, particularly hydrogen bond and generally includes at least one amino, carbonyl, hydroxyl or carboxyl, preferably at least two chemical functional groups.The candidate agent usually comprises ring carbon or heterocycle structure and/or fragrance or the many aromatic structures that replaced by one or more above-mentioned functional groups.The candidate agent also sees biomolecules, comprises peptide, sugar, lipid acid, steroid, purine, pyrimidine, derivative, analog or their combination.Special preferred peptide.

The candidate agent derives from various widely sources, comprises synthetic or natural compounds storehouse.For example, have big metering method to can be used for synthesizing various organic compound and biomolecules with orientation at random, method comprises the expression of randomization oligonucleotide.Perhaps, the natural compounds storehouse of bacterium, fungi, plant and animal form of extract can utilize or easily make.In addition, by chemistry, physics and the biochemistry method of routine, can modify the storehouse and the compound of natural or synthetic manufacturing at an easy rate.Can carry out orientation or chemically modified at random to known medicament, as acidylate, alkanisation, esterification, amidification, to produce analog.

In an embodiment preferred, the candidate biologically active agent is an albumen.Here " albumen " is meant at least 2 covalently bound amino acid, and it comprises albumen, polypeptide, oligopeptides and peptide.Albumen is made of natural amino acid and peptide bond, or is made of synthetic class peptide structure (peptidomimetic structures).Therefore, " amino acid " described herein or " peptide residue " had both referred to natural amino acid, also referred to synthetic amino acid, and for example, for the purposes of the present invention, height-phenylalanine, citrulline and nor-leucine are considered to amino acid." amino acid " also comprises subunit residue such as proline(Pro) and oxyproline.Side chain can be (R) or (S) configuration.In an embodiment preferred, amino acid is (S) or L-configuration.If use the non-natural side chain, then can use non--aminoacid replacement base, for example be prevention or prevention vivo degradation.

In an embodiment preferred, the candidate biologically active agent is native protein or naturally has a proteic fragment.Therefore, for example can use and contain proteic cell extract, perhaps at random or the albumen sexual cell extract of directed digestion.By this way, can prepare prokaryotic organism and the eukaryotic protein storehouse that is used for screening method of the present invention.In this embodiment, special preferred bacterium, fungi, virus and mammalian proteins storehouse are preferred with the latter, preferred especially people's albumen.

In an embodiment preferred, the candidate biologically active agent is about 5～about 30 amino acid whose peptides, preferred about 5～about 20 amino acid, preferred especially about 7～about 15 amino acid.Peptide may be that as above outline natural exists proteic digestion product, peptide or " edge " peptide at random at random.Here " peptide at random " or phraseological Equivalent are meant that each nucleic acid and peptide are made of random nucleotide and amino acid respectively basically.Since these normally chemosynthesis of peptide (or nucleic acid, state as follows) at random, they can be integrated into any Nucleotide or amino acid in any position so.Can design synthetic route producing albumen or nucleic acid at random, make to form all or most of possible combination, form candidate's activated protein agent storehouse at random thus along sequence length.

In one embodiment, the library is a completely random, does not all have sequence preferential or constant in any position.In an embodiment preferred, the library is a bias, also is some position in the sequence or keeps constant, perhaps only is selected from a limited number of possibility.For example, in an embodiment preferred, Nucleotide or amino-acid residue are at random in limiting kind, for example, be hydrophobic amino acid, hydrophilic residue, the resistive bias of space bit (little or big) residue, purpose is in order to produce nucleic acid binding domain, for producing the halfcystine of crosslinked usefulness, for the proline(Pro) in SH-3 district, for Serine, Threonine, tyrosine or the Histidine etc. of phosphorylation site, or in order to combine with purine etc.

In an embodiment preferred, the candidate biologically active agent is a nucleic acid." nucleic acid " described herein or " oligonucleotide " or phraseological Equivalent are meant that at least 2 Nucleotide are covalently bound.Nucleic acid of the present invention contains phosphodiester bond usually, although in some cases, as cited below, comprise nucleic acid analog with variable main chain, the latter for example contain phosphamide (Beaucage etc., Tetrahedron 49 (10): 1925 (1993) and citing document wherein; Letsinger, J.Org.Chem.35:3800 (1970); Sprinzl etc., Eur.J.Biochem.81:579 (1977); Letsinger etc., Nucl.Acids Res.14:3487 (1986); Sawai etc., Chem.Lett.805 (1984), Letsinger etc., J.Am.Chem.Soc.110:4470 (1988); With Pauwels etc., Chemica Scripta 26:141 91986)), thiophosphatephosphorothioate (Mag etc., Nucleic acids Res.19:1437 (1991); With United States Patent (USP) 5644048), phosphorodithioate (Briu etc., J.Am.Chem.Soc.111:2321 (1989), oxygen-methyl phosphoramidate (phophoroamidite) connects (referring to Eckstein, Oligonucleotide and Analogues:APractical Approach, Oxford University Press), with the peptide nucleic acid(PNA) main chain and be connected and (see Egholm, J.Am.Chem.Soc.114:1895 (1992); Meier etc., Chem.Int.Ed.Engl.31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson etc., Nature 380:207 (1996), all these documents all introduce reference).Other similar nucleic acid comprises nucleic acid (Denpcy etc., the Proc.Natl.Acad.Sci.USA 92:6097 (1995) of those positively charged main chains; Non--the ion main chain (United States Patent (USP) 5386023,5637684,5602240,5216141 and 4469863; Kiedrowshi etc., Angew.Chem.Intl.Ed.English 30:423 (1991); Letsinger etc., J.Am.Chem.Soc.110:4470 (1988); Letsinger etc., Nucleoside ﹠amp; Glycoside 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, " Carbohydrate Modifications inantisense Research ", Ed.Y S.Sanghui and P.Dan Cook; Mesmaeker etc., Bioorganic ﹠amp; Medicinal Chem.Lett.4:395 (1994); Jeffs etc., J.BiomolecularNMR 34:17 (1994); Tetrahedron Lett.37:743 (1996)) and non--ribose main chain, be included in United States Patent (USP) 5235033,5034506 and Chapters 6 and 7, ASC Symposium Series 580, " Carbohydrate Modifications in antisense Research ", those that describe among the Ed.Y S.Sanghui P.DanCook.The nucleic acid that contains one or more carbocyclic ring sugar is also included within the definition of nucleic acid (referring to Jenkins etc., Chem.Soc.Rev. (1995) pp169-176).Several nucleic acid analogs are at Rawls, C ﹠amp; Be described among E News June 2,1997 page 35.All these documents all are incorporated herein by reference with it.To these modifications that ribose-phosphoric acid ester main chain carries out, be for convenient interpolation extention such as marker, perhaps in order to increase the stability and the transformation period of molecule in physiological environment.The mixture that can prepare in addition, natural acid and analogue.Perhaps, the mixture and the natural mixture that has nucleic acid and analogue of preparation different IPs acid-like substance.By indicated such, nucleic acid can be strand or two strands, has perhaps both contained two strands and has also contained the single stranded sequence part.Nucleic acid can be DNA (both genome also cDNA), RNA or crossbred, its amplifying nucleic acid contains any combination of ribodesose and ribonucleotide, with the base arbitrary combination, comprise uridylic, VITAMIN B4, thymus pyrimidine, cytosine(Cyt), guanine, Trophicardyl, xathanine hypoxathanine, iso-cytosine, isoguanine etc.

As above the blanket property description of doing in the face of albumen is such, and nucleic acid candidate biologically active agent can be natural nucleic acid, random nucleic acid or " bias " random nucleic acid of existing.For example, as above do to describe like that, can use the digestion product of protokaryon or eukaryotic gene group in the face of albumen.

In an embodiment preferred, the candidate biologically active agent is the organic chemistry molecule, has put down in writing available various organic chemistry molecule in the document.

As mentioned above, in an embodiment preferred, can screen individual gene and gene product (albumen).In an embodiment preferred,, identified the gene relevant gene or the albumen of expressing, thereby described illness is relevant with these tissues with particular organization difference ground according to described in the following embodiment.Therefore, in one embodiment, screening is designed at first find and FGF-19 bonded candidate agent, these reagent that will search out then are used for estimating the active test of the adjusting active candidate agent of FGF-19.So, as will by those skilled in the art understand, can carry out a large amount of different tests.

Also can screen regulating the active reagent of FGF-19.In an embodiment preferred, can regulate the screening method of FGF-19 biologically active agent, comprise candidate bioactive agent is joined in the sample of FGF-19, and measure the bioactive change of FGF-19." the active adjusting of FGF-19 " comprises the variation of active increase, active reduction or active type.Therefore, in this embodiment, candidate agent should not only combine (although this may be unnecessary) but also influence described biology or the biochemical activity of FGF-19 with FGF-19.Method had both comprised above-mentioned in-vitro screening method, also comprised the screening method that changes the cell of FGF-19 existence, expression, distribution, activity or content in the body.

Therefore, in this embodiment, method comprises sample is combined with candidate bioactive agent, and estimates the active influence of FGF-19." FGF-19 protein-active " or phraseological Equivalent are meant the proteic at least a above-mentioned biological activity of FGF-19 here.

In an embodiment preferred, proteic active the increasing of FGF-19; In another preferred embodiment, proteic active decline of FGF-19.Thereby, in certain embodiments preferably as the biologically active agent of antagonist, preferred in other embodiments biologically active agent as agonist.

One aspect of the present invention uses the cell that contains the FGF-19 sequence in the drug screening test, detect the influence of drug candidate to FGF-19.Cell type comprises normal cell, tumour cell and adipocyte.

The active method of evaluation FGF-19 is known in the art, and do illustrative description in the following embodiments, described activity is as in the variation aspect glucose uptake, leptin release, metabolism, triglyceride level and free fatty acid levels, body weight and the body fat.

In an embodiment preferred, method comprises above-mentioned candidate bioactive agent is joined in the cell that contains FGF-19.Preferred cell type comprises almost any cell.Cell contains the proteic nucleic acid of coding FGF-19, the thing of preferably recombinating.In an embodiment preferred, the candidate agent storehouse is detected in many cells.

On the one hand, test is being evaluated under the situation of existence or shortage physiologic information thing or before or after being exposed to the physiologic information thing, described physiologic information thing is hormone, antibody, peptide, antigen, cytokine, somatomedin, action potential for example, and pharmacological agents comprises chemotherapeutics, radiotherapeutic agents, carcinogen or other cells (being cell-cells contacting).In another example, measure in the not same period of cell cycle progression.

FGF-19 sequence provided herein also can be used in diagnostic method.

The overexpression of FGF-19 shows undesired metabolic rate, and its low expression then shows lipophilia.And, can analyze in the sample of taking from the patient FGF-19 and whether make a variation or lose function.Usually, these class methods comprise and comparing picking up from patient's sample and expression and the contrast of FGF-19.

F. resist-FGF-19 antibody

The present invention further provides anti--FGF-19 antibody.Exemplary antibody comprises polyclonal antibody, monoclonal antibody, humanized antibody, bispecific antibody and allos coupling antibody.

1. polyclonal antibody

Anti--FGF-19 antibody comprises polyclonal antibody.The method for preparing polyclonal antibody is known for those skilled in the art.Polyclonal antibody can produce in Mammals, for example, causes immunizing agent by the one or many injection, if desired, can add adjuvant.Usually, subcutaneous or intraperitoneal multiple injection causes immunizing agent and/or adjuvant to Mammals.Cause immunizing agent and can comprise FGF-19 polypeptide or its fusion rotein.With cause immunizing agent with at immune Mammals have immunogenic albumen to carry out coupling be effective.The described example that causes immune protein includes but not limited to: keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin and Trypsin inhibitor SBTI.The example of operable adjuvant comprises Freund's complete adjuvant and MPL-TDM adjuvant (single phosphatidyl lipid A, synthetic trehalose double blank larynx bacterium acid esters (dicorynomycolate)).Need not loaded down with trivial details test, those skilled in the art just can select to cause immunization protocol.

