Mouse model construction method for psoriasis research based on dihydroxyisobutyrylation modification of SBSN 600-site lysine

Abstract

The invention relates to a mouse model construction method for psoriasis research based on dihydroxyisobutyrylation modification of SBSN 600 site lysine. The mouse model construction method comprises the following steps: 1, constructing a plasmid comprising an SBSN gene sequence and a DNA fragment of which the 600 site of the SBSN sequence is mutated from lysine (K) to threonine (T); 2, culturing the mouse embryonic stem cells; step 3, carrying out electric transformation on the embryonic stem cells; 4, screening the medicines; 5, calling and culturing the embryonic stem cells screened in the step 4; step 6, injecting the embryonic stem cells called and cultured in the step 4 into a mouse blastocyst, then culturing the mouse blastocyst, and transplanting the cultured blastocyst into the uterus of a pseudopregnant female mouse, so that the mouse generated by delivery of the female mouse is a chimeric mouse; and 7, breeding the chimeric mouse and identifying the genotype of the mouse through PCR (Polymerase Chain Reaction).

Description

Technical Field

[0001] The present invention relates to a method for constructing a mouse model based on dihydroxyisobutyrylation modification of lysine at position 600 of SBSN, and specifically to a method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine at position 600 of SBSN, and in particular to a method for constructing a mouse model for psoriasis research by combining gene editing technology construction with imiquimod induction. Background Art

[0002] Psoriasis is one of the most common immune-mediated chronic inflammatory skin diseases. Previous studies have suggested that psoriasis is related to family inheritance, infection, immune abnormalities, endocrine factors, and other factors. 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 parakeratinization, hyperkeratinization, abnormal proliferation, angiogenesis, and inflammatory cell infiltration of skin keratinocytes. It has a great impact on the patient's physical health and mental state, and is a huge challenge for my country's public health. Although current treatment measures are effective, they cannot achieve long-term relief.

[0003] Psoriasis is one of the diseases that are currently being studied in the field of dermatology, and the development of new drugs is particularly important. Animal models of psoriasis are important tools for the pathogenesis of psoriasis and the development of new drugs. Currently, the models used to study psoriasis include spontaneous mouse models, drug-induced models (such as mouse psoriasis models induced by topical application of imiquimod to the skin), genetically engineered mouse models, and allogeneic transplant 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 spontaneous mouse models can show psoriasis skin manifestations in a relatively short period of time, such as acanthosis and increased vascular dilation in the dermis, they rarely show T cell and neutrophil infiltration, cannot reflect all the characteristics of psoriasis, and lack response to psoriasis treatment drugs. Therefore, this type of model is only suitable for the study of anti-proliferative drugs, and is not suitable for the pathogenesis of psoriasis and the study of new drugs;

[0006] ② The genetically engineered mouse model was constructed based on the psoriasis susceptibility gene, which is of great help in studying the pathogenesis of psoriasis. However, the manifestation of psoriasis lesions is greatly affected by later factors such as transcription and translation, and there are differences in the consistency of the lesion model, which limits the research on the progression and treatment of psoriasis.

[0007] ③ The allogeneic transplant animal model is a model in which the skin of psoriasis patients is transplanted onto immunodeficient mice. This type of animal model can more completely simulate the complex immunological and histopathological changes of psoriasis and is a suitable tool for studying the T cell pathogenesis of psoriasis and drug development. However, this type of mouse is more susceptible to infection due to severe immune defects, so the cost of raising it is relatively high. Therefore, the promotion and application of this type of model also has certain limitations.

[0008] However, the construction time of drug-induced models needs to be further shortened, and the effect needs to be further improved. Summary of the invention

[0009] In view of the problems existing in the prior art, one of the purposes of the present invention is to construct a method for constructing a psoriasis mouse model that comprehensively reflects the changes in the microenvironment of psoriasis patients and is more consistent with human psoriasis in terms of skin appearance and inflammatory response.

[0010] To achieve this object, the present invention adopts the following technical solutions:

[0011] A method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN, comprising:

[0012] 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);

[0013] The second step is to culture mouse embryonic stem cells;

[0014] The third step is electroporation of embryonic stem cells, in which the plasmid obtained in the first step is electroporated into the mouse embryonic stem cells cultured in the second step;

[0015] The fourth step is drug screening, using drug screening to screen the embryonic stem cells electroporated in the third step, and screen out embryonic stem cells containing a DNA fragment in which the 600th position of the SBSN sequence is mutated from lysine (K) to threonine (T);

[0016] Step 5, extracting and culturing the embryonic stem cells screened in step 4;

[0017] Step 6, injecting the cultured embryonic stem cells obtained in step 4 into mouse blastocysts, then culturing the mouse blastocysts, and transplanting the cultured blastocysts into the uterus of pseudo-pregnant female mice, and the mice born from the female mice are chimeric mice;

[0018] 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 C57BL / 6J pure strain mice to obtain the gray mice derived from the aforementioned stem cells. Use the PCR method to identify the mice with positive results, and the mice with positive results are F1 generation positive heterozygous mice, that is, SBSNK600T Point mutation mice, mice with negative results are wild mice, that is, SBSN WT Mouse.

