CN1289854A - Human proteasome subunit protein and its coding sequence - Google Patents

Abstract

The present invention provides a novel human hPas protein expressed in human Cushing's adrenal adenoma and its coding sequence, the present invention also provides the preparation method of the protein and nucleic acid sequence, and the detection of human hPas nucleic acid sequence in samples and peptide methods.

Description

A kind of human proteasome subunit 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 hPas protein expressed in human Cushing's adrenal adenoma and its nucleic acid sequence. The present invention also relates to the preparation method and use of the protein and nucleic acid sequence.

The proteasome is a large protease complex whose function is to produce peptides that are mature molecules or N-terminal extended molecular precursors of almost all major histocompatibility antigen complexes of class I (MHC class I) chain ligand. (J Immunol 2000 Jul 15;165(2):768-78). The proteasome signaling pathway is also involved in regulating the level of retinoic acid receptors in human keratocytes, so that its degeneration, disorder and other mutations may lead to certain tumor diseases. (Cancer Res 2000 Apr 15;60(8):2247-52).

In the present invention, we cloned the homologous gene hPas of Sulfolobus solfataricus Pas gene in human Cushing's adrenal adenoma.

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

The first object of the present invention is to provide a new human gene hPas (Genbank Accession No. AF271782), which is a human hPas protein gene.

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

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

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

In one aspect of the present invention, an isolated DNA molecule is provided, the molecule includes: a nucleotide sequence encoding a polypeptide having human hPas protein activity, and the nucleotide sequence is identical to SEQ ID NO. The nucleotide sequence of the DNA molecule from nucleotide 332-1096 in 6 has at least 70% homology; or the nucleotide sequence can be under moderately stringent conditions with SEQ ID NO. In 6, the nucleotide sequence from nucleotide 332-1096 was hybridized. Preferably, said sequence code has SEQ ID NO. The polypeptide of the amino acid sequence shown in 7. More preferably, said sequence has SEQ ID NO. The nucleotide sequence from nucleotide 332-1096 in 6.

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

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, there is also provided a method for producing a polypeptide having human hPas protein activity, the steps are as follows:

(1) The nucleotide sequence encoding the polypeptide having human hPas protein activity is operably connected to the expression control sequence to form a human hPas protein expression vector, the nucleotide sequence and SEQ ID NO. The nucleotide sequence from nucleotide 332-1096 in 6 has at least 70% homology;

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

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

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

Preferably, the nucleic acid sequence used in the method has SEQ ID NO. Sequence of positions 332-1096 in 6.

The invention also provides an antibody specifically binding to the hPas 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 hPas protein (or polypeptide) coding sequence" refers to a nucleotide sequence encoding a polypeptide having human hPas protein activity, such as SEQ ID NO. The 332nd-1096th nucleotide sequence and its degenerate sequence in 6. The degenerate sequence refers to, located in SEQ ID NO. In the nucleotides 332-1096 of the coding frame of the 6 sequence, one or more codons are replaced by degenerate codons encoding the same amino acid. Due to the degeneracy of the codon, it is the same as SEQ ID NO. The degenerate sequences with a homology as low as about 70% of the 332-1096 nucleotide sequence in 6 can also encode SEQ ID NO. 7 for the sequence described. The term also includes the ability to combine under moderately stringent conditions, preferably under high stringent conditions with SEQ ID NO. A nucleotide sequence hybridized from a nucleotide sequence at nucleotide positions 332-1096 of 6. The term also includes those related to SEQ ID NO. The homology of the nucleotide sequence from nucleotide 332 to 1096 in 6 is at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.

The term also includes a protein that encodes a protein having the same function as natural human hPas, SEQ ID NO. Variant forms of open reading frame sequences in 6. 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 wet 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 hPas protein or polypeptide" refers to SEQ ID NO. 7 sequence peptides. The term also includes those having the same function as natural human hPas, SEQ ID NO. 7 variants of the sequence. 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 hPas protein.

Variant forms of the human hPas polypeptide of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybrids that can hybridize with human hPas DNA under high or low stringency conditions The encoded protein, and the polypeptide or protein obtained by using the antiserum against human hPas polypeptide. The present invention also provides other polypeptides, such as fusion proteins comprising human hPas polypeptide or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes soluble fragments of human hPas 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 hPas polypeptide sequence. 100 consecutive amino acids.

