CN112501175A - Psoriasis inhibiting gene and application thereof - Google Patents
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Abstract
The invention provides a psoriasis suppressor gene which is an N4BP1 gene. Simultaneously provides the application of the gene N4BP1 in preparing medicaments for treating psoriasis and the application in preparing a kit for detecting psoriasis. N4BP1 was highly expressed in keratinocytes and neutrophils of the skin. Following N4BP1 deletion, keratinocyte proliferation was accelerated and neutrophil counts and responses were enhanced. Thus N4BP1 is a key gene in the development of psoriasis and loss of function of N4BP1 may exacerbate disease progression. The invention provides a theoretical basis for the development research of psoriasis and the preparation of therapeutic drugs by over-expressing the N4BP1 gene or stabilizing and promoting the function of N4BP1 by using drugs.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a psoriasis suppressor gene and application thereof.
Background
Psoriasis is a chronic inflammatory skin disease with abnormal activation of immune cells and keratinocytes, which is closely related to genetics and environment. Psoriasis has various clinical manifestations, but mainly consists of silvery white scales attached to red plaques. Clinically, psoriasis can be classified into psoriasis vulgaris, psoriasis arthrosis, erythrodermic psoriasis, and pustular psoriasis.
Psoriasis, commonly known as "psoriasis", has a complex pathogenesis, and the exact cellular and molecular mechanisms of psoriasis are still not completely elucidated. At present, psoriasis is considered to be a strong hereditary autoimmune disease, which is mostly seen in the young. Psoriasis occurs in the human population at an incidence of about 2% and is a common disease of the dermatological family.
The pathogenesis of psoriasis is mainly characterized by two main cell types: keratinocytes and immune cells. Keratinocytes are the main cells of the human skin epidermis, and during the differentiation of the basal cell layer of the epidermis to the stratum corneum, they eventually lose the nucleus and organelles. The process of keratinocyte differentiation, i.e., keratinization, is believed to be a specific apoptotic process. During keratinization, a series of characteristic morphological changes are generated, and keratin is accumulated from a basal layer and finally fills the whole keratinocyte. In the normal process of neogenesis and homeostasis of the epithelium, the keratinization and keratinocyte differentiation processes must be tightly controlled to maintain a normal homeostasis of the keratinization process and epidermal renewal. When psoriasis develops, keratinocyte proliferation and differentiation become abnormal. Keratinocytes hyperproliferate, causing a significant thickening of the epidermal layer. Keratinocyte differentiation is significantly accelerated, forming a large amount of stratum corneum. These abnormalities of keratinocytes are due in part to genetic, environmental and other factors that cause the keratinocytes to undergo abnormal changes at the molecular level and in part to abnormal immune cell stimulation.
The most typical skin lesions of psoriasis are raised erythema with silvery-white scales, the major cells of which include neutrophils, T lymphocytes, keratinocytes, dendritic cells, macrophages, and mast cells. In addition to keratinocytes, these abnormally active immune cells secrete large amounts of inflammatory factors such as TNFa, IL-17, and IL-23. These cytokines stimulate the abnormal proliferation and differentiation of keratinocytes. Abnormally proliferating and differentiating keratinocytes further release factors that chemotaxis and activate immune cells forming positive feedback. Thus, the main cause of psoriasis is that the initiating factors activate keratinocytes or immune cells or both, and the cell-related activation of the activation leads to uncontrolled positive feedback, resulting in the development of psoriasis. Among them, cytokines play an extremely important role in causing abnormal activation of keratinocytes and immune cells. To date, TNFa, IL-17 and IL-23, 3 cytokines, are the more clinically and fundamentally studied molecules that play a central role in psoriasis. Antibodies against these 3 cytokines have shown superior efficacy in the treatment of clinical psoriasis. However, all current clinical treatments can only control and slow down the onset of psoriasis, and cannot radically cure the psoriasis. Therefore, the search for new core genes and the development of more effective methods and drugs remains a major focus in the research of psoriasis.
The traditional method for treating psoriasis mainly comprises physical therapy, local external therapy and systemic therapy. Topical drugs currently include mainly retinoids, vitamin D derivatives, corticosteroids, calcium inhibitors, and the like. Physical therapy includes light and laser therapy, etc. The systemic medicine mainly comprises methotrexate, abamectin, cyclosporine and the like.