[0019] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine at position 600 of SBSN further comprises:

[0020] Step 8: SBSN obtained in step 7 K600T The point mutation mice were induced with imiquimod to obtain a psoriasis-like mouse model.

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

[0022] The first step is to construct a plasmid comprising a DNA fragment of the SBSN gene sequence and a mutation from lysine (K) to threonine (T) at position 600 of the SBSN sequence; further comprising:

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

[0024] ② Fragment recovery: recover the PCR product obtained in step ① by gel electrophoresis;

[0025] ③ Ligation and transformation: connect the amplified fragment into the linearized vector pBR322 plasmid;

[0026] ④ Clone identification: After transformation, a single clone is selected for culture, and the plasmid is extracted by small-scale extraction. The plasmid is verified by enzyme digestion to confirm the correct cloned plasmid;

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

[0028] ⑥ Plasmid extraction: expand the amount of plasmid and obtain sufficient high-purity plasmid;

[0029] 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 in the linearized vector pBR322 plasmid connected to the SBSN sequence 600 position mutated from lysine (K) to threonine (T).

[0030] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine at position 600 of SBSN further comprises:

[0031] 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.

[0032] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine at position 600 of SBSN further comprises:

[0033] 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.

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

[0035] The second step, mouse embryonic stem cell culture, further includes:

[0036] The complete culture medium for embryonic stem cells 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.

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

[0038] 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:

[0039] 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 0.8ml of the above embryonic stem cell suspension, add about 35μg of the plasmid obtained in the first step, mix well and transfer to a sterile electroporation cup for electroporation at 250V and 500μF.

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

[0041] The fourth step is drug screening, using drug screening to screen the embryonic stem cells electroporated in the third step, and screen out embryonic stem cells containing a DNA fragment in which the 600th position of the SBSN sequence is mutated from lysine (K) to threonine (T), further comprising: the drug screening is positive and negative drug screening, and the embryonic stem cells obtained in the third step are selectively cultured with a culture medium containing selective drugs geneticin G418 and ganciclovir Ganciclovoir 24 hours after electroporation, and the culture medium is replaced 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.

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

[0043] Step 8: SBSN obtained in step 7 K600T The point mutation mice were induced with imiquimod to obtain a psoriasis-like mouse model, further comprising:

[0044] At the same time, SBSN WT Mice were induced with imiquimod.

[0045] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine at position 600 of SBSN further comprises:

[0046] After 5 days of imiquimod induction, SBSN K600T Point mutation mice and SBSN WT The mice were anesthetized to death and the local skin lesions were taken for identification:

[0047] After the specimens were fixed, paraffin sections were made, and the paraffin specimens were stained with HE. 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, while SBSN K600T The epidermis of point mutation mice was significantly thickened, with parakeratosis and hyperkeratosis, an increase in the number of spinous cells, and epidermal processes extending downward.

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

[0049] ① The present invention constructs a psoriasis mouse model based on the post-translational modification level, avoiding the influence of factors such as transcription and translation on the mouse phenotype, reducing the influence of non-experimental factors on the results to a certain extent, and ensuring the comparability of the results;

[0050] ② The present invention is the first to construct a psoriasis mouse model starting from dihydroxyisobutyrylation, which plays an important role in exploring the 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 for about 2 weeks;

[0052] ④ The mouse model constructed in the present invention has more severe skin lesions and inflammatory cell infiltration, which is most consistent with the characteristics of skin lesions in patients with psoriasis 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: Electrophoresis of PCR identification of 5' homology arm and 3' homology arm of F1 mice.

[0067] Figure 4 : Schematic diagram of the sequencing position verification of PCR products of F1 generation mice.

[0068] Reference numerals: 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: SBSN WT Wild-type mice and SBSN K600T Schematic diagram of skin lesion changes during imiquimod induction in point mutation mice, where: Figure 5-1 For SBSN WT Skin manifestations of wild-type mice on day 1 of imiquimod (IMQ) treatment;

[0075] Figure 5-2 For SBSN K600T Skin manifestations of point mutation mice on day 1 of IMQ treatment;

[0076] Figure 5-3 For SBSN WT Skin manifestations of wild-type mice on day 5 of IMQ treatment;

[0077] Figure 5-4 For SBSN K600T Skin manifestations of point mutation mice on day 5 of IMQ treatment.

