CN113577281A

CN113577281A - Agents and methods for modulating integrin beta subunit

Info

Publication number: CN113577281A
Application number: CN202010367306.6A
Authority: CN
Inventors: 陈剑峰; 张海龙; 郑雅娟
Original assignee: Center for Excellence in Molecular Cell Science of CAS
Current assignee: Center for Excellence in Molecular Cell Science of CAS
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2021-11-02
Also published as: WO2021218594A1

Abstract

Reagents and methods for modulating integrin beta subunits. The invention provides methods and uses of down-regulating integrin alpha E expression, reducing the ability of immune cells to migrate across blood vessels, inhibiting adhesion of immune cells to epithelial tissues, inhibiting homing of immune cells to gut-associated lymphoid tissues, and inhibiting the residence of immune cells in gut-associated lymphoid tissues, including down-regulating integrin beta subunit expression or activity. The invention also provides the use of an agent in the preparation of a medicament for down-regulating integrin alphaE expression, reducing the ability of immune cells to migrate across blood vessels, inhibiting the adhesion of immune cells to epithelial tissue, inhibiting the homing of immune cells to gut-associated lymphoid tissue, inhibiting the residence of immune cells in gut-associated lymphoid tissue, or inhibiting gut inflammation.

Description

Agents and methods for modulating integrin beta subunit

Technical Field

Background

Inflammatory Bowel Disease (IBD) is a type of Inflammatory response caused by immune dysregulation of the intestinal flora, mainly including Ulcerative Colitis (UC) and Crohn's Disease (CD). The onset of IBD is extremely common, approximately 40 million people per year worldwide, with the highest morbidity and mortality in developing countries. Common complications of intestinal inflammation are toxic intestinal dilatation, intestinal obstruction, intestinal perforation, gastrointestinal bleeding and colon cancer, which severely compromise human quality of life and life safety.

During the development and progression of IBD, mononuclear macrophages, neutrophils and inflammatory T cells all accumulate abnormally in the mucosal layer of the gut. First, destruction of the intestinal mucosa and epithelial structures causes a rapid response of innate immune cells, and mononuclear macrophages first home to the site of inflammation to secrete inflammatory factors (TNF-alpha, IL-1 beta, IL-6, and MCP-1) to exert a pro-inflammatory effect. Subsequently, the activated innate immune cells present antigens to the adaptive immune cells, and the activated inflammatory T cells (Th1 and Th17) further induce the migration of immune cells to an inflammation site, thereby performing the functions of bacteriostasis and mucosal epithelial repair. The above suggests that both innate and adaptive immune systems play important roles in the disease process. And over-activated immune cells can exacerbate the intestinal immune response.

The migration of lymphocytes from the circulatory system to specific tissue sites is a key link in the body's immunity and host defense. This process, in turn, relies on the binding of integrins to their cognate ligands, and is accomplished by two key steps, the regulation of lymphocyte rolling and stable adhesion. The integrin alpha 4 beta 7 is responsible for the directional migration of lymphocytes to intestinal tracts, can mediate the rolling of cells on the surface of vascular endothelium, can also mediate the stable adhesion of cells, and is also responsible for the transmission of transmembrane signals, and the interaction of the integrin alpha 4 beta 7 and the ligand Mucosal addressing 1 (MADCAM-1) plays an important role in the process of the tissue-specific migration of the lymphocytes. Under the condition that the lymphocyte is not stimulated, the integrin alpha 4 beta 7 on the cell membrane is in a low-activity conformation, shows low ligand affinity and can mediate the lymphocyte to roll and adhere on the surface of the vascular endothelium. When cells are subjected to an activator (chemokine)And cytokines) are activated, and a regulatory protein inside the cell induces a series of conformational changes of the integrin through a signal called amide-out signaling, i.e., a folding conformation with low activity changes to a stretching conformation with high activity, thereby completing the activation of the integrin. The activated integrin α 4 β 7 has high ligand affinity and mediates stable adhesion of lymphocytes. The integrin beta 17 subunit can also form heterodimers with the beta 0E subunit. Integrin α E β 7 is highly expressed on leukocytes in the intestinal mucosa, including Intraepithelial lymphocytes (IEL), dendritic cells, mast cells, and T Regulatory cells (Tregs). Integrin α E β 7 mediates adhesion of lymphocytes to intestinal epithelial cells through interaction with cadherin (E-cadherin), which is dependent primarily on Metal ion-dependent adhesion sites (MIDAS) of the α -I domain within the integrin α E subunit, allowing lymphocytes to reside in the gut. Chemokines have been reported to promote α E β 7⁺T lymphocytes enter the gut epithelium and increase adhesion to their ligand, E-cadherin. Integrin α E knockout mice show a significant decrease in cell number of IEL in the gut, suggesting its important function in maintaining gut lymphocyte and epithelial cell interactions. Therefore, the integrin beta 7 can regulate the homing of lymphocytes to the intestinal tract and regulate the retention of the lymphocytes in the intestinal tract, and the abnormal function of the integrin beta 7 (comprising two integrins of alpha 4 beta 7 and alpha E beta 7) is closely related to the occurrence and development of IBD and intestinal tumors.

Research reports that integrin beta 7 function loss can inhibit migration of lymphocytes to intestinal tract parts, so that the pathogenesis process of enteritis induced by T cells can be relieved. Based on this study, integrin beta 7 functional inhibitory antibodies were developed for clinical treatment of ulcerative colitis. Vedolizumab specifically blocks integrin α 4 β 7, and this monoclonal antibody has been approved for the treatment of adult ulcerative colitis and crohn's disease, but clinical data indicate that high doses of antibody exacerbate enteritis in some patients. In addition, another clinical data shows that the monoclonal antibody etrolizumab, which inhibits both α 4 β 7 and α E β 7, is not therapeutically effective in the high dose group, but in the low dose group. It is reported in the literature that the high dose group inhibits the migration of the inflammatory-suppressive Treg cells into the gut. Complete suppression of integrin beta 7 function (beta 7KO mice) results in the loss of intestinal Treg cells, exacerbating the acute intestinal inflammation mediated by innate immunity. The above reports indicate that over-inhibition of integrin beta 7 function has strong side effects in the treatment of IBD. Therefore, the development of new specific drugs for treating intestinal inflammation based on integrin structure and function has yet to be further studied.

Disclosure of Invention

Through research, the inventor finds that the activation of the integrin can be used for regulating the adhesion and homing of immune cells, the immune homeostasis of intestinal tracts of organisms and the generation and development of intestinal inflammation.

The invention provides a use of down-regulation of the expression or activity of integrin beta subunit in down-regulation of the expression of integrin alpha E, reduction of the ability of immune cells to migrate across blood vessels, inhibition of adhesion of immune cells to epithelial tissue, inhibition of homing of immune cells to gut-associated lymphoid tissue, and inhibition of residence of immune cells in gut-associated lymphoid tissue. In one or more embodiments, the use is a non-therapeutic or diagnostic use.

In one or more embodiments, the immune cell is a lymphocyte.

In one or more embodiments, the integrin beta subunit is an integrin beta 7 subunit.

The invention also provides methods of down-regulating integrin alpha E expression, reducing the ability of immune cells to migrate across blood vessels, inhibiting adhesion of immune cells to epithelial tissue, inhibiting homing of immune cells to gut-associated lymphoid tissue, inhibiting residence of immune cells in gut-associated lymphoid tissue, inhibiting gut inflammation, reducing gut graft versus host disease, inhibiting tumorigenesis, comprising the step of down-regulating integrin beta subunit expression or activity.

In one or more embodiments, the immune cell is a lymphocyte.

In one or more embodiments, the methods comprise reducing or inhibiting the interaction between an aromatic amino acid in the integrin beta subunit I domain and a metal cation of the SyMBS site.

In one or more embodiments, the method comprises reducing or inhibiting the interaction between phenylalanine at position 185 in the integrin beta subunit and a metal cation of the symmbs site.

In one or more embodiments, the methods comprise deletion or substitution mutagenesis of phenylalanine at position 185 of the β subunit.

In one or more embodiments, the methods comprise mutating phenylalanine (Phe, F) at position 185 of the beta subunit to tryptophan (Trp, W), histidine (His, H), and alanine (Ala, a)

In one or more embodiments, the method comprises mutating phenylalanine Phe at position 185 in the integrin beta subunit to alanine Ala, resulting in a complete disappearance of cationic pi interactions between the integrin beta subunit and SyMBS.

The invention also provides the use of an agent selected from the group consisting of:

(1) an integrin or β subunit thereof having reduced activity, or a nucleic acid sequence encoding the same, and optionally a knockout agent for a wild-type integrin or β subunit thereof,

(2) an agent that decreases integrin beta subunit activity,

(3) an agent that down-regulates integrin beta subunit expression.

In one or more embodiments, the side effect of the treatment of inflammatory bowel disease is a reduction in intestinal Treg cells.

In one or more embodiments, the intestinal inflammation is an adaptive immune-mediated chronic intestinal inflammation or an innate immune-mediated acute intestinal inflammation.

In one or more embodiments, the activity is a high affinity adhesion activity. Preferably, the activity is a stable adhesion activity mediated by a high affinity state of an integrin.

In one or more embodiments, the gut-associated lymphoid tissue is Peyer's patch, mesenteric mucosal lymph nodes, small intestinal intraepithelium, small intestinal mucosal lamina propria, and colon.

In one or more embodiments, (1) the integrin or β subunit thereof having reduced activity comprises an inactive or low activity wild-type integrin or β subunit thereof, or a mutant of an integrin or β subunit thereof having reduced or absent activation function.

In one or more embodiments, the integrin mutant comprises a mutation that results in reduced or absent interaction between an aromatic amino acid in the integrin beta subunit I domain and a metal cation of the symmbs site as compared to wild type.

In one or more embodiments, the integrin mutant comprises a mutation that results in a reduced or absent interaction between phenylalanine at position 185 in the integrin beta subunit and the metal cation of the SyMBS site as compared to wild type.

In one or more embodiments, the mutation is a mutation of F at position 185 of the integrin beta subunit or a mutation of the SyMBS site.

In one or more embodiments, the phenylalanine (Phe, F) at position 185 of the beta subunit of the integrin mutant is mutated to tryptophan (Trp, W), histidine (His, H), and alanine (Ala, a).

In one or more embodiments, the phenylalanine at position 185 of the beta subunit of the integrin mutant is mutated to a tryptophan. This mutation resulted in the complete disappearance of the cationic pi interaction between the aromatic system and SyMBS.

In one or more embodiments, the beta subunit of the integrin mutant has the sequence shown in SEQ ID No. 1 or 2 or a mutant having 80% sequence identity thereto and the mutation at position 185 is A, H or W.

In one or more embodiments, the integrin mutant has the sequence shown in SEQ ID No. 3 or 4 and the mutation at position 185 to A, H or W.

In one or more embodiments, the decreasing integrin beta subunit activity in (2) is decreasing or inhibiting cationic interaction between an integrin beta subunit aromatic amino acid and the SyMBS site; preferably, the interaction between the 185 th metal cation of the integrin beta subunit and the metal cation of the SyMBS site is reduced or inhibited.

In one or more embodiments, the agent that decreases integrin beta subunit activity is selected from the group consisting of:

modulators that stabilize integrin intracellular conserved sequences GEFKR and/or LLv-iHDR, such as modulators that allow GEFKR and LLv-iHDR to form hydrophobic and ionic bonds;

agents that enable the formation of alpha helical structures in both the α and β intracellular segments of integrins, such as agents that introduce peptide segments rich in acidic and basic amino acids into the α and β subunit intracellular segments;

agents that bind F at position 185 in the integrin beta subunit, such as folic acid, methylene blue, urea, ferrocene, hemin, hydrocortisone, 7-hydroxycoumarin, hydroxycamptothecin, and the like;

a reagent containing a high concentration of calcium ions, for example, a reagent containing 3 to 10mmol/L, 3.5 to 9mmol/L, 4 to 8mmol/L, 4.5 to 7mmol/L, 5 to 6mmol/L, or 5mmol/L of calcium ions;

an agent that inhibits binding of an intracellular protein to an integrin; said intracellular proteins are Talin, Kindlin and Paxillin; the agent comprises a protein that competitively binds with an alpha subunit and/or a beta subunit and a protein kinase that modulates the protein; in one or more embodiments, the proteins that compete for binding to the β subunit are DOK1, ICAP1, and finamin; in one or more embodiments, the protein that competes for binding to the alpha subunit is SHARPIN and MDG 1; in one or more embodiments, the protein kinase includes: ILK, Src, and Cas;

an agent that inhibits binding of an integrin to its ligand, preferably a β -subunit extracellular fragment or its coding sequence, that competitively binds to the ligand; the fragment has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% sequence identity to an extracellular portion of an integrin beta subunit; the fragment is preferably an amino acid sequence from positions 46-386 of the integrin beta subunit (extracellular MAdCAM-1 specific binding sequence).

In one or more embodiments, the agent that reduces integrin beta subunit activity is a knock-in agent for the integrin beta subunit or the SyMBS site that reduces or inhibits cationic interaction between the integrin beta subunit and the SyMBS site. Preferably, the knock-in causes a mutation at amino acid 185 of the β 7 subunit, for example to A, H or W, preferably to a. Preferably, the knock-in mutations the SyMBS locus. More preferably, the knock-in reagent comprises a nucleic acid molecule having the sequence shown in SEQ ID NO. 7.

In one or more embodiments, the agent that decreases integrin beta subunit activity is an antibody or nucleic acid sequence encoding a specific antibody to integrin or its beta subunit that decreases integrin beta subunit activity, or a small molecule compound having activity that inhibits integrin or its beta subunit.

In one or more embodiments, the agent that decreases integrin beta subunit activity is a cation chelator, such as EDTA.

In one or more embodiments, the agent that down-regulates expression of the integrin beta subunit is an siRNA, shRNA, or gene knock-out vector of the integrin beta subunit.

Also provided herein is a pharmaceutical composition comprising an agent as described in any embodiment herein and optionally a pharmaceutically acceptable carrier or excipient.

Also provided herein is a nucleic acid molecule comprising a sequence selected from the group consisting of:

(1) a nucleic acid sequence containing a sequence shown as SEQ ID NO. 7 and a LoxP site; and

(2) (1) the complement of said sequence.

In one or more embodiments, the nucleic acid sequence comprises a sequence of homology arms, wherein the 3' homology arm comprises SEQ ID No. 7.

In one or more embodiments, the nucleic acid sequence comprises, in order from 5 'to 3', a 5 'homology arm, a LoxP site, a marker gene, a LoxP site, and a 3' homology arm.

In one or more embodiments, the nucleic acid sequence comprises, in order from 5 'to 3', a 5 'homology arm, a LoxP site, frt, a marker gene, frt, a LoxP site, and a 3' homology arm.

In one or more embodiments, the homology arms can have an optional linker sequence with the LoxP sites.

Also provided herein is a vector comprising a nucleic acid sequence as described in the first aspect herein. In one or more embodiments, the vector is for use in homologous recombination.

Also provided herein is a genetically engineered host cell transformed with a vector according to the first aspect of the invention. In one or more embodiments, the host cell comprises in its genome a nucleic acid sequence as described in the first aspect herein.

In one or more embodiments, the host cell is a non-human mammalian cell.

In one or more embodiments, the host cell is a rodent cell.

In one or more embodiments, the host cell is a human somatic cell or an established embryonic stem cell.

In one or more embodiments, the host cell is a somatic cell or an embryonic stem cell of a non-human mammal.

Also provided herein is a method of constructing a transgenic mouse, the method comprising:

(1) there is provided a vector as described in the first aspect herein,

(2) transferring the vector into mouse embryonic stem cells, screening to obtain homologous recombination embryonic stem cell clone,

(3) injecting the embryonic stem cells obtained in the step (2) into a blastocyst of a mouse, transferring the blastocyst into a pseudopregnant mother mouse to obtain a chimeric mouse,

(4) mating the chimera mouse obtained in step (3) with a wild type mouse (e.g., C57/B6) to obtain a F185-mutated mouse having an integrin beta subunit of Flox site,

(5) crossing the F185-mutated mouse with the integrin beta subunit of Flox locus obtained in step (4) with a transgenic mouse expressing (e.g., constitutively expressing) Cre enzyme to obtain a first generation heterozygous mouse, and then crossing the heterozygous mouse to obtain a homozygous mutated mouse, i.e., the transgenic mouse,

optionally (6) the mice obtained in step (5) are mated with wild type mice (e.g., C57/B6) to purify the gene background.

In one or more embodiments, the construction method further comprises mating the homozygous mutant mouse obtained in step (6) with the homozygous control mouse obtained in this step, thereby expanding the population of homozygous mice.

In one or more embodiments, the transgenic mouse is characterized by an inactivity or a decreased activity of the integrin or its β subunit.

In one or more embodiments, the integrin is integrin α 4 β 7 and/or α E β 7.

Also provided herein is a transgenic mouse having no or reduced activity of an integrin or a β subunit thereof.

Also provided herein is the use of the integrin or beta subunit gene or protein as a target for screening drugs for treating or preventing intestinal disorders or ameliorating the therapeutic side effects of intestinal inflammatory diseases, or as a molecular index for clinical diagnosis of the course of intestinal disorders.

In one or more embodiments, the intestinal disease is a disease that benefits from inhibiting stable adhesion of lymphocytes, reducing the ability of immune cells to migrate across blood vessels, down-regulating integrin α E expression, inhibiting homing of immune cells to gut-associated lymphoid tissue, inhibiting intestinal inflammation. Preferably, the disease is intestinal inflammation. More preferably, the intestinal inflammation is adaptive immune-mediated chronic intestinal inflammation or innate immune-mediated acute intestinal inflammation.

Also provided herein is the use of a reagent for detecting a gene or protein of integrin or its beta subunit in the preparation of a kit for diagnosing an intestinal disease or determining the course of an intestinal disease.

Also provided herein is a detection kit comprising reagents for detecting a gene or protein of integrin or its beta subunit.

Drawings

FIG. 1 shows integrin Itgb7^F185A/F185AAnd constructing a strategy and identifying KI mice. (A) Mouse Itgb7 genomic sequence analysis. (B) Itgb7^F185A/F185AKI mouse plasmid construction strategy. (C) Genotype identification, wherein only 380bp strips are KI homozygotes; only the 360bp band was WT mouse; both bands were heterozygous mice. (D) A mouse genome fragment carrying the F185A mutation was amplified using KOD high fidelity DNA polymerase for sequencing identification. The results show the success of the codons TTT (Phe) encoding amino acid residue 185The mutation was GCG (Ala) and no further mutations were introduced.

