Plasmid for overexpressing IRF5 as well as construction method and application of plasmid

Abstract

The invention belongs to the technical field of biology, and particularly relates to an overexpression IRF5 plasmid as well as a construction method and application thereof. The invention provides a construction method of a plasmid for overexpression of IRF5 in zebra fish. The method comprises the following steps: carrying out reverse PCR on an empty vector to prepare a first linearized empty vector; amplifying IRF5 by taking the WT zebra fish cDNA as a template to obtain a first target gene product; adding a homologous arm to the first target gene product to obtain a homologous arm product; performing homologous recombination on the homologous arm product and a first linearized empty vector to obtain a first homologous recombination product; taking the first homologous recombination product as a template, and carrying out PCR (Polymerase Chain Reaction) amplification to obtain an IRF5 gene product with a promoter, namely a second target gene product; performing reverse PCR on the empty vector to prepare a second linearized empty vector; and carrying out homologous recombination on the second target gene product and a second linearized empty vector to obtain the plasmid. According to the invention, the IRF5 is cloned into the vector through homologous recombination, and the recombinant vector constructed by the method can be used for culturing the zebrafish over-expressing the IRF5.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to an overexpression IRF5 Plasmid and its construction method and application. Background Art

[0002] Interferon regulatory factor (IRF) is an important transcription factor that IFNs It binds to specific DNA fragments in gene promoters and interferon-stimulated genes to regulate the expression of target genes. IRF It plays an important biological role in innate immunity and adaptive immunity regulation, antiviral infection, cell proliferation, apoptosis, etc. Interferon regulatory factor 5, IRF5 Genes play an important role in the occurrence and development of various autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus and multiple sclerosis.

[0003] IRF5 The main functions include: 1. Participating in antiviral immune response, IRF5 Plays a central role in antiviral immunity, especially in initiating innate immune responses through the production of type I interferons. 2. Regulation of inflammatory responses, IRF5 Promotes the production of pro-inflammatory cytokines such as tumor necrosis factor α, interleukin 6 and interleukin 12. These cytokines are essential for initiating and maintaining inflammatory responses, attracting and activating immune cells to the site of infection; at the same time IRF5 It plays an important role in regulating the polarization of macrophages towards pro-inflammatory direction. IRF5 The polarization of macrophages into pro-inflammatory M1 macrophages can be regulated. This type of macrophage can kill pathogens and release a large number of pro-inflammatory factors, which is an important component of anti-infection immunity. IRF5 It is also related to the occurrence of a variety of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Systemic lupus erythematosus corresponds to Systemic lupus erythematosus, SLE, and rheumatoid arthritis corresponds to Rheumatic Arthritis, RA. 3. IRF5 It is also involved in tumor immune surveillance and can regulate the immune system's recognition and clearance of tumor cells. IRF5 It plays a key role in a variety of immune and inflammation-related diseases and is considered a potential target for the treatment of these diseases.

[0004] For research IRF5 Scientists have developed various IRF5Transgenic animal models, especially the commonly used mouse model. IRF5 Transgenic mice and knockout mice have been developed for studying viral infections, autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, tumor immunity, etc. Although the immune system of mice is similar to that of humans, there are species differences in some cytokines, receptors, and immune signaling pathways. Therefore, the results obtained in mice cannot always be directly extended to humans. Sometimes IRF5 Overexpression transgenic models may lead to unnatural overactive immune responses, resulting in pathological phenomena inconsistent with natural conditions, limiting their physiological relevance. Compared with mice, rats are closer to humans in some physiological functions, drug metabolism, immune responses, etc., and are suitable as models for certain diseases. However, compared with mice, genetic manipulation of rats is relatively difficult and more expensive. Therefore, IRF5 Transgenic rat models are rare and difficult to develop. At the same time, transgenic resources for rats are not as abundant as those for mice, and many molecular tools and methods are not as mature as those for mouse models, which restricts IRF5 Research in related fields.

[0005] The most commonly used model animals are rats, mice and zebrafish. IRF5 The system is mostly suitable for mice, but mice cannot simulate human inflammatory diseases well; while rats, which have a more similar immune system to humans, have overexpressed IRF5 Therefore, there is an urgent need to provide a method for overexpression in zebrafish. IRF5 New approach to the system. Summary of the invention

[0006] The present invention aims to provide an overexpression IRF5 A plasmid and its construction method and application are proposed, which can be used to overexpress in zebrafish IRF5 new method.

[0007] The technical solution adopted by the present invention is that the present invention provides a method for overexpressing in zebrafish IRF5 The method for constructing a plasmid comprises the following steps: The first linearized empty vector was prepared by reverse PCR using the synthetic nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, and the empty vector was used as a template. The empty vector was Tol2-ef1α-EGFP-PA. The primers as shown in SEQ ID NO.3 and SEQ ID NO.4 were synthesized, and the wild-type zebrafish cDNA was used as a template to amplify IRF5Gene, obtain the first target gene product. Synthesize the primers shown in SEQ ID NO.5 and SEQ ID NO.6, use the first target gene product as a template, add homology arms at both ends of the first target gene product by PCR, and obtain homology arm products. Perform homologous recombination between the homology arm product and the first linearized empty vector to obtain the first homologous recombination product, and the molar ratio of the homology arm product to the first linearized empty vector is 3:1. Synthesize the primers shown in SEQ ID NO.7 and SEQ ID NO.8, use the first homologous recombination product as a template, and PCR amplify to obtain the promoter. IRF5 Gene product, prepare the second target gene product. Synthesize the primers with nucleotide sequences as shown in SEQ ID NO.9 and SEQ ID NO.10, use the empty vector as template, and perform inverse PCR to prepare the second linearized empty vector, wherein the empty vector is Tol2-ef1α-EGFP-PA. Perform homologous recombination between the second target gene product and the second linearized empty vector to obtain the product for overexpression in zebrafish. IRF5 The molar ratio of the second target gene product to the second linearized empty vector is 3:1.

[0008] The present invention provides a method for overexpressing a gene in zebrafish prepared by the above-mentioned construction method. IRF5 of the plasmid.

