CN102807613A - Secreted protein with chemotactic activity and medicament usage thereof - Google Patents

Abstract

The present invention provides a protein with the amino acid sequence shown in SEQ ID NO: 1 (C17ORF87 secreted protein) having leukocyte and tumor cell chemotactic function, a polynucleotide encoding the protein, and a genetic engineering carrier containing the polynucleotide. It also relates to a pharmaceutical composition, containing the protein or polynucleotide, genetic engineering carrier and/or host cell, and pharmaceutically acceptable salt, carrier or excipient. It also relates to a method for detecting changes in the expression level of the protein or polynucleotide in vitro. Also, the protein or polynucleotide can be used to prevent and/or treat viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmune disease, tumor metastasis, immune regulation, stem cell proliferation and differentiation, bone Osteoarthritis, obesity, and insulin resistance, and commercially available reagents to study the above conditions. It also relates to the development of compounds, antibodies, polypeptide drugs and commercialized reagents using the protein or polynucleotide as a target.

Description

Secreted proteins with chemotactic activity and their medicinal uses

technical field

The present invention relates to the field of genetic engineering, in particular, the present invention relates to proteins with multiple functions and polynucleotides encoding them, genetic engineering vectors containing polynucleotides, and corresponding pharmaceutical compositions, and the present invention also relates to the proteins And the application of polynucleotide and pharmaceutical composition thereof.

Background technique

Cytokines are small molecular soluble proteins that are synthesized and secreted by various cells in the body and have various physiological activities and participate in pathological reactions. Chemokines are a class of cytokines with chemotactic effects, which play a role in the body's viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmune disease, tumor metastasis, immune regulation, stem cell proliferation and differentiation, bone It plays an important role in the process of obesity, obesity and insulin resistance. At present, chemokines have become one of the research hotspots at home and abroad.

The 10th anniversary of the completion of the Human Genome Framework Map, the current task is to clarify the functions of tens of thousands of genes and their products, gain a deep understanding of the genes and molecular mechanisms related to the occurrence and development of diseases, and discover disease susceptibility genes, disease resistance genes, and drug sensitivity genes , to discover new drug targets and new biotechnology drugs. These tasks will take far more time, greater investment, and heavier workload than gene sequence analysis, and are also more challenging.

Chemokine superfamily members have a certain homologous structure. According to the arrangement of the first 2 Cys of the conserved 4 and 6 Cys in the primary structure, the chemokine superfamily can be divided into CXC, CC, C and CX3C Four subfamilies, their function besides chemotactic activity, also has various biological effects such as cell activation. These chemokines specifically bind to seven transmembrane G protein-coupled receptors, transduce signals into cells through G protein, and exert their biological activities. So far, about 50 chemokines and 20 chemokine receptors have been discovered, among which multiple chemokines can exert chemotactic effects through the same chemokine receptor, and the same chemokine can also act on several Chemokine receptors exert chemotaxis.

In recent years, immunotherapy has undergone changes, and the transformation from basic to clinical to drug development has become a more important approach. By describing how immune system-associated chemokines and their ligands are organized to play a role in physiological and pathological activities, it is suggested that chemokines have strong clinical application prospects and can be used as adjuvant therapeutic agents or main therapy of immunotherapy.

The present inventor utilizes the human functional genomics database, bioinformatics analysis, molecular biology, cell biology and immunology technology to dig new genes with important physiological and pathological significance from the human gene database, in order to clarify the pathogenesis of diseases, It provides the basis for discovering disease markers, genomic drug targets (to develop compounds, antibodies, peptide drugs) or genetically engineered drugs. The detection methods of gene expression and its encoded product protein expression include reverse transcription-polymerase chain reaction, Western blot, ELISA, FACS, immunofluorescence, immunohistochemistry, immunocytochemistry and other detection methods. It can be applied to experimental research and clinical physiological and pathological diseases (viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmune disease, tumor metastasis, immune regulation, stem cell proliferation and differentiation, osteoporosis, obesity and Insulin resistance) gene expression in cells and tissues and detection of protein expression of its encoded product.

Genes with important physiological and pathological significance and their encoded product proteins can be used as drug targets. Compounds, antibodies, polypeptide drugs or genetic engineering drugs and commercial reagents targeting this molecule itself and its interacting molecules can be developed to be used in pathogenesis Mechanism research, disease marker research and clinical testing, and research and clinical drug treatment.

Contents of the invention

Using bioinformatics technology, the present invention successfully screened out a new cytokine with chemotactic activity from the new gene for the first time, that is, C17ORF87 (Chromosome 17 open reading frame87), whose nucleic acid sequence is registered in Gen-bank ^TM with the registration number of NM_207103.2. NM_207103.2 encodes 145 amino acids and has chemotactic activity in vitro. The experiments of protein expression, purification, N-terminal sequencing and chemotactic function detection in the present invention prove that the secreted protein with chemotactic activity of C17ORF87 is its C-terminal 145 amino acid sequence. The present invention also includes some other novel proteins successively obtained on the basis of C17ORF87.

In one aspect, the present invention provides a protein with chemotactic activity.

In another aspect, the present invention provides a polynucleotide encoding the protein of the present invention.

Another aspect of the present invention provides a genetic engineering vector containing the polynucleotide of the present invention.

In another aspect, the present invention provides a pharmaceutical composition, which contains the protein, polynucleotide and/or genetic engineering carrier of the present invention, and one or more pharmaceutically acceptable salts or pharmaceutically acceptable carrier or excipient.

Another aspect of the present invention provides that the protein or polynucleotide of the present invention can be used in the preparation of prevention and/or treatment of viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmune disease, tumor metastasis, immune regulation, The application of stem cell proliferation and differentiation, osteoporosis, obesity and insulin resistance drugs.

In another aspect, the present invention provides a method for detecting whether the expression level of the protein or polynucleotide of the present invention changes in a test sample.

In another aspect of the present invention, the protein or polynucleotide of the present invention is used in the preparation of commercial reagents to study viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmune disease, tumor metastasis, immune regulation, stem cell proliferation Applications in differentiation, osteoporosis, obesity and insulin resistance.

