WO2009070957A1 - Inhibitor of the interaction between blys and ngr and use thereof - Google Patents

Abstract

Provided are inhibitor of the interaction between BLyS and NgR and use of the inhibitor for manufacturing of a medicament for promoting neurite-outgrowth. The inhibitor can be selected from the group consisting of polypeptide, protein, small molecular compound, double stranded RNA, small RNA and RNA aptamer, preferably, the inhibitor is the polypeptide having the amino acid sequence shown in SEQ ID NO: 1. Also provided is a method of screening the inhibitor of the interaction between BLyS and NgR by use of alkaline phosphatase system (AP-system).

Description

 Inhibitors of interaction between BLyS and NgR and their applications

Technical field

 The present invention relates to a substance which blocks BLyS inhibition of neuronal neurite outgrowth information transmission and its use in nerve regeneration. Background technique

After the neuronal cells of the nervous system are damaged, they are usually divided into two types according to the injury site: one is cell body damage, in which case the cells die quickly; the other is axonal damage, and the damaged neuron cells can It lasts for a long time and can last for several years in the human body. Neuronal axons of the peripheral nervous system are relatively easy to regenerate after injury, but adult mammalian central neuron axons are difficult to regenerate after being damaged, mainly due to the central nervous system's own inhibitory environment. Recent studies have found that oligodendrocytes, which constitute the myelin of the neuron, express a factor that inhibits axonal growth and regulate axonal growth of damaged neurons, among which NogoA, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin Glycoprotein (Omgp) is a molecule with three significant inhibitory effects (He and Koprivica, 2004). ₀ NogoA, MAG and Omgp have no similarities in protein structure, but are capable of interacting with the same glycosylphosphatidylinesitol (GPI) domain. Nogo-66 receptor (NgR) specifically binds (McGee and Strittmatter, 2003). Further studies have shown that inhibition of NgR pathway by blocking the binding of molecules carrying central nervous system axonal regeneration inhibition information to NgR may be helpful in restoring the growth of injured neurons, and many in vitro and in vivo experiments provide relevant evidence. (Li and Strittmatter, 2003; Li et al., 2004; McKerracher and David, 2004; Atalay et al., 2007).

 In the development of central nervous system injury diseases, not only the damage of neuronal cells, but also the participation of inflammatory reactions. In the nervous system, the inflammatory response is mainly mediated by lymphocytes that enter the blood-brain barrier and glial cells of the nervous system itself. Most of the current clinical drugs are used to suppress the immune response caused by T cells and fail to achieve the desired therapeutic effect. For example, in the treatment of multiple sclerosis (MS) diseases, IFN-β can only relieve the symptoms of some patients. The disease cannot be cured, and the drug has no clinical effect on some patients. If you can find a substance that can affect the lymphocytes involved in the central nervous system to participate in the inflammatory response, and at the same time affect the axon growth of neuronal cells, it will treat the central nervous system injury disease with inflammatory reaction. Great application value.

In patients with multiple sclerosis, astrocytes in their brains are capable of secreting a large amount of B lymphocyte stimulator (BLyS) (Krumbholz et al., 2005). BLyS is a member of the Tumor Necrosis Factor (ΤΝϊ") family and has been named BAFF, TALL-K zTNF, THANK or TNFSF13B because it was discovered by many experimental groups. BLyS plays an important role in B cell development and antibody production. The effect can also promote the inflammatory response of lymphocyte Th1 (Sutherland et al., 2005). It is generally accepted that BLyS, like most TNF ligands, is active in soluble trimers. It has been found that BLyS has three receptors. Body, B-cell maturation antigen (BCMA), BLyS receptor 3 (BR3) and transmembrane activator And CAML-interactor (TACI), BLyS plays an important role in B cell growth and development through binding to these receptors (Cancro, 2004). B cells play an important role in autoimmune diseases, not only can produce autoantibodies, but also activate inflammation. BLyS is an important factor in maintaining the growth and development of B cells. Blocking the role of this molecule is considered to be an important direction for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Some BLyS antagonists (such as BR3-Fc, BCMA-Fc) has been used in clinical trials and the results are optimistic. Therefore, what role BLYS plays in the nervous system has attracted much attention.

 The present invention clarifies the role of BLyS in inhibiting the growth of neuronal processes in the nervous system, and finds that the new receptors for BLyS, NgR, BLyS, can inhibit the growth of neurites through the receptor NgR, thereby providing a therapeutic effect by blocking the inhibition of BLyS. The theoretical basis of systemic disease.

 Related literature is:

 Atalay B, Bavbek M, Cekinmez M, Ozen 0, Nacar A, arabay G, Gulsen S (2007) Antibodies neutralizing Nogo-A increase pan-cadherin expression and motor recovery Following spinal cord injury in rats. Spinal Cord.

 Cancro MP (2004) The BLyS family of ligands and receptors: an archetype for niche-specific homeostatic regulation. Immunol Rev 202:237-249.

 He Z, Koprivica V (2004) The Nogo signaling pathway for regeneration block. Annu Rev Neurosci 27:341-368.

 Krumbholz M, Theil D, Derfuss T, Rosenwald A, Schrader F, Monoranu CM, Kalled SL, Hess DM, Serafini B, Aloisi F, Wekerle H, Hohlfeld R, Meinl E (2005) BAFF is produced by astrocytes and up-regulated In multiple sclerosis lesions and primary central nervous system lymphoma. J Exp Med 201:195-200.

 Lagenaur C, Lemmon V (1987) An LI -like molecule, the 8D9 antigen, is a potent substrate for neurite extension. Proc Natl Acad Sci U S A 84:7753-7757.

 Li S, Strittmatter SM (2003) Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci 23:4219-4227.

 Li S, et al. (2004) Blockade of Nogo-66, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein by soluble Nogo-66 receptor promotes axonal sprouting and recovery after spinal injury. J Neurosci 24:10511-10520.

 McGee AW, Strittmatter SM (2003) The Nogo-66 receptor: focusing myelin inhibition of axon regeneration. Trends Neurosci 26: 193-198.

 Mc erracher L, David S (2004) Easing the brakes on spinal cord repair. Nat Med 10:1052-1053.

 Sambrook J, et al. (2000) Molecular Cloning: A Laboratory Manual (Third Edition): Cold Spring Harboratory Press.

 Sutherland AP, Ng LG, Fletcher CA, Shum B, Newton RA, Grey ST, Rolph MS, Mackay F, Mackay CR (2005) BAFF augments certain Thl - associated inflammatory responses. J Immunol 174: 5537-5544.

