HMGB1 gene targeting shRNA target, shRNA and application thereof, and construction method of transgenic mouse model

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an HMGB1 gene targeting shRNA target, shRNA and application thereof, and a construction method of a transgenic mouse model. The siRNA target of the targeted HMGB1 gene, the shRNA obtained through construction and the recombinant plasmid can specifically and efficiently inhibit expression of the HMGB1 gene, a mouse model for expressing the shRNA-HMGB1 is successfully constructed through a transgenic technology, expression of the HMGB1 can be obviously reduced, the problem that newborn mice cannot survive without HMGB1 expression is ingeniously solved, and the siRNA target of the targeted HMGB1 gene, the shRNA obtained through construction and the recombinant plasmid can be used for preparing a mouse model for expressing the shRNA-HMGB1 and the mouse model for expressing the shRNA-HMGB1. The whole organ of the obtained transgenic mouse model has the expression of the shRNA-HMGB1, and the transgenic mouse model can be used for researching the effect of the HMGB1 in multiple organs and multiple diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a shRNA target site targeting HMGB1 gene, shRNA and application thereof, and a method for constructing a transgenic mouse model. Background Art

[0002] RNA interference (RNAi) is a very effective technology for specifically inhibiting the expression of target genes. Double-stranded RNAs (dsRNAs) formed by homologous sequences of target gene mRNA can activate the RNAi pathway. Short hairpin RNA transcription adopts a stable neck-loop structure scheme; cloned oligonucleotides can easily express shRNA, which is a more suitable and reproducible method for activating RNAi in mammalian cell lines.

[0003] Currently, an important method for studying gene function is gene knockout. Gene inactivation is achieved by interrupting the coding sequence of the target gene. Animal models carrying heterozygous and homozygous gene knockout can determine a specific gene function, but generating animal gene knockout models requires a lot of manpower, material and financial resources; another method to inhibit gene expression is gene silencing. The cellular process of RNAi has been used to effectively silence gene expression. RNAi is activated by the introduction of double-stranded RNA, the sequence of which is homologous to the sequence of the target gene. Endogenous RNA is digested into small interfering RNAs (siRNAs) composed of 21-23nt. siRNAs form an RNA-induced silencing complex (RISC) with the nuclease complex. RISC base pairs with the target endogenous gene and eliminates mRNA. Compared with traditional gene knockout methods, specific gene silencing can be achieved quickly and easily in animal and cell line models.

[0004] The HMGB1 gene plays a very important role in the development of the embryo. The functional diversity of the HMGB1 gene determines that this gene is indispensable. Mice with systemic knockout of HMGB1 will die within 24 hours after birth; the preparation cycle of tissue-specific knockout mice is long, time-consuming and laborious. There are also problems with silencing the expression of HMGB1 using RNAi technology, that is, if the expression level of HMGB1 in newborn mice is too low, it will also affect the growth and development of mice. Therefore, how to silence the expression of HMGB1 is an important issue that needs to be solved urgently. Summary of the invention

[0005] In view of the above problems, the object of the present invention is to provide a shRNA target site targeting the HMGB1 gene, shRNA and its application, and a method for constructing a transgenic mouse model.

[0006] The technical contents of the present invention are as follows:

[0007] The present invention provides a shRNA target targeting HMGB1 gene. The sequences of the shRNA target targeting HMGB1 gene are shown in SEQ ID NO.1-5, which are named shRNA-HMGB1-1, shRNA-HMGB1-2, shRNA-HMGB1-3, shRNA-HMGB1-4, and shRNA-HMGB1-5, respectively.

