CN1249346A - Human gene sequence, its encoded polypeptide and its preparing process - Google Patents

Abstract

The present invention discloses a new human gene nucleotide sequence, specifically, the cDNA sequence of human WSB1. The protein coded by said sequence is the homolog of mouse WSB1. The present invention also relates to the polypeptide coded by said nucleotide sequence and the application and preparing process of said polynucleotide and said polypeptide.

Description

New human gene sequence, its coded polypeptide and preparing process

The invention relates to the field of genetic engineering, in particular to a novel human gene nucleotide sequence. More particularly, the invention relates to the cDNA sequence of human WSB1, which is a homologue of murine WSB 1. The present invention also relates to the polypeptide coded by the nucleotide sequence, the application of these polynucleotides and polypeptides, and the production process of said polynucleotides and polypeptides.

WSB1 belongs to a member of a subfamily (WD-40) in the cytokine signaling repressor family (SOC). WSB1, WSB2 are two genes found in mice, which are thought to be involved in the inhibition of cytokine signaling. Cytokine signaling is a common phenomenon found in all eukaryotes and prokaryotes. Especially in eukaryotes, cytokines are a class of factors with important biological functions, which regulate and control the secretion of important proteins in vivo, which play important roles in the development and differentiation of organisms. Cytokines exert their function of inducing cell division and differentiation through a signaling pathway called JAK-STAT. The cytokine signaling repressor can be induced by a plurality of cytokines which can activate JAK-STAT pathway, and has an inhibiting effect on the JAK-STAT pathway.

Currently, research is still underway for members of the WSB subfamily, and relatively early on for the SOC (cytokine signaling repressor family). According to the difference of central functional areas contained in the members in the SOC family, the SOC family is divided into four subfamilies, namely CIS, WSB, SRPY and ASB. The earliest member of the SOC gene family was CIS, which was discovered in mice by Yoshimura A. et al in 1995 (EMMO J.1995, 14, 2816-2826). Then, genes of CIS family members SOCS-1, SOCS-2, etc. were isolated and cloned in 1997 by Starr R et al (Nature1997, 387 (6636): 917-. Recently, clones of members of the SPRY and ASB families were obtained from mice, respectively. WSB1 is a gene found in mice. It is a gene cloned from mouse spermatocyte cDNA library by Hilton D.J. et al in 1998 (Proc. Natl. Acad. Sci. USA 1998, 95: 114-. WSB1 and WSB2 belong to the SOC family with some of the genes mentioned above, and it has been found that although they differ somewhat in domain, they function very similarly and act as regulators of protein transport in cells.

However, prior to the present invention, no members belonging to the WSB subfamily have been found in humans.

It is an object of the present invention to provide a novel polynucleotide sequence encoding a human protein homologous to mouse WSB1, the human gene of the present invention homologous to mouse WSB1 being designated human WSB1(GenBank accession No. af064257) (note that the sequences registered prior to this application were not publicly disclosed for some time because of privacy applied).

It is another object of the present invention to provide a novel protein, designated human WSB1 protein.

It is still another object of the present invention to provide a method for producing the novel human WSB1 protein using recombinant technology.

The invention also provides the application of the nucleotide sequence and protein of the human WSB 1.

In one aspect of the invention, there is provided an isolated DNA molecule comprising: a nucleotide sequence for coding a polypeptide with the activity of human WSB1 protein, wherein the nucleotide sequence has at least 70 percent of homology with the nucleotide sequence from nucleotide 31 to 1296 in SEQ ID NO. 3; or the nucleotide sequence can be hybridized with the nucleotide sequence from 31 to 1296 of nucleotides in SEQ ID NO.3 under moderate stringent conditions. Preferably, the sequence encodes a polypeptide having the sequence shown in SEQ ID NO. 4. More preferably, the sequence has the nucleotide sequence from 31 to 1296 in SEQ ID NO. 3.

In another aspect of the present invention, there is provided an isolated human WSB1 protein polypeptide comprising: a polypeptide having the amino acid sequence of SEQ ID No.4, or an active fragment or active derivative thereof. Preferably, the polypeptide is a polypeptide having the sequence of SEQ ID No. 4.

In another aspect of the present invention, there is provided a vector containing the isolated DNA described above.

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

In another aspect of the present invention, there is provided a method for producing a polypeptide having the activity of human WSB1 protein, the method comprising:

(a) operably linking a nucleotide sequence encoding a polypeptide with the activity of the human WSB1 protein to an expression regulatory sequence to form a human WSB1 protein expression vector, wherein the nucleotide sequence has at least 70 percent of homology with the nucleotide sequence from 31 to 1296 of nucleotides in SEQ ID NO. 3;

(b) transferring the expression vector in the step (a) into a host cell to form a recombinant cell of the human WSB1 protein;

(c) culturing the recombinant cell of step (b) under conditions suitable for expression of the human WSB1 protein polypeptide;

(d) isolating the polypeptide with the activity of the human WSB1 protein.