 Glossary:

 AP alkaline phosphatase alkaline alkaline enzyme;

 BSA bovine serum albumin;

 BCIP 5-bromo-4-chloro-3-indole phosphate disodium salt;

BLyS B lymphocyte stimulator; cDNA complementary DNA complementary deoxyribonucleotides;

COS-7 cells are derived from the cell line of African green monkey kidney cells and are capable of expressing large τ antigens;

DAF decay accelerating factor;

 DRG dorsal root ganglion X avian embryo dorsal root neurons;

 The Fc Fc fragment of IgG, when fused to a protein of interest as a protein tag, can be used for detecting the expression of a protein of interest as well as improving the stability of expression of a protein of interest;

 FLAG refers specifically to DYKDDD polypeptide and can be used as a protein tag;

 kDa kilo Dalton, Thousand Daltons;

 Laminin laminin, which is coated with a cell culture dish to enhance cell adhesion;

 NBT nitrogen chloride tetrazolium blue;

 One of the best substrate combinations for NBT/BCIP alkaline phosphatase staining, which can be shown by staining the alkaline phosphatase staining signal;

 NgRHl Nogo-66 receptor homologue 1;

 NgRH2 Nogo-66 receptor homologue 2;

 A fragment of Nogo-66 NogoA protein outside the cell membrane also has the effect of inhibiting neurite outgrowth;

 PCR Polymerase Chain Reaction, a molecular biology technique used to amplify specific DNA fragments and can be considered as special DNA copies in vitro;

 Phalloidin, which specifically binds to the cytoskeletal actin Poly-L-lysine polylysine, which is coated with a cell culture dish to enhance cell adhesion;

 PI-PLC phosphatidylinositol-specific phospholipase C, which specifically releases GPI-anchored proteins from the cell membrane;

 RhoA A small molecule of GTPase that regulates cytoskeletal actin;

TACI transmembrane activator and C AML-interactor ₀

 The present invention discloses that BLyS inhibits the growth and regeneration of neurites by binding to NgR, and also regulates the signaling pathway of neurite outgrowth by RhoA.

 By inhibiting the binding of BLyS to NgR, BLyS inhibits the transmission of growth information of neuronal neurite outgrowth by blocking the binding of BLyS to NgR.

 The present invention also provides the use of the above-mentioned substance which inhibits the binding of BLyS and NgR in the preparation of a medicament for promoting neurite outgrowth.

 The substance which inhibits binding of BLyS to NgR may be a polypeptide, a protein, a small molecule of a compound, a double-stranded RNA, a small RA or an RNA aptamer, and preferably has an amino terminal position 1-47 amino acid of GenBank Accession No. NP-075380. The polypeptide of the sequence shown by the residue is designated as sNgR, and the amino acid sequence is shown in SEQ ID No: 1.

One of ordinary skill in the art can know from the common knowledge that the amino acid sequence represented by SEQ ID No: 1 has been substituted, deleted or added with one or several amino acids and has the amino acid represented by SEQ ID No: 1 which inhibits the binding activity of BLyS and NgR. The sequence-derived polypeptide can also achieve the object of the present invention. 艮卩, by blocking the binding of BLyS to NgR, BLyS can inhibit the growth of neuronal neurite outgrowth. Passing the block.

 The present invention still further provides a composition comprising the above-mentioned substance which inhibits the binding of BLyS to NgR. The above compositions may also comprise a pharmaceutically acceptable carrier such as water, an aqueous buffer, a pharmaceutically acceptable salt and/or hyaluronic acid.

 The above compositions may also contain suitable excipients and/or suitable additives which are pharmaceutically acceptable. Suitable excipients include stabilizers, antioxidants, permeability modifiers, buffers and/or pH modifiers. Suitable additives include suitable buffers, additional chelating masking agents (such as DTPA or DTPA bisamide) or calcium chelating complexes (such as DTPA, CaNaDTPA bisamide) or calcium or sodium salts (calcium chloride, calcium ascorbate, glucose). Calcium acid or calcium lactate). The compositions of the invention may be packaged in liquid form or lyophilized.

 The compositions of the present invention may be used in conventional pharmaceutically acceptable non-toxic carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate.

 The above composition may be introduced into the body by injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue; or may be mixed or wrapped by other substances. After importing into the body. The effective dosage is based on the purpose of promoting the growth of injured neurites. When NgRl-447 is an effective medicinal ingredient, the specific dosage of 4 mg NgRl-447/kg body weight can be referred to.

 The present invention also provides a substance which blocks a BLyS-related nerve cell growth signaling pathway by inhibiting the binding of BLyS to a nerve cell growth signaling pathway; and blocking a BLyS-related neuronal cell growth signaling pathway by binding to BLyS A substance that blocks the growth signaling pathways involved in BLyS and RhoA by inhibiting the binding of BLyS to the neuronal growth signaling pathway.

 The present invention also provides a method for screening a human fetal brain cDNA library using a fusion protein of AP and BLyS (AP-BLyS) as a probe to obtain NgR. The above method for screening human fetal brain cDNA library with AP-BLyS to obtain NgR: Using alkaline phosphatase-labeled BLyS fusion protein (AP-BLyS) as a soluble probe, the COS-7 cells transfected with the human brain cDNA library were subjected to the above method. Screening, alkaline phosphatase staining was used to detect whether AP-BLyS binds to COS-7 cells expressing a protein in a human fetal brain cDNA library.

 It is known to those skilled in the art that the above-mentioned substances which block BLyS inhibition of neuronal neurite outgrowth information transmission can be used for the preparation of diseases for treating nervous system damage (such as multiple sclerosis, myelitis, Alzheimer's disease, muscular atrophic lateral sclerosis). Drugs for disease, Parkinson's disease, spinal cord injury, sciatic nerve injury or facial nerve injury, etc.).

 The above substances which block BLyS inhibition of neuronal neurite outgrowth information transmission can be made into a kit for convenient application.

Experiments have shown that BLyS can inhibit the growth and regeneration of central neurites by transmitting information in combination with NgR and play a role in the signaling pathway by which RhoA regulates nerve cell growth. Based on this fact, it will be apparent to those skilled in the art that substances which inhibit the binding of BLyS to NgR and substances which block the signaling pathway of RhoA regulation of nerve cell growth by inhibiting the binding of BLyS to downstream components can be used to promote damage to neurites. Growing. To inhibit the binding of BLyS to NgR and/or to inhibit Drugs that bind BLyS to downstream components and block RhoA signaling pathways that regulate neural cell growth are also active ingredients that can be used to promote the growth and regeneration of damaged neurites. DRAWINGS

 Figure 1 shows the interaction between AP-BLyS and NgR:

 a is the binding of AP-BLyS to COS-7 cells expressing NgR;

 b is Flag-BLyS specifically blocking the binding of AP-BLyS to NgR-expressing COS-7 cells;

 c is the binding of AP to COS-7 cells expressing NgR;

 d is the binding of AP-BLyS to COS-7 cells expressing TACI;

 e is the binding of AP-BLyS to COS-7 cells expressing DAF;

 f is the binding of AP-BLyS to COS-7 cells expressing the NgR homologous protein NgRH1; g is the binding of AP-BLyS to COS-7 cells expressing the NgR homologous protein NgRH2; h is AP-BLyS and expressing NgR Binding of COS-7 cells with the co-receptor p75; i is the binding of AP-BLyS to COS-7 cells expressing the NgR co-receptor Lingol; j is the saturation of AP-BLyS binding to COS-7 cells expressing NgR curve.

 Figure 2 shows the results of in vitro co-immunoprecipitation of soluble protein NgR and BLyS.

 Figure 3 shows the effect of BLyS on the growth cone structure of chick embryo dorsal root neurons.

 Figure 4 shows the growth of neurites in chick embryo dorsal root neurons of BLyS inhibiting embryos for 12 days.

 Figure 5 shows that sNgR-Fc blocks the binding of AP-BLyS to COS-7 cells expressing NgR.