In recent years, fundamental breakthrough is made in the field of psoriasis treatment, and better clinical treatment effect is achieved aiming at single-molecule biological agents. For example, the antibodies against TNF-a include etanercept, infliximab, adalimumab, certolizumab ozogamicin, and golimumab. The antibody aiming at the IL-23 is ubsunumab. The antibodies against IL-17 include Securiyumumab, Eizumab, and the like. These biologics work well in clinical treatment, but still a large proportion of patients respond poorly to the drugs or relapse within a short time after treatment. More importantly, these biological agents also do not cure psoriasis radically, but only control and slow down the symptoms.
The medicine for treating psoriasis has great progress, but all the current treatment methods can only control or relieve the disease and can not eradicate the psoriasis. The above shows that the pathogenesis of psoriasis needs to be deeply researched, unknown important genes in psoriasis are searched, and a new treatment method is further developed, so that the treatment effect of psoriasis can be further improved fundamentally, or the aim of eradicating psoriasis is fulfilled.
As shown in fig. 1, N4BP1 is highly expressed in skin in all tissues of human. As shown in fig. 2, the expression of N4BP1 was highest in neutrophils in all immune cell sub-classes. However, the N4BP1 gene is still a gene which is less studied, and the function of the gene is less studied. It has been shown that N4BP1 can inhibit viral replication by degrading the RNA of the HIV virus. Other functions for N4BP1 are almost unknown. The target of the existing biological preparation for treating psoriasis is mainly related to the function of T cells, and although the control and the relief of the disease are effectively realized, the radical treatment of the disease is not realized. It is suggested that in addition to T cells, there are other factors or other cells involved in the initiation mechanism of psoriasis in the pathogenesis of psoriasis. Therefore, the invention aims to provide a psoriasis inhibiting gene and application thereof in preparing a medicament for treating psoriasis.
Disclosure of Invention
The invention aims to solve the technical problems and provides a psoriasis inhibiting gene and application thereof.
In order to solve the above technical problems, embodiments of the present invention provide a psoriasis suppressor gene, which is N4BP1 gene.
The invention also provides application of the psoriasis suppressor gene N4BP1 in preparing a medicament for treating psoriasis.
The invention also provides application of the psoriasis suppressor gene N4BP1 in preparing a psoriasis detection kit.
The invention also provides a method for detecting psoriasis inhibition by the gene N4BP1, which comprises the following steps:
(1) 4BP1 and N4BP1 knockout mice
(1-1) HeLa cell and N4BP1 overexpression Hela cell genetically modified by HeLa cellGood DMEM high blood sugar medium (containing 10% fetal bovine serum and 1% antibiotics) at 37 deg.C and 5% CO2Culturing under the condition; respectively transfecting over-expression unloaded plasmid and over-expression N4BP1 plasmid by using liposome 2000, and replacing a culture medium after 6h of transfection; after transfection for 48h, puromycin is used for screening cells which are successfully transfected, and the cells are cultured for 2 weeks;
(1-2) to obtain an N4BP1 knockout mouse, we designed two sgRNAs that specifically recognize both ends of the second exon, and a preparation procedure of an N4BP1 knockout mouse was carried out by Shanghai model organism center, Inc. Mixing N4BP1+/-Hybridization of mice to produce N4BP1-/-Mice, and backcrossed over 10 generations on a C57BL/6 genetic background;
(2) establishing psoriasis mouse model
Shaving off the hair on the back of a mouse aged 8-10 weeks by using an electric razor, taking 1.875mg of imiquimod cream, continuously smearing the imiquimod cream on the backs of an N4BP1 knockout mouse and a wild type mouse for 11 days respectively, measuring the thickness of the skin on the back by using a vernier caliper every day, killing the mouse by breaking the neck within a preset time, and taking the skin on an affected part for subsequent experiments;
