CN1278005A - Human nucleolar chromosome protein and coding series thereof - Google Patents

Abstract

The present invention provides a novel human hN027 protein expressed in the human hypothalamus and its coding sequence, the present invention also provides a preparation method for the protein and nucleic acid sequence, and a method for detecting human hN027 nucleic acid sequence and polypeptide in a sample .

Description

A kind of human nucleolus protein and its coding sequence

The invention relates to the fields of molecular biology, enzymology, physiology, genetic engineering and the like. Specifically, the present invention relates to a hNO27 protein expressed in human hypothalamus and its nucleic acid sequence. The present invention also relates to the preparation method and use of the protein and nucleic acid sequence.

NO27 protein is a nucleolar chromosome protein cloned from Xenopus laevis. In the present invention, we cloned the homologous gene hNO27 of Xenopus laevis NO27 gene in human hypothalamus.

Before the publication of the present invention, the human hNO27 protein sequence and its nucleic acid sequence mentioned in this patent application have not been disclosed or reported.

The first object of the present invention is to provide a new human gene hNO27 (Genbank Accession No.AF261137), which is a human hNO27 protein gene.

The second object of the present invention is to provide a new human protein hNO27.

The third object of the present invention is to provide a method for producing the above-mentioned novel human hNO27 protein and nucleic acid sequence using recombinant technology.

The present invention also provides the application of the human hNO27 protein polypeptide and coding sequence.

In one aspect of the present invention, an isolated DNA molecule is provided, which comprises: a nucleotide sequence encoding a polypeptide having human hNO27 protein activity, and the nucleotide sequence is identical to that of SEQ ID NO.6 from The nucleotide sequence of the nucleotide 111-857 DNA molecule has at least 70% homology; or the nucleotide sequence can be obtained from the nucleotide sequence of SEQID NO.6 under moderately stringent conditions The 111-857 nucleotide sequence is hybridized. Preferably, the sequence encodes a polypeptide having the amino acid sequence shown in SEQ ID NO.7. More preferably, said sequence has the nucleotide sequence from nucleotide 111-857 in SEQ ID NO.6.

In another aspect of the present invention, an isolated human hNO27 protein polypeptide is provided, which includes: a polypeptide having the amino acid sequence of SEQ ID NO.7, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof thing. Preferably, the polypeptide is a polypeptide having the sequence of SEQ ID NO.7.

In another aspect of the present invention, a vector comprising the above-mentioned DNA molecule is also provided.

In another aspect of the present invention, a host cell transformed with the above vector is also provided. In one example the host cell is E. coli; in another example, the host cell is a eukaryotic cell.

In another aspect of the present invention, a method for producing a polypeptide having human hNO27 protein activity is also provided, the steps of which are as follows:

(1) The nucleotide sequence encoding the polypeptide having human hNO27 protein activity is operably connected to the expression control sequence to form a human hNO27 protein expression vector, and the nucleotide sequence and SEQ ID NO.6 are derived from nucleosides The nucleotide sequence at positions 111-857 of acid has at least 70% homology;

(2) transferring the expression vector in step (1) into a host cell to form a recombinant cell of human hNO27 protein;

(3) cultivating the recombinant cells in step (2) under conditions suitable for expressing the human hNO27 protein polypeptide;

(4) Isolating a polypeptide having human hNO27 protein activity.

Preferably, the nucleic acid sequence used in the method has the sequence of positions 111-857 in SEQ ID NO.6.

The invention also provides an antibody specifically binding to the hNO27 protein polypeptide.

In the present invention, "isolated", "purified" or "substantially pure" DNA means that the DNA or fragment has been separated from the sequences flanking it in its natural state, and also means that the DNA or fragment has been Separated from the components that naturally accompany nucleic acids and that have been separated from the proteins that accompany them in cells.

In the present invention, the term "human hNO27 protein (or polypeptide) coding sequence" refers to a nucleotide sequence encoding a polypeptide having human hNO27 protein activity, such as the 111-857th nucleotide sequence in SEQ ID NO.6 and its degenerate sequence. The degenerate sequence refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the nucleotides 111-857 of the coding frame of the sequence of SEQ ID NO.6 . Due to the degeneracy of codons, a degenerate sequence with as low as about 70% homology with the 111-857th nucleotide sequence in SEQ ID NO.6 can also encode the sequence described in SEQ ID NO.7 . The term also includes a nucleotide sequence capable of hybridizing to the nucleotide sequence from nucleotides 111-857 in SEQ ID NO.6 under moderately stringent conditions, preferably under highly stringent conditions. The term also includes at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 70% homology with the nucleotide sequence from nucleotide 111-857 in SEQ ID NO.6 At least 95% nucleotide sequence.

The term also includes variants of the open reading frame sequence of SEQ ID NO. 6 that encode a protein that has the same function as native human hNO27. These variations include (but are not limited to): the deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , insertion and/or substitution, and addition of several (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) at the 5' and/or 3' end ) nucleotides.

In the present invention, "substantially pure" protein or polypeptide means that it accounts for at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (by dry weight) of the total substance of the sample. or display weight). Purity can be measured by any suitable method, such as measuring the purity of the polypeptide by column chromatography, PAGE or HPLC. A substantially pure polypeptide is substantially free of components that accompany it in its native state.

In the present invention, the term "human hNO27 protein or polypeptide" refers to a polypeptide having the sequence of SEQ ID NO.7 having human hNO27 protein activity. The term also includes variant forms of the sequence of SEQ ID NO. 7 that have the same function as native human hNO27. These variations include (but are not limited to): deletions and insertions of several (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at 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 human hNO27 protein.

Variants of the human hNO27 polypeptide of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybrids that can hybridize with human hNO27 DNA under high or low stringent conditions The encoded protein, and the polypeptide or protein obtained by using the antiserum against human hNO27 polypeptide. The present invention also provides other polypeptides, such as fusion proteins comprising human hNO27 polypeptide or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes soluble fragments of human hNO27 polypeptides. Typically, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 80 contiguous amino acids of the human hNO27 polypeptide sequence. 100 consecutive amino acids.