 Figure 6 shows that sNgR-Fc blocks BLyS inhibition of neurite outgrowth in chick embryo dorsal root neurons.

 Figure 7 shows the inhibition of BLyS on the growth of neurites in chick embryo dorsal root neurons by PI-PLC.

 Figure 8 shows that BLyS affects the growth of neurite outgrowth in chick embryo dorsal root neurons by activating RhoA. detailed description

 Specific experimental procedures for the following examples can be accomplished by one of ordinary skill in the art in accordance with Molecular Cloning (Sambrook J, 2000).

 Example 1. AP-BLyS inhibits the growth of damaged neurites

 1. AP-BLyS and NgR bind to each other and cannot directly interact with other members of the NgR receptor complex.

 1. Preparation of plasmid

 1) Construction of NgR expression vector pcDNA-NgR

The human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 2 and SEQ ID NO: 3 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Xba1. Ligation with pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba1, and ligated the product into E. coli DH5a competent cells. According to sequencing, the amplified fragment (the coding gene of NgR) is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No. NM-023004, encoding GenBank. Accession No. NP- 075380 amino acid at position 1-473 from the amino terminus. The recombinant vector containing the NgR-encoding gene was named pcDNA-NgR.

 2) Construction of NgRH1 expression vector pcDNA-NgRHl

The human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 4 and SEQ ID NO: 5 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Xba I. Ligation was carried out with pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I, and the ligation product was transformed into E. coli DH5a competent cells. According to sequencing, the amplified gene of the fragment iNg i is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No. NM-178570, and encodes the amino terminal of GenBank Accession No. NP-848665. - 420 amino acids. The heavy expression vector containing the N _g R /7 coding gene was named pcDNA-NgRH1.

 3) Construction of NgRH2 expression vector pcDNA-NgRH2

 The human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 6 and SEQ ID NO: 7 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Xba I. Ligation was carried out with pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I, and the ligation product was transformed into E. coli DH5a competent cells. According to sequencing, the amplified gene of the fragment NgRH2 is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No. NM-178568, encoding the amino terminal of the GenBank Accession No. NP-848663. 442 amino acids. The re-expression vector containing the gene encoding the Λ^ 2 was named pcDNA-NgRH2.

 4) Construction of 73⁄4C7 expression A pcDNA-TACI

 The human spleen cDNA library (Invitrogen, Cat. 10425-015) was used as a template, and SEQ ID NO: 8 and SEQ ID NO: 9 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Xba I. The pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I was ligated, and the ligated product was transformed into E. coli DH5 (x competent cells. According to sequencing, the amplified fragment was The coding gene of 73⁄4C is identical to the sequence of the degenerate ribonucleotide of the gene coding region of GenBank Accession No. NM-012452, and encodes the amino acid residue at position 1-194 from the amino terminus of GenBank Accession No. NP-036584. The volume group expression vector of the 73⁄4 C/encoding gene was named pcDNA-TACI.

 5) Construction of Z3⁄44F expression vector pcDNA-DAF

 The human spleen cDNA library (Invitrogen, Cat. 10425-015) was used as a template, and SEQ ID NO: 10 and SEQ ID NO: 11 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Xba I. The pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I was ligated, and the ligation product was transformed into E. coli DH5a competent cells. According to sequencing, the amplified fragment (the coding gene of DAF) is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No. NM_000574.3, and encodes the amino terminal of GenBank Accession No. NP-5000565. -382 amino acid residues. One set of expression vectors containing the coding gene was named pcDNA-DAF.

6) Construction of Lingo 1 expression vector pcDNA-Lingol The human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 12 and SEQ ID NO: 13 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Hind III. Ligation was carried out with pcDNA3.1 (- ) (Invitrogen, Cat. V790-20) digested with EcoR I and Hind III, and the ligation product was transformed into E. coli DH5a competent cells. According to sequencing, the amplified fragment (the coding gene of '«go7) is identical to the sequence of the 3⁄4 coding region deoxyribonucleotide of GenBank Accession No. NM-032808.5, encoding the amino terminal of GenBank Accession No. NP_116197. Amino acid residue at position 1-620. The recombinant expression vector containing the Lingo 1 encoding gene was named pcDNA-Lingol.

 7) /?7_5 expression vector construction of pcDNA-p75

 Using human brain cDNA) i (Invitrogen, Cat. SL.B4HB3MA) as a template, SEQ ID NO: 14 and SEQ ID NO: 15 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and then The pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I was ligated, and the ligation product was transformed into E. coli DH5a competent cells. According to sequencing, the amplified fragment (the coding gene of p75) is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No. M14764, and encodes the 1-888 position from the amino terminus of GenBank Accession No. Ml 4764. Amino acid residues. The recombinant expression vector containing the p75-encoding gene was named pcDNA-p75.

 8) Construction of expression vector pFLAG-AP-CMV1

 The human placenta cDNA library (Invitrogen, Cat. SL. NB2HP) was used as a template, SEQ ID NO: 16 and SEQ ID NO: 17 were used as primers for PCR amplification, and the PCR amplification products were digested with Not I and used with Not I. The digested pFLAG-CMV1 (Sigma, Cat. E7273) was ligated and the ligation product was transformed into E. coli DH5a competent cells. According to sequencing, the gene encoding the amplified fragment has the same sequence as the deoxyribonucleotide of the gene coding region of GenBank Accession No. X55958, and encodes the amino acid residue of GenBank Accession No. CAA39425. The recombinant expression vector containing the /4 cadaver-encoding gene was named pFLAG-AP-CMV1.

 9) SL; ^ expression vector pFLAG-AP-BLyS-CMVl construction

 The human spleen cDNA library (Invitrogen, Cat. 10425-015) was used as a template, and SEQ ID NO: 18 and SEQ ID NO: 19 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Sal I. The recombinant expression vector pFLAG-AP-CMV1 of the above step 8) digested with EcoR I and Sal I was ligated, and the ligation product was transformed into E. coli DH5a competent cells. According to the sequencing, the amplified coding gene is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No. NM_006573, and encodes the amino acid residue at positions 137-285 from the amino terminus of GenBank Accession No. NP-006564. base. The recombinant expression vector containing the coding gene was designated as pFLAG-AP-BLyS-CMV1.

 10) Construction of expression vector pFLAG-AP-Nogo-66-CMV1

The human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 20 and SEQ ID NO: 21 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Sal I. The recombinant expression vector pFLAG-AP-CMV1 of the above step 8) digested with EcoR I and Sal I was ligated, and the ligation product was transformed into E. coli DH5a competent cells. Root According to sequencing, the amplified fragment (N O-encoding gene) is identical to the sequence of GenBank Accession No. AF 148537 from the 5' end of the 3304-3501 deoxyribonucleotide, encoding GenBank Accession No. XP- Amino acid residues at positions 1-55-1120 from the amino terminus of 001496876. The recombinant expression vector containing the Nogo-66 encoding gene was named pFLAG-AP-Nogo66-CMV 1.