Another aspect of the present invention provides the use of the protein or polynucleotide of the present invention or the interacting molecule of the protein of the present invention as a target to develop compounds, antibodies, polypeptide drugs and commercial reagents.

According to some embodiments of the present invention, the present invention provides a protein comprising:

(1) a protein of the amino acid sequence shown in SEQ ID NO: 1; or

(2) A protein having at least 80% homology to the protein described in (1), whose function is the same or similar to that of the protein described in (1).

Wherein, the protein of the amino acid sequence shown in SEQ ID NO: 1 is the C-terminal 145 amino acid sequence of the C17ORF87 protein (the registration number of the nucleic acid sequence of the C17ORF87 protein in Gen-bank ^TM is NM_207103.2); in the present invention is called C17ORF87 secreted protein.

The protein of the present invention also includes at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, particularly preferably at least 98%, and more particularly preferably at least 80% of the amino acid sequence shown in SEQ ID NO: 1. Sequences of at least 99% homology (sequence match). These sequences may have the same, similar or different biological functions as the sequence shown in SEQ ID NO: 1 of the present invention, preferably have the same or similar functions.

In some preferred embodiments of the present invention, the protein of the present invention is:

(1) a protein of the amino acid sequence shown in SEQ ID NO: 1; or

(2) A protein having at least 90% homology to the protein described in (1), whose function is the same as that of the protein described in (1).

Furthermore, according to some embodiments of the present invention, the present invention also provides a polynucleotide comprising:

(1) a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 1; or

(2) A polynucleotide having at least 80% homology with the polynucleotide described in (1), and the protein encoded by it has the same function as the protein encoded by the polynucleotide described in (1).

The amino acid sequence shown in SEQ ID NO: 1 is the 145 amino acid sequence of C17ORF87 secretory protein. The polynucleotide sequence of the present invention may only encode the C17ORF87 secreted protein, or may add non-coding sequences, such as introns, non-coding sequences at the 5' or 3' end of the coding sequence, on the basis of the coding sequences of the above proteins. The polynucleotide sequences of the invention are preferably provided in isolated form. A polynucleotide of the invention is in "isolated" form in that it has been separated not only from the proteins that accompany it in a cell, but also from the sequences that flank it in nature.

In some embodiments of the present invention, the present invention also includes at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, especially Polynucleotide sequences with at least 95%, more particularly preferably at least 98%, homology are preferred. Preferably, the polynucleotide of the present invention is a polynucleotide having at least 80% homology with the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof, and the protein encoded by the polynucleotide is identical to that of SEQ ID NO: 1. The protein shown by ID NO: 1 has the same function.

In addition, according to some embodiments of the present invention, the present invention also relates to a genetic engineering vector, which contains a polynucleotide encoding the secreted protein of the present invention. The genetic engineering vectors can be common vectors, expression vectors and the like.

In addition, according to some embodiments of the present invention, the present invention also provides that the C17ORF87 secreted protein is used in the preparation of prevention and/or treatment of viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmune disease, tumor metastasis, Applications in drugs for immune regulation, stem cell proliferation and differentiation, osteoporosis, obesity and insulin resistance.

The present invention proves through experiments that the C17ORF87 secreted protein of the present invention has chemotactic activity comparable to that of chemokines, and it is derived from human itself, so the C17ORF87 secreted protein has wide application in the prevention and/or treatment of various diseases prospect.

In addition, according to some embodiments of the present invention, the present invention also provides that the C17ORF87 secreted protein is useful in the preparation of commercial reagents to study viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmune disease, tumor metastasis, immune regulation, stem cell proliferation and differentiation, osteoporosis, obesity and insulin resistance.

In addition, according to some embodiments of the present invention, the present invention also provides a method for detecting the expression level of the protein or polynucleotide of the present invention in a sample from a subject in vitro, the method is reverse transcription-polymerase Chain reaction, western blot or ELISA, FACS, immunofluorescence, immunohistochemistry, immunocytochemistry detection methods.

Description of drawings

Figure 1 shows the expression results of C17ORF87 secreted protein detected by Western Blotting. After adding Anti-c-myc antibody, IRDyeTM 800-labeled anti-mouse IgG secondary antibody was added and scanned by Odyssey Imaging System. The picture shows the supernatant of C17ORF87(NM_207103.2)-myc-his overexpression, and there is a clear band at 28kd.

Figures 2A-2E show chemotaxis of C17ORF87 secreted protein supernatants. Among them: Figure 2A shows that C17ORF87 (NM_207103.2) has a chemotactic effect on PBMC, while the supernatant expressed in pcDB is used as a control, it can be seen that C17ORF87 has obvious chemotactic effect; Figure 2B shows that C17ORF87 has a chemotactic effect on guinea pig neutrophils, while pcDB The expression supernatant is used as a control, and it can be seen that C17ORF87 has an obvious chemotaxis effect; Figure 2C shows that C17ORF87 has a chemotaxis effect on U937, and the pcDB expression supernatant is used as a control, and C17ORF87 has an obvious chemotaxis effect; Figure 2D shows that C17ORF87 has a chemotaxis effect on Jurkat, pcDB expression supernatant was used as a control, and C17ORF87 had obvious chemotactic effect; Figure 2E shows that C17ORF87 has a chemotactic effect on THP-1, and pcDB expression supernatant was used as a control, and C17ORF87 had obvious chemotactic effect.

Figure 3 shows the N-terminal sequencing results of C17ORF87-myc-his6 eukaryotic protein, specifically showing the 10 amino acid sequencing results of a clear band at 28kd at the N-terminal of C17ORF87(BC039396.1)-myc-his.

