WO2000026361A1

WO2000026361A1 - New protein associated with leptin receptor for obesity and the encoding sequence thereof and the methods for producing same and the uses of the same

Info

Publication number: WO2000026361A1
Application number: PCT/CN1999/000167
Authority: WO
Inventors: Yumin Mao; Yi Xie; Yan Huang
Original assignee: Shanghai Biorigin Gene Development Co., Ltd.
Priority date: 1998-10-30
Filing date: 1999-10-25
Publication date: 2000-05-11
Also published as: CN1253178A

Abstract

The present invention relates to new protein OB-RGRP2 associated with leptin receptor for obesity and the encoding sequence thereof. The protein is associated with leptin receptor for obesity, and is highly homology with OB-RGRP2. The present invention also relates to the methods for producing the nucleic acid and the protein, and relates to the uses of the same.

Description

The present invention belongs to the field of biotechnology and genetic engineering. More specifically, the present invention relates to a new obostatin receptor-related protein OB-RGRP2. And its nucleic acid coding sequence, method for producing the nucleic acid sequence and protein polypeptide, and uses of the polypeptide and nucleic acid sequence.

Obesity is the most common nutritional disease in developed western countries. More than 30% of adults weigh more than 20% of their standard weight. As a result, obesity has become an important public health issue. In addition, obesity is often closely related to diabetes, hypertensive disease, hyperlipidemia, cardiovascular and cerebrovascular disease and even cancer. Therefore, people have invested a lot of manpower and material resources in the research of obesity. However, the molecular mechanism of obesity has been unclear.

It has been thought for many years that obesity is genetically and genetically related. In recent years, an ob gene, a gene closely related to obesity, has been cloned from a mouse with obesity. This gene encodes a hormone factor that regulates the energy balance of the human body, known as obesin (leptin, also translated as wasting). ), And later, homologous OB genes were also cloned in humans [Zhang Y, et al. Nature 1994; 372: 425]. The human OB gene was mapped to 7q31.3. The OB gene encodes a prostatin protein consisting of 167 amino acid residues. After removing the N-terminal 21 amino acid long signal peptide, the mature obstatin contains 146 amino acids. Studies have shown that obostatin is mainly secreted by white adipose tissue cells.

Studies have also shown that obesitin has multiple biological effects [Halaas JL, et al. Science 1995; 269: 543]:

(1) Regulate the balance of energy metabolism. This is one of the most important functions of obostatin, which mainly regulates food intake and stimulates energy consumption (including generation of calories). There are two sites and pathways:

A. Action on the central nervous endocrine system: The main target site is the hypothalamus. Obstatin enters the central nervous system through the blood-brain barrier through osmotic transport. The molecule acting downstream of obestatin may be hypothalamic neuropeptide Y (NPY). NPY can stimulate food intake, while obstatin can inhibit NPY synthesis. In addition, other factors or hormones such as adrenocorticotropic hormone (ACTH), glycogen-like peptides, etc. may also be related to the activity of obesin.

B. Effect on peripheral tissues: Obostatin plays a role in regulating energy balance by regulating lipid metabolism. Obestatin can directly inhibit the synthesis of fatty acids and triglycerides in cells, and at the same time increase the oxidation of lipids, resulting in a decrease in the content of fatty acids and triglycerides in cells. Further research shows that the increase in oxidation in mitochondria is not accompanied by an increase in ATP synthesis, that is, most of the energy added by oxidation is converted into heat.

(2) Obestatin also has important regulatory effects on other systems. For example A. For the reproductive system, obostatin plays an important role in embryo development and maturation. B. For the hematopoietic system, obstatin can directly affect the early hematopoietic process. And stimulate the development and differentiation of red and granulocyte progenitor cells. This suggests that obostatin has therapeutic value for hematological diseases, for example, it can be used to isolate OB-R positive early hematopoietic progenitor cells, which is of great significance for hematopoietic stem cell transplantation. It can also be used to treat anemia and enhance macrophage function and immunity.

Obstatin works through the mediation of obostatin receptors (referred to as "OB-R") on the cell surface. OB-R is a transmembrane protein that has a high degree of homology to the gpl30 subunit of the interleukin-6 receptor and belongs to the cytokine class I family [Tartaglia LA, et al. Cell 1995; 83: 1263].

