Method for constructing a mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification

Abstract

The application relates to a mouse model construction method for psoriasis research based on SBSN 600 lysine dihydroxyisobutyryl modification, which comprises the following steps: first step, constructing a plasmid comprising a DNA fragment of a SBSN gene sequence, wherein 600 sites of the SBSN sequence are mutated from lysine (K) to threonine (T); second step, culturing mouse embryonic stem cells; third step, electrically transforming the embryonic stem cells; fourth step, drug screening; fifth step, culturing the embryonic stem cells screened in the fourth step; sixth step, injecting the embryonic stem cells cultured in the fourth step into mouse blastocysts, then culturing the mouse blastocysts, and transplanting the cultured blastocysts into the uterus of a pseudopregnant female mouse, wherein the mouse delivered by the female mouse is a chimera mouse; and seventh step, breeding the chimera mouse and identifying the mouse genotype through PCR.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for constructing a mouse model based on the dihydroxyisobutyrylation modification of lysine at position 600 of SBSN, in particular to a method for constructing a mouse model based on the dihydroxyisobutyrylation modification of lysine at position 600 of SBSN for psoriasis research, and especially to a method for constructing a mouse model for psoriasis research by combining gene editing technology and imiquimod induction. BACKGROUND

[0002] Psoriasis is a common chronic inflammatory skin disease mediated by immunity. Previous studies suggest that psoriasis is related to family inheritance, infection, immune abnormalities, endocrine factors, etc. There is evidence that the imbalance of immune cells, especially T cells, plays a key role in the pathogenesis of psoriasis. The pathological features are mainly characterized by parakeratosis, hyperkeratosis, abnormal proliferation, vascularization and inflammatory cell infiltration, which has a great impact on the physical health and mental state of patients, and is a great challenge to public health in China. Although the current treatment measures are effective, they cannot achieve long-term remission.

[0003] Psoriasis is one of the diseases that are currently focused on in the field of dermatology, and the development of new drugs is particularly important, and the psoriasis animal model is an important tool for the study of the pathogenesis of psoriasis and the development of new drugs. At present, the models used to study psoriasis include spontaneous mouse models, drug-induced models (such as topical skin application of imiquimod to induce psoriasis in mice), genetically engineered mouse models, and allogeneic transplantation mouse models.

[0004] Although there are many animal models of psoriasis, they all have shortcomings in fully simulating the characteristics of psoriasis:

[0005] ① Although the spontaneous mouse model can exhibit psoriasis skin manifestations such as acanthosis and increased dermal vascular dilation in a relatively short period of time, it rarely exhibits T cell and neutrophil infiltration, and cannot reflect all the characteristics of psoriasis, and it lacks response to psoriasis treatment drugs, so this type of model is only suitable for the study of anti-proliferative drugs, and is not suitable for the study of the pathogenesis of psoriasis and new drugs;

[0006] ② The genetically engineered mouse model is constructed from psoriasis-susceptible genes, which is of great help to the study of the pathogenesis of psoriasis, but the manifestation of psoriasis lesions is greatly affected by post-transcriptional and post-translational factors, and the consistency of the lesion model varies, thereby limiting the study of the progression and treatment of psoriasis;

[0007] The third method is to transplant the skin of a psoriasis patient into an immunodeficient mouse, and the animal model can simulate the complex immunological and histopathological changes of psoriasis, and is a suitable tool for researching the pathogenesis of T cells of psoriasis and drug development, but the mouse is susceptible to infection due to the serious deficiency of immune function, and the breeding cost is high, so the promotion and application of the model also have certain limitations.

[0008] The construction time of the drug-induced model needs to be further shortened, and the effect needs to be further improved. SUMMARY

[0009] In view of the problems in the prior art, one of the purposes of the present application is to construct a method for constructing a mouse model for psoriasis, which can comprehensively reflect the changes in the microenvironment of a psoriasis patient and is more consistent with human psoriasis in skin appearance and inflammatory response.

[0010] To achieve this purpose, the following technical solutions are used in the present application:

[0011] A mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyryl modification, comprising:

[0012] Step 1: Constructing a plasmid comprising a SBSN gene sequence, and the SBSN sequence 600 sites are mutated from lysine (K) to threonine (T);

[0013] Step 2, mouse embryonic stem cell culture;

[0014] Step 3, embryonic stem cell electroporation, the plasmid obtained in step 1 is electroporated into the mouse embryonic stem cells cultured in step 2;

[0015] Step 4, drug screening, the embryonic stem cells electroporated in step 3 are screened by drug screening to obtain embryonic stem cells containing a DNA fragment in which the SBSN sequence 600 sites are mutated from lysine (K) to threonine (T);

[0016] Step 5, the embryonic stem cells screened in step 4 are taken and cultured;

[0017] Step 6, the embryonic stem cells taken and cultured in step 4 are injected into mouse blastocysts, and then the mouse blastocysts are cultured and transplanted into the uterus of a pseudopregnant female mouse, and the mouse delivered by the female mouse is a chimeric mouse;

[0018] Step 7, breeding of the chimeric mouse and PCR identification of the genotype of the mouse: selecting a male mouse with a chimeric rate of more than 50% to mate with a C57BL / 6J inbred mouse to obtain the aforementioned gray mouse derived from the stem cells, and using a PCR method for identification, and the mouse with a positive result is an F1 positive heterozygote mouse, which is an SBSNK600T Point mutation mouse, the mouse with negative result is wild mouse, that is SBSN WT Mouse.

[0019] The mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification further comprises the following steps:

[0020] Step 8: The SBSN 600 lysine dihydroxyisobutyrylation modification of the mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification is further included. K600T The point mutation mouse is induced by imiquimod to obtain a mouse model of psoriasis.