2. monoclonal antibody

Perhaps, resist-FGF-19 antibody is monoclonal antibody.Can use hybridoma method to prepare monoclonal antibody, as Kohler and Milstein at Nature, the method for describing among the 256:495 (1975).In hybridoma method, usually with causing immunizing agent immune mouse, hamster or other suitable host animal, with cause generation maybe can produce will with the lymphocyte that causes immunizing agent specificity bonded antibody.Perhaps, at external primed lymphocyte.

Cause immunizing agent and generally include FGF-19 polypeptide or its fusion rotein.Generally speaking, derive from people's cell if desired, then use peripheral blood lymphocyte (" PBLs "), the cell in non-human mammal source so just uses splenocyte or lymph-node cell if desired.Then, use suitable fusion reagent such as polyoxyethylene glycol, lymphocyte and immortal cell line are merged, thereby form hybridoma [Goding, monoclonal antibody: Principles and Practice, Academic Press, (1986) 59-103 page or leaf].The mammalian cell that immortal cell line normally transforms, particularly rodents, ox and myeloma cell anthropogenous.Usually use rat or mouse myeloma cell line.Can cultivate hybridoma in appropriate media, described substratum preferably contains one or more and suppresses infinite multiplication cell growth of not merging or the material of surviving.For example, parent cell lacks enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the substratum of hybridoma generally includes xanthoglobulin, aminopterin and thymidine (" HAT substratum "), and these materials suppress the growth of HGPRT-deficient cells.

Preferred immortal cell line is that those effectively merge, support antibody-founder cell of filtering out high level expression antibody stably, and to the clone of substratum such as HAT sensitivity.Preferred immortal cell line is a rat bone marrow tumour system, and the latter can derive from for example Salk Institute CellDistribution Center, San Diego, California and American Type Culture Collection, Manassas, Virginia.Human myeloma and mouse-people are hybridized myeloma cell line and also have been used to produce human monoclonal antibodies [Kozbor, J.Immunol., 133:3001 (1984); Brodeur etc., Monoclone antibodyProduction Techniques and Applications, Marcel Dekker, Inc., NewYork, (1987) pp.51-63].

Next, can examine and determine the monoclonal antibody that whether has direct anti-FGF-19 in the substratum of cultivating hybridoma.Preferably, with immunoprecipitation or external, measure the binding specificity of the monoclonal antibody of hybridoma generation in conjunction with testing as radioimmunology analysis (RIA) or Enzyme Linked Immunoadsorbent Assay (ELISA).These technology and test analysis method are known in the art.Use for example Scatchard analysisof Munson and Pollard, Anal.Biochem., 107:220 (1980) disclosed method can be measured the binding affinity of monoclonal antibody.

Identify after the required hybridoma, the clone is carried out the subclone processing and adopts standard method to make it growth [Goding, ibid] through limiting dilution assay.Be used for this purpose suitable culture base and for example comprise the improved Eagle ' of Dulbecco ' s s substratum and RPMI-1640 substratum.Perhaps, hybridoma can be grown with the ascites form in mammalian body.

Use routine immunization sphaeroprotein purification process, for example albumin A-sepharose, hydroxyapatite chromatography method, gel electrophoresis, dialysis or affinity chromatography, can be from substratum or ascites isolated or purified by subclone excretory monoclonal antibody.

Also can use recombinant DNA method manufacture order clonal antibody, for example the method for in United States Patent (USP) 4816567, describing.Use routine techniques (for example, use can with the gene specific bonded oligonucleotide probe of coding murine antibody heavy chain and light chain), separate the DNA of code book invention monoclonal antibody at an easy rate and carry out sequencing.Hybridoma of the present invention is as the preferred source of this DNA.In case isolate this DNA, just it can be implanted in the expression vector, use latter's transfection host cell then, described host cell such as monkey COS cell, Chinese hamster ovary (CHO) cell or do not produce the myeloma cell of immunoglobulin (Ig) so that in recombinant host cell synthetic monoclonal antibody.Also can modify DNA, for example the encoding sequence of people's heavy chain and constant region of light chain is by corresponding mouse sequence displacement [United States Patent (USP) 4816567; Morrison etc., ibid], perhaps that all or part of encoding sequence of non--immunoglobulin polypeptides is covalently bound to the encoding sequence of immunoglobulin (Ig).This type of non--immunoglobulin polypeptides can replace the constant region of antibody of the present invention, maybe can replace the variable region of an antigen binding site of antibody of the present invention, to produce chimeric bivalent antibody.

Antibody can be univalent antibody.The method for preparing univalent antibody is known in the art.For example, there is a method to relate to the recombinant expressed of light chain immunoglobulin and modification back heavy chain.Usually in any site in Fc district with the heavy chain brachymemma, thereby prevent that heavy chain is crosslinked.Perhaps, replace relevant cysteine residues or cysteine residues to lack crosslinked with other amino-acid residue to prevent.

In vitro method also is suitable for preparing univalent antibody.Use this area routine techniques can finish antibody digestion and generation antibody fragment, particularly Fab fragment.

3. people's antibody and humanized antibody

The present invention is anti--and FGF-19 antibody further comprises humanized antibody or people's antibody.Non--people (for example, mouse) humanized antibody be gomphosis immunoglobulin, immunoglobulin chain or its fragment (as Fv, Fab, Fab ', F (ab ') ₂Or other antigens of antibody are in conjunction with subsequence), they comprise the minmal sequence of non-human immunoglobulin.Humanized antibody comprises that the CDR residue that receptor's complementary determining region (CDR) residue in the human normal immunoglobulin (receptor's antibody) is had inhuman source species antibody (donor antibody) such as the mouse of required specificity, avidity and performance, rat, rabbit replaces.In some instances, the Fv framework region residue of human normal immunoglobulin is replaced by corresponding non-human residue.Humanized antibody also can comprise all non-existent residue in receptor's antibody or donor CDR or the framework sequence.Usually, humanized antibody consist essentially of at least one, common two variable regions whole, CDR whole or wherein, and FR sequence whole or all be the human normal immunoglobulin sequence basically basically all corresponding to the corresponding section of non-human immunoglobulin.Humanized antibody is also optional to comprise constant region for immunoglobulin (Fc), is generally at least a portion of the constant region of human normal immunoglobulin.See Jones etc. for details, nature, 321:522-525 (1986); Riechmann etc., natural 332:323-329 (1988); And Presta, Curr.Op.Struct.Biol.2:593-596 (1992).

Humanization is non--and the method for people's antibody is that those skilled in the art are known.Usually, import one or more in the humanized antibody and be derived from inhuman amino-acid residue.These non-human amino-acid residues often are called " introduction " residue, and they are usually from " introduction " variable region.The humanization process is substantially as Winter and colleague (Jones etc., nature, 321:522-525 (1986); Riechmann etc., nature, 332:323-327 (1988); Verhoeyen etc., science, 239:1534-1536 (1988)) described, the corresponding sequence that replaces human antibodies with one or more CDR sequences is carried out.Therefore, such " humanization " antibody is chimeric antibody (United States Patent (USP) 4816567), and wherein the seldom part of complete human variable region is replaced by non-human species's corresponding sequence.In the practice, humanized antibody is people's antibody normally, some of them CDR residue and have part FR residue and replaced by the residue in similar site in the rodents antibody.

Use various techniques known in the art can prepare people's antibody, described method comprises phage display library [Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991); Marks etc., J.Mol.Biol., 222:581 (1991)].Also can use the Cole etc. and the technology of descriptions such as Boemer to prepare human monoclonal antibodies [Cole etc., Monoclonal Antibody and Cancer Therapy, Alan R.Liss, (1985) the 7th pages, with Boemer etc., J.Immunol., 1471:86-95 (1991)].Similarly, by the locus of human normal immunoglobulin is inserted transgenic animal for example mouse produce people's antibody, the endogenous immunoglobulin genes of described host animal is by part or all of deactivation.After the attack, observe people's antibody and produce, this all to being seen very similar at human body, comprises gene rearrangement, assembling and antibody repertoire in all fields.About progress is described in for example following patent and scientific publication thing: United States Patent (USP) 5545807; 5545806; 5569825; 5625126; 5633425; 5661016; Marks etc., Bio/Technologv 10,779-783 (1992); Lonberg etc., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild etc., Nature Biotechnologv 14,845-51 (1996); Neuberger, Nature Biotechnology 14,826 (1996); Lonberg and Huszar, Intem.Rev.Immunol.1365-93 (1995).

4. bispecific antibody

Bispecific antibody is a monoclonal antibody, preferably has people's antibody or humanized antibody antibody at least two kinds of antigenic binding specificities of difference.In the present invention, a kind of binding specificity is at FGF-19, and is another kind of at other antigen, preferred pin pair cell surface protein or acceptor or receptor subunits.

The method for preparing bispecific antibody is known in the art.The traditional preparation process method of bi-specific antibody is based on two kinds of coexpressions that heavy chain immunoglobulin-light chain is right, and wherein these two chains have not homospecificity (Millstein etc., Nature, 305:537-539 (1983)).Because the random assignment of heavy chain immunoglobulin light chain, these hybridomas (quadroma (quadroma)) may produce the mixture of 10 kinds of different antibodies molecules, wherein have only a kind of correct dual specific structure that has.Purifying (being undertaken by the affinity chromatography step usually) to described correct molecule is very complicated, and output is very low.Similarly method is seen WO93/08829 and Traunecker etc., EMBOJ, 10:3655-3659 (1991).

Antibody variable region and constant region for immunoglobulin sequence with required binding specificity (antibody-antigen binding site) can be merged.The preferred immunoglobulin heavy chain constant region with at least a portion, CH2 and the CH3 district that comprise hinge area of this fusion merges.Preferably make and contain light chain and appear at least in a kind of fusion in conjunction with first CH (CH1) in required site.Can be with coding heavy chain immunoglobulin syzygy, and in case of necessity, the DNA of coding light chain immunoglobulin inserts different expression vectors, cotransfection is to suitable host living beings.Produce the further detail content of bispecific antibody, for example see also Suresh etc., Methods in Enzymology, 121:210 (1986).

According to WO 96/27011 described another kind of method, can transform the interface between a pair of antibody molecule, the per-cent maximum of the feasible heterodimer that from reconstitution cell is cultivated, obtains.Preferred interface comprises at least a portion of antibody constant region CH3 structural domain.In the method, the little side chain of one or more amino acid that comes from the first antibody molecule interface is replaced by larger side chain (as tyrosine or tryptophane).The complementation " ditch " identical or close with described bulky side chain size can be by replacing the amino acid bulky side chain and forming on the interface of second antibody molecule with little side chain (as L-Ala or Threonine).This makes the undesired end product of rate ratio such as the dimeric height of homotype of heterodimer.

Bispecific antibody can prepare full length antibody or antibody fragment (as F (ab ') ₂Bispecific antibody).The existing document of technology for preparing bi-specific antibody from antibody fragment.For example, bi-specific antibody can utilize chemistry to connect preparation.Brennan etc. have described among the science 229:81 (1985) complete antibody have been prepared segmental method through proteolysis.These fragments are reduced when dimercapto recombiner Sodium metaarsenite exists, thus the sulfydryl of stabilize adjacent, and the formation of prevention intermolecular disulfide bond.Fab ' the fragment that generates is converted into sulphur nitrobenzoate (TNB) derivative.Wherein a kind of Fab '-TNB derivative is reduced into Fab '-mercaptan through mercaptoethylamine, mixes with other Fab '-TNB derivative of equimolecular quantity to form bi-specific antibody again.So the bi-specific antibody that produces can be used as used reagent in the selectivity immobilization of enzyme.