Figure 2 shows that the integrin beta 7F185A mutation results in a decreased ability of lymphocytes to migrate to intestinal tract-associated tissue sites. (A) H & E staining showed that KI and KO mice had essentially intact intestinal structures, but finer villi and decreased numbers of lymphocytes within the villi, compared to WT mice. (B) The colon structures of KI and KO mice were essentially intact. (C, D) the Peltier's knot (PP) on the small intestine of KI and KO mice became small and the number of lymphocytes in PP decreased.

FIG. 3 shows that the integrin beta 7F185A mutation inhibits lymphocyte adhesion and migration. (A) FACS detection of WT, beta 7 heterozygotes (Itgb 7)^+/-+/-), beta 7 gene Knock-down (KD), KI and KO mouse spleen lymphocyte surface integrin alpha 4 and beta 7 expression. The dashed lines indicate the control group, i.e., no staining for α 4 and β 7 antibodies. The solid line indicates the expression of α 4 and β 7. (B) Flow cell technology (Flow chamber) examined WT, +/-, KD, KI and KO mouse spleen lymphocytes for adhesion to the integrin ligand MAdCAM-1. The cells are stimulated by three conditions of no chemokine and chemokine (CCL21 or CCL 25). The cells were stressed at 1dyn/cm (Shear stress) respectively²Or 2dyn/cm²Through the flow chamber. Dark grey and blank columns represent stably adherent cells (firm adhesive cells) and Rolling adherent cells (Rolling cells), respectively. (C, D) the ability of WT, +/-, KD, KI and KO mouse spleen lymphocytes to migrate through the perforations on the ligands MAdCAM-1(C) and ICAM-1(D) was tested using a serum-induced cell perforation migration assay. P<0.001; ns means no significant difference (not significant).^AAAP<0.001(Student's t-test at A-D). Data were counted as mean ± standard deviation. Asterisks indicate total number of adherent cells.

FIG. 4 shows that the integrin beta 7F185A mutation did not affect the adhesion of alpha E alpha 07 to E-cadherin. (A) FACS measures the expression of cell surface integrins α 5E and α 67 on Jurkat T- α 1E α 27WT and Jurkat T- α 3E α 47F 185A. The dashed lines indicate the control group, i.e., no staining for α E and β 7 antibodies. The solid line indicates the expression of α E and β 7. The numbers in the boxes represent the mean fluorescence intensity. (B) Flow Chamber assay Jurkat T-alpha E beta 7WT and Jurkat T-alpha E beta 7F185A cells andadhesion of ligand E-cadherin. The mechanism of chemokine-regulated integrin beta 7 mutations was studied by three conditions, namely, addition of chemokine (CCL21 or CCL25) and addition of antibody M290 that inhibits binding. The cells are stressed at 1dyn/cm²Through the flow chamber. P<0.001; ns means no significant difference (not significant). Data were counted as mean ± standard deviation.

FIG. 5 shows that the integrin beta 7F185A mutation results in alpha E beta 7⁺Lymphocytes were significantly reduced and the expression of integrin alphae was down-regulated. (A) FACS measures α E expression in WT, +/-, KI and KO mouse spleen lymphocytes. (B) FACS measures expression of IEL, LPL α E in WT, +/-, KI and KO mice in the small intestine and colon. The numbers in the boxes of (A, B) represent the proportion of α E β 7-positive cells. Intraepithelial lymphocytes (IEL); mucosal lymphocytes (LPL).

FIG. 6 shows that loss of integrin beta 7 activation function inhibits migration of spleen lymphocytes to gut-associated immune tissues. (A-C) the rate of migration of splenic lymphocytes from KI (A), KO (B) and +/- (C) mice to different lymphoid tissues in recipient mice. FACS 18 hours after injection measures the ratio of KI, KO or +/-lymphocytes to WT lymphocytes in a particular tissue. Peripheral Blood (PB); peripheral Lymph Nodes (PLN); mesenteric Lymphnodes (MLN); peyer's patch, PP); small Intestine (SI); colon (Colon); spleen (Spleen, SP); liver (Liver, LIV) and Lung (Lung, LUN). P <0.001(Student's t-test); ns means no significant difference (not significant). Data were counted as mean ± standard deviation.

FIG. 7 shows Itgb7^F185A/F185AKI mouse CD4⁺CD45RB^highThe ability of T cells to induce enteritis is diminished. (A) Rag1^-/-The recipient mice were given 1X 10 tail vein injections, respectively⁵WT, KI or KO CD4⁺CD45RB^highT cells, followed by 12 weeks to observe the body weight change of recipient mice. (B) Tail vein injection of each group of mice CD4⁺CD45RB^highpost-T cell, Rag1^-/-Enteritis attack in recipient miceAnd (5) carrying out score statistics. (C) Tail vein injection of each group of mice CD4⁺CD45RB^highpost-T cell, Rag1^-/-Recipient mouse colon distal H&And E, dyeing results. (D) Immunofluorescence detection of Rag1^-/-Recipient mouse colon distal CD4⁺T cell infiltration. And counting the CD4 in each fluorescence image⁺The number of T cell infiltrates. The scale bar is 100 μm. P<0.005，***P<0.001; ns means no significant difference (not significant). Data were counted as mean ± standard deviation.

FIG. 8 shows Itgb7^F185A/F185AKI mice did not increase susceptibility to DSS-induced acute intestinal inflammation. (A) Mice were treated with medication for five days, followed by ten days of recovery from normal drinking water, and the weight changes of the WT, KI and KO groups of mice were observed. (B) And (4) carrying out statistics on the mortality of the mice in each group after the DSS treatment. (C) And (4) counting the enteritis incidence scores of the mice in each group after the DSS treatment. (D) A Real-time quantitative nucleic acid amplification detection system (qPCR) detects the secretion of proinflammatory factors IL-6, TNF-alpha and IL-1 beta in intestinal tissues of mice. (E) DSS treatment of distal colon H in each group of mice on the tenth day&And E, dyeing results. (F) Treg cells in the mouse intestinal tract are in CD4⁺The proportion in T cells and the total number of Treg cells. (G) And (3) carrying out immunofluorescence detection and counting ICAM-1 expression and macrophage infiltration of intestinal epithelial cells of mice on the fourth day of DSS treatment.

Detailed Description

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) may be combined with each other to constitute a preferred embodiment.

SyMBS (the synthetic binding site), MIDAS (metal ion-dependent addition site) and ADMIDAS (the ad jacent to MIDAS) are the three metal ion binding sites of integrin beta subunit I domain. SyMBS functions primarily through cooperation with MIDAS and is a positive regulatory site for integrins, whose activation depends on the binding of SyMBS to metal ions. Previous work on integrin α 4 β 7 found that SyMBS functions by forming a cation-pi interaction with the benzene ring of F185 in its vicinity (Pan et al, 2010). This cation-pi interaction links the integrin beta subunit metal ion binding site to another regulatory element of integrin, sdl (specific metal binding site) (Pan et al, 2010). Loss of cation-pi interactions severely affects high affinity α 4 β 7-mediated stable cell adhesion, but has little effect on low affinity α 4 β 7-mediated rolling cell adhesion (Pan et al, 2010).

In the invention, the expression of integrin alpha E, the ability of immune cells to migrate across blood vessels, the adhesion of immune cells to epithelial tissues, the homing of immune cells to intestinal tract-associated lymphoid tissues, the residence of immune cells in intestinal tract-associated lymphoid tissues and/or intestinal inflammation can be further regulated and controlled by regulating and controlling the activity of integrin beta subunits. It was found that human beings activate mutant mouse Itgb7 by constructing integrin beta 7^F185A/F185A(the mutation inhibits the activation of the integrin beta 7), and the discovery shows that the inhibition of the activation of the integrin beta 7, namely the partial deletion of the function of the integrin beta 7, can reduce the ability of the immune cells to migrate across blood vessels, inhibit the homing of the immune cells to the intestinal tract-associated lymphoid tissue, and can inhibit the adhesion of the immune cells to the epithelial tissue and the retention of the immune cells in the intestinal tract-associated lymphoid tissue due to the reduction of the expression of the integrin alpha E. More importantly, the inhibition of integrin beta 7 activation does not cause intestinal Treg cell loss compared with the complete inhibition of integrin beta 7 function, and avoids the side effect of innate immunity-mediated intestinal exacerbation caused by the complete inhibition of integrin beta 7 function (the advantage of integrin beta 7 activation as an IBD treatment strategy). Therefore, the inhibition of integrin beta 7 activation can inhibit adaptive immune-mediated chronic enteritis and can also slow down congenital immune-mediated acute enteritis, reduce the side effect of the existing integrin beta 7 inhibition antibody in IBD treatment, and provide a new idea for clinical treatment and new drug development.

Accordingly, provided herein is a method of modulating the expression of integrin alphae, the ability of immune cells to migrate across blood vessels, the adhesion of immune cells to epithelial tissues, the homing of immune cells to gut-associated lymphoid tissues, the residence of immune cells in gut-associated lymphoid tissues, the inhibition of adaptive immune-mediated enteritis, and not the increase in susceptibility to innate immunity-mediated enteritis by modulating integrin beta subunit activity. Herein, "modulation" includes up-regulation including promotion, increase and/or enhancement and down-regulation including inhibition, reduction, attenuation, disruption and/or reduction. Herein, "gut-associated lymphoid tissue" includes, but is not limited to, Peyer's Patch (PP), mesenteric mucosal lymph nodes, small intestinal intraepithelial, small intestinal mucosal lamina propria, and colon. As used herein, "homing" refers to the directional migration of immune cells from the blood to the lymphoid organs through the capillary hyperendothelial veins, as well as the exudation of immune cells to sites of inflammation. Herein, a "membrane" is a perforated chamber that mimics the process of passage of lymphocytes through blood vessels in vivo.

Herein, "targeting" refers to the specific migration of immune cells to secondary lymphoid organs, sites of inflammation or tumor tissue, etc., to enhance immune surveillance, maintain immune homeostasis or promote an immune response. Herein, "individual", "subject" or "patient" refers to a mammal, in particular a human.

Herein, an integrin or integrin is a transmembrane heterodimer consisting of two non-covalently associated transmembrane subunits, the α and β subunits. The extracellular domain of integrins can bind to extracellular ligands such as matrix proteins, and intracellular structures initiate downstream processes. Binding of the integrin to the ligand requires a divalent cation, such as Ca²⁺、Mg²⁺And so on. Exemplary integrin beta subunits are the integrin beta 7 subunits shown in

SEQ ID NO

1 or 2. Alpha subunits capable of forming heterodimers with the integrin beta subunit include alpha 4 and alpha E. The integrin alpha subunit has the sequence shown in

SEQ ID NO

3 or 4 as integrin alpha 4 subunit, and

SEQ ID NO

5 or 6 as integrin alpha E subunit. It is to be understood that, herein, unless specifically mentioned, integrins refer broadly to integrins of various origins.

Mutants of the integrin are also included herein. The integrin mutants referred to herein include mutants in which the alpha subunit of the wild-type integrin is retained, and mutations occur only in the beta subunit or SyMBS sites. The mutation of the β subunit or the SyMBS site may be an insertion, substitution or deletion, so long as the mutation modulates the cationic interaction between the integrin β subunit and the SyMBS site and optionally does not affect the binding of the integrin to its ligand. In certain embodiments, the integrin mutants comprise mutants that have undergone a mutation that results in a failure to mediate cationic interaction between the β subunit and the SyMBS site. The mutation may be a substitution mutation, particularly a substitution mutation of F at position 185 of the β subunit, such as a mutation to a.

The regulation of the interaction between the beta subunit and the SyMBS can be realized by regulating the beta subunit and/or regulating the SyMBS. For example, the interaction between an aromatic amino acid in the β subunit I domain and a metal cation of the SyMBS site can be down-regulated by decreasing the activity of the integrin, thereby down-regulating the expression of integrin α E, decreasing the ability of immune cells to migrate across blood vessels, inhibiting the adhesion of immune cells to epithelial tissues, inhibiting the homing of immune cells to gut-associated lymphoid tissues, inhibiting the residence of immune cells in gut-associated lymphoid tissues, or inhibiting gut inflammation. The present invention is not intended for treatment or diagnosis. Methods of reducing the activity of an integrin include, but are not limited to, the use of (1) reduced activity integrins or the β subunit thereof, or nucleic acid sequences encoding the same, and optionally a knockout agent for the wild-type integrin or the β subunit thereof, (2) an agent that reduces the activity of the β subunit of an integrin.

Reduced activity integrins or integrin beta subunits described herein are any form of reduced or deleted high affinity adhesion activity integrin or beta subunit, including inactive or low activity wild type integrins or beta subunits, or reduced or deleted activation function integrin or beta subunit mutants, such as mutants comprising a mutation in the integrin beta subunit or SyMBS site. Thus, the coding sequence for the β subunit or the SyMBS site expressing the mutation can be integrated into the genome of the cell of interest by a knock-in vector (replacement vector, targeting vector, etc.), replacing the coding sequence for the wild-type β subunit or the SyMBS site, thereby allowing the cell of interest to express the mutant β subunit or the SyMBS site with reduced interaction (e.g., more than 50% less) as compared to the unmutated protein, while retaining other biological activities of the β subunit and the integrin. Exemplary mutant integrins include, but are not limited to, mutants in which the β subunit or the SyMBS site is mutated, resulting in reduced or absent interaction between aromatic amino acids in the β subunit I domain and the metal cation of the SyMBS site as compared to wild type. Exemplary mutations include, but are not limited to, a mutation of F at position 185 of the beta subunit. For example, the amino acid residue F at position 185 may be deleted, or F may be substituted with other amino acids, especially with other amino acid residues not belonging to the same amino acid class as F. For example, mutations that reduce the interaction between the β subunit and the SyMBS site can be: phe (symbol F) phenylalanine at position 185 was mutated to Trp (symbol W) tryptophan and histidine (symbol H).

Herein, the classes of amino acids can be broadly classified into: (1) nonpolar amino acids (hydrophobic amino acids) including alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), and methionine (Met); (2) polar amino acids (hydrophilic amino acids), including (a) polar uncharged amino acids, which are glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), and glutamine (Gln); (b) polar positively charged amino acids (basic amino acids) are lysine (Lys), arginine (Arg) and histidine (His); and (c) polar negatively charged amino acids (acidic amino acids) which are aspartic acid (Asp) and glutamic acid (Glu).

The agents that reduce integrin beta subunit activity described herein are agents that reduce or inhibit the interaction between aromatic amino acids in the integrin beta subunit I domain and metal cations of the SyMBS site. Such agents may be knock-in agents for the integrin beta subunit or the SyMBS site that result in reduced or inhibited interaction between the aromatic amino acid in the integrin beta subunit I domain and the metal cation of the SyMBS site, for example, by mutating the beta subunit (e.g., amino acid 185) or the SyMBS site. The knock-in agent may be a knock-in vector as described above, which has a nucleic acid molecule having a nucleic acid sequence of the β subunit or the SyMBS site to be knocked in, or a complementary sequence thereof. An exemplary nucleic acid molecule for knock-in has the nucleic acid sequence shown in SEQ ID NO 7. SEQ ID NO 7 is an exemplary knock-in sequence, the base sequence of a homologous fragment downstream of P349, in which the "TTT" (phenylalanine) of the wild type is mutated to "GCG" (alanine). The nucleic acid sequence may also contain a sequence of homology arms for homologous recombination and a linker sequence between the homology arms and the sequence to be knocked in. In certain embodiments, elements of homology arms, knock-in sequences, LoxP sites, and linker sequences are shown in figure 1. The skilled worker is aware of the sequences of these elements or other elements required for knock-in by homologous recombination or methods for obtaining the sequences, for example via the NCBI website.

Agents described herein that reduce integrin beta subunit activity also include, but are not limited to, modulators that stabilize integrin intracellular conserved sequences GEFKR and LLv-iHDR, agents that form integrin alpha and beta intracellular segments into alpha helical structures, agents that bind F at position 185 in integrin beta subunit, agents that reduce integrin beta subunit activity that are or contain high concentrations of calcium ions (e.g., at least 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L, 10mmol/L), agents that inhibit binding of intracellular proteins to integrins, agents that inhibit binding of integrins to their ligands, gene knock-in agents, cation chelators, integrin or antibodies specific for its beta subunit or nucleic acid sequences encoding the same that reduce integrin beta subunit activity, or a small molecule compound having activity of inhibiting integrin or its beta subunit. The antibody may not significantly affect the interaction of the integrin with its ligand.

Herein, intestinal diseases are diseases that benefit from down-regulation of integrin alphae expression, decreased ability of immune cells to migrate across blood vessels, inhibition of adhesion of immune cells to epithelial tissue, inhibition of homing of immune cells to gut-associated lymphoid tissue, inhibition of residence of immune cells in gut-associated lymphoid tissue, such as intestinal inflammation, which is adaptive immune-mediated chronic intestinal inflammation or innate immune-mediated acute intestinal inflammation. By reducing the activity of integrin beta subunit, migration and homing of immune cells to intestinal tract-associated lymphoid tissue can be inhibited, thereby inhibiting inflammation. In addition, since integrin α E forms a heterodimer with β 7 subunit, specific adhesion and retention of cells to epithelial tissues is mediated by interaction with the epithelial cell ligand E-cadherin. Based on the discovery of the inventor, the expression of the beta 7 subunit is reduced by reducing the activity of the integrin beta 7 subunit, and then the expression of the integrin alpha E is reduced, so that the residence of immune cells in intestinal tract-related lymphoid tissues can be inhibited, the inflammation is inhibited, the intestinal graft-versus-host disease (Gut graft-cover-host disease) is reduced, and the tumorigenesis is inhibited. This is probably due to the inability of the excess α E subunit to form a functional form, resulting in instability of the α E subunit, which is degraded by intracellular proteases.

To attenuate or disrupt the interaction between aromatic amino acids in the domain of the integrin beta subunit I and metal cations of the SyMBS site within cells, a gene knockout or knock-down vector can be introduced into a cell to knock-out or knock-down the expression of an integrin and/or a beta subunit in the cell, and/or knocking out or knocking down the expression of the cell integrin and/or beta subunit by adopting gene editing technology such as ZFN, TALEN or CRISPR/Cas9 and the like, and/or knockout or knock-down of integrin and/or beta subunit expression by interfering RNA mediated gene silencing, and/or a gene insertion vector can be introduced into an immune cell to attenuate or eliminate the interaction between the aromatic amino acids in the beta subunit I domain and the metal cation of the SyMBS site while knocking out the coding sequence of the integrin and/or beta subunit, or integrating the expression cassette of the beta subunit with the mutation at the 185 th amino acid into the genome of the cell. ZFN, TALEN and CRISPR/Cas9 technologies suitable for use in the present invention are well known in the art. Each technique realizes the knockout of a target gene through the combined action of a DNA recognition domain and an endonuclease.