Figure 4 shows RT-PCR identification of changes in C17ORF87 transcription levels in PBMCs stimulated by different concentrations of LPS (Lipopolysaccharides). In the figure, the 1st lane is the molecular weight standard; the 2nd, 3rd, and 4th lanes are the PBMCs of three different individuals, and the 5th, 6th, 7th, 8th, 9th, and 10th lanes are the PBMCs respectively with LPS 10ng/ml, 1μg/ml , 5μg/ml respectively stimulated 6h, 24h reverse transcription cDNA as a template. Among them, A shows the electrophoresis results of 40 rounds of amplification using C17ORF87 (F, R) as primers, and a clear band can be seen at 438 bp; B shows the electrophoresis results of 22 rounds of amplification using GAPDH (F1, R1) A clear band can be seen here, which is the internal standard.

Figure 5 shows that the purified C17ORF87 prokaryotic protein sample was subjected to 12.5% SDS-PAGE electrophoresis and directly stained with Coomassie brilliant blue. In the figure, the arrow points to C17ORF87-his6 prokaryotic protein. The first lane is BSA 400μg; the second lane is BSA 200μg; the third lane is BSA 150μg; the fourth lane is BSA 100μg; the fifth lane is C17ORF87 loading 2μl.

Figure 6 shows the chemotactic effect of C17ORF87 prokaryotic protein on THP-1 cells. Among them, the chemotactic effect of 100ng/ml prokaryotic protein of C17ORF87 is the most obvious.

Figure 7 shows the expression profile of C17ORF87 in various human tissues. In the figure, lane 9 is the molecular weight standard; lanes 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, and 17 are human brain and thymus respectively. , heart, lung, liver, pancreas, small intestine, colon, spleen, peripheral blood lymphocytes, skeletal muscle, kidney, prostate, testis, ovary, placenta. Among them, A shows the electrophoresis results of 40 rounds of amplification using C17ORF87 (F, R) as primers, and a clear band can be seen at 438 bp; B shows the electrophoresis results of 25 rounds of amplification using GAPDH (F1, R1) A clear band can be seen here, which is the internal standard. The results showed that C17ORF87 was expressed in thymus, small intestine, spleen and peripheral blood lymphocytes, but not in other tissues.

Figure 8 shows the subcellular localization of C17ORF87 in cells. Among them, A shows the localization of pEGFP-N1-C17ORF87 fusion protein in the cell membrane of Hela cells. B shows the result of transfection with pEGFP-N1. C shows the localization of overexpressed pcDNA3.1-C17ORF87-myc-his in the cell membrane of Hela cells, D shows the result of overexpressed pcDNA3.1-myc-his6.

Detailed ways

In the following, some specific implementations will be described in more detail with respect to the aforementioned subject matter of the invention. It should be understood, however, that these descriptions and the following examples are not intended to limit the scope of the claimed invention.

The present invention constructs C17ORF87 eukaryotic expression plasmid pCDNA3.1-C17ORF87(NM_207103.2)-MYC-HIS6 and 293T eukaryotic expression system, and obtains secreted and expressed C17ORF87 recombinant protein after expression and purification. The secreted and expressed C17ORF87 supernatant has chemotactic effects on human peripheral blood mononuclear cells, guinea pig neutrophils and U937, Jurkat, THP-1 cells. The C17ORF87 protein band was further separated by SDS-PAGE, and N-terminal sequencing was carried out to obtain a C17ORF87 secreted protein, which was the mature form of the C17ORF87 secreted protein discovered for the first time in the world in the experiment of the present invention. At the same time, the present invention constructs the prokaryotic expression plasmid of the secreted protein, and performs prokaryotic expression to obtain the C17ORF87 prokaryotic protein. This protein has chemotactic effect on THP-1 cells.

Among the proteins provided by the present invention, the protein with the amino acid sequence shown in SEQ ID NO: 1 is the C-terminal 145 amino acid sequence of the C17ORF87 protein (the registration number of the nucleic acid sequence of the C17ORF87 protein in Gen-bank ^TM is NM_207103. 2); referred to as C17ORF87 secreted protein in the present invention. The protein of the present invention also includes a homology (sequence matching) of more than 80%, preferably more than 85%, more preferably more than 90%, even more preferably more than 95%, and particularly preferably more than 95% with the amino acid sequence shown in SEQ ID NO: 1. 98%, more particularly preferably more than 99% of the sequences. These sequences may have the same, similar or different biological functions as the sequence shown in SEQ ID NO: 1 of the present invention, preferably have the same or similar functions.

The secreted C17ORF87 protein or fragment thereof of the present invention may be natural, synthetic, semi-synthetic, or recombinantly produced. The C17ORF87 secreted protein of the present invention can be used in Applied Biosystem synthesizer or ^PioneerTM according to the method described by Steward and Young (Steward, JMand Young, JD, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, Ill., (1984)) The peptide synthesizer is synthesized by solid-phase chemistry technique. Generally, these methods involve the sequential addition of one or more amino acids or appropriately protected amino acids to a growing peptide chain. Usually, the amino or carboxyl group of the first amino acid is protected with a suitable protecting group, and then the protected amino acid is attached to an inert solid phase support, and then the corresponding amino or carboxyl group is properly protected under conditions suitable for the formation of an amide bond. the next amino acid in . The protecting group is then removed from the newly added amino acid residue and the operation repeated if necessary by adding the next amino acid, suitably protected. When all amino acids are linked in the correct order, any remaining protecting groups and solid support are removed sequentially or simultaneously to obtain the final protein. By simple modification of this standard procedure, it is possible to add more than one amino acid to a growing chain at a time. In a preferred embodiment of the present invention, an automatic synthesizer is used to prepare the protein of the present invention. Among them, amino acids in which the α-amino group is protected by an acid- or base-sensitive group are used. This protecting group should be stable under the conditions of peptide bond formation, and be easily removed without disrupting the growing peptide chain and without causing racemization of any chiral centers therein. Suitable protecting groups are 9-fluorenylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 2-cyano-tert-butoxycarbonyl and the like. The 9-fluorenylmethoxycarbonyl group (Fmoc) is particularly preferred for the synthesis of the peptides of the invention. The protein of the present invention can also be produced by encoding the recombinant DNA sequence in the host cell according to conventional bioengineering methods (see Examples 1-4 for details). In Examples 1-4, the C17ORF87 coding sequence was inserted into the expression system, expressed and purified to obtain the secreted and expressed C17ORF87 recombinant protein, and further separated by SDS-PAGE to obtain the C17ORF87 protein band, and N-terminal sequencing was performed to obtain the C17ORF87 recombinant protein of the present invention. protein. Those skilled in the art will know that the polynucleotide sequence encoding the protein of the present invention can be directly used to produce the C17ORF87 secreted protein of the present invention, for example, the polynucleotide sequence encoding the protein of the present invention (sequence shown in SEQ ID NO: 1 or its fragments) are directly inserted into the expression system, expressed and purified to obtain the protein of the present invention. Alternatively, mRNA derived from the DNA constructs of the invention can be used to produce the desired protein in a cell-free translation system.