Recent progress has promoted Fab '-SH fragment from colibacillary direct recovery, and this fragment can form bi-specific antibody through chemical coupling.Shalaby etc., The Journal of Experimental Medicine has been described full-length human bi-specific antibody F (ab ') among the 175:217-225 (1992) ₂The generation of molecule.Each Fab ' fragment secretes respectively from intestinal bacteria, and external direct chemical coupling forms bi-specific antibody.So the bi-specific antibody of preparation can combine with the cell and the normal human T-cell of overexpression ErbB2 acceptor, can also cause the lytic activity of human cell's poison lymphocyte to HBT's cell.

Directly the multiple technologies of preparation and separation bispecific antibody fragment are also existing from reconstitution cell is cultivated describes.For example, available leucine zipper prepares bi-specific antibody.Kostelny etc., Journal of Immunology, 148 (5): 1547-1553 (1992)).To be connected by gene fusion with the Fab ' part of two kinds of different antibodies from the proteic leucine zipper peptide of Fos and Jun.Make the homodimer of antibody be reduced into monomer, reoxidized the heterodimer that forms antibody then at hinge area.This method also can be used for preparing the antibody morphism dimer.By Hollinger etc., NAS's journal, 90:6444-6448 (1993)) " bivalent antibody " technology of describing provides the another kind of method for preparing bispecific antibody fragment.Contain variable region of heavy chain (V in the described fragment _H), it is by joint and variable region of light chain (V _L) link to each other, this joint is very short, makes can't match between two structural domains of same chain.Therefore, the V on the same fragment _HAnd V _LStructural domain be forced to another fragment on complementary V _LAnd V _HThe structural domain pairing, thus two antigen binding sites formed.Reported the another kind of strategy for preparing bi-specific antibody with strand Fv (sFv) dimer in addition.See Gruber etc., Journal of Immunology, 152:5368 (1994).Also considered the above antibody of divalence.As preparing three-specific antibody.Tutt etc., Journal of Immunology, 147:60 (1991).

Illustrative bi-specific antibody can with two kinds of different epi-position combinations on the specified FGF-19 polypeptide of this paper.Perhaps, can with resist-FGF-19 polypeptide arm and white corpuscle on the arm of trigger molecule combine, thereby strengthen at the cytophylaxis mechanism of expressing specific FGF-19 polypeptide cell, described trigger molecule such as TXi Baoshouti molecule (CD2, CD3, CD28 or B7), or IgG Fc acceptor (Fc γ R) is as Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16).Bi-specific antibody also can be used for cellulotoxic preparation is positioned on the cell of expressing specific FGF-19 polypeptide.These antibody have the FGF-19-brachium conjunctivum and in conjunction with cellulotoxic preparation or the haptenic arm of radio isotope, as EOTUBE, DPTA, DOTA or TETA.Another relevant bispecific antibody is in conjunction with the FGF-19 polypeptide and further combined with tissue factor (TF).

5. allos coupling antibody

Allos coupling antibody is also included within the scope of the invention.Allos link coupled antibody is made up of two covalently bound antibody.Have viewpoint to think, this antibody-like can be used for the immunocyte undesired cell (United States Patent (USP) 4676980) that leads, also can be used for treating HIV infect (WO91/00360, WO92/200373, EP03089).Can consider to use currently known methods in the albumen chemosynthesis, comprise relating to the use linking agent, at external preparation allos coupling antibody.For instance, use the disulfide exchange reaction or, can make up immunotoxin by forming thioether bond.The example that is used for the suitable reagent of this purpose comprises immune thiolate and methyl-4-sulfydryl, sulfydryl butyrimidate and for example in United States Patent (USP) 4676980, describe those.

6. effector function is engineered

Aspect effector function, may wish to improve antibody of the present invention, to strengthen the effect when treating (for example treating cancer).For example, can in the Fc zone, introduce cysteine residues, thereby in this zone, form interchain disulfide bond.The homodimer that makes like this has the complement-mediated cell killing ability of improved internalization ability and/or raising and depends on the cytotoxicity (ADCC) of antibody.Referring to Caron etc., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.J.Immunol.148:2918-2922 (1992).Also can be as Wolff etc., as described in the Cancer Research 53:2560-2565 (1993), prepare anti-tumor activity enhanced homotype dimerization antibody with the functional linking agent of different dimerization.Perhaps, antibody can be had two Fc district through engineered one-tenth, thereby may strengthen complement cracking and ADCC ability.See Stevenson etc., Anti-Cancer Drng Design 3:219-230 (1989).

7. immune conjugate

The invention still further relates to immune conjugate, wherein contain and cell toxicity medicament link coupled antibody, described cell toxicity medicament such as chemotherapeutics, toxin (for example the enzyme activity toxin of small molecules toxin or bacterium, fungi, plant or animal-origin comprises its fragment and/or variant) or radio isotope (promptly radiating conjugate).

The chemotherapeutics that is applicable to this para-immunity conjugate of preparation is above being described.Adaptable enzyme activity toxin and fragment thereof comprise: diphtheria toxin A chain, the non-binding active fragments of diphtheria toxin, exotoxin A chain (from pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, the bent toxin of α-broom, tung oil tree (Aleutites fordii) albumen, oleanolic acid albumen, dyers' grapes (Phytolaca Americana) albumen (PAPI, PAPII, PAP-S), balsam pear (momordica charantia) supressor, curcin, crotin, Saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin, mitogillin (mitogellin), restrictocin, phenomycin, enomycin and trichothecene (tricothecenes).Multiple radio isotope can be used for preparing radioactivity link coupled antibody, and example comprises Bi ²¹², I ¹³¹, In ¹³¹, Y ⁹⁰And Re ¹⁸⁶

Antibody can be connected by multiple bifunctional protein coupling agent with the conjugate of cellulotoxic preparation; described bifunctional protein coupling agent is as N-succinimido-3-(2-pyridyl dimercapto) propionic ester (SPDP); succinimido-4-(N-maleimide aminomethyl) hexanaphthene-1-carboxylicesters; imino-sulfane (iminothiolane) (IT); the dual-function derivative of imido-ester (as imino-dimethyl adipate hydrochloride); active ester class (as two succinimido suberates); aldehydes (as glutaraldehyde (glutareldehyde)); two-triazo-compound (as two (right-the triazobenzene formyl radical) hexanediamines); two-diazo compound derivative (as two-(right-the diazobenzene formyl radical)-quadrols); vulcabond is (as tolylene 2; the 6-vulcabond); with two-active fluorine cpd (as 1; 5-two fluoro-2, the 4-dinitrobenzene).For example, the ricin immunotoxin can be as Vitetta etc., the described preparation of science 238:1098 (1987).C ¹⁴The 1-isothiocyanic acid phenmethyl of mark-3-methyl diethylidene three ammonia pentaacetates (MX-DTPA) are that the radioactive nuleus thuja acid is coupled to one of coupling agent of antibody.See WO94/11026.

In another embodiment, antibody can with " acceptor " (as avidin) coupling of using in the pre-target of tumour, this antibody-acceptor conjugate is applied to the patient, remove unconjugated conjugate in the circulation with scavenging agent afterwards, " part " (as the avidin) of cellulotoxic preparation (as the radioactive nuleus thuja acid) that given coupling again.

8. immunoliposome

Antibody disclosed herein also can be made into immunoliposome.The liposome that contains antibody can prepare by means known in the art, as Epstein etc., the journal 82:3688 of NAS (1985); Hwang etc., the journal 77:4030 of NAS (1980); United States Patent (USP) 4485045 and on October 23rd, 4544545 and 1997 disclosed WO97/38731.The liposome that has increased cycling time is disclosed in United States Patent (USP) 5013566.

Useful especially liposome can utilize the lipid composition that comprises phosphatidylcholine, cholesterol and PEG deutero-phosphatidylethanolamine (PEG-PE) to make through reverse phase evaporation.Liposome is pressed through the filter membrane that limits the aperture, obtains to have the liposome of required diameter.Fab ' the fragment of antibody of the present invention can be as Martin etc., journal of biological chemistry, and 257:286-288 (1982) is described like that, through disulfide exchange reaction and liposome coupling.Can choose wantonly and in described liposome, comprise a kind of chemotherapeutics.See Gabizon etc., J.National Cancer Inst, 81 (19) 1484 (1989).

9. the pharmaceutical compositions of antibody

The FGF-19 polypeptid specificity bonded antibody of identifying with this paper, and with other molecule of aforementioned disclosed shaker test evaluation, form that can pharmaceutical compositions is used and is treated various illnesss.

If the FGF-19 polypeptide is in cell and uses complete antibody as inhibitor, then preferred internalization antibody.Yet also available lipofection thing or liposome are delivered to antibody or antibody fragment in the cell.When using antibody fragment, preferably with the minimum inhibitor fragment of target protein binding domains specific combination.For example, based on the antibody variable region sequence, can be designed to peptide molecule to keep and target protein sequence bonded ability.This type of peptide can chemosynthesis and/or is produced with recombinant DNA technology.Referring to for example Marasco etc., Proc.Natl.Acad.Sci.USA, 90:7889-7893 (1993).Preparation described here also comprises more than one active compounds necessary for controlling specific adaptations is levied, those compounds that preferably have the complementary activity that has no adverse effect each other.Perhaps or in addition, composition can contain the reagent that strengthens its function, such as cytotoxic agent, cytokine, chemotherapeutics or growth inhibitor.These molecules are suitable to the effective amount of institute's therapeutic purpose is united.

Also active ingredient can be encapsulated in the microcapsule according to for example condensation technique or the preparation of interfacial polymerization technology, for example, respectively at colloidal state drug delivery system (liposome for example, the albumin microsphere spheroid, microemulsion, nano particle and Nano capsule) or the macro emulsification agent in Walocel MT 20.000PV or gelatin microcapsule and poly--(methyl methacrylate) microcapsule.These technology are disclosed in Remington ' s PharmaceuticalSciences, during ibid.

The preparation that is used for vivo medicine-feeding must be aseptic.This can realize easily by the degerming membrane filtration.

Also can prepare controlled release preparation.The suitable example of controlled release preparation comprises half permeability matrix of the solid-state hydrophobic polymer that contains antibody, and described matrix is the goods with definite shape, as film or microcapsule.The controlled release preparation example comprises that polyester, hydrogel are (as poly-(2-hydroxyethyl-methacrylic acid ester) or poly-(vinyl alcohol), polylactide (United States Patent (USP) 3,773,919), the multipolymer of L-L-glutamic acid and γ ethyl-L-glutamate, nondegradable ethylene-ethyl acetate, degradable poly lactic coglycolic acid such as LUPRON DEPOT ^TM(the injectable microsphere of forming by poly lactic coglycolic acid and sour Leuprolide (leuprolide)), and poly-D-(-)-3-hydroxybutyric acid.Although polymkeric substance such as ethane-acetic acid ethyenyl ester and lactic-co-glycolic acid can continue to discharge molecule 1 more than 00 day, it is shorter that some hydrogel discharges the proteic time.When encapsulated antibody stopped in vivo for a long time, their can sex change or cohesion owing to be exposed to 37 ℃ of moisture under, thereby causes biological activity loss, and immunogenicity may change.Can come the reasonable strategy of design stabilityization according to the mechanism that relates to.For example, if finding the mechanism of cohesion is to exchange by the sulfo-disulfide linkage to have formed intermolecular S-S key, then can be by modifying sulfhydryl residue, freeze-drying in acidic solution, controlling moisture degree, adopting suitable additive and exploitation specific polymers substrate composition to reach stabilization.