[0021] The mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification,

[0022] The first step of the construction comprises the SBSN gene sequence, and the SBSN sequence 600 point position is mutated from lysine (K) to threonine (T) DNA fragment plasmid; further comprising:

[0023] ① Fragment PCR amplification: two DNA fragments corresponding to the homologous arms of the DNA fragments amplified from the C57BL / 6J mouse genome about 100bp above and below the SBSN mutation site 600 are amplified by PCR;

[0024] ② Fragment recovery: the PCR products obtained in step ① are subjected to gel electrophoresis recovery;

[0025] ③ Ligation transformation: the amplified fragments are ligated into the linearized vector pBR322 plasmid;

[0026] ④ Clone identification: after transformation, single colonies are picked and cultured, and plasmid is extracted by small extraction, and the plasmid is subjected to enzyme digestion verification to confirm the correct cloned plasmid;

[0027] ⑤ Sequencing verification: after identifying the correct positive clone, further sequencing verification is performed;

[0028] ⑥ Plasmid extraction: the amount of plasmid is expanded to obtain sufficient high-purity plasmid;

[0029] ⑦ Plasmid linearization: using NotI endonuclease treatment, the circular plasmid is de-circularized into linear plasmid, and after enzyme reaction, DNA is recovered by ethanol precipitation, thereby obtaining linearized vector pBR322 plasmid connected with SBSN sequence 600 point position mutated from lysine (K) to threonine (T) DNA.

[0030] The mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification further comprises the following steps:

[0031] The resistance gene fragment is connected to the 600th lysine of the SBSN sequence in the linearized vector pBR322 plasmid by homologous recombination.

[0032] The method for constructing the mouse model for psoriasis research based on the dihydroxyisobutyrylation modification of the 600th lysine of the SBSN further comprises the following steps:

[0033] The resistance gene fragment is a positive selection resistance gene and / or a negative selection resistance gene, the positive selection refers to a selection that can survive under drug conditions, and the negative selection refers to a selection that cannot survive under drug conditions.

[0034] The method for constructing the mouse model for psoriasis research based on the dihydroxyisobutyrylation modification of the 600th lysine of the SBSN,

[0035] The second step of culturing the mouse embryonic stem cells further comprises the following steps:

[0036] The complete culture medium of the embryonic stem cells comprises 10-6 mol / L β-mercaptoethanol, 2 mM glutamine, 0.1 mM non-essential amino acids, 100 U / ml penicillin, 50 mg / L streptomycin, 15% ES fetal bovine serum, and 1000 U / ml LIF.

[0037] The method for constructing the mouse model for psoriasis research based on the dihydroxyisobutyrylation modification of the 600th lysine of the SBSN,

[0038] The third step of electrically transforming the embryonic stem cells further comprises the following steps:

[0039] The embryonic stem cells in the logarithmic growth phase are digested with 0.125% trypsin-EDTA and counted, and an appropriate amount of PBS is added to make the cell density reach about 1.5×10 7 / m1, 0.8 ml of the above embryonic stem cell suspension is taken, about 35 μg of the plasmid obtained in the first step is added, and the mixture is transferred to a sterile electric transformation cup for electric transformation at an electric parameter of 250 V and 500 μF.

[0040] The method for constructing the mouse model for psoriasis research based on the dihydroxyisobutyrylation modification of the 600th lysine of the SBSN,

[0041] Fourth step, drug screening, using drug screening on the third step of the electric transfer of embryonic stem cells, screening out the embryonic stem cells containing the DNA fragment of SBSN sequence 600 sites lysine (K) mutation into threonine (T), further comprising: the drug screening is positive and negative drug screening, the third obtained embryonic stem cells are replaced with culture solution containing selection drugs geneticin G418 and ganciclovir after 24h of electric transfer, and are selectively cultured, the culture solution is replaced every day, and after 7-8 days of selective culture, the resistant embryonic stem cells grow into visible clones, and then the clones are picked.

[0042] The method for constructing the mouse model for psoriasis research based on the dihydroxyisobutyrylation modification of lysine at the 600th site of SBSN,

[0043] The eighth step is to obtain SBSN K600T The point mutation mouse is induced by imiquimod to obtain a psoriasis-like mouse model, and the method further comprises the following steps:

[0044] Meanwhile, the SBSN WT Mouse is induced by imiquimod.

[0045] The method for constructing the mouse model for psoriasis research based on the dihydroxyisobutyrylation modification of lysine at the 600th site of SBSN, further comprises the following steps:

[0046] After 5 days of imiquimod induction, the SBSN K600T Point mutation mouse and the SBSN WT Mouse are anesthetized to death, and local skin lesion sites are identified:

[0047] After the sample is fixed, paraffin sections are prepared, the paraffin sample is subjected to HE staining, and the skin histopathology is observed under a microscope, and it can be seen that the SBSN WT Mouse has a thinner epidermal layer after imiquimod induction, while the SBSN K600T Point mutation mouse has a significantly thickened epidermal layer, incomplete keratinization and hyperkeratosis, increased number of prickle cell layers, and downward extension of epidermal processes.

[0048] Compared with the prior art, the present application has at least the following beneficial effects:

[0049] ① The present application constructs a psoriasis mouse model from the post-translational modification level, avoids the influence of transcription, translation and other factors on the mouse phenotype, to a certain extent, reduces the influence of non-experimental factors on the results, and ensures the comparability of the results;

[0050] ② The present application first constructs a psoriasis mouse model from dihydroxyisobutyrylation, which plays an important role in developing dihydroxyisobutyrylation modification of psoriasis;

[0051] ③ The modeling cycle of the present invention is short, and obvious psoriasis skin manifestations can appear on the 5th day of imiquimod induction, while the modeling cycle reported in the literature previously lasted about 2 weeks;

[0052] ④ The skin lesions and inflammatory cell infiltration of the mouse model constructed by the present invention are more severe, which is most consistent with the characteristics of skin lesions in psoriasis patients and meets the requirements for animal models in the primary stage of psoriasis research. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 :SBSN K600T Schematic diagram of the strategy for constructing point mutation mice.