In a specific embodiment of the invention, the isolated polynucleotide of the invention has a full length of 1325 nucleotides, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located between nucleotides 31 and 1296.

In the present invention, "isolated", "purified" or "substantially pure" DNA means that the DNA or fragment has been isolated from the sequences which flank it in its natural state, and that the DNA or fragment has been separated from components which accompany the nucleic acid in its natural state, and from the proteins which accompany it in the cell.

In the present invention, the term "human WSB1 protein (or polypeptide) encoding sequence" refers to a nucleotide sequence encoding a polypeptide having the activity of human WSB1 protein, such as the nucleotide sequence of positions 31-1296 in SEQ ID NO.4 and degenerate sequences thereof. The degenerate sequence is a sequence which is produced by substituting one or more codons in the nucleotides from the coding box 31 to 1296 of the sequence of SEQ ID No.4 with a degenerate codon which codes for the same amino acid. Due to the degeneracy of the codons, a degenerate sequence with a homology as low as about 70% to the nucleotide sequence 31-1296 of SEQ ID NO.4 also codes for the sequence depicted in SEQ ID NO. 4. The term also includes nucleotide sequences that hybridize under moderately stringent conditions, more preferably under highly stringent conditions, to the nucleotide sequence of SEQ ID NO.3 from nucleotide 31 to nucleotide 1296. The term also includes nucleotide sequences having at least 70%, preferably at least 80%, more preferably at least 90% homology to the nucleotide sequence from nucleotides 31 to 1296 of SEQ ID NO. 3.

The term also includes variants of the open reading frame sequence of SEQ ID No.3 that encode proteins having the same function as human WSB 1. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 90, preferably 1 to 60, more preferably 1 to 20, most preferably 1 to 10) nucleotides, and addition of several (usually less than 60, preferably less than 30, more preferably less than 10, most preferably less than 5) nucleotides at the 5 'and/or 3' end.

In the present invention, a "substantially pure" protein or polypeptide means that it constitutes at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (by dry weight or wet weight) of the total material of the sample. Purity can be measured by any suitable method, such as 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 WSB1 protein polypeptide" refers to a polypeptide having the sequence of SEQ id No.4 having the activity of human WSB1 protein. The term also includes variants of the sequence of SEQ ID NO.4 that have the same function as human WSB 1. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of human WSB1 protein.

Variants of the polypeptide include: homologous sequences, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to human WSB1 DNA under high or low stringency conditions, and polypeptides or proteins obtained using antisera to human WSB1 polypeptides. The invention also provides other polypeptides, such as fusion proteins comprising a human WSB1 polypeptide or fragment thereof. In addition to the nearly full-length polypeptide, the invention also includes soluble fragments of the human WSB1 polypeptide. Typically, the fragment has at least about 10 contiguous amino acids, typically 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 100 contiguous amino acids of the human WSB1 polypeptide sequence.

The invention also provides analogues of human WSB1 protein or polypeptide. These analogs may differ from the native human WSB1 polypeptide by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

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

The invention also includes antisense sequences of the coding sequence of the human WSB1 polypeptide. Such antisense sequences are useful for inhibiting the expression of human WSB1 in a cell.

The present invention also includes a nucleotide molecule useful as a probe, which molecule typically has from 8 to 100, preferably from 15 to 50, consecutive nucleotides of the nucleotide sequence of human WSB 1. The probe can be used to detect the presence of a nucleic acid molecule encoding human WSB1 in a sample.

The invention also includes a method of detecting the nucleotide sequence of human WSB1, which comprises hybridizing the probe described above to a sample and detecting whether the probe has bound. Preferably, the sample is the product of a PCR amplification, wherein the PCR amplification primers correspond to the coding sequence for the human WSB1 polypeptide and may be located on both sides or in the middle of the coding sequence. Primers are typically 20-50 nucleotides in length.

In the present invention, various carriers known in the art, such as commercially available carriers, can be used.

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

In another aspect, the invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the polypeptide encoded by human WSB1 DNA or a fragment thereof. Herein, "specificity" means that the antibody binds to the human WSB1 gene product or fragment. Preferably, those antibodies that bind to the human WSB1 gene product or fragment, but do not recognize and bind to other unrelated antigenic molecules. The antibodies of the present invention include those molecules that bind to and inhibit human WSB1 protein, as well as those antibodies that do not affect the function of human WSB1 protein. The invention also includes those antibodies that bind to the human WSB1 gene product in modified or unmodified form.

The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)₂A fragment; an antibody heavy chain; light body resistanceA chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies that have murine antibody binding specificity but retain portions of the antibody from a human.