 11) Construction of FZ^G^^ expression vector pFLAG-BLyS-CMV1

 The recombinant expression vector pFLAG-AP-BLyS-CMV1 constructed in the above step 9) was digested with the restriction enzymes EcoR I and Sal I, and the BLyS fragment recovered by the gel was pFLAG-digested with EcoR I and Sal I. CMV1 was ligated and the resulting recombinant expression vector was named pFLAG-BLyS-CMVl o

 12) Construction of Fc expression vector pcDNA-Fc

 The human spleen cDNA library (Invitrogen, Cat. 10425-015) was used as a template, and SEQ ID NO: 22 and SEQ ID NO: 23 were used as primers for PCR amplification, and the PCR amplification products were digested with Xba I and Xba l The digested pcDNA3.1 (+) (Invitrogen, Cat. V790-20) was ligated and the ligation product was transformed into E. coli DH 5α competent cells. According to sequencing, the amplified fragment (the coding gene of Fc) has the same sequence as the deoxyribonucleotide of the gene coding region of GenBank Accession No. BC073782, and encodes the amino acid position 254-475 of GenBank Accession No. AAH73782. Amino acid residues. The recombinant expression vector containing the Fc-encoding gene was named pcDNA-Fc.

 13) Construction of sNgR-Fc expression vector pcDNA-sNgR-Fc

 A human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 24 and SEQ ID NO: 25 were used as primers for PCR amplification, and a fragment of the NgR-encoding gene which does not contain a GPI domain portion was amplified. (designated as sNgK), the PCR amplification product was digested with EcoR I and Xba I and ligated with pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I to ligate the product. Transformation of E. coli DH5 a competent cells. According to sequencing, the amplified gene encoding NgR is identical to the sequence of GenBank Accession No. NM-023004 from the 5' end of 202-1542 deoxyribonucleotides, encoding the amino group of GenBailk Accession No. NP_075380. Amino acid residues at positions 1-447. The expression vector containing the sNgR-encoding gene was named pcDNA-sNgR.

 The recombinant expression vector pcDNA-Fc constructed in the above step 12) was digested with Xba I, and the Fc fragment recovered from the gel was ligated with the above pcDNA-sNgR digested with Xba1, and the resulting recombinant expression vector was named as pcDNA-sNgR-Fc.

 14) Construction of sNgRH2-Fc expression vector pcDNA-sNgRH2-Fc

A human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 26 and SEQ ID NO: 27 were used as primers for PCR amplification, and a fragment of the NgRH2 encoding gene which does not contain a domain portion was amplified ( Named sNgR /2), the PCR amplification products were digested with EcoR I and Xba I and ligated with pcDNA3.1 ( +) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I. The product was transformed into E. coli DH5a competent cells. According to sequencing, the amplified gene encoding the fragment Ng 2 is identical to the sequence of the 1st to 1242 deoxyribonucleotides from the 5' end of the gene coding region of GenBank Accession No. NM-012452. The amino acid residues at positions 1-415 from the amino terminus of GenBank Accession No. NP_848663. The recombinant expression vector containing the sNgRH2 encoding gene I was named pcDNA-sNgRH2. The recombinant expression vector pcDNA-Fc constructed in the above step 12) was digested with Xba I, and the Fc fragment recovered in the gel was ligated with the above-mentioned pcDNA-sNgRH2 digested with Xba I, and the resulting recombinant expression vector was named as pcDNA-sNgRH2-Fc.

 2. Transiently transfected with calcium phosphate, HEK 293T cells, expressed protein

HEK 293T cells were cultured in a 010 cm cell culture plate at a cell density of 30-50%. The recombinant expression vectors pFLAG-AP-CMV1, pFLAG-AP-BLyS-CMV1, pFLAG-AP-Nogo-66-CMV1, pFLAG-BLyS-CMV pcDNA-sNgRH2-Fc and pcDNA constructed in the above step 1 were respectively prepared as follows. -sNgR-Fc was transfected into HEK 293T cells for transient expression of the protein. 4 μg of the above recombinant expression vector was diluted into 170 μΐ 2×HBS (50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HP0 ₄ , 11 7.10), and 170 μΐ 250 mM CaCl _{2 was} added dropwise to the above diluted recombinant expression vector. , fully mixed. The diluted recombinant expression vector and the mixture of CaCl ₂ were added to the cell supernatant, and after 4 hours, the culture solution was aspirated, and the cells were treated with 10% DMSO (diluted in PBS) for 2 min, and replaced with fresh normal cell culture. The solution is placed in a cell culture incubator. After 48-72 h, the cell supernatant was collected and protease inhibitor (final concentration 1 g/ml aprotinin, 1 Mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), 4 ° 4,000 was added. 111 centrifugation 10 1^11, the collected cell supernatant was poured into a 19 ml ultrafiltration tube (Centricon tubes Plus-20, Millipore, Beijing, China), and the horizontal rotor was centrifuged at 4 ° C, 3,800 rpm. Protein concentration. The concentrated protein was subjected to immunoblot detection. The results showed that the size of sNgR-Fc expressed by cells transfected into the recombinant expression vector pcDNA-sNgR-Fc was approximately 73 kDa; the size of sNgRH2-Fc expressed by cells transfected into the recombinant expression vector pcDNA-sNgRH2-Fc was approximately 72 kDa. Transfer into recombinant expression vector

The size of AP-BLyS expressed by cells of pFLAG-AP-BLyS-CMV1 was 75 kDa; the size of AP-Nogo66 expressed by cells transfected into recombinant expression vector pFLAG-AP-Nogo-66-CMV1 was 65.7 kDa, neurite outgrowth The Nogo66 used in the inhibition assay was AP-Nogo66 prepared by this method; the FLAG-BLyS expressed by the cells transfected into the recombinant expression vector pFLAG-BLyS-CMV1 was 16.7 kDa.

 3, protein purification

 (1) The sNgR-Fc and sNgRH2-Fc expressed in the above step 2 were purified by immobilized Protein A agarose (RepliGen, Massachusetts) as follows:

 a) The concentrated cell supernatant containing sNgR-Fc or sNgRH2-Fc protein stored at -80 °C was thawed at 4 °C while Protein A beads (RepliGen, Massachusetts), 4 was prepared. The column was washed with PBS buffer 14, 000 g for 30 s to remove the ethanol in the preservation solution under C conditions.

 b) The concentrated cell supernatant containing sNgR-Fc or sNgRH2-Fc protein was dispensed into a 1.5 ml centrifuge tube and centrifuged at 12 000 g for 10 min to remove the insoluble fraction.

 c) Add the appropriate amount of agarose gel (binding capacity 18-43mg agar gel / ml protein solution), bind for about 90 min at 4 °C, or combine for 1 h under ice bath.

d) Wash at 4 °C twice with 100 mM Tris buffer (pH 8.0), centrifuge for 14 sec at 14 000 g each time, and wash twice with 10 mM Tris buffer (pH 8.0). 14, 000 g centrifugation Wash for 30 s. _ e) Add 0.1 M glycine (pH 3.0) to the column, incubate for 10 min at room temperature, and wash with 14 000 g M heart for 30 s, 4. C collects the product (elution); depending on the efficiency with which the protein product is eluted, a second elution can be performed, in the same manner as above, preferably within 20 minutes. 1/10 volume of 1 M Tris (pH 8.5) was added to the eluted product to balance the pH, and 50 x cocktail (Roche, Cat. 1697498) was added.