[0054] Reference numerals: 1001: wild type allele; 1002: exon 1; 1003: exon 4; 1004: targeting vector.

[0055] 1005:5 ， -Homology arm (5 ， -homology arm); 1006: Frt; 1007: pGK-Neo-polyA;

[0056] 1008:3 ， -Homology arm (3 ， -homology arm);

[0057] 1009: MCI-TK-polyA; 1010: Targeted allele;

[0058] 1011: Targeted allele (Neo Removed);

[0059] *: Mutation.

[0060] Figure 2 : Schematic diagram of the F1 generation mouse construction strategy.

[0061] Reference numerals: 2001: wild type allele; 2002: exon 1; 2003: exon 4;

[0062] 2004; Targeted allele (Neo Removed);

[0063] 2005:5， - homology arm (5 ， - homology arm); 2006: Frt;

[0064] 2007: 3 ， - homology arm (3 ， - homology arm); *: mutation;

[0065] →: primer location; -: PCR product.

[0066] Figure 3-1 , Figure 3-2 , Figure 3-3 : F1 generation mouse 5' homology arm and 3' homology arm PCR identification electrophoresis map.

[0067] Figure 4 : F1 generation mouse PCR product sequencing location verification schematic.

[0068] Figure legend: 3001: Wild type allele; 3002: exon 1; 3003: exon 4;

[0069] 3004; Targeted allele (Neo Removed);

[0070] 3005: 5 ， - homology arm (5 ， - homology arm); 3006: Frt;

[0071] 3007: 3 ， - homology arm (3 ， - homology arm); *: mutation;

[0072] →: primer location; -: PCR product.

[0073] -: PCR sequenced region.

[0074] Figure 5-1 SBSN WT Wild type mouse skin on the first day of imiquimod (IMQ) treatment;

[0075] Figure 5-2 SBSN K600TThe skin of the point mutation mouse on the first day of IMQ treatment shows;

[0076] Figure 5-3 SBSN WT The skin of the wild type mouse on the fifth day of IMQ treatment shows;

[0077] Figure 5-4 SBSN K600T The skin of the point mutation mouse on the fifth day of IMQ treatment shows.

[0078] Figure 6-1 SBSN WT The skin of the wild type mouse on the fifth day of IMQ treatment shows; Figure 6-2 SBSN K600T The skin of the point mutation mouse on the fifth day of IMQ treatment shows.

[0079] Figure 7 Part of the flow chart of the psoriasis mouse constructed by the present application;

[0080] The figure mark: 7001: the first step, the gene targeting of ES cell; 7002: the separation and culture of ES cell;

[0081] 7003: ES cell; 7004: electric conversion; 7005: a small amount of cell carrying the middle target gene; 7006: the middle target gene;

[0082] 7007: the construction of the targeting vector; 7008: the vector; 7009: the inserted DNA fragment; 7010: pGK-Neo-polyA;

[0083] 7011: MCI-TK-polyA; 7012: 5 ， -homology arm (5 ， -homology arm);

[0084] 7013: 3 ， -homology arm (3 ， -homology arm); 7014: ES cell electric conversion; 7015: the target gene;

[0085] 7016: homologous recombination; 7017: the middle target gene; 7018: drug screening; 7019: the middle target ES cell expansion.

[0086] Figure 8 Part of the flow chart of the psoriasis mouse constructed by the present application;

[0087] 8001: gene knock-in mouse; 8002: ES cell blastocyst cavity injection;

[0088] 8003: injection of target ES cells into the blastocyst cavity of a mouse; 8004: transplantation of the injected blastocyst into the uterus of a pseudopregnant mouse;

[0089] 8005: chimeric inner cell mass; 8006: birth and breeding of chimeric mice; 8007: birth of chimeric mice;

[0090] 8008: mating of male chimeric mice with wild-type mice to obtain gene knock-in mice; 8009: chimeric mice;

[0091] 8010: wild-type mice; 8011: gene knock-in mice. DETAILED DESCRIPTION

[0092] The technical solutions of the present application will be further described below in combination with the drawings and through specific embodiments.

[0093] To better illustrate the present application and facilitate understanding of the technical solutions of the present application, the specific embodiments of the present application are as follows:

[0094] The inventors of the present patent have found that the dihydroxyisobutyryl modification of the amino acid at position 510 of SBSN is up-regulated in the skin lesions of psoriasis patients, and that changes in the level of this modification can promote the proliferation of keratinocytes and inhibit the apoptosis of keratinocytes, which is consistent with the clinical characteristics of psoriasis. At the same time, the lysine at position 510 of human SBSN corresponds to the lysine at position 600 of C57BL / 6J mice.

[0095] Based on the research results, to solve the problems existing in the prior art, the inventors of the present patent obtained a heterozygous mouse with lysine (K) at position 600 of SBSN gene mutated to threonine (T) through genetic engineering technology, and then successfully constructed a psoriasis mouse model by combining local external use of imiquimod induction. The model of the present application has a shorter modeling time, more severe lesion and inflammatory cell infiltration, and is more consistent with the characteristics of psoriasis in skin pathology.