Two main types of receptors have been found: OB-RL and OB-RS. OB-RL consists of more than 3,000 amino acid residues, and its intracellular part is about 303 amino acids long. It contains domains related to intracellular signaling, such as the motif associated with Jak and the motif that activates STAT. OB-RS lacks the STAT motif, but its extracellular part is exactly the same as OB-RL. There are currently at least four OB-RSs of varying length.

Another shorter obstatin receptor also lacks the transmembrane part, which is a soluble component present in the blood and may be a binding protein for obstatin. Its source may be formed by the extracellular part of OB-RS entering the blood after protease action.

OB-RL is mainly expressed in the hypothalamus and is expressed in small amounts in other tissues. OB-RS is expressed to varying degrees in various tissues, with the highest expression in lungs and kidneys. The differences in the structure and expression profiles of the two OB-Rs determine their functional differences. It is currently believed that the effect of obesin is mainly mediated by OB-RL.

One cause of obesity is obstruction of the obstatin gene, which results in obstructed synthesis or loss of vitality, but the incidence of obesity caused by this obstatin gene defect is extremely low. Most obesity may be caused by leptin resistance. It is now believed that there are two main reasons for obestatin resistance: A. Obstatin is blocked from the blood through the blood-brain barrier into the hypothalamus, so that the obstatin secreted by adipose tissue cannot effectively pass the blood-brain barrier, however, the central The nervous system is still sensitive to astatin. It can be speculated that OB-R activators that can freely cross the blood-brain barrier may be of great value in the treatment of this obesity. B. Obesity The receptors or factors behind the receptors have decreased or lost their functions, which leads to resistance. The decline or loss of function in any link of the obstatin receptor and downstream signaling pathways may be related to the onset of obesity. It can be seen that OB-R is one of the important links of obesity, and also an important target site for the treatment and prevention of obesity [Tartaglia LA. J Bio Chem 1997; 272: 6093].

Recently, some scholars cloned a gene related to the OB-R gene. The gene contains a 396bp open reading frame (ORF), which encodes a protein of 131 amino acids, and is named OB-RGRP (OB-R Gene related protein). The sequence of the first two exons of OB-RGRP (164bp) is 100% homologous to the sequence of 5'-untranslated region of OB-R, but the sequence after 165bp is completely different from OB-R [Bailleul B, et al. Nucleic Acids Res 1997; 25: 2752]. Therefore, the author proposes that OB-RGRP and OB-R belong to the same gene locus, are controlled by the same promoter, and are formed by different exon splicing processes. Transcription products.

The homology comparison analysis of OB-RGRP revealed high homology with the C30B5.2 protein of the nematode. There are two domains that are highly homologous: (1) Domain 1 from +9 to +27, which is composed of highly hydrophobic and non-polar amino acids; (2) from +65 to +88 The domain 2. These domains may be transmembrane structures. In many cytokine receptor (including OB-R) families, there is a Pro-X-Pro sequence near the transmembrane structure (where X represents any amino acid residue) behind a group of hydrophobic residues This sequence is called boxl. If two Pros are replaced by Ser, the receptor-mediated JAK2 tyrosine phosphorylation disappears. A similar boxl (Pro ₄₆ -Ile-Pro ₄₈ ) exists in OB-RGRP. It is known that OB-RL can guide the JAK / STAT signaling pathway, but OB-RS cannot guide this pathway. And both OB-Rs contain the boxl sequence, so this shows that boxl is not enough to activate the JAK / STAT system. However, both OB-Rs can induce the expression of downstream signal molecule genes under the stimulation of obestatin, which suggests that OB-RGRP may be an important synergistic protein in the process of obestatin signaling, especially for the OB-RS mediator. Signal transduction.

Since OB-R belongs to the class I cytokine receptor family, other class I cytokines, such as interferon and interleukin 6 receptors, which have similar structures and functions as OB-R and similar signaling pathways, may also have similar synergistic proteins. .

However, prior to the present application, no novel astatin receptor related protein and coding sequence according to the present invention have been published or disclosed. The first object of the present invention is to provide a new nucleic acid coding sequence, which encodes a new member of the obesin receptor-related protein family. The new human gene of the present invention is named OB-RGRP2

A second object of the present invention is to provide a novel obstatin receptor-related protein, which is named OB-RGRP2 protein.

A third object of the present invention is to provide a method for producing said new human OB-RGRP2 nucleic acid sequence and polypeptide using recombinant technology.

A fourth object of the present invention is to provide the use of human OB-RGRP2 nucleic acid sequences and polypeptides.