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, the purified human WSB1 gene product, or antigenic fragments thereof, can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing human WSB1 or antigenic fragments thereof can be used to immunize animals to produce antibodies. The antibody of the present invention may also be a monoclonal antibody. Such Monoclonal Antibodies can be prepared using hybridoma technology (see 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; Hammerling et al, In Monoclonal Antibodies and T Cell hybrids, Elsevier, N.Y., 1981). The antibodies of the present invention include antibodies that block the function of human WSB1 as well as antibodies that do not affect the function of human WSB 1. The antibodies of the invention can be obtained by conventional immunization techniques using fragments or functional regions of the human WSB1 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to an unmodified form of the human WSB1 gene product can be produced by immunizing an animal with a gene product produced in a prokaryotic cell (e.g., e.coli); antibodies that bind to post-translationally modified forms (e.g., glycosylated or phosphorylated proteins or polypeptides) can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell).

The full-length sequence of the nucleotide sequence of the human WSB1 or a fragment thereof can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method. For the PCR amplification method, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a known method as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

In one embodiment of the present invention, the cDNA nucleotide sequence of human WSB1 was obtained by synthesizing a forward primer A1: 5 '-GTATTCCCGGAATCAGACGGTGC-3' and reverse primer A2: 5 '-GTCCCTACTACTAGGGAAGGCAG-3', PCR was performed to obtain a 1325bp (A1/A2) target fragment. The full-length cDNA sequence of SEQ ID NO.3 is obtained after sequencing.

WSB1, WSB2 belong to members of a subfamily (WSB) of the cytokine signaling repressor family (SOC). WSB1 and WSB2 are believed to be involved in the inhibition of cytokine signaling. Cytokines are a class of factors with important biological functions, which regulate the secretion of important proteins in vivo, which play important roles in the development and differentiation of organisms. The cytokine signaling repressor can be induced by a plurality of cytokines which can activate JAK-STAT pathway, and has an inhibiting effect on the JAK-STAT pathway. The human WSB1 gene of the present invention is highly homologous to mouse WSB1, and also has higher homology to WSB2, which indicates that it is a homologous gene of mouse WSB1 in human, and has similar biology as WSB1 (and WSB 2).

In the drawings, there is shown in the drawings,

FIG. 1 is a diagram showing homology comparison of nucleic acid sequences of human WSB1(hWSB1) and mouse WSB1(mWSB1) according to the present invention. Wherein identical nucleotides are denoted by "|".

FIG. 2 is a diagram showing the homology comparison of the amino acid sequences of the human WSB1 protein (hWSB1) and the mouse WSB1 protein (mWSB1) of the present invention. Where identical amino acids are marked with "|" between the two sequences and similar amino acids are marked with "·". Similar amino acids are: a, S, T; d, E; n, Q; r, K; i, L, M, V; f, Y and W.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

Example 1

Cloning and determination of cDNA sequence of human WSB1

1. Amplification with primers

Using a human testis λ gt11cDNA library (purchased from Clontech) as a template, a pair of oligonucleotides as primers-a 1: 5 '-GTATTCCCGGAATCAGACGGTGC-3' (SEQ ID NO: 1) is a forward primer, oligonucleotide A2: 5 '-GTCCCTACTACTAGGGAAGGCAG-3' (SEQ ID NO: 2) was used as a reverse primer for PCR. PCR conditions for primer pair A1, A2 were 93 ℃ for 4 minutes followed by 35 cycles at 93 ℃ for 1 minute, 70 ℃ for 1 minute and 72 ℃ for 1 minute, and final extension at 72 ℃ for 5 minutes; the PCR fragment obtained by electrophoresis was amplified to a desired fragment length of about 1300 base pairs using A1 and A2 as primer sets.

Sequencing of PCR products

The PCR amplification products obtained as above were ligated with pGEM-T vector (Promega), respectively, Escherichia coli JM103 was transformed, plasmids were extracted with QIAprep Plasmid kit (QIAGEN), the inserted fragments were subjected to directed serial deletion with double-stranded nested deletion kit (Pharmacia), and the deletions were subjected to PCRAnd performing quick identification and sequencing. Using SequiTherm EXCEL^TMDNA sequencing kit (Epicentre Technologies) sequences the successively truncated deletants, and finally uses computer software to splice the sequence to obtain a full-length cDNA sequence, which is 1325bp in total, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is positioned at 31-1296 nucleotides.

The amino acid sequence of the human WSB1 is deduced according to the obtained full-length cDNA sequence, and the amino acid sequence is detailed in SEQ ID NO.4, wherein the total number of the amino acid residues is 421.

Example 2

Homology comparison

The full-length cDNA sequence of human WSB1 and its encoded protein were used to perform nucleic acid and protein homology searches in the Non-redundant GenBank + EMBL + DDBJ + PDB database and the Non-redundant GenBank CDStranslations + PDB + SwissProt + Spupdate + PIR database using the BLAST program. As a result, they were found to have very high homology with mouse WSB1 gene and its encoded protein, about 87% identity at nucleic acid level (FIG. 1), and 92% and 95% identity and similarity at protein level (FIG. 2), respectively, so that human WSB1 of the present invention is a homolog of mouse WSB1 in humans, and has the same or similar function as WSB family members (especially mouse WSB 1).