The modulation methods described herein may be in vitro or in vivo. In certain embodiments, the present invention provides methods of down-regulating the ability of immune cells to migrate to gut-associated lymphoid tissue. The method comprises subjecting an immune cell in vitro to one or more of the following treatments such that the immune cell has an inhibition or attenuation of the interaction between an aromatic amino acid in the integrin beta subunit I domain and a metal cation of the symmbs site as compared to its corresponding wild-type cell (i.e., an immune cell isolated directly from the subject): an agent that causes an immune cell to express an integrin mutein having reduced or eliminated interaction between an aromatic amino acid in its β subunit I domain and a metal cation of the SyMBS site. The knockout, knock-down or expression of the mutated integrin can be achieved using the expression vectors, integration vectors or ZFNs, TALENs or CRISPR/Cas9, etc., as described previously, as the agent. In certain embodiments, the integrin mutein is an integrin protein having a mutation at position 185 of the β subunit. By reduced migratory capacity is meant that the migratory capacity of immune cells treated by the methods described herein to gut-associated lymphoid tissue is reduced, e.g., by at least 10%, at least 20%, at least 30% or at least 50% as compared to immune cells not treated by the methods described herein. As previously mentioned, the ability of the immune cells of the present application to migrate can be assessed using methods well known in the art for testing the ability of tumor cells to migrate. For example, the evaluation can be performed using the methods described in section 1.2.6 herein below.

Also provided herein is a tool cell having an extracellular fragment of a β -subunit or a coding sequence thereof, which competitively binds to a ligand in vivo with an immune cell, and inhibits migration of the immune cell to the intestinal tract. Or the tool cell has a peptide segment capable of expressing a specific recognition site for an activated integrin, and an extracellular segment of the beta subunit that binds to the activated integrin and inhibits its ability to competitively bind to the ligand from having at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% sequence identity with the extracellular portion of the beta subunit of the integrin. In one or more embodiments, the fragment is an amino acid sequence from positions 46-386 of the integrin beta subunit. In certain embodiments, also provided herein is a pharmaceutical composition comprising a tool cell as described herein and optionally a pharmaceutically acceptable carrier or excipient. Herein, the pharmaceutically acceptable carrier, excipient or stabilizer is non-toxic to the immune cells and the recipient of the immune cells at the dosages and concentrations employed, and may include various types of carriers or excipients commonly used in the delivery of live immune cells in immune cell therapy as is well known in the art. The immune cells are present in the pharmaceutical composition in a therapeutically effective amount. The therapeutically effective amount of the immune cells described herein can be determined according to the type of immune cell, the age, sex, severity of the disease, etc., of the patient receiving the immune cell. The immune cells described herein can be administered by conventional administration means, e.g., conventional immune cell therapy can be used to effect reinfusion of the immune cells.

In certain embodiments, also provided herein is a method of treating intestinal inflammation, reducing intestinal graft versus host disease, or inhibiting tumorigenesis, the method comprising the step of reducing the interaction between an aromatic amino acid in the integrin beta subunit I domain and a metal cation of the symmbs site in immune cells of a subject. In certain embodiments, the immune cell: expression vectors containing mutations in the integrin beta subunit or SyMBS sites that inhibit the interaction of aromatic amino acids in the integrin beta subunit I domain with metal cations of the SyMBS site.

The invention also includes the coding sequences of the various mutants described herein and their complements, as well as nucleic acid constructs comprising the coding sequences or complements. Herein, a nucleic acid construct is an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue. The nucleic acid construct comprises a coding sequence described herein or a complement thereof, and one or more regulatory sequences operatively linked to the sequences. The control sequence may be an appropriate promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the amino acid sequence to be expressed. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is ligated to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator which is functional in the host cell of choice may be used herein.

In certain embodiments, the nucleic acid construct is a vector. In particular, the coding sequences described herein can be cloned into many types of vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The vector may be an expression vector, or a cloning vector.

Generally, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, methanol oxidase promoter of Pichia pastoris and other known promoters which can control the expression of genes in prokaryotic or eukaryotic cells or viruses. Marker genes can be used to provide phenotypic traits useful for selection of transformed host cells, including but not limited to dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli. When the polynucleotides described herein are expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells. Methods well known to those skilled in the art can be used to construct expression vectors containing the polynucleotide sequences described herein and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

Also included herein are host cells comprising a polynucleotide sequence described herein or a nucleic acid construct thereof. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; filamentous fungal cells, or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells, or Bowes melanoma cells.

The vectors herein can be introduced into host cells by conventional methods including microinjection, particle gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, and the like.

The invention also includes the use of an agent for detecting the expression or activity of a gene or protein of integrin or its beta subunit in the preparation of a kit for diagnosing a bowel disease or determining the course of a bowel disease, which is a disease benefiting from the inhibition of stable adhesion of lymphocytes, the reduction of the ability of immune cells to migrate across blood vessels, the down-regulation of the expression of integrin alphae, the inhibition of homing of immune cells to gut-associated lymphoid tissue. Such as an antibody that specifically recognizes activin. Methods for detecting integrin activity are known in the art, such as X-ray crystallography, nuclear magnetic resonance, Fluorescence Resonance Energy Transfer (FRET), soluble ligand binding assays, cell adhesion assays, cell spreading assays, and cell migration assays.

The invention constructs a homologous recombinant vector PL-124 containing F185A point mutation and SEQ ID NO 7, transfects ES cells of 129 mice with the plasmid and screens positive ES cells, and then injects the positive ES cells into blastocysts of 129 mice to obtain chimera mice. KI mice containing F185A point mutation were obtained by mating chimeric mice, and then mated with EIIa-Cre tool mice to excise the LoxP sequence, resulting in β 7 knock-in (KI) mice with loss of cation- π interaction.

The present invention is described in further detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations which become apparent in light of the teachings provided herein. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.

Examples

Experimental materials and methods

1.1 Experimental materials

1.1.1 common buffer formulations

TBS：20mM Tris-HCl(pH 7.4)，150mM NaCl，1mM CaCl₂，1mM MgCl₂；

Cell lysis buffer: TBS, 1% Triton X-100, 0.05% Tween 20, Complete Protease Inhibitor Cocktail tables, PhosSTOP Phosphase Inhibitor Cocktail tables;

2 x SDS protein loading buffer: 100mM Tris-HCl (pH6.8), 4% SDS, 0.2% bromophenol blue, 20% glycerol, 10% beta-Me;

tris-glycine protein electrophoresis buffer: 25mM Tris, 250mM glycine, 0.1% SDS;

protein membrane transfer buffer: 3g Tris, 14.4g glycine, 200ml methanol, Milli-Q H₂O is constant volume to 1L;

TBS：20mM Tris-HCl(pH 7.4)，150mM NaCl，1mM CaCl₂，1mM MgCl₂；

TBST buffer: 8.8g NaCl, 6g Tris, 0.5ml Tween-20, pH 7.5, Milli-Q H₂O is constant volume to 1L;

PBS：8g NaCl，0.2g KCl，3.63g Na₂HPO₄·3H₂O，0.24g KH₂PO₄，Milli-Q H₂o is subjected to constant volume to 1L, the pH value is 7.4, and filtration sterilization is carried out;

2×HBS：8.0g NaCl，0.37g KCl，201mg Na₂HPO₄.7H₂o, 1.0g glucose, 5.0g HEPES, Milli-Q H₂O is added to 500ml with pH 7.05, filtered and sterilized, and stored at 4 ℃;

LB culture solution: 10g peptone, 10g NaCl, 5g yeast extract;

buffer used for flow laboratory experiments:

Ca²⁺&Mg²⁺-free HBSS：137mM NaCl，5.4mM KCl，0.4mM KH₂PO₄，0.3mM Na₂HPO₄，4.2mM NaHCO₃5.6mM glucose, pH 7.4;

coating buffer solution: PBS, 10mM NaHCO₃，pH9.0；

Blocking buffer: coating buffer plus 2% BSA;

washing buffer solution: 0.2g BSA, 40ml HBSS, 400. mu.l EDTA (0.5M, pH 8.0);

Buffer A：0.225g BSA，45ml HBSS；

buffer used for Fc-Tag fusion protein purification:

protein a binding buffer: 0.1M Tris-HCl, pH 8.0;

protein a elution buffer: 0.1M Glycine, pH 3.0;

protein a neutralization buffer: 1M Tris-HCl, pH 8.0.

Erythrocyte lysate:

pH 7.25 (used after filtration and autoclaving, stored at 4 ℃ C.) 0.15M NH4Cl, 1mM NaHCO3, 0.1mM EDTA.

Intestinal immune cell separation solution:

Pre-digestion buffer:1mM EDTA,2mM HEPES,5％FBS,1mM DTT；

Digestion buffer:0.5mg/ml Collogenase D,0.5mg/ml DNase,3mg/ml Dispase II,10mM HEPES,5％FBS；

percoll separating medium 40% and 70% Percoll separating medium.

1.1.2 basic chemical reagents

1.1.3 antibodies

1.1.4 enzymes

Name (R)	Purchased from
		KOD plus Neo	TOYOBO
Restriction enzyme	New England Biolabs
		T4 ligase	Fermentas
M-MLV reverse transcriptase	Promega

1.1.5 kits and others

Name (R)	Purchased from
		ECL substrate	Pierce
DNA small quantity recovery kit	Tiangen (root of heaven)
		BCA protein quantitative kit	Biyuntian (a Chinese character)
PhosStop Phosphatase Inhibitor Cocktail Tablets	Roche
		Complete Protease Inhibitor Cocktail Tablets	Roche
CellTrace Violet Cell Proliferation kit	Thermo Fisher Scientific
		CellTrace Yellow Cell proliferation kit	Thermo Fisher Scientific
Human/mouse chemokines CCL21	R&D Systems
		Human/mouse chemokines CCL25	R&D Ststems
Treg cell staining kit	eBioscience
		DAPI	Sigma
SYBR Premix ExTaq kit	TaKaRa
		Mouse MAdCAM-1/Fc	Laboratory purification
Mouse ICAM-1/Fc	Laboratory purification
		Human MadCAM-1/Fc	Laboratory purification
Mouse E-cadherin/Fc	Laboratory purification
		Chromatographic qualitative analysis filter paper	Xinhua tea
Nitrocellulose membrane	Whatman
		0.22/0.45 mu m acetate fiber sterilization filter membrane	Pall
Protein A/G-sepharose	Amersham Pharmacia
		Insulin needle	BD

1.1.6 Main Instrument

1.1.7 cell lines

Name (R)	Culture solution
		293T	DMEM/10％FBS/PSG
Jurkat T	1640/10％FBS/PSG
		Hybridoma cells producing FIB504antibody	1640/10％FBS/PSG/NaPyr
Hybridoma cells producing M290antibody	1640/10％FBS/PSG/NaPyr

1.1 Experimental methods

1.2.1 construction of integrin beta 7F85A KI mice

(1) Strategy for constructing Itgb 7F185A KI plasmid

The PL-124 plasmid contains two Loxp sequences in the same orientation, contains a neo resistance gene in the middle, and is a common vector for genetic engineering KI and knock-out (KO). The vector contains a plurality of enzyme cutting sites, and is beneficial to inserting the required exogenous genes. Homologous recombination integrates the Loxp and neo sequences into the mouse genome by inserting upstream and downstream homologous sequences on both sides of Loxp, respectively. Finally, the Loxp and neo genes were excised at the mouse level by Cre recombinase, leaving only one Loxp sequence.

Construction of upstream homologous sequences: 2970bp of P348 upstream homologous fragment specifically amplified from the genomic DNA of the C57/B6 strain mouse was integrated into the PL124 plasmid vector as the upstream homologous sequence using SacII and NotI enzyme cleavage sites.

Constructing a downstream homologous sequence: 1) specifically amplifying P349 and P350 downstream homologous fragments with the lengths of 360bp and 1600bp respectively from the genome DNA of the C57/B6 strain mouse; 2) integrating a P349 sequence into a PMD-T19 vector by utilizing ClaI and SacII enzyme cleavage sites, and simultaneously introducing an F185A point mutation; 3) the P350 sequence is integrated into another PMD-T19 vector by utilizing SacII and SalI enzyme cutting sites; 4) integrating a P349 sequence into a PMD-T19-P350 vector to construct a PMD-T19-P349-P350 plasmid; 5) P349-P350 was integrated downstream of Loxp of the PL124 vector as a downstream homologous sequence using ClaI and SalI cleavage. The upstream and downstream homologous sequences are sequenced to confirm that the sequences are correct, and then the construction of the Itgb 7F185A KI plasmid is completed.

(2) Itgb7 KI ES cell line establishing strategy

The constructed F185A KI plasmid PL124-248- (349-350) is digested and linearized with Sal I, and then transformed into 129 strain mouse ES cells. After entering ES cells, plasmids replace wild sequences between upstream and downstream homologous sites with mutant sequences through homologous recombination. KI-positive ES cells were screened by the neomycin resistance gene. Genomic DNA from the selected resistant ES cells was sequenced, and ES clones carrying the F185A mutation were selected.

(3) Itgb7 KI mouse strain establishment strategy

Positive ES cells were injected into mouse blastocysts by microinjection, and the blastocysts were implanted into the uterus of a pseudopregnant female mouse. The positive ES cells and blastocysts develop chimeric mice containing KI gene sequence tissues at the same time. If KI-positive ES cells are chimeric into the mouse reproductive system, then the germ cells of the chimera may contain the F185A point mutation. The yellow coat color of the 129 strain mice is the dominant gene, so mating the chimera with the C57/B6 mice gives rise to both gray and black color in F1 generation pups. It is possible that the gray mouse juveniles contain the genetic genes of 129 lines of ES cells. F1 yellow young mouse is selected to extract genome DNA by tail shearing for genotype identification, and heterozygote containing KI sequence is obtained.

After obtaining the F1 generation heterozygote, the heterozygote needs to be further mated with mice which systemically express Cre enzyme, and Loxp and other sequences such as neo positioned between the Loxp and the other sequences are cut off so as to reduce the influence of exogenous insertion sequences on the genome. Mice can then be subcultured according to Mendelian's Law of heritage and homozygotes or heterozygotes identified for subsequent experiments.

Mating male mice and female mice heterozygous for the F1 generation to obtain 1/4 homozygotes in the F2 generation according to Mendelian's law of inheritance, namely that both alleles are KI fragments containing F185A point mutation; 1/2, wherein one allele is KI fragment and the other allele is WT gene; 1/4, i.e., both alleles are WT genes.

On the other hand, since ES cells are derived from 129 strain mice, F1 generation mice contain both a genetic background of 129 and a genetic background of C57/B6, and it is difficult to control the genetic quality. Therefore, further continuous hybridization with mice of C57/B6 strain for 5-6 generations is needed to ensure that the genotype background of the mice is relatively pure.

1.2.2 plasmid construction

I. PCR amplification of fragments of interest

Mu.l of 5 ng/. mu.l template DNA and 34. mu.l ddH were added to a 50. mu.l reaction system₂O，5μl 10×KOD buffer，5μl 2mM dNTP，2μl 25mM Mg²⁺Mu.l 5. mu.M primer A, 3. mu.l 5. mu.M primer B, 1. mu.l KOD. The PCR reaction conditions were as follows: firstly, denaturation is carried out for 10min at 94 ℃; the cycle was 27 times with 94 ℃ 30sec, 60 ℃ 30sec, 68 ℃ 3min 10sec, and finally 68 ℃ extension 10 min. Mu.l of the PCR product was isolated with 1% agarose gel and the band size was determined.

II. Enzyme cutting electrophoresis separation recovery of plasmid and PCR product

Adding corresponding enzyme digestion reaction buffer solution, vector DNA or PCR product and restriction endonuclease into 10-20 μ l reaction system, allowing the mixture to act at 37 deg.C for 2-4h, adding DNA loading buffer solution, and performing 1% agarose gel electrophoresis separation. After the correct PCR band was identified, the PCR product was recovered with a Tiangen gum recovery kit and finally dissolved in 30. mu.l of deionized water.

III ligation of the insert to the vector

To a 10. mu.l reaction system, ligation buffer, T4 ligase, the resulting insert and an appropriate amount of vector were added, and the reaction was carried out overnight at 16 ℃.

Coli DH5 alpha competent cell transformed with the plasmid

(1) 100 μ l of competent cells taken out of a-80 ℃ refrigerator were placed on ice until they were thawed;

(2) adding the prepared conversion product;

(3) mixing gently, and standing on ice for 30 min;

(4) heat shock is carried out for 45s at 42 ℃;

(5) standing on ice for 2 min;

(6) adding 900 mul of liquid LB culture medium;

(7) mixing the mixture gently, and placing the mixture in a water bath at 37 ℃ for 30 min.

After resuspension is completed, the EP tube is placed in a centrifuge and centrifuged for 5min at 5000rpm, so that the thalli are enriched at the bottom of the tube. Excess supernatant was aspirated off in a clean bench, and about 100. mu.l of the supernatant was left, and the cells were gently aspirated to suspend. The suspension was aspirated off in its entirety and applied to LB (Amp)⁺) On a flat plate. After the glass beads are coated with the bacterial liquid uniformly, the glass beads are discarded. The plate was placed upside down in a 37 ℃ biochemical incubator and incubated overnight for 12 h.

V, miniprep of plasmid DNA

Transfer about 5ml of overnight grown bacteria to 1.5ml EP tube, centrifuge at 10000rpm for 1min, remove supernatant, use the residual wall of a small solution on the shaker suspension bacteria. Adding 200 μ l Sol I, shaking, mixing, adding 200 μ l Sol II, slightly inverting for two times, adding 200 μ l Sol III, inverting, mixing, and centrifuging at 12000rpm for 5 min. The supernatant was transferred to a new tube and 300. mu.l isopropanol was added, mixed well at room temperature for 15min and centrifuged at 12000rpm for 15 min. Discarding supernatant, washing precipitate with 70% ethanol, air drying, and dissolving in 50 μ l Milli-Q H₂O。

1.2.3 cell culture and transfection

HEK (human embryonic kidney) 293T cells were cultured using DMEM supplemented with 10% fetal bovine serum (Gibco), 50U/ml penicillin and 50. mu.g/ml streptomycin. The incubator was maintained at 37 ℃ and a carbon dioxide concentration of 5%.