A fifth object of the present invention is to provide an antibody against OB-RGRP2. In one aspect of the present invention, an isolated nucleic acid molecule is provided, which comprises: a nucleotide sequence encoding a polypeptide having human OB-RGRP2 protein activity, the nucleotide sequence is in accordance with 85-480 of SEQ ID No. 1. Position nucleotide sequence has at least 70% homology; or the nucleotide sequence can hybridize with the nucleotide sequence at positions 85-480 in SEQ ID No. 1 under highly stringent conditions. Preferably, the nucleotide sequence encodes a polypeptide having the amino acid sequence shown in SEQ ID No. 2. More preferably, the nucleotide sequence has SEQ ID No. 1 A sequence of 85-480 bits.

In another aspect of the present invention, an isolated OB-RGRP2 polypeptide is provided, which includes: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof . Preferably, the polypeptide has the amino acid sequence of SEQ ID No. 2.

In another aspect of the present invention, a vector containing the aforementioned nucleic acid molecule is provided.

In another aspect of the invention, a host cell transformed with the vector is provided.

In another aspect of the present invention, a method for preparing a polypeptide having human OB-RGRP2 protein activity is provided. The method includes:

(a) A nucleotide sequence encoding a polypeptide having OB-RGRP2 protein activity is operably linked to an expression control sequence to form an OB-RGRP2 protein expression vector. The nucleotide sequence is the same as in SEQ ID N0.1. Sequences at positions 85-480 have at least 70% homology;

(b) transferring the expression vector in step (a) into a host cell to form a recombinant cell of the OB-RGRP2 protein;

(c) culturing the recombinant cells in step (b) under conditions suitable for expression of the OB-RGRP2 protein polypeptide;

(d) Isolating a polypeptide having OB-RGRP2 protein activity.

In another aspect of the present invention, an antibody capable of specifically binding the above-mentioned OB-RGRP2 polypeptide is provided. The novel obesin receptor-related protein provided by the present invention is named OB-RGRP2. On the one hand, OB-RGRP2, as a synergistic protein of obesitin (OB-R), can promote the signal transduction of obesin; on the other hand, OB-RGRP2 has the role of cooperating with other class I cytokine receptors in cell development. And differentiation, metabolism, and regulation of immune function have important functions. OB-RGRP2 dysfunction can cause obesity, diseases of the hematopoietic system, diabetes, immune disorders, malignant tumors, etc. Anti-OB-RGRP2 antibodies can be used to diagnose or treat OB-RGRP2-related diseases

One of the features of the present invention is an isolated or purified nucleic acid (polynucleotide) sequence, which encodes an OB-RGRP2 polypeptide having an amino acid sequence shown in SEQ ID NO: 2. Due to the degeneracy of the codons, the degenerate sequence with as little as about 70% homology to the 85-480 sequence in SEQ ID N0.1 can also encode the amino acid sequence described in SEQ ID N0.2. Preferably, the nucleic acid sequence is substantially the same as the sequence shown in SEQ ID NO: 1. As used herein, "substantially the same" refers to homology of at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99%.

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. Single-stranded DNA can be a coding or non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence (85-480bp) shown in SEQ ID NO: 1 or it may be a degenerate variant. In the present invention, "degenerate variant" refers to a coding device Nucleic acid sequence of a protein or peptide having the sequence of SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1. _

The invention also includes isolated or purified nucleic acid fragments that can hybridize to the sequence of SEQ ID NO: 1 (especially the open reading frame sequence) under stringent conditions. In the present invention, the "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20 nucleotides, more preferably at least 40 nucleotides, and most preferably at least 60 nucleotides. "Hybridization" refers to binding to a specific nucleic acid sequence. As known to those skilled in the art, "stringent conditions" generally means: (1) hybridization and elution at low ionic strength and high temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42'C, etc .; or (3) at least 95% identity between the two sequences , Better to cross at least 97%. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding OB-RGRP2.

The coding region sequence of the polynucleotide of the present invention may be an allelic variant of the coding region sequence shown in SEQ ID NO: 1. The prior art has shown that allelic variants are of one or more (usually 1-60, more preferably 1-20, most preferably 1-10) nucleotides in a polynucleotide. The replacement form formed by substitution, deletion, or insertion also includes adding several at the 5 and / or 3 ends (usually within 60, preferably within 30, more preferably within 10, most preferably Is within 5) nucleotides. These substitutions do not substantially alter the function of the encoded protein or polypeptide.