[0078] Figure 6: SBSN WT Wild-type mice and SBSN K600T Schematic diagram of HE staining observation of imiquimod-induced tissue sections in point mutation mice.

[0079] in, Figure 6-1 For SBSN WT Schematic diagram of HE observation of skin lesion tissue sections of wild-type mice on the 5th day after IMQ treatment; Figure 6-2 For SBSN K600T Schematic diagram of HE observation of skin lesion tissue sections of point mutation mice on the 5th day after IMQ treatment.

[0080] Figure 7 : Partial flow chart of the construction of psoriasis mice of the present invention;

[0081] Reference numerals: 7001: first step, gene targeting of ES cells; 7002: isolation and culture of ES cells;

[0082] 7003: ES cells; 7004: electroporation; 7005: a small number of cells carrying the target gene; 7006: the target gene;

[0083] 7007: Construction of targeting vector; 7008: Vector; 7009: Inserted DNA fragment; 7010: pGK-Neo-polyA;

[0084] 7011:MCI-TK-polyA;7012:5 ， -Homology arm (5 ， -homology arm);

[0085] 7013:3 ， -Homology arm (3 ， -homology arm); 7014: ES cell electroporation; 7015: target gene;

[0086] 7016: Homologous recombination; 7017: Targeted gene; 7018: Drug screening; 7019: Targeted ES cell expansion.

[0087] Figure 8 : Partial flow chart of the construction of psoriasis mice of the present invention;

[0088] 8001: Knock-in mice; 8002: ES cell blastocyst injection;

[0089] 8003: Targeted ES cells were injected into the mouse blastocyst cavity; 8004: The injected blastocysts were transplanted into the uterus of pseudo-pregnant mice;

[0090] 8005: Chimeric inner cell mass; 8006: Birth and breeding of chimeric mice; 8007: Birth of chimeric mice;

[0091] 8008: Male chimeric mice mated with wild-type mice to obtain gene knock-in mice; 8009: Chimeric mice;

[0092] 8010: wild-type mice; 8011: knock-in mice. DETAILED DESCRIPTION

[0093] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementation methods.

[0094] In order to better illustrate the present invention and facilitate understanding of the technical solution of the present invention, the specific implementation methods of the present invention are as follows:

[0095] The inventor of this patent has devoted himself to research and found that the amino acid at position SBSN510 is upregulated by dihydroxyisobutyrylation in the skin lesions of psoriasis patients, and found that changes in the level of this modification can promote keratinocyte proliferation and inhibit keratinocyte apoptosis, which is consistent with the clinical characteristics of psoriasis. At the same time, the lysine at position SBSN510 in humans corresponds to the lysine at position SBSN600 in C57BL / 6J mice.

[0096] Based on the research results, in order to solve the problems existing in the prior art, the inventors of this patent obtained heterozygous mice with lysine (K) mutated to threonine (T) at position 600 of the SBSN gene through genetic engineering technology, and then combined with topical imiquimod induction to successfully construct a psoriasis mouse model. The model of the present invention has a shorter modeling time, more severe skin lesions and inflammatory cell infiltration, and is more consistent with the characteristics of psoriasis in terms of skin pathological manifestations.

[0097] The main inventive concept of the present invention is to utilize the principle of homologous recombination and adopt embryonic stem cell (ESC) targeting to knock in the K600T expression frame at the exon 1 (Exon 1) site of the SBSN gene, that is, to mutate the lysine (K) at position 600 of the SBSN protein to threonine (T), in order to simulate the high dihydroxyisobutyrylation modification of lysine at position 510 of human SBSN, so as to obtain SBSN K600TMice heterozygous for the point mutation.

[0098] The brief process is as follows: An ES cell targeting vector was constructed by the Infusion method. The vector contains a 2.9kb 5' homology arm, K600T (KI element), PGK-Neo-polyA, a 3.0kb 3' homology arm and an MC1-TK-polyA negative screening 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, the positive ES cell clones were injected into the blastocysts of C57BL / 6J mice to obtain chimeric mice. High-ratio chimeric mice were mated with Flp mice to obtain 6 positive F1 generation Neo-de heterozygous mice. The construction strategy is as follows Figure 1 As shown, imiquimod ointment was then applied topically to the skin to further induce the psoriasis mouse model.