Those of ordinary skill in the art will know that the protein or its fragments of the present invention can form fusion proteins with other proteins or their fragments. Other proteins or fragments thereof are generally known, and some are commercially available as vectors, or may be synthesized by conventional methods or cloned from known organisms.

The amino acid sequence shown in SEQ ID NO: 1 is the 145 amino acid sequence of C17ORF87 secretory protein. The polynucleotide sequence of the present invention may only encode the C17ORF87 secreted protein, or may add non-coding sequences, such as introns, non-coding sequences at the 5' or 3' end of the coding sequence, on the basis of the coding sequences of the above proteins. The polynucleotide sequences of the invention are preferably provided in isolated form. A polynucleotide of the invention is in "isolated" form in that it has been separated not only from the proteins that accompany it in a cell, but also from the sequences that flank it in nature.

The present invention also includes at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, particularly preferably at least 95%, and more particularly preferably Polynucleotide sequences of at least 98% homology. In particular, it relates to polynucleotides that hybridize to polynucleotides of C17ORF87 secreted protein under stringent conditions, and the term "stringent conditions" means that the hybridization occurs at least 95% homology between the sequences. Such sequences may be naturally occurring or artificially produced, and may include allelic variants of the polynucleotide sequence of the secreted C17ORF87 protein, and may also include deletions, insertions and substitutions of bases in the polynucleotide sequence of the secreted C17ORF87 protein. The protein encoded by such a sequence may be functionally the same, similar, or different from the C17ORF87 secreted protein of the present invention, but preferably encodes a protein having substantially the same biological activity as the C17ORF87 secreted protein. Therefore, preferably, the polynucleotide of the present invention is a polynucleotide having at least 80% homology with the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof, and the protein encoded by the polynucleotide Has the same function as the protein shown in SEQ ID NO:1.

The polynucleotide sequence of the present invention can be DNA or RNA, wherein DNA includes cDNA, genomic DNA and synthetic DNA, DNA can be double-stranded or single-stranded form, and single-stranded DNA can be coding strand or non-coding strand (antisense chain). The antisense strand of the present invention can be the complementary sequence of the sequence shown in SEQ ID NO:1. Those of ordinary skill in the art know that the antisense strand or a part thereof (antisense oligonucleotide) can be used to inhibit the expression of the C17ORF87 secreted protein of the present invention in cells. The nucleotide sequence of the C17ORF87 secretory protein of the present invention can be from any species, especially mammals, including cattle, sheep, pigs, mice, horses, preferably humans.

The polynucleotide sequence as shown in SEQ ID NO: 1 is a polynucleotide encoding the protein of the present invention, which is from human. Therefore, it is preferred that the polynucleotide of the present invention comprises a polynucleotide as shown in SEQ ID NO: 1 or its complementary sequence.

The genetic engineering carrier involved in the present invention contains the polynucleotide encoding the secreted protein of the present invention. The genetic engineering vectors can be common vectors, expression vectors and the like. Among them, ordinary vectors are mainly used for the establishment of various genomic libraries and cDNA libraries, and they usually contain two or more marker genes, one of which is used to select transformants (transformants), and the other gene is used to check vectors Whether there is foreign DNA inserted in it. Expression vectors are mainly used to study gene expression or to mass-produce some useful transcripts or proteins, and some can also be used for the establishment of cDNA libraries. In addition to the characteristics of ordinary vectors, such vectors should also contain appropriate promoters, ribosome binding sites, terminators, etc. In order to facilitate the localization of the expression product in cells, an appropriate leader sequence can be added upstream of the protein coding sequence.

Selection of suitable vectors and promoters is known to those of ordinary skill in the art. Those of ordinary skill in the art will be aware of methods for constructing vectors containing the polynucleotides of the invention together with appropriate transcriptional and translational regulatory elements. Specifically, commercially available expression vectors suitable for prokaryotic cells generally have selectable markers and cell replication origins, bacterial promoters such as lacI, T7, λPL, and trp, and the known cloning vector pBR322 (ATCC 37017). other genetic elements. Such commercially available vectors include pGEM (Promega) and pKK223-3 (Pharmacia). An appropriate vector derived from pBR322 can be selected according to the appropriate promoter selected and the structural gene sequence to be expressed. The GST prokaryotic expression system can also be used in the present invention. Carriers suitable for eukaryotic cells have eukaryotic cell promoters such as CMV, SV40, etc. Such vectors include pMT-hIL-3 (Ma Dalong, Di Chunhui, Pang Jian, etc., High Technology Communication 11: 26-29 (1991 )), pQE-9 (Qiagen), pD10, pNH18A (Stratagene), pKK233-3, pDR540, pRIT5 (Pharmacia), and pcDNA3, pCI, pWLNEO, pSG (Stratagene), pSVL (Pharmacia). In Example 1 of the present invention, the C17ORF87 coding sequence was inserted into the pCDNA3.1-myc-his6 (Invitrogen Company) expression vector to construct the pCDNA3.1-C17ORF87-myc-his6 expression plasmid.