In one example of the present invention, a nucleic acid molecule having the sequence shown in SEQ ID No. 1 is provided, wherein the open reading frame is located at nucleotides 85-480.

The invention also provides an isolated or purified OB-RGRP2 polypeptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 2.

In the present invention, the term "OB-RGRP2 protein and / or polypeptide" refers to a polypeptide having the amino acid sequence shown in SEQ ID N0.2 having OB-RGRP2 protein activity. The term also includes variants of the SEQ ID NO. 2 sequence that have the same function as the human OB-RGRP2 protein. These variants include (but are not limited to): several (usually 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions, insertions and / or substitutions, and One or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids are added to the terminal and / or N-terminus. As is well known to those skilled in the art, substitution of amino acids with similar or similar properties generally does not change the function of the protein. As another example, adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the OB-RGRP2 protein.

Variants of the polypeptide include: homologous sequences, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize to OB-RGRP2 DNA under high or low stringency conditions, and A polypeptide or protein obtained using an antiserum against an OB-RGRP2 polypeptide. The present invention also provides other polypeptides, such as a fusion protein comprising an OB-RGRP2 polypeptide or a fragment thereof. In addition to almost full-length polypeptides, the invention also provides soluble fragments of the OB-RGRP2 polypeptide. Generally, the fragment has at least about 10 consecutive amino acids, preferably at least about 30 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids in the OB-RGRP2 polypeptide sequence.

The invention also provides analogs of OB-RGRP2 protein or polypeptide. The difference between these analogs and the natural OB-RGRP2 polypeptide can be a difference in the amino acid sequence, a difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as by site-directed mutagenesis or other known molecular biology techniques. Analogs may also include analogs having residues other than natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, hydrazone-amino acids).

Modified (usually unchanged primary structure) forms include: Chemically derived forms of polypeptides in vivo or in vitro such as acetylated or carboxylated. Modifications also include glycosylation, such as those produced by glycosylation modification in the synthesis and processing of polypeptides or in further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that undergoes glycosylation, such as mammalian glycosylase or deglycosylation enzyme.

In the present invention, "conservative variant polypeptide of OB-RGRP2" means that there are at most 10, preferably at most 8 and more preferably at most 5 amino acids compared with the amino acid sequence of SEQ ID No. 2 being similar in nature Or similar amino acids are substituted to form a polypeptide. These conservatively mutated polypeptides are preferably produced by substitution according to Table 1.

Table 1

In the present invention, "isolated or purified" means that the substance has been separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but if the same polynucleotide or polypeptide is separated from other substances that exist in the natural state, it is isolated. Or purified.

The present invention also includes a vector containing a nucleic acid molecule of the open reading frame sequence in SEQ ID NO: 1 or a variant or a fragment thereof, a host cell genetically engineered with the vector, and the present invention obtained by a recombinant DNA technology. Protein or peptide products.

The invention also provides a method for diagnosing a disease associated with OB-RGRP2 dysfunction, including (but Not limited to) detecting the expression of OB-RGRP2 or mutation of the OB-RGRP2 gene in a tissue or cell. OB-RGRP2 expression can be determined by measuring the mRNA or protein level of OB-RGRP2 in tissues or cells.

The present invention also provides a method for treating diseases associated with OB-RGRP2 dysfunction, which includes, but is not limited to, administering an effective amount of a complex to a cell to activate or inhibit the production or activity of OB-RGRP2. The types of the complex include: compounds; ligands, polypeptides or antibodies that bind to OB-RGRP2; antisense oligonucleotides; ribozymes; and mixtures thereof. Methods for cloning the OB-RGRP2 gene of the present invention are commonly used by those skilled in the art. A feasible approach is to use mRNA derived from mammalian tissues (such as embryonic brain or liver or lung, etc.) to synthesize cDNA by reverse transcription, and construct a corresponding cDNA library, and then obtain the OB-RGRP2 gene from the library. clone.

There are many mature techniques for extracting mRNA, and related kits are also commercially available. The method of constructing a cDNA library is also a conventional method (Sambrook, et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). In addition, cDNA libraries for a variety of tissues can be obtained directly from commercial sources such as Clontech.