[0096] The main inventive concept of the present application is to use the principle of homologous recombination and the method of embryonic stem cell (ESC) targeting to knock in K600T expression frame at the Exon 1 site of SBSN gene, i.e. to mutate lysine (K) at position 600 of SBSN protein to threonine (T), aiming to simulate the high dihydroxyisobutyryl modification of lysine at position 510 of human SBSN, so as to obtain a heterozygous mouse with point mutation. K600T

[0097] ​Briefly, the ES cell targeting vector was constructed by the method of Infusion, which contains 2.9 kb 5' homology arm, K600T (KI element), PGK-Neo-polyA, 3.0 kb 3' homology arm and MC1-TK-polyA negative selection marker. The vector was linearized and transfected into cells. After long fragment PCR identification, a total of 1 positive clone with correct homologous recombination was obtained. After amplification of the positive ES cell clone, it was injected into the blastocyst of C57BL / 6J mouse to obtain a chimeric mouse. The chimeric mouse with a high proportion was mated with Flp mouse to obtain 6 positive F1 generation Neo heterozygous mice. The construction strategy is shown in Figure 1 The mouse model for psoriasis research was constructed based on the dihydroxyisobutyrylation modification of SBSN 600 lysine, and then the mouse model was further induced by topical application of imiquimod ointment.

[0098] A mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification, comprising:

[0099] Step 1: Construct a plasmid containing a DNA fragment of SBSN gene sequence, and the 600th position of the SBSN sequence is mutated from lysine (K) to threonine (T);

[0100] Step 2: Culture mouse embryonic stem cells;

[0101] Step 3: Electroporation of embryonic stem cells, the plasmid obtained in step 1 is electroporated into the mouse embryonic stem cells cultured in step 2;

[0102] Step 4: Drug screening, the embryonic stem cells electroporated in step 3 are screened by drug screening to obtain embryonic stem cells containing a DNA fragment of SBSN sequence with the 600th position mutated from lysine (K) to threonine (T);

[0103] Step 5: Culture of the embryonic stem cells screened in step 4;

[0104] Step 6: Inject the embryonic stem cells cultured in step 4 into mouse blastocysts, then culture the mouse blastocysts, and transplant the cultured blastocysts into the uterus of a pseudopregnant female mouse, and the mouse delivered by the female mouse is a chimeric mouse;

[0105] Step 7: Chimeric mouse breeding and PCR identification of mouse genotype: select a male mouse with a chimeric rate of more than 50% and mate it with a C57BL / 6J inbred mouse to obtain a gray mouse derived from the aforementioned stem cells. The result is positive for the F1 generation positive heterozygous mouse, i.e. the SBSN K600T point mutation mouse, and the result is negative for the wild type mouse, i.e. the SBSN WT mouse.

[0106] The mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification further comprises the following steps:

[0107] The eighth step is to obtain a psoriasis-like mouse model by inducing imiquimod on the SBSNK600T point mutation mouse obtained in the seventh step.

[0108] The mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification further comprises the following steps:

[0109] The first step comprises a plasmid containing the SBSN gene sequence, and the DNA fragment of the SBSN sequence at position 600 is mutated from lysine (K) to threonine (T); further comprising:

[0110] ① Fragment PCR amplification: PCR amplification of two homologous arm DNA fragments corresponding to the DNA fragments of about 100 bp above and below the SBSN mutation site 600 from the C57BL / 6J mouse genome;

[0111] ② Fragment recovery: The PCR products obtained in step ① are subjected to gel electrophoresis recovery;

[0112] ③ Ligation and transformation: ligate the amplified fragments into the linearized vector pBR322 plasmid;

[0113] ④ Clone identification: After transformation, single colonies are picked and cultured, and plasmid is extracted by small extraction, and the plasmid is subjected to enzyme digestion to verify the correct clone plasmid;

[0114] ⑤ Sequencing verification: After identifying the correct positive clone, further sequencing verification is performed;

[0115] ⑥ Plasmid extraction: Expand the plasmid amount to obtain sufficient high-purity plasmid;

[0116] ⑦ Plasmid linearization: Use NotI endonuclease to treat, so that the circular plasmid is de-circularized into linear plasmid, and after the enzyme reaction is completed, the DNA is recovered by ethanol precipitation, thereby obtaining the linearized vector pBR322 plasmid connected with the DNA of SBSN sequence at position 600 mutated from lysine (K) to threonine (T).

[0117] The mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification further comprises the following steps:

[0118] In the linearized vector pBR322 plasmid connected with the DNA of SBSN sequence at position 600 mutated from lysine (K) to threonine (T), the resistance gene fragment is connected by homologous recombination.

[0119] The mouse model construction method for psoriasis research based on SBSN 600 lysine dihydroxyisobutyryl modification further comprises:

[0120] The resistance gene fragment is a positive selection resistance gene and / or a negative selection resistance gene, and the positive selection refers to a selection that can survive under drug conditions, and the negative selection refers to a selection that cannot survive under drug conditions.

[0121] The mouse model construction method for psoriasis research based on SBSN 600 lysine dihydroxyisobutyryl modification,

[0122] The second step of mouse embryonic stem cell culture further comprises:

[0123] The complete culture medium of the embryonic stem cells comprises 10-6 mol / L beta-mercaptoethanol, 2 mM glutamine, 0.1 mM non-essential amino acids, 100 U / ml penicillin, 50 mg / L streptomycin, 15% ES fetal bovine serum, and 1000 U / ml LIF.

[0124] The mouse model construction method for psoriasis research based on SBSN 600 lysine dihydroxyisobutyryl modification,

[0125] The third step of the embryonic stem cell electroporation further comprises:

[0126] The embryonic stem cells in the logarithmic growth phase are digested with 0.125% trypsin-EDTA and counted, and an appropriate amount of PBS is added to make the cell density reach about 1.5x107 / ml. 0.8 ml of the above embryonic stem cell suspension is taken, about 35 ug of the plasmid obtained in the first step is added, and after mixing, it is transferred to a sterile electroporation cup for electroporation at an electric parameter of 250V and 500uF.