I. Cell recovery and culture (293T cells as an example)

(1) Taking out the 293T cells from the liquid nitrogen tank, and thawing in a water bath at 37 ℃;

(2) taking 4ml of fresh DMEM culture solution into a 15ml sterile tube, slowly adding 1ml of culture solution into a cell freezing tube, gently blowing and uniformly mixing, transferring into the 15ml sterile tube, and centrifuging for 3min at 1200 rmp. Discarding the supernatant;

(3) gently resuspending the cells with 1ml of fresh DMEM culture solution, transferring the cells to a 10cm cell culture dish, and supplementing the cell culture solution to 10 ml;

(4) placing at 37 ℃ and 5% CO₂And culturing in an incubator with saturated humidity.

II. Cell passage (293T cell as an example)

(1) After the cells grow over the culture dish, discarding the old culture solution;

(2) 3ml of 1 XPBS was added, washed gently once to remove residual culture medium, after which the 1 XPBS was discarded;

(3) adding 1ml pancreatin, standing for 10s, and gently tapping by hand to dissociate cells from the culture dish;

(4) adding 3ml of DMEM complete culture solution, stopping digestion, and blowing to obtain monoclone;

(5) transferring the cells into a 15ml centrifuge tube, and centrifuging for 3min at 1200 rmp;

(6) the supernatant was discarded and 10ml of the culture broth was blown up and mixed well. If 1:10 passages, taking 1ml and adding the 1ml into a culture dish containing 9ml of culture solution; if 1: 3, adding 3ml after passage;

(7) placing at 37 ℃ and 5% CO₂And culturing in an incubator with saturated humidity.

III transient transfection of 293T cells by calcium phosphate method

(1) Dividing cells one day before transfection to make cells reach 60-70% density before transfection;

(2) 1h before transfection, changing into a culture solution containing 25 mu M chloroquine;

(3) in a 15ml centrifuge tube, 10. mu.g of DNA was added and MilliQ-H was used₂O constant volume is 1095 mu L, then 155 mu L of 2M CaCl is added₂. Then, 1250. mu.L of 2 XHBS was added dropwise thereto and gently mixed with the solution. The mixture was then added dropwise directly to the cells. Note that after 2 × HBS addition, it is completed within 1-2 min. And ensuring that the liquid drops are uniformly sprinkled on the whole surface of the plate;

(4) after 7-11h of incubation, very fine, dusty particles will be seen. Then, rinsing once, and changing into a culture medium without chloroquine;

(5) cells were harvested 48-72h after transfection and used for subsequent experiments.

IV, Lentiviral infection of T cells

Calcium phosphate transfection method for transfection 293T cells for lentivirus packaging, the proportion of transfection plasmids is (10 cm dish as an example): pCDH vector 20. mu.g, psPAX 215. mu.g and pMD2. G6. mu.g. After 72 hours of transfection, the culture medium containing the lentivirus supernatant was collected with a 10ml syringe, the supernatant was filtered through a 0.45 μm filter, and the cells of interest were infected after ultracentrifugation and concentration. The ultracentrifugation conditions were 20000rpm, 2h, 4 ℃. After centrifugation, the pellet was removed and re-dissolved in 100-. When infecting, respectively using cell culture solution: resuspending cells in a lentivirus culture solution at a ratio of 1:1, adding polybrene (polybrene) with a final concentration of 8 μ g/ml into cell supernatant, mixing well, culturing at 37 deg.C for 24h, replacing fresh virus culture solution, and continuing infection. And (5) detecting by flow cytometry after 72 h.

1.2.4 flow cytometry (FACS) assay for integrin expression

(1) Take 5X 10⁵The individual cells were centrifuged in a 1.5ml Ep tube at 3000rpm for 3min at 4 ℃ and washed once with PBS;

(2) cell pellets were directly dissolved in 300. mu.l PBS and placed at 4 ℃ (Mock); b. the cell pellet was dissolved in 50. mu.l PBS, 5. mu.g/ml antibody was added, mixed gently, and left to stand at 4 ℃ for 30 min. Adding 1ml PBS, mixing, centrifuging at 4 deg.C 3000rmp for 3min, discarding supernatant, repeating once, and suspending the cells in 300 μ l PBS;

(3) the cell suspension was transferred to a cell collection tube and analyzed using a flow cytometer Calibur or lsii (bd).

1.2.5 flow cell System for detecting integrin and ligand-mediated cell adhesion Capacity

I. Cell processing

(1) Taking T immune cells in a 15ml centrifuge tube at 1200rpm for 3 min;

(2) washed twice with 5ml of washing buffer (HBSS, 5mM EDTA, 2% BSA, pH 8.0), 1200rpm, 3 min;

(3)5ml of buffer A (HBSS, 2% BSA, pH 8.0), 1200rpm, 3min, repeated once;

(4) the cells were dissolved in 1ml of buffer A and counted, adjusting the final cell concentration to 1X 10⁷One per ml.

II. Cell adhesion in flow cell

The principle is as follows:

to study integrin-mediated cell adhesion function, we established a flow cell system in vitro and investigated the interaction of integrins β 7 and β 2 with their ligands MAdCAM-1, E-cadherin or ICAM-1, respectively. The cells enter from an inlet tube, flow through a flow chamber, and then exit from another tube. A programmable pump is connected to the tube to control the flow rate of the liquid. The ligand for integrin was coated on this plastic petri dish. Firstly, EDTA is used to chelate metal ions in solution, then corresponding metal ions are added into suspension liquid in which cells are suspended, the cells are sucked into a flow chamber, and the wall shear stress is 1dyn/cm²Or 2dyn/cm². The experimental process is recorded in a video recording mode and used for later data analysis.

The method comprises the following steps:

(1) coating ligand: taking a clean plastic plate, dropping 20 mu l of MAdCAM-1, E-cadherin or ICAM-1 with the diameter of about 5mm at the central point of the bottom of the plate, placing the plate in a wet box, and incubating for 1h at 37 ℃;

(2) blocking of hetero-proteins: the plate was removed, washed three times with coating buffer (to keep the ligand surface always covered with liquid) at the marked place, 20. mu.l of blocking buffer was added to the circle, and incubated for 1h at 37 ℃;

(3) washing twice with HBSS, then installing a flow chamber system, laying a layer of HBSS on the flow chamber system, and pumping air by a vacuum pump to prevent air leakage;

(4) add 50. mu.l of treated cells to 450. mu.l of buffer A, add 2. mu.l of divalent cations, mix well, place in flow cell system for detection, start pump program and video recording program simultaneously, record the movement state of cells for analysis.

1.2.6 cell migration assay

Both sides of a perforated chamber (Transwell chambers, 5 μm pore size for mouse primary cells and 8 μm pore size for Jurakt T cells, Millipore) were coated with 10 μ g/ml MAdCAM-1 or ICAM-1 protein, respectively, overnight at 4 ℃ and blocked with blocking buffer at 37 ℃ for 1 h. Cell resuspension in the absence of added bloodCell count to 1X 10 in clear RPMI 1640 medium⁶At one ml, 200. mu.l of cells were added to the Well upper layer. 600 μ l of RPMI 1640 culture solution containing 10% FBS is added to the lower layer of the Well, the liquid level inside and outside the Well is ensured, and the Well is kept still and incubated for 6h at 37 ℃. After incubation, cells were fixed with 3.7% paraformaldehyde, and cells in the upper Well layer were dipped away with a cotton swab and cells migrating to the lower Well layer were counted by DAPI staining to relatively quantify the transmembrane migration ability of the cells.

1.2.7 DSS induces acute colitis

Adding 2.0% (wt/vol) DSS into drinking water, with molecular weight of 36-50KD, for 5 days (day 0-5), and changing to normal drinking water day (day 6-14) to induce acute colitis. Mice were evaluated daily for body weight, diarrhea and bloody stools.

Disease activity index (Disease activity index) and tissue damage score are as follows; the mice were assessed for appearance by a combined stool consistency score (score 0, normal; score 1, stool wet/sticky; score 2, soft; score 3, diarrhea), whether there was blood in the stool (score 0, no blood; score 1, blood in the stool or around the anus; score 2, heavy bleeding), score 0, normal; score 1, crinkle or change in gait; score 2, lethargy or moribundity).

The severity of colitis was assessed by staining sections, using established criteria based on crypt damage and ulceration, with crypt damage scores as follows: 0min, the crypts are intact; 1 minute, one third of the structure is damaged; 2min, two thirds of the structure is destroyed; 3, the crypts are completely destroyed; 4 points, the epithelial surface was altered by erosion; 5 points, confluent erosions. Ulcers were scored as follows: 0 point, no ulcer; 1 minute, 1-2 ulcer foci; 2 minutes, 2-4 ulcer foci; score 3, ulcer confluent or extensive. Colon tissue was analyzed by histology and quantitative PCR.

1.2.8 metastatic colitis of T cell

Sorter sorting CD4 in mouse spleen⁺CD45RB^high

T cell, 1X 10⁵CD4 of one WT, KI or KO mouse⁺CD45RB^high

T cells were injected into Rag1 via tail vein^-/-In recipient mice. Mice were weighed weekly and mice status and hematochezia recorded. After euthanasia after 12 weeks, colon histology and quantitative PCR analysis.

1.2.9 real-time quantitative PCR

Total cellular RNA was extracted with TRIzol (Invitrogen) and Reverse-transcribed into cDNA (Reverse Transcriptase M-MLV, RNase H-, TaKaRa) using oligo dT as a primer. Quantitative real-time PCR was performed using the SYBR green method (Applied Biosystems, Foster City, Calif., USA). The content of GAPDH was used as an internal control. The reaction procedure was as follows: 1 cycle was run at 94 ℃ for 4min, then 45cycles of 94 ℃ for 30 seconds, 58 ℃ for 30 seconds and 72 ℃ for 40 seconds run 45 cycles.

1.2.10 intestinal immune cell isolation

Isolation procedure of Intraepithelial immune cells (IEL):

(1) after euthanasia, the intestinal tract was removed and the adipose tissue and Peyer's knots were cleared;

(2) washing the intestinal tract with PBS until the intestinal tract is clear, longitudinally cutting the intestinal tract, and cutting into 5 mm-sized segments;

(3) pre-digest buffer to digest intestinal tissue 2 times with shaking for 20 minutes each time, 37 ℃;

(4) after twice digestion, the supernatants were pooled, 1000g centrifuged for 10min and discarded;

(5) washing twice with PBS, 10 minutes each time, 1000 g;

(6) resuspending the cells in 8ml of 40% Percoll separation medium, placing the cell suspension on 5ml of 80% Percoll separation medium, and centrifuging at 650g and 20 ℃ for 20 minutes;

(7) cells at the boundary between 40% and 80% were collected after centrifugation, i.e., IEL.

Separation step of mucosal Lamina propria immune cells (LPL):

(1) digesting the intestinal tissue remained after IEL separation for 1h by shaking with a digestion buffer at 37 ℃;

(2) after digestion, 1000g is centrifuged for 10 minutes, and then the supernatant is discarded;

(3) washing twice with PBS, 10 minutes each time, 1000 g;

(4) resuspending the cells in 8ml of 40% Percoll separation medium, placing the cell suspension on 5ml of 80% Percoll separation medium, and centrifuging at 650g and 20 ℃ for 20 minutes;

(5) after centrifugation, the cells at the junction of 40% and 80% were collected, i.e., LPL.

1.2.11 Paraffin sections, H & E staining

Tissue selection:

(1) taking intestinal tract tissue between colon (far end) and cecum 0.5cm above anus;

(2) pbs (ice cold) rinse inside and outside;

(3) the distal colon was excised and fixed in 4% PFA for 4 h. (shaking table at 4 ℃ in 15ml centrifuge tube);

(4) washing with 15ml Tube and PBS at 4 deg.C for 3.5 min;

(5) 75% ethanol overnight.

Dewatering and fixing:

(1) 75% ethanol for 20 min;

(2) 95% ethanol for 20min, (during which 80 deg.C wax is melted);

(3) 100% ethanol for 20 min;

(4) 50% ethanol + 50% xylene. 2min, adding and shaking;

(5) xylene for 1 min;

(6) benzene wax (50% + 50%), 65 deg.C, 15 min;

(7) wax I, 1.5 h;

(8) wax II, 1.5h, during which the embedding machine was opened.

Embedding and slicing:

(1) and taking the wax II and the sample to the embedding machine at the same time, and putting the mold into the wax tray on the left side at the same time. Taking the mold, flushing wax, and adding the sample. Putting on ice, cooling, embedding, righting with tweezers, and covering with cover. Cooling on ice at 4 deg.C for 10 min;

(2) putting the paraffin block into the temperature of minus 20 ℃ for about 10min in advance;

(3) adjusting a slicing machine to cut the slices in parallel to a thickness of 3-5 microns;

(4) continuously slicing, namely slicing 3 glass slides, and recording the sequence;

(5) spreading the slices at 37 ℃;

(6) taking out the slide, and paying attention to avoid generating bubbles (PLL coating glass slide);

(7) baking the slices at 37 deg.C overnight.

H & E staining:

xylene I, 5 min; xylene II, 5 min; 100% ethanol for 5 min; 100% ethanol for 5 min; 95% ethanol for 5 min; 75% ethanol for 5 min; dd H₂O, 5 min; hematoxylin, 45 s; ddH 2O is cleaned for 2-3 min; the differentiation reagent is used for 1-3 s; dd H2O flowing water for 30min, (do not face the sheet, the flow rate cannot be too fast); eosin A, 1 min; eosin B, 25 s; 95% ethanol for 5 min; 100% ethanol for 5 min; 100% ethanol for 5 min; xylene I, 5 min; xylene II, 5 min; neutral gum was fixed and air dried overnight in a fume hood.

1.2.12 frozen sections, immunofluorescent staining

Tissue selection:

(1) taking the part 0.5cm above the anus, from the colon (far end) to the ileocecal part;

(2) pbs (ice cold) rinse inside and outside;

(3) colonic tissue was excised and fixed in 4% PFA for 4 h. (shaking table at 4 ℃ in 15ml centrifuge tube);

(4)4℃，15ml Tube，PBS wash 3.5minutes；

(5) dehydrating 30% sucrose for 4 h;

(6) OCT embedding;

freezing and slicing: 8 μm (-80 ℃ for frozen storage)

And (3) immunofluorescence staining:

(1) freezing and storing the sample with the thickness of 8 mu m at-80 ℃ in a refrigerator;

(2) precooling acetone in a refrigerator at the temperature of minus 20 ℃, and fixing an ice-cut sample for 15 min;

(3) volatilizing acetone at room temperature, and forming a pen drawing circle;

(4)PBS wash 3.5min；

(5)10％goat serum+1％BSA in PBS(Blocking buffer)blocking for 1h,RT；

(6) primary anti-in blocking buffer, statining for 2h in RT or 4 ℃ overlap;

(7)PBS wash 3.5min；

(8) a secondary antibody in blocking buffer, statingfor 2h in RT;

(9)PBS wash 3.5min；

(10)DAPI in PBS，stainingfor 15min RT；

(11)PBS wash 3.5min；

(12) sealing the fluorescent sealing agent;

1.2.13 competitive homing assay in vivo

2×10⁷Spleen immune cells of heterozygote, KI or KO are labeled with Yellow dye and mixed with the same amount of WT spleen immune cells labeled with Violet dye, and are injected into WT mice through tail vein, after 18 hours of injection, the mice are euthanized, and the immune cells of spleen, peripheral blood, peripheral lymph nodes, intestinal mucosal lymph nodes, Peyer's node, small intestine, colon, bone marrow, liver or lung are taken, and the proportion of Yellow and Violet labeled immune cells in different tissue organs is detected by flow cytometry, and the homing index is calculated as [ Yellow⁺]_tissue/[Violet⁺]_tissue.

1.2.14 knock-down of beta 7 integrin from WT spleen immune cells

Plasmid construction: beta 7shRNA primer (CCCGTCTTCTAGTGTTCACTT) constructed on pCHD vector

Lentivirus packaging and infection of immune cells: calcium phosphate transfection method for transfection 293T cells for lentivirus packaging, the proportion of transfection plasmids is (10 cm dish as an example): pCDH vector 20. mu.g, psPAX 215. mu.g and pMD2. G6. mu.g. After 72 hours of transfection, the culture medium containing the lentivirus supernatant was collected with a 10ml syringe, the supernatant was filtered through a 0.45 μm filter, and the cells of interest were infected after ultracentrifugation and concentration. The ultracentrifugation conditions were 20000rpm, 2h, 4 ℃. After centrifugation, the pellet was removed and re-dissolved in 100-. When infecting, respectively using cell culture solution: resuspending cells in a lentivirus culture solution at a ratio of 1:1, adding polybrene (polybrene) with a final concentration of 8 μ g/ml into cell supernatant, mixing well, culturing at 37 deg.C for 24h, replacing fresh virus culture solution, and continuing infection. And (5) detecting by flow cytometry after 72 h.

1.2.15 protein expression and purification

293T cell expression purification of mouse E-cadherin protein

(1) Transfecting 293T cells by a calcium phosphate method;

(2) culturing until the culture solution turns yellow, and collecting the culture solution (about 3-4 days after transfection);

(3) purifying the Binding buffer diluted culture solution by the same volume of Protein A, and stirring while adding the Binding buffer during dilution, wherein the pH value is 8.0;

(4) centrifuging at 15000rpm for 30 minutes at 4 ℃;

(5) taking the supernatant, and then pouring the supernatant into a Protein A column;

(6) protein A purification Binding buffer column washing 15 column volumes;

(7) eluting 7 column volumes of Protein A purification Elution buffer, and adding the Protein A purification Neutralization buffer into a collecting pipe in advance according to a ratio of 1: 10;

(8) the eluate was collected and concentrated while changing the buffer to TBS and stored at-80 ℃.