The pharmaceutical composition provided by the present invention may comprise the protein of the present invention, the polynucleotide encoding the protein of the present invention, the genetic engineering vector containing the polynucleotide and/or the host cell. In addition, in addition to the above-mentioned active ingredients, One or more pharmaceutically acceptable salts or pharmaceutically acceptable carriers or excipients may also be included. The selection of these pharmaceutically acceptable salts or pharmaceutically acceptable carriers or excipients depends, for example, on the route of administration of the drug.

The present invention proves through experiments that the C17ORF87 secreted protein of the present invention can be used to prepare and prevent and/or treat viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmune disease, tumor metastasis, immune regulation, stem cell proliferation Differentiated drugs.

Early studies found that chemokines and chemokine receptors play an important role in mediating the acute and chronic authentication process. Recent studies have shown that chemokines and chemokine receptors are involved in growth and development; maintenance of homeostasis; osteoporosis; obesity and insulin resistance; viral infection; immune response; migration of bone marrow chief cells and autoimmune diseases and other pathophysiological processes play an important role in. Recent studies have also shown that chemokines and chemokine receptors play an important role in the process of tumorigenesis, development and metastasis.

Diseases such as viral infection, allergic disease, inflammation, transplant rejection, brain disease, autoimmune disease or tumor metastasis are closely related to chemokines and their receptors, desensitization of agonists, neutralizing antibodies, etc. main treatment. The present invention proves through experiments that the C17ORF87 secreted protein of the present invention has chemotactic activity comparable to that of chemokines, and it is derived from human itself, so the C17ORF87 secreted protein has wide application in the prevention and/or treatment of various diseases prospect.

The present invention also provides the application of the C17ORF87 secreted protein of the present invention in the preparation of commercial reagents for studying viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmune disease, tumor metastasis, immune regulation, stem cell proliferation and differentiation.

The present invention also provides a method for in vitro detection of the expression level of the protein or polynucleotide of the present invention in a sample from a test subject, the method is reverse transcription-polymerase chain reaction, Western blot or ELISA, FACS , immunofluorescence, immunohistochemistry, and immunocytochemistry detection methods.

The expression level of the protein or polynucleotide of the present invention can be detected by any method known in the art. Preferably, reverse transcription-polymerase chain reaction (RT-PCR) is used to detect the expression level of the polynucleotide at the nucleic acid level; or a specific monoclonal or polyclonal antibody is used to detect the expression of the polynucleotide at the protein level Level, such as Western blotting or ELISA, FACS, immunofluorescence, immunohistochemistry, immunocytochemistry detection methods. The test sample can be obtained from various cells or body fluids from subjects. PT-PCR mainly includes the following steps: extracting total RNA, adding a primer complementary to the 3' end of mRNA, synthesizing cDNA under the action of reverse transcriptase; and using cDNA as a template, adding another primer complementary to cDNA ( The two primers are located on different exons to avoid genomic DNA contamination) for PCR amplification. Western blotting is mainly divided into three stages: the first stage is SDS-polyacrylamide gel electrophoresis for protein samples such as antigens; the second stage is electrotransfer: transfer the separated bands in the gel to nitrocellulose membrane; The third stage is chromogenic detection: nitrocellulose membrane (equivalent to the solid-phase carrier coated with antigen), after reacting with specific antibody and enzyme-labeled secondary antibody in turn, add an enzyme reaction substrate that can form chromogenic substance , to stain the bands. Enzymes for labeling secondary antibodies include horseradish peroxidase (HRP) and alkaline phosphatase (AP). Glucose oxidase, β-D-galactosidase, urease and the like can also be used. In a specific embodiment of the present invention, horseradish peroxidase is used as the enzyme for labeling the secondary antibody. Those of ordinary skill in the art know that different substrates can be used for different enzymes, and the substrates for HRP include, but not limited to, phenylenediamine (OPD), tetramethylbenzidine (TMB) and ABTS. The substrate of alkaline phosphatase generally adopts p-nitrophenyl phosphate (p-NPP), and AP also has a fluorescent substrate (4-methylumbelliferone phosphate). Commonly used enzyme-labeled secondary antibodies are commercially available.

The present invention also provides the application of the C17ORF87 secreted protein or polynucleotide or C17ORF87 interacting molecules of the present invention as target development compounds, antibodies, polypeptide drugs and commercialized reagents.

Example

Example 1, construction of pcDNA3.1-C17ORF87-myc-his6 fusion protein expression plasmid

The pcDNA3.1-C17ORF87-myc-his6 plasmid was constructed to express the C17ORF87-myc-his6 fusion protein.

1. Method:

The C17ORF87 coding sequence (SEQ ID NO: 2) was inserted into the pcDNA3.1-myc-his6 (Invitrogen Company) expression vector to construct the pcDNA3.1-C17ORF87-myc-his6 expression plasmid. After the correctness of the plasmid was verified by sequencing, the plasmid was amplified, and the plasmid was extracted with the Axygen plasmid extraction kit for cell transfection.

2. Results:

The sequence of the coding region was correct by DNA sequencing.

Example 2, plasmid transfection cells to obtain C17ORF87 expression supernatant

1. Method:

HEK 293T cells were adjusted to a concentration of 6×10 ⁵ cells/2ml and cultured in a 6-well plate at 37°C in 5% CO for 24 hours for _transfection , pcDNA3.1-C17ORF87(NM_207103.2)-myc-his6 DNA 2μg, vigofect ( Grass Biotechnology Co., Ltd.) 2 μl, the mixture was slowly dropped into the prepared cells, and the pcDNA3.1-myc-his6 empty vector transfected cells were set as a control, and the serum-free medium HEKG was changed after 6 hours, and cultured at 37°C Forty-eight hours, the post-transfection supernatant was harvested.

Use Western Blotting to check the expression of the target protein:

After 12.5% SDS-PAGE electrophoresis, the protein was transferred to the nitrocellulose membrane with Tris-glycine electrotransfer solution, after the Anti-c-myc antibody (MBL company) was added to act, then IRDyeTM 800-labeled anti-mouse IgG (LICOR Bioscience ), directly detected by the Odyssey Imaging System detection system.