[0099] A method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN, comprising:

[0100] 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);

[0101] The second step is to culture mouse embryonic stem cells;

[0102] The third step is electroporation of embryonic stem cells, in which the plasmid obtained in the first step is electroporated into the mouse embryonic stem cells cultured in the second step;

[0103] The fourth step is drug screening, using drug screening to screen the embryonic stem cells electroporated in the third step, and screen out embryonic stem cells containing a DNA fragment in which the 600th position of the SBSN sequence is mutated from lysine (K) to threonine (T);

[0104] Step 5, extracting and culturing the embryonic stem cells screened in step 4;

[0105] Step 6, injecting the cultured embryonic stem cells obtained in step 4 into mouse blastocysts, then culturing the mouse blastocysts, and transplanting the cultured blastocysts into the uterus of pseudo-pregnant female mice, and the mice born from the female mice are chimeric mice;

[0106] Step 7: Breeding of chimeric mice and identification of mouse genotypes by PCR: Select male mice with a coat color chimerism rate greater than 50% and mate them with C57BL / 6J pure strain mice to obtain the gray mice derived from the aforementioned stem cells. Use the PCR method to identify that the mice with positive results are F1 generation positive heterozygous mice, that is, SBSNK600T point mutation mice, and the mice with negative results are wild mice, that is, SBSNWT mice.

[0107] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine at position 600 of SBSN further comprises:

[0108] Step 8: The SBSNK600T point mutation mice obtained in step 7 were induced with imiquimod to obtain a psoriasis-like mouse model.

[0109] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN,

[0110] The first step is to construct a plasmid comprising a DNA fragment of the SBSN gene sequence and a mutation from lysine (K) to threonine (T) at position 600 of the SBSN sequence; further comprising:

[0111] ① Fragment PCR amplification: PCR amplify two DNA fragments of homology arms corresponding to the DNA fragments of about 100 bp above and below the SBSN mutation site from the C57BL / 6J mouse genome;

[0112] ② Fragment recovery: recover the PCR product obtained in step ① by gel electrophoresis;

[0113] ③ Ligation and transformation: connect the amplified fragment into the linearized vector pBR322 plasmid;

[0114] ④ Clone identification: After transformation, a single clone is selected for culture, and the plasmid is extracted by small-scale extraction. The plasmid is verified by enzyme digestion to confirm the correct cloned plasmid;

[0115] ⑤ Sequencing verification: After the correct positive clone is identified, further sequencing verification is performed;

[0116] ⑥ Plasmid extraction: expand the amount of plasmid and obtain sufficient high-purity plasmid;

[0117] 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 in the linearized vector pBR322 plasmid connected to the SBSN sequence 600 position mutated from lysine (K) to threonine (T).

[0118] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine at position 600 of SBSN further comprises:

[0119] 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.

[0120] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine at position 600 of SBSN further comprises:

[0121] 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.

[0122] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN,

[0123] The second step, mouse embryonic stem cell culture, further includes:

[0124] The complete culture medium for embryonic stem cells 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.

[0125] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN,

[0126] 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:

[0127] Embryonic stem cells in the logarithmic growth phase are digested with 0.125% trypsin-EDTA and counted. An appropriate amount of PBS is added to make the cell density reach about 1.5×107 / 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.

[0128] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN,

[0129] The fourth step is drug screening, using drug screening to screen the embryonic stem cells electroporated in the third step, and screen out embryonic stem cells containing a DNA fragment in which the 600th position of the SBSN sequence is mutated from lysine (K) to threonine (T), further comprising: the drug screening is positive and negative drug screening, and the embryonic stem cells obtained in the third step are selectively cultured with a culture medium containing selective drugs geneticin G418 and ganciclovir Ganciclovoir 24 hours after electroporation, and the culture medium is replaced 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.

[0130] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN,

[0131] Step 8: Inducing the SBSNK600T point mutation mice obtained in step 7 with imiquimod to obtain a psoriasis-like mouse model, further comprising:

[0132] At the same time, SBSNWT mice were induced with imiquimod.

[0133] The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine at position 600 of SBSN further comprises:

[0134] Five days after imiquimod induction, the SBSNK600T point mutation mice and SBSNWT mice were anesthetized to death and the local skin lesions were taken for identification:

[0135] After the specimens were fixed, paraffin sections were made, and the paraffin specimens were stained with HE. The pathological changes of skin tissue were observed under a microscope. It can be seen that the epidermis of the skin of SBSNWT mice was thinner after imiquimod induction, while the epidermis of SBSNK600T point mutation mice was significantly thickened, with incomplete keratinization and hyperkeratinization, an increase in the number of spinous cells, and epidermal processes extending downward.

[0136] As a preferred embodiment, an implementation method with a high success rate and a more preferred technical effect for realizing the present invention is further described in detail below. Those skilled in the art should know that the following implementation method is not the only implementation method of the present invention, but is only a further exemplary enumeration for facilitating understanding of the concept of the present invention.