 (2) The AP-BLyS, FLAG-BLyS and AP-Nogo-66 expressed in the above step 2 were purified by anti-FLAG M2 affinity gel (Sigma, St. Louis, MO). The specific method is as follows:

 a) The above proteins stored at -80 °C were thawed at 4 °C, and anti-FLAG M2 affinity gel (Sigma, Cat. A2220) was prepared with Ji-inch, and TBS buffer (50 mM) at 4 °C. Tris, 150 mM NaCl, pH 7.4) Centrifuge for 5 min three times at 1000 g to remove glycerol from the agarose preservation solution.

 b) Dispense the above proteins in a 1.5 ml centrifuge tube and centrifuge at 12 000 g for 10 min to remove the insoluble fraction.

 c) Add the appropriate amount of agarose gel (binding capacity > 0.6mg agar gel / ml protein solution), shake it slowly on a shaker at 4 °C, and combine for about 90 minutes.

 d) Wash the anti-FLAG M2 affinity gel with the above proteins in TBS buffer, and centrifuge at 5 °C for 1 000 § 5 °C at 4 °C for 4 times.

 e) Prepare FLAG protein elution solution: Dilute 5 mg/ml FLAG peptide (Sigma, Cat. F4799) in TBS buffer to a final concentration of 150 ng/μΐ and add protease inhibitor 50xcocktail (Roche, Cat. 1697498) ). When eluting, add an equal volume of FLAG protein elution solution to the gel and incubate at 40 °C for 40-50 min. Finally, 8 000 g was centrifuged 30 see and the product was collected. Store at -80 °C.

 4, expressive cloning method

 (1) Screening of human fetal brain cDNA library

 a) The backbone of the vector is pRK5 (BD Biosciences Pharmingen, Cat. 556104), and a linker is inserted through EcoR I/Xba I to introduce MIu I and Not I sites for easy ligation to the library. The sequence of Linker is shown in SEQ ID NO: 28 and SEQ ID NO: 29, and the recombinant vector ligated into Linker was named pRK5L. pRK5L was linearized with Not I /Mlu I, then dephosphorylated and purified using a DNA purification kit (Promega, Cat. A9281) to obtain a carrier concentration of approximately 100 ng^l.

 b) The human fetal brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was digested with Not I /Mlu I, and the digested library fragments were separately recovered using a gel recovery kit (QIANGEN).

c) The linearized and orthoylated pRK5L vector prepared in step a) was ligated with a cDNA library using T4 ligase and transformed with high-efficiency competent cell DH5a (Allele Biotech, Cat. ABP-CE-CC02020). The bacteria were then applied to a 015 cm plate of SOB medium (2% Bacto Tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgC12, 10 mM MgS04, 15 g/l Agar). 20 pieces were placed in an incubator at 37 °C overnight. When the colony grows large enough, count the number of colonies and scrape off all the colonies. Both were performed on ice to prevent amplification of the library. Finally, the bacterial solution was stored at -80 °C;

 (2) Method for expressing cloned library

 The recombinant human brain cDNA library was divided into different pools and transfected into COS-7 cells by transfection reagent Polyfect (QIAGEN, Cat. 301105). The recombinant expression vector pFLAG-AP-BLyS-CMV1 was used in HEK 293T. The protein product AP-BLyS expressed in the cell was used as a recombinant probe, incubated with COS-7 cells expressing the cDNA pool, and stained by alkaline phosphatase.

(See step (3) for details.) Observe the binding of AP-BLyS to molecules expressed on the surface of COS-7 cells. If AP-BLyS is able to bind to a molecule in the pool, the COS-7 cells expressing the molecule turn brownish black after color development by the substrate.

 (3) In the experiments of a and c in Fig. 1, pcDNA-NgR was transfected into COS-7 cells by lipofection, and after transfection for 36 hours, alkaline phosphatase was performed using AP-BLyS as a probe. dyeing. The specific dyeing steps are as follows:

 a) COS-7 cells in good growth state were passaged to poly-L-lysine (Sigma, Cat. P7890) coated cell culture plates one day prior to transfection with a cell density of 30-40%. After passage for 24 h, the recombinant expression vector pcDNA-NgR was transfected into the above COS-7 cells using polyfect (QIAGEN, Cat. 301105) according to the instructions for transfection reagent. For 12-well plates, the amount of plasmid per well was 750 ng and the transfection reagent was 5 μΐ; for 24-well plates, the amount of plasmid per well was 300 ng and the transfection reagent was 2.5 μl.

b) After transfection for 36-48h, when the cell density is about 80%, the cells are washed twice with HBHA solution (20 mM Hepes, pH 7.0, 0.1% NaN ₃ , 0.5 mg/ml BSA), first added to the blocking solution. (DMEM, 10% calf serum, 50 mM Hepes, pH 7.0), and then add the alkaline phosphatase-tagged protein AP-BLyS and AP expressed in step 2 above, and incubate at 23-25 °C for 1.5-2 h. .

 c) Wash the cells of step b) above for 6 times with pre-cooled HBHA solution for 7 min each time.

 d) Freshly prepare the fixative, dissolve the formaldehyde in HBS (20 mM Hepes, pH 7.0, 150 mM NaCl) to a final concentration of 1.8%, fix the cells for about 10 min at room temperature, and wash the cells on HBS. liquid.

 e) The cells were heated in a 65 °C electrothermal incubator for 2 h to inactivate the endogenous alkaline phosphatase. One

f) Wash the cells twice with staining buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl ₂ ) and then incubate the cells in this buffer for 10 min to accommodate high pH environments.

 g) Freshly prepare the staining solution. Dissolve NBT/BCIP (Roche, Cat 1681451) in the staining buffer to cover the cell surface. Staining is performed in the dark, and the staining of the cells is observed in real time. If the staining is weak, it can be incubated overnight. The dyeing reaction can be terminated by washing twice with PBS.

 (4) In the competition experiment of b in Figure 1, the recombinant expression vector pcDAN-NgR constructed in the above step 1 was transfected into COS-7 cells, and then the recombinant proteins FLAG-BLyS and AP-BLyS were separately added to the COS-7. The cells were incubated, and alkaline phosphatase staining was carried out according to the method of (3) above.

 (5) In the positive control experiment of d in Figure 1, the recombinant expression vector pcDNA-TACI constructed in the above step 1 was transfected into COS-7 cells by lipofection, transfected for 36 h, and then treated with AP-BLyS. The method of the above (3) is performed by alkaline phosphatase staining.

(6) In the negative control experiment of e in Figure 1, the recombinant expression vector constructed in the above step 1 pcDNA-DAF was transfected into COS-7 cells by lipofection. After transfection for 36 h, alkaline phosphatase staining was performed by AP-BLyS according to the above method (3).

 (7) In the experiment of f in Figure 1, the recombinant expression vector pcDNA-NgRH1 constructed in the above step 1 was transfected into COS-7 cells by lipofection, and transfected for 36 h, using AP-BLyS according to the above ( 3) The method is performed by alkaline phosphatase staining.

 (8) In the experiment of g in Figure 1, the recombinant expression vector pcDNA-NgRH2 constructed in the above step 1 was transfected into COS-7 cells by lipofection, transfected for 36 h, and then treated with AP-BLyS according to the above ( 3) The method is performed by alkaline phosphatase staining.