[0009] The present invention also provides a method for overexpressing in zebrafish IRF5 Plasmids were used to overexpress IRF5 Application of transgenic zebrafish.

[0010] For overexpression in zebrafish IRF5 Plasmids were used to cultivate overexpression IRF5 To produce transgenic zebrafish, the steps are as follows: The overexpression IRF5 The plasmid and Tol2 transposase mRNA were mixed in a mass ratio of 1:1 and introduced into zebrafish fertilized eggs. The fertilized eggs were screened and identified, hybridized and purified, and the resulting zebrafish were overexpressed. IRF5 of zebrafish.

[0011] Preferably, the cell stage of the fertilized egg is from the 1-cell stage to the 4-cell stage.

[0012] Preferably, the transgenic zebrafish is used to screen drugs for preventing or treating viral infections.

[0013] Preferably, the transgenic zebrafish is used to screen drugs for preventing or treating immune system diseases.

[0014] Preferably, the immune system disease is systemic lupus erythematosus or rheumatoid arthritis.

[0015] Compared with the prior art, the present invention has the following advantages: the present invention provides a method for overexpressing IRF5 The method for constructing a plasmid comprises the following steps: synthesizing primers with nucleotide sequences such as those shown in SEQ ID NO.1 and SEQ ID NO.2, using an empty vector as a template, and performing inverse PCR to prepare a first linearized empty vector, wherein the empty vector is Tol2-ef1α-EGFP-PA; synthesizing primers with nucleotide sequences such as those shown in SEQ ID NO.3 and SEQ ID NO.4, using wild-type zebrafish cDNA as a template, and amplifying IRF5 gene, obtain the first target gene product; synthesize the primers shown in SEQ ID NO.5 and SEQ ID NO.6, use the first target gene product as a template, add homology arms at both ends of the first target gene product by PCR, and obtain homology arm products; homologous recombination is performed between the homology arm product and the first linearized empty vector to obtain the first homologous recombination product, and the molar ratio of the homology arm product to the first linearized empty vector is 3:1; synthesize the primers shown in SEQ ID NO.7 and SEQ ID NO.8, use the first homologous recombination product as a template, and PCR amplify to obtain a promoter IRF5 Gene product, prepare a second target gene product; synthesize the primers shown in SEQ ID NO.9 and SEQ ID NO.10, use the empty vector as a template, and perform inverse PCR to prepare a second linearized empty vector, wherein the empty vector is Tol2-ef1α-EGFP-PA; homologously recombine the second target gene product with the second linearized empty vector to obtain a target gene product for overexpression in zebrafish IRF5 The molar ratio of the second target gene product to the second linearized empty vector is 3:1. IRF5 The recombinant vector constructed by the method of the present invention can be used to cultivate overexpression IRF5 of zebrafish.

[0016] Existing technology for cultivating overexpression IRF5 Animal models of overexpression prepared by the method of the present invention are often time-consuming, difficult to operate, costly and have a low success rate. IRF5 Animal models have the advantages of short construction time, low construction cost, and higher success rate than mice and rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Picture 1 Overexpression IRF5Representative images of transgenic zebrafish, A, C and E: representative images of transgenic zebrafish of F0 generation 1 day, 2 days and 3 days after fertilization, respectively; B, D and F: fluorescent images of transgenic zebrafish of F0 generation 1 day, 2 days and 3 days after fertilization, respectively; G: an enlarged image of F; H: a representative image of transgenic zebrafish of F1 generation 3 days after fertilization; I: a fluorescent image of transgenic zebrafish of F1 generation 3 days after fertilization; J: an enlarged image of I.

[0018] Picture 2 For overexpression IRF5 Verification of transgenic zebrafish expression and related factor expression, A is overexpression IRF5 qPCR identification of transgenic zebrafish; B is overexpression IRF5 Expression levels of related inflammatory factors in transgenic zebrafish.

[0019] Picture 3 HE detection images, A: wild-type zebrafish; B and C: 20-fold and 40-fold magnifications of the arrowed areas; D and E: 20-fold and 40-fold magnifications of the arrowed areas; F: overexpression IRF5 Transgenic zebrafish; G and H: The arrowed areas are magnified 20 times and 40 times, respectively; I and J: The arrowed areas are magnified 20 times and 40 times, respectively.

[0020] Picture 4 For overexpression IRF5 Red fluorescence labeling analysis of transgenic zebrafish, A~E: Representative images of WT wild-type zebrafish injected with BL21-mcherry at 0hpi, 6hpi, 12hpi, 18hpi, and 24hpi; a~e: Red fluorescence images corresponding to A~E; F~J: Overexpression IRF5 Representative images of zebrafish injected with BL21-mcherry at 0hpi, 6hpi, 12hpi, 18hpi, and 24hpi; f~j: red fluorescence images corresponding to figures F~J.

[0021] Picture 5 For overexpression IRF5 Survival diagram of transgenic zebrafish, A is the zebrafish survival curve; B is the bacterial fluorescence analysis.

[0022] Picture 6 Characterization diagram of dual fluorescent transgenic zebrafish, A and B are respectively: fluorescence diagram and representative pictures of Tg, mpx:Dsred; C~E: fluorescence diagram and representative pictures of dual fluorescent transgenic zebrafish; F is qPCR detection IRF5 Expression level; G is the analysis of neutrophil fluorescence intensity. DETAILED DESCRIPTION

[0023] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0024] The inventive concept of the present invention is as follows: IRF5 is an important transcription factor that participates in the regulation of various immune responses. Its abnormal expression is closely related to various diseases, such as autoimmune diseases and infectious diseases. IRF5 Its specific role in immune cell development, activation and cytokine secretion can be systematically analyzed.