In the present invention, "human hPas conservative variant polypeptide" refers to the sequence of SEQ ID NO. Compared with the amino acid sequence of 7, at most 10, preferably at most 8, more preferably at most 5 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. 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 hPas protein or polypeptide. The difference between these analogs and the natural human hPas polypeptide may be the difference in the 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 hPas polypeptide of the present invention, the human hPas coding sequence can be operably linked to the expression control sequence, thereby forming a human hPas 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 hPas, 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 hPas. For example, the purified human hPas gene product or its antigenic fragments are injected into animals to produce polyclonal antibodies. Likewise, cells expressing human hPas or antigenic fragments thereof can also be used to immunize animals to produce antibodies. Antibodies prepared according to the present invention may also be monoclonal antibodies, which may be prepared using hybridoma technology (for example, 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 hPas, and can also be an antibody that does not affect the function of human hPas. Each type of antibody can be produced by immunizing fragments or functional domains of human hPas gene products, and human hPas gene products and fragments thereof can be produced by recombinant methods or synthesized by polypeptide synthesizers. Antibodies that bind to unmodified forms of the human hPas gene product can be used in prokaryotic cells such as E. The gene product produced in coli is obtained by immunizing animals. 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 hPas antibody of the present invention can be used to identify cells expressing human hPas protein or polypeptide, such as Jurkat T cells. For example, a human hPas-specific antibody can be labeled with a detectable molecule such as fluorescein isothiocyanate (FITC), and the human hPas-specific antibody can be contacted with a cell sample, and then detected by fluorescence microscopy or flow cytometry. Cells bound by human hPas-specific antibodies.

In addition to detecting human hPas 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 Cushing's adrenal adenoma and dissolved in lysis buffer containing detergent. Cell extracts were then separated by SDS-polyacrylamide gel electrophoresis (with purified human hPas polypeptide as a positive control), followed by transfer to nitrocellulose by electrophoretic hybridization. For immunodetection of human hPas polypeptides 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 hPas gene product can also be analyzed by Northern blot technique, that is, the presence and quantity of RNA transcript of human hPas in cells can be analyzed.

Northern blot analysis of human hPas DNA and Western blot analysis of human hPas-specific antibodies can be used in combination to confirm the expression of human hPas in biological samples. Human hPas 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 hPas nucleotide coding sequence, preferably 15-50 consecutive nucleotides acid. The probe can be used to detect whether there is a nucleic acid molecule encoding human hPas in a sample.

The present invention also provides a method for detecting whether there is a human hPas 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 hPas 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 hPas nucleotide sequence and amino acid sequence of the present invention, human hPas 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 array of human cDNAs or genomic DNAs related to the human hPas gene, a human cDNA or genomic DNA library can be screened with DNA probes that are made against all or part of human hPas with 32P under low stringency conditions radioactively labeled. The most suitable cDNA library for screening is that from human Cushing's adrenal adenoma. 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 also commercially available, for example, from Clontech, Palo Alto, Cal. . This screening method can identify the nucleotide sequences of gene families related to human hPas.

Screening for human hPas homologues based on nucleotide similarity can be accomplished as follows. Human Cushing's adrenal adenoma 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 hPas gene sequence. To complete the identification of clones with DNA insertion sequences at least 70% homologous to the human hPas 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 hPas gene using DNA restriction enzyme analysis and DNA sequencing. The distribution of tissue expression can be analyzed using the Northern blotting technique described above.

Human hPas homologues can also be identified using antibodies against human hPas proteins or polypeptides. For example, commercially available or constructed expression libraries derived from cells or tissues such as Cushing's adrenal adenoma 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 specific human hPas antibodies. 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 hPas gene. Tissue expression profiles of newly identified genes can be analyzed similarly as described above.

The full-length human hPas nucleotide 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 hPas 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 synthesis of peptides 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, through fluorescence in situ hybridization (FISH), cDNA clones are hybridized with chromosomes in metaphase, and chromosome localization can be accurately performed. 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.

Using the human hPas protein of the present invention, through various conventional screening methods, substances that interact with human hPas, or receptors, inhibitors or antagonists, etc. can be screened out.

When the human hPas 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 hPas 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 preparation should match the mode of administration. The human hPas 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 hPas 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 micrograms/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 the homology comparison (FASTA) diagram of the amino acid sequences (GenPept Accession No. CAB57566) of the human hPas protein of the present invention and the Sulfolobus solfataricus Pas 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, generally according to routine conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (NewYork: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions.

Example 1

Cloning of human hPas gene

1. Tissue isolation (Cushing's adrenal adenoma isolation)

Cushing's adrenal adenomas were obtained from 5 normal adult male donors. Cushing's adrenal adenomas were removed within four hours after death and immediately placed in liquid nitrogen for cryopreservation.