In the present invention, "human hNO27 conservative variant polypeptide" means that compared with the amino acid sequence of SEQ ID NO.7, at most 10, preferably at most 8, more preferably at most 5 amino acids are similar or similar in nature Amino acids are substituted to form polypeptides. These conservative variant polypeptides are preferably produced by substitutions according to Table 1.

Table 1 initial residue representative replacement preferred substitution 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 His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) 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; Leu

The invention also includes analogs of human hNO27 protein or polypeptide. The difference between these analogs and the natural human hNO27 polypeptide may be the difference in amino acid sequence, or the 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 random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids, cosmids and the like. When producing the human hNO27 polypeptide of the present invention, the human hNO27 coding sequence can be operably linked to the expression control sequence, thereby forming a human hNO27 protein expression vector.

As used herein, "operably linked" refers to the condition that some portion of a linear DNA sequence is capable of affecting the activity of other portions of the same linear DNA sequence. For example, a signal peptide (secretion leader) DNA is operably linked to a polypeptide DNA if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide; if a promoter controls the transcription of the sequence, it is operably linked to A coding sequence; a ribosome binding site is operably linked to a coding sequence if it is placed in a position to enable its translation. Generally, "operably linked to" means adjacent, and with respect to a secretory leader it means adjacent in reading frame.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cells are eukaryotic cells, such as CHO cells, COS cells and the like.

The invention also provides antibodies specifically binding to human hNO27, including polyclonal antibodies and monoclonal antibodies.

In the present invention, a series of methods known in the art can be used to prepare antibodies specific to human hNO27. For example, the purified human hNO27 gene product or its antigenic fragments are injected into animals to produce polyclonal antibodies. Likewise, cells expressing human hNO27 or antigenic fragments thereof can also be used to immunize animals to produce antibodies. Antibodies produced according to the present invention may also be monoclonal antibodies, which can be produced using hybridoma technology (e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6 : 511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976). The antibody of the present invention includes an antibody that can suppress the function of human hNO27, and can also be an antibody that does not affect the function of human hNO27. Each type of antibody can be produced by immunizing the fragment or functional domain of the human hNO27 gene product, and the human hNO27 gene product and its fragment can be produced by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies that bind unmodified forms of the human hNO27 gene product can be obtained by immunizing animals with the gene product produced in prokaryotic cells such as E. coli. Antibodies that bind to post-translationally modified forms, such as glycosylated or phosphorylated proteins or polypeptides, can be obtained by immunizing animals with gene products produced in eukaryotic cells, such as yeast or insect cells.

The human hNO27 antibody of the present invention can be used to identify cells expressing human hNO27 protein or polypeptide, such as Jurkat T cells. For example, a human hNO27-specific antibody can be labeled with a detectable molecule such as fluorescein isothiocyanate (FITC), and the human hNO27-specific antibody can be contacted with a cell sample, and then detected by fluorescence microscopy or flow cytometry. Cells bound by human hNO27-specific antibody.

In addition to detecting human hNO27 on the cell surface, the protein can also be analyzed by Western blotting. Cell lysates can be extracted from cultured cells or tissue samples from patients such as the hypothalamus and dissolved in lysis buffer containing detergent. Cell extracts were then separated by SDS polyacrylamide gel electrophoresis (with purified human hNO27 polypeptide as a positive control), followed by transfer to nitrocellulose by electrophoretic hybridization. For immunodetection of human hNO27 polypeptide by Western blot, typical antibody-binding detection methods, such as autoradiography or alkaline phosphatase detection methods, can be used. Inoculation serum or irrelevant monoclonal antibodies can be used as controls for non-specific reactions.

The expression of human hNO27 gene product can also be analyzed by Northern blot technique, that is, the presence or absence and quantity of RNA transcript of human hNO27 in cells can be analyzed.

Northern blot analysis of human hNO27 DNA and Western blot analysis of human hNO27-specific antibodies can be used in combination to confirm the expression of human hNO27 in biological samples. Human hNO27 DNA can also be used for Southern blot analysis or in situ hybridization analysis to locate the gene on the chromosome, and genetic linkage analysis can be performed to find out other possible disease-related genes.

In addition, the present invention also provides a nucleic acid molecule that can be used as a probe, which generally has 8-100 consecutive nucleotides of the human hNO27 nucleotide coding sequence, preferably has 15-50 consecutive nucleotides acid. The probe can be used to detect whether there is a nucleic acid molecule encoding human hNO27 in a sample.

The present invention also provides a method for detecting whether there is a human hNO27 nucleotide sequence in a sample, which comprises using the above-mentioned probe to hybridize with the sample, and then detecting whether the probe is combined. Preferably, the sample is a product of PCR amplification, wherein the PCR amplification primers correspond to the human hNO27 nucleotide coding sequence and can be located on both sides or in the middle of the coding sequence. Primers are generally 15-50 nucleotides in length.

In addition, according to the human hNO27 nucleotide sequence and amino acid sequence of the present invention, human hNO27 homologous genes or homologous proteins can be screened on the basis of nucleic acid homology or expressed protein homology.

In order to obtain a dot matrix of human cDNAs or genomic DNAs related to the human hNO27 gene, human cDNA or genomic DNA libraries can be screened with DNA probes that use ³² P to all or part of human hNO27 under low stringency conditions obtained by radioactive labeling. The most suitable cDNA library for screening is that from the human hypothalamus. cDNA libraries from other human tissues involved in endocrine or specific human cell lines may also be used for screening purposes. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the art of molecular biology. In addition, many such cDNA libraries are commercially available, for example, from Clontech, Palo Alto, Cal. This screening method can identify the nucleotide sequences of gene families related to human hNO27.