[0025] In the prior art, methods for constructing transgenic models of mice and rats include embryonic stem cell method and microinjection method. In terms of construction cycle, the construction of transgenic models of mice and rats usually takes 6 months to 1 year, including steps such as gene construction, injection, screening and phenotypic analysis; although mice and rats are widely used in biomedical research, their transgenic models are not flexible enough in some cases, such as it is difficult to achieve rapid gene function research or drug screening; at the same time, the success rate of transgenic mice is usually between 10% and 30%. In contrast, the construction time of zebrafish transgenic models is usually shortened to a few weeks to a few months, which is significantly faster than mice and rats; zebrafish models are suitable for high-throughput screening and real-time imaging, and can conduct drug response and gene function research more quickly. In addition, its development process is transparent, making observation and analysis more intuitive; at the same time, the transgenic success rate of zebrafish can reach 50% to 80%, which is usually high, which means that more individuals can successfully carry the required transgenes. Compared with mice and rats, the construction time of zebrafish transgenic models is shortened by 50% to 80%, the application range is wider, and the success rate is increased by 20% to 70%. These advantages make zebrafish an efficient and flexible research model, particularly suitable for rapid screening and real-time observation.

[0026] Overexpression in zebrafish IRF5 System ， It can systematically analyze its role in early embryonic development and immune system regulation, use model organisms to conduct efficient gene function research, accelerate drug screening and high-throughput research, and reveal IRF5 mechanisms in inflammatory responses, make up for the limitations of mammalian models, promote research on developmental and disease-related gene regulation, and optimize gene editing efficiency and stability.

[0027] Based on this, the present invention aims to construct a method for zebrafish IRF5 Expression system, generate an overexpression IRF5 The zebrafish model focuses on providing a method for overexpression of IRF5The preparation method of the plasmid is as follows: using an empty vector as a template, inverse PCR is used to prepare a first linearized empty vector, wherein the empty vector is Tol2-ef1α-EGFP-PA; using wild-type zebrafish cDNA as a template, amplify IRF5 gene, obtain the first target gene product; using the first target gene product as a template, adding homology arms at both ends of the first target gene product by PCR to obtain a homology arm product; homologous recombination of the homology arm product with the first linearized empty vector to obtain a first homologous recombination product; using the first homologous recombination product as a template, PCR amplification to obtain a promoter-containing IRF5 gene product, prepare a second target gene product; use the empty vector as a template, and perform inverse PCR to prepare a second linearized empty vector, wherein the empty vector is Tol2-ef1α-EGFP-PA; perform homologous recombination between the second target gene product and the second linearized empty vector to obtain a target gene product for overexpression in zebrafish IRF5 of the plasmid.

[0028] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0029] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0030] Example 1 One for overexpression in zebrafish IRF5 The steps for constructing the plasmid are as follows: S1: Synthesize the nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, use the empty vector as a template, and perform inverse PCR to prepare the first linearized empty vector, the method is as follows: According to the original pTol2[Exp]-ef1α-EGFP-PA plasmid vector map information, a reverse primer was designed in the blank region after ef1α-EGFP for reverse PCR to obtain a linearized Tol2-ef1α-EGFP-PA vector and prepare a first linearized empty vector. The primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0031] Tol2 Vector-F1: 5'-CACACCTCCCCTGAACCTGAAA-3', SEQ ID NO.1.

[0032] Tol2 Vector-R1: 5'-GGTGGCCGAGGAATTCTTTGCCA-3', SEQ ID NO. 2.

[0033] The PCR reaction system and amplification program are shown in Tables 1 and 2, and the reaction system is 20 μL.

[0034] Table 1 PCR reaction system involved in S1

[0035] Table 2 PCR amplification procedures involved in S1

[0036] Use agarose gel electrophoresis to check whether the band size is correct, take pictures, cut the gel for recovery, measure the concentration and mark it, and store it at -20℃.

[0037] S2: Synthesize primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, and amplify the wild-type zebrafish cDNA as a template. IRF5 Gene, obtain the first target gene product, the method is as follows: Look up zebrafish at NCBI IRF5 Gene CDS sequence, based on zebrafish IRF5 Gene CDS sequence, synthetic primers, primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. Extract wild-type zebrafish total RNA, reverse transcribe into cDNA; use this cDNA as a template, PCR to obtain zebrafish IRF5 Gene.

[0038] S2.1: Extract total RNA from zebrafish as follows: Ten 3-day-old zebrafish fry were selected and placed in a 1.5 mL EP tube. After removing excess water, they were washed twice with phosphate buffer and dried again.

[0039] Add 1 mL of Trizol reagent to the tube, and use a pipette to repeatedly blow and shake to ensure that the larval fish tissue is fully broken and evenly mixed, then let it stand on ice for 10 minutes to complete cell lysis.

[0040] 200 μL of chloroform was added to the sample, equivalent to 1 / 5 of the original volume, and then the sample was shaken vigorously for 30 seconds and placed on ice for 10 minutes; then centrifuged at 12,000 g for 15 minutes at 4°C.

[0041] Gently take out the centrifuge tube. The sample will separate into three layers: the upper layer is a colorless aqueous phase, the middle layer is a white protein layer, and the lower layer is a red organic phase. Use a pipette to gently take the upper aqueous phase, about 500 μL, and transfer it to a new 1.5 mL centrifuge tube.

[0042] Add an equal volume of pre-chilled isopropanol to the aqueous phase and invert upside down to mix.

[0043] The mixture was refrigerated at -20°C for 30 min; then centrifuged at 12,000 g for 20 min at 4°C. The formation of a white precipitate was usually observed.

[0044] Remove the supernatant and slowly add 1 mL of 75% ethanol prepared with DEPC water along the opposite side of the precipitate to clean the tube cap and tube wall, tap the bottom of the tube gently, and let it stand for 5 min.

[0045] Centrifuge at 12,000 g for 5 min at 4°C.

[0046] Discard the supernatant and centrifuge at 4°C for 30 seconds. Use a pipette to completely remove the liquid on the tube wall.

[0047] Lay a clean paper towel on the clean bench, open the EP tube cap, place the tube upside down on the paper towel and let it stand for about 10 minutes. Be careful to avoid excessive drying of the precipitate. Wait until the white precipitate turns into a colorless and transparent colloidal state.

[0048] Add 10 μL of pre-cooled DEPC water to the precipitate, pipette gently to aid dissolution, and then heat in a metal water bath at 56°C for 10 min to completely dissolve.