 (9) In the experiment of h in Figure 1, the recombinant expression vector pcDNA-p75 constructed in the above step 1 was transfected into COS-7 cells by liposome, transfected for 36 h, and then treated with AP-BLyS (3). The method is performed by alkaline phosphatase staining.

 (10) In the experiment of i in Figure 1, the recombinant expression vector pcDNA-Lingol constructed in the above step 1 was transfected into COS-7 cells by liposome, transfected for 36 h, and then treated with AP-BLyS according to the above (3). The method was performed by alkaline phosphatase staining.

 The results of the staining test are shown in Figure 1. In Figure 1a, AP-BLyS binds to COS-7 cells expressing NgR; in Figure 1 b, FLAG-BLyS blocks AP-BLyS binding to NgR; Figure 1 c, when AP acts as a probe , can not bind to NgR; Figure I d, TACI as a known receptor for BLyS, can bind to AP-BLyS, is the experimental positive control; Figure 1 e, DAF as another protein of the GPI protein family, Can not bind to AP-BLyS, is the negative control of the experiment; Figure I f, Figure 1 g, the other two homologous molecules NgR NgRHl and NgRH2 are transiently expressed in COS-7 cells, AP-BLyS can not Direct binding; In Figure lh, Figure 1 i, the other two components of the NgR receptor complex, p75 and Lingol, were expressed on the surface of COS-7 cells, respectively, and the results were also negative, and AP-BLyS could not directly bind to it.

(11) The quantitative curve of the binding of COS-7 cell lysate expressing NgR to AP-BLyS was used as the saturation curve of protein binding. The specific method is as follows: Six different concentrations of alkaline phosphatase recombinant probe AP-BLyS were selected. Co-incubation with COS-7 cells expressing NgR, respectively, for staining, specific cell culture, transfection and staining with a) -c) of the above step (3) (select cell culture plates with a diameter of 60 mm, without poly- L-lysine coating). The cells were treated with a 300 μM cell lysate (10 mM Tris, pH 8.0, 1% Triton) at 4 °C for 60 min on a shaker to maximize the cell lysate on the cell surface. The broken cells were blown down with a pipette tip, centrifuged at 10,000 rpm, and the precipitate was discarded. The supernatant was incubated in a 65 °C water bath for 1 h. Cell lysis supernatants stained with different concentrations of alkaline phosphatase recombinant probe AP-BLyS were added to a 96-well ELISA (Nunc) plate, and 100 μM substrate solution [15 ml 2 Μ diethanolamine (15 ml 2 ) was simultaneously added to the gun. Amresco, Cat. 0238), pH 9.8, 1 M magnesium chloride 15 μΐ, 100 mg p-nitrophenyl phosphate disodium (Sigma, Cat. 2765)], immediately measured the absorbance at OD _{4 () 5} nm. The results are shown in Figure 1 j, and the mosaic of the figure is a scatchard analysis. It indicates that the binding ability of NgR to BLyS is 4.60 ± 1.15 nM.

 5, immunoprecipitation experiment

Since BLyS exists in a soluble active form, it is difficult to detect the binding of BLyS to NgR in vivo. To further demonstrate that BLyS can interact with NgR in HEK 293T cells The soluble proteins FLAG-BLyS and sNgR-Fc were expressed in large amounts, and co-immunoprecipitation experiments were performed in vitro by concentration. The specific experimental procedures are as follows: 5 μ _Ε sNgR-Fc and 3 μ _δ FLAG-BLyS are co-dissolved in 1.5 ml TBS (10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5), and the protease inhibitor cocktail ( Roche Applied Science, Mannheim, Germany), 1 h at room temperature. Add Protein A beads or Anti-FLAG M2 beads to the reaction solution, 4. C acts for 1 h. The agar gel of Protein A combined with sNgR-Fc was washed 3 times with 100 mM Tris (pH 8.0) and 3 times with 10 mM Tris (pH 8.0); Anti-FLAG M2 combined with FLAG-BLyS The agarose gel was washed 5 times with TBS and centrifuged at 800 °g for 5 min each time at 4 °C. The above-prepared sample was added to 5x loading buffer (0.1 M Tris-cl; 4% SDS; 0.2% bromophenol blue, 20% glycerol, 0.1 M DTT), and the sample treated with Anti-FLAG M2 agar gel The sample buffer does not contain DTT to prevent antibodies in the agarose gel from eluting. Samples treated with Protein A agarose and samples treated with Anti-FLAG M2 agarose were incubated for 3 min at 100 °C on a heater and used for immunoblotting. sNgR-Fc was detected with horseradish peroxidase-labeled anti-human Fc (Beijing Dingguo Biotechnology Co., Ltd.), and FLAG-BLyS was detected with anti-FLAG (Sigma, Cat. F 1804).

 In Fig. 2, "10" indicates that the substance is added to the reaction system; "-" indicates that the substance is not added to the reaction system; from left to right, lane 1 indicates that no target protein is added to the reaction system, and only Protein A agar is added. At the time of gelation, anti-human Fc and anti-FLAG did not recognize any protein, indicating that the system can be used in this experiment; the lane 2 is a protein of sNgR-Fc that binds to Protein A agarose and reacts with anti-human Fc. Lane 3; the lane 3 indicates that the FLAG-BLyS protein cannot bind to the Protein A agar gel, and only the FLAG-BLyS protein and the Protein A agar gel cannot be immunoprecipitated to any protein; in lane 4, from the top, the first band is a protein band of sNgR-Fc that binds to Protein A agarose and reacts with anti-human Fc. The second band is FLAG that is simultaneously immunoprecipitated by Protein A agar gel and recognized by anti-FLAG by binding to sNgR-Fc. -BLyS protein band; Lane 5 indicates that no protein of interest is added to the reaction system, and anti-human Fc and anti-FLAG do not recognize any protein when only Anti-FLAG M2 beads are added. Can be used in this experiment; Lane 6 indicates that sNgR-Fc protein cannot bind to Anti-FLAG M2 beads, only sNgR-Fc protein and Anti-FLAG M2 beads can not be immunoprecipitated to any protein; Lane 7 is capable of Anti-FLAG M2 beads bind to the FLAG-BLyS protein band recognized by anti-FLAG; in lane 8, from the top, the first band is immunoprecipitated by Anti-FLAG M2 beads by binding to FLAG-BLyS The sNgR-Fc protein band, which is recognized by anti-human Fc, and the second band, is a FLAG-BLyS protein band that is immunoprecipitated by Anti-FLAG M2 beads and can be recognized by anti-FLAG.

 The results showed that when immunoprecipitation with Protein Abeads, anti-Fc detected sNgR-Fc protein, anti-FLAG detected FLAG-BLyS protein; when anti-FLAG M2 beads were used for immunoprecipitation, anti-FLAG was detected. The FLAG-BLyS protein, anti-human Fc, detected the sNgR-Fc protein precipitated by FLAG-BLyS, as shown in Figure 2. The above results indicate that soluble BLyS can directly interact with NgR in vitro.