Obtaining the OB-RGRP2 gene from a cDNA library includes (but is not limited to) the following two technical routes: one is to first screen the library containing OB-RGRP2 cDNA clones, and then perform DNA sequence analysis on these positive clones; the other is to first determine the library All clones inserted the 5 'and 3' sequences of the DNA fragments, and then found the clones containing the OB-RGRP2 cDNA, and performed the sequence analysis of the full-length cDNA.

Methods for screening libraries include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) development or loss of function of a marker gene; (3) determination of OB-RGRP2 transcript levels; (4) Detection of protein expression by applying immunological techniques or measuring biological activity. The above methods can be used alone or in combination.

In the method (1), the probe used for the hybridization is the same as any part of the OB-RGRP2 DNA fragment (the fragment of the sequence shown in SEQ ID No. 1), and the length of the probe is at least 10 consecutive nucleotides Preferably, it is at least about 20-30 consecutive nucleotides, more preferably at least about 50-60 consecutive nucleotides, and most preferably at least about 100 nucleotides. The length of the probe is usually within 5 kb, preferably within 2 kb, more preferably within 1 kb, and most preferably within 500 bp. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

In the method (4), the detection of the protein product of the OB-RGRP2 gene expression can be performed by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). The DNA sequence can be determined by the conventional dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467), and the primers used for sequencing can be known cDNA sequences (such as the sequence of SEQ ID No. 1) design.

In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.

If the complete 5 'end coding sequence is not available (which is a common problem in cDNA cloning), then RACE (rapid amplification of cDNA ends) is required.

Once the full-length cDNA sequence is obtained, the sequence can be analyzed by a computer, such as the position of the open reading frame, possible signal peptides, homology comparison of the transmembrane structure and its gene or protein structure, etc.

In addition, after the full-length cDNA sequence is obtained, relevant primers can be designed and synthesized by conventional methods, and the coding sequence of the protein of the present invention can be obtained by PCR amplification.

The OB-RGRP2 polynucleotide sequence of the present invention can be used to express or produce a recombinant OB-RGRP2 protein or polypeptide. Generally there are the following steps:

( _a ) A nucleotide sequence encoding a polypeptide having OB-RGRP2 protein activity is operably linked to an expression control sequence to form an OB-RGRP2 protein expression vector. The nucleotide sequence is the same as in SEQ ID N0.1. Sequences at positions 85-480 have at least 70% homology;

(d) Isolating a polypeptide having OB-RGRP2 protein activity.

In step (a), the nucleotide sequence used to encode a polypeptide having OB-RGRP2 protein activity may be a DNA fragment having a coding region of SEQ ID No. 1 or a variant thereof.

"Expression control sequences" include at least one promoter, at least one stop codon, and any other DNA sequence necessary or preferred for proper transcription and subsequent translation of the vector nucleic acid sequence, such as a leader sequence, an enhancer, and the like.

As used herein, "operably linked" refers to a condition where certain parts of a linear DNA sequence can affect the activity of other parts of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secret leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, it is operably linked to Coding sequence; if the ribosome binding site is placed at a position where it can be translated, it is operably linked to the coding sequence. In general, "operably linked" means adjacent, and for a secretion leader sequence, it means adjacent in a reading frame.

An important feature of expression vectors is the inclusion of appropriate promoters and translation control elements. Multiple expressions It is commercially available. Commonly used vectors include (but are not limited to): plasmids, phages, cosmids, yeast expression vectors, viruses, Ti plasmids, etc., but the most commonly used vectors are preferred plasmids. Commonly used host cells include (but are not limited to): bacteria, yeast, insect cells, animal cells, or plant cells, but the most commonly used host cells are bacteria, especially E. coli. Another more commonly used expression system is mammalian cell line, such as Hela cell line, COS cell line or CHO cell line.

Methods known to those skilled in the art can be used to construct recombinant expression vectors containing the OB-RGRP2 coding sequence. These methods include: in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (see: Sambrook, et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).

Recombinant OB-RGRP2 protein or polypeptide has many uses. These uses include (but are not limited to): direct use as a drug to treat diseases caused by OB-RGRP2 hypofunction or loss, and for screening antibodies, peptides or other ligands that promote or antagonize OB-RGRP2 function. For example, antibodies can be used to activate or inhibit the function of OB-RGRP2. In addition, screening the peptide library with the expressed recombinant OB-RGRP2 protein can be used to find polypeptide molecules with therapeutic value that can inhibit or stimulate the function of OB-RGRP2.