[0049] Take 1 μL of the sample to determine the RNA concentration and store the remaining RNA in a -80 °C freezer or use it immediately for reverse transcription reaction.

[0050] S2.2: Reverse transcription to obtain zebrafish cDNA, the method is as follows: According to the instructions of the first-strand cDNA synthesis kit, in order to obtain higher synthesis efficiency, the following components were added to a 1.5 mL centrifuge tube, as shown in Table 3.

[0051] Table 3 Component 1 for synthesizing cDNA

[0052] Mix well, incubate in a 65°C water bath for 5 min, and then put on ice for 2 min.

[0053] Continue to add the following ingredients, as shown in Table 4.

[0054] Table 4 Component 2 for synthesizing cDNA

[0055] Mix gently and incubate at 42°C for 30 min.

[0056] Heat in an 85°C metal bath for 5 seconds to inactivate gDNA Remover and EasyScript RT Enzyme Mix. Measure the concentration and label the aliquots. Store in a -20°C refrigerator.

[0057] S2.3: PCR acquisition IRF5 Target gene, the method is as follows: Amplify target gene IRF5 CDS region: Download the IRF5 gene sequence from NCB, design primers in the CDS region or outside the CDS region, use the zebrafish cDNA obtained by reverse transcription as a template, and amplify the target gene coding region with primers IRF5-F and IRF5-R. The primer sequences are as follows: IRF5-F: 5'-CCGTTTGGACACTTTTGCGT-3', SEQ ID NO. 3.

[0058] IRF5-R: 5'-AGGCCACCTCCCTATTCTGT-3', SEQ ID NO.4.

[0059] The PCR reaction system and reaction procedure are shown in Tables 5 and 6: Reaction system 20 μL.

[0060] Table 5 PCR reaction system involved in S2.3

[0061] Table 6 PCR reaction procedures involved in S2.3

[0062] Agarose gel electrophoresis detection: First prepare 1% agarose gel and 1×TAE buffer; set the voltage to 100V and the current to 300mA, and perform electrophoresis for 30min; after the electrophoresis, use the UV gel imaging system to check whether the size of the DNA band is as expected, and record the image data.

[0063] S2.4: Recover the target gene as follows: Use agarose gel electrophoresis to separate the target DNA fragment from other fragments as much as possible. Use a clean surgical blade to cut out the agarose gel block containing the target DNA fragment, remove as much agarose as possible that does not contain the target DNA, put it into a 1.5 mL centrifuge tube, and weigh it.

[0064] According to the weight of the gel block, add 300 μL Buffer B2 for every 100 mg agarose. If the weight of the gel block is less than 100 mg, add water to make up to 100 mg.

[0065] Place the centrifuge tube containing agarose in a 50°C water bath and heat for 10 min, stirring occasionally, until the agarose is completely melted.

[0066] The melted agarose solution was transferred to the adsorption column and centrifuged at 8000 g for 30 s; the liquid in the collection tube was then discarded and the adsorption column was returned to the original collection tube.

[0067] Add 300 μL of Buffer B2 to the adsorption column and centrifuge at 9000 g for 30 s; discard the liquid in the collection tube again and put the adsorption column back into the original collection tube.

[0068] Add 500 μL of washing solution to the adsorption column and centrifuge at 9000g for 30 seconds; then discard the liquid in the collection tube and return the adsorption column to the original collection tube. Repeat once. Place the empty adsorption column and collection tube into the centrifuge and centrifuge at 9000g for 1 minute; this step is necessary because any residual ethanol will adversely affect the purity of the DNA and subsequent experiments.

[0069] Add 40 μL of elution solution preheated to 65°C to the center of the adsorption membrane, let stand at room temperature for 2 minutes, and then centrifuge at 9000g for 1 minute; collect the resulting DNA solution, i.e., the first target gene product, determine its concentration and mark it, and then store it at -20°C for subsequent experiments.

[0070] S3: Synthesize primers with nucleotide sequences such as those shown in SEQ ID NO.5 and SEQ ID NO.6, use the first target gene product as a template, and add homology arms at both ends of the first target gene product by PCR to obtain homology arm products, as follows: according to IRF5 Gene synthesis primers for IRF5 The 5' end of the gene is added with about 20 bp of the same sequence as the two ends of the linearized vector. The primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6. After PCR, IRF5 Perform agarose gel electrophoresis, use ultraviolet gel imaging system to check whether the size of DNA bands is consistent with expectations, and record image data.

[0071] IRF5 homology arm-F: 5'-CAAAGAATTCCTCGGCCACCCCGTTTGGACACTTTTGCGT-3', SEQ ID NO. 5.

[0072] IRF5 homology arm-R: 5'-CAGGTTCAGGGGGAGGTGTGAGGCCACCTCCCTATTCTGTTG-3', SEQ ID NO. 6.

[0073] The PCR reaction system is shown in Table 7.

[0074] Table 7 PCR reaction system involved in S3

[0075] The PCR reaction program is shown in Table 8.

[0076] Table 8 PCR reaction procedures involved in S3

[0077] S4: homologous recombination is performed between the homology arm product and the first linearized empty vector to obtain the first homologous recombination product, as follows: S4.1: Homologous recombination ligation, the method is as follows: According to the one-step operation guide of the rapid cloning kit, the homology arm product and the first linearized empty vector were mixed at a molar ratio of 3:1. The recommended optimal amount of the vector is 0.03 pmol = 0.02 × the number of vector base pairs, in ng. The reaction system is shown in Table 9.

[0078] Table 9 Homologous recombination reaction system of homology arm products and first linearized empty vector

[0079] Once the reaction system is set up, mix the components by gently pipetting up and down, then centrifuge briefly to pool the mixture at the bottom of the tube.

[0080] Place the mixed reaction tube into the PCR instrument and react at 50°C for 20 minutes.

[0081] After the reaction is completed, the reaction tube is immediately placed on ice to cool for 5 minutes. This is to prevent high temperature from affecting the sensitivity of the cells and thereby reducing the conversion efficiency.

[0082] The recombinant product can be used for transformation immediately, or stored in a -20°C refrigerator and taken out for transformation when needed.