G uses anti--FGF-19 antibody

The present invention is anti--and FGF-19 antibody serves many purposes.For example, anti--FGF-19 antibody can be used for the diagnositc analysis of FGF-19, for example, detects the expression of FGF-19 at specific cells, tissue or serum.Can use various diagnositc analysis test known in the art, as CBA, direct and indirect sandwich test, and the immunoprecipitation test.Zola, monoclonal antibody: technical manual, the 147-158 page or leaf (CRCPress, Inc.1987).But also can be with test section on the described antibody labeling.But should directly or indirectly producing, the test section can record signal.For example, can to record part be radio isotope as ³H, ¹⁴C, ³²P, ³⁵S or ¹²⁵I, fluorescence or chemiluminescence compound such as fluorescein isothiocyanate, rhodamine or luciferin, perhaps enzyme such as alkaline phosphatase, beta-galactosidase enzymes or horseradish peroxidase.Known in the art but antibody and any method of test section link coupled all can be used, be included in Hunter etc., Nature, 144:945 (1962); David etc., Biochemistry, 13:1014 (1974); Pain etc., J.Immunol.Meth., 40:219 (1981); And Nygren, the method for describing among J.Histochem. and the Cytochem., 30:407 (1982).

Anti--FGF-19 antibody also is applicable to affinity purification FGF-19 from reconstitution cell culture or natural origin.In this process, use means known in the art will resist FGF-19 antibody to be fixed on suitable carrier such as Sephadex resin or the filter paper.Then, with fixed antibody with containing the sample bag quilt of the FGF-19 that desires purifying, next with can be basically with in the sample with immobilization carrier bonded FGF-19 outside the suitable solvent wash vehicle of all substances removal.At last, with another suitable solvent wash vehicle that can from antibody, disengage FGF-19.

Following embodiment just provides for purpose of description, is not to be intended to limit by any way protection scope of the present invention.

All patents and the reference quoted as proof in this specification sheets all are hereby incorporated by with it.

Embodiment

Unless stated otherwise, otherwise the commercially available reagent of being mentioned among the embodiment, according to the explanation use of manufacturers.The source of the cell that defines with the ATCC registration number in following embodiment and whole specification sheets is in American type culture collection, Manassas, and VA carries out preservation.

Embodiment 1

The cDNA clone who separates coding people FGF-19

The est sequence registration number is that the mouse fibroblast cell somatomedin (FGF-15) of AF007268 is used to retrieve various public est databases (as GenBank, Dayhoff etc.).The program that uses a computer BLAST or BLAST2[Altschul etc., Methods In Enzymology, 266:460-480 (1996)] comparison est sequence 6 frameworks translate and the ECD protein sequence is retrieved.Result for retrieval hits GenBank ESTAA220994, and the latter has been accredited as STRATAGENE NT2 neurone precursor 937230.The sequence of AA220994 is also referred to as DNA47412 here.

Based on the DNA47412 sequence, synthetic oligonucleotide :) as the clone's who separates the FGF-19 complete encoding sequence probe 1) so that contain the cDNA library of target sequence, with 2 with PCR method identification.Forward and inverse PCR primer generally contain 20～30 Nucleotide, and often are designed to produce the PCR product of the about 100-1000bp of length.Probe sequence length is generally 40-55bp.In some cases, when consensus sequence during greater than about 1-1.5kbp, then synthetic other oligonucleotide.In order to screen the library of several full-length clones, according to Ausubel etc. at Current Protocols in Molecular Biology, the method for describing during ibid, adopt the pcr amplification method with the PCR primer to screening DNA from the library.Then, use probe oligonucleotides and primer to one of, separate the clone of the target gene of encoding with positive library.

Synthetic pcr primer thing (forward and reverse):

Forward PC primer5 '-ATCCGCCCAGATGGCTACAATGTGTA-3 ' (SEQ ID NO:3) and

The inverse PCR primer5 '-CCAGTCCGGTGACAAGCCCAAA-3 ' (SEQ ID NO:4).

In addition, by the synthetic property oligonucleotide hybridization probe of the DNA47412 sequence construct with following nucleotide sequence:

Hybridization probe

5′-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA-3′(SEQ?ID?NO：5)。

The RNA that is used for the construction cDNA library separates from the human fetal retinal tissue.Be used to separate cDNA clone's cDNA library, can use to be purchased reagent and to adopt standard method to make up, described reagent is as available from Invitrogen, San Diego, CA.With the few dT guiding cDNA that contains the NotI site, its blunt end is connected with SalI half kinases adapter, use the NotI cracking, tell suitable size, and direction is cloned in the suitable cloning vector unique XhoI and NotI site (as pRKB or pRKD in accordance with regulations with gel electrophoresis; PRK5B is the pRKSD precursor that does not contain the Sfil site; See Holmes etc., Science, 253:1278-1280 (1991)) locate.

Above-mentioned isolating clone is carried out dna sequencing, obtain the full length DNA sequence (this paper is decided to be DNA49435-1219[Fig. 1, SEQ ID NO:1]) of total length FGF-19 polypeptide and the deutero-protein sequence of FGF-19 polypeptide.

The full-length clone of above-mentioned evaluation contains single open reading frame, and it has tangible translation initiation site at Nucleotide 464-466 place, and at Nucleotide 1112-1114 place termination signal (Fig. 1, SEQ ID NO:1) is arranged.Long 216 amino acid of the polypeptide precursor of this prediction, about 24003 dalton of theoretical molecular estimate pI about 6.99.Be shown in the analytical proof of the total length FGF-19 sequence of Fig. 2 (SEQ ID NO:2), have a plurality of important polypeptide structure territories in the sequence shown in Figure 2, wherein the position in those important polypeptide structure territories and top described be approaching.Karyomit(e) is drawn to be proved, the FGF-19-coding nucleic acid is corresponding to human chromosome 11q13.1, and q13.1 is with.Cloned DNA 49435-1219 on November 21st, 1997 in the ATCC preservation, the ATCC preserving number is 209480.

Dayhoff database (version 35.45 SwissProt 35) is analyzed, utilized ALIGN-2 sequence comparative analysis, had sequence identity: AF007268_1, S54407, P_W52596, FGF2_XENLA, P_W53793, AB002097_1, P_R27966, HSU67918_1, S23595 and PR_70824 between its proof FGF-19 aminoacid sequence and the following Dayhoff sequence full length sequence shown in Fig. 2 (SEQ IDNO:2).

Embodiment 2

FGF-19 is as the application of hybridization probe

Following method is described the application of the nucleotide sequence of coding FGF-19 as hybridization probe.

The DNA that comprises the encoding sequence of total length or ripe FGF-19 is as the probe of (as those of the natural variant of coding FGF-19) of homologous dna in screening people tissue cDNA library or the people's tissue gene group library.

Under the height stringent condition, hybridize, and washing contains the film of any one library DNA.Derive from the radio-labeled probe of FGF-19 and the hybridization of filter membrane, in following solution, carry out: 50% methane amide, 5x SSC, 0.1%SDS in 42 ℃, 0.1% trisodium phosphate, 50mM sodium phosphate, pH6.8,2x Denhardt ' s solution and 10% dextran sulfate were hybridized 20 hours.The filter membrane washing then is to carry out in the aqueous solution of 0.1x SSC and 0.1%SDS in 42 ℃.

Then, use standard technique known in the art, can identify the DNA that those have expected sequence identity with respect to the DNA of coding total length native sequences FGF-19.

Embodiment 3

The expression of FGF-19 in intestinal bacteria

This embodiment describes the FGF-19 by the not glycosylation form of recombinant expressed preparation in intestinal bacteria.

Use selected PCR primer, the dna sequence dna of coding FGF-19 is tentatively increased.Primer should contain the restriction endonuclease sites corresponding to restriction enzyme site in the selected expression vector.Can use various expression vectors.An example of appropriate carrier is that pBR322 (derives from intestinal bacteria; See Bolivar etc., Gene, 2:95 (1977)), it contains penbritin and tetracycline resistance gene.This carrier is with digestion with restriction enzyme and dephosphorylation.Then the sequence of pcr amplification is connected in this carrier.Carrier preferably includes following sequence: sequence, trp promotor, poly Histidine leader sequence (comprising first 6 STII codon, poly Histidine sequence and enteropeptidase cracking site), FGF-19 coding region, λ transcription terminator and the argU gene of coding antibiotics resistance gene.

Then, use Sambrook etc., the method that ibid describes transforms selected e. coli strains with the connection mixture.Transformant is identified by its energy for growth on the LB plate, is selected the antibiotics resistance bacterium colony then.Isolated plasmid dna, and confirm with restriction analysis and dna sequencing.

But grow overnight in clone's liquid medium within of selecting (as having added antibiotic LB meat soup).Subsequently, overnight culture is used to inoculate more massive cultivation.Cell grows to required optical density(OD) then, expresses promotor during this period and is opened.

Culturing cell is after a few hours, centrifugal cell harvesting.Use all ingredients known in the art can dissolve the little group of centrifugal gained cell, then, use metal to sting zygostyle purifying dissolved FGF-19 albumen under the proteic condition that allows to combine closely.

Through following operation, FGF-19 can be intestinal bacteria with poly histidine mark formal representation.Use selected PCR primer, the DNA of preliminary amplification coding FGF-19.Primer contain corresponding to the restriction enzyme site of restriction enzyme site in the selected expression vector and other for provide effectively, reliable translation initiation, fast purifying and carry out proteolysis with enteropeptidase and remove useful sequence on metal chelating column.Next, the sequence of the poly histidine mark of pcr amplification is connected in the expression vector, the latter is used to transform escherichia coli host (W3110 fuhA (tonA) Ion galErpoHts (htpRts) clpP (lacIq) based on bacterial strain 52.Transformant is shaking culture in 30 ℃ of LB that containing the 50mg/ml Pyocianil at first, reaches 3-5 until O.D.600.Then, culture (is mixed 3.57g (NH with the CRAP substratum in 500mL water ₄) ₂SO ₄, 0.71g Trisodium Citrate 2H ₂O, 1.07g KCI, 5.36g Difco yeast extract, 5.36g Sheffield hycase SF, and 110mM MPOS, pH7.3,0.55% (w/v) glucose and 7mM MgSO4) dilute 50-100 doubly, in 30 ℃ of about 20-30 of shaking culture hours.Sampling is analyzed with SDS-PAGE and is confirmed to express, and culture in enormous quantities is centrifuged into cell mass.Frozen cell group is until purifying and refolding (refolding).

In the 7M guanidine of 10 times of volumes (w/v), 20mM Tris, pH8 buffer reagent, suspending again derives from the intestinal bacteria mashed prod of 0.5～1L fermented product (the little group of 6-10g).Add solid sodium sulfite and sodium tetrathionate and make final concentration be respectively 0.1M and 0.02M, spend the night at 4 ℃ of stirred solutions.This step produces metaprotein, and all cysteine residues of the latter are sealed by sulfitolization.Solution was gone up 40000rpm centrifugal 30 minutes at Beckman ultracentrifuge (Ultracentifuge).Supernatant liquor is with the metal chelating column damping fluid of 3-5 times of volume (6M guanidine, 20mM Tris, pH7.4) dilution, and filter so that the supernatant liquor clarification by 0.22 micron filter.Sample on the clarifying extracting solution is extremely used on the metal chelating column damping fluid equilibrated 5ml Qiagen Ni-NTA metal chelating column.Wash post with another pH7.4 damping fluid that contains 50mM imidazoles (Calbiochem, Utrol grade).With the buffer solution eluted protein that contains the 250mM imidazoles.Compile the fraction that contains desirable proteins and in 4 ℃ of storages.The delustring that utilization calculates based on the Argine Monohydrochloride sequence (extinction) coefficient is by the optical density estimation protein concentration at 280nm place.