(3) hematoxylin-eosin staining
(3-1) cutting the skin tissue of the affected part into small pieces of 1cm x 1cm, soaking in 4% paraformaldehyde for 24 hr, taking out the pieces the next day, performing alcohol gradient dehydration, and embedding in paraffin to obtain tissue blocks;
(3-2) cutting the tissue blocks into slices with the thickness of 5um, dewaxing, hydrating, dyeing with hematoxylin for 30min, dyeing with eosin for 5s, dehydrating the dyed slices with alcohol, performing xylene transparency, sealing the slices with resin, and observing under a mirror;
(4) preparation of mouse embryonic fibroblasts
(4-1) closing the N4BP1 heterozygote mice for 13-15 days, taking out pregnant mice, breaking the neck, killing, taking out fetal mice, rinsing twice in PBS (phosphate buffer solution) with antibiotics in advance, and placing in a culture dish with the diameter of 10 cm;
(4-2) cutting off the head of the fetal rat, putting the fetal rat into a 1.5ml centrifuge tube for subsequent genotype identification, cutting the rest part by using scissors, and directly adding a modified DMEM high-blood-sugar culture medium (containing 10% fetal calf serum and 1% antibiotics) into a culture dish;
(4-3) MEF cells were placed at 37 ℃ and 5% CO2 Medium culture, changing liquid every two days;
(5) RNA extraction and real-time fluorescent quantitative PCR
(5-1) taking about 30mg of mouse skin tissue, putting the mouse skin tissue into a 1.5ml centrifuge tube, adding 1ml of Trizol, homogenizing by a homogenizer, and extracting;
(5-2) digesting the cells to be RNA-extracted, centrifuging, collecting, adding 1ml of Trizol, and standing at room temperature for five minutes; adding chloroform, centrifuging and taking upper layer RNA; adding isopropanol to extract RNA; centrifuging, washing twice with 75% alcohol, and dissolving in DEPC water;
(5-3) determining the purity and concentration of total RNA using a Nano Drop 1000 spectrophotometer (Thermo Fisher Science);
(5-4) reverse transcription of 1. mu.g of total RNA using HiScript II one-step RT-PCR kit; CDNA is diluted by 5 times, and gene expression is quantitatively detected by an SYBR Green PCR kit (# Q112-02/03, Vazyme);
(6) RNA binding protein immunoprecipitation
(6-1) washing twice with precooled PBS to remove the culture medium, then adding 10ml of PBS, adding formaldehyde (37% stock solution) into the solution to reach the final solution concentration of 1%, and slowly rotating and incubating for 10 minutes at room temperature; the crosslinking reaction (pH =7.0) was stopped by adding glycine solution to a final concentration of 0.25M, followed by incubation at room temperature for 5 minutes with gentle shaking; adding 5-10ml of precooled PBS to wash the cells, and scraping the cells in 2ml of precooled PBS;
(6-2) centrifuging to collect cells, and washing twice with precooled PBS; collecting cells, adding RIPA lysis buffer containing PMSF, and performing ultrasonic lysis; after rotating for 1h, centrifuging at 13000rpm and 4 ℃; taking supernatant, pretreating with protein A, centrifuging, and incubating with anti-FLAG antibody (# F3165, Sigma) for 2 h; then, protein a beads were collected and washed; resuspending the immunoprecipitated sample in a cross-linking agent at 70 ℃ for 45min, and carrying out reverse cross-linking; after reverse cross-linking, RNA was extracted with Trizol.
The technical scheme of the invention has the following beneficial effects: the invention discovers that N4BP1 is highly expressed in keratinocytes and neutrophils of skin, and after N4BP1 is deleted, the proliferation of the keratinocytes is accelerated, and the quantity and the reflection of the neutrophils are enhanced. Thus N4BP1 is a key gene in the development of psoriasis and loss of function of N4BP1 may exacerbate disease progression. The invention provides a theoretical basis for the development research of psoriasis and the preparation of therapeutic drugs by over-expressing the N4BP1 gene or stabilizing and promoting the function of N4BP1 by using drugs.