Second, experimental results

2.1 integrin Itgb7^F185A/F185AConstruction and identification of KI mice

The method comprises the following steps of constructing a Knock-in mouse by using a genetic engineering means: the mouse integrin beta 7 gene is located on the long arm of

chromosome

12, 14 exons are spliced and translated into an integrin beta 7 subunit encoding a sequence of 807 amino acid residues, and phenylalanine Phe at position 185 is located in the middle of the fourth exon (fig. 1, a). Firstly, a plasmid vector PL-124 containing two pairs of upstream and downstream homologous sequences, F185A point mutation and a drug-resistant gene neo is constructed, then the plasmid is transfected into ES cells of 129 mice, a wild type sequence is replaced by homologous recombination, the F185A point mutation is inserted into an integrin beta 7 genome, and positive ES cells are screened by adding drugs (figure 1, B). And finally injecting the positive ES cells into 129 mouse blastocysts, and obtaining a chimeric mouse if the ES cells containing the F185A point mutation are integrated into a reproductive system. The chimera mouse mating offspring can obtain KI mouse with F185A point mutation with certain probability, and then mating with EIIa-Cre tool mouse to excise LoxP sequence. The KI mice obtained required serial mating with C57BL/6J wild-type mice for more than 5 generations to purify the gene background.

Mating male mice and female mice heterozygous for the F1 generation to obtain 1/4 homozygotes in the F2 generation according to Mendelian's law of inheritance, namely that both alleles are KI fragments containing F185A point mutation; 1/2, wherein one allele is KI fragment and the other allele is WT gene; 1/4, i.e., both alleles are WT genes. And (3) clipping mouse tail extracted genome DNA as a template to perform PCR amplification reaction. The WT primer can be designed to amplify a gene sequence of 360bp, and the KI primer can amplify a gene fragment of 380 bp. In conclusion, the genotype identification can only amplify a 380bp gene segment into a KI homozygote mouse; only 360bp gene segments can be amplified to be WT mice; the heterozygote mouse can be obtained by amplifying the two fragments (figure 1, C).

In order to ensure that the sequence of the gene fragment after homologous recombination replacement only contains the F185A point mutation without introducing other mutations, the related fragment in the genome is amplified by using a high-fidelity KOD enzyme for sequencing identification. The agarose gel electrophoresis results indicated that the PCR bands were of the correct size (FIG. 1, C). The results of gene sequencing showed that the F185A point mutation was introduced and no other mutations were introduced in the KI mouse gene (fig. 1, D).

2.2 integrin beta 7F185A mutation results in a diminished ability of immune cells to home to the gut

Migration and homing of immune cells to the villi of the small intestine are key steps in the mucosal immunity of the intestinal tract, and play important roles in maintaining immune homeostasis and regulating inflammatory response. H&The E staining results showed that the small intestine and colon structures of WT, KI and KO mice were essentially identical (FIG. 2, A, B). However, the KI mouse Peyer's patch was smaller than the WT mouse Peyer's patch but larger than the KO mouse Peyer's patch (fig. 2, C, D). Table 1 shows that the flow cytometry (FACS) technique detects T cells (CD 3) in each lymphoid tissue of WT, KI and KO mice⁺) And B cells (CD 19)⁺) The number of (2). Peripheral Blood (PB); peripheral Lymph Nodes (PLN); mesenteric Lymphnodes (MLN); bone marrow (Bone marrow, BM); intraepithelial lymphocytes (IEL); mucosal lymphocytes (Lamina)propria lymphocytes, LPL); spleen (spleens); peyer's Patch (PP), Small Intestine (SI); colon (Colon) and Thymus (Thymus).^AP<0.01；^AAP<0.005(Student's t-test)。^BIndicating the removal of the cecum. ND means Not detectable (Not detected). Data were counted as mean ± standard deviation.

The results show that the lack of integrin α 4 β 7 activation function inhibits the homing of T cells and B cells to gut-associated immune tissues including Peyer's patch, small intestinal intraepithelial, small intestinal mucosal lamina propria, and colon, but does not affect the homing of immune cells to spleen, thymus, bone marrow, and peripheral lymph nodes (table 1), unlike other gut-associated immune tissues, mesenteric mucosal lymph nodes are not affected by integrin α 4 β 7 expression and F185A mutation. Meanwhile, the integrin β 7F185A mutation did not significantly affect the relative proportion of T, B cells in each lymphoid organ (table 1). The above data indicate that migration of immune cells to the gut and Peyer's patch homing is dependent on integrin α 4 β 7 function: when integrin α 4 β 7 function is absent, there is a significant reduction in the number of immune cells in the gut and in the Peyer's patch; meanwhile, the F185A mutation affects the normal function of the integrin alpha 4 beta 7, and the migration and homing of part of immune cells to the small intestine and the Peler's patch are inhibited.

TABLE 1 lymphocyte distribution

2.3 integrin beta 7F185A mutation inhibits adhesion and migration of immune cells to MAdCAM-1

In comparison with WT mice, we found that integrin β 7 expression was reduced by about 50% on splenic immune cell membranes of KI mice, and KO mice expressed almost no β 7 (fig. 3, a, table 2). At the same time, the expression of integrin α 4 was also reduced accordingly (fig. 3, a, table 2). Heterozygote Itgb7^+/-(+/-) mice also had about 33% downregulation in expression due to the presence of only a single copy of the integrin beta 7 gene (FIG. 3, A, Table 2). In order to avoid the influence of the reduction of the expression quantity of the KI mouse integrin on the biological function,in addition to using heterozygote mice as a control group, we also Knocked Down (KD) the expression level of integrin β 7 on immune cells of WT mice in vitro to be consistent with that of KI mice (fig. 3, a, table 2), as an important control group for the following experiments.

TABLE 2

There is a cation-pi interaction between phenylalanine (F185) in integrin beta 7 and the metal cation of the SyMBS site to modulate the activation function of integrin. To verify the effect of point mutations on the affinity of integrin ligands on the surface of immune cell membranes, we isolated primary spleen immune cells and tested the interaction of integrin α 4 β 7 with its ligand MAdCAM-1 using the Flow Chamber technology (Flow Chamber) system. Integrins bind adherently to ligands in a low affinity conformation without chemokine stimulation. At this time, the same number of WT, +/-, KD and KI mouse spleen immune cells bound to the ligand, and KO mouse immune cells did not express integrin α 4 β 7 and could not bind to the ligand (FIG. 3, B). Upon stimulation with added chemokines such as CCL25 and CCL21, the integrin α 4 β 7 on the cell membrane surface of the splenic immune cells of WT mice was activated and bound to the ligand in a high affinity conformation. At this time, more WT mouse splenic immune cells stably adhered to their ligands, while KI splenic immune cells were not activated by cytokines and their adhesion binding to ligands was not enhanced (fig. 3, B), while the levels of integrin β 7 expression of +/-and KD also decreased, but were still activated by chemokines and increased stable adhesion to ligands (fig. 3, B). The data show that the activation function of integrin α 4 β 7 is inhibited after F185A KI point mutation.

In addition, serum-induced cell punch migration experiments showed that WT spleen immune cells passed through the wells coated with their ligand MAdCAM-1 into the bottom chamber in greater numbers than KI immune cells, with no significant difference from +/-and KD immune cell numbers, with KO immune cells having the weakest transmembrane motility (fig. 3, C). While the number of splenic immune cells in each group of mice passed through the ligand ICAM-1 coated wells into the bottom chamber was substantially the same (FIG. 3, D). The data indicate that loss of integrin α 4 β 7 activation function severely inhibits integrin α 4 β 7-mediated ability of immune cells to translocate across membranes.

2.4 deletion of the integrin beta 7 activating function results in alpha E beta 7⁺Immune cell reduction and alpha E expression down-regulation

In addition to integrin α 4 β 7, β 17 subunit can also form heterodimers with integrin β 0E. Integrin beta 2E beta 37 mainly with intestinal epithelial E-cadherin binding, maintain immune cells in the intestinal tract in the residence. We first over-expressed mouse integrin β 4E β 57 and mutant β 6E β 77F185A on the immortalized human T immune cell line Jurkat T cell line and at the same time over-expressed the receptor CCR9 for the chemokine CCL25 (Jurkat T cells have expression of the receptor CCR7 for the chemokine CCL21 themselves). FACS detection of equal expression levels of β 3E and β 17 on Jurkat T- β 8E β 97WT and Jurkat T- β 0E β 7F185A (FIG. 4, A), adhesion to ligand E-cadherin was further detected at similar expression levels. Flow chamber results show that both Jurkat T- β 5E β 27WT and Jurkat T- β 6E β 47F185A cells showed similar adhesion behavior to E-cadherin before and after stimulation with the chemokines CCL21 and CCL25, and that this binding was specifically inhibited by the blocking antibody M290 (fig. 4, B). While Jurkat T cells that do not overexpress β 7E β 87 are unable to bind E-cadherin. This result indicates that the β 7-F185A mutation does not inhibit the chemokine-activated α E β 7 from adhering to E-cadherin, since this adhesion is primarily dependent on α E integrins, and can adhere to E-cadherin as long as α E expression is normal, and chemokines can increase this adhesion. However, we found α E β 7 in spleen cells of KI mice⁺Has a significantly reduced proportion of immune cells (FIG. 5A), while the small intestine and colon have alpha E beta 7 in IEL and LPL⁺Also significantly decreased immune cells (fig. 5, B), and further analysis found α E β 7⁺The expression of alpha E of the immune cells is significantly reduced (Table 3, the numbers in the table represent alpha E beta 7)⁺Mean fluorescence intensity of α E on lymphocytes (MFI)), which indicates that loss of integrin β 7 activation function results in a significant decrease in integrin α E expression on immune cells, and thus α E β 7 expression in the gut⁺Immune cells are reduced.

TABLE 3

2.5 deletion of integrin beta 7 activating function inhibits homing of immune cells to gut-associated immune tissue

Integrin α 4 β 7 mediates low affinity and high affinity adhesion in the binding of immune cells to their ligand MAdCAM-1. Immunocytes cannot be adhered to MAdCAM-1 after the function of the integrin alpha 4 beta 7 is lost, so that the intestinal immune system of KO mice is remarkably reduced. To study the effect of integrin alpha 4 beta 7 activation-mediated deletion of high affinity adhesion function on immune cell homing in vivo, we established an in vivo immune cell competitive homing experimental system: marking the spleen immune cells of WT, KI, +/-and KO mice with different fluorescent dyes, and injecting the same amount of marked immune cells into CD45.1 receptor mice through tail vein; the fluorescence labeled cells enter different lymph organs along with the homing of blood circulation, and the mice are sacrificed 18 hours later; the relative proportion of fluorescently labeled positive cells was analyzed by FACS to analyze the competitive migration and homing of WT, KI, +/-and KO mouse immune cells to different tissue organs.

The experimental data show that: in the Peripheral Blood (PBL) of CD45.1 receptor mice, the number of WT and KI fluorescence labeling positive immune cells is equivalent, indicating that the initial cell amount is the same; migration homing of WT and KI fluorescence-labeled positive immune cells to Spleen (SP), Peripheral Lymph Node (PLN), Bone Marrow (BM), Liver (LIV) and Lung (LUN) of CD45.1 recipient mice was consistent; compared with WT immune cells, KI immune cells have significantly reduced ability to migrate into the Mesenteric Lymph Node (MLN), Peyer's Patch (PP), Small Intestine (SI) and Colon (Colon) of CD45.1 recipient mice (FIG. 6, A); indicating that integrin α 4 β 7 activation-mediated loss of high affinity adhesion function inhibits homing of immune cells into gut-associated lymphoid tissues.

The KO immune cells gave similar results to KI immune cells. Compared with WT immune cells, the homing ability of KO immune cells to intestinal tract-associated lymphoid tissues of CD45.1 receptor mice is also significantly reduced (FIG. 6, B), indicating that the expression and activation functions of integrin alpha 4 beta 7 play an important role in the homing of immune cells to intestinal tract-associated lymphoid tissues. Whereas the immune cells of the heterozygote (+/-) mice functioned normally due to integrin activation, and did not affect their homing to gut-associated lymphoid tissues (FIG. 6, C). The results show that the integrin alpha 4 beta 7 plays an important role in homing of immune cells to intestinal tract-associated immune tissues, and the deletion of high-affinity adhesion function mediated by the activation of the integrin alpha 4 beta 7 inhibits the homing of immune cells to intestinal tract-associated immune tissues.

2.6 integrin beta 7F185A mouse CD4⁺CD45RB^highImpaired ability of T cells to induce chronic enteritis

Integrin α 4 β 7-mediated immune cell homing plays an important role in the pathogenesis of enteritis. Furthermore, the method detects the induction of Rag1 by KI T cells by using a T cell induced enteritis model^-/-The ability of the recipient mouse to develop enteritis. CD4 was first obtained by FACS sorting⁺CD45RB^highT cells, which were injected into Rag1 via tail vein^-/-In recipient mice. Subsequent twelve week body weight observations revealed WT CD4⁺CD45RB^highT cell induced Rag1^-/-The recipient mice lost weight significantly, while KI and KO CD4⁺CD45RB^highT cells can not induce Rag1^-/-The recipient mice lost weight (fig. 7, a). With KI and KO CD4⁺CD45RB^highT cell comparison, WT CD4⁺CD45RB^highThe T cell-induced gut inflammation index was significantly increased (fig. 7, B). H&E staining showed Rag1^-/-Recipient mice received tail vein injection of WT CD4⁺CD45RB^highAfter T cells, the destruction of the colonic fold structure is severe and the degree of immune cell infiltration is increased. And KI CD4⁺CD45^RBhighT cell injection into Rag1^-/-After the tail vein of the recipient mouse, Rag1^-/-The intestinal architecture of the mice remained essentially normal with a weaker degree of associated inflammatory responses (fig. 7, C).

In CD4⁺CD45RB^highT cell induced Rag1^-/-CD4 in the pathogenesis of intestinal inflammation in recipient mice⁺T cell targeting receptor mouse gutThe homing of the tract tissue plays an important role in the development of intestinal inflammation. Rag1 by immunofluorescence staining of CD4^-/-Recipient mice intestinal tissue sections and quantification of CD4 from fluorescent staining⁺T cells, we found that WT CD4 was injected in the tail vein⁺CD45RB^highpost-T cell, Rag1^-/-The colon tissue of the recipient mouse has a large amount of migration and infiltration of CD 4T cells. And tail vein injection of KI or KO CD4⁺CD45RB^highpost-T cell, Rag1^-/-There was no abnormal migratory infiltration of CD 4T cells in the colon tissue of the recipient mice (fig. 7, D). The above results indicate that the fusion protein binds to WT CD4⁺CD45RB^highT cell differential, KI and KO CD4⁺CD45RB^highT cells do not induce Rag1^-/-Occurrence and development of intestinal inflammation in recipient mice. The migration of KI inflammatory T cells to intestinal tissue is inhibited, which does not cause an imbalance in intestinal immune homeostasis. The result shows that the lack of activation function of the integrin alpha 4 beta 7 inhibits the generation and the development of chronic intestinal inflammation mediated by adaptive immunity.

2.7 integrin beta 7F185A mice are more resistant to DSS-induced acute enteritis

To investigate the function and mechanism of action of integrin α 4 β 7 activation in innate immunity-mediated intestinal inflammation, we chose a DSS-induced enteritis model. 2% DSS (molecular weight 36,000-50,000) was added to the drinking water of mice for five days, followed by ten days with normal drinking water. The data indicate that KO mice lost weight more significantly, while WT and KI mice lost weight only slightly (fig. 8, a). While 80% of KO mice died due to severe dehydration caused by enteritis, WT and KI mice did not die (fig. 8, B). The intestinal inflammation index was significantly increased in KO mice compared to KI mice (FIG. 8, C), and the proinflammatory factors IL-6, TNF- α and IL-1 β were significantly increased in colon tissue, indicating an enhanced inflammatory response (FIG. 8, D). H & E staining indicated severe disruption of the colonic fold structure and increased immune cell infiltration in KO mice, while the intestinal structure remained essentially normal in KI mice (fig. 8, E). Combining the above data, KO mice were more sensitive to DSS-induced acute enteritis, while KI and WT mice were consistent and more resistant to DSS-induced acute enteritis.

In previous work, we found that the reduction of Treg cells in the gut leads to upregulation of the intestinal epithelial cell ICAM-1 expression, further mediates innate immune macrophage infiltration and plays an important role in DSS-induced colitis. We found that although there was a partial reduction of Treg cells in KI mice, sufficient Treg cells remained to suppress aberrant activation of innate immunity (fig. 8, F). By immunofluorescent staining of colon tissue of mice on day four with ICAM-1 and F4/80, we found that ICAM-1 expression in intestinal epithelial cells was significantly upregulated in DSS-treated KO mice, accompanied by infiltration of large numbers of macrophages (FIG. 8, G); DSS-treated WT and KI mice had no infiltration of macrophages and no upregulation of associated inflammatory factor expression. The above results indicate that KO mice are more sensitive to DSS-induced acute enteritis, and the development and progression of this inflammation is mainly dependent on the migratory infiltration of macrophages mediated by the high expression of intestinal epithelial cells ICAM-1 and the secretion of macrophage proinflammatory factors. Unlike KO mice, WT and KI mice were more resistant to DSS-induced acute enteritis, suggesting that loss of integrin α 4 β 7 activation function does not contribute to exacerbation of acute intestinal inflammation mediated by innate immunity.

In conclusion, we found that the lack of integrin beta 7 activation function can not only inhibit the migration of immune cells to intestinal tract-related immune organs, but also reduce the residence of immune cells in intestinal tract, and simultaneously inhibit the functions of both α 4 beta 7 and α E beta 7 integrins. Thereby inhibiting the occurrence and the development of spontaneous intestinal inflammation mediated by abnormal infiltration of T cells; meanwhile, unlike the biological function of completely inhibiting integrin beta 7, inhibition of the activation function can retain enough Treg cells in the intestinal tract, thereby inhibiting adaptive immune-mediated colitis without increasing the susceptibility of DSS-induced congenital acute intestinal inflammation. Based on the fact, the development of the drug for specifically inhibiting the activation function of the integrin beta 7 can become a new direction for treating the intestinal inflammation, and the research provides theoretical basis and guidance for the treatment of the intestinal inflammatory diseases and the drug screening.