Screening for human hNO27 homologues based on nucleotide similarity can be accomplished as follows. A human hypothalamus cDNA library, such as Clontech Cat.#74XX (Clontech, Palo Alto, Cal.) can be screened using a randomly primed DNA probe containing all or part of the human hNO27 gene sequence. To complete the identification of clones with DNA insertion sequences at least 70% homologous to the human hNO27 sequence, a hybridization solution with a hybridization temperature of 55° C. can be used, followed by washing with 0.5×SSC and 0.1% SDS. The cloned DNA insert identified in this way can be further evaluated for similarity to the human hNO27 gene by DNA restriction enzyme analysis and DNA sequencing. The distribution of tissue expression can be analyzed using the Northern blotting technique described above.

Human hNO27 homologues can also be identified with antibodies against human hNO27 protein or polypeptide. For example, commercially available or constructed expression libraries derived from cells or tissues such as the hypothalamus can be screened using standard methods. The library is poured into a plate, and the colony is transferred to a nitrocellulose membrane, allowing the expressed recombinant protein to bind to the membrane. Typical antibody binding and detection can then be performed with a specific human hNO27 antibody. The DNA insert in the clone identified by this method can be further analyzed by DNA restriction enzyme analysis and DNA sequencing to evaluate its similarity to the human hNO27 gene. Tissue expression profiles of newly identified genes can be analyzed similarly as described above.

The human hNO27 nucleotide full-length sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial chemical synthesis. Before this application, in the prior art, it was completely possible to obtain the nucleic acid sequence encoding the human hNO27 protein of the present invention by first synthesizing multiple small polynucleotide fragments and then connecting them. Then, the nucleic acid sequence can be introduced into various existing DNA molecules (such as vectors) and cells in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition to recombinant production, fragments of the proteins of the invention can also be produced by direct peptide synthesis using solid-phase techniques (Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963) J. Am Chem. Soc 85:2149-2154). Protein synthesis in vitro can be performed manually or automatically. For example, peptides can be synthesized automatically using an Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA). Fragments of a protein of the invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

The coding sequence of the protein of the present invention can be used for gene mapping. For example, by fluorescent in situ hybridization (FISH), cDNA clones can be hybridized with metaphase chromosomes to accurately locate chromosomes. The technique can use cDNAs as short as about 500 bp; cDNAs as long as about 2000 bp or longer can also be used. For this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise location on a chromosome, it will be possible to correlate the sequence's physical location on the chromosome with genetic map data. Such genetic map data are available, for example, through the Mendelian Human Genetics Database (available online through the Johns Hopkins University Welch Medical Library). Linkage analysis is then used to identify associations between genes and diseases that have been mapped to the same chromosomal region.

Next, it is necessary to determine the differences in cDNA or genome sequence between diseased and healthy individuals. If a mutation is present in some or all affected individuals but not in normal individuals, the mutation may be the cause of the disease.

Utilizing the human hNO27 protein of the present invention, through various conventional screening methods, substances interacting with human hNO27, or receptors, inhibitors or antagonists can be screened out.

When the human hNO27 protein of the present invention and its antibody, inhibitor, antagonist or receptor are administered (administered) therapeutically, various effects can be provided. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

Taking the human hNO27 protein of the present invention as an example, it can be used in combination with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions contain a therapeutically effective amount of protein and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The human hNO27 protein of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other auxiliary agents. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When the human hNO27 protein polypeptide of the present invention is used as a drug, a therapeutically effective dose of the polypeptide can be administered to mammals, wherein the therapeutically effective dose is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dosage is about 10 microgram/kg body weight to about 1 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Table 2 is a homology comparison (FASTA) table of the nucleotide sequences (GenBankAccession No. AB018189) of the human hNO27 protein of the present invention and the Xenopus laevis NO27 protein.

Table 3 is the homology comparison (FASTA) table of the amino acid sequences (GenPeptAccession No. BAA33761) of human hNO27 protein of the present invention and Xenopus laevis NO27 protein. Among them, the same amino acid is marked with a single amino acid character between the two sequences, and the similar amino acid is marked with "+".

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.

Example 1

Cloning of human hNO27 gene

1. Tissue isolation (hypothalamus isolation)

The hypothalamus was obtained from 5 normal adult male donors. The hypothalamus was removed within four hours after death and immediately placed in liquid nitrogen for cryopreservation.

2. Isolation of mRNA (mRNA isolation)

Take out the tissue, grind it with a mortar, add it into a 50ml tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to a new 50ml tube and extract total RNA (TRIzol Reagents, Gibco, NY, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis. The mRNA in the total RNA was separated and quantified using a cellulose column with Oligo d(T).

3. Construction of cDNA library

Using mRNA as a template, double-stranded cDNA is synthesized, and the reverse transcription primer is shown in SEQ ID NO.1. After blunting the ends, add adapters containing EcoRI cutting points, and the adapter sequences are shown in SEQ ID NO.2 and 3, respectively. After phosphorylation of the EcoRI terminus, it was digested with XhoI restriction endonuclease for 1.5 hours before fragment isolation. Fragments with a length >500bp were screened through the column, extracted with phenol-chloroform, precipitated with ethanol, dissolved in sterile water, connected to the Uni-ZAP XR vector (Stratagene, CA9203, USA), and Zap-cDNA Gigapack III Gold Cloning Kit (Stratagene , CA9203, USA) were packaged, and the host bacteria used XL 1-Blue MRF (Stratagene, CA9203, USA) bacteria. Plate and measure titer.

4. Sequencing and Database Constructing

The clones with foreign fragments inserted in the library were selected, and the plasmids were extracted after amplification (Qiagen, Germany). T3 and T7 were used as universal primers at the 3' and 5' ends, and terminators were fluorescently labeled (Big-Dye, Perkin- Elmer, USA) method, EST large-scale sequencing was performed on an ABI377 sequencer (Perkin-Elmer, USA). The sequencing results were removed from the carrier sequence by FACTURA software and transferred to SUN Ultra 450 Server for further processing. All the sequence information is then searched in the existing database (Genebank+EMBL) with BLAST and FASTA software in the GCG software package (Wisconsin group, USA), and sequences with no homology or homology lower than 95% are regarded as new Gene database.