Drawings
FIG. 1 is a schematic representation of the high expression of N4BP1 in skin in all human tissues;
FIG. 2 is a graph showing that the expression of N4BP1 is highest in neutrophils in all immune cell sub-types;
FIG. 3 is a schematic representation of N4BP1 deficient mice developing more severe psoriasis under IMQ drug induction in the present invention;
FIG. 4 is a schematic diagram of the increase of psoriasis-associated gene expression in mice deficient in N4BP1 under IMQ drug induction in accordance with the present invention;
FIG. 5 is a schematic diagram of the significant down-regulation of the expression of a psoriasis-associated gene CXCL1 after over-expression of N4BP1 in the present invention;
FIG. 6 is a schematic diagram showing that the expression of psoriasis-associated genes JunB and FosB is significantly reduced after N4BP1 is overexpressed in the invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
The invention provides a psoriasis suppressor gene which is an N4BP1 gene. Simultaneously provides the application of the gene N4BP1 in preparing medicaments for treating psoriasis and the application in preparing a kit for detecting psoriasis.
The inhibition of psoriasis development by overexpression of the N4BP1 gene, or the stabilization and promotion of N4BP1 function by drugs is illustrated by the following specific experimental procedures.
A method for detecting psoriasis inhibition by a gene N4BP1, which is characterized by comprising the following steps:
(1) n4BP1 knockout cell lines (study of the molecular mechanisms of N4BP1 in inhibiting psoriasis) and N4BP1 knockout mice (study of the in vivo effects and molecular mechanisms of N4BP1 in inhibiting psoriasis)
(1-1) HeLa cells were cultured in modified DMEM high-glucose medium (containing 10% fetal bovine serum and 1% antibiotics) at 37 ℃ and 5% CO2Culturing under the condition; respectively transfecting over-expression unloaded plasmid and over-expression N4BP1 plasmid by using liposome 2000, and replacing a culture medium after 6h of transfection; after transfection for 48h, puromycin is used for screening cells which are successfully transfected, and the cells are cultured for 2 weeks;
(1-2) in order to obtain an N4BP1 knockout mouse, two sgRNAs which specifically recognize two ends of a second exon are designed, and when the No. 2 exon of an N4BP1 gene is knocked out, a frame shift mutation occurs in the N4BP1 gene, so that a functional N4BP1 protein cannot be generated. The preparation process of the N4BP1 knockout mouse is completed by Shanghai model organism center, Inc. Mixing N4BP1+/-Hybridization of mice to produce N4BP1-/-Mice, and backcrossed over 10 generations on a C57BL/6 genetic background.
(2) Establishing psoriasis mouse model
Shaving the hair of the back of a mouse with the age of 8-10 weeks by using an electric razor, taking 1.875mg of imiquimod cream, continuously smearing the imiquimod cream on the back of an N4BP1 knockout mouse and the back of a wild type mouse for 11 days respectively, measuring the thickness of the skin on the back by using a vernier caliper every day, killing the mouse by breaking the neck within a preset time, and taking the skin on an affected part for subsequent experiments.
(3) Hematoxylin-eosin staining
(3-1) cutting the skin tissue of the affected part into small pieces of 1cm x 1cm, soaking in 4% paraformaldehyde for 24 hr, taking out the pieces the next day, performing alcohol gradient dehydration, and embedding in paraffin to obtain tissue blocks;
(3-2) cutting the tissue blocks into slices with the thickness of 5um, dewaxing, hydrating, dyeing with hematoxylin for 30min, dyeing with eosin for 5s, dehydrating the dyed slices with alcohol, performing xylene transparency, sealing the slices with resin, and observing under a mirror;
(4) preparation of mouse embryonic fibroblasts
(4-1) closing the N4BP1 heterozygote mice for 13-15 days, taking out pregnant mice, breaking the neck, killing, taking out fetal mice, rinsing twice in PBS (phosphate buffer solution) with antibiotics in advance, and placing in a culture dish with the diameter of 10 cm;