2. Results:

Use Western Blot to check the expression of the target protein:

The results are shown in Figure 1. After adding Anti-c-myc antibody, IRDyeTM 800-labeled anti-mouse IgG secondary antibody was added to react, and after scanning by Odyssey Imaging System, there was a clear band at 28kd (Figure 1).

Example 3, the separation of human peripheral blood mononuclear cells

1. Method:

Concentrated white blood cells were provided by Beijing Blood Center, and human peripheral blood mononuclear cells were obtained using lymphocyte separation medium (Shanghai Huajing Bio-Technology Co., Ltd.). Culture with RPMI 1640 (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin.

2. Results:

Human peripheral blood mononuclear cells were obtained and cultured in vitro for future use.

Embodiment 4, preparation of guinea pig neutrophils

1. Method:

One adult guinea pig was taken and injected intraperitoneally with normal saline containing 0.17% glycogen. After 13 hours, the peritoneal cavity was washed with PBS buffer solution, and the peritoneal fluid was collected at 1500 rpm/min for 10 min. The supernatant was discarded. Cleave red blood cells with 3-5ml 37°C pre-warmed (Tris-NH ₄ CL) erythrocyte lysis solution with pH 7.2, then add serum-free RPMI1640, and wash twice. Dissolve the purified neutrophils in serum-free RPMI 1640, count the cells, adjust the cell concentration to 5x10 ⁵ /ml, and set aside.

2. Results:

The guinea pig neutrophils are obtained, and the purity can reach more than 97%.

Example 5, Chemotactic function of C17ORF87 supernatant

1. Method:

Chemotaxis function detection of C17ORF87 supernatant: Chemotaxis experiments were performed in a 48-well chemotaxis chamber (Neutroprobe; Cabin John, MD, USA). The supernatant used for chemotaxis detection was added to the lower well of the small chamber (28 μl/well), then diluted with Hepes RPMI 1640 and the same medium was resuspended to 1×10 ⁶ cells/ml, and added to the upper well of the small chamber In the middle (55 μl/well), the upper and lower wells are separated by a polycarbonate filter membrane without polyvinylpyrrolidone. PBMC and guinea pig neutrophils use a filter membrane with a pore size of 3 μm, and U937, Jurkat, and THP-1 use a filter membrane with a pore size of 5 μm. Put the chamber into an incubator at 37°C, 5% CO ₂ , and incubate for 3h. At the end of chemotaxis, PBMC, U937, Jurkat, and THP-1 cells in the lower well of the suction chamber were measured using luciferase-containing reagent Cell-Titer Glo (Promega) to measure their chemiluminescence values in response to cell counts. The transfection supernatant of pcDNA3.1 empty vector was used as negative control.

At the end of guinea pig neutrophil chemotaxis, the filter membrane was removed from the chamber, washed with a 3-step staining kit, fixed, and stained. The migrated cells in each well were randomly selected from 5 high-magnification fields of view and counted. The transfection supernatant of pcDNA3.1 empty vector was used as a control.

Chemotaxis index CI was the number of migrated cells divided by the number of cells in the control group. All experiments were repeated at least 3 times. CI>2 is significant.

2. Results:

As shown in Figure 2A-2E, C17ORF87 culture supernatant has positive effects on PBMC (Figure 2A), guinea pig neutrophils (Figure 2B), U937 (Figure 2C), Jurkat (Figure 2D), THP-1 (Figure 2E) Chemotaxis. Example 6, Purification and N-terminal sequencing of C17ORF87-myc-his6 protein

1. Method:

HEK 293T cells were digested and centrifuged and adjusted to a cell density of 1.5×10 ⁷ , seeded in a 150 mm diameter plate, and transfected at the same time. pcDNA3.1-C17ORF87-myc-his6 DNA 12.5μg, PEI (Viglas Biotechnology Co., Ltd.) 50μg, the mixture was slowly dropped into the prepared cells, and the pcDNA3.1-myc-his6 empty vector was transfected at the same time For the control cells, the serum-free medium HEKG was changed after 16 hours, cultured at 37°C, 5% CO ₂ for 48 hours, and the supernatant after transfection was harvested. 2000rpm/min, 10 minutes, 0.22μm filter membrane filtration. Take Ni Sepharose 6 Fast Flow (GE healthcare) column material, equilibrate with 20 times the volume of water, 5mM imidazole 200mM NaCl equilibrium solution, wash the column with miscellaneous protein washing solution after the supernatant passes through the column, and then wash with protein washing solution containing 1M imidazole take off. After purification and elution, the sample was transferred to PVDF membrane by 12.5% SDS-PAGE electrophoresis, stained with Coomassie brilliant blue, decolorized with methanol/glacial acetic acid and cut out the same band as the positive position detected by Western blot for protein N-terminal sequencing ( Commissioned by Shanghai Jikang Biotechnology Co., Ltd. for sequencing).

2. Results:

As shown in Figure 3, the purified protein samples were subjected to 12.5% SDS-PAGE electrophoresis, transferred to PVDF membranes and then sequenced at the N-terminal. C17ORF87(NM_207103.2)-myc-his has a clear band at 28kd. Sent to Shanghai Jikang Biotechnology Co., Ltd. for N-terminal sequencing. The N-terminal sequencing result of the protein is M-D-T-F-T-V-Q-D-S-T.

Example 7, Changes of C17ORF87 transcription level in PBMC stimulated by different concentrations of LPS (Lipopolysaccharides)

1. Method:

The PBMCs obtained in Example 3 were respectively stimulated with LPS 10ng/ml, 1 μg/ml, and 5 μg/ml for 6h and 24h respectively, and then the Trizol reagent of Invitrogen Company was used to extract the total RNA of the cells, and the reverse transcription kit (Invitrogen) was used to carry out reverse transcription and PCR. With C17ORF87 upstream F (5' ATGGAT ACT TTC ACA GTT CAG GAT TC 3' (SEQ ID NO: 3)), downstream R (5' GGC GGT CAA AAT GAT GCT TTT TCA G 3' (SEQ ID NO: 4)) As primers, 40 rounds of amplification were performed. After PCR products were subjected to 1% agarose gel electrophoresis, EB staining was photographed by UV.