WSB1 belongs to a member of the cytokine signal repressor family (SOC) subfamily (WSB). In vivo, cytokines primarily play a role in regulating and controlling the secretion of important proteins by cells, and these proteins also play an important role in the development and differentiation of organisms. The cytokine protein product exerts its function of inducing cell division and differentiation through a signaling pathway called JAK-STAT. The repressor of cytokine signaling can be induced by a number of cytokines that activate the JAK-STAT pathway and has inhibitory effects on the JAK-STAT pathway (Nature 1995, 377(19), 591-594). All members of the SOC family share similar structural features, with their N-termini containing amino acid components of varying lengths, and the C-termini having a non-discriminating, fancy structure, the SOCS box. SOC families are divided into four subfamilies, depending on the composition of the central domain: the CIS subfamily containing the SH2 domain, the WSB subfamily containing the WD-40 domain, the SSB subfamily containing the SPRY domain, and the ASB subfamily containing the ankyrin repeat domain (J Leukoe Biol1998, 63 (6): 665-. The function of human WSB1 can be deduced from the functions of these genes or proteins based on the principle that the structure determines the function and the structure is similar.

It is known that signal transduction of cytokines and inhibition thereof play an important role in regulating secretion and transport of important proteins in organisms, and thus WSB1 plays an important role in organisms.

WSB family members contain both WD-40 repeats and SOCS domains. Its function in the organism is also determined by these two functional regions. In particular, there are 2 characteristic sequences of the WD-40 repeat consisting of 15 amino acids in the amino acid sequence of human WSB 1:

——(L/I/V/M/S/T/A/C)-(L/I/V/M/F/Y/W/S/T/A/G/C)-(L/I/M/S/T/A/G)-(L/I/V/M/S/T/A/G/C)-X2-(D/N)-X2-(L/I/V/M/W/S/T/A/C)-X-(L/I/V/M/F/S/T/A/G)-W-(D/E/N/)-(L/I/V/M/F/S/T/A/G/C/N)

[ note: x in the sequence is any amino acid, the number of the "2" and the like is the number of the amino acids, and the "(L/I/V/M/S/T/A/C)" indicates that one amino acid is arbitrarily selected from the 8 amino acids ].

Sequence segments in the proteins of the invention that fit the above pattern are: LATGLNNGRIKIWDV (positions 140 and 155 in SEQ ID NO. 4), LVSASRDKTLRVWDL (positions 183 and 198 in SEQ ID NO. 4). This further demonstrates that human WSB1 of the present invention is a member of the WD-40 family.

In addition, it was found that 1 GH-WD repeat sequence was also present in human WSB1 of the present invention

-X_6-94-(GH-X_23-41-WD)-

For a description of this GH-WD repeat sequence, see Eva j. neer et al, Nature1994, 371 (22): 297-300. Wherein,X_6-94represents a variable length of 6 to 94 amino acids, X_23-41Representing a variable length of 23-41 amino acids, preferably with a spacing between GH and WI of 27 ± 2 amino acids.

The sequence segments of the protein of the invention which conform to the above-mentioned pattern are: GHHHDVVACDFSPDGALLATASYDTRVVIWD (position 253-284 in SEQ ID NO. 4), a GH-WD repeat sequence, i.e., a WD repeat sequence, wherein GH and WD are separated by 27 amino acids, which further demonstrates that human WSB1 of the present invention is a member of the WD-40 family.

The WD-40 family is very widely distributed, from the nucleus to the cytoplasmic membrane, and is also considered to be a component of the cytoskeleton. Yeast is considered to be the best mode of studying the function of the WD-40 repeat, which is involved in protein interactions. The WD-40 repeat typically interacts with the repeats of the TPR family members, thereby modulating protein interaction and transport. It was found that the interaction of WD-40 with the TPR repeat is accomplished by inhibiting the transcriptional expression of nucleic acids (FEBS Lett.1992307(2) 131-. In summary, human WSB belongs to a member of the WD-40 family and has the above-described related functions of the WD-40 family members.

In addition, members of the SOC family each contain a SOCs box, and inhibitors of cytokine signaling mainly play a role in inhibiting cytokine signaling in the organism. Research shows that the function of the protein is probably to inhibit the normal transport of the protein by combining with a special sequence of a receptor cell so as to prevent the protein from combining with the receptor. When the secreted proteins have exceeded their normal levels in the cell, negative feedback is generated by the cytokine repressor protein to control the secretion of these proteins by the cytokine. The SOCS box is probably the functional region where these proteins play a role, and after the target protein is combined with the SOCS box, the SOC protein makes a correct judgment to determine the speed of protein production, and the signal transduction protein transports the target protein to a certain position in the cell and stabilizes the target protein at the correct position of the cell. The signaling repressor protein may also inhibit the transport of the protein by binding to the kinase through the SOCS box, inhibiting the catalytic activity of the kinase (Proc. Natl. Acad. Sci. USA 1998, 95: 114-. The C-terminus of the human WSB1 polypeptide of the invention also contains a region of high homology to the SOCS box, suggesting that it may also have the same or similar function as a member of the SOC family.