[0008] The present invention also provides a shRNA targeting the HMGB1 gene, which is synthesized based on the above shRNA target, and has two complementary sense strand and antisense strand sequences respectively;

[0009] The structure of the sense strand sequence is: 5' end - XhoI restriction enzyme cleavage site + target sequence sense strand + hairpin loop + target sequence antisense strand + terminator + MluI restriction enzyme cleavage site + residue - 3' end;

[0010] The antisense strand has a HindIII restriction enzyme site added to the 5' end, which then forms a sequence that is reverse complementary to the sense strand sequence;

[0011] The sense strand sequence is reversely complementary to the antisense strand sequence;

[0012] The sense sequence of the hairpin loop is TTCAAGAGA, and the antisense sequence is TCTCTTGAA;

[0013] The terminator is composed of 6 bp of thymine T;

[0014] The sequences of the sense strand and antisense strand synthesized based on shRNA-HMGB1-1 are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively;

[0015] The sequences of the sense strand and antisense strand synthesized based on shRNA-HMGB1-2 are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively;

[0016] The sequences of the sense strand and antisense strand synthesized based on shRNA-HMGB1-3 are shown in SEQ ID NO.10 and SEQ ID NO.11, respectively;

[0017] The sequences of the sense strand and antisense strand synthesized based on pshRNA-HMGB1-4 are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively;

[0018] The sequences of the sense strand and antisense strand synthesized based on shRNA-HMGB1-5 are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively.

[0019] The present invention also provides a recombinant plasmid targeting the HMGB1 gene, wherein the recombinant plasmid is a recombinant plasmid obtained by constructing the above shRNA sequence into a plasmid vector;

[0020] The plasmid vector includes a pSingle-tTS-shRNA vector;

[0021] The recombinant plasmids were named pSingle-tTS-shRNA-HMGB1-1, pSingle-tTS-shRNA-HMGB1-2, pSingle-tTS-shRNA-HMGB1-3, pSingle-tTS-shRNA-HMGB1-4, and pSingle-tTS-shRNA-HMGB1-5.

[0022] The present invention also provides a method for constructing the above-mentioned recombinant plasmid targeting the HMGB1 gene, comprising the following steps:

[0023] 1) synthesizing the above-mentioned shRNA targeting the HMGB1 gene;

[0024] 2) annealing the shRNA oligonucleotides from step 1) to form double-stranded DNA fragments;

[0025] 3) Double enzyme digestion and recovery and purification of the expression vector;

[0026] 4) connecting the double-stranded DNA fragment to the vector purified in step 3) to obtain a recombinant plasmid;

[0027] 5) Transforming and expressing the recombinant plasmid through competent cells;

[0028] The expression vector includes a pSingle-tTS-shRNA vector.

[0029] The present invention also provides the use of the above siRNA or shRNA or recombinant plasmid targeting the HMGB1 gene in the preparation of an HMGB1 gene expression inhibitor.

[0030] The present invention also provides the use of the above siRNA or shRNA or recombinant plasmid targeting the HMGB1 gene in the preparation of a drug for treating organ fibrosis.

[0031] The present invention also provides a method for constructing a HMGB1 low-expression transgenic mouse model, comprising the following steps:

[0032] 1) amplifying the above recombinant plasmid;

[0033] The primer sequences for the amplification are shown in SEQ ID NOs. 16 to 21;

[0034] 2) The recombinant plasmid was digested with three enzymes to recover the vector and extract the target gene shRNA-HMGB1;

[0035] 3) The target gene is transferred into recipient female mice by injection to obtain a transgenic mouse model expressing shRNA-HMGB1;

[0036] The primer sequences used for identifying transgenic mice are shown in SEQ ID NOs. 22-23.

[0037] The beneficial effects of the present invention are as follows:

[0038] The shRNA target, shRNA and recombinant plasmid targeting the HMGB1 gene of the present invention can specifically and efficiently inhibit the expression of the HMGB1 gene. The Knockout Single Vector Inducible RNAi System is used to successfully construct a mouse model expressing shRNA-HMGB1 through transgenic technology. The Tet-on system is used to add doxycycline to the drinking water so that the mouse can specifically downregulate the expression of HMGB1 at a specific time, and the expression of HMGB1 can be significantly downregulated, thereby overcoming the expression barrier of the HMGB1 gene during the development period of newborn mice, and cleverly avoiding the problem that newborn mice without HMGB1 expression cannot survive. The whole body organs of the obtained transgenic mouse model have the expression of shRNA-HMGB1. The transgenic mouse model can be used to study the role of HMGB1 in multiple organs and multiple diseases (such as fibrotic diseases of organs). The tool mouse can be used to obtain animal models of multiple organ diseases, providing a research platform for subsequent research on the function of HMGB1 in immune damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the sense and antisense strand sequences of shRNA-HMGB1 oligonucleotide;