[0083] S4.2: Transformation screening of recombinant products, the method is as follows: Thaw DH5α competent cells on ice, take 10 μL of recombinant plasmid, add it to 100 μL of competent cells, shake gently to mix evenly, and then place it on ice for 30 minutes.

[0084] The mixture was placed at 42°C for 60 seconds for heat shock treatment and then quickly transferred to ice for 2 minutes for cooling.

[0085] 900 μL of antibiotic-free LB medium was added to the mixture, and the mixture was incubated at 37° C. for 2 min to promote cell recovery; the mixture was then shaken and cultured at 37° C. and 200 rpm / min for 1 hour.

[0086] In a sterile environment, transfer the culture medium to a 1.5 mL centrifuge tube, centrifuge at 5000 rpm / min for 1 min, and remove 900 μL of supernatant; then use the remaining culture medium to resuspend the bacteria, and use a sterile spreading stick to evenly spread it on the LB solid culture medium containing ampicillin; after the spreading is completed, wait for the culture medium to absorb the bacterial solution, then invert the culture dish and culture it overnight at 37°C.

[0087] Pick at least 5 smooth, moist, and independent monoclonal colonies from the transformed LB plate and inoculate them into 1 mL of Amp + The cells were cultured in resistant LB liquid medium at 37°C with shaking at 225 rpm for about 5 h.

[0088] Take out 1 μL from 1 mL of bacterial solution and put it into a new 0.5 ml EP tube. Keep the rest at 4°C for later use. Add 19 μL of ddH2O to dilute it and take out 1 μL as a template.

[0089] PCR was performed to identify whether the recombinant plasmid was successfully connected. The PCR reaction system and reaction procedure are shown in Tables 10 and 11.

[0090] Table 10 PCR reaction system involved in S4.2

[0091] Table 11 PCR reaction procedures involved in S4.2

[0092] Agarose gel electrophoresis was used to detect whether the band size was correct and photos were taken. If the band size was correct, the connection was successful, and the first homologous recombination product, pTol2[Exp]-ef1α-EGFP-IRF5, was obtained.

[0093] After successful plasmid ligation, 10 μL of the remaining 1 mL of bacterial solution was taken out and added to 5 mL of Amp + The cells were cultured in resistant LB liquid medium at 37°C and 225 rpm overnight for 16 h.

[0094] The first homologous recombination product was extracted using a plasmid miniprep kit.

[0095] S5: Synthesize primers with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8, use the first homologous recombination product as a template, and perform PCR amplification to obtain a promoter-containing IRF5Gene product, preparing the second target gene product, the method is as follows: The first homologous recombination product pTol2[Exp]-ef1α-EGFP-IRF5 was used as a template, and the primers ef1α-IRF5-SV40-F and ef1α-IRF5-SV40-R were used for colony PCR amplification to obtain the ef1α-IRF5-SV40 fragment; the primer sequences are shown in SEQ ID NO. 7 and SEQ ID NO. 8. The PCR reaction system and PCR reaction program are shown in Tables 12 and 13.

[0096] Ef1α-IRF5-SV40-F: 5'-TCTTAGGTACCGAGCTCTAGGGATAACAGGTAATCAGGGGGATCATCTAATCAAGC-3', SEQ ID NO. 7.

[0097] Ef1α-IRF5-SV40-R: 5'-CGAGCAGAGGTGTAAAAAGTACTC-3', SEQ ID NO. 8.

[0098] Table 12 PCR reaction system involved in S5

[0099] Table 13 PCR reaction procedures involved in S5

[0100] Use agarose gel electrophoresis to check whether the band size is correct, take pictures, cut the gel for recovery, measure the concentration and mark it, then the second target gene product is obtained and stored at -20℃.

[0101] S6: Synthesize the primers with nucleotide sequences as shown in SEQ ID NO.9 and SEQ ID NO.10, use the empty vector as template, and perform inverse PCR to prepare the second linearized empty vector, the method is as follows: Using the original plasmid pTol2-ef1α-EGFP-PA as a template, the Tol2 plasmid was amplified with primers SEQ ID NO.9 and SEQ ID NO.10 to linearize the Tol2 plasmid. The PCR reaction system and amplification procedure are shown in Tables 14 and 15.

[0102] Tol2 Vector-F2: 5'-GTACTTTTTACACCTCTGCTCGACCATATGGGAGAGCTCCCAA-3', SEQ ID NO. 9.

[0103] Tol2 Vector-R2: 5'-ACCCTGTTATCCCTAGAGCTCGGTACCTAAGATACATTGATGAG-3', SEQ ID NO. 10.

[0104] Table 14 PCR reaction system involved in S6

[0105] Table 15 PCR amplification procedures involved in S6

[0106] Use agarose gel electrophoresis to check whether the band size is correct, take pictures, cut the gel for recovery, measure the concentration and mark it, then you will get the second linearized empty vector, and store it at -20℃.

[0107] S7: Homologous recombination of the second target gene product with the second linearized empty vector to obtain a vector for overexpression in zebrafish IRF5 The method is as follows: According to the rapid cloning kit, Shanghai Yisheng Biotechnology Co., Ltd., catalog number: 10911ES50, the second target gene product and the second linearized empty vector were mixed at a molar ratio of 3:1. The recommended optimal amount of the vector is 0.03 pmol = 0.02 × the number of vector base pairs, ng. The reaction system is shown in Table 16.

[0108] Table 16 Homologous recombination reaction system of the second target gene product and the second linearized empty vector

[0109] Once the reaction system is set up, mix the components by gently pipetting up and down, then centrifuge briefly to pool the mixture at the bottom of the tube.

[0110] Place the mixed reaction tube into the PCR instrument and react at 50°C for 20 minutes.

[0111] After the reaction is completed, immediately place the reaction tube on ice to cool for 5 minutes. This is to prevent high temperature from affecting the sensitivity of the cells and thereby reducing the conversion efficiency.

[0112] The recombinant product can be used for transformation immediately, or stored in a -20°C refrigerator and taken out for transformation when needed.