[0137] The specific implementation methods of this patent are further described in detail as follows:

[0138] Basic information of the mutated gene:

[0139] The name of the gene mutated in the method of the present invention (Ensembl number) is SBSN (ENSMUSG00000046056).

[0140] Ensembl website link for mutation target gene:

[0141] http: / / www.ensembl.org / Mus_musculus / Gene / Summary?db=core;g= ENSMUSG00000046056; r=7:30450958-30455556; t=ENSMUST00000080518

[0142] Transcript targeted by the protocol (Ensembl number): ENSMUST00000080518.14

[0143] Exon targeted by site-directed knock-in: exon 1

[0144] Step 1: Plasmid construction process

[0145] That is: a primer sequence containing the SBSN 600 site mutation was designed, and the C57BL / 6J mouse genomic DNA was used as a template to amplify the homologous arm DNA fragment about 100 bp upstream and downstream of the mutation site.

[0146] Original backbone plasmid name: pBR322

[0147] ① Fragment amplification: Two DNA fragments with homology arms corresponding to the DNA fragments about 100 bp above and below 600 of the SBSN mutation site were amplified from the C57BL / 6J mouse genome.

[0148] ② Fragment recovery: The PCR product obtained in step ① was recovered by gel electrophoresis, and was recovered and purified using an Omega gel purification recovery kit, and the concentration was determined.

[0149] ③ Ligation transformation: connect the amplified fragment into the linearized vector pBR322 plasmid, use Takara Infusion ligase, competent strain: DH5α. Apply the transformed strain to the ampicillin resistance plate and culture at 37℃ overnight.

[0150] The ligase can be selected from: Manufacturer: Takara, Product Name: HD Cloning Plus

[0151] Item number: 638910, website: https: / / www.takarabiomed.com.cn /

[0152] ④ Clone identification: After transformation, pick a single clone for culture, extract the plasmid by microextraction, and perform enzyme digestion verification on the plasmid to confirm the correct cloned plasmid.

[0153] ⑤ Sequencing verification: After the correct positive clone is identified, the bacterial liquid or plasmid is sent to a sequencing company for Sanger sequencing verification (Biogen).

[0154] ⑥Plasmid extraction: This step is to expand the amount of plasmid and obtain sufficient high-purity plasmid.

[0155] 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 in the linearized vector pBR322 plasmid connected to the SBSN sequence 600 position mutated from lysine (K) to threonine (T).

[0156] Preferably, for the convenience of plasmid screening, the DNA further comprises a resistance gene fragment. The resistance gene fragment is also seamlessly cloned into the pBR322 plasmid by homologous recombination, that is, the NotI linearized pBR322-MK-SBSN-K600T plasmid DNA is finally obtained.

[0157] The pBR322-MK-SBSN-K600T plasmid refers to: a DNA fragment in which the 600th position of the SBSN sequence is mutated from lysine (K) to threonine (T) and a resistance screening marker MK are connected in the linearized vector pBR322 plasmid. Preferably, the resistance screening marker MK is a positive resistance screening marker or a positive and negative resistance screening marker, and can also be a positive and negative resistance screening marker. The positive and negative resistance screening markers are preferably 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.

[0158] 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.

[0159] The pBR322-MK-SBSN-K600T plasmid vector preferably comprises a 2.9 kb 5' homology arm, K600T (KI element), PGK-Neo-polyA, a 3.0 kb 3' homology arm and a MC1-TK-polyA negative selection marker.

[0160] (All molecular biology experiments are standard experiments. For detailed steps, please refer to the "Molecular Cloning Experiment Guide" ISBN9787030386069. For commercial kits, please refer to the kit instructions).

[0161] Primers, sequencing and other services are provided by Sangon Biotech (Shanghai) Co., Ltd.

[0162] Amplification enzyme:

[0163] Manufacturer: TOYOBO Product Name: KOD-Multi&Epi

[0164] Item No.: KME-101

[0165] Website: http: / / www.bio-toyobo.cn /

[0166] Ligase:

[0167] Manufacturer: Takara

[0168] Product Name: HD Cloning Plus

[0169] SKU: 638910

[0170] Website: https: / / www.takarabiomed.com.cn /

[0171] Endonuclease:

[0172] Manufacturer: NEB

[0173] Product Name: NotI-HF

[0174] Item No.: R3189L

[0175] Website: http: / / www.neb-china.com /

[0176] Gel Recovery Kit:

[0177] Manufacturer: Omega

[0178] Product Name: DNA Fragment Gel Recovery Kit

[0179] Item No.: D2500-01

[0180] Plasmid Extraction Kit

[0181] Manufacturer: Omega

[0182] Product name: Plasmid Mini Kit II(50)

[0183] Item No.: D6945-01

[0184] Plasmid extraction kit product information:

[0185] Manufacturer: MACHEREY-NAGEL

[0186] Product name: Endotoxin-free plasmid DNA purification

[0187] SKU: NucleoBond Xtra Midi EF.