Sample is slowly diluted with freshly prepared refolding damping fluid, thereby make refolding proteins, the formation of described refolding damping fluid is: pH8.620mM Tris, 0.3M NaCl, 2.5M urea, 5mM halfcystine, 20mM glycine and 1mM EDTA.The volume of selecting refolding liquid is so that final concentration of protein is 50～100 micrograms/ml.Stirred refolding liquid gently 12-36 hour in 4 ℃.Adding TFA is 0.4% (pH is about 3) to come cancellation refolding liquid to final concentration.Be further purified before the white matter, solution filters by 0.22 micron filter, and adding acetonitrile to final concentration is 2-10%.Albumen to refolding on Poros R1/H reversed-phase column carries out chromatographic analysis, and the moving phase damping fluid that uses is 0.1%TFA, with 10～80% acetonitrile gradient wash-outs.The sample aliquot that has the A280 place to absorb is analyzed on sds page, collects the proteic fraction that contains even refolding.Usually, the albumen of the correct refoldings of great majority elute when minimum acetonitrile concentration, and this is because these proteinoids are the most closely, and their hydrophobic interior is masked thereby avoid interacting with reversed-phase resin.Accumulation type albumen elutes when high acetonitrile concentration usually.Except the albumen of desired form and the albumen of false folding being made a distinction, this anti-phase step also can be removed intracellular toxin from sample.

Collection contains the fraction of required folding FGF-19 polypeptide, removes acetonitrile with the direct featheriness solution of nitrogen gas stream.Through dialysis or by carrying out gel-filtration, and albumen is mixed with the 20mM Hepes solution of pH6.8 with 0.14M sodium-chlor and 4% N.F,USP MANNITOL, and carries out filtration sterilization with preparing damping fluid equilibrated G25 Superfine (Pharmacia) resin.

Embodiment 4

The expression of FGF-19 in mammalian cell

This embodiment describes the FGF-19 by the potential glycosylation form of recombinant expressed preparation in mammalian cell.

Carrier pRK5 (see EP 307247, open day is on March 15th, 1989) is as expression vector.Randomly, use such as Sambrook etc., the method for attachment of describing during ibid is connected to FGF-19DNA among the pRK5 with selected restriction enzyme, and described restriction enzyme allows to insert FGF-19DNA.Consequent carrier is called pRK5-FGF-19.

In one embodiment, selected host cell is 293 cells.Grow to fusion in the substratum of people's 293 cells (ATCC CCL1573) in tissue culturing plate, described substratum is as having added foetal calf serum and optional nutritive ingredient and/or antibiotic DMEM.The DNA of about 10 μ g pRK5-FGF-19DNA and about 1 μ g coding VARNA gene [Thimmappaya etc., Cell, 31:543 (1982)] mixes, and is dissolved in 500 μ l 1mM Tris-HCI, 0.1mM EDTA, 0.227M CaCl ₂In.Dropwise 5 00 μ l50mM HEPES (pH7.35), 280mM NaCI, 1.5mM NaPO in this mixture ₄, 25 ℃ of 10 minutes formation precipitations.This throw out that suspends adds 293 cells, and 37 ℃ left standstill about 4 hours.The sucking-off substratum adds the PBS solution 2ml of 20% glycerine with 30 seconds times.Wash 293 cells with not containing blood serum medium then, add fresh culture, about 5 days of insulation cell.

After the transfection about 24 hours, the reject substratum replaced substratum (separately) or contains 200 μ Ci/ml ³⁵S-halfcystine and 200 μ Ci/ml ³⁵The substratum of S-methionine(Met).Be incubated after 12 hours, the collection condition nutrient solution concentrates on rotary filter, and last sample is to the 15%SDS gel.Make gel drying behind the electrophoresis, and on film, expose for some time, thus the existence that discloses the FGF-19 polypeptide.Further incubation contains the culture (in serum free medium) of transfection type cell, detects culture in selected biological detection method.

In another technology, use Somparyrac etc. are at Proc.Natl.Acad.Sci., and the T 500 method of describing among the 12:7575 (1981) can be with instantaneous introducing 293 cells of FGF-19.293 cells grow to maximum density in rotary flask, add 700 μ g pRK5-FGF-19DNA.The cell that picks up from rotary flask concentrates by centrifugal carrying out the first time, washs with PBS.In the little group of cell with DNA-dextran throw out incubation 4 hours.Cell, is packed into once more and is contained tissue culture medium (TCM), in the rotary flask of 5 μ g/ml Sigma I8405s and 0.1 μ g/ml ox transferrin with the tissue culture medium (TCM) washing with 20% glycerin treatment 90 seconds.After about 4 days, the centrifugal nutrient solution that should adjust filters and removes cell and fragment.Then, the sample that contains the FGF-19 of expression can be used any method selected, as the dialysis and/or column chromatography concentrates and purifying.

In another embodiment, FGF-19 can express in Chinese hamster ovary celI.Use known agent such as CaPO ₄Or deae dextran, pRK5-FGF19 can be transfected into Chinese hamster ovary celI.As mentioned above, the incubation cell culture, substratum with substratum (separately) or contain the radio-labeled thing as ³⁵The substratum of S-methionine(Met) is replaced.After having measured the existence of FGF-19 polypeptide, available serum free medium is replaced this substratum.Preferably, the culture incubation is gathered in the crops the nutrient solution of adjusting after about 6 days.Then, this contains the nutrient solution of the FGF-19 of expression with the concentrated also purifying of the method for any selection.

Also can in host CHO cell, express the FGF-19 of epi-position-mark.FGF-19 can clone out from the pRK5 carrier Central Asia.Can carry out PCR to this subclone inset, make it and the epi-position mark selected such as poly Histidine-mark merge in the same reading frame on rhabdovirus expression vector.Then, the FGF-19 inset subclone of poly histidine mark can be gone into contain the driving carrier of SV40 of the selection marker DHFR of stable clone (as be used for selecting).At last, with the driving carrier transfection CHO cell of this SV40 (as mentioned above).Can be by the above-mentioned mark that carries out, to confirm expression.Then, with the method for any selection, as use Ni ²⁺-Chinese blister beetle mixture affinity chromatography can concentrate and purifying contains the substratum of the FGF-19 of the poly histidine mark of having expressed.

Also can in CHO and/or COS cell, express FGF-19, or in Chinese hamster ovary celI, express FGF-19 by another stably express method by instant expression method.

Stably express in Chinese hamster ovary celI can make with the following method.With protein expression is IgG construct (immunoadhesin), sequence of every kind of proteic soluble form (for example extracellular domain) of wherein encoding and IgG1 constant region sequence merge, and described IgG1 constant region comprises hinge area, CH2 district and CH2 district and/or is the form of poly histidine mark.

Behind the pcr amplification, use Ausubel etc. are at Current Protocols of Molecular Biology.Unit 3.16, and standard technique described in John Wiley and the Sons (1997) is gone into each DNA subclone in the CHO expression vector.Make up the CHO expression vector, make it have 5 of target dna ' and 3 ' consistency restriction site, so that the shuttling back and forth of cDNA.That uses carrier in Chinese hamster ovary celI is expressed in Lucas etc., and (1774-1779 has description in (1996) to Nucl.Acids Res.24:9, and uses SV40 early promoter/enhanser to drive the expression of target cDNA and Tetrahydrofolate dehydrogenase (DHFR).DHFR expresses and allows to select to keep stable plasmid after the transfection.

Use is purchased transfection reagent Superfect  (Quiagen), Dosper  or Fugene  (Boehringer Mannheim), and the required plasmid DNA of 12 micrograms is introduced about 1,000 ten thousand Chinese hamster ovary celIs.Press Lucas etc., ibid described method culturing cell.In each ampoule freezing about 3 * 10 ⁷Individual cell is in order to further growth as described below and propagation.

The ampoule that contains plasmid DNA is put into water-bath to thaw and vortex mixed.With transfer pipet the content immigration is filled in the centrifuge tube of 10mL substratum centrifugal 5 minutes of 1000rpm.The sucking-off supernatant liquor is with 10mL selective medium (foetal calf serums of filtering PS20 of 0.2 μ m and 5%0.2 μ m diafiltrations) suspension cell.Then with the cell five equilibrium to the 100mL turner that contains the 90mL selective medium.After 1-2 days, with cell transfer to the 250mL turner that fills 150mL selective growth substratum, and in 37 ℃ of incubations.After 2-3 days, in 250mL, 500mL and 2000mL turner, inoculate 3 * 10 ⁵Cell/mL.Cell culture medium is replaced with fresh culture in centrifugal back, resuspension in producing substratum.Although can use any suitable CHO substratum, what reality was used is to produce substratum described in the United States Patent (USP) of issuing on January 16th, 1,992 5122469.3L produces in the turner and inoculates 1.2 * 10 ⁶Cell/mL.At 0 day, measure cell count PH ieThe 1st day,, begin the filtered air of spraying from the turner sampling.The 2nd day, from turner, to take a sample, temperature transfers to 33 ℃, adds 500g/L glucose 30mL and 10% antifoam 0.6mL (for example 35% aqueous emulsion of dimethyl polysiloxane fluid, DowCorning 365 Medical Grade Emulsion).In the whole process of production, adjust pH in case of necessity and make it to remain on about 7.2.After 10 days, or reduce to 70% when following, centrifugal cell harvesting culture and by 0.22 μ m membrane filtration until vigor.Filtrate is stored in 4 ℃ or go up column purification immediately.

For the construct of poly histidine mark, use Ni-NTA post (Qiagen) purifying protein.Before the purifying, adding imidazoles to concentration in the nutrient solution of adjusting is 5mM.In 4 ℃, with the nutrient solution of this adjustment with 4-5ml/min speed pump to 6ml Ni-NTA post, the latter is with the damping fluid 20mM Hepes, the pH7.4 balance that contain 0.3M NaCI and 5mM imidazoles.Behind the dress post, wash post, with the level pad eluted protein that contains the 0.25M imidazoles with other level pad.Subsequently, make highly purified albumen desalination to the storage damping fluid that contains 10mM Hepes, 0.14MNaCl and 4% N.F,USP MANNITOL pH6.8 with 25ml G25Superfine (Pharmacia) post, and store in-80C.

According to following step, purifying immunoadhesin (containing Fc) construct from the nutrient solution of adjusting.The nutrient solution of adjusting is pumped into 5ml albumin A post (Pharmacia), and the latter has used the 20mM sodium phosphate buffer balance of pH6.8.Behind the dress post, before with pH3.5 100mM citric acid wash-out, thoroughly wash post with balance liquid earlier.By the 1ml fraction being focused in the test tube that contains 275 μ L pH9 1M Tris damping fluids, and the albumen of wash-out is neutralized immediately.Then, according to the front at the proteic description of poly-his mark with highly purified albumen desalination to storage buffer.Carry out the N-terminal amino acid sequencing with sds polyacrylamide gel electrophoresis with the Edman degradation technique, measure homogeneity.

Embodiment 5

The expression of FGF-19 in yeast

Following method method is described FGF-19 recombinant expressed in yeast.