[0127] The mouse model construction method for psoriasis research based on SBSN 600 lysine dihydroxyisobutyryl modification,

[0128] The fourth step of drug screening further comprises: the drug screening is positive and negative drug screening, and the embryonic stem cells obtained in the third step are cultured in a culture solution containing the selection drugs geneticin G418 and ganciclovir after 24 hours of electroporation for selective culture. The culture solution is replaced every day, and after 7-8 days of selective culture, the resistant embryonic stem cells grow into visible clones, which can be picked.

[0129] The method for constructing a mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification,

[0130] Step eight: obtain a psoriasis-like mouse model by imiquimod induction on the SBSN K600T point mutation mouse obtained in step seven, further comprising:

[0131] Meanwhile, the SBSN WT mouse is induced by imiquimod.

[0132] The method for constructing a mouse model for psoriasis research based on SBSN 600 lysine dihydroxyisobutyrylation modification, further comprises:

[0133] After 5 days of imiquimod induction, the SBSN K600T point mutation mouse and the SBSN WT mouse are narcotized to death to identify the local skin lesion site:

[0134] After fixing the specimen, paraffin sections are prepared, the paraffin specimen is stained by HE, and the skin histopathology changes are observed under a microscope, and it can be seen that the epidermal layer of the SBSN WT mouse after imiquimod induction is thinner, while the epidermal layer of the SBSN K600T point mutation mouse is significantly thickened, and incomplete keratinization and hyperkeratosis can be seen, the number of prickle cell layers is increased, and the epidermal process extends downward.

[0135] As a preferred embodiment, the following further detailed description of the implementation of the present application has a high success rate and a more preferred technical effect. Those skilled in the art should know that the following embodiments are not the only embodiments of the present application, but are only further exemplary lists for the purpose of understanding the concept of the present application.

[0136] The specific embodiments of the present patent are further described in detail as follows:

[0137] Basic information of the mutant gene:

[0138] The name of the mutant gene in the method of the present application (Ensembl number) is SBSN (ENSMUSG00000046056).

[0139] Ensembl website link of the target gene for mutation:

[0140] http: / / www.ensembl.org / Mus_musculus / Gene / Summary?db=core;g=ENSMUSG00000046056;r=7:30450958-30455556;t=ENSMUST00000080518 Figure 2

[0141] Transcript targeted by the scheme (Ensembl number): ENSMUST00000080518.14

[0142] Exon targeted by the site-directed knock-in: exon 1

[0143] First step, plasmid construction process

[0144] That is: design a primer sequence containing a SBSN 600 site mutation, use C57BL / 6J mouse genomic DNA as a template, and amplify a homologous arm DNA fragment of about 100bp upstream and downstream of the mutation site.

[0145] Original backbone plasmid name: pBR322

[0146] ①Fragment amplification: two DNA fragments corresponding to the homologous arms of the SBSN mutation site 600bp upstream and downstream are amplified from the C57BL / 6J mouse genome.

[0147] ②Fragment recovery: The PCR products obtained in step ① were subjected to gel electrophoresis recovery and purified using an Omega gel purification kit, and the concentration was determined.

[0148] ③Ligation and transformation: The amplified fragments were ligated into linearized vector pBR322 plasmid, using Takara Infusion ligase, and competent bacterial strain: DH5α. The transformed strain was smeared onto ampicillin-resistant plates and incubated at 37°C overnight.

[0149] The ligase can be selected from: manufacturer: Takara, product name: HD Cloning Plus

[0150] Catalog No.: 638910, website: https: / / www.takarabiomed.com.cn /

[0151] ④Clone identification: After transformation, single colonies were picked and cultured, and plasmid was extracted by small plasmid extraction, and the plasmid was digested to verify the correct cloned plasmid.

[0152] ⑤Sequencing verification: After identifying the correct positive clone, the bacterial solution or plasmid was sent to a sequencing company for sanger sequencing verification (Shengong).

[0153] ⑥Plasmid extraction: This step is to expand the amount of plasmid and obtain sufficient high-purity plasmid.

[0154] ⑦Plasmid linearization: Use NotI endonuclease to treat, so that the circular plasmid is de-circularized into linear plasmid. After the enzyme reaction is completed, the DNA is recovered by ethanol precipitation, thereby obtaining linearized vector pBR322 plasmid containing SBSN sequence 600 point mutation from lysine (K) to threonine (T) DNA.

[0155] Preferably, for the convenience of plasmid screening, the DNA further comprises a resistance gene fragment. The resistance gene fragment is also cloned into the pBR322 plasmid by homologous recombination, i.e. the final NotI linearized pBR322-MK-SBSN-K600T plasmid DNA is obtained.

[0156] The pBR322-MK-SBSN-K600T plasmid refers to a linearized vector pBR322 plasmid connected with a SBSN sequence 600 point DNA fragment mutated from lysine (K) to threonine (T) and a resistance screening marker MK, preferably the resistance screening marker MK is a positive resistance screening marker or a positive and negative resistance screening marker, or a positive and negative resistance screening marker. The positive and negative resistance screening marker preferably comprises two resistances PGK-Neo-polyA and MCI-TK-polyA, which are promoter-resistance-terminator, respectively. Neo refers to the resistance gene of neomycin, PGK-Neo is a positive screening resistance gene, and MC1-TK is a negative screening resistance gene.

[0157] Positive and negative drug screening: positive screening refers to screening that can survive under drug conditions, and negative screening refers to screening that cannot survive under drug conditions.

[0158] The pBR322-MK-SBSN-K600T plasmid vector preferably comprises a 2.9 kb 5' homologous arm, a K600T (KI element), a PGK-Neo-polyA, a 3.0 kb 3' homologous arm, and a MC1-TK-polyA negative screening marker.