[0040] Figure 2 This is the PCR identification result of the recombinant plasmid colony;

[0041] Figure 3 The figure is the result of double enzyme digestion identification of the recombinant plasmid;

[0042] Figure 4 This is the result of sequencing and identification of the recombinant plasmid nucleotide sequence;

[0043] Figure 5This is a diagram showing the PCR screening results of effective RNAi target sequences of pSingle-tTS-shRNA-HMGB1-5;

[0044] Figure 6 Schematic diagram of triple restriction enzyme digestion of pSingle-tTS-shRNA-HMGB1-5;

[0045] Among them, A: the main components and restriction sites of the pSingle-tTS-shRNA-HMGB1-5 recombinant vector; B: the main components of the target DNA after triple restriction digestion;

[0046] Figure 7 Schematic diagram of toe numbering for shRNA-HMGB1 transgenic mice;

[0047] Figure 8 This is a diagram of the identification results of transgenic mice;

[0048] Fig. 9 This is the result of Western Blot detection of HMGB1 expression. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0050] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0051] The reagents used in the present invention are as follows:

[0052] 1.LB medium: Tryptone 10g, Yeast extract 15g, NaCl 10g, adjust to pH 7.5, make up to 1000mL, sterilize with high pressure steam, and store at 4℃.

[0053] 2.LB solid medium: Tryptone 10g, Yeast extract 15g, NaCl 10g, agar powder 15g, adjust to pH 7.5, dilute to 1000mL, sterilize with high pressure steam. After cooling slightly, add antibiotics, inject into sterile culture plate, wait for cooling, and store at 4℃.

[0054] 3.LB recombinant plasmid screening plate: Add ampicillin when plating LB solid culture medium, with a final concentration of 100 μg / mL.

[0055] 4. PBS buffer: KCl 0.2 g, KH2PO4 0.24 g, NaCl 8.0 g, Na2HPO4·12H2O 2.08 g, ddH2O to 1000 mL;

[0056] Autoclave and store at 4°C.

[0057] 5.50×TAE Buffer (pH 8.5): Tris base 242 g, EDTA 37.2 g, acetic acid 57.1 mL, ddH2O to 1000 mL;

[0058] Stir well to mix and store at room temperature.

[0059] 6.5×TBE electrophoresis buffer: Tris base 54 g, boric acid 27.5 g, EDTA 3.72 g, ddH2O to 800 mL;

[0060] Adjust to pH 8.3 and make up to 1000 mL with ddH2O.

[0061] 7.10×TBS: Tris·base 34.2 g, NaCl 80 g, concentrated HCl adjusted to pH 7.6, ddH2O to 1000 mL, autoclave;

[0062] 1×TBS / T: Dilute 10×TBS 10-fold and add 1 mL of Tween-20 per 1000 mL of TBS.

[0063] 8.1mol / L Tris Cl: Tris·base 12.11g, concentrated HCL adjusted to pH 6.8, ddH2O to 100mL.

[0064] 9.1.5mol / L Tris Cl: Tris·base 18.165g, concentrated HCl adjusted to pH 8.8, ddH2O to 100mL.

[0065] 10.10% SDS: SDS 10 g, adjusted to pH 7.2 with concentrated HCl, and made up to 100 mL with ddH2O.

[0066] 11. Transfer solution: Tris.HCl 3.375g, Glycine 15.014g, Methanol 200mL, HCL adjusted to pH 8.5, ddH2O to 1000mL.