2. Results:

As shown in Figure 4: the first lane is the molecular weight standard; the second, third, and fourth lanes are the PBMCs of three different individuals, and the fifth, sixth, seventh, eighth, nineth, and tenth lanes are the PBMCs respectively with LPS 10ng/ml, 1 μg/ml, 5 μg/ml respectively stimulated 6h, 24h reverse transcription cDNA as a template, with C17ORF87 (F, R); (B) F1 (5'ACCACAGTCCATGCCATCAC 3'(SEQ ID NO: 5)) of GAPDH, R2( 5'TCCACCACCCCTGTTGCTGTA 3' (SEQ ID NO: 6)) is the RT-PCR that primer carries out. The results of RT-PCR showed that the expression of C17ORF87 was low in PBMC, and the expression increased after LPS stimulation, and the expression reached the peak at 6 hours and 24 hours respectively after stimulation with 100 μg/ml LPS.

Example 8, construction of pET32a-C17ORF87-his6 fusion protein prokaryotic expression plasmid

The pET32a-C17ORF87-his6 fusion protein prokaryotic expression plasmid was constructed for expressing the C17ORF87-his6 prokaryotic protein.

1. Method:

The C17ORF87 coding sequence (SEQ ID NO: 2) was inserted into the pET32a (Novagen Company) expression vector to construct the pET32a-C17ORF87-his6 prokaryotic expression plasmid. After the correctness of the plasmid was verified by sequencing, the plasmid was amplified, and the plasmid was extracted with Viglas Mini Kit for subsequent prokaryotic expression host bacterial transfection.

2. Results:

The sequence of the coding region was correct by DNA sequencing.

Example 9, C17ORF87-his6 prokaryotic protein expression and purification

1. Method:

After the pET32a-C17ORF87-his6 prokaryotic expression plasmid was transfected into Rosseta host bacteria, cultured in LA medium at 280rpm/min at 37°C, when the bacteria grew to OD 0.6, added IPTG with a final concentration of 20μM at 250rpm/min at 25°C for overnight induction , discard the medium after centrifugation, resuspend in PBS 7.4, and sonicate the cells. Harvest the supernatant after crushing the bacterial cells, centrifuge at 12000 rpm/min for 10 minutes, and filter with a 0.22 μm filter membrane. Take Ni Sepharose 6 HP (GE healthcare) column material, equilibrate with 20 times the volume of water, and equilibrate with 20mM imidazole 500mM NaCl equilibrium solution. After the supernatant passes through the column, first wash the column with miscellaneous protein washing solution, and then wash with protein washing solution containing 500mM imidazole take off. After purification, the eluted sample was concentrated by ultrafiltration through an ultrafiltration tube (milipore 3000D). Finally, DEAE (GE healthcare) was used to remove endotoxin, BCA was quantified and then frozen at -80°C for subsequent functional experiments.

2. Results:

As shown in Figure 5, the purified protein samples were subjected to 12.5% SDS-PAGE electrophoresis and directly stained with Coomassie brilliant blue. The C17ORF87-his6 indicated by the arrow in the figure is the target protein band.

The result of BCA quantification showed that the concentration of purified C17ORF87-his6 was 200 μg/ml.

Example 10, Chemotactic effect of C17ORF87-his6 prokaryotic protein on THP-1 cells

Chemotaxis function detection of C17ORF87-his6 prokaryotic protein: Chemotaxis experiments were carried out in a 48-well chemotaxis chamber (Neutroprobe; Cabin John, MD, USA). The protein used for chemotaxis detection was diluted with Hepes RPMI 1640 0.1% BSA to 1ng/ml, 10ng/ml, 100ng/ml, 1000ng/ml, 10000ng/ml and added to the lower hole of the small chamber (27μl/hole), and then used Dilute Hepes RPMI 1640 0.1% BSA and resuspend the same medium to 4×10 ⁶ cells/ml, add to the upper well of the small chamber (55 μl/well), and use polycarbonate filter membrane without polyvinylpyrrolidone in the upper and lower wells Separately, THP-1 uses a filter membrane with a pore size of 5 μm. Put the chamber into an incubator at 37°C, 5% CO ₂ , and incubate for 4.5h. At the end of chemotaxis, the filter membrane was removed from the chamber, washed with a 3-step staining kit, fixed, and stained. The migrated cells in each well were randomly selected from 5 high-magnification fields of view and counted. Hepes RPMI 1640 0.1% BSA without protein group was used as control.

Chemotaxis index CI was the number of migrated cells divided by the number of cells in the control group. All experiments were repeated at least 3 times. CI>2 is significant.

2. Results:

As shown in Figure 6, C17ORF87 prokaryotic protein has chemotactic effect on THP-1 and the effect is most obvious at 100ng/ml.

Example 11, the expression profile of C17ORF87 in various human tissues

1. Method:

Human tissue cDNA libraries were purchased from Clontech. With C17ORF87 upstream F (5' ATG GAT ACT TTC ACA GTT CAG GAT TC 3' (SEQ ID NO: 3)), downstream R (5' GGC GGT CAA AAT GAT GCT TTT TCA G 3' (SEQ ID NO: 4) ) as primers, amplified for 40 rounds. After PCR products were subjected to 1% agarose gel electrophoresis, EB staining was photographed by UV.

2. Results:

As shown in Figure 7: the ninth lane is the molecular weight standard; the first, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, and 17 lanes are human The cDNA libraries of brain, thymus, heart, lung, liver, pancreas, small intestine, colon, spleen, peripheral blood lymphocytes, skeletal muscle, kidney, prostate, testis, ovary and placenta are respectively based on (A)C17ORF87(F, R ); (B) RT-PCR with primers F1 (5'ACCACAGTCCATGCCATCAC 3' (SEQ ID NO: 5)) and R2 (5' TCCACCACCCTGTTGCTGTA 3' (SEQ ID NO: 6)) of GAPDH.