From the above, it is possible that members of the WSB subfamily play a dual role in this process as well. It plays a role in regulating and guiding the correct protein transport in organisms. Plays an important role in the processes of various physiological functions (such as cell development, differentiation, proliferation and immunity) depending on JAK-STAT pathway. This also provides the basis for further studies on the in vivo effect of signal factor repressors.

In addition to being a member of this family for further functional studies, human WSB1 of the present invention can also be used to produce fusion proteins with other proteins, such as immunoglobulins. Furthermore, the present inventors WSB1 can also fuse or exchange fragments with other members of the family to produce novel proteins. For example, the N-terminus of our WSB1 was exchanged with the N-terminus of mouse WSB1 to generate a new protein with higher activity or new properties.

Antibodies against WSB1 of the present inventors were used to screen other members of the family or to affinity purify related proteins (e.g. other members of the family).

In addition, the WSB1 nucleic acid (coding sequence or antisense sequence) of the present inventors can be introduced into cells to increase the expression level of human WSB1 or to inhibit the overexpression of human WSB 1. The human WSB1 protein or its active polypeptide fragment can be used for treating or alleviating the relative diseases caused by the deletion, non-function or abnormality of human WSB 1. In addition, the nucleic acid sequences or antibodies according to the invention can be used for relevant diagnostic or prognostic determinations.

Example 3

Expression of human WSB1 in E.coli

In this example, the cDNA sequence encoding human WSB1 was amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' ends of the DNA sequence to obtain human WSB 1cDNA as an insert.

The 5' oligonucleotide primer sequences used in the PCR reaction were:

5＇-CAGAGTCGACATGGCCAGCTTTCCCCCGAG-3＇(SEQ ID NO.5)，

the primer contains the restriction site of SalI restriction endonuclease, which is followed by 20 nucleotides of the coding sequence of human WSB1 beginning with the start codon;

the 3' end primer sequence is as follows:

5’-CCAAGTCGACCTAAATACGATACGAGAGAAAC-3’(SEQ ID NO.6)，

the primer contains the restriction enzyme cutting site of SalI restriction enzyme, a translation terminator and a partial coding sequence of human WSB 1.

The restriction enzyme cleavage site on the primer corresponds to that on the bacterial expression vector pQE-9(Qiagen Inc., Chatsworth, Calif.), which encodes for antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a Ribosome Binding Site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector and the insert were digested with SalI, and the insert was subsequently ligated to the pQE-9 vector and the open reading frame was kept starting at the bacterial RBS. Coli strain purchased from Qiagen under the trade name M15/rep4, M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and carries kanamycin resistance (Kan)^r). Transformants were selected on LB plates containing Amp and Kan, plasmids were extracted, and sequencing verified that the cDNA fragment of human WSB1 had been correctly inserted into the vector.

Positive transformant clones containing the desired construct were cultured overnight (O/N) in LB liquid medium supplemented with Amp (100. mu.g/ml) and Kan (25. mu.g/ml). The overnight (O/N) cultures were diluted at a dilution rate of 1: 100 to 1: 250 and inoculated into a large volume of medium and cells were cultured to 600 optical densities (OD600) of 0.4 to 0.6, and IPTG ("isopropylthio-. beta. -D-galactoside") was added to a final concentration of 1 mM. IPTG-induced initiation of P/O leads to increased levels of gene expression by inactivation of the lacI repressor. The cells were further cultured for 3-4 hours, followed by centrifugation (6000 Xg, 20 minutes). The inclusion bodies were sonicated, the cells were collected and the cell pellet was dissolved in 6M guanidine hydrochloride. After clarification, the solubilized human WSB1 was purified from solution by nickel-chelate column chromatography under conditions that allowed for tight binding of the 6-His tag-containing protein. Human WSB1 was eluted from the column with 6M guanidine hydrochloride (pH 5.0). Several methods can be used to denature precipitated proteins from guanidine hydrochloride. Alternatively, a dialysis step is used to remove guanidine hydrochloride or to isolate the purified protein from the nickel-chelating column. The purified protein was bound to a second column with a decreasing linear guanidine hydrochloride gradient. Proteins denature when bound to the column and are subsequently eluted with guanidine hydrochloride (ph 5.0). Finally, the soluble proteins were dialyzed against PBS and then the proteins were stored in a stock solution with a final concentration of 10% (w/v) glycerol.

The size of the expressed protein was identified to be about 48kDa by electrophoresis on a 12% SDS-PAGE gel.

In addition, amino acids of 10 amino acids each of the N-and C-termini of the protein were sequenced by a conventional method, and found to be identical to the sequence of SEQ ID NO. 4.

Example 4

Expression of human WSB1 in eukaryotic cells (CHO cell line)

In this example, the cDNA sequence encoding human WSB1 was amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' ends of the DNA sequence to obtain human WSB 1cDNA as an insert.