[0159] (Molecular biology experiments are standard experiments, and detailed steps can be referred to Molecular Cloning Experiment Guide, ISBN 9787030386069. Commercial kits are involved, and kit instructions can be referred to.)

[0160] Primers, sequencing, etc. are all from Shenguo Bioengineering (Shanghai) Co., Ltd.

[0161] Amplification enzyme:

[0162] Manufacturer: TOYOBO Product name: KOD-Multi & Epi

[0163] Part number: KME-101

[0164] Website: http: / / www.bio-toyobo.cn /

[0165] Ligase:

[0166] Manufacturer: Takara

[0167] Product name: HD Cloning Plus

[0168] Item No. 638910

[0169] Website: https: / / www.takarabiomed.com.cn /

[0170] Endonuclease:

[0171] Manufacturer: NEB

[0172] Product Name: NotI-HF

[0173] Item No. R3189L

[0174] Website: http: / / www.neb-china.com /

[0175] Gel recovery kit:

[0176] Manufacturer: Omega

[0177] Product Name: DNA Fragmentation Gel Recovery Kit

[0178] Item No. D2500-01

[0179] Plasmid Mini Kit

[0180] Manufacturer: Omega

[0181] Product Name: Plasmid Mini Kit II (50)

[0182] Item No. D6945-01

[0183] Plasmid Midi Kit Product Information:

[0184] Manufacturer: MACHEREY-NAGEL

[0185] Product Name: Endotoxin-free plasmid DNA purification

[0186] Item No. NucleoBond Xtra Midi EF.

[0187] Second step, embryonic stem cell culture: after the culture dish was coated with 0.1% gelatin, the mitomycin C treated feeder cells (2x106 feeder cells were inoculated per 10 cm dish) were inoculated, and after overnight culture, the embryonic stem cells were inoculated. The complete culture medium of embryonic stem cells was DMEM containing 10-6 mol / L β-mercaptoethanol, 2 mM glutamine, 0.1 mM non-essential amino acids, 100 U / ml penicillin, 50 mg / L streptomycin, 15% ES fetal bovine serum, 1000 U / ml LIF.

[0188] Third step, ES cell electroporation: ES cells in logarithmic growth phase were digested with 0.125% trypsin-EDTA and counted, and then added with PBS to make the cell density about 1.5 x 10 7 7 cells per milliliter). 0.8 ml of the above ES cell suspension was taken and added with about 35 μg of pBR322-MK-Sbsn-K600T plasmid DNA linearized with Notl, mixed and then transferred to a sterile electroporation cup for electroporation at 250 V and 500 μF. After resuspension, the mixture was evenly distributed into three 10 cm petri dishes coated with feeder cells.

[0189] Fourth step, positive and negative drug screening: after 24 h of electroporation, the ES cells were cultured in a medium containing the selection drug geneticin G418 (final concentration 250 mg / L) and ganciclovir (final concentration 2 μmol / L). The medium was changed every day, and after 7-8 days of selective culture, the resistant ES cells grew into visible clones which were then picked up.

[0190] Fifth step, picking up of double-resistant cell clones and culture: the resistant clones were picked up and digested in a 96-well plate (concave bottom) containing 30 μl of 0.1% trypsin-EDTA for about 3 min, and then gently blown to disperse the cells, which were then transferred to a 96-well culture plate for culture. When the cells grew to 60%-80%, they were subcultured into two 96-well plates at a ratio of 1:2, and one part was frozen and the other part was used for extraction of genomic DNA.

[0191] Sixth step, blastocyst injection of ES cells and embryo transplantation: the injection was performed in DMEM complete medium without LIF, and about 15 ES cells were injected into each blastocyst. After injection, the blastocysts were cultured in DMEM complete medium without LIF at 37°C and 5% CO2 for about 1 h, and then transplanted into the uterus of 2.5-day pseudopregnant female mice at 8-10 per side. The pseudopregnant female mice were bred in the SPF animal room of Shanghai South Model Organisms Center, and the offspring were chimeric mice.

[0192] Seventh step, breeding of chimeric mice and PCR identification of mouse genotype: male mice with a chimeric rate of more than 50% were selected and mated with C57BL / 6J inbred mice to obtain ES cell-derived gray mice. The positive heterozygous mice were identified by PCR. The positive F1 generation positive heterozygous mice were SBSN K600T point mutation mice, and the F1 generation negative mice were SBSN WT ​Wild type mice.

[0193] F1 generation mice identification strategy as shown in Figure 4

[0194] Cut 0.5 cm from the end of mouse tail, then extract DNA, and perform PCR identification according to the following method:

[0195] F1 generation mice 5' homologous arm identification method:

[0196] Primer information: Forward 5'-TTCCCCACCTACTGGCTCTT-3';

[0197] Reverse 5'-AGCTTAAGGCAATGCGGTCT-3'

[0198] PCR reaction body series is listed in Table 1 below.

[0199] Table 1

[0200]

[0201] KOD-Multi & Epi-*(TOYOBO, Code No: KME-101) PCR reaction conditions are listed in Table 2 below.

[0202] Table 2

[0203]

[0204] F1 generation mice 3' homologous arm identification method:

[0205] Primer information: Forward 5'-GAGTGGTCCAGTTAGCCACC-3';

[0206] Reverse 5'-TGCTGCAAAATCTTTTCATCCTCTT-3' PCR reaction body series is listed in Table 3 below.