(4-2) cutting off the head of the fetal rat, putting the fetal rat into a 1.5ml centrifuge tube for subsequent genotype identification, and cutting the rest part by using scissors to change the tissues of the fetal rat into single cells. Adding modified DMEM high-blood-sugar medium (containing 10% fetal calf serum and 1% antibiotics) into the culture dish containing the single cells, and changing the cells into MEFs cells after the cells grow and expand;
(4-3) MEF cells were placed at 37 ℃ and 5% CO2 Medium culture, changing liquid every two days;
(5) RNA extraction and real-time fluorescent quantitative PCR
(5-1) taking about 30mg of mouse skin tissue or cultured MEFs cells, putting the cells into a 1.5ml centrifuge tube, adding 1ml of Trizol, homogenizing by a homogenizer, and extracting;
(5-2) digesting the cells to be RNA-extracted, centrifuging, collecting, adding 1ml of Trizol, and standing at room temperature for five minutes; adding chloroform, centrifuging and taking upper layer RNA; adding isopropanol to extract RNA; centrifuging, washing twice with 75% alcohol, and dissolving in DEPC water;
(5-3) determining the purity and concentration of total RNA using a Nano Drop 1000 spectrophotometer (Thermo Fisher Science);
(5-4) reverse transcription of 1. mu.g of total RNA using HiScript II one-step RT-PCR kit; CDNA is diluted by 5 times, and gene expression is quantitatively detected by an SYBR Green PCR kit (# Q112-02/03, Vazyme);
the mRNA levels were referenced to 18S, and the primer sequences were as follows:
| Name | Oligo sequences (5’ – 3’) |
| Human/Mouse 18S, forward primer: | GAACGAGACTCTGGCATGCTA |
| Human/Mouse 18S, reverse primer: | CACGCTGAGCCAGTCAGTGTA |
| Human JUNB, forward primer: | CAGGAGGGCTTCGCCGACGGC |
| Human JUNB, reverse primer: | AGTAGCTGCTGAGGTTGGTGT |
| Human JUNC, forward primer: | GAACGTGACAGATGAGCAGGA |
| Human JUNC, reverse primer: | CCGGCGGCTCGCTGTGCAGGC |
| Human FOSB, forward primer: | GAGGAGAAGCGAAGGGTGCGC |
| Human FOSB, reverse primer: | CCCGGTTTGTGGGCCACCAGC |
| Human FOSC, forward primer: | AGCTGACTGATACACTCCAAG |
| Human FOSC, reverse primer: | CAGGCAGGTCGGTGAGCTGCC |
| Human CXCL1, forward primer: | CAAAGTGTGAACGTGAAGTCCC |
| Human CXCL1, reverse primer: | GCTTTCCGCCCATTCTTGAG |
| Human TNF-α, forward primer: | TCTGGGCAGGTCTACTTTGG |
| Human TNF-α, reverse primer: | TGAGCCAGAAGAGGTTGAGG |
| Mouse CXCL1, forward primer: | CCTATCGCCAATGAGCTGC |
| Mouse CXCL1, reverse primer: | CTCGCGACCATTCTTGAGTG |
| Mouse CCL20, forward primer: | CGTCTGCTCTTCCTTGCTTT |
| Mouse CCL20, reverse primer: | ACAGTCGTAGTTGCTTGCTG |
| Mouse S100A8, forward primer: | GTTGTCTCCATAGCCCGAGG |
| Mouse S100A8, reverse primer: | TCCAGTTCAGACGGCATTGTC |
| Mouse S100A9, forward primer: | ACTGGGCTTACACTGCTCTT |
| Mouse S100A9, reverse primer: | AGGTGTCGATGATGGTGGTT |
(6) RNA binding protein immunoprecipitation
(6-1) washing the cultured cells or tissues twice with precooled PBS to remove the culture medium, then adding 10ml of PBS, adding formaldehyde (37% stock solution) to the solution to a final solution concentration of 1%, and slowly rotating and incubating for 10 minutes at room temperature; the crosslinking reaction (pH =7.0) was stopped by adding glycine solution to a final concentration of 0.25M, followed by incubation at room temperature for 5 minutes with gentle shaking; adding 5-10ml of precooled PBS to wash mouse embryonic fibroblasts and scraping the cells in 2ml of precooled PBS;
(6-2) centrifugally collecting mouse embryonic fibroblasts, and washing the mouse embryonic fibroblasts twice by using precooled PBS; collecting cells, adding RIPA lysis buffer containing PMSF, and performing ultrasonic lysis; after rotating for 1h, centrifuging at 13000rpm and 4 ℃; taking supernatant, pretreating with protein A, centrifuging, and incubating with anti-FLAG antibody (# F3165, Sigma) for 2 h; then, protein a beads were collected and washed; resuspending the immunoprecipitated sample in a cross-linking agent at 70 ℃ for 45min, and carrying out reverse cross-linking; after reverse cross-linking, RNA was extracted with Trizol.