At first, make up Yeast expression carrier, so that FGF-19 is subjected to the driving of ADH2/GAPDH promotor and produces or secretion in cell.With the DNA of coding FGF-19 and the suitable restriction enzyme site that promotor is inserted selected plasmid, with the cell inner expression of guiding FGF-19.In order to secrete FGF-19, the dna clone of coding FGF-19 can be gone in the selected plasmid, DNA, natural FGF-19 signal peptide or other signal peptide of Mammals of the ADH2/GAPDH promotor of inserting with DNA of encoding in addition, perhaps for example yeast α-factor or saccharase secretion signal/leader sequence and catenation sequence (if desired) so that FGF-19 express.

Then, available above-mentioned expression plasmid transformed yeast cell such as yeast strains AB 110, and in the fermention medium of selecting, cultivate.By precipitating with 10% Tricholroacetic Acid and separating with SDS-PAGE, then use the Coomassie blue stain gel, can detect the supernatant liquor of transformed yeast.

Subsequently,, then use the pillar filter enrichment medium of selecting, separate therefrom and purification of Recombinant FGF-19 through centrifugal and from fermention medium, remove yeast cell.Use the column chromatography resin of selecting, can be further purified the enriched material that contains FGF-19.

Embodiment 6

The expression of FGF-19 in the insect cell of baculovirus-infection

Following method is described the expression of reorganization FGF-19 in the insect cell of baculovirus-infection.

The encoding sequence of FGF-19 merges the upstream at the contained epi-position mark of rhabdovirus expression vector.This class epi-position mark comprises poly histidine mark and immune globulin white marker (as the Fc district of IgG).Can use various plasmids, comprise being purchased plasmid such as pVL1393 (Novagen).In brief, if use with 5 ' and 3 ' district complementary primer be extracellular protein through the required part of the encoding sequence of pcr amplification FGF-19 or FGF-19 encoding sequence as the sequence or the albumen of coding transmembrane protein extracellular domain, the proteic sequence of encoding mature.5 ' primer can add flank (selection) restriction enzyme site.Go in the expression vector with the digestion with restriction enzyme product and the subclone of those selections then.

Recombinant baculovirus is to produce like this: use lipofection reagent (available from GIBCO-BRL), with above-mentioned plasmid and BaculoGold ^TMViral DNA (Pharmingen) be total to-is transfected in fall army worm (Spodoptera frugiperda) (" the Sf9 ") cell (ATCC CRL 1711).28 ℃ after incubation 4-5 days, the virus that results discharge is used for further amplification.At Baculovirus Express Vectors:A Laboratory Manual, the description among the Oxford:OxfordUniversity Press (1994) is carried out according to O ' Reilley etc. for viral infection and protein expression.

Next, but the FGF-19 of the poly histidine mark that purifying is expressed, for example, by following Ni ²⁺-chelating affinity chromatography carries out.The preparation of the extract of the Sf9 cell of recombinant virus-infection, at Nature, method is carried out described in the 362:175-179 (1993) according to Rupert etc.Say that briefly washing Sf9 cell is at ultrasonic damping fluid (25mL Hepes, pH7.9; 12.5mM MgCl ₂0.1mM EDTA; 10% glycerine; 0.1%NP-40; 0.4M resuspension KCl), in the ice bath ultrasonic 2 times, each 20 second.Product after centrifugal removing is ultrasonic, supernatant liquor filling damping fluid (50mM phosphoric acid salt, 300mM NaCl, 10% glycerine, pH7.8) 50 times of dilutions are filtered through 0.45 μ m filter.Preparation Ni ²⁺-NTA agarose column (available from Qiagen), bed volume 5mL uses the 25mL water washing, fills the damping fluid balance with 25mL.With filtering cell extract upper prop, wash post to A with the speed of per minute 0.5mL with the filling damping fluid ₂₈₀Baseline begins to collect fraction at this point.Then, with second lavation buffer solution (50mM phosphoric acid salt; 300mM NaCl, 10% glycerine pH6.0) is washed post, and this process wash-out goes out the albumen of non-specific combination.Reach A heavily again ₂₈₀Behind the baseline, wash post with the imidazoles gradient of 0～500mM in second lavation buffer solution.Collect the 1mL fraction, with SDS-PAGE and silver dyeing or with alkaline phosphatase (Qiagen) coupling type Ni ²⁺-NTA carries out the Western trace, analyzes.Gather the Histidine that contains wash-out ₁₀The fraction of the FGF-19 of-mark is at filling the damping fluid dialysis.

Perhaps, use known chromatographic technique, comprise for example albumin A or Protein G column chromatography, the FGF-19 of IgG mark (or Fc mark) is carried out purifying.

Embodiment 7

Preparation is in conjunction with the antibody of FGF-19

This embodiment describe can with the MONOCLONAL ANTIBODIES SPECIFIC FOR of FGF-19 specific combination.

The technology of manufacture order clonal antibody is that those skilled in the art are known, and at for example Goding, and going out has description during ibid.Operable immunogen comprise purifying FGF-19, contain the fusion rotein of FGF-19 and at the cell of cell surface expression reorganization FGF-19.Need not loaded down with trivial details experiment, those skilled in the art just can make one's options to immunogen.

Mouse such as Balb/c are through subcutaneous or peritoneal injection 1-100 microgram original immunity of emulsive FGF-19 immunity in complete Freund ' s adjuvant.Perhaps, immunogen the MPL-TDM adjuvant (RibiImmunochemical Research, Hamilton, MT) in emulsification, be injected into the metapedes pad of animal.After 10～12 days, be used in the mouse after to immunity of emulsive immunogen in the selected adjuvant once more and carry out booster immunization.After this several weeks, also can increase immunization and to the mouse booster immunization.Through after-eye socket bloodletting mode regularly gathers the mice serum sample, carries out elisa assay, the antibody of-FGF-19 anti-to detect.

After having detected suitable antibody titers, the last intravenous injection FGF-19 of antagonist " positive " animal.After 3 or 4 days, put to death mouse, get its splenocyte.Then splenocyte and selected rat bone marrow tumour cell system (as P3 X 63 AgU.1, deriving from ATCC CRL No. 1597) are merged (using 35% polyoxyethylene glycol).Be inoculated in the 96 hole tissue culturing plates merging the hybridoma that produces, contain HAT (xanthoglobulin, aminopterin and thymidine) substratum on the described culture plate to suppress the propagation of non--fused cell, marrow heterozygote and splenocyte heterozygote.

Screen hybridoma according to the anti-FGG-19 activity among the ELISA.Screening can be secreted the mensuration of " positive " hybridoma of required anti-FGF1-19 monoclonal antibody, is this area general knowledge.

Can give homology Balb/c mouse peritoneal injection positive hybridoma cell, contain the ascites of anti--FGF-19 monoclonal antibody with generation.Perhaps, make hybridoma in tissue culture flasks or roll in the bottle and grow.Use ammonium sulfate precipitation, that continues uses gel exclusion chromatography, can finish the Purification of Monoclonal Antibodies to producing in the ascites.Perhaps, use is based on the bonded affinity chromatography of antibody and albumin A or Protein G.

Embodiment 8

Use specific antibody purifying FGF-19 polypeptide

Can use the various standard techniques in protein purification field, carry out purifying natural or reorganization FGF-19 polypeptide.For example, but the immune-affinity chromatography purifying of the specific antibody of application target FGF-19 polypeptide former-FGF-19 polypeptide, ripe FGF-19 polypeptide or preceding-FGF-19 polypeptide.Generally speaking, immune affinity column be by will resist-antibody and the activatory chromatographic resin covalent coupling of FGF-19 polypeptide make up.

Use ammonium sulfate precipitation or by (N.J.) purifying on can prepare polyclonal immunoglobulin from immune serum for Pharmacia LKBBiotechnology, Piscataway at the immobilization albumin A.Similarly, with ammonium sulfate precipitation or the chromatography on the immobilization albumin A, can from mouse ascites, prepare monoclonal antibody.Partially purified immunoglobulin (Ig) is covalently attached to chromatographic resin such as CnBr-activatory SEPHAROSE ^TM(Pharmacia LKB Biotechnology).Antibody and resin covalent coupling, thereby sealing resin, the resin that is produced according to the indication wash-out of manufacturers.

Contain the cell fraction of solubility FGF-19 polypeptide by preparation, can in the purifying of FGF-19 polypeptide, use this immune affinity column.This preparation realizes through the differential centrifugation of adding stain remover or intact cell or the subcellular fraction that uses approach well known to obtain by dissolving.Perhaps, the solubility FGF-19 polypeptide that contains signal sequence can be secreted in the substratum at cell growth place with significant quantity.

Make the agent flow that contains solubility FGF-19 polypeptide through immune affinity column, making that (the high ionic strength buffers liquid when for example, having stain remover) washes post under the condition of preferential absorption FGF-19 polypeptide.Then, make antibody/FGF-19 polypeptide in conjunction with the splitted condition under (for example, low pH damping fluid such as the about 2-3 of pH, or high density chaotropic agent such as urea or thiocyanate ion) wash-out, and collection FGF-19 polypeptide.

Embodiment 9

Drug screening

By use FGF-19 polypeptide or its binding fragment in multiple drug screening technology, the present invention is particularly useful for SCREENED COMPOUND.It can be free form in the solution that this class detects used FGF-19 polypeptide or fragment, also can be to be fixed on the solid carrier, to result from cell surface or to be positioned at cell.One of drug screening method is to utilize the eucaryon or the prokaryotic host cell of being expressed FGF-19 polypeptide or segmental recombinant nucleic acid stable conversion.The medicine of anti-this type of transformant of screening in CBA.No matter viable cell or machine made this type of cell are activated or fixed, all can be used for standard in conjunction with test.Can measure the formation of the mixture between FGF-19 polypeptide for example or its fragment and the tested reagent.Perhaps, can check the minimizing that mixture forms between the FGF-19 polypeptide that caused by tested reagent and its target cell or the target acceptor.

Therefore, the invention provides screening of medicaments or any other can influence the compositions and methods of FGF-19 polypeptide-relative disease or illness.These methods comprise this class reagent are contacted with FGF-19 polypeptide or its fragment, and use the existence of mixture between this reagent of well known methods analyst (I) and FGF-19 polypeptide or the fragment, or the (ii) existence of the mixture between FGF-19 polypeptide or fragment and the cell.In this type of CBA, FGF-19 polypeptide or fragment are labeled usually.After the suitable incubation, free FGF-19 polypeptide or fragment are separated with combining form FGF-19 polypeptide or fragment, free or do not form the amount of the marker of mixture, can weigh the binding ability of particular agent and FGF-19 polypeptide or to the obstruction ability of FGF19 polypeptide/cell complexes.

Another technology that is used for drug screening provides high-level efficiency to screen the compound that has at the suitable binding affinity of polypeptide, describes in detail and sees among the disclosed WO 84/03564 on September 13rd, 1984.Briefly say, go up synthetic a large amount of different little peptide in solid substrate (as continuously connected fastener or other surface) and inspect compound.As be used for the FGF-19 polypeptide, peptide is inspected compound and reaction of FGF-19 polypeptide and washing.Detect bonded FGF-19 polypeptide with well known method.Also can be on the used plate of aforementioned drug screening technology with the FGF-19 polypeptide direct coated of purifying.In addition, non--neutralizing antibody can be used for catching described peptide and described peptide is fixed on the solid carrier.

The present invention has also considered the test of use competitive drug screening assay, in described test, can combine with FGF-19 polypeptide or its fragment with test compound competition ground with FGF-19 polypeptid specificity bonded neutralizing antibody.By this way, antibody can be used for detecting the existence that has any peptide of one or more identical epi-positions with the FGF-19 polypeptide.