The 5' oligonucleotide primer sequences used in the PCR reaction were:

5 '-CAGAGAATTCATGGCCAGCTTTCCCCCGAG-3' (SEQ ID NO.7), the primer containing the restriction site for the EcoRI restriction endonuclease followed by 20 nucleotides of the coding sequence for human WSB1 beginning with the start codon;

the 3' end primer sequence is as follows:

5'-CCAATCTAGACTAAATACGATACGAGAGAAAC-3' (SEQ ID NO.8) the primer contains the XbaI restriction enzyme cleavage site, a translation terminator and part of the coding sequence of human WSB 1.

The restriction enzyme cleavage site on the primer corresponded to that on the CHO cell expression vector pcDNA3, which encoded antibiotic resistance (Amp)^rAnd Neo^r) A bacteriophage origin of replication (fl ori), a viral origin of replication (SV40 ori), a T7 promoter, a viral promoter (P-CMV), an Sp6 promoter, an SV40 promoter, an SV40 tailing signal and corresponding polyA sequence, a BGH tailing signal and corresponding polyA sequence.

The pcDNA3 vector and the insert were digested with EcoRI, XbaI and subsequently ligated into the pcDNA3 vector. Coli DH5 a strain was subsequently transformed with the ligation mixture. Transformants were selected on LB plates containing Amp and clones containing the desired construct were grown overnight (O/N) in LB liquid medium supplemented with Amp (100. mu.g/ml). Extracting plasmid, using HindIII enzyme to identify size and direction of inserted fragment, and sequencing to verify that cDNA fragment of WSB1 has been correctly inserted into vector.

Transfection of CHO cells with plasmids was performed by Lipofectin using the Lipofectin kit (GiBco Life). 48 hours after transfection, cells were collected and cell supernatants were assayed for protease expression by 2-3 weeks of continuous G418 pressure screening. Removing G418, and continuously subculturing; and (4) carrying out extreme dilution on the mixed clone cells, and selecting cell subclones with higher protein activity. The above-mentioned positive subclones were cultured in a large amount according to a conventional method. After 48 hours, the cells and supernatant were collected and disrupted by sonication. The active peak of the protein was collected on a pre-equilibrated Superdex G-75 column using a 50mM Tris-HCl (pH7.6) solution containing 0.05% Triton as an equilibration solution and an eluent. Then, the protein was subjected to gradient elution using 50mM Tris-HCl (pH8.0) solution containing 0-1M NaCl as an eluent by using a DEAE-Sepharose column equilibrated with 50mM Tris-HCl (pH8.0), and the active peak of the protein was collected. Then, the expressed protein solution was dialyzed against PBS (pH 7.4). And finally freeze-drying and storing.

The size of the expressed protein was identified to be 48kDa by electrophoresis on 12% SDS-PAGE gel.

In addition, amino acids of 10 amino acids each of the N-and C-termini of the protein were sequenced by a conventional method, and found to be identical to the sequence of SEQ ID NO. 4.

Example 5

Preparation of antibodies

The recombinant proteins obtained in examples 3 and 4 were used to immunize animals to produce antibodies as follows. The recombinant protein is separated by chromatography and is ready for use. Separation can also be carried out by SDS-PAGE gel electrophoresis, the bands being excised from the gel and emulsified with equal volumes of complete Freund's adjuvant. Mice were injected intraperitoneally with 50-100. mu.g/0.2 ml of emulsified protein. After 14 days, mice were boosted by intraperitoneal injection with 50-100. mu.g/0.2 ml of the same antigen emulsified with incomplete Freund's adjuvant. Boosters were performed every 14 days, at least three times. The specific reactivity of the antiserum obtained was evaluated by its ability to precipitate the translation product of the human WSB1 gene in vitro.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing (1) general information: (i) the applicant: university of double denier (ii) title of invention: a new human gene sequence, its coded polypeptide and its preparing process (iii) sequence number: (2) SEQ ID NO: 1 information (i) sequence characteristics

(A) Length: 23 bases

(B) Type (2): nucleic acids

(C) Chain property: single strand

(D) Topological structure: linear (ii) molecular type: oligonucleotide (xi) sequence description: SEQ ID NO: 1: GTATTCCCGG AATCAGACGG TGC 23(2) SEQ ID NO: 2 information (i) sequence characteristics

(A) Length: 23 bases

(B) Type (2): nucleic acids

(C) Chain property: single strand

(D) Topological structure: linear (ii) molecular type: oligonucleotide (xi) sequence description: SEQ ID NO: 2GTCCCTACTA CTAGGGAAGG CAG 23(2) information of SEQ ID NO. 3: (i) sequence characteristics:

(A) length: 1325bp

(B) Type (2): nucleic acids

(C) Chain property: single strand

(D) Topological structure: linear (ii) molecular type: cdna (xi) sequence description: information of SEQ ID No.31 GTATTCCCGG AATCAGACGG TGCCCCATAG ATGGCCAGCT TTCCCCCGAG GGTCAACGAG61 AAAGAGATCG TGAGATCACG TACTATAGGT GAACTTTTAG CTCCTGCAGC TCCTTTTGAC121 AAGAAATGTG GTCGTGAAAA TTGGACTGTT GCTTTTGCTC CAGATGGTTC ATACTTTGCT181 TGGTCACAAG GACATCGCAC AGTAAAGCTT GTTCCGTGGT CCCAGTGCCT TCAGAACTTT241 CTCTTGCATG GCACCAAGAA TGTTACCAAT TCAAGCAGTT TAAGATTGCC AAGACAAAAT301 AGTGATGGTG GTCAGAAAAA TAAGCCTCGT GAACATATTA TAGACTGTGG AGATATAGTC361 TGGAGTCTTG GTTTTGGGTC ATCAGTTCCA GAAAAACAGA GTCGCTGTGT AAATATAGAA421 TGGCATCGCT TCAGATTTGG ACAAGATCAG CTACTTCTTG CTACAGGGTT GAACAATGGG481 CGTATCAAAA TATGGGATGT ATATACAGGA AAACTCCTCC TTAACTTGGT AGATCATACT541 GAAGTGGTCA GAGATTTAAC TTTTGCTCCA GATGGAAGCT TGATCCTGGT GTCAGCTTCA601 AGAGACAAAA CTCTCAGAGT ATGGGACCTG AAAGATGATG GAAACATGAT GAAAGTATTG661 AGGGGGCATC AGAATTGGGT GTACAGCTGT GCATTCTCTC CTGACTCTTC TATGCTGTGT721 CCAGTCGGAG CCAGTAAAGC AGTTTTCCTT TGGAATATGG ATAAATACAC CATGATACGG781 AAACTAGAAG GACATCACCA TGATGTGGTA GCTTGTGACT TTTCTCCTGA TGGAGCATTA841 CTGGCTACTG CATCTTATGA TACTCGAGTA TATATCTGGG ATCCACATAA TGGAGACATT901 CTGATGGAAT TTGGGCACCT GTTTCCCCCA CCTACTCCAA TATTTGCTGG AGGAGCAAAT961 GACCGGTGGG TACGATCTGT ATCTTTTAGC CATGATGGAC TGCATGTTGC AAGCCTTGCT1021 GATGATAAAA TGGTGAGGTT CTGGAGAATT GATGAGGATT ATCCAGTGCA AGTTGCACCT1081 TTGAGCAATG GTCTTTGCTG TGCCTTCTCT ACTGATGGCA GTGTTTTAGC TGCTGGGACA1141 CATGACGGAA GTGTGTATTT TTGGGCCACT CCACGGCAGG TCCCTAGCCT GCAACATTTA1201 TGTCGCATGT CAATCCGAAG AGTGATGCCC ACCCAAGAAG TTCAGGAGCT GCCGATTCCT1261 TCCAAGCTTT TGGAGTTTCT CTCGTATCGT ATTTAGAAGA TTCTGCCTTC CCTAGTAGTA1321 GGGAC (2) SEQ ID No. 4: (i) sequence characteristics:

(A) length: 421 amino acids

(B) Type (2): amino acids

(D) Topological structure: linear (ii) molecular type: polypeptide (xi) sequence description: SEQ ID NO.41 Met Ala Ser Phe Pro Pro Arg Val Asn Glu Lys Glu Ile Val Arg16 Ser Arg Thr Ile Gly Glu Leu Leu Ala Pro Ala Ala Pro Phe Asp31 Lys Lys Cys Gly Arg Glu Asn Trp Thr Val Ala Phe Ala Pro Asp46 Gly Ser Tyr Phe Ala Trp Ser Gln Gly His Arg Thr Val Lys Leu61 Val Pro Trp Ser Gln Cys Leu Gln Asn Phe Leu Leu His Gly Thr76 Lys Asn Val Thr Asn Ser Ser Ser Leu Arg Leu Pro Arg Gln Asn91 Ser Asp Gly Gly Gln Lys Asn Lys Pro Arg Glu His Ile Ile Asp106 Cys Gly Asp Ile Val Trp Ser Leu Gly Phe Gly Ser Ser Val Pro121 Glu Lys Gln Ser Arg Cys Val Asn Ile Glu Trp His Arg Phe Arg136 Phe Gly Gln Asp Gln Leu Leu Leu Ala Thr Gly Leu Asn Asn Gly151 Arg Ile Lys Ile Trp Asp Val Tyr Thr Gly Lys Leu Leu Leu Asn166 Leu Val Asp His Thr Glu Val Val Arg Asp Leu Thr Phe Ala Pro181 Asp Gly Ser Leu Ile Leu Val Ser Ala Ser Arg Asp Lys Thr Leu196 Arg Val Trp Asp Leu Lys Asp Asp Gly Ash Met Met Lys Val Leu211 Arg Gly His Gln Asn Trp Val Tyr Ser Cys Ala Phe Ser Pro Asp226 Ser Ser Met Leu Cys Pro Val Gly Ala Ser Lys Ala Val Phe Leu241 Trp Asn Met Asp Lys Tyr Thr Met Ile Arg Lys Leu Glu Gly His256 His His Asp Val Val Ala Cys Asp Phe Ser Pro Asp Gly Ala Leu271 Leu Ala Thr Ala Ser Tyr Asp Thr Arg Val Tyr Ile Trp Asp Pro286 His Asn Gly Asp Ile Leu Met Glu Phe Gly His Leu Phe Pro Pro301 Pro Thr Pro Ile Phe Ala Gly Gly Ala Asn Asp Arg Trp Val Arg316 Ser Val Ser Phe Ser His Asp Gly Leu His Val Ala Ser Leu Ala331 Asp Asp Lys Met Val Arg Phe Trp Arg Ile Asp Glu Asp Tyr Pro346 Val Gln Val Ala Pro Leu Ser Asn Gly Leu Cys Cys Ala Phe Ser361 Thr Asp Gly Ser Val Leu Ala Ala Gly Thr His Asp Gly Ser Val376 Tyr Phe Trp Ala Thr Pro Arg Gln Val Pro Ser Leu Gln His Leu391 Cys Arg Met Ser Ile Arg Arg Val Met Pro Thr Gln Glu Val Gln406 Glu Leu Pro Ile Pro Ser Lys Leu Leu Glu Phe Leu Ser Tyr Arg421 Ile (2) SEQ ID NO: 5 information (i) sequence characteristics