Figure 3 shows N4BP1 deficient mice, which, upon IMQ drug induction, develop more severe psoriasis. Wherein, FIG. 3A shows photographs of IMQ-induced dermatitis in N4BP1 wild-type and deficient mice; figure 3B shows IMQ-induced dermatitis thickness in N4BP1 wild-type and deficient mice; figure 3C shows IMQ-induced dermatitis in N4BP1 wild-type and deficient mice, organized to show keratinocyte proliferation.
Figure 4 shows that N4BP1 deficient mice have increased psoriasis-associated gene expression under IMQ drug induction. Wherein, fig. 4A shows that CXCL1, CCL20, S100a8, S100a9 genes are significantly increased in N4BP1 deficient mice after IMQ-induced dermatitis in N4BP1 wild-type and deficient mice; FIG. 4B shows N4BP1 wild-type and defective MEF cells, with CXCL1, CCL20, S100A8, significantly increased gene expression in N4BP1 defective MEF cells after IL-17 treatment; fig. 4C shows N4BP1 wild-type and deficient MEF cells, with CXCL1, CCL20, S100a8, with significantly increased gene expression in N4BP1 deficient MEF cells after R848 treatment.
Fig. 5 shows that after N4BP1 is overexpressed, the expression of psoriasis-associated gene CXCL1 is significantly down-regulated. Fig. 5A shows that CXCL1 is significantly downregulated after Hela cells stably overexpress N4BP 1; fig. 5B shows that CXCL1 mRNA stability was significantly down-regulated after Hela cells stably over-expressed N4BP 1; FIGS. 5C and 5D show that the binding of FLAG-N4BP1 and CXCL1 mRNA was observed after Hela cells stably overexpressed Flag-N4BP 1.
Fig. 6 shows that the expression of psoriasis-associated genes JunB and FosB is significantly down-regulated after overexpression of N4BP 1. FIG. 6A shows that JunB and FosB are significantly down-regulated after Hela cells stably over-express N4BP 1; FIG. 6B shows that the stability of mRNA of JunB and FosB is significantly down-regulated after Hela cells stably over-express N4BP 1; FIGS. 6C and 6D show that FLAG-N4BP1 was associated with JunB and FosB mRNA after Hela cells stably overexpressed Flag-N4BP 1.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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<211> 21
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 7
Gly Ala Gly Gly Ala Gly Ala Ala Gly Cys Gly Ala Ala Gly Gly Gly
1 5 10 15
Thr Gly Cys Gly Cys
20
<210> 8
<211> 21
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 8
Cys Cys Cys Gly Gly Thr Thr Thr Gly Thr Gly Gly Gly Cys Cys Ala
1 5 10 15
Cys Cys Ala Gly Cys
20
<210> 9
<211> 21
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 9
Ala Gly Cys Thr Gly Ala Cys Thr Gly Ala Thr Ala Cys Ala Cys Thr
1 5 10 15
Cys Cys Ala Ala Gly
20
<210> 10
<211> 21
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 10
Cys Ala Gly Gly Cys Ala Gly Gly Thr Cys Gly Gly Thr Gly Ala Gly
1 5 10 15
Cys Thr Gly Cys Cys
20
<210> 11
<211> 22
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 11
Cys Ala Ala Ala Gly Thr Gly Thr Gly Ala Ala Cys Gly Thr Gly Ala
1 5 10 15
Ala Gly Thr Cys Cys Cys
20
<210> 12
<211> 20
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
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Gly Cys Thr Thr Thr Cys Cys Gly Cys Cys Cys Ala Thr Thr Cys Thr
1 5 10 15
Thr Gly Ala Gly
20
<210> 13
<211> 20
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 13
Thr Cys Thr Gly Gly Gly Cys Ala Gly Gly Thr Cys Thr Ala Cys Thr
1 5 10 15
Thr Thr Gly Gly
20
<210> 14
<211> 20
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 14
Thr Gly Ala Gly Cys Cys Ala Gly Ala Ala Gly Ala Gly Gly Thr Thr
1 5 10 15
Gly Ala Gly Gly
20
<210> 15
<211> 19
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 15
Cys Cys Thr Ala Thr Cys Gly Cys Cys Ala Ala Thr Gly Ala Gly Cys