The present invention also provides an antibody or antibody fragment capable of specifically binding to OB-RGRP2. Antibody fragments are

Fab, Fab ', F ( _a b') ₂ or F _r fragments. The antibody may be a single chain, humanized or chimeric antibody.

A variety of methods are available for the production of antibodies against the OB-RGRP2 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.

Anti-OB-RGRP2 antibodies can be used in immunohistochemical techniques to detect OB- in biopsy samples

The presence or absence of RGRP2. Monoclonal antibodies that bind to OB-RGRP2 can also be labeled with radioisotopes and injected into the body to track the location and distribution of OB-RGRP2. This radio-labeled antibody can be used as a non-invasive diagnostic method for localization of related cells, such as determining whether tumor cells have metastasized.

The antibodies of the present invention can be used to treat or prevent diseases related to OB-RGRP2. Administration of an appropriate dose of antibody can stimulate or block the production or activity of OB-RGRP2.

Antibodies can also be designed to target specific toxins in the body. For example, OB-RGRP2 high affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group on the antibody with a mercapto crosslinker such as SPDP (N-succinimide-3- (2-pyridinedithio) -propionate), through the exchange of disulfide bonds To bind toxins to antibodies. This hybrid antibody can be used to kill OB-RGRP2 positive cells.

Polyclonal antibodies can be produced by immunizing animals such as rabbits, mice and rats with OB-RGRP2 protein or peptide. A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like. OB-RGRP2 monoclonal antibodies can be produced using hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497). Chimeric antibodies that combine human constant regions and non-human variable regions can be produced using existing technologies (Morrison et al, PNAS, 1985, 81: 6851). In addition, the existing technology for producing single chain antibodies (US Patent No. 4946778) can also be used to produce single chain antibodies against OB-RGRP2.

Polypeptide molecules capable of binding to OB-RGRP2 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase.

The OB-RGRP2 polynucleotide can also be used for the diagnosis and treatment of OB-RGRP2 related diseases. For diagnostic purposes, OB-RGRP2 polynucleotides can be used to detect whether OB-RGRP2 is expressed or whether OB-RGRP2 expression is abnormal (such as in a disease state). For example, the OB-RGRP2 DNA sequence can be used to hybridize biopsy samples to determine the abnormal expression of OB-RGRP2. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available. In addition, OB-RGRP2 specific primers can be used to detect OB-RGRP2 transcripts by RNA-polymerase chain reaction (PCR) in vitro amplification. The specific primer can be designed according to the sequence of SEQ ID No. 1, and its length is usually 15-60 nucleotides, preferably 20-50 nucleotides, and more preferably 25-40 nucleotides. acid.

In addition, detection of mutations in the OB-RGRP2 gene can also be used to diagnose OB-RGRP2-related diseases. OB-RGRP2 mutations include: point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type OB-GRP2 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations sometimes affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

The OB-RGRP2 polynucleotide can also be used in a variety of therapeutic applications. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by OB-RGRP2 non-expression or abnormal / inactive OB-RGRP2 expression. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant OB-RGRP2, which is used to inhibit endogenous OB-RGRP2 activity. For example, a variant OB-RGRP2 may be a truncated OB-RGRP2 lacking a signaling domain. Therefore, although this variant OB-RGRP2 can bind to downstream substrates, it lacks signaling activity. In this way, the recombinant gene therapy vector is used to treat diseases caused by abnormal expression or activity of OB-RGRP2. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the OB-RGRP2 gene into cells. Methods for constructing a recombinant viral vector carrying the OB-RGRP2 gene can be found in the existing literature (Sambrook, et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). In addition, the recombinant OB-RGRP2 gene can be packaged in liposomes and transferred into cells. Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit translation of OB-RGRP2 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RNA, DNA, and ribozymes can be obtained by any conventional techniques for synthesizing RNA or DNA, such as the widely used technique of solid-phase phosphate amide synthesis for oligonucleotide synthesis. Antisense RNA molecules can also be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA, where the DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, so that the oligonucleotide uses a phosphorothioate bond instead of a phosphodiester bond.

Methods for introducing polynucleotides into tissues or cells are various conventional techniques known in the art, including (but not limited to): directly injecting polynucleotides into tissues in vivo; or in vitro The polynucleotide (such as a virus, a phage, or a plasmid) is first introduced into the cell, and then the cell is transplanted into the body. In the drawings, FIG. 1 is a diagram showing a homology comparison of the amino acid sequences of human OB-RGRP2 and OB-RGRP according to the present invention. Among them, the same amino acid is marked with a single character of the amino acid between the two sequences, and the similar amino acid is marked with "+".