5. Cloning of Full-length cDNA

On the basis of the obtained new gene fragment sequence information, the cDNA full-length clone is carried out in two stages:

(1) "Electronic Cloning"

Use the new gene fragment sequence as a probe to search the dbEST database, and consider the expressed sequence tag (Expressed Sequence Tag, “EST” for short) sequence with an overlapping sequence > 50bp and a homology of more than 98% as the same sequence (consensus sequence), take it out and use AUTOASSEMBLER software for splicing, part of EST can extend the probe sequence. Then use STRIDER software to analyze whether the extended sequence has a complete open reading frame (Open Reading Frame, ORF), and use BLAST to search Genbank or SwissProt to determine whether the sequence is homologous to other species at the nucleotide and amino acid levels , to help determine the full-length integrity of the obtained gene. By means of electronic cloning, the full-length sequence of the human hNO27 gene can usually be obtained.

(2) Rapid amplification of cDNA ends (Rapid Amplification of cDNA Ends, RACE)

If the complete cDNA full-length has not been obtained by the "electronic cloning" method, then design primers at the 5' or 3' end of the existing sequence, and carry out in the human hypothalamic Marathon-Ready cDNA library (Clontech Lab, Inc, USA) Long range PCR reaction. The PCR products were then cloned and sequenced. Use AUTOASSEMBLER and STRIDER software to analyze whether the extended sequence has a complete ORF, if not, repeat the above process until the full length is obtained.

(3) RT-PCR

For the known sequences at the 5' and 3' ends, if there is still a gap in the middle that cannot be obtained from the existing public database or its own database, RT-PCR may be considered. Primers were designed at the 5' end of the sequence, and Oligo-dT was used as a primer at the 3' end to amplify in the hypothalamic total RNA library. The products were then cloned and sequenced. Finally splice and get the full length.

By using the above three methods in combination, the full-length coding sequence of the candidate human hNO27 protein was obtained. On the basis of splicing to obtain the full length (including at least the complete open reading frame), further design primer R1: 5'-CTCCACCGTCAACTCAGGTT-3' (SEQ ID NO.4) as the forward primer, oligonucleotide R2: 5' -CACAAGCTGGTGGCAAGTAA-3'(SEQ ID NO.5) is the reverse primer, and the total RNA of the hypothalamus is used as the template for RT-PCR amplification. The PCR conditions of R1/R2 are 94°C for 5 minutes, followed by 94°C for 30 minutes 35 cycles of 30 seconds at 60°C and 1 minute at 72°C, with a final extension at 72°C for 5 minutes. The PCR amplification product was detected by electrophoresis, and the length of the amplified fragment obtained was 998bp. Then, the PCR amplification product was cloned and sequenced according to conventional methods to obtain the sequence shown in SEQ ID NO.6.

Example 2

Sequence information and homology analysis of human hNO27 gene:

The novel human hNO27 full-length cDNA of the present invention (GenBank Accession No.AF261137. It has not been disclosed before this application) has a length of 998bp, and the detailed sequence is shown in SEQ ID NO.6, wherein the open reading frame is located at 111-857 nucleotides . The amino acid sequence of human hNO27 was deduced according to the full-length cDNA, with a total of 248 amino acid residues, a molecular weight of 26396.57, and a pI of 12.24. See SEQ ID NO.7 for the detailed sequence.

The full-length cDNA sequence of hNO27 and its encoded protein were used to perform nucleotide and protein homology in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using the BLAST program After searching, it was found that it has a certain homology with the NO27 gene of Xenopus laevis. At the nucleotide level, it has 69.3% identity with the mRNA full coding sequence (GenBank Accession No.AB018189) of the Xenopus NO27 gene (Appendix 2), and at the amino acid level, it has the same (GenPept Accession No.BAA33761) amino acid residues 1-248 have 77.6% identity and 88.2% similarity (Supplementary Table 3). It can be seen from the above that the hNO27 gene and the Xenopus NO27 gene have high homology in both nucleic acid and protein levels, and it can be considered that the two also have a high similarity in function.

The human hNO27 of the present invention can be used as a member of the family for further functional research, and can also be used to produce fusion proteins with other proteins, such as immunoglobulins to produce fusion proteins. In addition, the human hNO27 of the present invention can also be fused or exchanged fragments with other members of the family to produce new proteins. For example, the N-terminal of the human hNO27 protein of the present invention is exchanged with the N-terminal of the Xenopus NO27 protein to produce a new protein with higher activity or new properties.

The antibody against human hNO27 of the present invention is used for screening other members of this family, or for affinity purification of related proteins (such as other members of this family).

In addition, the human hNO27 nucleic acid (coding sequence or antisense sequence) of the present invention can be introduced into cells to increase the expression level of human hNO27 or inhibit the overexpression of human hNO27. The human hNO27 protein or its active polypeptide fragments of the present invention can be administered to patients to treat or alleviate related diseases caused by the lack, non-function or abnormality of human hNO27. In addition, the nucleic acid sequence or antibody based on the present invention can also be used for relevant diagnosis or prognosis.

Since the human hNO27 protein of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and/or Lower side effects (eg less or no immunogenicity in humans).