1 5 10 15
Thr Gly Cys
<210> 16
<211> 20
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 16
Cys Thr Cys Gly Cys Gly Ala Cys Cys Ala Thr Thr Cys Thr Thr Gly
1 5 10 15
Ala Gly Thr Gly
20
<210> 17
<211> 20
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 17
Cys Gly Thr Cys Thr Gly Cys Thr Cys Thr Thr Cys Cys Thr Thr Gly
1 5 10 15
Cys Thr Thr Thr
20
<210> 18
<211> 20
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 18
Ala Cys Ala Gly Thr Cys Gly Thr Ala Gly Thr Thr Gly Cys Thr Thr
1 5 10 15
Gly Cys Thr Gly
20
<210> 19
<211> 20
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 19
Gly Thr Thr Gly Thr Cys Thr Cys Cys Ala Thr Ala Gly Cys Cys Cys
1 5 10 15
Gly Ala Gly Gly
20
<210> 20
<211> 21
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 20
Thr Cys Cys Ala Gly Thr Thr Cys Ala Gly Ala Cys Gly Gly Cys Ala
1 5 10 15
Thr Thr Gly Thr Cys
20
<210> 21
<211> 20
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 21
Ala Cys Thr Gly Gly Gly Cys Thr Thr Ala Cys Ala Cys Thr Gly Cys
1 5 10 15
Thr Cys Thr Thr
20
<210> 22
<211> 20
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 22
Ala Gly Gly Thr Gly Thr Cys Gly Ala Thr Gly Ala Thr Gly Gly Thr
1 5 10 15
Gly Gly Thr Thr
20
Claims (4)
1. A psoriasis suppressor gene which is an N4BP1 gene.
2. An application of psoriasis suppressor gene N4BP1 in preparing medicine for treating psoriasis is disclosed.
3. An application of psoriasis suppressor gene N4BP1 in preparing a psoriasis detection kit.
4. A method for detecting psoriasis inhibition by a gene N4BP1, which is characterized by comprising the following steps:
(1) 4BP1 and N4BP1 knockout mice
(1-1) HeLa cells in modified DMEM high-glucose Medium at 37 ℃ and 5% CO2Under the condition ofCulturing; respectively transfecting over-expression unloaded plasmid and over-expression N4BP1 plasmid by using liposome 2000, and replacing a culture medium after 6h of transfection; after transfection for 48h, puromycin is used for screening cells which are successfully transfected, and the cells are cultured for 2 weeks;
(1-2) mixing N4BP1+/-Hybridization of mice to produce N4BP1-/-Mice, and backcrossed over 10 generations on a C57BL/6 genetic background;
(2) establishing psoriasis mouse model
Shaving off the hair on the back of a mouse aged 8-10 weeks by using an electric razor, taking 1.875mg of imiquimod cream, continuously smearing the imiquimod cream on the backs of an N4BP1 knockout mouse and a wild type mouse for 11 days respectively, measuring the thickness of the skin on the back by using a vernier caliper every day, killing the mouse by breaking the neck within a preset time, and taking the skin on an affected part for subsequent experiments;
(3) hematoxylin-eosin staining
(3-1) cutting the skin tissue of the affected part into small pieces of 1cm x 1cm, soaking in 4% paraformaldehyde for 24 hr, taking out the pieces the next day, performing alcohol gradient dehydration, and embedding in paraffin to obtain tissue blocks;
(3-2) cutting the tissue blocks into slices with the thickness of 5um, dewaxing, hydrating, dyeing with hematoxylin for 30min, dyeing with eosin for 5s, dehydrating the dyed slices with alcohol, performing xylene transparency, sealing the slices with resin, and observing under a mirror;
(4) preparation of mouse embryonic fibroblasts
(4-1) closing the N4BP1 heterozygote mice for 13-15 days, taking out pregnant mice, breaking the neck, killing, taking out fetal mice, rinsing twice in PBS (phosphate buffer solution) with antibiotics in advance, and placing in a culture dish with the diameter of 10 cm;