[0188] Step 2: Embryonic stem cell culture: culture dishes are coated with 0.1% Gelatin and inoculated with trophoblast cells treated with mitomycin C (2×106 trophoblast cells are inoculated per 10 cm dish). After overnight culture, embryonic stem cells can be inoculated. The complete culture medium for embryonic stem cells is 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, and 1000 U / ml LIF.

[0189] Step 3: Electroporation of embryonic stem cells: ES cells in the logarithmic growth phase were digested with 0.125% trypsin-EDTA and counted. An appropriate amount of PBS (a commonly used buffer, after digestion, cells are placed in PBS for easy cell counting) was added to make the cell density reach about 1.5×10 7 / m1 (i.e., each milliliter has 1.5×10 7 Take 0.8 ml of the above ES cell suspension, add about 35 μg of pBR322-MK-Sbsn-K600T plasmid DNA linearized with NotI, mix well and transfer to a sterile electroporation cup for electroporation at 250 V and 500 μF. After resuspending, evenly distribute to three 10 cm culture dishes with trophoblast cells laid on them for culture.

[0190] Step 4, positive and negative drug screening: 24 hours after electroporation, ES cells are replaced with culture medium containing the selective drugs Geneticin G418 (final concentration of 250 mg / L) and Ganciclovir (final concentration of 2 umol / L) for selective culture. The culture medium is replaced every day. After 7-8 days of selective culture, resistant ES cells can be picked when they grow into clones visible to the naked eye.

[0191] Step 5. Picking and culturing double-resistant cell clones: Pick resistant clones and place them in a 96-well plate (concave bottom) containing 30 μl 0.1% trypsin-EDTA for digestion for about 3 minutes. Gently blow to disperse the cells and transfer them to a 96-well culture plate for culture. After the cells have grown to 60%-80%, subculture them in two 96-well plates at a ratio of 1:2. After they have grown to fullness, one portion is frozen and the other is used to extract genomic DNA.

[0192] Step 6, blastocyst injection and embryo transplantation of embryonic stem cells: DMEM complete medium without LIF is used for injection. Each blastocyst is injected with about 15 embryonic stem cells. After injection, the blastocyst is cultured in DMEM complete medium without LIF at 37°C and 5% CO2 for about 1 hour. The injected embryos are transplanted into the uterus of 2.5-day pseudo-pregnant female mice, with 8-10 embryos transplanted on each side. The pseudo-pregnant female mice are raised in the SPF animal room of the Shanghai Southern Model Organism Research Center, and the offspring born naturally are chimeric mice.

[0193] Step 7: Breeding chimera mice and identifying mouse genotypes by PCR: Select male mice with a coat color chimerism rate greater than 50% and mate them with C57BL / 6J pure strain mice to obtain ES cell-derived gray mice. Use the same ES cell identification method to identify positive heterozygous mice by PCR. The positive F1 generation positive heterozygous mice are SBSN. K600T Point mutation mice, F1 generation negative mice are SBSN WTWild-type mice.

[0194] F1 generation mouse identification strategy Figure 2 As shown:

[0195] Cut 0.5 cm from the end of the mouse tail, extract DNA, and perform PCR identification as follows: Identification method for the 5' homology arm of F1 generation mice:

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

[0197] Reverse 5'-AGCTTAAGGCAATGCGGTCT-3'

[0198] The PCR reactions are listed in Table 1 below.

[0199] Table 1

[0200]

[0201] KOD-Multi&Epi-*(TOYOBO, Code No: KME-101)

[0202] The PCR reaction conditions are listed in Table 2 below.

[0203] Table 2

[0204]

[0205] Method for identifying 3' homology arms of F1 mice:

[0206] Primer information: Forward 5′-GAGTGGTCCAGTTAGCCACC-3′;

[0207] Reverse 5'-TGCTGCAAAATCTTTTCATCCTCTT-3'

[0208] The PCR reactions are listed in Table 3 below.