 2. BLyS inhibits the growth of chicken embryo neurons

1. Effect on the structure of neuron growth cone Chicken embryo dorsal root neurons (DRG) explants isolated from 12-day embryos were cultured on coverslips coated with poly-L-lysine (PLL) (Sigma, Cat. P7890) and Laminin (Sigma, Cat. L2020). After 16-24 h, when the growth cone area of the axon was obvious, PBS, 2 g/ml Nogo-66 and 0.5 g/ml BLyS (Peprotech, Cat. 310-13) were added at 37 °C for 1 h. , fixed with 4% paraformaldehyde fixative (4 g paraformaldehyde per 100 ml PBS buffer, 25 g sucrose, pH 7.4) for 1 h at room temperature. The coverslips coated with chicken embryo DRG explatns were carefully removed and stained with phalloidin, Alexa Fluor 488 nM (Cambrex, Cat. PA3010), which specifically binds to the cytoskeleton. The confocal microscope can clearly observe the damage structure of the growth cone. The Nikon ECLIPSE TE2000-U fluorescence microscope is used to randomly select different fields of view. The photographs are taken under a 30-inch objective lens to measure the damage of the growth cone. The results are shown in Figure 3. . The growth of the stained DRG neurons in the growth cone can be seen in Figure 3-a. Figure 3-b is the statistical result of three replicate experiments. The number of damaged growth cones is counted according to the double-blind standard. Statistics were performed using the student's t test method. */? < 0.04, compared to the PBS treated group; **/? < 0.009, compared to the PBS treated group. The experimental results show that the degree of damage in the growth cone of DRG explants treated with BLyS is significantly increased.

 2. Effect on the growth of neuronal neurites

Embryo DRG isolated from embryos for 12 days, after trypsinization, cultured on coverslips coated with PLL and inhibitory proteins (1 μ _§ Nogo-66 or 200 ng BLyS), cultured in 24-well cell culture plates 16 -18 h The cells were fixed with the above paraformaldehyde fixative for 1 h at room temperature, and then blocked with a blocking solution (0.1 g Gelatin, 1 g BSA and 0.02 g NaN ₃ , pH 7.4 per 100 ml PBS buffer) for 1 h at room temperature; Add anti-β III tubulin (Millipore, Cat. MAB5544) specific for neurons. Incubate for 1 h at room temperature, wash 5 times with PBS for 5 min each time; add anti-mouse conjugated Rhodamine C Beijing Zhongqiao Jinqiao Bio Company) Incubate for 1 h at room temperature, wash 5 times with PBS for 5 min each time; Finally, place the coverslip on absorbent paper, dry in air, and seal. Different fields of view were randomly selected under a fluorescence microscope (the intersections could not be repeated between the fields of view), and photographs were taken under a microscope of Nikon ECLIPSE TE2000-U fluorescence microscope lOx. The length of the DRG neurite was measured using RS image software and the results are shown in FIG. The structure of DRG neurites under different treatment conditions can be seen from Figure 4-a. Figure 4-b is the statistical result of three replicates of the experiment in Figure 4-a. Each experimental group randomly takes 100 DRG neurites. The length of the double-blind method was statistically analyzed using the Student's t test method.

 0.0001, relative to the experimental group treated with PBS. The experimental results show that, similar to Nogo-66, BLyS can significantly inhibit the growth of neurites.

 Example 2. BLyS inhibits neurite outgrowth by binding to NgR, and sNgR inhibits BLyS inhibition of neurite outgrowth by blocking the binding of BLyS and NgR.

 I. sNgR-Fc inhibits the binding of AP-BLyS to NgR

The pcDNA-NgR recombinant vector was transfected into COS-7 cells, and the same amount of AP, AP-Nogo66 and AP-BLyS probes were combined with NgR-expressing COS-7 cells to perform alkaline phosphatase staining quantitative experiments. For the procedure, refer to the method of step 1/4/(11) in the first embodiment. The NgR-expressing COS-7 cells incubated with the recombinant alkaline phosphatase-labeled probe were lysed, and the alkaline phosphatase bound to NgR was measured at OD 405 nm. The experimental results are shown in Figure 5. When sNgR-Fc is present, the binding ability of AP-BLyS to NgR is significantly decreased, indicating that sNgR-Fc can block BLyS. Combined with NgR. The experiment was repeated three times and statistically calculated using the Student's t test method, * _P < 0.04.

 Second, BLyS affects the growth of chicken embryo neurites through NgR

 The embryonic dorsal root neurons of 12.5-day-old embryos were isolated and cultured in a cover glass coated with polylysine and the following proteins expressed in the above Example 1 after trypsinization: protein-free PBS solution, dissolved 100 ng sNgR-Fc in PBS, 1 Nogo-66 in PBS, 1 Nogo-66 in PBS, 200 ng BLyS in PBS, 200 ng BLyS in PBS, and 100 ng sNgR-Fc or 100 a mixture of ng sNgRH2-Fc. After culture for 18 h, the cells were fixed with the above 4% paraformaldehyde fixative for 1 h at room temperature; then blocked with the above blocking solution for 1 h at room temperature; add anti-β III tubulin specific for nerve cells and incubate for 1 h at room temperature, wash with PBS 5 times, 5 min each time; add anti-mouse conjugated Rhodamine for 1 h at room temperature, wash 5 times with PBS for 5 min each time; Finally, place the coverslip on absorbent paper, dry in air, seal sheet. Different fields of view were randomly selected under a fluorescence microscope (the intersections could not be repeated between the fields of view), and the Nikon ECLIPSE TE2000-U fluorescence microscope was photographed under the objective lens. The length of the radii of the dorsal root neurons was measured by RS image software, and the results are shown in Fig. 6. Statistical analysis was performed using Student's t test method, **/? < 0.0001, compared to the experimental group treated with PBS. The results showed that sNgR-Fc blocked the inhibition of BLyS induction, while sNgRH2-Fc, which also carries the Fc protein fragment, could not block the inhibition induced by BLyS, suggesting that sNgR-Fc specifically blocks BLyS-induced inhibition of neurite outgrowth. effect. In combination with the experimental results in the above, the substance capable of blocking the BLyS-induced inhibition of neurite outgrowth is sNgR, a polypeptide of the sequence represented by amino acid residues 1-447 of the NgR full-length amino terminus.

 To further verify whether BLyS affects neurite outgrowth through NgR, phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that specifically releases GPI-anchored proteins from cell membranes, was used for experiments.

Chicken embryo DRG isolated from 12-day embryos was trypsinized and cultured on coverslips coated with the following proteins expressed in Example 1 above: protein-free PBS solution, 1 μ _δ Nogo-66 in PBS And 200 ng BLyS in PBS. After 3 h of culture, a neuron medium was added while adding 1 U/ml PI-PLC (Sigma, Cat. 5542). After 19 h, the cells were fixed with the above-mentioned paraformaldehyde fixative for 1 h at room temperature; then blocked with the above blocking solution for 1 h at room temperature; the anti-β III tubulin specific for the nerve cell was added and incubated for 1 h at room temperature, and washed with PBS for 5 times. 5 min; add anti-mouse conjugated Rhodamine for 1 h at room temperature, wash 5 times with PBS for 5 min each time; Finally, place the coverslip on absorbent paper, dry in air and seal. Different fields of view were randomly selected under a fluorescence microscope (the intersections could not be repeated between the fields of view), and the Nikon ECLIPSE TE2000-U fluorescence microscope was used to observe the photographs. The length of the DRG ganglion was measured using RS image software and the results are shown in FIG. Statistical analysis was performed using the Student's Uest method. < 0.04, relative to the experimental group treated with PBS. The results showed that PI-PLC can block the inhibition of ganglion growth by BLyS, and further explain the information that BLyS inhibits neurite outgrowth through NgR transmission.