(4-2) cutting off the head of the fetal rat, putting the fetal rat into a 1.5ml centrifuge tube for subsequent genotype identification, cutting the rest part into pieces by using scissors, and directly adding an improved DMEM high blood sugar culture medium into a culture dish;
(4-3) MEF cells were placed at 37 ℃ and 5% CO2 Medium culture, changing liquid every two days;
(5) RNA extraction and real-time fluorescent quantitative PCR
(5-1) taking 30mg of mouse skin tissue, putting the mouse skin tissue into a 1.5ml centrifuge tube, adding 1ml of Trizol, homogenizing by a homogenizer, and extracting;
(5-2) digesting the cells to be RNA-extracted, centrifuging, collecting, adding 1ml of Trizol, and standing at room temperature for five minutes; adding chloroform, centrifuging and taking upper layer RNA; adding isopropanol to extract RNA; centrifuging, washing twice with 75% alcohol, and dissolving in DEPC water;
(5-3) measuring the purity and concentration of the total RNA by using a spectrophotometer;
(5-4) reverse transcription of 1. mu.g of total RNA using HiScript II one-step RT-PCR kit; CDNA is diluted by 5 times, and a PCR kit is used for quantitatively detecting gene expression;
(6) RNA binding protein immunoprecipitation
(6-1) washing twice with precooled PBS to remove the culture medium, then adding 10ml of PBS, adding formaldehyde into the solution to reach the final solution concentration of 1%, and slowly rotating and incubating for 10 minutes at room temperature; adding glycine solution to a final concentration of 0.25M to terminate the crosslinking reaction, then incubating for 5 minutes at room temperature, and gently shaking; adding 5-10ml of precooled PBS to wash the cells, and scraping the cells in 2ml of precooled PBS;
(6-2) centrifuging to collect cells, and washing twice with precooled PBS; collecting cells, adding RIPA lysis buffer containing PMSF, and performing ultrasonic lysis; after rotating for 1h, centrifuging at 13000rpm and 4 ℃; taking supernatant, pretreating with protein A, centrifuging, and incubating with anti-FLAG antibody for 2 h; then, protein a beads were collected and washed; resuspending the immunoprecipitated sample in a cross-linking agent at 70 ℃ for 45min, and carrying out reverse cross-linking; after reverse cross-linking, RNA was extracted with Trizol.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170343534A1 (en) * | 2012-08-15 | 2017-11-30 | The Procter & Gamble Company | Systems, Models and Methods for Identifying and Evaluating Skin-Active Agents Effective for Treating an Array of Skin Disorders |
| US20190085402A1 (en) * | 2010-07-23 | 2019-03-21 | President And Fellows Of Harvard College | Methods for Detecting Signatures of Disease or Conditions in Bodily Fluids |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190085402A1 (en) * | 2010-07-23 | 2019-03-21 | President And Fellows Of Harvard College | Methods for Detecting Signatures of Disease or Conditions in Bodily Fluids |
| US20170343534A1 (en) * | 2012-08-15 | 2017-11-30 | The Procter & Gamble Company | Systems, Models and Methods for Identifying and Evaluating Skin-Active Agents Effective for Treating an Array of Skin Disorders |
Non-Patent Citations (4)
| Title |
|---|
| ALEXANDER D. GITLIN等: "Integration of innate immune signaling by caspase-8 cleavage of N4BP1", 《NATURE》 * |
| DAICHI YAMASOBA等: "N4BP1 restricts HIV-1 and its inactivation by MALT1 promotes viral reactivation", 《NATURE MICROBIOLOGY》 * |
| PETER DONNELLY 等: "Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity", 《NATURE GENETICS》 * |
| SAEEDA MUNIR等: "Association analysis of GWAS and candidate gene loci in a Pakistani population with psoriasis", 《MOLECULAR IMMUNOLOGY》 * |
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