[0209] Table 3

[0210] Reaction Component Volume(μl) Double deionized water (ddH2O) 8.05 2xPCR Buffer 10 Primer I (20 pmol / μl) 0.3 Primer II (20 pmol / μl) 0.3 KOD-Multi&Epi-* 0.35 Genomic DNA 1 Total volume 20

[0211] KOD-Multi&Epi-*(TOYOBO, Code No: KME-101)

[0212] PCR reaction conditions:

[0213]

[0214] Homologous recombination positive mouse PCR identification scheme:

[0215] Mice with positive homologous recombination in the 5' arm should amplify a 4.1kb fragment, and wild-type mice should amplify a 7.9kb fragment; mice with positive homologous recombination in the 3' arm should amplify a 3.6kb fragment, and wild-type mice should amplify a 7.8kb fragment.

[0216] The results of PCR electrophoresis for the 5' and 3' homology arms of F1 mice are shown in Figure 3. Sequencing confirmed that all of them were positive.

[0217] Results of PCR identification and sequencing of F1 generation mice:

[0218] The PCR identification products of F1 positive mice were sequenced, and a total of 4 sequencing reactions were performed. The corresponding regions of the sequencing reactions were as follows: Figure 4 Among them, 2 sequencing reactions were performed for 5' homology arm identification and PCR product sequencing, which were marked as 1 and 2 respectively; 2 sequencing reactions were performed for 3' homology arm identification and PCR product sequencing, which were marked as 3 and 4 respectively.

[0219] Step 8: SBSN obtained in step 7 K600T Point mutation mice and SBSN WT The psoriasis-like mouse model was obtained by imiquimod (IMQ)-induced treatment in mice.

[0220] The specific induction method is: K600T Point mutation mice and SBSN WT The back of wild-type mice was depilated, with an area of ​​2cm*2cm. 62.5mg of 5% imiquimod cream was applied to the back of the mice daily for 5 days. Obvious psoriasis lesions appeared in the depilated area on the back. The changes of lesions during the modeling period are shown in Figure 5. There was no obvious change in the depilated area on the back in the first two days of modeling. SBSN was started on the third day. K600T The skin of heterozygous mice with point mutations was slightly red, while that of wild-type mice was unchanged; on the 4th day, the mutant mice developed obvious erythema, some lesions were covered with scales, and the lesions were moderately raised, while the skin of wild-type mice was slightly red; Figure 5-3 and 5-4 As shown, on day 5, mice were compared with wild-type mice (see Figure 5-3 ) compared to mutant mice (see Figure 5-4 ) The erythema deepens, the surface is covered with thick scales, and the lesions are significantly thickened, which is consistent with psoriasis-like changes.

[0221] To further verify the similarity of mouse skin lesions in histopathological manifestations with psoriasis lesions, HE staining was used to identify mouse skin lesions. K600T Point mutation mice and SBSN WTThe mice were anesthetized to death and the local skin lesions were taken for identification:

[0222] After the specimens were fixed, paraffin sections were made, and the paraffin specimens were stained with HE, and the pathological changes of the skin tissue, especially the thickness of the epidermis, were observed under a microscope. The results are shown in Figure 6. It can be seen that the epidermis of the skin of wild-type mice after imiquimod induction was thinner, while the epidermis of mutant mice was significantly thickened, with parakeratosis and hyperkeratosis, an increase in the number of spinous cells, and epidermal processes extending downward, which were highly similar to the histopathological changes of skin lesions in patients with psoriasis.

[0223] Figure 5: SBSN WT Wild-type mice and SBSN K600T Schematic diagram of skin lesion changes during imiquimod induction in point mutation mice, where: Figure 5-1 For SBSN WT Skin manifestations of wild-type mice on day 1 of imiquimod (IMQ) treatment; Figure 5-2 For SBSN K600T Skin manifestations of point mutation mice on day 1 of IMQ treatment; Figure 5-3 For SBSN WT Skin manifestations of wild-type mice on day 5 of IMQ treatment; Figure 5-4 For SBSN K600T Skin manifestations of point mutation mice on day 5 of IMQ treatment.

[0224] Figure 6: SBSN WT Wild-type mice and SBSN K600T Schematic diagram of HE staining observation of imiquimod-induced tissue sections in point mutation mice. Figure 6-1 For SBSN WT Schematic diagram of HE observation of skin lesion tissue sections of wild-type mice on the 5th day after IMQ treatment. Figure 6-2 For SBSN K600T Schematic diagram of HE observation of skin lesion tissue sections of point mutation mice on the 5th day of IMQ treatment. HE staining showed that the epidermis of wild-type mice was thinner after IMQ treatment, while the epidermis of mutant mice was significantly thickened, with parakeratosis and hyperkeratosis, an increase in the number of spinous cells, and epidermal processes extending downward.

[0225] Figure 7 , 8: Partial flow chart of the construction of psoriasis mice of the present invention.

[0226] Based on the results of previous studies, the present invention is the first in the world to construct a psoriasis mouse animal model based on the dihydroxyisobutyrylation modification level of protein combined with imiquimod stimulation.