Sequence listing

<110> center of outstanding innovation in molecular cell science of Chinese academy of sciences

<120> Agents and methods for modulating integrin beta subunit

<130> 201097

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<170> SIPOSequenceListing 1.0

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<211> 798

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<213> Homo sapiens

<400> 1

Met Val Ala Leu Pro Met Val Leu Val Leu Leu Leu Val Leu Ser Arg

1 5 10 15

Gly Glu Ser Glu Leu Asp Ala Lys Ile Pro Ser Thr Gly Asp Ala Thr

20 25 30

Glu Trp Arg Asn Pro His Leu Ser Met Leu Gly Ser Cys Gln Pro Ala

35 40 45

Pro Ser Cys Gln Lys Cys Ile Leu Ser His Pro Ser Cys Ala Trp Cys

50 55 60

Lys Gln Leu Asn Phe Thr Ala Ser Gly Glu Ala Glu Ala Arg Arg Cys

65 70 75 80

Ala Arg Arg Glu Glu Leu Leu Ala Arg Gly Cys Pro Leu Glu Glu Leu

85 90 95

Glu Glu Pro Arg Gly Gln Gln Glu Val Leu Gln Asp Gln Pro Leu Ser

100 105 110

Gln Gly Ala Arg Gly Glu Gly Ala Thr Gln Leu Ala Pro Gln Arg Val

115 120 125

Arg Val Thr Leu Arg Pro Gly Glu Pro Gln Gln Leu Gln Val Arg Phe

130 135 140

Leu Arg Ala Glu Gly Tyr Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu

145 150 155 160

Ser Tyr Ser Met Lys Asp Asp Leu Glu Arg Val Arg Gln Leu Gly His

165 170 175

Ala Leu Leu Val Arg Leu Gln Glu Val Thr His Ser Val Arg Ile Gly

180 185 190

Phe Gly Ser Phe Val Asp Lys Thr Val Leu Pro Phe Val Ser Thr Val

195 200 205

Pro Ser Lys Leu Arg His Pro Cys Pro Thr Arg Leu Glu Arg Cys Gln

210 215 220

Ser Pro Phe Ser Phe His His Val Leu Ser Leu Thr Gly Asp Ala Gln

225 230 235 240

Ala Phe Glu Arg Glu Val Gly Arg Gln Ser Val Ser Gly Asn Leu Asp

245 250 255

Ser Pro Glu Gly Gly Phe Asp Ala Ile Leu Gln Ala Ala Leu Cys Gln

260 265 270

Glu Gln Ile Gly Trp Arg Asn Val Ser Arg Leu Leu Val Phe Thr Ser

275 280 285

Asp Asp Thr Phe His Thr Ala Gly Asp Gly Lys Leu Gly Gly Ile Phe

290 295 300

Met Pro Ser Asp Gly His Cys His Leu Asp Ser Asn Gly Leu Tyr Ser

305 310 315 320

Arg Ser Thr Glu Phe Asp Tyr Pro Ser Val Gly Gln Val Ala Gln Ala

325 330 335

Leu Ser Ala Ala Asn Ile Gln Pro Ile Phe Ala Val Thr Ser Ala Ala

340 345 350

Leu Pro Val Tyr Gln Glu Leu Ser Lys Leu Ile Pro Lys Ser Ala Val

355 360 365

Gly Glu Leu Ser Glu Asp Ser Ser Asn Val Val Gln Leu Ile Met Asp

370 375 380

Ala Tyr Asn Ser Leu Ser Ser Thr Val Thr Leu Glu His Ser Ser Leu

385 390 395 400

Pro Pro Gly Val His Ile Ser Tyr Glu Ser Gln Cys Glu Gly Pro Glu

405 410 415

Lys Arg Glu Gly Lys Ala Glu Asp Arg Gly Gln Cys Asn His Val Arg

420 425 430

Ile Asn Gln Thr Val Thr Phe Trp Val Ser Leu Gln Ala Thr His Cys

435 440 445

Leu Pro Glu Pro His Leu Leu Arg Leu Arg Ala Leu Gly Phe Ser Glu

450 455 460

Glu Leu Ile Val Glu Leu His Thr Leu Cys Asp Cys Asn Cys Ser Asp

465 470 475 480

Thr Gln Pro Gln Ala Pro His Cys Ser Asp Gly Gln Gly His Leu Gln

485 490 495

Cys Gly Val Cys Ser Cys Ala Pro Gly Arg Leu Gly Arg Leu Cys Glu

500 505 510

Cys Ser Val Ala Glu Leu Ser Ser Pro Asp Leu Glu Ser Gly Cys Arg

515 520 525

Ala Pro Asn Gly Thr Gly Pro Leu Cys Ser Gly Lys Gly His Cys Gln

530 535 540

Cys Gly Arg Cys Ser Cys Ser Gly Gln Ser Ser Gly His Leu Cys Glu

545 550 555 560

Cys Asp Asp Ala Ser Cys Glu Arg His Glu Gly Ile Leu Cys Gly Gly

565 570 575

Phe Gly Arg Cys Gln Cys Gly Val Cys His Cys His Ala Asn Arg Thr

580 585 590

Gly Arg Ala Cys Glu Cys Ser Gly Asp Met Asp Ser Cys Ile Ser Pro

595 600 605

Glu Gly Gly Leu Cys Ser Gly His Gly Arg Cys Lys Cys Asn Arg Cys

610 615 620

Gln Cys Leu Asp Gly Tyr Tyr Gly Ala Leu Cys Asp Gln Cys Pro Gly

625 630 635 640

Cys Lys Thr Pro Cys Glu Arg His Arg Asp Cys Ala Glu Cys Gly Ala

645 650 655

Phe Arg Thr Gly Pro Leu Ala Thr Asn Cys Ser Thr Ala Cys Ala His

660 665 670

Thr Asn Val Thr Leu Ala Leu Ala Pro Ile Leu Asp Asp Gly Trp Cys

675 680 685

Lys Glu Arg Thr Leu Asp Asn Gln Leu Phe Phe Phe Leu Val Glu Asp

690 695 700

Asp Ala Arg Gly Thr Val Val Leu Arg Val Arg Pro Gln Glu Lys Gly

705 710 715 720

Ala Asp His Thr Gln Ala Ile Val Leu Gly Cys Val Gly Gly Ile Val

725 730 735

Ala Val Gly Leu Gly Leu Val Leu Ala Tyr Arg Leu Ser Val Glu Ile

740 745 750

Tyr Asp Arg Arg Glu Tyr Ser Arg Phe Glu Lys Glu Gln Gln Gln Leu

755 760 765

Asn Trp Lys Gln Asp Ser Asn Pro Leu Tyr Lys Ser Ala Ile Thr Thr

770 775 780

Thr Ile Asn Pro Arg Phe Gln Glu Ala Asp Ser Pro Thr Leu

785 790 795

<210> 2

<211> 806

<212> PRT

<213> Mus musculus

<400> 2

Met Val Asp Ser Ser Thr Val Leu Ile Phe Leu Leu Val Leu Gly Gly

1 5 10 15

Gly Gln Ser Glu Leu Asp Thr Lys Ile Thr Ser Ser Gly Glu Ala Ala

20 25 30

Glu Trp Glu Asp Pro Asp Leu Ser Leu Gln Gly Ser Cys Gln Pro Val

35 40 45

Pro Ser Cys Gln Lys Cys Ile Leu Ser His Pro Ser Cys Ala Trp Cys

50 55 60

Lys Gln Leu Asn Phe Thr Ala Ser Gly Glu Ala Glu Ala Arg Arg Cys

65 70 75 80

Ala Arg Arg Glu Glu Leu Leu Ala Arg Gly Cys Pro Ala Gln Glu Leu

85 90 95

Glu Glu Pro Arg Gly Arg Gln Glu Val Leu Gln Asp Lys Pro Leu Ser

100 105 110

Gln Gly Asp Arg Gly Glu Gly Ala Thr Gln Leu Ala Pro Gln Arg Ile

115 120 125

Arg Val Thr Leu Arg Pro Gly Glu Pro Gln Lys Phe Arg Val Arg Phe

130 135 140

Leu Arg Ala Ala Gly Tyr Pro Val Asp Leu Tyr Tyr Leu Met Asp Leu

145 150 155 160

Ser Tyr Ser Met Lys Asp Asp Leu Glu Arg Val Arg Gln Leu Gly His

165 170 175

Ala Leu Leu Val Arg Leu Gln Glu Val Thr His Ser Val Arg Ile Gly

180 185 190

Phe Gly Ser Phe Val Asp Lys Thr Val Leu Pro Phe Val Ser Thr Val

195 200 205

Pro Ser Lys Leu His His Pro Cys Pro Ser Arg Leu Glu Arg Cys Gln

210 215 220

Pro Pro Phe Ser Phe His His Val Leu Ser Leu Thr Gly Asp Ala Gln

225 230 235 240

Ala Phe Glu Arg Glu Val Gly Arg Gln Asn Val Ser Gly Asn Leu Asp

245 250 255

Ser Pro Glu Gly Gly Phe Asp Ala Ile Leu Gln Ala Ala Leu Cys Gln

260 265 270

Glu Gln Ile Gly Trp Arg Asn Val Ser Arg Leu Leu Val Phe Thr Ser

275 280 285

Asp Asp Thr Phe His Thr Ala Gly Asp Gly Lys Leu Gly Gly Ile Phe

290 295 300

Met Pro Ser Asp Gly Arg Cys His Leu Asp Ser Asn Gly Val Tyr Thr

305 310 315 320

Asn Ser Ala Glu Phe Asp Tyr Pro Ser Val Gly Gln Val Ala Gln Ala

325 330 335

Leu Thr Ala Ala Asn Ile Gln Pro Ile Phe Ala Val Thr Gly Ala Thr

340 345 350

Leu Pro Val Tyr Gln Glu Leu Arg Gln Leu Ile Pro Lys Ser Ala Val

355 360 365

Gly Glu Leu Ser Glu Asp Ser Ser Asn Val Val Gln Leu Ile Met Asp

370 375 380

Ala Tyr Asp Ser Leu Ser Ser Thr Val Thr Leu Glu His Ser Pro Leu

385 390 395 400

Pro Pro Gly Val Ser Ile Ser Phe Glu Ser His Cys Lys Gly Pro Glu

405 410 415

Lys Thr Glu Gly Glu Ala Gly Asp Arg Gly Gln Cys Asn Asp Val Arg

420 425 430

Val Asn Gln Thr Val Asp Phe Trp Val Thr Leu Gln Ala Thr His Cys

435 440 445

Leu Pro Glu Ala His Val Leu Arg Leu Trp Ala Leu Gly Phe Ser Glu

450 455 460

Glu Leu Thr Val Glu Leu His Thr Val Cys Asp Cys Asn Cys Gly Asp

465 470 475 480

Ala Gln Pro His Ala Pro Tyr Cys Ser Asp Gly Gln Gly Asp Leu Gln

485 490 495

Cys Gly Ile Cys Ser Cys Ala Pro Gly Arg Leu Gly Gln Leu Cys Glu

500 505 510

Cys Ser Glu Ala Asp Leu Ser Ser Pro Asp Leu Glu Ser Gly Cys Arg

515 520 525

Ala Pro Asn Gly Thr Gly Pro Leu Cys Ser Gly Lys Gly Arg Cys Gln

530 535 540

Cys Gly Arg Cys Ser Cys Ser Gly Gln Ser Ser Gly Arg Leu Cys Glu

545 550 555 560

Cys Asp Asp Ala Ser Cys Glu Arg His Glu Gly Ile Leu Cys Gly Gly

565 570 575

Phe Gly His Cys Gln Cys Gly Val Cys His Cys His Ala Asn His Thr

580 585 590

Gly Arg Ala Cys Glu Cys Ser Lys Ser Val Asp Ser Cys Val Ser Pro

595 600 605

Glu Gly Gly Leu Cys Ser Gly His Gly Tyr Cys Lys Cys Asn Arg Cys

610 615 620

Gln Cys Leu Asp Gly Tyr Tyr Gly Ala Leu Cys Asp Gln Cys Leu Gly

625 630 635 640

Cys Lys Ser Pro Cys Glu Gln Tyr Arg Asp Cys Ala Glu Cys Gly Ala

645 650 655

Phe Gly Thr Gly Pro Leu Ala Ala Asn Cys Ser Val Val Cys Ala Asp

660 665 670

Val Asn Val Thr Leu Thr Leu Ala Pro Asn Leu Asp Asp Gly Trp Cys

675 680 685

Lys Glu Arg Thr Ile Asp Asn Gln Leu Phe Phe Phe Leu Val Glu His

690 695 700

Ala Ala Ser Gly Ile Val Leu Arg Val Arg Pro Gln Glu Lys Gly Val

705 710 715 720

Asp His Thr Arg Ala Ile Ile Leu Gly Cys Thr Gly Gly Ile Val Ala

725 730 735

Val Gly Leu Gly Leu Val Leu Ala Tyr Arg Leu Ser Val Glu Ile Tyr

740 745 750

Asp Arg Arg Glu Tyr Arg Arg Phe Glu Lys Glu Gln Gln Gln Leu Asn

755 760 765

Trp Lys Gln Asp Asn Asn Pro Leu Tyr Lys Ser Ala Ile Thr Thr Thr

770 775 780

Val Asn Pro Arg Phe Gln Gly Thr Asn Gly Arg Ser Pro Ser Leu Ser

785 790 795 800

Leu Thr Arg Glu Ala Asp

805

<210> 3

<211> 1032

<212> PRT

<213> Homo sapiens

<400> 3

Met Ala Trp Glu Ala Arg Arg Glu Pro Gly Pro Arg Arg Ala Ala Val

1 5 10 15

Arg Glu Thr Val Met Leu Leu Leu Cys Leu Gly Val Pro Thr Gly Arg

20 25 30

Pro Tyr Asn Val Asp Thr Glu Ser Ala Leu Leu Tyr Gln Gly Pro His

35 40 45

Asn Thr Leu Phe Gly Tyr Ser Val Val Leu His Ser His Gly Ala Asn

50 55 60

Arg Trp Leu Leu Val Gly Ala Pro Thr Ala Asn Trp Leu Ala Asn Ala

65 70 75 80

Ser Val Ile Asn Pro Gly Ala Ile Tyr Arg Cys Arg Ile Gly Lys Asn

85 90 95

Pro Gly Gln Thr Cys Glu Gln Leu Gln Leu Gly Ser Pro Asn Gly Glu

100 105 110

Pro Cys Gly Lys Thr Cys Leu Glu Glu Arg Asp Asn Gln Trp Leu Gly

115 120 125

Val Thr Leu Ser Arg Gln Pro Gly Glu Asn Gly Ser Ile Val Thr Cys

130 135 140

Gly His Arg Trp Lys Asn Ile Phe Tyr Ile Lys Asn Glu Asn Lys Leu

145 150 155 160

Pro Thr Gly Gly Cys Tyr Gly Val Pro Pro Asp Leu Arg Thr Glu Leu

165 170 175

Ser Lys Arg Ile Ala Pro Cys Tyr Gln Asp Tyr Val Lys Lys Phe Gly

180 185 190

Glu Asn Phe Ala Ser Cys Gln Ala Gly Ile Ser Ser Phe Tyr Thr Lys

195 200 205

Asp Leu Ile Val Met Gly Ala Pro Gly Ser Ser Tyr Trp Thr Gly Ser

210 215 220

Leu Phe Val Tyr Asn Ile Thr Thr Asn Lys Tyr Lys Ala Phe Leu Asp

225 230 235 240

Lys Gln Asn Gln Val Lys Phe Gly Ser Tyr Leu Gly Tyr Ser Val Gly

245 250 255

Ala Gly His Phe Arg Ser Gln His Thr Thr Glu Val Val Gly Gly Ala

260 265 270

Pro Gln His Glu Gln Ile Gly Lys Ala Tyr Ile Phe Ser Ile Asp Glu

275 280 285

Lys Glu Leu Asn Ile Leu His Glu Met Lys Gly Lys Lys Leu Gly Ser

290 295 300

Tyr Phe Gly Ala Ser Val Cys Ala Val Asp Leu Asn Ala Asp Gly Phe

305 310 315 320

Ser Asp Leu Leu Val Gly Ala Pro Met Gln Ser Thr Ile Arg Glu Glu

325 330 335

Gly Arg Val Phe Val Tyr Ile Asn Ser Gly Ser Gly Ala Val Met Asn

340 345 350

Ala Met Glu Thr Asn Leu Val Gly Ser Asp Lys Tyr Ala Ala Arg Phe

355 360 365

Gly Glu Ser Ile Val Asn Leu Gly Asp Ile Asp Asn Asp Gly Phe Glu

370 375 380

Asp Val Ala Ile Gly Ala Pro Gln Glu Asp Asp Leu Gln Gly Ala Ile

385 390 395 400

Tyr Ile Tyr Asn Gly Arg Ala Asp Gly Ile Ser Ser Thr Phe Ser Gln

405 410 415

Arg Ile Glu Gly Leu Gln Ile Ser Lys Ser Leu Ser Met Phe Gly Gln

420 425 430

Ser Ile Ser Gly Gln Ile Asp Ala Asp Asn Asn Gly Tyr Val Asp Val

435 440 445

Ala Val Gly Ala Phe Arg Ser Asp Ser Ala Val Leu Leu Arg Thr Arg

450 455 460

Pro Val Val Ile Val Asp Ala Ser Leu Ser His Pro Glu Ser Val Asn

465 470 475 480

Arg Thr Lys Phe Asp Cys Val Glu Asn Gly Trp Pro Ser Val Cys Ile

485 490 495

Asp Leu Thr Leu Cys Phe Ser Tyr Lys Gly Lys Glu Val Pro Gly Tyr

500 505 510

Ile Val Leu Phe Tyr Asn Met Ser Leu Asp Val Asn Arg Lys Ala Glu

515 520 525

Ser Pro Pro Arg Phe Tyr Phe Ser Ser Asn Gly Thr Ser Asp Val Ile

530 535 540

Thr Gly Ser Ile Gln Val Ser Ser Arg Glu Ala Asn Cys Arg Thr His

545 550 555 560

Gln Ala Phe Met Arg Lys Asp Val Arg Asp Ile Leu Thr Pro Ile Gln

565 570 575

Ile Glu Ala Ala Tyr His Leu Gly Pro His Val Ile Ser Lys Arg Ser

580 585 590

Thr Glu Glu Phe Pro Pro Leu Gln Pro Ile Leu Gln Gln Lys Lys Glu

595 600 605

Lys Asp Ile Met Lys Lys Thr Ile Asn Phe Ala Arg Phe Cys Ala His

610 615 620

Glu Asn Cys Ser Ala Asp Leu Gln Val Ser Ala Lys Ile Gly Phe Leu

625 630 635 640

Lys Pro His Glu Asn Lys Thr Tyr Leu Ala Val Gly Ser Met Lys Thr

645 650 655

Leu Met Leu Asn Val Ser Leu Phe Asn Ala Gly Asp Asp Ala Tyr Glu

660 665 670

Thr Thr Leu His Val Lys Leu Pro Val Gly Leu Tyr Phe Ile Lys Ile

675 680 685

Leu Glu Leu Glu Glu Lys Gln Ile Asn Cys Glu Val Thr Asp Asn Ser

690 695 700

Gly Val Val Gln Leu Asp Cys Ser Ile Gly Tyr Ile Tyr Val Asp His

705 710 715 720

Leu Ser Arg Ile Asp Ile Ser Phe Leu Leu Asp Val Ser Ser Leu Ser

725 730 735

Arg Ala Glu Glu Asp Leu Ser Ile Thr Val His Ala Thr Cys Glu Asn

740 745 750

Glu Glu Glu Met Asp Asn Leu Lys His Ser Arg Val Thr Val Ala Ile

755 760 765

Pro Leu Lys Tyr Glu Val Lys Leu Thr Val His Gly Phe Val Asn Pro

770 775 780

Thr Ser Phe Val Tyr Gly Ser Asn Asp Glu Asn Glu Pro Glu Thr Cys

785 790 795 800

Met Val Glu Lys Met Asn Leu Thr Phe His Val Ile Asn Thr Gly Asn

805 810 815

Ser Met Ala Pro Asn Val Ser Val Glu Ile Met Val Pro Asn Ser Phe