(A) Length: 30 bases

(B) Type (2): nucleic acids

(C) Chain property: single strand

(D) Topological structure: linear (ii) molecular type: oligonucleotide (xi) sequence description: SEQ ID NO: 5: CAGAGTCGAC ATGGCCAGCT TTCCCCCGAG 30(2) SEQ ID NO: 6 information (i) sequence characteristics

(A) Length: 32 bases

(B) Type (2): nucleic acids

(C) Chain property: single strand

(D) Topological structure: linear (ii) molecular type: oligonucleotide (xi) sequence description: SEQ ID NO: 6: CCAAGTCGAC CTAAATACGA TACGAGAGAA AC 32(2) SEQ ID NO: 7 information (i) sequence characteristics

(A) Length: 30 bases

(B) Type (2): nucleic acids

(C) Chain property: single strand

(D) Topological structure: linear (ii) molecular type: oligonucleotide (xi) sequence description: SEQ ID NO: 7: CAGAGAATTC ATGGCCAGCT TTCCCCCGAG 30(2) SEQ ID NO: 8 information (i) sequence characteristics

(A) Length: 32 bases

(B) Type (2): nucleic acids

(C) Chain property: single strand

(D) Topological structure: linear (ii) molecular type: oligonucleotide (xi) sequence description: SEQ ID NO: 8: CCAATCTAGA CTAAATACGA TACGAGAGAA AC 32

Claims (14)

1. An isolated DNA molecule comprising: a nucleotide sequence for coding a polypeptide with the activity of human WSB1 protein,

the nucleotide sequence has at least 70 percent of homology with the nucleotide sequence from 31 to 1296 of nucleotides in SEQ ID NO. 3; or

The nucleotide sequence can be hybridized with the nucleotide sequence from 31 to 1296 of nucleotides in SEQ ID NO.3 under moderate stringency conditions.

2. The DNA molecule of claim 1 wherein said sequence encodes a polypeptide having the sequence shown in SEQ ID No. 4.

3. The DNA molecule of claim 1, wherein said sequence comprises the nucleotide sequence of positions 31-1296 of SEQ ID No. 3.

4. An isolated human WSB1 protein polypeptide, comprising: a polypeptide having the amino acid sequence of SEQ ID No.4, or an active fragment or active derivative thereof.

5. The polypeptide of claim 4, wherein the polypeptide has the sequence of SEQ ID No. 4.

6. A vector comprising the DNA of claim 1.

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

8. The host cell of claim 7, wherein the cell is E.coli.

9. The host cell of claim 7, wherein the cell is a eukaryotic cell.

10. A method of producing a polypeptide having the activity of human WSB1 protein, the method comprising:

(a) operably linking a nucleotide sequence encoding a polypeptide with the activity of the human WSB1 protein to an expression regulatory sequence to form a human WSB1 protein expression vector, wherein the nucleotide sequence has at least 70 percent of homology with the nucleotide sequence from 31 to 1296 of nucleotides in SEQ ID NO. 3;

(b) transferring the expression vector in the step (a) into a host cell to form a recombinant cell of the human WSB1 protein;

(c) culturing the recombinant cell of step (b) under conditions suitable for expression of the human WSB1 protein polypeptide;

(d) isolating the polypeptide with the activity of the human WSB1 protein.

11. The method of claim 10, wherein the sequence is SEQ ID No.3 from nucleotide 31 to 1296.

12. An antibody that specifically binds to the human WSB1 protein polypeptide of claim 4.

13. A nucleotide molecule which is an antisense sequence of the DNA molecule of claim 1.

14. A probe molecule comprising about 8 to 100 contiguous nucleotides of the DNA molecule of claim 1.

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