Example 3

Tissue expression profile of human hNO27 gene

Electronic Northern Expression Profiles. According to Ton C. et al.'s method (Ton C et al., Biochem BiophysRes Commun 1997 Dec 18; 241 (2): 589-594; Hwang DM, et al., Circulation 1997 Dec 16; 96 (12): 4146-4203 ), the human hNO27 cDNA sequence was searched by BLAST in the humanEST database in the GCG software package. Among the obtained human ESTs, there are hundreds of ESTs with probability values <10e-10 and identity >95%, which can be regarded as the The transcription and expression of the gene in the tissue, from which the tissue spectrum of the expression of the gene is obtained, and it is found that it is expressed in tissues such as lymph, pancreas, placenta, infant heart, fetal liver, testis, fetal brain, etc., indicating that it is in the human body play an important role in these tissues and organs.

Example 4

Preparation and Purification of Human hNO27 Polypeptide

In this example, the full-length human hNO27 coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

Prokaryotic expression of human hNO27 polypeptide in the form of GST fusion protein in Escherichia coli.

Construction of prokaryotic expression vector and transformation of Escherichia coli

According to the full-length coding sequence of human hNO27 (SEQ ID NO.6), primers were designed to amplify the complete coding reading frame (corresponding to about 20 or more nucleotides at the 5' and 3' ends of the coding sequence, respectively), and Restriction endonuclease sites were introduced into the anti-primers (this depends on the pGEX-2T vector selected), so as to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the human hNO27 gene was cloned into the pGEX-2T vector (Pharmacia, Piscataway, NJ) under the premise of ensuring the correct reading frame. The identified expression vector was transformed into Escherichia coli DH5α using the CaCl ₂ method, and the engineered strain DH5α-pGEX-2T-hNO27 containing the pGEX-2T-hNO27 expression vector was screened and identified.

Isolation and Identification of Engineering Bacteria Expressing GST-hNO27 Recombinant Protein

Pick the DH5α-pGEX-2T-hNO27 engineering bacteria of a single colony in 3 ml LB medium containing 100 μg/ml ampicillin and culture overnight with shaking, and draw the culture solution into a new LB medium (containing 100 μg/ml ampicillin) at a concentration of 1:100 /ml ampicillin) for about 3 hours, and when the OD ₆₀₀ reached 0.5, IPTG was added to a final concentration of 1 mmol/L to continue culturing at 37°C for 0, 1, 2, and 3 hours, respectively. Centrifuge 1ml of bacterial solution with different culture time, add lysate (50μl of 2×SDS loading buffer, 45μl of distilled water, 5μl of dimercaptoethanol) to the bacterial sediment, suspend the bacterial sediment, boil in boiling water bath for 5 minutes, 10000rpm Centrifuge for 1 minute, add the supernatant to 12% SDS-PAGE gel electrophoresis. After staining, it is observed that the amount of protein with expected molecular weight increases with the increase of IPTG induction time, which is the engineering bacteria expressing GST-hNO27 fusion protein.

Extraction and Purification of GST-hNO27 Fusion Protein

The engineering bacteria DH5α-pGEX-2T-hNO27 expressing GST-hNO27 fusion expression protein were induced according to the above method. The induced bacteria were centrifuged and precipitated, and 20ml of PBS was added to resuspend the bacteria for every 400ml of bacteria, and the bacteria were sonicated. Add 50% glutathione Sepharose 4B saturated with PBS in an amount of 20 microliters per milliliter to the complete ultrasonic liquid, shake at 37°C for 30 minutes, and centrifuge at 10,000 rpm for 10 minutes to precipitate glutathione bound to GST-hNO27 Sepharose 4B, discard the supernatant. Wash twice by adding 100 μl PBS to the precipitate obtained from the ultrasonic solution, then add 10 μl reduced glutathione eluent to the precipitate obtained from the ultrasonic solution, place at room temperature for 10 minutes, and centrifuge at 10,000 rpm for 10 minutes. The supernatant is Eluted fusion protein. Repeat the elution twice. The eluted supernatant was stored at -80°C, and subjected to SDS-PAGE electrophoresis to detect the purification effect. The protein band at 26kDa is human hNO27 protein.

Example 5

Eukaryotic expression of human hNO27 protein or polypeptide in insect cells

1. Construction of human hNO27 baculovirus expression vector and transfection to Sf9 insect cell line

According to the full-length coding sequence of human hNO27 (SEQ ID NO.6), design primers to amplify the complete coding reading frame, and introduce restriction endonuclease sites on the forward and reverse primers respectively (this may vary depending on the carrier selected). ) to construct expression vectors. Using the amplification product obtained in Example 1 as a template, after PCR amplification, the human hNO27 cDNA was cloned into pVL1392 vector (Invitrogen, Carlsbad, CA) under the premise of ensuring the reading frame. Add 3 μg of the identified expression vector, 1 μg of wild-type linear baculovirus DNA (BaculoGold ^TM ACMNPV DNA, Pharmingen, San Diego, CA) and 25 μl of Lipofection (Gibco-BRL, NY) to 1 ml of serum-free insect medium, shake Mix for 15 seconds and incubate at room temperature for 15 minutes. Take 1ml (2×10 ⁶ ) of Sf9 insect cell suspension in a 60mm tissue culture plate, change the transfection medium after 1 hour of attachment, discard the medium after incubating at room temperature for 15 minutes, and add the prepared DNA vector transfection mixture , Seal the culture plate with Parafilm, shake and culture at room temperature 27°C for 4 hours, then change the complete medium and culture for 3 days, and collect the supernatant for use.

2. Screening and identification of insect cell lines transformed into recombinant expression vectors

Insect cells 3 days after transfection were made into cell suspension (2×10 ⁶ /1ml) with fresh medium, 1ml of cell suspension was placed in a 60mm tissue culture dish, 3ml of medium was added, and 100μl of culture supernatant collected, Adhere to the wall for 1 hour, discard 2ml of medium, continue to culture at room temperature for 1 hour, discard all the medium, add 3ml of preheated semi-solid medium containing 20μl of 4% X-gal, pick white cell clones after 5-7 days of culture Cultured in a 96-well culture plate for 3-5 days, and then aspirate the supernatant to infect Sf9 insect cells.