[0067] 12. Western-Blot blocking buffer: 5 g skim milk powder, 1×TBS / T 100 mL, mix well, and store at 4°C.

[0068] 13. 30% acrylamide gel buffer: 29 g acrylamide, 1 g N,N′-methylenebisacrylamide, ddH2O to 100 mL;

[0069] Filter through 0.45 μm filter membrane and store in brown bottle at 4°C.

[0070] 14.5× Tris-glycine electrophoresis buffer: Tris.base 15.1 g, glycine 94 g, 10% SDS 50 mL, ddH2O to 1000 mL.

[0071] 15.5% SDS polyacrylamide gel electrophoresis separation gel (10 mL): 30% acrylamide solution 0.83 mL, 1.0 mol / L Tris.HCl (pH 6.8) 0.63 mL, 10% SDS 0.05 mL, 10% ammonium persulfate 0.05 mL, ddH2O 3.4 mL, TEMED 0.005 mL.

[0072] 16.5×SDS-PAGE loading buffer: 1 mol / L Tris·HCl (pH 6.8) 0.6 mL, 10% SDS 2 mL, 1% bromofen blue 1 mL, 50% glycerol 5 mL, 2-mercaptoethanol 0.5 mL, ddH2O to 10 mL.

[0073] Example

[0074] 1. Design of shRNA

[0075] 1.1 Select shRNA target sequence

[0076] Mouse HMGB1 nucleotide sequence (NCBI Reference Sequence: NM_010439.3) was obtained through the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ). TM The website's online shRNA design software was used to design and select appropriate shRNA oligonucleotide sequences. The sequences of the selected five shRNA targets, shRNA-HMGB1-1, shRNA-HMGB1-2, shRNA-HMGB1-3, shRNA-HMGB1-4, and shRNA-HMGB1-5, are shown in SEQ ID NOs. 1-5, respectively.

[0077] 1.2 Design of shRNA oligonucleotides

[0078] Two complementary oligonucleotide sequences are synthesized for each shRNA target sequence, and the oligonucleotide sequence inserted into the pSingle-tTS-shRNA vector must have a unique sequence structure.

[0079] The sequence structure is as follows:

[0080] 1) Add an XhoI restriction enzyme site to the 5'-end of the sense strand and a HindIII restriction enzyme site to the 5'-end of the antisense strand. These two restriction enzyme sites can directly clone the annealed oligonucleotides into the XhoI / HindIII restriction enzyme sites of the pSingle-tTS-shRNA vector;

[0081] 2) Add 19 bp of the target sense strand downstream of the XhoI restriction site on the sense strand;

[0082] 3) 9 bp hairpin loop sequence;

[0083] 4) 19 bp target antisense strand;

[0084] 5) RNA polymerase III terminator sequence containing 6 Ts;

[0085] 6) Add MLuI (5'-ACGCGT-3') restriction enzyme site to verify whether the inserted sequence is correct;

[0086] 7) Finally, an adenine residue (A) is added.

[0087] Starting from the 5'-end, a typical sense chain oligonucleotide should consist of the following parts: a complete XhoI (5bp) cloning site, a 19bp sense chain, a 9bp hairpin loop, a 19bp anti-sense chain, a 6bp thymine (T) terminator, a 6bp restriction enzyme site MLuI, and the last one is an adenine residue (A).

[0088] The sense sequence of the hairpin loop is TTCAAGAGA, and the antisense sequence is TCTCTTGAA;

[0089] Invitrogen (USA) was commissioned to synthesize the sense and antisense strands of each oligonucleotide. The oligonucleotide sequence constructed in the pSingle-tTS-shRNA vector must have a unique sequence structure such as Figure 1 As shown;

[0090] The sequences of the sense strand and antisense strand synthesized based on shRNA-HMGB1-1 are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively;

[0091] The sequences of the sense strand and antisense strand synthesized based on shRNA-HMGB1-2 are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively;

[0092] The sequences of the sense strand and antisense strand synthesized based on shRNA-HMGB1-3 are shown in SEQ ID NO.10 and SEQ ID NO.11, respectively;

[0093] The sequences of the sense strand and antisense strand synthesized based on shRNA-HMGB1-4 are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively;

[0094] The sequences of the sense strand and antisense strand synthesized based on shRNA-HMGB1-5 are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively.