FIG. 2 is a homology comparison diagram of the amino acid sequence of human OB-RGRP2 and C30b5 of the nematode. Among them, the same amino acid is marked with a single character of the amino acid between the two sequences, and similar amino acids are marked with "+". The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions (such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)), or Follow the conditions recommended by the manufacturer. Example 1: Cloning of the OB-RGRP2 gene

Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiegene). 2 micrograms of poly (A) mRNA was formed into cDNA by reverse transcription. The cDN A fragment was inserted into the multicloning site of the vector using the UniZ APxR vector kit (purchased from Stratagene), and the DH5 bacteria were transformed to form a cDNA library. A total of 3028 clones were obtained. The dideoxy method was used to determine the sequences at the 5 'and 3' ends of all clones. The determined cDNA sequence was compared with the existing public DNA sequence database, and it was found that the DNA sequence of one clone (018el l) had a homology with human OB-RGRP. 70.2%. A series of primers were synthesized to determine the DNA sequence of the 018el 1 clone in both directions. Computer analysis showed that the full-length cDNA contained in the 018el l clone was a new DNA sequence (as shown in SEQ ID No. 1). There was a 396bp ORF from 85bp to 480bp, encoding a new 131 amino acid sequence. The protein has the amino acid sequence shown in SEQ ID No. 2. The protein was named OB-RGRP2 protein.

OB-RGRP 2 contains 131 amino acids, which is exactly the same length as OB-RGRP. Protein-level homology analysis showed that OB-RGRP2 and OB-RGRP were 67% identical and 80% similar (Figure 1). It has 48% identity and 78% similarity to C30b5 protein of nematodes (Figure 2). Structural analysis showed that OB-RGRP2 has two domains (positions 9-27 and 65-88) mainly composed of hydrophobic amino acids, which are presumed to be transmembrane structures, so OB-RGRP2 is a transmembrane protein. There is a LRRs motif in domain 1 that is rich in leucine and its function is mainly involved in protein-protein interactions. Downstream of domain 1, there is also a Pro-X-Pro motif (amino acids Pro-lie Pro at positions 46-48 in SEQ ID No. 2), which is related to the function of Jak2. The above results show that OB-RGRP2 and 0B-RGRP belong to the same gene family and have similar functions as OB-RGRP, that is, as a synergistic protein of OB-R, it promotes the signal transduction of obesin; in addition, it may also have synergy with other cytokines The role of the receptor, so its function is more extensive than OB-RGRP. It plays an important role in cell development and differentiation, metabolism, and regulation of immune function.

In addition to being used as a member of the family for further functional research, the human OB-RGRP2 of the present invention can also be used to produce fusion proteins with other proteins, such as fusion proteins with immunoglobulins. In addition, the inventor OB -RGRP2 can also be fused or exchanged with other members of the family to produce new proteins. For example, the N-terminus of the present inventor OB-RGRP2 is exchanged with the N-terminus of human OB-RGRP to produce a new protein with higher activity or new characteristics.

The antibody against the present inventor OB-RGRP2 is used for screening other members of the family, or for affinity purification of related proteins (such as other members of the family).

For example, the inventor's OB-RGRP2 nucleic acid (coding sequence or antisense sequence) can be introduced into cells to increase the expression level of human OB-RGRP2 or inhibit the overexpression of human OB-RGRP2. The human OB-RGRP2 protein or an active polypeptide fragment thereof of the present invention can be administered to a patient to treat or alleviate related disorders, especially some obesity-related disorders, caused by human OB-RGRP2 deletion, nonfunction or abnormality. In addition, the nucleic acid sequence or antibody based on the present invention can also be used for related diagnosis or prognosis. Example 2: Cloning of OB-RGRP2 by RT-PCR

CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.