2. 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

Use mRNA as a template to synthesize double-stranded cDNA, and reverse transcription primers are shown in SEQ ID NO. 1. After filling in the blunt end, add a linker containing EcoRI cutting point, and the linker sequence is shown in SEQ ID NO. 2 and 3. After phosphorylation of the EcoRI terminus, it was digested with XhoI restriction endonuclease for 1.5 hours, and then the fragments were separated. 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 carried out on ABI 377 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 hPas 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, primers are designed at the 5' or 3' end of the existing sequence, and the human Cushing's adrenal adenoma Marathon-Ready cDNA library (ClontechLab, Inc, USA ) for long-range PCR reactions. 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 the Cushing's adrenal adenoma 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 hPas 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'-CTGTTGGACTATGCCCCACT-3' (SEQ ID NO.4) as the forward primer, oligonucleotide R2: 5' -CACACACCTGCACTCACACA-3' (SEQ ID NO.5) was used as a reverse primer, and the total RNA of Cushing's adrenal adenoma was used as a template for RT-PCR amplification. The PCR conditions of R1/R2 were 94°C for 5 minutes, followed by It was subjected to 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 1 minute, 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 was 1292 bp. Then clone and sequence the PCR amplification product according to the conventional method to obtain the SEQ ID NO. The sequence shown in 6.

Example 2

Sequence information and homology analysis of human hPas gene:

The novel human hPas full-length cDNA of the present invention (GenBank Accession No. AF271782. It has not been disclosed before this application) is 1292bp in length, and the detailed sequence is shown in SEQ ID NO. 6, wherein the open reading frame is located at 332-1096 nucleotides. The amino acid sequence of human hPas was deduced according to the full-length cDNA, with a total of 254 amino acid residues, a molecular weight of 29505.64, and a pI of 6.99. For the detailed sequence, see SEQ ID NO. 7.

The full-length protein sequence of hPas was searched for protein homology in the Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database with BLAST program, and it was found that it had certain homology with the Pas gene of Sulfolobus solfataricus. At the amino acid level, it has 17.9% identity and 53.7% similarity with amino acid residues 24-202 of Sulfolobus solfataricus Pas protein (GenPept Accession No. CAB57566) (Supplementary Table 2). It can be seen from the above that the hPas gene and the Sulfolobus solfataricus Pas gene have high homology at the protein level, and it can be considered that the two are also highly similar in function.

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

Antibodies against human hPas of the present invention are 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 hPas nucleic acid (coding sequence or antisense sequence) of the present invention can be introduced into cells to increase the expression level of human hPas or inhibit the overexpression of human hPas. The human hPas 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 hPas. 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 hPas protein of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and/or better activity when administered to humans than homologous proteins derived from other species (such as Sulfolobus solfataricus). Low side effects (eg, less or no immunogenicity in humans).

Example 3

Tissue expression profile of human hPas gene

Electronic Northern Expression Profiles. Press Ton C． The method of et al. (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), will The hPas cDNA sequence is searched by BLAST in the humanEST database in the GCG software package. Among the obtained human ESTs, there are hundreds of ESTs with a probability value <10e-10 and an identity >95%, which can be regarded as the gene in the tissue. The transcript expression version of the gene was obtained, and the tissue profile of the expression gene was obtained. It is expressed in tissues such as liver, testis, fetal brain, etc., indicating that it plays an important role in these tissues and organs of the human body.

Example 4

Preparation and purification of human hPas polypeptide

In this example, the full-length human hPas 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 hPas 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 hPas (SEQ ID NO.6), primers were designed to amplify the complete coding reading frame (corresponding to more than 20 nucleotides at the 5' and 3' ends of the coding sequence respectively), and the 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 hPas 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 by the CaCl2 method, and the engineering strain DH5α-pGEX-2T-hPas containing the pGEX-2T-hPas expression vector was screened and identified.

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

Pick a single colony of DH5α-pGEX-2T-hPas engineering bacteria and shake it overnight in 3ml of LB medium containing 100μg/ml ampicillin, and pipette the culture solution into a new LB medium (containing 100μg /ml ampicillin) for about 3 hours, until OD ₆₀₀ reached 0.5, added IPTG to a final concentration of 1 mmol/L and continued to culture at 37°C for 0, 1, 2, 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-hPas fusion protein.