[0095] 2. pSingle-tTS-shRNA vector cloning

[0096] 2.1 Nucleotide annealing to form double-stranded DNA fragments

[0097] 1) Dissolve the oligonucleotide in sterile water to a final concentration of 100 μM;

[0098] 2) Mix the sense strand oligonucleotide and the antisense strand oligonucleotide in a ratio of 1:1, with the final concentration of each oligonucleotide being 50 μM, and mix well;

[0099] 3) 95°C, 30 seconds, start intermolecular annealing;

[0100] 4) 72°C, 2 min;

[0101] 5) 37°C, 2 min;

[0102] 6) 25°C, 2 min;

[0103] 7) Place on ice.

[0104] The annealed oligonucleotides were stored at -20°C for future use.

[0105] 2.2 Enzyme digestion reaction of pSingle-tTS-shRNA vector

[0106] The pSingle-tTS-shRNA vector was double-digested with XhoI and HindIII restriction endonucleases, and the reaction system is shown in Table 1:

[0107] Table 1 Double enzyme digestion reaction system

[0108]

[0109] Incubate in a 37°C water bath for 6 hours. After the reaction is complete, the digestion products are detected by agarose electrophoresis. The results show that the fragments obtained by double digestion are basically the same size as the expected fragments, indicating that the pSingle-tTS-shRNA vector was successfully constructed.

[0110] 2.3 Double enzyme digestion vector recovery

[0111] EZgene TM The double-enzyme-digested pSingle-tTS-shRNA vector was recovered and purified using the Gel Extraction Kit.

[0112] The recovery and purification steps are as follows:

[0113] 1) Cut the agarose gel containing the pSingle-tTS-shRNA vector, and try to weigh as little gel as possible, and add Buffer GC at a ratio of gel: Buffer GC (m:v) = 1:1;

[0114] 2) Incubate at 55°C for 8 min, shake 2-3 times in a water bath, and after complete dissolution, cool to room temperature, add 15 μL of solution B, and mix thoroughly;

[0115] 3) Place the carrier / Buffer GC mixture in a centrifugal column, let stand for 2 min, and centrifuge at 10,000 g for 1 min;

[0116] 4) Discard the filtrate, add 500 mL Wash Buffer to the centrifuge column, centrifuge at 10,000 g for 30 seconds, and discard the filtrate;

[0117] 5) Repeat step 4) once;

[0118] 6) Centrifuge the empty centrifugal column at 13000 g for 2 min to remove residual alcohol;

[0119] 7) Place the centrifuge column in a new nuclease-free 1.5 mL sterile centrifuge tube and leave it for 10 minutes until the alcohol evaporates;

[0120] 8) Take 30 μL of preheated (60°C) sterile ddH2O and place it in the center of the centrifuge column. Let it stand for 2 minutes. Centrifuge at 15000g for 1 minute. The eluted solution is the desired DNA solution. Transfer the DNA solution to the centrifuge column and centrifuge again for the second elution.

[0121] 9) The purified vector solution was stored at -20°C for future use.

[0122] 2.4 Ligation of shRNA-HMGB1 fragment and pSingle-tTS-shRNA expression vector

[0123] The ligation reaction of shRNA-HMGB1 and pSingle-tTS-shRNA was carried out in a 200 μL centrifuge tube. The reaction system is shown in Table 2.

[0124] Table 2 Ligation reaction system

[0125]

[0126] After mixing, centrifuge at 16°C overnight. Store the ligation product at -20°C for later use.