820 825 830

Ser Pro Gln Thr Asp Lys Leu Phe Asn Ile Leu Asp Val Gln Thr Thr

835 840 845

Thr Gly Glu Cys His Phe Glu Asn Tyr Gln Arg Val Cys Ala Leu Glu

850 855 860

Gln Gln Lys Ser Ala Met Gln Thr Leu Lys Gly Ile Val Arg Phe Leu

865 870 875 880

Ser Lys Thr Asp Lys Arg Leu Leu Tyr Cys Ile Lys Ala Asp Pro His

885 890 895

Cys Leu Asn Phe Leu Cys Asn Phe Gly Lys Met Glu Ser Gly Lys Glu

900 905 910

Ala Ser Val His Ile Gln Leu Glu Gly Arg Pro Ser Ile Leu Glu Met

915 920 925

Asp Glu Thr Ser Ala Leu Lys Phe Glu Ile Arg Ala Thr Gly Phe Pro

930 935 940

Glu Pro Asn Pro Arg Val Ile Glu Leu Asn Lys Asp Glu Asn Val Ala

945 950 955 960

His Val Leu Leu Glu Gly Leu His His Gln Arg Pro Lys Arg Tyr Phe

965 970 975

Thr Ile Val Ile Ile Ser Ser Ser Leu Leu Leu Gly Leu Ile Val Leu

980 985 990

Leu Leu Ile Ser Tyr Val Met Trp Lys Ala Gly Phe Phe Lys Arg Gln

995 1000 1005

Tyr Lys Ser Ile Leu Gln Glu Glu Asn Arg Arg Asp Ser Trp Ser Tyr

1010 1015 1020

Ile Asn Ser Lys Ser Asn Asp Asp

1025 1030

<210> 4

<211> 1032

<212> PRT

<213> Mus musculus

<400> 4

Met Ala Ala Glu Ala Arg Cys Arg Pro Arg Ser Arg Gly Ile Ala Leu

1 5 10 15

Arg Glu Ala Val Met Leu Leu Leu Tyr Phe Gly Val Pro Thr Gly His

20 25 30

Ser Tyr Asn Leu Asp Pro Glu Asn Ala Leu Leu Tyr Gln Gly Pro Ser

35 40 45

Gly Thr Leu Phe Gly Tyr Ser Val Val Leu His Ser His Gly Ser Lys

50 55 60

Arg Trp Leu Ile Val Gly Ala Pro Thr Ala Ser Trp Leu Ser Asn Ala

65 70 75 80

Ser Val Val Asn Pro Gly Ala Ile Tyr Arg Cys Gly Ile Arg Lys Asn

85 90 95

Pro Asn Gln Thr Cys Glu Gln Leu Gln Leu Gly Ser Pro Ser Gly Glu

100 105 110

Pro Cys Gly Lys Thr Cys Leu Glu Glu Arg Asp Asn Gln Trp Leu Gly

115 120 125

Val Thr Leu Ser Arg Gln Pro Gly Glu Asn Gly Ser Ile Val Thr Cys

130 135 140

Gly His Arg Trp Lys Asn Ile Phe Tyr Met Lys Ser Asp Asn Lys Leu

145 150 155 160

Pro Thr Gly Ile Cys Tyr Val Met Pro Ser Asp Leu Arg Thr Glu Leu

165 170 175

Ser Lys Arg Met Ala Pro Cys Tyr Lys Asp Tyr Thr Arg Lys Phe Gly

180 185 190

Glu Asn Phe Ala Ser Cys Gln Ala Gly Ile Ser Ser Phe Tyr Thr Gln

195 200 205

Asp Leu Ile Val Met Gly Ala Pro Gly Ser Ser Tyr Trp Thr Gly Thr

210 215 220

Val Phe Val Tyr Asn Ile Thr Thr Asn Gln Tyr Lys Ala Phe Val Asp

225 230 235 240

Arg Gln Asn Gln Val Lys Phe Gly Ser Tyr Leu Gly Tyr Ser Val Gly

245 250 255

Ala Gly His Phe Arg Ser Pro His Thr Thr Glu Val Val Gly Gly Ala

260 265 270

Pro Gln His Glu Gln Ile Gly Lys Ala Tyr Ile Phe Ser Ile Asp Glu

275 280 285

Asn Glu Leu Asn Ile Val Tyr Glu Met Lys Gly Lys Lys Leu Gly Ser

290 295 300

Tyr Phe Gly Ala Ser Val Cys Ala Val Asp Leu Asn Ala Asp Gly Phe

305 310 315 320

Ser Asp Leu Leu Val Gly Ala Pro Met Gln Ser Thr Ile Arg Glu Glu

325 330 335

Gly Arg Val Phe Val Tyr Ile Asn Ser Gly Met Gly Ala Val Met Val

340 345 350

Glu Met Glu Arg Val Leu Val Gly Ser Asp Lys Tyr Ala Ala Arg Phe

355 360 365

Gly Glu Ser Ile Ala Asn Leu Gly Asp Ile Asp Asn Asp Gly Phe Glu

370 375 380

Asp Ile Ala Ile Gly Ala Pro Gln Glu Asp Asp Leu Arg Gly Ala Val

385 390 395 400

Tyr Ile Tyr Asn Gly Arg Val Asp Gly Ile Ser Ser Thr Tyr Ser Gln

405 410 415

Arg Ile Glu Gly Gln Gln Ile Ser Lys Ser Leu Arg Met Phe Gly Gln

420 425 430

Ser Ile Ser Gly Gln Ile Asp Ala Asp Asn Asn Gly Tyr Val Asp Val

435 440 445

Ala Val Gly Ala Phe Gln Ser Asp Ser Ala Val Leu Leu Arg Thr Arg

450 455 460

Pro Val Val Ile Val Glu Ala Ser Leu Ser His Pro Glu Ser Val Asn

465 470 475 480

Arg Thr Lys Phe Asp Cys Thr Glu Asn Gly Leu Pro Ser Val Cys Met

485 490 495

His Leu Thr Leu Cys Phe Ser Tyr Lys Gly Lys Glu Val Pro Gly Tyr

500 505 510

Ile Val Leu Phe Tyr Asn Val Ser Leu Asp Val His Arg Lys Ala Glu

515 520 525

Ser Pro Ser Arg Phe Tyr Phe Phe Ser Asn Gly Thr Ser Asp Val Ile

530 535 540

Thr Gly Ser Ile Arg Val Ser Ser Ser Gly Glu Lys Cys Arg Thr His

545 550 555 560

Gln Ala Phe Met Arg Lys Asp Val Arg Asp Ile Leu Thr Pro Ile His

565 570 575

Val Glu Ala Thr Tyr His Leu Gly His His Val Ile Thr Lys Arg Asn

580 585 590

Thr Glu Glu Phe Pro Pro Leu Gln Pro Ile Leu Gln Gln Lys Lys Glu

595 600 605

Lys Asp Val Ile Arg Lys Met Ile Asn Phe Ala Arg Phe Cys Ala Tyr

610 615 620

Glu Asn Cys Ser Ala Asp Leu Gln Val Ser Ala Lys Val Gly Phe Leu

625 630 635 640

Lys Pro Tyr Glu Asn Lys Thr Tyr Leu Ala Val Gly Ser Met Lys Thr

645 650 655

Ile Met Leu Asn Val Ser Leu Phe Asn Ala Gly Asp Asp Ala Tyr Glu

660 665 670

Thr Thr Leu Asn Val Gln Leu Pro Thr Gly Leu Tyr Phe Ile Lys Ile

675 680 685

Leu Asp Leu Glu Glu Lys Gln Ile Asn Cys Glu Val Thr Glu Ser Ser

690 695 700

Gly Ile Val Lys Leu Ala Cys Ser Leu Gly Tyr Ile Tyr Val Asp Arg

705 710 715 720

Leu Ser Arg Ile Asp Ile Ser Phe Leu Leu Asp Val Ser Ser Leu Ser

725 730 735

Arg Ala His Glu Asp Leu Ser Ile Ser Val His Ala Ser Cys Glu Asn

740 745 750

Glu Gly Glu Leu Asp Gln Val Arg Asp Asn Arg Val Thr Leu Thr Ile

755 760 765

Pro Leu Arg Tyr Glu Val Met Leu Thr Val His Gly Leu Val Asn Pro

770 775 780

Thr Ser Phe Val Tyr Gly Ser Ser Glu Glu Asn Glu Pro Glu Thr Cys

785 790 795 800

Met Ala Glu Lys Leu Asn Leu Thr Phe His Val Ile Asn Thr Gly Ile

805 810 815

Ser Met Ala Pro Asn Val Ser Val Lys Ile Met Val Pro Asn Ser Phe

820 825 830

Leu Pro Gln Asp Asp Lys Leu Phe Asn Val Leu Asp Val Gln Thr Thr

835 840 845

Thr Gly Gln Cys His Phe Lys His Tyr Gly Arg Glu Cys Thr Phe Ala

850 855 860

Gln Gln Lys Gly Ile Ala Gly Thr Leu Thr Asp Ile Val Lys Phe Leu

865 870 875 880

Ser Lys Thr Asp Lys Arg Leu Leu Tyr Cys Met Lys Ala Asp Gln His

885 890 895

Cys Leu Asp Phe Leu Cys Asn Phe Gly Lys Met Glu Ser Gly Lys Glu

900 905 910

Ala Ser Val His Ile Gln Leu Glu Gly Arg Pro Ser Ile Leu Glu Met

915 920 925

Asp Glu Thr Ser Ser Leu Lys Phe Glu Ile Lys Ala Thr Ala Phe Pro

930 935 940

Glu Pro His Pro Lys Val Ile Glu Leu Asn Lys Asp Glu Asn Val Ala

945 950 955 960

His Val Phe Leu Glu Gly Leu His His Gln Arg Pro Lys Arg His Phe

965 970 975

Thr Ile Ile Ile Ile Thr Ile Ser Leu Leu Leu Gly Leu Ile Val Leu

980 985 990

Leu Leu Ile Ser Cys Val Met Trp Lys Ala Gly Phe Phe Lys Arg Gln

995 1000 1005

Tyr Lys Ser Ile Leu Gln Glu Glu Asn Arg Arg Asp Ser Trp Ser Tyr

1010 1015 1020

Val Asn Ser Lys Ser Asn Asp Asp

1025 1030

<210> 5

<211> 1179

<212> PRT

<213> Homo sapiens

<400> 5

Met Trp Leu Phe His Thr Leu Leu Cys Ile Ala Ser Leu Ala Leu Leu

1 5 10 15

Ala Ala Phe Asn Val Asp Val Ala Arg Pro Trp Leu Thr Pro Lys Gly

20 25 30

Gly Ala Pro Phe Val Leu Ser Ser Leu Leu His Gln Asp Pro Ser Thr

35 40 45

Asn Gln Thr Trp Leu Leu Val Thr Ser Pro Arg Thr Lys Arg Thr Pro

50 55 60

Gly Pro Leu His Arg Cys Ser Leu Val Gln Asp Glu Ile Leu Cys His

65 70 75 80

Pro Val Glu His Val Pro Ile Pro Lys Gly Arg His Arg Gly Val Thr

85 90 95

Val Val Arg Ser His His Gly Val Leu Ile Cys Ile Gln Val Leu Val

100 105 110

Arg Arg Pro His Ser Leu Ser Ser Glu Leu Thr Gly Thr Cys Ser Leu

115 120 125

Leu Gly Pro Asp Leu Arg Pro Gln Ala Gln Ala Asn Phe Phe Asp Leu

130 135 140

Glu Asn Leu Leu Asp Pro Asp Ala Arg Val Asp Thr Gly Asp Cys Tyr

145 150 155 160

Ser Asn Lys Glu Gly Gly Gly Glu Asp Asp Val Asn Thr Ala Arg Gln

165 170 175

Arg Arg Ala Leu Glu Lys Glu Glu Glu Glu Asp Lys Glu Glu Glu Glu

180 185 190

Asp Glu Glu Glu Glu Glu Ala Gly Thr Glu Ile Ala Ile Ile Leu Asp

195 200 205

Gly Ser Gly Ser Ile Asp Pro Pro Asp Phe Gln Arg Ala Lys Asp Phe

210 215 220

Ile Ser Asn Met Met Arg Asn Phe Tyr Glu Lys Cys Phe Glu Cys Asn

225 230 235 240

Phe Ala Leu Val Gln Tyr Gly Gly Val Ile Gln Thr Glu Phe Asp Leu

245 250 255

Arg Asp Ser Gln Asp Val Met Ala Ser Leu Ala Arg Val Gln Asn Ile

260 265 270

Thr Gln Val Gly Ser Val Thr Lys Thr Ala Ser Ala Met Gln His Val

275 280 285

Leu Asp Ser Ile Phe Thr Ser Ser His Gly Ser Arg Arg Lys Ala Ser

290 295 300

Lys Val Met Val Val Leu Thr Asp Gly Gly Ile Phe Glu Asp Pro Leu

305 310 315 320

Asn Leu Thr Thr Val Ile Asn Ser Pro Lys Met Gln Gly Val Glu Arg

325 330 335

Phe Ala Ile Gly Val Gly Glu Glu Phe Lys Ser Ala Arg Thr Ala Arg

340 345 350

Glu Leu Asn Leu Ile Ala Ser Asp Pro Asp Glu Thr His Ala Phe Lys

355 360 365

Val Thr Asn Tyr Met Ala Leu Asp Gly Leu Leu Ser Lys Leu Arg Tyr

370 375 380

Asn Ile Ile Ser Met Glu Gly Thr Val Gly Asp Ala Leu His Tyr Gln

385 390 395 400

Leu Ala Gln Ile Gly Phe Ser Ala Gln Ile Leu Asp Glu Arg Gln Val

405 410 415

Leu Leu Gly Ala Val Gly Ala Phe Asp Trp Ser Gly Gly Ala Leu Leu

420 425 430

Tyr Asp Thr Arg Ser Arg Arg Gly Arg Phe Leu Asn Gln Thr Ala Ala

435 440 445

Ala Ala Ala Asp Ala Glu Ala Ala Gln Tyr Ser Tyr Leu Gly Tyr Ala

450 455 460

Val Ala Val Leu His Lys Thr Cys Ser Leu Ser Tyr Ile Ala Gly Ala

465 470 475 480

Pro Arg Tyr Lys His His Gly Ala Val Phe Glu Leu Gln Lys Glu Gly

485 490 495

Arg Glu Ala Ser Phe Leu Pro Val Leu Glu Gly Glu Gln Met Gly Ser

500 505 510

Tyr Phe Gly Ser Glu Leu Cys Pro Val Asp Ile Asp Met Asp Gly Ser

515 520 525

Thr Asp Phe Leu Leu Val Ala Ala Pro Phe Tyr His Val His Gly Glu

530 535 540

Glu Gly Arg Val Tyr Val Tyr Arg Leu Ser Glu Gln Asp Gly Ser Phe

545 550 555 560

Ser Leu Ala Arg Ile Leu Ser Gly His Pro Gly Phe Thr Asn Ala Arg

565 570 575

Phe Gly Phe Ala Met Ala Ala Met Gly Asp Leu Ser Gln Asp Lys Leu

580 585 590

Thr Asp Val Ala Ile Gly Ala Pro Leu Glu Gly Phe Gly Ala Asp Asp

595 600 605

Gly Ala Ser Phe Gly Ser Val Tyr Ile Tyr Asn Gly His Trp Asp Gly

610 615 620

Leu Ser Ala Ser Pro Ser Gln Arg Ile Arg Ala Ser Thr Val Ala Pro

625 630 635 640

Gly Leu Gln Tyr Phe Gly Met Ser Met Ala Gly Gly Phe Asp Ile Ser

645 650 655

Gly Asp Gly Leu Ala Asp Ile Thr Val Gly Thr Leu Gly Gln Ala Val

660 665 670

Val Phe Arg Ser Arg Pro Val Val Arg Leu Lys Val Ser Met Ala Phe

675 680 685

Thr Pro Ser Ala Leu Pro Ile Gly Phe Asn Gly Val Val Asn Val Arg

690 695 700

Leu Cys Phe Glu Ile Ser Ser Val Thr Thr Ala Ser Glu Ser Gly Leu

705 710 715 720

Arg Glu Ala Leu Leu Asn Phe Thr Leu Asp Val Asp Val Gly Lys Gln

725 730 735

Arg Arg Arg Leu Gln Cys Ser Asp Val Arg Ser Cys Leu Gly Cys Leu

740 745 750

Arg Glu Trp Ser Ser Gly Ser Gln Leu Cys Glu Asp Leu Leu Leu Met

755 760 765

Pro Thr Glu Gly Glu Leu Cys Glu Glu Asp Cys Phe Ser Asn Ala Ser

770 775 780

Val Lys Val Ser Tyr Gln Leu Gln Thr Pro Glu Gly Gln Thr Asp His

785 790 795 800

Pro Gln Pro Ile Leu Asp Arg Tyr Thr Glu Pro Phe Ala Ile Phe Gln

805 810 815

Leu Pro Tyr Glu Lys Ala Cys Lys Asn Lys Leu Phe Cys Val Ala Glu

820 825 830

Leu Gln Leu Ala Thr Thr Val Ser Gln Gln Glu Leu Val Val Gly Leu

835 840 845

Thr Lys Glu Leu Thr Leu Asn Ile Asn Leu Thr Asn Ser Gly Glu Asp

850 855 860

Ser Tyr Met Thr Ser Met Ala Leu Asn Tyr Pro Arg Asn Leu Gln Leu

865 870 875 880

Lys Arg Met Gln Lys Pro Pro Ser Pro Asn Ile Gln Cys Asp Asp Pro

885 890 895

Gln Pro Val Ala Ser Val Leu Ile Met Asn Cys Arg Ile Gly His Pro

900 905 910

Val Leu Lys Arg Ser Ser Ala His Val Ser Val Val Trp Gln Leu Glu

915 920 925

Glu Asn Ala Phe Pro Asn Arg Thr Ala Asp Ile Thr Val Thr Val Thr

930 935 940

Asn Ser Asn Glu Arg Arg Ser Leu Ala Asn Glu Thr His Thr Leu Gln

945 950 955 960

Phe Arg His Gly Phe Val Ala Val Leu Ser Lys Pro Ser Ile Met Tyr

965 970 975

Val Asn Thr Gly Gln Gly Leu Ser His His Lys Glu Phe Leu Phe His

980 985 990

Val His Gly Glu Asn Leu Phe Gly Ala Glu Tyr Gln Leu Gln Ile Cys

995 1000 1005

Val Pro Thr Lys Leu Arg Gly Leu Gln Val Val Ala Val Lys Lys Leu

1010 1015 1020

Thr Arg Thr Gln Ala Ser Thr Val Cys Thr Trp Ser Gln Glu Arg Ala

1025 1030 1035 1040

Cys Ala Tyr Ser Ser Val Gln His Val Glu Glu Trp His Ser Val Ser

1045 1050 1055

Cys Val Ile Ala Ser Asp Lys Glu Asn Val Thr Val Ala Ala Glu Ile

1060 1065 1070

Ser Trp Asp His Ser Glu Glu Leu Leu Lys Asp Val Thr Glu Leu Gln

1075 1080 1085

Ile Leu Gly Glu Ile Ser Phe Asn Lys Ser Leu Tyr Glu Gly Leu Asn

1090 1095 1100

Ala Glu Asn His Arg Thr Lys Ile Thr Val Val Phe Leu Lys Asp Glu

1105 1110 1115 1120

Lys Tyr His Ser Leu Pro Ile Ile Ile Lys Gly Ser Val Gly Gly Leu

1125 1130 1135

Leu Val Leu Ile Val Ile Leu Val Ile Leu Phe Lys Cys Gly Phe Phe

1140 1145 1150

Lys Arg Lys Tyr Gln Gln Leu Asn Leu Glu Ser Ile Arg Lys Ala Gln

1155 1160 1165

Leu Lys Ser Glu Asn Leu Leu Glu Glu Glu Asn

1170 1175

<210> 6

<211> 1167

<212> PRT

<213> Mus musculus

<400> 6

Met Lys Trp Leu Phe His Thr Leu Leu Cys Met Ala Ser Leu Lys Pro

1 5 10 15

Gln Gly Ala Phe Asn Leu Asp Val Asp Trp Ala Trp Val Thr Ala Leu

20 25 30

Gln Pro Gly Ala Pro Ala Val Leu Ser Ser Leu Leu His Gln Asp Pro

35 40 45

Ser Asn Asn Gln Thr Cys Leu Leu Val Ala Arg Arg Ser Ser Asn Arg

50 55 60

Asn Thr Ala Ala Leu Tyr Arg Cys Ala Ile Ser Ile Ser Pro Asp Glu