Extraction and Purification of GST-hPas Fusion Protein

The engineered strain DH5α-pGEX-2T-hPas expressing GST-hPas fusion expression protein was induced by 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 completely broken bacteria, shake at 37°C for 30 minutes, and centrifuge at 10,000 rpm for 10 minutes to precipitate glutathione bound to GST-hPas 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 30kDa is human hPas protein.

Example 5

Eukaryotic expression of human hPas protein or polypeptide in insect cells

1. Construction of Human hPas Baculovirus Expression Vector and Transfection to Sf9 Insect Cell Line

According to the full-length coding sequence of human hPas (SEQ ID NO. 6), primers for amplifying the complete coding reading frame were designed, and restriction endonuclease sites were introduced on the forward and reverse primers (this may vary depending on the vector selected). ) to construct expression vectors. Using the amplification product obtained in Example 1 as a template, after PCR amplification, the human hPas 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 with recombinant expression vector

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 performed. 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 with anti-human hPas 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 secondary antibody solution of biotin-labeled anti-human hPas primary antibody, 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 hPas were picked.

3. Extraction and purification of human hPas protein

The supernatant of Sf9 cell clones highly expressing human hPas 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, pH 7.8), 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, and add 2ml of 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 mM imidazole to obtain a purified protein. The eluate was stored at 4°C, and the purity of the extracted human hPas protein was detected by SDS-PAGE electrophoresis. The protein band at 30kDa is human hPas protein.

Example 6

Preparation of anti-human hPas antibody

1. Preparation of immunized mice and splenocytes: the human hPas protein obtained in Example 5 and Example 6 was separated by chromatography and then used for later use, or it could be separated by SDS-PAGE gel electrophoresis, and the electrophoresis band was separated from Gel and emulsified with an equal volume of complete Freund's adjuvant. Take 6-8 weeks old Balb/C female mice, and inject 50-100μg/0.2ml emulsified protein intraperitoneally to the mice. After 14 days, the same antigen emulsified with incomplete Freund's adjuvant was used to immunize the mice again with a dose of 50-100 μg/0.2ml, and it was 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. Feeder cells were prepared according to the method on page 371 of "Tissue Culture and Molecular Cytological Techniques" (supra).

3. Cell fusion was carried out according to the method on page 213 of "Tissue Culture and Molecular Cytology Techniques" (supra).

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 hPas protein is used for preliminary screening of antibody activity. Commonly used methods include: immunofluorescence test, emission immunoassay assay (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) SEQ ID NO. Information for 1 (i) Sequence features:

(A) Length: 50bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topological structure: linear (ii) Molecular type: oligonucleotide (iii) Sequence description: SEQ ID NO. 1GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT 50

(2) SEQ ID NO. 2 information

(i) Sequence features:

(A) Length: 13bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(iii) Sequence description: SEQ ID NO. 2

AATTCGGCAC GA G 13(3) SEQ ID NO. 3 information

(i) Sequence features:

(A) Length: 9bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(iii) Sequence description: SEQ ID NO. 3

GCCGTGCTC 9(4) SEQ ID NO. 4 Information (i). Sequence features:

(A) Length: 20bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topological structure: linear (ii). Molecular type: oligonucleotide (Ⅲ). Sequence description: SEQ ID NO. 4CTGTTGGACTATGCCCCACT 20(5) SEQ ID NO. 5 information (ⅰ). Sequence features:

(A) Length: 20bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topological structure: linear (ii). Molecular type: oligonucleotide (Ⅲ). Sequence description: SEQ ID NO. 5CACACACCTGCACTCACACA 20(6) SEQ ID NO. Information for 6 (i) Sequence characteristics:

(A) Length: 1292bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topological structure: linear (ii) Molecular type: nucleotide (iii) Sequence description: SEQ ID NO. 61 CTGTTGGACT ATGCCCCACT  GCCAGGAAAC AGGCGCCGGA AGGTTCTCTG51 ACAAGATCTC GCTTTCCTAG  GGCGGTGAAG GCGTTCAAAG GTCGGGAAGG101 GGCGCTGGGA GAAGCGGGGC  AGCGCTGAGC CATGCTCGCG AACTGTGGGT151 CTGTCTGTGA AGAGACCCAG  TTTCGTGGGA CCACGGTGGC GCCTGCGCTG201 GGAGGTGAGC TTGTGACAGA  GCGAAAACTA CAATTCCCAG CATTCCTGTG251 GTGCCAGAAC TACCTTGCCC  GAAAGCCTGT GCGAGATTTA CCCCGTCTTC301 CGCCTCCCTC CCACCGGAAA  ACTCTGAGGA CATGAATAGT CGCCAGGCTT351 GGCGGCTCTT TCTCTCCCAA  GGCAGAGGAG ATCGTTGGGT TTCAAGGCCC401 CGCGGGCATT TCTCGCCGGC  CCTGCGGAGA GAGTTCTTCA CTACCACAAC451 CAAGGAGGGA TATGATAGGC  GGCCAGTGGA TATAACTCCT TTAGAACAAA501 GGAAATTAAC TTTTGATACC  CATGCATTGG TTCAGGACTT GGAAACTCAT551 GGATTTGACA AAACACAAGC  AGAAACAATT GTATCAGCGT TAACTGCTTT601 ATCAAATGTC AGCCTGGATA  CTATCTATAA AGAGATGGTC ACTCAAGCTC651 AACAGGAAAT AACAGTACAA  CAGCTAATGG CTCATTTGGA TGCTATCAGG701 AAAGACATGG TCATCCTAGA  GAAAAGTGAA TTTGCAAATC TGAGAGCAGA751 GAATGAGAAA ATGAAAATTG  AATTAGACCA AGTTAAGCAA CAACTAATGC 801  ATGAAACCAG TCGAATCAGA GCAGATAATA AACTGGATAT  CAACTTAGAA851  AGGAGCAGAG TAACAGATAT GTTTACAGAT CAAGAAAAGC  AACTTATGGA901  AACAACTACA GAATTTACAA AAAAGGATAC TCAAACCAAA  AGTATTATTT951  CAGAGACCAG TAATAAAATT GACGCTGAAA TTGCTTCCTT  AAAAACACTG1001  ATGGAATCTA ACAAACTTGA GACAATTCGT TATCTTGCAG  CTTCAGTGTT1051  TACTTGCCTG GCAATAGCAT TGGGATTTTA TAGATTCTGG  AAGTAGTATT1101  AATGCTCATC CTGCTGTGGC TGTTGGCTTC TTAGAACACC  AAACCGGGAG1151  AGATTTACTT TGAACATTGT CAGTTGCAGC AAAAATTTAC  TACACAAGAT1201  TATTCGAAGT GTATACGGAC TAAAAGAGGA AGTGTTTTAG  AATGAGAAGA1251  GATACTGTGT CTTTATTGTG TGTGTGTGAG TGCAGGTGTG  TG(7)SEQID NO． Information for 7 (i) Sequence features:

(A) Length: 254 amino acids

(B) Type: amino acid

(C) chain: single chain

(D) Topological structure: linear (ii) Molecular type: polypeptide (iii) Sequence description: SEQ ID NO. 7：1  MNSRQAWRLF  LSQGRGDRWV  SRPRGHFSPA  LRREFFTTTT  KEGYDRRPVD51  ITPLEQRKLT  FDTHALVQDL  ETHGFDKTQA  ETIVSALTAL  SNVSLDTIYK101  EMVTQAQQEI  TVQQLMAHLD  AIRKDMVILE  KSEFANLRAE  NEKMKIELDQ151  VKQQLMHETS  RIRADNKLDI  NLERSRVTDM  FTDQEKQLME  TTTEFTKKDT201  QTKSIISETS  NKIDAEIASL  KTLMESNKLE  TIRYLAASVF  TCLAIALGFY251  RFWK

h．pep：人hPas蛋白氨基酸序列s．pep：S．solfataricusPas蛋白氨基酸序列(GenBank Accession No．CAB57566)

h. pep: amino acid sequence of human hPas protein s. pep: S． Amino acid sequence of solfataricusPas protein (GenBank Accession No. CAB57566)

Claims (11)

1. isolated dna molecular, it is characterized in that, it comprises: coding has the nucleotide sequence of the polypeptide of people hPas protein active, and shows at least 70% homology from the nucleotides sequence of Nucleotide 332-1096 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 332-1096 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 332-1096 position.

4. isolated people hPas 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 people hPas protein active, is characterised in that its step is as follows:

(1) nucleotide sequence that coding is had a polypeptide of people hPas protein-active operationally is connected in expression regulation sequence, form people hPas protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 332-1096 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 people hPas;

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

(4) isolate polypeptide with people hPas protein-active.

9. energy and the described people hPas of claim 7 protein polypeptide 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 people hPas 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 people hPas nucleotide coding sequence, and can be positioned at the both sides or the centre of this encoding sequence, primer length is 15～50 Nucleotide.