Embodiment 10

Reasonably medicinal design

The target of rational drug design be productive target biologically active polypeptides (being the FGF-19 polypeptide) analog or with the interactional small molecules of polypeptide for example agonist, antagonist or inhibitor.Wherein any example can be used for forming medicine, and described medicine is to have more the activity or the FGF-19 polypeptide of stable form more, and perhaps medicine strengthens or disturbs function (referring to Hodgson, Bio/Technologv, 9:19-21 (1991)) in the FGF-19 polypeptide body.

In one approach, with x-ray crystallography, computer model design or the most common these two combination, measure the three-dimensional structure of FGF-19 polypeptide or FGF-19 polypeptide-inhibitor complexes.Be the avtive spot of description scheme and mensuration molecule, must determine the shape and the electric charge of FGF-19 polypeptide.The method that is of little use is by the model based on the homologous protein structure, to obtain the useful information about the FGF-19 polypeptide structure.Two kinds of situations are all used relevant structural information to come like the design class FGF-19 polypeptide sample molecule or are used to identify effective inhibitor.The useful example of rational drug design comprises Braxton and Wells, Biochemistrv, having that 31:7796-7801 (1992) is confirmed improves active or stable molecule, perhaps Athauda etc., J.Biochem., the molecule of the described inhibitor of 113:742-746 (1993), agonist or antagonist as native peptides.

Also having a kind of may be to separate the target specific antibody, screens by aforesaid functional analysis, solves its crystalline structure then.By and large, this approach produces medicine parent nucleus (pharmacore), can be based on this parent nucleus design medicine.By producing, can get around the albumin crystal conformation fully at anti--idiotype antibody (anti--idiotype) functional, pharmacologically active antibody.As the mirror image of mirror image, estimate that the binding site of anti--idiotype antibody is the analogue of original acceptor.Therefore, anti--idiotype antibody can be used for identifying and isolated peptides from the peptide storehouse of chemistry or biology generation.Subsequently, isolating peptide useful as drug parent nucleus.

The present invention can provide the FGF-19 polypeptide of q.s to finish such as this type of the analysis and research of X-line crystallography.In addition, the information of FGF-19 polypeptid acid sequence provided herein will provide guidance to those personnel that use a computer the modelling technique replacement x-ray crystallography or the modelling technique that also uses a computer except that using x-ray crystallography.

Embodiment 11

The body weight of FGF-19 transgenic mouse, Leptin level, food ration, voided volume, oxygen-consumption and The research of triglyceride level and free fatty acid levels

As mentioned above, FGF-19 be confirmed as recently with fibroblast growth factor-related secretory protein growth factor family in a member.The feature of FGF-19 is with 4 interactions of FGF acceptor but does not show as mitogen herein.For further studying this proteic function, produced the transgenic mouse of expressing human FGF-19.

More specifically, the cDNA with coding people FGF-19 is cloned in the plasmid that contains the myosin light chain promotor.This promotor is enough to make this transgenosis generation muscle specific to be transcribed.5 of FGF-19cDNA ' end also comprises acceptor splicing site and donor to increase expression level, and 3 of FGF-19cDNA ' end comprises donor splicing site and acceptor splicing site and polyadenylic acid additional signal, to increase transcriptional level and the Transcription Termination site is provided.

Use suitable restriction enzyme, from the bacteria carrier sequence, discharge and comprise MLC promotor, 5 ' acceptor splicing site and donor, FGF-19cDNA, the DNA in 3 ' acceptor splicing site and donor and Transcription Termination site (transgenosis) passes through fractional separation and purifying subsequently on sepharose.The DNA of purifying is injected in the protokaryon of fertilization mouse ovum, produces transgenic mouse, and identifies (Genetic Modification of Animals by the described method of document; Tim Stewart; In Exploring GeneticMechanisms 565-598 page or leaf; M Singer in 1997 and P Berg write; University ScienceBooks; Sausalito, Calif).Mouse grows to following measurement water intake, food consumption, voided volume and hematocrit after 6 ages in week.Same mouse is measured leptin, triglyceride level and free fatty acids after growing to for 8 ages in week.

By following result finding is discussed, the consumption rate of these mouse shows that its ingestion of food and metabolic rate increase.The increase although ingest, the body weight of these mouse than other not the body weight of the brood mouse of transgenosis significantly reduce.Body weight reduces the result that obesity seemingly alleviates, because the amount of leptin and people and rodentine fatty tissue is closely related, and leptin reduces in transgenic mouse.For further supporting this point, the linear measure evaluation from the nose to the buttocks shows that transgenic mouse is the normality linear growth.As for body temperature, height (bone is long) and hematology detected value, these animals are all normal.Ingesting consistent with increase is that the voided volume of transgenic mouse increases.Because these mouse do not show and drink Geng Duoshui, and through normal plasma cell specific volume mensuration confirmation also performance dehydration, the voided volume of Zeng Jiaing may be the metabolism of ingesting that is derived from increasing so.Because FGF-19 reduces fat and does not change muscle quality or long bone formation, is the effective therapeutical agent in obesity and the associated conditions treatment so show FGF-19.

More specifically, under the different fasting and the condition of ingesting, in the body weight of various time point weighing MLC-FGF-19 transgenic mouses.Particularly, 6 weeks were organized female FGF-19 transgenic mouse and the brood mouse of Qi Fei-transgenosis during ages to each, weighed 24 hours the time ad libitum access, fasting 6 hours and 24 hours and after finishing fasting in 24 hours.As shown in Figure 3A, under all conditions, FGF-19 transgenic mouse (solid bar) body weight is lower than its wild-type, the brood mouse of transgenosis (stippling rod) not.

Fig. 3 B shows the analysis of leptin in the serum of mouse on the same group shown in Fig. 3 A.The leptin that reduces in the FGF-19 transgenic mice is consistent with lose weight (Fig. 3 A) that it causes owing to obesity alleviates.

Monitor one group 6 age in week transgenic mouse ingest (Fig. 4 A), take the photograph water (Fig. 4 B), urinate (Fig. 4 C) and hematocrit (Fig. 4 D) situation.As seen in Fig., FGF-19 transgenic mouse (solid bar) consumes more foods than the brood mouse of wild-type, but water uptake does not increase.Although water consumption does not change, transgenic mouse produces the urine amount increases (Fig. 4 C).Increase although produce the urine amount, can prove that by the normal plasma cell specific volume dehydration (Fig. 4 D) does not appear in transgenic mouse.

Can explain that weight loss (Fig. 3) increases (Fig. 4) with food consumption with the metabolic rate increase.Measure metabolic rate by measuring oxygen-consumption.As shown in Figure 5,24 hours bright, half-light is in the cycle fasting 24 hours and after finishing fasting in 24 hours, and the metabolic rate of FGF-19 transgenic mouse is all seen increase.

Rising fat and triglyceride level and free fatty acids is the Hazard Factor of cardiovascular disorder.Because FGF-19 reduces the factor (fat (Fig. 3)) in the risk factors of cardiovascular diseases, therefore, whether also reduces other Hazard Factor for FGF-19 and also studies.As Fig. 6 finding, triglyceride level and free fatty acids (FFA) level also descends in the FGF-19 transgenic mouse.

Embodiment 12

The FGF-19 infusion causes ingestion of food increase and oxygen-consumption to increase

In order to confirm that in the seen effect of FGF-19 transgenic mouse be to be caused by FGF-19 albumen, by the driving implantable pump administering mode of osmotic pressure, to each group non--transgenosis FvB mouse input reorganization FGF-19 (1 mg/kg/ day, vein).Shown in Fig. 7 A-B, compare with the mouse of simple input carrier fluid, administered recombinant people FGF-19 causes the increase of ingesting.And oxygen-consumption is measured and is shown that the FGF-19 input also causes metabolic rate to increase.

Embodiment 13

FGF-19 reduces glucose uptake and increase Leptin discharges from adipocyte

For further research FGF-19 changes metabolic mechanism, recombinant human FGF-19 is joined former being commissioned to train of rat fat cell support, and detect the situation that glucose uptake and leptin discharge from these cells.Shown in Fig. 8 A, B, FGF-19 increases the release of leptin in former generation rat fat cell, and reduces glucose and take in.

Embodiment 14

The glucose tolerance of high fat diet FGF-19 transgenic mouse and the research of fat pad weight

Usually, it is fat that the mouse of high fat diet (and people) body weight will increase and become, and glucose tolerance descends or suffers from diabetes.Accept FGF-19 whether to fat and glucose tolerance is influential in order to check, basically the method for describing in Metabolism nineteen ninety-five the 44th volume the 5th phase 645-651 page or leaf at Metabolism 1993 the 42nd volumes o. 11th 405-1409 page or leaf and Surwit etc. according to Rebuffe-Scrive etc., be sodium content to be changed into meet normal diet standard (diet is from ResearchDiets Inc.Catalog no.D12330N), make a group transgenic mice and its not genetically modified brood mouse (mouse age and sex are suitable) feed high fat diet.

After normal mice grain or high fat diet fed for 10 weeks, mouse (female transgenic mouse and its be the brood mouse of transgenosis not) was accepted the glucose tolerance test.Every mouse intraperitoneal injection 1.0mg glucose/kg body weight like this is after the injection, according to glucose concn in the certain hour measuring space blood.Glucose level in the curve display mouse body among Figure 10, the female not transgenic mouse that proves 8/9 feed high fat diet is confirmed as diabetes, and (glucose level was greater than 200mg/dl in 2 hours; (World Book of Diabetes in Practice.Vo] 3; Ed Krall, L.P.; And the transgenic mouse of 0/5 the identical diet of feed is suffered from diabetes Elsevier)).

High fat diet feeding male mouse 6 or 10 weeks back execution animal are measured obese degree by the weight of measuring specific fat depot.As shown in Figure 9, the transgenic mouse of feed high fat diet more not the brood mouse of transgenosis have significantly few fat.

The material preservation

Following material American type culture collection (the American Type CultureCollection, 10801 University Blvd., Manassas, VA 20110-2209, USA) (ATCC) preservation:

Material The ATCC preserving number Preservation day

DNA49435-1219 209480 1997.11.21

This preservation is to carry out according to the budapest treaty of the microbial preservation that is used for patented procedure of international recognition and article (budapest treaty) thereof.This guarantees that the preservation culture keeps survival in the time of 30 years preservation days.This makes and can obtain at the preservation thing of budapest treaty in the time limit by ATCC, and obedience Genentech, Inc. and the agreement between the ATCC, the latter guarantees that the public is based on the related U.S. patent of promulgation or based on open to any U.S. or foreign patent application person, and can permanent and unrestrictedly utilize the thing that goes down to posterity of preservation culture, whichsoever first, council's rule according to 35 USC 122 of the United States Patent (USP) trade mark council and institute's foundation (comprises 37 CFR 1.14, especially referring to 886 OG 638) the regulation granted entitlements, guarantee that individuality obtains subculture.

The application's consignor agrees, if preservation culture material is dead or lose or destroyed in the culturing process under optimum conditions, and should be in the back that have notice rapidly with another same material replacement preserved material.According to the regulation of patent law, the availability of preserved material, shall not be construed as is the permission that carries out an invention to running counter to any Governmental Authority institute's entitle.

The specification sheets of writing previously has been enough to make those skilled in the art to implement the present invention.The present invention is not subjected to the restriction of preservation construct scope, because the preservation scheme is intended to the explanation as a particular aspects of the present invention, any construct with identical functions is also included within the scope of the invention.The preserved material of this paper neither constitutes permits deficiency that this printed instructions comprised so that the adding of the content that any aspect of the present invention (comprising its optimal mode) is implemented, and the protection domain that also is not interpreted as claim is defined as specific illustrations.In fact,, the present invention described above is carried out various changes change, be conspicuous to those skilled in the art, and fall into protection scope of the present invention except shown in this paper and described.