[0207] Table 3

[0208]

[0209]

[0210] KOD-Multi & Epi-*(TOYOBO, Code No: KME-101)

[0211] PCR reaction conditions:

[0212]

[0213] ​PCR identification scheme of homologous recombination positive mice:

[0214] 5' arm homologous recombination positive mice should amplify a 4.1 kb fragment, wild type mice should amplify a 7.9 kb fragment; 3' arm homologous recombination positive mice should amplify a 3.6 kb fragment, wild type mice should amplify a 7.8 kb fragment.

[0215] The electrophoresis results of PCR identification of F1 generation mice 5' and 3' homologous arm are shown in Figure 3. Sequencing confirms that they are all positive.

[0216] Sequencing alignment results of PCR identification of F1 generation mice:

[0217] Sequencing of PCR identification products of F1 generation positive mice, a total of 4 sequencing reactions were performed. The corresponding regions of the sequencing reactions are shown in Figure 5-1 to Figure 5-4 . Among them, 2 sequencing reactions were performed for PCR product sequencing of 5' homologous arm identification, marked as: 1, 2; 2 sequencing reactions were performed for PCR product sequencing of 3' homologous arm identification, marked as: 3, 4.

[0218] Step 8, SBSN K600T point mutant mice and SBSN WT Mice were induced by imiquimod (IMQ) to obtain a psoriasis-like mouse model.

[0219] The specific induction method is: SBSN K600T point mutant mice and SBSN WT Wild type mice were depilated on the back, with an area of 2 cm*2 cm, and 5% imiquimod cream 62.5 mg was applied daily on the back of the mice for 5 days. Obvious psoriasis skin lesion performance appeared on the depilated part of the back, and the skin lesion changes during modeling are shown in Figure 5-3 . There was no obvious change in the depilated part of the back two days before modeling, and SBSN K600T point mutant heterozygote mice had a slight redness of the skin, while wild type mice had no change; on the 4th day, mutant mice had obvious erythema, and some skin lesions were covered with scales, and the skin lesions were moderately raised, while wild type mice had a slight redness of the skin; as shown in Figure 5-3 and 5-4 , on the 5th day, compared with wild type mice (see Figure 5-4 ), mutant mice (see Figure 6-1 ) had deepened erythema, thick layer of scales on the surface, and significantly thickened skin lesions, which were consistent with psoriasis-like changes.

[0220] To further verify the similarity of the skin lesions of mice in histopathology to psoriasis lesions, HE staining was used to identify the skin lesions of mice. After 5 days of imiquimod induction, SBSN K600T point mutant mice and SBSNWT The mice were anesthetized to death to identify the local skin lesion site:

[0221] After fixing the specimen, paraffin sections were prepared, and the paraffin specimen was subjected to HE staining, and the skin histopathology, especially the epidermis thickness, was observed under a microscope. The results are shown in Figure 5-1 As shown in Fig. 6-4, it can be seen that the epidermis of the wild-type mice is thinner after being induced by imiquimod, while the epidermis of the mutant mice is significantly thicker, and incomplete keratinization and hyperkeratosis can be seen, and the number of prickle cell layers is increased, and the epidermis extends downward, which has a high similarity with the histopathological changes of the skin lesions of psoriasis patients.

[0222] Figure 5-2 SBSN WT The skin of the wild-type mice on the first day of imiquimod (IMQ) treatment showed that Figure 5-3 SBSN K600T The skin of the point mutation mice on the first day of IMQ treatment showed that Figure 5-4 SBSN WT The skin of the wild-type mice on the fifth day of IMQ treatment showed that Figure 6-1 SBSN K600T The skin of the point mutation mice on the fifth day of IMQ treatment showed that

[0223] Figure 6-2 SBSN WT The skin of the wild-type mice on the fifth day of IMQ treatment showed that Figure 7 SBSN K600T The skin of the point mutation mice on the fifth day of IMQ treatment showed that HE staining showed that the epidermis of the wild-type mice was thinner after IMQ treatment, while the epidermis of the mutant mice was significantly thicker, and incomplete keratinization and hyperkeratosis could be seen, and the number of prickle cell layers was increased, and the epidermis extended downward.

[0224] ​ 8: Partial flow chart of construction of psoriasis mouse model according to the present application.

[0225] Based on the previous research results, the present application is the first to construct a psoriasis mouse animal model from the level of protein dihydroxyisobutyryl modification combined with imiquimod stimulation.

[0226] Compared with the previously reported methods for constructing a psoriasis mouse model, the present application has the following advantages:

[0227] ① The present technology constructs a psoriasis mouse model from the level of post-translational modification, avoiding the influence of transcription, translation and other factors on the mouse phenotype, to a certain extent, reducing the influence of non-experimental factors on the results, and ensuring the comparability of the results;

[0228] The technology is first to construct a psoriasis mouse model from dihydroxy isobutyryl, and has important role in developing dihydroxy isobutyryl modification of psoriasis;

[0229] The technology has short modeling cycle, and obvious psoriasis skin performance appears on the 5th day of imiquimod induction, while the modeling cycle of the previously reported literature lasts for about 2 weeks.

[0230] The mouse model constructed by the technology has more serious skin lesion and inflammatory cell infiltration, is most consistent with the skin lesion characteristics of psoriasis patients, and meets the requirements of animal models in the primary stage of psoriasis research.

[0231] The applicant declares that the detailed process of the present application is illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed process, that is, it does not mean that the present application must rely on the above-mentioned detailed process to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of the selected method, step, etc. of the present application, and increase of auxiliary steps, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0232] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0233] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

[0234] In addition, various different embodiments of the present application can also be combined in any manner, as long as it does not deviate from the idea of the present application, and it should be considered as disclosed content of the present application.