After purification of Qiagen's kit, PCR amplification was performed with the following primers: forward primer F1 5-CCGCCGTAGCGCGTCTTG-3 at the beginning of SEQ ID No. 1; reverse primer R1: 5- GCATAGCAATTATTTTCCAG-3 is located at 1532-155 lbp of SEQ ID No. 1. Amplification reaction conditions: 50μ1 reaction volume containing 50mmol / L Cl, 10mmol / L Tris-Cl, (pH8.5), 1.5mmol / L MgC12, 20 (^ mol L dNTP, 25pmol primer, 2.5U Taq DNA polymerase. Reaction on a PE9600 DNA thermal cycler under the following conditions for 25 cycles: 94'C 30 seconds; 55 ° C, 30 seconds; 72 ° C 2 minutes. Simultaneously set β-actin during RT-PCR. The positive control and the template blank were negative controls. After purification of the amplified product QIAGEN kit, the TA cloning kit was used to connect to the pCR vector (Invitrogen), and the DNA sequence was determined. Results The DNA sequence of the PCR product and SEQ ID NO: 1 L-1551bp are identical. Example 3: In vitro expression, isolation and purification of recombinant OB-RGRP2

A pair of primers were designed at the start codon and the stop codon of the OB-RGRP2 gene.

The 5 ends have BamHI and EcoRI restriction sites. OB-RGRP2 gene coding region was obtained by PCR amplification using plasmid 018e 11 or a commercially available cDNA library as a template. The amplified fragment was inserted into the expression vector PGEX-2T (purchased from Pharmacia Biotech) after enzymatic digestion, and transformed into E. coli DH5ct. On a LB plate containing ampicillin and IPTG, 5 white recombinant transformants were selected and carried out. The DNA sequence analysis showed that the sequence of the gene coding region in 018e ll was exactly the same. The recombinant clone can express the GST-OBRGRP2 fusion protein, and the clone is designated as pOBRGRP2.

Pick a loop of E. coli DH5cc (pOBRGRP2) strain, inoculate it in 20ml LB medium (containing 100 μg / ml ampicillin), shake culture at 37 ° C overnight as the seed solution, take the seed solution and transfer it to 4 liters at 2% inoculation amount LB medium, cultured at 37 'C with shaking to A _{6 (KΓ} 0.7 (logarithmic growth phase), add IPTG to a final concentration of 0.4 mmol / L, and culture at 25' C for 12 hours. Centrifuge to collect bacteria The body was washed with lxPBS with buffer solution A (16mM Na2HPO4, 4mM NaH2PO4, pH 6.5) at 10ml / g bacterial cells, sonicated in an ice bath, and the supernatant was collected after centrifugation. The supernatant was passed through glutathione-Sepharose 4B chromatography column, washed with buffer A, and then eluted with an eluate containing 5mM reduced glutathione. SDS-PAGE showed that the GST-OBRGRP2 fusion protein product was about 44kDa. Implementation Example 4: Production of anti-OB-RGRP2 antibodies

A peptide synthesizer (PE-ABI) was used to synthesize the following OB-RGRP2-specific polypeptides: NH2-Leu-Pro-Ile-Val-Phe-Ala-Arg-Ala-His-Leu-COOH. The polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas. Immunochemistry, 1969; 6:43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine the titer of antibody in rabbit serum. Positive from antibodies with protein A-Sepharose Total IgG was isolated from rabbit serum. The peptide was bound to a cyanogen bromide-activated Seph _a rose 4B column, and the anti-peptide antibody was separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to OB-RGRP2.

All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

Rights request

An isolated nucleic acid molecule, characterized in that it comprises: a nucleotide sequence encoding a polypeptide having human OB-RGRP2 protein activity, the nucleotide sequence and the nucleus at positions 85-480 in SEQ ID No. 1 The nucleotide sequence is at least 70% homologous; or the nucleotide sequence is capable of hybridizing to the nucleotide sequence at positions 85-480 in SEQ ID No. 1 under highly stringent conditions.

The molecule according to claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence shown in SEQ ID No. 2.

The molecule according to claim 1, wherein the nucleotide sequence has a sequence of positions 85 to 480 in SEQ ID No. 1.

4. An isolated OB-RGRP2 polypeptide, comprising: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof.

The polypeptide according to claim 4, wherein the polypeptide is a polypeptide having the amino acid sequence of SEQ ID No. 2.

6. A vector comprising the nucleic acid molecule according to claim 1.

7. A host cell transformed with the vector of claim 6.

8. A method for preparing a polypeptide having human OB-RGRP2 protein activity, characterized in that the method includes:

(d) Isolating a polypeptide having OB-RGRP2 protein activity.

An antibody capable of specifically binding to the OB-RGRP2 polypeptide according to claim 4.

10. A nucleic acid molecule comprising the continuous 10-2000 nucleotides in the nucleic acid molecule according to claim 1.

-16- Replacement page (Article 26)