[0113] The transformation of the recombinant product is as follows: Thaw DH5α competent cells on ice, take 10 μL of recombinant plasmid, add it to 100 μL of competent cells, shake gently to mix evenly, and then place it on ice for 30 minutes.

[0114] The mixture was heat-shocked at 42°C for 60 seconds and then quickly transferred to ice for cooling for 1 to 2 minutes.

[0115] 900 μL of antibiotic-free LB medium was added to the mixture, and the mixture was incubated at 37° C. for 2 min to promote cell recovery; the mixture was then shaken and cultured at 37° C. and 200 rpm / min for 1 hour.

[0116] In a sterile environment, transfer the culture medium to a 1.5 mL centrifuge tube, centrifuge at 5000 rpm / min for 1 min, and remove 900 μL of supernatant; then use the remaining culture medium to resuspend the bacteria, and use a sterile spreading stick to evenly spread it on the LB solid culture medium containing ampicillin; after the spreading is completed, wait for the culture medium to absorb the bacterial solution, then invert the culture dish and culture it overnight at 37°C.

[0117] The screening and verification of recombinant plasmids are as follows: Pick at least 5 smooth, moist, and independent monoclonal colonies from the transformed LB plate and inoculate them into 1 mL of Amp + The cells were cultured in resistant LB liquid medium at 37°C with shaking at 225 rpm for about 5 h.

[0118] Take 1 μL from 1 mL of bacterial solution and put it into a new 0.5 mL EP tube. Keep the rest at 4°C for later use. Add 19 μL of ddH2O to dilute it. Take 1 μL as a template. Perform PCR with primers SEQ ID NO.11 and SEQ ID NO.12 to identify whether the recombinant plasmid is successfully connected.

[0119] Second recombination identification-F: 5'-CATGGTCCTGCTGGAGTT-3', SEQ ID NO.11.

[0120] Recombinant identification-R: 5'-CGAGCAGAGGTGTAAAAAGTACTC-3', SEQ ID NO. 12. The PCR reaction system and reaction procedure are shown in Tables 17 and 18.

[0121] Table 17 PCR reaction system involved in S7

[0122] Table 18 PCR reaction procedures involved in S7

[0123] Check whether the band size is correct by agarose gel electrophoresis and take pictures. If the band size is correct, the connection is successful.

[0124] After successful plasmid ligation, 10 μL of the remaining 1 mL of bacterial solution was taken out and added to 5 mL of Amp + The cells were cultured in resistant LB liquid medium at 37°C and 225 rpm overnight for 16 h.

[0125] The recombinant plasmid was extracted using a plasmid extraction kit.

[0126] Overexpression IRF5 Plasmid sequencing was performed as follows: The obtained recombinant plasmid samples were sent to Shanghai Bioengineering for sequencing analysis, and the standard vector connection verification primer-R was used to confirm the connection. The sequencing data was checked by Snapgene software, and its sequence alignment function was used to confirm whether there was a base variation in the sequence. If the alignment was correct, the correct plasmid sample was saved, and the overexpression was obtained. IRF5 The plasmid was placed in a low temperature environment of -80℃ for later use.

[0127] Endotoxin-free extraction overexpression IRF5 The plasmid pTol2[Exp]-ef1α-EGFP-ef1α-IRF5 was recombined as follows: Extract overexpression IRF5 The plasmid pTol2[Exp]-ef1α-EGFP-ef1α-IRF5 glycerol strain was seeded from frozen stock at a ratio of 1:1000 into the Amp + The cells were cultured in 10 mL LB medium at 37°C and 225 rpm / min on a shaker for 16 hours. The next day, the culture was transferred to a 2 mL centrifuge tube and centrifuged at 12000 rpm / min for 1 min. The supernatant was discarded and the bacterial pellet was retained.

[0128] CP4 adsorption column equilibration steps: inject 500 μL BL equilibration solution, centrifuge at 12000 rpm / min for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube for use.

[0129] Pour 500 μL of L1 buffer into the centrifuge tube containing the bacteria and use a vortex to fully suspend the bacteria.

[0130] Add 500 μL P2 lysis buffer to the centrifuge tube containing bacteria and shake gently 8 times until the bacteria are completely lysed. The bacterial solution should be clear and sticky at this time. Limit the operation to no more than 5 minutes to avoid damage to the plasmid DNA.

[0131] Following the previous step, add 500μL P4 precipitation solution to the tube and shake it quickly 8 times to mix it thoroughly. After standing at room temperature for 10 minutes, centrifuge at 12000rpm / min for 10 minutes. The precipitate will form at the bottom of the tube. After adding P4, operate quickly to prevent the precipitate from partially dissolving. If there is still white precipitate in the supernatant, centrifuge it again to remove it.

[0132] The supernatant was slowly added to the CS filter column, placed in a collection tube, centrifuged at 12000 rpm / min for 2 min, and the filtrate was collected in a new 2 mL centrifuge tube.

[0133] Add one-third of the volume of isopropanol to the filtrate, mix by inversion, and add to the CP4 adsorption column in batches. The capacity is limited to 700 μL. Centrifuge at 12000 rpm / min for 1 min at room temperature, discard the waste liquid, and return the adsorption column to the collection tube.

[0134] Inject 500 μL of PD deproteinized solution into the adsorption column, centrifuge at 12,000 rpm / min for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.

[0135] Add 600 μL of PW washing solution containing anhydrous ethanol to the adsorption column, let it stand at room temperature for 2 to 5 minutes, then centrifuge at 12000 rpm / min for 1 minute, discard the waste liquid, and return the adsorption column to the collection tube. Repeat this step once.

[0136] The adsorption column was collected into a collection tube, centrifuged at 12000 rpm / min for 2 min, and left at room temperature with the lid open for 5 min to ensure that the residual washing liquid in the adsorption material was dry.

[0137] Transfer the adsorption column to a new 1.5mL NF tube, add 100μL of NF water with a pH value of 7.0 to 8.5 to the center of the adsorption membrane, place at room temperature for 2 minutes, centrifuge at 12000rpm / min for 1 minute, and collect the plasmid. Determine the plasmid concentration, dilute it to 50ng / μL appropriately for microinjection, and store the remaining plasmid at -80℃.