Claims

1. A method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation of lysine 600 in SBSN, comprising: Step 1: Construct a plasmid containing the SBSN gene sequence and a DNA fragment in which position 600 of the SBSN sequence is mutated from lysine (K) to threonine (T); The second step is to culture mouse embryonic stem cells; The third step is embryonic stem cell electroporation, where the plasmid obtained in the first step is electroporated into the mouse embryonic stem cells cultured in the second step; The fourth step is drug screening, in which the embryonic stem cells electroporated in the third step are screened for embryonic stem cells containing a DNA fragment in which position 600 of the SBSN sequence is mutated from lysine (K) to threonine (T); Step 5: The embryonic stem cells screened in step 4 are retrieved and cultured; Step 6: injecting the cultured embryonic stem cells obtained in Step 4 into mouse blastocysts, culturing the mouse blastocysts, and transplanting the cultured blastocysts into the uterus of pseudo-pregnant female mice. The mice born from the female mice are chimeric mice. Step 7: Breeding chimeric mice and identifying mouse genotypes by PCR: Select male mice with a coat color chimerism rate greater than 50% and mate them with purebred C57BL / 6J mice to obtain the aforementioned stem cell-derived gray mice. Mice with positive results are identified by PCR, and mice with positive results are F1 generation positive heterozygous mice, i.e., SBSN. K600T Point mutation mice, mice with negative results are wild mice, that is, SBSN WT mice; Step 8: SBSN obtained in step 7 K600T Imiquimod was used to induce psoriasis in point mutation mice.

2. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 in SBSN according to claim 1, The first step comprises constructing a plasmid comprising a DNA fragment containing the SBSN gene sequence and a mutation at position 600 of the SBSN sequence from lysine (K) to threonine (T); further comprising: ① Fragment PCR amplification: Two DNA fragments corresponding to the DNA fragments of about 100 bp above and below the SBSN mutation site were amplified from the C57BL / 6J mouse genome by PCR; ② Fragment recovery: The PCR product obtained in step ① is recovered by gel electrophoresis; ③ Ligation and transformation: ligate the amplified fragment into the linearized vector pBR322 plasmid; ④ Clone identification: After transformation, single clones were picked for culture, plasmids were extracted by microextraction, and enzyme digestion was performed to confirm the correct cloned plasmids; ⑤ Sequencing verification: After the correct positive clone is identified, further sequencing verification is performed; ⑥ Plasmid extraction: expand the amount of plasmid and obtain sufficient high-purity plasmid; ⑦ Plasmid linearization: Use NotI endonuclease to decircle the circular plasmid into a linear plasmid. After the enzyme reaction is completed, the DNA is recovered by ethanol precipitation to obtain the DNA of the linearized vector pBR322 plasmid with the SBSN sequence 600 position mutated from lysine (K) to threonine (T) connected to it.

3. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation of lysine 600 in SBSN according to claim 2, further comprising: The resistance gene fragment is connected to the DNA in which the 600th position of the SBSN sequence is mutated from lysine (K) to threonine (T) in the linearized vector pBR322 plasmid by homologous recombination.

4. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation of lysine 600 in SBSN according to claim 3, further comprising: The resistance gene fragment is a positive screening resistance gene and / or a negative screening resistance gene. Positive screening refers to screening that can survive under drug conditions, and negative screening refers to screening that cannot survive under drug conditions.

5. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation of lysine 600 in SBSN according to any one of claims 1 to 4, The second step, mouse embryonic stem cell culture, further includes: Embryonic stem cell complete culture medium contains 10-6 mol / L β-mercaptoethanol, 2 mM glutamine, 0.1 mM non-essential amino acids, 100 U / ml penicillin, 50 mg / L streptomycin, 15% ES fetal bovine serum, and 1000 U / ml LIF.

6. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation of lysine 600 in SBSN according to any one of claims 1 to 4, The third step is electroporation of embryonic stem cells, wherein the plasmid obtained in the first step is electroporated into the mouse embryonic stem cells cultured in the second step, further comprising: Embryonic stem cells in the logarithmic growth phase were digested with 0.125% trypsin-EDTA and counted. An appropriate amount of PBS was added to make the cell density reach about 1.5×10 7 / m1, take 0.8 ml of the above embryonic stem cell suspension, add about 35 μg of the plasmid obtained in the first step, mix well and transfer to a sterile electroporation cup for electroporation at 250 V and 500 μF.

7. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation of lysine 600 in SBSN according to any one of claims 1 to 4, The fourth step is drug screening, wherein the embryonic stem cells electroporated in the third step are screened for embryonic stem cells containing a DNA fragment in which position 600 of the SBSN sequence is mutated from lysine (K) to threonine (T), further comprising: The drug screening is positive and negative drug screening. The embryonic stem cells obtained in the third step are selectively cultured in a culture medium containing the selective drugs Geneticin G418 and Ganciclovir 24 hours after electroporation. The culture medium is changed every day. After 7-8 days of selective culture, the resistant embryonic stem cells can be picked when they grow into clones visible to the naked eye.

8. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation of lysine 600 in SBSN according to any one of claims 2 to 4, Step 8: SBSN obtained in step 7 K600T The point mutation mice are induced with imiquimod to obtain a psoriasis-like mouse model, further comprising: At the same time, SBSN WT Mice were induced with imiquimod.

9. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation of lysine 600 in SBSN according to claim 8, further comprising: After 5 days of imiquimod induction, SBSN K600T Point mutation mice and SBSN WT Mice were anesthetized and killed, and local skin lesions were collected for identification: After the specimens were fixed, paraffin sections were made and HE staining was performed on the paraffin specimens. The pathological changes of skin tissue were observed under a microscope, and SBSN was visible. WT The epidermis of mice skin was thinner after imiquimod induction, and SBSN K600T The epidermis of point mutation mice was significantly thickened, with parakeratosis and hyperkeratosis, an increase in the number of spinous cells, and downward extension of epidermal processes.

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