The results showed that C17ORF87 was expressed in thymus, small intestine, spleen and peripheral blood lymphocytes, but not in other tissues.

Example 12, Subcellular localization of C17ORF87

1. Method:

The eukaryotic cell expression plasmid pEGFP-N1-C17ORF87 was constructed. The eukaryotic cell expression plasmid pcDB-C17ORF87-myc-his and the eukaryotic expression vector pEGFP-N1 were digested with Xho I and Kpn I, respectively, and the digested C17ORF87 fragment was connected to the vector at 16°C for 8 hours, and transformed into Escherichia coli DH5α, transformants were grown on LB plate medium containing kanamycin, the grown colonies were selected, plasmids were extracted, identified by PCR, positive clones were selected, and verified by sequencing, pEGFP-N1-C17ORF87 had a GFP tag at the C-terminus and Expression plasmid with signal peptide.

Transfection of HeLa cells by electroporation and transfection: HeLa cells were subcultured in HepesRPMI 1640 medium containing 10% calf serum at a rate of 4.0×10 ⁵ cells/10mm plate, and cultured in an incubator at 5% CO ₂ at 37°C for 24 Hour. Digest HeLa cells, wash cells twice with Hepes RPMI 1640 medium, adjust cell concentration to 2-3×10 ⁶ /ml, take 300 μl/sample of cells, add 10 μg DNA, mix well and transfer to 2 mm electroporation cup, electric shock condition 123v, 20ms; use the same total amount of pEGFP-N1 plasmid to express green fluorescent protein to detect the transfection efficiency. Place at room temperature for 5 minutes after transfection, suck out and resuspend in 7ml of Hepes RPMI 1640 medium containing 10% calf serum, spread on 10mm plate, culture for 6-8 hours and replace with fresh medium after cell adherence. 24 hours after transfection, the transfection efficiency of pEGFP-N1 wells was detected under a fluorescence microscope.

In another experiment, the steps of treating cells before electroporation of HeLa cells were the same as before. Take 300 μl/sample of cells, add 10 μg pcDNA3.1-C17ORF87-myc-his6 DNA, mix well and transfer to a 2mm electroporation cup, and the electric shock conditions are 123v, 20ms; At the same time, a pcDNA3.1-myc-his6 empty vector transfection cell control was set. Transiently transfected cells were cultured on coverslips for 48 hours. Cells were then fixed (30 minutes at room temperature, 4% paraformaldehyde), permeabilized (5 minutes, 0.1% (vol/vol) Triton X-100), and blocked (3% FBS). All solutions were prepared in PBS. Stained with anti-myc antibody, mouse IgG, and FITC-labeled goat anti-mouse Ig in turn. Laser confocal microscope observation. The same total amount of pEGFP-N1 plasmid was used to express green fluorescent protein to detect the transfection efficiency. After transfection, place at room temperature for 5 minutes, aspirate and resuspend in 7ml of Hepes RPMI 1640 medium containing 10% calf serum, spread on 10mm plate, culture for 6-8 hours and replace with fresh medium after cells adhere. 24 hours after transfection, the transfection efficiency of pEGFP-N1 wells was detected under a fluorescence microscope.

2. Results:

The results are shown in Figure 8: Among them, A shows that the pEGFP-N1-C17ORF87 fusion protein is localized in the cell membrane of Hela cells; C shows that the overexpression of pcDNA3.1-C17ORF87-myc-his6 is localized in the cell membrane of Hela cells; B and D are pEGFP -Control results of N1 and pcDNA3.1-myc-his6 empty vector transfected cells.

The invention has been illustrated herein by means of some specific embodiments, and many details have been described for the purpose of illustration. For those skilled in the art, the present invention may include some other specific implementation manners, and appropriate adjustments and changes may be made to the disclosed content of the invention without departing from the gist of the present invention.

Claims (11)

1. A protein with leukocyte and tumor cell chemotactic function, comprising: (1) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (2) A protein having at least 80% homology to the protein described in (1), whose function is the same or similar to that of the protein described in (1). 2. The protein of claim 1, which is: (1) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (2) A protein having at least 90% homology to the protein described in (1), whose function is the same as that of the protein described in (1). 3. A polynucleotide comprising: (1) a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 1; or (2) A polynucleotide having at least 80% homology with the polynucleotide described in (1), and the protein encoded by it has the same function as the protein encoded by the polynucleotide described in (1). 4. The polynucleotide according to claim 3, comprising a sequence shown in SEQ ID NO: 1 or its complementary sequence. 5. A genetic engineering carrier comprising the polynucleotide according to claim 3 or 4. 6. A pharmaceutical composition, which contains the protein according to claim 1 or 2, the polynucleotide encoding the protein and/or the genetic engineering carrier containing the polynucleotide, and one or more drugs that can acceptable salts or pharmaceutically acceptable carriers or excipients. 7. A protein as claimed in claim 1 or 2 or a polynucleotide encoding the protein is used in the preparation of prevention and/or treatment of viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmunity The application of medicines in diseases, tumor metastasis, immune regulation, stem cell proliferation and differentiation, osteoporosis, obesity and insulin resistance. 8. A method for detecting the expression level of the protein of claim 1 or 2 or the polynucleotide encoding the protein in a sample from a subject in vitro. 9. The method according to claim 8, which is a detection method of reverse transcription-polymerase chain reaction or Western blot, ELISA, FACS, immunofluorescence, immunohistochemistry, or immunocytochemistry. 10. A protein as claimed in claim 1 or 2 or a polynucleotide encoding the protein is used in the preparation of commercial reagents to study viral infection, allergic disease, inflammatory response, transplant rejection, brain disease, autoimmune disease, Applications in tumor metastasis, immune regulation, stem cell proliferation and differentiation, osteoporosis, obesity and insulin resistance. 11. A protein as claimed in claim 1 or 2 or the application of encoding said protein and its interacting molecules as target development compounds, antibodies, polypeptide drugs and commercialized reagents.

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