[0138] Example 2 Overexpression IRF5 The application of plasmids includes the following experiments.

[0139] Overexpression in zebrafish IRF5 System ， Its role in early embryonic development and immune system regulation can be systematically analyzed.

[0140] Experiment 1: Using the protein prepared in Example 1 for overexpression in zebrafish IRF5 Plasmid construction for overexpression IRF5 To produce transgenic zebrafish, the steps are as follows: S1: Prepare Tol2 transposase mRNA as follows: S1.1: Obtain high-concentration pCS-TP plasmid as follows: Take out the pCS-TP plasmid at a concentration of 50 ng / μL and DH5α competent cells from the -80°C freezer and place them on ice to thaw.

[0141] Take 5 μL of pCS-TP plasmid with a pipette and add it to 100 μL of DH5α competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, and then stand on ice for 2 minutes. This transformation process is designed to increase the number of pCS-TP plasmids in DH5α cloning bacteria.

[0142] To the mixture, 200 μL of resistance-free LB medium was added, and the mixture was cultured at 37° C. with shaking at 225 rpm / min for 30 min.

[0143] The converted product is evenly coated on a surface containing Amp + On LB solid medium, absorb the bacterial solution, invert the plate, and culture overnight at 37°C; The next day, three single clones were selected from the solid culture medium and inoculated into the Amp + The culture was shaken in LB liquid medium at 37°C and 225 rpm / min for more than 12 h; then 10 mL of bacterial solution was collected for plasmid extraction without removing endotoxin, eluted with 50 μL NF water, the concentration was determined and stored at -20°C.

[0144] S1.2: Linearization of pCS-TP plasmid, as follows: use Xba The pCS-TP plasmid carrying the transposase gene was linearized with restriction enzyme I. The specific restriction enzyme reaction system is shown in Table 19.

[0145] Table 19 Linearization reaction system of pCS-TP plasmid

[0146] After the plasmid was treated with enzymes for 2 h at 37 °C, 1 μL of the sample was taken for agarose gel electrophoresis to verify whether the linearization of the plasmid was complete.

[0147] S1.3: Recover the pCS-TP linearized plasmid as follows: Transfer the remaining enzyme digestion reaction solution to a clean 1.5 ml centrifuge tube, add 5 times the volume of Buffer B3, and mix thoroughly.

[0148] Transfer all the mixed liquid into the adsorption column and centrifuge at 8000g for 30s; pour out the liquid in the collection tube and place the adsorption column into the same collection tube.

[0149] Add 500 μL Wash Solution to the adsorption column and centrifuge at 9000 g for 30 seconds; pour out the liquid in the collection tube and place the adsorption column in the same collection tube. Repeat once.

[0150] The empty adsorption column together with the collection tube was centrifuged at 9000 g for 1 min.

[0151] Add 40 μL of Elution Buffer preheated to 60°C to the center of the adsorption membrane, incubate at 60°C for 2 minutes, and centrifuge at 9000g for 1 minute; measure the concentration of the resulting DNA solution, mark it, and store it at -20°C or use it for subsequent experiments.

[0152] S1.4: Tol2 transposase mRNA, i.e., pCS-TP mRNA, is obtained by in vitro transcription as follows: In vitro transcription was performed according to the instructions of the mMESSAGE mMACHINE™ SP6 Transcription Kit. The reaction system is shown in Table 20.

[0153] Table 20 In vitro transcription to obtain Tol2 transposase mRNA reaction system

[0154] The reaction was carried out at a constant temperature of 37°C for 2 hours. After the reaction was completed, 1 μL of TURBO DNase was added to the system and incubated for another 15 minutes to remove the residual DNA template.

[0155] In order to purify the obtained TP mRNA, the lithium chloride precipitation method was selected: 50 μL of LiCl solution was added after the transcription reaction, mixed thoroughly, and then refrigerated at -20°C for at least 30 min.

[0156] Next, the mixture was centrifuged at 12,000 rpm for 15 min at 4°C to precipitate the RNA.

[0157] Gently remove the supernatant and wash the precipitate once with 1 ml of a solution containing 70% ethanol prepared with NF water, and then centrifuge at the same 4°C, 12,000 rpm conditions for 15 min to remove incompletely bound nucleic acid molecules.

[0158] The supernatant was discarded, and the precipitate was air-dried at room temperature for 10 min, and then the RNA was dissolved in 20 μL NF water. The RNA concentration was measured and adjusted to 100 ng / μL for subsequent microinjection, and the aliquots were stored in a -80°C low-temperature refrigerator for preservation.

[0159] S2: Overexpression IRF5The plasmid and Tol2 transposase mRNA were mixed in a mass ratio of 1:1 and introduced into zebrafish fertilized eggs. The fertilized eggs were screened and identified, hybridized and purified, and the resulting zebrafish were overexpressed. IRF5 The method is as follows: Microinjection, overexpression at a concentration of 50ng / μL IRF5 The plasmid and Tol2 transposase mRNA at a concentration of 50 ng / μL were mixed in an equal volume ratio of 1:1 and injected into the animal pole of WT zebrafish embryos at the 1-cell stage to the 4-cell stage. The total amount of the mixture was adjusted according to the expected number of eggs to be injected. When the embryo was at the single-cell stage and the cell morphology was regular, the injection needle was inserted into the animal pole of the embryo under microscopic observation for intracytoplasmic injection. The injection volume of each egg was 1 nL. The injected embryos were transferred to a culture dish containing embryo culture water E2 water and placed in a constant temperature and light incubator at 28.5°C for culture, maintaining 14h light / 10h dark.

[0160] Observe the fluorescence expression from 1 to 5 days after injection, take photos and record them every day, and screen the fluorescence for 3 consecutive days. Discard embryos with no green fluorescence, weak fluorescence, or deformity. Picture 1 As shown. F0 expressing strong green fluorescence in multiple parts of the body are selected and raised to sexual maturity, and mated with AB wild-type zebrafish to produce F1. If the F1 fry can normally express strong green fluorescence in multiple parts of the body, the F0 can be used as a founder fish. The transgenic fish can be seen with obvious whole-body green fluorescence at 2dpi, and after 3dpi, it is a scaly green fluorescence all over the body.