Infected cells were collected for Western identification. After the cells were lysed, SDS-PAGE electrophoresis was carried out, and the gel after electrophoresis was transferred to the nitrocellulose membrane in Pharmacia's Multiphor II semi-dry electrotransfer instrument, and the nitrocellulose membrane was placed in the blocking solution for blocking for 1 hour, and then in Block in the anti-human hNO27 antibody solution for 1 hour, shake and wash with TBS solution for 5 minutes, a total of 2 times, then place the membrane in the biotin-labeled anti-human hNO27 primary antibody secondary antibody solution, shake for 1 hour, wash with TBS, Add avidin-alkaline phosphatase complex to react for 30 minutes, wash twice with TBS, add freshly prepared chromogenic solution to develop color and observe protein bands.

Sf9 cell clones that highly express human hNO27 were picked.

3. Extraction and purification of human hNO27 protein

The supernatant of Sf9 cell clones highly expressing human hNO27 was used to infect Sf9 cells in large quantities. After 48 hours of infection, the cells were collected and washed with PBS. Add 20ml of cell lysate (0.5% Triton X-100, 20mM Na ₃ PO ₄ (sodium phosphate, pH7.8), 500mM NaCl, 1mM Na ₃ VO ₄ (sodium vanadate), 1mM Pefabloc, for every 2×10 ⁸ cells 1 μg/ml pepsin, leupeptin and aprotinin) to break the cells, centrifuge at 12000×g for 20min to remove cell debris, add 2ml NTA-agarose (Qiagen, Germany) to the supernatant for every 2× ¹⁰⁸ cells , adsorption at 4°C for 1 hour. Then, it was washed twice with His buffer containing 100 nM imidazole, and eluted with a buffer containing 20 mM N,N'-dipiperazine, 500 mM NaCl, and 300 nM imidazole to obtain a purified protein. The eluate was stored at 4°C, and the purity of the extracted human hNO27 protein was detected by SDS-PAGE electrophoresis. The protein band at 26kDa is human hNO27 protein.

Example 6

Preparation of anti-human hNO27 antibody

1. Preparation of immunized mice and splenocytes: the human hNO27 protein obtained in Example 5 and Example 6 is separated by chromatography and then used for later use, or can be separated by SDS-PAGE gel electrophoresis, and the electrophoresis strip The bands were cut from the gel and emulsified with an equal volume of complete Freund's adjuvant. Take 6-8 week-old Balb/C female mice, and inject them intraperitoneally with 50-100 μg/0.2ml emulsified protein. After 14 days, the mice were boosted with the same antigen emulsified with incomplete Freund's adjuvant at a dose of 50-100 μg/0.2ml, and used for fusion after 3-5 days. Among them, for the preparation of spleen cells, see E Zheng, editor-in-chief, "Tissue Culture and Molecular Cytology", Beijing Publishing House, p. 210.

2. Prepare feeder cells according to the method in "Tissue Culture and Molecular Cytology Techniques" (supra), p. 371.

3. Carry out cell fusion according to the method in "Tissue Culture and Molecular Cytology Techniques" (supra), p. 213.

4. Antibody detection: After 10-15 days of cell fusion, it is necessary to check each hole. Once vigorous hybrid cell colony growth is found, human hNO27 protein is used for preliminary screening of antibody activity. Commonly used methods include: immunofluorescence test, Emission immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). Immediately after the wells with antibody activity were detected, the clones were cultured and the antibodies were isolated.

The sequences and symbols involved in the present invention are listed as follows:

(1) Information on SEQ ID NO.1

(i) Sequence features:

(A) Length: 50bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(iii) Sequence description: SEQ ID NO.1GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT 50

(2) Information of SEQ ID NO.2

(i) Sequential features:

(A) Length: 13bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(iii) Sequence description: SEQ ID NO.2

AATTCGGCAC GA G 13

(3) Information of SEQ ID NO.3

(i) Sequential features:

(A) Length: 9bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: oligonucleotide

(iii) Sequence description: SEQ ID NO.3GCCGTGCTC 9(4) Information on SEQ ID NO.4

(i). Sequence features:

(A) Length: 20bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii).Molecular type: oligonucleotide

(iii). Sequence description: SEQ ID NO.4CTCCACCGTCAACTCAGGTT 20(5) information of SEQID NO.5

(i). Sequence features:

(A) Length: 20bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii).Molecular type: oligonucleotide

(iii). Sequence description: SEQ ID NO.5CACAAGCTGGTGGCAAGTAA 20(6) Information on SEQ ID NO.6

(i) Sequential features:

(A) Length: 998bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: Nucleotide