65 70 75 80

Ile Ala Cys Gln Pro Val Glu His Ile Cys Met Pro Lys Gly Arg Tyr

85 90 95

Gln Gly Val Thr Leu Val Gly Asn His Asn Gly Val Leu Val Cys Ile

100 105 110

Gln Val Gln Ala Arg Lys Phe Arg Ser Leu Asn Ser Glu Leu Thr Gly

115 120 125

Ala Cys Ser Leu Leu Thr Pro Asn Leu Asp Leu Gln Ala Gln Ala Tyr

130 135 140

Phe Ser Asp Leu Glu Gly Phe Leu Asp Pro Gly Ala His Val Asp Ser

145 150 155 160

Gly Asp Tyr Cys Arg Ser Lys Gly Gly Ser Thr Gly Glu Glu Lys Lys

165 170 175

Ser Ala Arg Arg Arg Arg Thr Val Glu Glu Glu Asp Glu Glu Glu Asp

180 185 190

Gly Thr Glu Ile Ala Ile Val Leu Asp Gly Ser Gly Ser Ile Glu Pro

195 200 205

Ser Asp Phe Gln Lys Ala Lys Asn Phe Ile Ser Thr Met Met Arg Asn

210 215 220

Phe Tyr Glu Lys Cys Phe Glu Cys Asn Phe Ala Leu Val Gln Tyr Gly

225 230 235 240

Ala Val Ile Gln Thr Glu Phe Asp Leu Gln Glu Ser Arg Asp Ile Asn

245 250 255

Ala Ser Leu Ala Lys Val Gln Ser Ile Val Gln Val Lys Glu Val Thr

260 265 270

Lys Thr Ala Ser Ala Met Gln His Val Leu Asp Asn Ile Phe Ile Pro

275 280 285

Ser Arg Gly Ser Arg Lys Lys Ala Leu Lys Val Met Val Val Leu Thr

290 295 300

Asp Gly Asp Ile Phe Gly Asp Pro Leu Asn Leu Thr Thr Val Ile Asn

305 310 315 320

Ser Pro Lys Met Gln Gly Val Val Arg Phe Ala Ile Gly Val Gly Asp

325 330 335

Ala Phe Lys Asn Asn Asn Thr Tyr Arg Glu Leu Lys Leu Ile Ala Ser

340 345 350

Asp Pro Lys Glu Ala His Thr Phe Lys Val Thr Asn Tyr Ser Ala Leu

355 360 365

Asp Gly Leu Leu Ser Lys Leu Gln Gln His Ile Val His Met Glu Gly

370 375 380

Thr Val Gly Asp Ala Leu Gln Tyr Gln Leu Ala Gln Thr Gly Phe Ser

385 390 395 400

Ala Gln Ile Leu Asp Lys Gly Gln Val Leu Leu Gly Thr Val Gly Ala

405 410 415

Phe Asn Trp Ser Gly Gly Ala Leu Leu Tyr Ser Thr Gln Asn Gly Arg

420 425 430

Gly Cys Phe Leu Asn Gln Thr Ala Lys Glu Asp Ser Arg Thr Val Gln

435 440 445

Tyr Ser Tyr Leu Gly Tyr Ser Leu Ala Val Leu His Lys Ala His Gly

450 455 460

Val Ser Tyr Val Ala Gly Ala Pro Arg His Lys Leu Arg Gly Ala Val

465 470 475 480

Phe Glu Leu Arg Lys Glu Asp Arg Glu Glu Asp Ala Phe Val Arg Arg

485 490 495

Ile Glu Gly Glu Gln Met Gly Ser Tyr Phe Gly Ser Val Leu Cys Pro

500 505 510

Val Asp Ile Asp Met Asp Gly Thr Thr Asp Phe Leu Leu Val Ala Ala

515 520 525

Pro Phe Tyr His Ile Arg Gly Glu Glu Gly Arg Val Tyr Val Tyr Gln

530 535 540

Val Pro Glu Gln Asp Ala Ser Phe Ser Leu Ala His Thr Leu Ser Gly

545 550 555 560

His Pro Gly Leu Thr Asn Ser Arg Phe Gly Phe Ala Met Ala Ala Val

565 570 575

Gly Asp Ile Asn Gln Asp Lys Phe Thr Asp Val Ala Ile Gly Ala Pro

580 585 590

Leu Glu Gly Phe Gly Ala Gly Asp Gly Ala Ser Tyr Gly Ser Val Tyr

595 600 605

Ile Tyr Asn Gly His Ser Gly Gly Leu Tyr Asp Ser Pro Ser Gln Gln

610 615 620

Ile Arg Ala Ser Ser Val Ala Ser Gly Leu His Tyr Phe Gly Met Ser

625 630 635 640

Val Ser Gly Gly Leu Asp Phe Asn Gly Asp Gly Leu Ala Asp Ile Thr

645 650 655

Val Gly Ser Arg Asp Ser Ala Val Val Leu Arg Ser Arg Pro Val Val

660 665 670

Asp Leu Thr Val Ser Met Thr Phe Thr Pro Asp Ala Leu Pro Met Val

675 680 685

Phe Ile Gly Lys Met Asp Val Lys Leu Cys Phe Glu Val Asp Ser Ser

690 695 700

Gly Val Ala Ser Glu Pro Gly Leu Arg Glu Met Phe Leu Asn Phe Thr

705 710 715 720

Val Asp Val Asp Val Thr Lys Gln Arg Gln Arg Leu Gln Cys Glu Asp

725 730 735

Ser Ser Gly Cys Gln Ser Cys Leu Arg Lys Trp Asn Gly Gly Ser Phe

740 745 750

Leu Cys Glu His Phe Trp Leu Ile Ser Thr Glu Glu Leu Cys Glu Glu

755 760 765

Asp Cys Phe Ser Asn Ile Thr Ile Lys Val Thr Tyr Glu Phe Gln Thr

770 775 780

Ser Gly Gly Arg Arg Asp Tyr Pro Asn Pro Thr Leu Asp His Tyr Lys

785 790 795 800

Glu Pro Ser Ala Ile Phe Gln Leu Pro Tyr Glu Lys Asp Cys Lys Asn

805 810 815

Lys Val Phe Cys Ile Ala Glu Ile Gln Leu Thr Thr Asn Ile Ser Gln

820 825 830

Gln Glu Leu Val Val Gly Val Thr Lys Glu Val Thr Met Asn Ile Ser

835 840 845

Leu Thr Asn Ser Gly Glu Asp Ser Tyr Met Thr Asn Met Ala Leu Asn

850 855 860

Tyr Pro Arg Asn Leu Gln Phe Lys Lys Ile Gln Lys Pro Val Ser Pro

865 870 875 880

Asp Val Gln Cys Asp Asp Pro Lys Pro Val Ala Ser Val Leu Val Met

885 890 895

Asn Cys Lys Ile Gly His Pro Ile Leu Lys Arg Ser Ser Val Asn Val

900 905 910

Ser Val Thr Trp Gln Leu Glu Glu Ser Val Phe Pro Asn Arg Thr Ala

915 920 925

Asp Ile Thr Val Thr Ile Ser Asn Ser Asn Glu Lys Ser Leu Ala Arg

930 935 940

Glu Thr Arg Ser Leu Gln Phe Arg His Ala Phe Ile Ala Val Leu Ser

945 950 955 960

Arg Pro Ser Val Met Tyr Met Asn Thr Ser Gln Ser Pro Ser Asp His

965 970 975

Lys Glu Phe Phe Phe Asn Val His Gly Glu Asn Leu Phe Gly Ala Val

980 985 990

Phe Gln Leu Gln Ile Cys Val Pro Ile Lys Leu Gln Asp Phe Gln Ile

995 1000 1005

Val Arg Val Lys Asn Leu Thr Lys Thr Gln Asp His Thr Glu Cys Thr

1010 1015 1020

Gln Ser Gln Glu Pro Ala Cys Gly Ser Asp Pro Val Gln His Val Lys

1025 1030 1035 1040

Glu Trp His Ser Val Val Cys Ala Ile Thr Ser Asn Lys Glu Asn Val

1045 1050 1055

Thr Val Ala Ala Glu Ile Ser Val Gly His Thr Lys Gln Leu Leu Arg

1060 1065 1070

Asp Val Ser Glu Leu Pro Ile Leu Gly Glu Ile Ser Phe Asn Lys Ser

1075 1080 1085

Leu Tyr Glu Gly Leu Asn Ala Glu Asn His Arg Thr Lys Ile Thr Val

1090 1095 1100

Ile Phe Leu Lys Glu Glu Glu Thr Arg Ser Leu Pro Leu Ile Ile Gly

1105 1110 1115 1120

Ser Ser Ile Gly Gly Leu Leu Val Leu Val Val Ile Ile Ala Ile Leu

1125 1130 1135

Phe Lys Cys Gly Phe Phe Lys Arg Lys Tyr Gln Gln Leu Asn Leu Glu

1140 1145 1150

Ser Thr Arg Arg Ala Gln Leu Lys Ala Asp Ser Leu Leu Gln Asp

1155 1160 1165

<210> 7

<211> 360

<212> DNA

<213> Artificial Sequence

<400> 7

catctaggtg tatcatctga gtatttagca gggactctta aatcctgtcg atgccttgtg 60

atctttggca cagctcccat cgctttctgc tttcaggttt tggctccttc gtggacaaaa 120

cggtgctgcc cgcggtaagc accgtgccct ccaagcttca ccacccatgc cccagccgac 180

tggagcgttg tcagccaccc ttcagctttc accacgtgct gtccctcacc ggggacgctc 240

aagccttcga gagagaggtg ggacgccaga atgtctctgg caacctggac tcacccgaag 300

gcggctttga tgccattttg caggctgccc tctgccaggt gagatagggt taggatctga 360

Claims

1. A non-therapeutic or diagnostic method of down-regulating integrin α E expression, reducing the ability of immune cells to migrate across blood vessels, inhibiting adhesion of immune cells to epithelial tissue, inhibiting homing of immune cells to gut-associated lymphoid tissue, and inhibiting the residence of immune cells in gut-associated lymphoid tissue in vitro comprising down-regulating integrin β subunit expression or activity.

2. The method of claim 1, wherein the method comprises reducing or inhibiting the interaction between an aromatic amino acid in the integrin beta subunit I domain and a metal cation of the SyMBS site,

preferably, the method comprises reducing or inhibiting the interaction between phenylalanine at position 185 in the integrin beta subunit and a metal cation of the SyMBS site,

more preferably, the method comprises mutating phenylalanine at position 185 of the beta subunit.

3. Use of an agent for down-regulating integrin alphae expression, reducing immune cell migration across blood vessels, inhibiting immune cell adhesion to epithelial tissue, inhibiting homing of immune cells to gut-associated lymphoid tissue, inhibiting residence of immune cells in gut-associated lymphoid tissue, inhibiting gut inflammation, reducing gut graft versus host disease, inhibiting tumorigenesis, or ameliorating a side effect of a treatment for a gut inflammatory disease, or for use in the manufacture of a medicament for treating or preventing a gut disease that would benefit from down-regulating integrin alphae expression, reducing immune cell migration across blood vessels, inhibiting adhesion of immune cells to epithelial tissue, inhibiting homing of immune cells to gut-associated lymphoid tissue, inhibiting immune cell retention in gut-associated lymphoid tissue, said agent selected from the group consisting of:

(1) reduced activity of an integrin or β subunit thereof, or a nucleic acid sequence encoding the same, and optionally a knockout agent for a wild-type integrin or β subunit thereof, preferably, a reduced activity of an integrin or β subunit thereof comprises an inactive or low activity wild-type integrin or β subunit thereof, or a mutant of an integrin or β subunit thereof with reduced or absent activation function, more preferably, the mutant of an integrin or β subunit thereof comprises a mutation that results in reduced or absent interaction between an aromatic amino acid in integrin β subunit I domain and a metal cation of the SyMBS site as compared to wild-type,

(2) an agent that decreases integrin beta subunit activity,

(3) an agent that down-regulates the expression of an integrin beta subunit,

preferably, the intestinal inflammation is adaptive immune-mediated chronic intestinal inflammation or innate immune-mediated acute intestinal inflammation,

preferably, the integrin beta subunit is integrin beta 7 subunit.

4. The use of claim 3, wherein the agent that decreases integrin beta subunit activity is one or more of the agents that decrease integrin beta subunit aromatic amino acids and SyMBS sites that decrease or inhibit cationic interactions, preferably wherein the agent that decreases integrin beta subunit activity is selected from the group consisting of:

(ii) regulators of the stable integrin intracellular conserved sequences GEFKR and/or LLv-iHDR,

an agent capable of forming alpha helix structures on the α and β intracellular segments of integrin,

an agent that binds an aromatic amino acid in the integrin beta subunit I domain,

contains calcium ions with a concentration of at least 1mmol/L,

an agent that inhibits binding of an intracellular protein to an integrin, preferably said agent comprises: a protein that competitively binds with the intracellular protein, and a protein kinase that modulates the protein that competitively binds with the intracellular protein,

an agent that inhibits binding of integrin to its ligand, preferably an extracellular fragment of the beta subunit or a coding sequence thereof, that is capable of competing for binding to the ligand, said fragment having at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% sequence identity to the extracellular portion of the beta subunit of integrin,

(ii) a knock-in agent for the integrin beta subunit or SyMBS locus that reduces or inhibits cationic interaction between the integrin beta subunit and the SyMBS locus,

an antibody or nucleic acid sequence encoding the same specific for integrin or its beta subunit that decreases integrin beta subunit activity, or a small molecule compound that inhibits integrin or its beta subunit activity,

a cation chelating agent, preferably EDTA.

5. A nucleic acid molecule comprising a sequence selected from:

(1) a nucleic acid sequence containing a sequence shown in SEQ ID NO. 7 and a LoxP site; and

(2) (1) the complement of said sequence.

6. A vector comprising the nucleic acid sequence of claim 5.

7. A genetically engineered host cell transformed with the vector of claim 5.

8. A method of constructing a transgenic mouse, the method comprising:

(1) providing a vector according to claim 6, wherein said vector is selected from the group consisting of,

9. The use of integrin or its beta subunit gene or protein as target in screening drugs, or as molecular index in clinical diagnosis of intestinal disease course development, the drug is used for treating or preventing intestinal disease or improving intestinal inflammatory disease treatment side effects,

preferably, the intestinal disease is a disease benefiting from the inhibition of stable adhesion of lymphocytes, a reduction of the ability of immune cells to migrate across blood vessels, a down-regulation of the expression of integrin alphae, an inhibition of homing of immune cells to intestinal associated lymphoid tissue, preferably the disease is intestinal inflammation.

10. The application of the reagent for detecting the gene or protein of the integrin or beta subunit thereof in the preparation of a kit for diagnosing the intestinal diseases or judging the course development of the intestinal diseases,

preferably, the intestinal disease is a disease benefiting from the inhibition of stable adhesion of lymphocytes, a reduction of the ability of immune cells to migrate across blood vessels, a down-regulation of the expression of integrin alphaE, an inhibition of homing of immune cells to gut-associated lymphoid tissue, an inhibition of intestinal inflammation,

more preferably, the disease is intestinal inflammation.