[0161] Embryonic cells with obvious green fluorescence and normal morphology were cultured normally in a culture room with a water temperature of 28.5°C, a pH value of 8.0, and a cycle of 14 hours of daylight and 10 hours of darkness until they were 3 months old, i.e. the F0 generation.

[0162] The F0 generation of IRF5 overexpressing transgenic zebrafish were tail-cut and sequenced to screen for overexpression IRF5 Transgenic zebrafish were mated with WT fish to generate F1 generation, and F1 generation with strong green fluorescence was screened.

[0163] The F1 generation was identified by fluorescence screening and sequencing. IRF5 Transgenic zebrafish strains were successfully constructed, such as Picture 1 As shown, the F0 generation showed fluorescent mosaic expression, and the F1 generation showed fluorescent constitutive expression.

[0164] Experiment 2: Overexpression IRF5 Functional verification of transgenic zebrafish, the method is as follows: The F1 generation of IRF5-overexpressing transgenic zebrafish were taken out of the water and placed in ice water. After the fish became tetanic, they were quickly taken out and their tails were cut. They were cultured separately in clean water for 1 week and then placed in normal culture water.

[0165] RNA was extracted from each fish tail and reverse transcribed as an independent sample. qPCR was used to verify whether the IRF5 gene was overexpressed. The overexpressed F1 generation was the stably inherited transgenic zebrafish Tg, ef1α: EGFP; ef1α: IRF5 The transgenic fish showed obvious green fluorescence at 2 dpi, and after 3 dpi, the fish showed constitutive green fluorescence all over the body. Picture 1 As shown. Validated by qPCR IRF5 High expression, related inflammatory factors IL-1, IL-8, TNF-α expression levels increased, such as Picture 2 shown.

[0166] HE staining revealed IRF5 The intestinal development of transgenic zebrafish was significantly slower than that of the control group, the muscle tissue was irregularly arranged, and the inflammatory cell infiltration increased, such as Picture 3 shown.

[0167] Bacterial infection can cause continuous death of zebrafish. Red fluorescent labeled Escherichia coli BL21-mcherry was injected into the yolk sac of zebrafish. IRF5 The antibacterial ability of transgenic zebrafish and WT wild-type zebrafish was compared to further explore the function of IRF5. Picture 4 and Picture 5 As shown, the survival analysis showed that: IRF5 Compared with the wild-type zebrafish WT-Model group, the transgenic zebrafish IRF5-Model group showed IRF5 Transgenic zebrafish can avoid death caused by lethal bacterial infection. At the same time, 18 hours after infection, the fluorescence intensity of bacteria in the IRF5-Model group was significantly reduced compared with the WT-Model group. IRF5 Protective effect of overexpression in zebrafish against bacterial infection.

[0168] Neutrophils, as the first line of defense of innate immunity, play an important role in bacterial infection. The double fluorescent transgenic zebrafish were generated by crossing the Tg transgenic strain with the red neutrophil transgenic zebrafish Tg,mpx:Dsred purchased from the National Zebrafish Resource Center (CZRC). IRF5 Effects of overexpression on the immune system, such as Picture 6 As shown, the results show IRF5 Overexpression led to an increase in the level of neutrophils in zebrafish, which may be related to enhanced antibacterial ability.

[0169] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0170] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for overexpression in zebrafish IRF5 The method for constructing a plasmid is characterized in that: The following steps are involved: The nucleotide sequences are synthesized as shown in SEQ ID NO.1 and SEQ ID NO.2, and the empty vector is used as a template, and the first linearized empty vector is prepared by inverse PCR, wherein the empty vector is Tol2-ef1α-EGFP-PA; The primers with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4 were synthesized, and wild-type zebrafish cDNA was used as a template to amplify IRF5 Gene, obtaining the first target gene product; Synthesize primers with nucleotide sequences as shown in SEQ ID NO.5 and SEQ ID NO.6, use the first target gene product as a template, add homology arms at both ends of the first target gene product by PCR, and obtain homology arm products; The homologous arm product is homologously recombined with the first linearized empty vector to obtain a first homologous recombination product, wherein the molar ratio of the homologous arm product to the first linearized empty vector is 3:1; The primers with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8 were synthesized, and the first homologous recombination product was used as a template to obtain a promoter-containing gene by PCR amplification. IRF5 gene product, preparing a second target gene product; The nucleotide sequences are synthesized as primers shown in SEQ ID NO.9 and SEQ ID NO.10, and an empty vector is used as a template, and a second linearized empty vector is prepared by inverse PCR, wherein the empty vector is Tol2-ef1α-EGFP-PA; The second target gene product is homologously recombined with the second linearized empty vector to obtain a vector for overexpression in zebrafish IRF5 The molar ratio of the second target gene product to the second linearized empty vector is 3:

1.

2. A method for overexpression in zebrafish prepared by the construction method as claimed in claim 1 IRF5 of the plasmid.

3. The method as claimed in claim 2 for overexpression in zebrafish IRF5 Plasmids were used to overexpress IRF5 Application of transgenic zebrafish.

4. The use according to claim 3, characterized in that For overexpression in zebrafish IRF5 Plasmids were used to cultivate overexpression IRF5 To produce transgenic zebrafish, the steps are as follows: The overexpressed IRF5 The plasmid and Tol2 transposase mRNA were mixed in a mass ratio of 1:1 and introduced into zebrafish fertilized eggs. The fertilized eggs were screened and identified, hybridized and purified, and the resulting zebrafish were overexpressed. IRF5 of zebrafish.

5. The use according to claim 4, characterized in that The cell stage of the fertilized egg is 1 cell stage to 4 cell stage.

6. The use according to claim 3, characterized in that The transgenic zebrafish is used to screen drugs for preventing or treating viral infection.

7. The use according to claim 3, characterized in that The transgenic zebrafish is used to screen drugs for preventing or treating immune system diseases.

8. The use according to claim 7, characterized in that The immune system disease is systemic lupus erythematosus or rheumatoid arthritis.

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