[0227] Compared with the previously reported methods for constructing psoriasis mouse models, the present invention has the following advantages:

[0228] ① This technology constructs a psoriasis mouse model based on the post-translational modification level, avoiding the influence of factors such as transcription and translation on the mouse phenotype, and to a certain extent reducing the influence of non-experimental factors on the results, ensuring the comparability of the results;

[0229] ② This technology is the first to construct a psoriasis mouse model starting from dihydroxyisobutyrylation, which plays an important role in exploring the dihydroxyisobutyrylation modification of psoriasis;

[0230] ③ The modeling cycle of this technology is short. Obvious psoriasis skin manifestations can appear on the 5th day of imiquimod induction, while the modeling cycle in previous literature reports lasted about 2 weeks;

[0231] ④ The mouse model constructed in the present invention has more severe skin lesions and inflammatory cell infiltration, which is most consistent with the characteristics of skin lesions in patients with psoriasis and meets the requirements for animal models in the primary stage of psoriasis research.

[0232] The applicant declares that the present invention illustrates the detailed process of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed process, that is, it does not mean that the present invention must rely on the above-mentioned detailed process to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the methods and steps selected by the present invention, addition of auxiliary steps, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0233] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0234] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0235] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of 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 electroporation of embryonic stem cells, in which 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, using drug screening to screen the embryonic stem cells electroporated in the third step, and screen out embryonic stem cells containing a DNA fragment in which the 600th position of the SBSN sequence is mutated from lysine (K) to threonine (T); Step 5, extracting and culturing the embryonic stem cells screened in step 4; Step 6, injecting the cultured embryonic stem cells obtained in step 4 into mouse blastocysts, then culturing the mouse blastocysts, and transplanting the cultured blastocysts into the uterus of pseudo-pregnant female mice, and 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 C57BL / 6J pure strain mice to obtain the gray mice derived from the aforementioned stem cells. Use the PCR method to identify the mice with positive results, and the mice with positive results are F1 generation positive heterozygous mice, that is, SBSN K600T Point mutation mice, mice with negative results are wild mice, that is, SBSN WT Mouse.

2. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN according to claim 1, further comprising: Step 8: SBSN obtained in step 7 K600T The point mutation mice were induced with imiquimod to obtain a psoriasis-like mouse model.

3. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN according to claim 1, The first step is to construct a plasmid comprising a DNA fragment of the SBSN gene sequence and a mutation from lysine (K) to threonine (T) at position 600 of the SBSN sequence; further comprising: ① Fragment PCR amplification: PCR amplify two DNA fragments of homology arms corresponding to the DNA fragments of about 100 bp above and below the SBSN mutation site from the C57BL / 6J mouse genome; ② Fragment recovery: recover the PCR product obtained in step ① by gel electrophoresis; ③ Ligation and transformation: connect the amplified fragment into the linearized vector pBR322 plasmid; ④ Clone identification: After transformation, a single clone is selected for culture, and the plasmid is extracted by small-scale extraction. The plasmid is verified by enzyme digestion to confirm the correct cloned plasmid; 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 in the linearized vector pBR322 plasmid connected to the SBSN sequence 600 position mutated from lysine (K) to threonine (T).

4. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN as claimed in claim 3, 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.

5. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN as claimed in claim 4, 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.

6. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN according to any one of claims 1 to 5, 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.

7. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN according to any one of claims 1 to 5, 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 0.8ml of the above embryonic stem cell suspension, add about 35μg of the plasmid obtained in the first step, mix well and transfer to a sterile electroporation cup for electroporation at 250V and 500μF.

8. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN according to any one of claims 1 to 5, The fourth step is drug screening, using drug screening to screen the embryonic stem cells electroporated in the third step to select 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 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.

9. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN according to any one of claims 2 to 5, Step 8: SBSN obtained in step 7 K600T The point mutation mice were induced with imiquimod to obtain a psoriasis-like mouse model, further comprising: At the same time, SBSN WT Mice were induced with imiquimod.

10. The method for constructing a mouse model for psoriasis research based on dihydroxyisobutyrylation modification of lysine 600 of SBSN as claimed in claim 9, further comprising: After 5 days of imiquimod induction, SBSN K600T Point mutation mice and SBSN WT The mice were anesthetized to death and the local skin lesions were taken for identification: After the specimens were fixed, paraffin sections were made, and the paraffin specimens were stained with HE. 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, while SBSN K600T The epidermis of point mutation mice was significantly thickened, with parakeratosis and hyperkeratosis, an increase in the number of spinous cells, and epidermal processes extending downward.

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