[0127] The obtained recombinant plasmids were named pSingle-tTS-shRNA-HMGB1-1, pSingle-tTS-shRNA-HMGB1-2, pSingle-tTS-shRNA-HMGB1-3, pSingle-tTS-shRNA-HMGB1-4, and pSingle-tTS-shRNA-HMGB1-5, and the nucleic acid sequences were as shown in SEQ ID NOs. 6-15, respectively.

[0128] 3. Preparation of DH5α competent cells

[0129] 1) Culture DH5α on LB medium plates at 37°C overnight;

[0130] 2) Inoculate a single DH5α colony into 3 mL LB liquid medium and culture overnight at 37°C with shaking at 250 rpm;

[0131] 3) Take 50 μL of DH5α bacterial solution and transfer it to 5 mL of LB liquid medium. Cultivate at 250 rpm and 37°C until OD 600 =0.3-0.4;

[0132] 4) Incubate on ice for 15 min, dispense into 2 mL centrifuge tubes, centrifuge at 4000 g, 4°C, for 10 min to collect the cells;

[0133] 5) Discard the supernatant, invert the centrifuge tube for 1 min, and add 400 μL of pre-cooled 0.1 mol / L CaCl2 to suspend the bacteria;

[0134] 6) Ice bath for 30 min, 4000 rpm, 4°C, 7 min;

[0135] 7) Discard the supernatant, add 200 μL of pre-cooled 0.1 mol / L CaCl2, carefully suspend the cells, and store at 4°C.

[0136] 4. Transformation of Recombinant Plasmid

[0137] Take 10μL of the above recombinant plasmid to 200μL pre-cooled competent cells, gently rotate the centrifuge tube, mix, ice bath for 30min, heat shock at 42℃ for 90sec, quickly transfer to ice, ice bath for 2-3min, transfer competent cells to 800μLL LB liquid culture medium without Amp, 150rpm, 37℃, shake culture for 1h. Take 200μL bacterial solution and evenly spread it on the LB plate containing 50μg / mL Amp. Place it upright in a 37℃ incubator until the bacterial solution is completely absorbed, then invert the plate and culture overnight.

[0138] 5. Screening of recombinant transformants

[0139] A small single colony was picked from the above culture plate, inoculated into 5 mL of LB liquid culture medium containing 50 μg / mL Amp, and cultured at 37° C. with shaking overnight.

[0140] 6. Identification of recombinant plasmid

[0141] 6.1 PCR identification of the bacterial culture in step 5

[0142] Primer sequences:

[0143] U6F (SEQ ID NO. 16): 5'-GAGAACGATGTCGAGTTTA-3';

[0144] HMGB1-1R (SEQ ID NO.17): 5'-TTATTCTCTTGAAATAACG-3';

[0145] HMGB1-2R (SEQ ID NO.18): 5'-ATGTTCCTTGAAACATC-3';

[0146] HMGB1-3R (SEQ ID NO.19): 5'-ATACTAAGTTTCCTAGTA-3';

[0147] HMGB1-4R (SEQ ID NO.20): 5'-TTCTTCTCTTGAAAGAAG-3';

[0148] HMGB1-5R (SEQ ID NO. 21): 5'-ACATTAAGTTCTCTAATG-3'.

[0149] The reaction system of bacterial liquid PCR identification is shown in Table 3 and Table 4:

[0150] Table 3 Reaction system

[0151]

[0152] Table 4 Reaction parameters

[0153]

[0154] After the PCR reaction was completed, 3 μL of PCR product was taken for agarose gel electrophoresis.

[0155] The genomic DNA of the above-mentioned cultured bacterial solution was used as a template to identify the recombinant plasmid. The estimated size of the PCR product was 313 bp. The electrophoresis results were as follows: Figure 2 (M: DNA marker; 1, 2: pSingle-Tts-shRNA-HMGB1-1; 3, 4: pSingle-Tts-shRNA-HMGB1-2; 5, 6 pSingle-Tts-shRNA-HMGB1-3; 7, 8: pSingle-Tts-shRNA-HMGB1-4; 9, 10: pSingle-Tts-shRNA-HMGB1-5). It can be seen from the figure that the size of the obtained PCR fragment is 313 bp, which is consistent with the expected size, indicating that the target fragment has been correctly cloned into the expression vector pSingle-tTS-shRNA.