 The above experimental results indicate that BLyS affects the growth of neurons by specific binding to NgR, and sNgR specifically blocks the inhibition of neurite outgrowth induced by BLyS.

Third, BLyS affects the growth of chicken embryo neuron by activating RoA From the above experiments, BLyS can affect the growth cone of neurons, and the shape of the growth cone is mainly controlled by cytoskeletal proteins. Studies have shown that RhoA activation has a great influence on the growth cone of neurons, and studies have used RhoA inhibitors to promote neuronal regeneration. We used the ROCK-specific inhibitor Y27632 (Sigma, Cat. 0503) on the RhoA pathway to detect neurite outgrowth by inhibiting RhoA.

 Chicken embryo DRG isolated from 12-day embryos was trypsinized and cultured on coverslips coated with the following proteins of different protein expression in Example 1 above: protein-free PBS solution, 1 μg Nogo- dissolved in PBS 66 and 200 ng BLyS in PBS, 15 μΜ Υ27632 was added to the culture solution. After 18-19 h, the above paraformaldehyde fixative was fixed at room temperature for 1 h; then blocked with the above blocking solution at room temperature for 1 h; add anti-β III tubulin specific for nerve cells for 1 h at room temperature, and wash 5 times with PBS. 5 min each time; add anti-mouse conjugated Rhodamine for 2 h at room temperature, wash 5 times with PBS for 5 min each time; Finally, place the coverslip on absorbent paper, dry in air and seal. Different fields of view were randomly selected under a fluorescence microscope (the intersections could not be repeated between the fields of view), and photographs were taken under a 10x objective lens of a Nikon ECLIPSE TE2000-U fluorescence microscope. The length of the DRG neurite was measured by RS image software and the results are shown in Fig. 7. The growth of neurites in the dorsal root neurons of stained chicken embryos can be seen in Figure 8-a; Figure 8-b is the statistical results of three replicate experiments. The experimental results indicate that Y27632 can block the inhibition of neurite outgrowth by BLyS. In another aspect, BLyS affects the growth of neuronal processes by activating the RhoA pathway.

 When analyzing the data for statistics, the photos of the different experimental groups of each batch are regrouped, and the names of the original encoded files are retained. For experiments on the effects of neuron growth cones, the scrambled pictures were opened in Adobe photoshop software to see the damage and intact number of growth cones. For the sacral growth experiments, the counting of ganglion lengths was performed in the RS image software (Roper Scientific, Tucson, AZ), and the method was referenced (Lagenaur and Lemmon, 1987). Pick up neurites that are clearly not intersecting the surroundings, measured from the middle of the cell body to the tip of the neurite. The number of each photo is counted, corresponding to the original encoded file name, and the number of each experimental group is counted. The mean value of the lesion growth cone or neurite length was calculated using Graph Prism 5.0 software and plotted, and the error bars represent SEM.

Claims

Rights request 1. Blocking the substance that BLyS inhibits the transmission of neuronal neurite outgrowth information.  The substance for blocking BLyS inhibition of neuronal neurite outgrowth information transmission according to claim 1, wherein the substance is a substance which inhibits binding of BLyS to NgR.  The substance for inhibiting BLyS inhibiting neuronal neurite outgrowth information transmission according to claim 2, wherein the substance that inhibits binding of BLyS to NgR is a polypeptide, a protein, an antibody, a small molecule of a compound, a double-stranded RNA, Small RNA or RNA aptamers.  The substance for blocking BLyS inhibition of neuronal neurite outgrowth information transmission according to claim 3, wherein the substance which inhibits binding of BLyS to NgR is  (a) a polypeptide having the sequence of SEQ ID No: 1; or  (b) A polypeptide derived from the amino acid sequence of SEQ ID No: 1 which has been substituted, deleted or added with one or more amino acids and has an activity of inhibiting BLyS and NgR binding.  The use of a substance for blocking BLyS inhibition of neuronal neurite outgrowth information according to any one of claims 1 to 4 for the preparation of a medicament for promoting neurite outgrowth.  The use of a substance for blocking BLyS inhibition of neuronal neurite outgrowth information transmission according to any one of claims 1 to 4 for the preparation of a medicament for treating a nervous system injury disease.  The application according to claim 6, wherein the nervous system damage disease refers to multiple sclerosis, myelitis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, spinal cord injury, sciatic nerve injury. Or facial nerve damage.  A kit comprising a substance for blocking BLyS inhibition of neuronal neurite outgrowth information transmission according to any one of claims 1 to 4.  A composition comprising a substance for blocking BLyS inhibition of neuronal neurite outgrowth information transmission according to any one of claims 1 to 4.  10. Composition according to claim 9, characterized in that it further comprises a pharmaceutically acceptable carrier, a pharmaceutically acceptable salt and/or hyaluronic acid.  11. Composition according to claim 9, characterized in that it comprises pharmaceutically acceptable suitable excipients and / or suitable additives.  12. Composition according to claim 11, characterized in that the excipient is a stabilizer, an antioxidant, a permeability regulator, a buffer and/or a pH regulator. 13. The composition according to claim 11, wherein the additive is suitable Buffer, additional chelation masking agent or calcium chelate complex or calcium or sodium salt.  14. The composition of claim 11 further comprising a pharmaceutically acceptable non-toxic carrier.  15. The composition according to claim 14, wherein the pharmaceutically acceptable non-toxic carrier is pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, Glucose, sucrose or magnesium carbonate.  16. The composition of claim 9 in liquid form packaged or lyophilized.  17. A method of introducing a composition according to any one of claims 9-16 into a body by injection, spray, nasal drops, eye drops, infiltration, absorption, physical or chemical mediated methods.  A method for screening a substance for blocking BLyS inhibition of neuronal neurite outgrowth information transmission according to any one of claims 1 to 4.  The screening method according to claim 18, wherein the screening method refers to a method of screening a human fetal brain cDNA library with AP-BLyS to obtain NgR.  The screening method according to claim 19, wherein the method for screening a human fetal brain cDNA library by using AP-BLyS to obtain NgR is as follows: using an alkaline phosphatase-labeled BLyS fusion protein as a soluble probe, COS-7 cells transfected with a fetal brain cDNA library were screened, and alkaline phosphatase staining was used to detect whether AP-BLyS binds to COS-7 cells expressing a protein in a human fetal brain cDNA library.  21. The substance for blocking BLyS inhibition of neuronal neurite outgrowth information transmission according to claim 1, wherein the substance means blocking BLyS-related by inhibiting binding of BLyS to a substance of a nerve cell growth signaling pathway. A substance that stimulates the signaling pathway of nerve cells.  The substance for blocking BLyS inhibiting neuronal neurite outgrowth information transmission according to claim 1, wherein the substance refers to a substance that blocks a BLyS-related nerve cell growth signal pathway by binding to BLyS. .  The substance for blocking BLyS inhibiting neuronal neurite outgrowth information transmission according to claim 1, wherein the substance means blocking BLY and RhoA by inhibiting binding of BLyS to a substance of a nerve cell growth signaling pathway. A substance related to the growth pathway of nerve cells.