(iii) Sequence description: SEQ ID NO.6

1 CTCCACCGTC AACTCAGGTT CGGCCGGTCC CCGGCCCGCC TGCCGGAGCC

51 GTGGTGGCAG CCCCGGGAGG AGCACTGGCG TCTGTTTCCT TCGATTCTCG

101 GGATTCGAAG ATGGCTGCAC AGTCAGCGCC GAAAGTTGTG CTAAAAAGCA

151 CCACCAAGAT GTCTCTAAAT GAGCGCTTTA CTAATATGCT GAAGAACAAA

201 CAGCCGACGC CAGTGAATAT TCGGGCTTCG ATGCAGCAAC AACAGCAGCT

251 AGCCAGTGCC AGAAACAGAA GACTGGCCCA GCAGATGGAG AATAGACCCT

301 CTGTCCAGGC AGCATTAAAA CTTAAGCAGA GCTTAAAAGCA GCGCCTGGGT

351 AAGAGTAACA TCCAGGCACG GTTAGGCCGA CCCATAGGGG CCCTGGCCAG

401 GGGAGCAATC GGAGGACGAG GCCTACCCAT AATCCAGAGA GGCTTGCCCA

451 GAGGAGGACT ACGTGGGGGA CGTGCCACCA GAACCCTACT TAGGGGCGGG

501 ATGTCACTCC GAGGTCAAAA CCTGCTCCGA GGTGGACGAG CCGTAGCTCC

551 CCGAATGGGC TTAAGAAGAGGTGGTGTTCG AGGTCGTGGA GGTCCTGGGA

601 GAGGGGGCCT AGGGCGTGGA GCTATGGGTC GTGGCGGAAT CGGTGGTAGA

651 GGTCGGGGTA TGATAGGTCG GGGAAGAGGG GGCTTTGGAG GCCGAGGCCG

701 AGGCCGTGGA CGAGGGAGAG GTGCCCTTGC TCGCCCTGTA TTGACCAAGG

751 AGCAGCTGGA CAACCAATTG GATGCATATA TGTCGAAAAC AAAAGGACAC

801 CTGGATGCTG AGTTGGATGG CTACATGGCG CAGACAGATC CCGAAACCAA

851 TGATTGAAGC CTGCCCATCC TCCCATGAGA GACTCTTGTT AGTCAACACA

901 TCTGTAAATA ACCTTGAGAT AACAGATGAG AAGAAATCTG ATTGATGCTG

951 Information of GATGGACCTA TCACAATAGG CTGTGGACTT ACTTGCCACC AGCTTGTG(7) SEQ ID NO.7

(i) Sequential features:

(A) Length: 248 amino acids

(B) Type: amino acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: polypeptide

(iii) Sequence description: SEQ ID NO.7:

1 MAAQSAPKVV LKSTTKMSLN ERFTNMLKNK QPTPVNIRAS MQQQQQLASA

51 RNRRLAQQME NRPSVQAALK LKQSLKQRLG KSNIQARLGR PIGALARGAI

101 GGRGLPIIQR GLPRGGLRGG RATRTLLRGG MSLRGQNLLR GGRAVAPRMG

151 LRRGGVRGRG GPGRGGLGRG AMGRGGIGGR GRGMIGRGRG GFGGRGRGRG

201 RGRGALARPV LTKEQLDNQL DAYMSKTKGH LDAELDAYMA QTDPETND

Table 269.3% identity in 783 nt overlap

70 80 90 100 110 120h.seq CCCCGGGAGGAGCACTGGCGTCTGTTTCCTTCGATTCTCGGGATTCGAAGATGGCTGCAC

||| |||||||| || |x.seq TAGTGAAGCCATTCAGGACTCCAGGGCCGCTCGCTTTCTCAGACTTCAAGATGGCCGCCC

10 20 30 40 50 60

130 140 150 160 170 180h.seq AGTCAGCGCCGAAAGTTGTGCTAAAAAGCACCACCAAGATGTCTCTAAATGAGCGCTTTA

|| |||||||||||||||||||||||||||||||||||||||||||||||x .seq CATCCGCGTCTAAAGTTGTGCTAAAGAGTACCACAAAGTTGTCGCTGAATGAGCGCTTTA

70 80 90 100 110 120

190 200 210 220 230 240h.seq CTAATATGCTGAAGAACAAACAGCCGACGCCAGTGAATATTCGGGCTTCGATGCAGCAAC

||||||||||||||||||||||||||||||||||||||||||||||||x. seq CTAACATGCTGAAGAATAAACAGCCGATGCCGGTGAATATCCGCGCCAGCTTGCAGCAGC

130 140 150 160 170 180

250 260 270 280 280 290h.seq AACAGCAGC---TAGCCAGTGCCAGAAACAGAAGACTGGCCCAGCAGATGGAGAATAGAC

||||||| ||||||||| ||||||||||||||||||||||||||||||||x .seq AGCAGCAGCACACGGCCAGTGCTAAGAACAGAAGACTTGCTCAGCAGATGGAGAACAGAC

190 200 210 220 230 240

300 310 320 330 340 350h.seq CCTCTGTCCAGGCAGCATTAAAACTTAAGCAGAGCTTAAAGCAGCGCCTGGGTAAGAGTA

||||||||||||||||||||||||||||||||||||||||||||x.seq

250 260 270 280 290 300

360 370 380 390 400 410h.seq ACATCCAGGCACGGTTAGGCCGACCCATAGGGGCCCTGGCCAGGGGAGCAATCGGAGGAC

|||||||||||||||||||||||||||||||||||||x.seq ATATCCAGGCCCGGCTGGGAAGGCCAATGGGAGGTCTTGTACGGGGAGCCATAGGAATGA

310 320 330 340 350 360

420 430 440 450 460 470h.seq GAGGCCTACCCATAATCCAGAGAGGCTTGCCCAGAGGAGGACTACGTGGGGGACGTGCCA

||||||||||||||||||||||||||||||||x.seq GAGGAGCAGGAATGGGACTACGAGGAGTACAGAGAGGGGTGATGCGTGGCGGTCGTGGGG

370 380 390 400 410 420

480 490 500 510 520 530h.seq CCAGAACCCTACTTAGGGGCGGGATGTCACTCCGAGGTCAAAAACCTGCTCCGAGGTGGAC

|||||||||||||||||||||||||||||x.seq GAAGAGGAATGGCCAGGGGATCCATGGCTATGAGGGGGCAGAAC---CTCAGA---GGAC

430 440 450 460 470 480

540 550 560 570 580 590h.seq GAGCCGTAGCTCCCGAATGGGCTTAAGAAGAGG--TG-GTGTTCGAGGTCGTGGAGGTC

||| | || || ||||||| | || ||||| || | | ||||| || ||

490 500 510 520 530 540

600 610 620 630 640h.seq CTGG------GAGAGGGGGCCTAGGGCGTGGAGCTATGGGT---CGTGGCGGAATCGGTG

|| || ||||||| | | || || | |||||| | || ||||| ||||

550 560 570 580 590 600

650 660 670 680 690 700h.seq GTAGAGGTCGGGGTATGATAGGTCGGGGAAGAGGGGGCTTTGGAGGCCGAGGCCGAGGCC

||||||||| || || | |||||| ||||||||| ||||| ||||

610 620 630 640 650

710 720 730 740 750 760h.seq GTGGACGAGGGAGAGGTGCCCTTGCTCGCCCTGTATTGACCAAGGAGCAGCTGGACAACC

| || |||| || |||| | | | ||||| || | || ||||||||||||||||||x.seq GAGGGAGAGGAAGGGGTG TGACCCGCCCAGTTATAACAAAGGAGCAGCTGGATAACC

660 670 680 690 700 710

770 780 790 800 810 820h.seq AATTGGATGCATATATGTCGAAAACAAAAGGACACCTGGATGCTGAGTTGGATGCCTACA

||||||||||||||||||||||||||||||||||||||||||||||||||| x.seq AACTTGATGCCTATATGTCTAAAACGAGGGGGCACCTGGATGCCGAGCTGGATGCCTACA

720 730 740 750 760 770

830 840 850 860 870 880h.seq TGGCGCAGACAGATCCCGAAACCAATGATTGAAGCCTGCCCATCCTCCCATGAGAGACTC

|||| ||| |||| || || |||| || ||||x.seq TGGCACAGGCAGACCCAGAGTCCAACGACTGAATGTTTTATAGATTTCTAAGGATCAGA

780 790 800 810 820 830

890 900 910 920 930 940h.seq TTGTTAGTCAACACATCTGTAAATAACCTTGAGATAACAGATGAGAAGAAATCTGATTGAx.seq CTTGCCAGCATGCACATACCTGTGCCATGTCTCCTTGCCCGTA

840 850 860 870 880 890h.seq: Nucleic acid sequence of human hNO27 protein (GenBank Accession No.AF261137)x.seq: Nucleic acid sequence of Xenopus laevis NO27 protein (GenBank Accession 18 Accession 09)

Table 377.6% identity in 254 aa overlap, 88.2% similarity in 254 aa overlap

10 20 30 40 50 59h.pep MAAQSAPKVVLKSTTKMSLNERFTNMLKNKQPTPVNIRASMQQQQQ-LASARNRRLAQQM

||| || |||||||||+|||||||||||||||||||||+|||||||+||| |||||x.pep MAAPSASKVVLKSTTKLSLNERFTNMLKNKQPMPVNIRASLQQQQQHTASAKNRRLAQQM

10 20 30 40 50 60

60 70 80 90 100 110 119h.pep ENRPSVQAALKLKQSLKQRLGKSNIQARLGRPIGALARGAIGGRGLPIIQRGLPRGGL RG

||||||||||||||||||||||||||||||+|+|+||||| || + ||+ || +||x.pep ENRPSVQAALKLKQSLKQRLGKSNIQARLGRPMGGLVRGAIGMRGAGMGLRGVQRGVMRG

70 80 90 100 110 120

120 130 140 150 160 170 179h.pep GRATRTLLRGGMSLRGQNLLRGGRAVAPRMGLRRGGVRGRGGPGRGGLGRGAMGRGG

||+ | + ||+|++||||| || |+ + |||||||+|||||||||+ ||||||||+|| x.pep GRGGRGMARGSMAMRGQNL-RG-RGGVSRMGLRRG-MRGRGGPGRGMM-RGAMGRGGMGG

130 140 150 160 170

180 190 200 210 220 230h.pep RGRGMIGRGRGGF----G-GRGRGRGRGRGALARPVLTKEQLDNQLDAYMSKTKGHLDAE

|| || +|||||+ | ||||||||||| ++|||+|||||||||||||||+|||| ||x.pep RG-GMGARGRGGMTRGRGFGRGRGRGRGRG-VTRPVITKEQLDNQLDAYMSKTRGHLDAE

180 190 200 210 220 230

                                               

|||||||+|||+||x.pep LDAYMAQADPESND

      240h.pep: Human hNO27 protein amino acid sequence x.pep: Xenopus laevis NO27 protein amino acid sequence (GenBank Accession No.BAA33761)

Claims (11)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of human hN 027 protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 111-857 position among described nucleotide sequence and the SEQ ID NO.6; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.6 in from the nucleotide sequence hybridization of Nucleotide 111-857 position.

2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID NO.7.

3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.6 nucleotide sequence from Nucleotide 111-857 position.

4. isolated human hN 027 protein polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.7 aminoacid sequence, or its reactive derivative.

5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.7 polypeptide of sequence.

6. a carrier is characterized in that, it comprises the described DNA of claim 1.

7. one kind with the described carrier transformed host cells of claim 6, it is characterized in that it comprises prokaryotic cell prokaryocyte and eukaryotic cell.

8. a generation has the method for the polypeptide of human hN 027 protein active, is characterised in that its step is as follows:

(1) nucleotide sequence that coding is had a polypeptide of human hN 027 protein-active operationally is connected in expression regulation sequence, form the human hN 027 protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 111-857 position among described nucleotide sequence and the SEQ ID NO.6;

(2) change the expression vector in the step (1) over to host cell, form the proteic reconstitution cell of human hN 027;

(3) under the condition that is fit to expressing human hNO27 protein polypeptide, the reconstitution cell in the culturing step (2);

(4) isolate polypeptide with human hN 027 protein-active.

9. energy and the described human hN 027 protein polypeptide of claim 7 specificity bonded antibody is characterized in that it comprises polyclonal antibody and monoclonal antibody.

10. a nucleic acid molecule is characterized in that, it comprises 8-100 continuous nucleotide in the described dna molecular of claim 1.

11. one kind is used for the method whether test sample exists the human hN 027 nucleotide sequence, it is characterized in that it comprises with described probe of claim 10 and sample hybridizes, whether detection probes combination has taken place then, this sample is the product behind the pcr amplification, wherein the pcr amplification primer is corresponding to the human hN 027 nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence, primer length is 15～50 Nucleotide.