Claims (77)

1. isolated nucleic acid molecule, it comprises the dna molecular with the FGF-19 polypeptide of (a) encoding, described polypeptide comprises about 1 or about 23 sequences to about 216 amino acids residues among Fig. 2 (SEQ ID NO:2), or (b) complementary strand of (a) described dna molecular, have DNA at least about 80% sequence identity.

2. the described isolated nucleic acid molecule of claim 1 comprises among Fig. 1 (SEQ ID NO:1) about 464 or about 530 to about 1111 nucleotide sequence.

3. the described isolated nucleic acid molecule of claim 1 comprises nucleotide sequence shown in Fig. 1 (SEQ ID NO:1).

4. the described isolated nucleic acid molecule of claim 1 comprises in the code pattern 2 (SEQ ID NO:2) about 1 or about 23 nucleotide sequences to the sequence of about 216 amino acids residues.

5. isolated nucleic acid molecule, it comprise with (a) coding by being the dna molecular of the coded identical mature polypeptide of people's albumen cDNA of 209480 (DNA49435-1219) on November 21st, 1997 at the ATCC of ATCC preservation preserving number, perhaps (b) (a) complementary strand of described dna molecular has the DNA at least about 80% sequence identity.

6. the described isolated nucleic acid molecule of claim 5, it comprises that coding is by being the DNA of the coded identical mature polypeptide of people's albumen cDNA of 209480 (DNA49435-1219) on November 21st, 1997 at the ATCC of ATCC preservation preserving number.

7. isolated nucleic acid molecule, it comprises and the dna molecular of full-length polypeptide encoding sequence that (a) on November 21st, 1997 at the ATCC of ATCC preservation preserving number is people's albumen cDNA of 209480 (DNA49435-1219), perhaps (b) (a) complementary strand of described encoding sequence has the DNA at least about 80% sequence identity.

8. the described isolated nucleic acid molecule of claim 7, it comprises that on November 21st, 1997 was the full-length polypeptide encoding sequence of people's albumen cDNA of 209480 (DNA49435-1219) at the ATCC of ATCC preservation preserving number.

9. the isolated nucleic acid molecule of coding FGF-19 polypeptide, it comprise can with 1 or about 23 DNA in the code pattern 2 (SEQ IDNO:2) to the complementary strand hybridization of the nucleotide sequence of about 216 amino acids.

10. the described isolated nucleic acid molecule of claim 9, wherein 1 or about 23 nucleic acid to about 216 amino acids comprise shown in Fig. 1 (SEQ ID NO:1) 464 or about 530 to about 1111 Nucleotide in the code pattern 2 (SEQ ID NO:2).

11. the described isolated nucleic acid molecule of claim 9, wherein hybridization occurs under strict hybridization and the wash conditions.

12. isolated nucleic acid molecule, it contains among (a) coding and Fig. 2 (SEQ ID NO:2) from 1 or about 23 when the sequence of about 216 amino-acid residues is compared, the DNA of positive at least 80% the polypeptide of marking, or (b) complementary strand of (a) described DNA.

13. contain isolated nucleic acid molecule at least about 22 Nucleotide, its following generation: under stringent hybridization condition, test dna molecule and (a) coding are contained among Fig. 2 (SEQ ID NO:2) from 1 or about 23 dna moleculars to the FGF-19 polypeptide of about 216 amino acid residue sequences, or (b) complementary strand of (a) described dna molecular is hybridized, and separates this test dna molecule.

14. the described isolated nucleic acid molecule of claim 13, itself and the sequence identity that (a) or (b) has at least about 80%.

15. contain the carrier of arbitrary nucleic acid molecule in the claim 1 to 14.

16. claim 15 described carrier, wherein said nucleic acid molecule link to each other with can being operated by the control sequence that the host cell that transformed with this carrier is discerned.

17. nucleic acid molecule with ATCC registration number 209480 (DNA49435-1219) preservation.

18. contain the host cell of the described carrier of claim 15.

19. the described host cell of claim 18, wherein said cell is a Chinese hamster ovary celI.

20. the described host cell of claim 18, wherein said cell is intestinal bacteria.

21. the described host cell of claim 18, wherein said cell is a yeast cell.

22. prepare the method for FGF-19 polypeptide, be included under the suitable condition of expressing described FGF-19 polypeptide and cultivate the described host cell of claim 18, and from cell culture, reclaim described FGF-19 polypeptide.

23. isolating FGF-19 polypeptide, comprise with Fig. 2 (SEQ ID NO:2) in from 1 or about 23 to the sequence of about 216 amino acids residues aminoacid sequence at least about 80% sequence identity.

24. the described isolating FGF-19 polypeptide of claim 23, it contains among Fig. 2 (SEQ ID NO:2) from 1 or about 23 to about 216 amino-acid residue.

25. isolating FGF-19 polypeptide, itself and on November 21st, 1997 are that cDNA in the carrier of 209480 (DNA49435-1219) preservation inserts sub-encoded polypeptide and has sequence identity at least about 80% with the ATCC preserving number at ATCC.

26. the described isolating FGF-19 polypeptide of claim 25, its be by on November 21st, 1997 be that cDNA in the carrier of 209480 (DNA49435-1219) preservation inserts the son coding with the ATCC preserving number at ATCC.

27. isolating FGF-19 polypeptide, when with Fig. 2 (SEQ ID NO:2) in 1 or about 23 to about 216 aminoacid sequence relatively the time, it has at least 80% positive scoring.

28. isolating FGF-19 polypeptide, it contains 1 or about 23 sequence or its fragments to about 216 amino acids residues among the Fig. 2 (SEQ ID NO:2) that is enough to provide anti--FGF-19 antibody combining site.

29. isolated polypeptide, it is by being prepared as follows (i) under stringent condition, make test dna molecule and (a) coding contain among Fig. 2 (SEQ ID NO:2) 1 or about 23 dna moleculars to the FGF-19 polypeptide of the sequence of about 216 amino acids residues, or (b) complementary strand of (a) described dna molecular is hybridized, (ii) under the condition of the described polypeptide of suitable expression, cultivate and contain the host cell of described test dna molecule and (iii) from cell culture, reclaim described polypeptide.

30. the described isolated polypeptide of claim 29, wherein said test dna and the sequence identity that (a) or (b) has at least about 80%.

31. contain the chimeric molecule that FGF-19 polypeptide and allogeneic amino acid sequence merge.

32. the described chimeric molecule of claim 31, wherein said allogeneic amino acid sequence is the epi-position flag sequence.

33. the described chimeric molecule of claim 31, the Fc district that wherein said allogeneic amino acid sequence is an immunoglobulin (Ig).

34. with FGF-19 polypeptid specificity bonded antibody.

35. the described antibody of claim 34, wherein said antibody is monoclonal antibody.

36. the described antibody of claim 34, wherein said antibody is humanized antibody.

37. the described antibody of claim 34, wherein said antibody is antibody fragment.

38.FGF-19 the agonist of polypeptide.

39.FGF-19 the antagonist of polypeptide.

40. composition of matter contains (a) FGF-19 polypeptide that exists with form of mixtures with pharmaceutically acceptable carrier, (b) agonist of FGF-19 polypeptide, (c) antagonist of FGF-19 polypeptide, or (d) anti--FGF-19 antibody.

41. can with the screening method of FGF19 bonded biologically active agent, comprising:

A) in the FGF-19 sample, add candidate bioactive agent; With

B) detect combining of described candidate agent and described FGF-19, wherein take place combination then show be can with FGF-19 bonded biologically active agent.

42. can regulate the screening method of the active biologically active agent of FGF-19, described method comprises the steps:

A) in the FGF-19 sample, add candidate bioactive agent; With

(b) detect the bioactive change of FGF-19, wherein changing then shows it is to regulate the active biologically active agent of FGF-19.

43. the described method of claim 42, wherein said biological activity are to reduce the picked-up of adipocyte to glucose.

44. the described method of claim 42, wherein said biological activity are to increase leptin to discharge from adipocyte.

45. identify the method for FGF-19 acceptor, described method comprises: FGF-19 is combined with the component that contains the cytolemma material,, determine that then described acceptor is the FGF-19 acceptor if the acceptor on described FGF-19 and the described cytolemma material forms mixture.

46. the described method of claim 45, wherein FGF-19 and described receptors bind, and described method further comprises described FGF-19 and the crosslinked step of acceptor.

47. the described method of claim 45, wherein said component is a cell.

48. the described method of claim 45, wherein said component are the cytolemma extract products.

49. the method for inducing leptin to discharge from adipocyte, described method comprise the FGF-19 that uses the significant quantity that can induce leptin release to described cell.

50. the described method of claim 49, wherein said FGF-19 uses with the albumen form.

51. the described method of claim 49, wherein said FGF-19 uses with the nucleic acid form.

52. the induced lipolysis cell reduces the method for glucose uptake, described method comprises the FGF-19 that uses the significant quantity that can induce the glucose uptake minimizing to described cell.

53. the described method of claim 52, wherein said FGF-19 uses with the albumen form.

54. the described method of claim 52, wherein said FGF-19 uses with the nucleic acid form.

55. control individual fat method, described method comprises the composition that contains FGF-19 to the described fat significant quantity of described individual administering therapeutic.

56. the described method of claim 55, wherein said Bariatric further cause to the treatment of obesity related disorders.

57. the described method of claim 55, wherein said FGF-19 uses with the albumen form.

58. the described method of claim 55, wherein said FGF-19 uses with the nucleic acid form.

59. the described method of claim 55, wherein said composition further comprises pharmaceutically acceptable carrier.

60. the described method of claim 55, aminoacid sequence has the amino acid sequence identity at least about 85% shown in wherein said FGF-19 and Fig. 2 (SEQ ID NO:2).

61. alleviate the method for individual TBW, described method comprises to described individuality uses significant quantity FGF-19.

62. the described method of claim 61, wherein said FGF-19 uses with the albumen form.

63. the described method of claim 61, wherein said FGF-19 uses with the nucleic acid form.

64. the described method of claim 61, wherein said FGF-19 uses with pharmaceutically acceptable carrier.

65. alleviating, the described method of claim 61, wherein said TBW comprise that described body fat reduces.

66. the described method of claim 61, aminoacid sequence has the amino acid sequence identity at least about 85% shown in wherein said FGF-19 and Fig. 2 (SEQ ID NO:2).

67. the method for at least a triglyceride level and free fatty acid levels in the reduction individuality, described method comprises the FGF-19 that uses significant quantity to described individuality.

68. the described method of claim 67, wherein said FGF-19 uses with the albumen form.

69. the described method of claim 67, wherein said FGF-19 uses with the nucleic acid form.

70. the described method of claim 67, wherein said FGF-19 uses with pharmaceutically acceptable carrier.

71. the described method of claim 67, aminoacid sequence has the amino acid sequence identity at least about 85% shown in wherein said FGF-19 and Fig. 2 (SEQ ID NO:2).

72. increase the method for individual metabolic rate, described method comprises the FGF-19 that uses significant quantity to described individuality.

73. the described method of claim 72, wherein said FGF-19 uses with the albumen form.

74. the described method of claim 72, wherein said FGF-19 uses with the nucleic acid form.

75. the described method of claim 72, wherein said FGF-19 uses with pharmaceutically acceptable carrier.

76. the described method of claim 72, aminoacid sequence has the amino acid sequence identity at least about 85% shown in wherein said FGF-19 and Fig. 2 (SEQ ID NO:2).

77. contain the genetically modified genomic rodent that comprises the FGF-19 that encodes.

Images