[0156] 6.2 Double enzyme digestion identification of recombinant plasmid

[0157] 1) Take 5 mL of the bacterial culture solution from step 5 and use Plus SV Minipreps DNA Purification System kit was used to extract and purify the recombinant plasmid according to the kit instructions and was appropriately optimized. The extraction and purification steps are as follows:

[0158] 2) Take 5 mL of bacterial solution into a 10 mL centrifuge tube, centrifuge at 10 000 g for 5 min, and discard the supernatant;

[0159] 3) Add 250 μL Cell Resuspension Solution to resuspend the cells;

[0160] 4) Add 250 μL Cell Lysis Solution, mix well, shake the centrifuge tube vigorously (do not use a vortex shaker) 4 times, leave at room temperature, and incubate until the bacterial suspension is transparent;

[0161] 5) Add 10 μL Alkaline Protease Solution, mix well, shake the centrifuge tube vigorously 4 times, and incubate at room temperature for 5 minutes.

[0162] 6) Add 350 μL Neutralization Solution, mix immediately, and shake vigorously 4 times;

[0163] 7) Centrifuge the bacterial lysate at room temperature, 14000g, for 10 min.

[0164] 8) Take the supernatant and transfer it to a centrifuge column.

[0165] 9) 14000g, 1 min, room temperature, discard the filtrate.

[0166] 10) Add 750 μL Column Wash Solution, incubate at 14,000 g for 1 min at room temperature, and discard the filtrate.

[0167] 11) Repeat step 9);

[0168] 12) 14000g, 2min, room temperature, to remove residual alcohol;

[0169] 13) Transfer the spin column to a new sterile, nuclease-free 1.5 mL centrifuge tube;

[0170] 14) Add 100 μL sterile ddH2O, let stand at room temperature for 2 min, 14000g, 1 min, room temperature. The eluate is the desired recombinant plasmid solution;

[0171] 15) Store at -20℃ for future use.

[0172] The recombinant plasmid pSingle-tTS-shRNA-HMGB1-X was subjected to double restriction enzyme digestion identification, and the restriction enzyme digestion reaction system is shown in Table 5:

[0173] Table 5 Enzyme digestion reaction system

[0174]

[0175]

[0176] Incubate in 37°C water bath for 6 h. After the reaction is complete, take 3 μL of the digested product for agarose electrophoresis detection.

[0177] The recombinant plasmid DNA was extracted from the transformants identified as positive clones by colony PCR. The recombinant plasmids of the transformants identified as positive clones by PCR were double-digested with Mlu I and BamH I to further confirm that the inserted fragment was the target fragment. Figure 3(M: DNA marker; 1: pSingle-tTS-shRNA-HMGB1-1; 2: pSingle-tTS-shRNA-HMGB1-2; 3: pSingle-tTS-shRNA-HMGB1-3; 4: pSingle-tTS-shRNA-HMGB1-4; 5: pSingle-tTS-shRNA-HMGB1-5). As can be seen from the figure, the fragments obtained by double enzyme digestion are basically the same size as the expected fragments, which can further confirm that the target fragment has been correctly connected to the pSingle-tTS-shRNA vector.

[0178] 6.3 Sequencing and identification of recombinant plasmids

[0179] The recombinant plasmids that were positive clones identified by bacterial liquid PCR and double enzyme digestion were commissioned to Invitrogen for sequencing.

[0180] The recombinant plasmids that were positive clones after bacterial liquid PCR and double enzyme digestion were sequenced for nucleotide sequence identification. Figure 4 (AE are pSingle-tTS-shRNA-HMGB1-1-5, respectively). The sequencing results showed that the cloned target fragment was correctly inserted into the pSingle-tTS-shRNA vector.

[0181] 7. Screening of effective shRNA target sequences

[0182] 7.1 Cultivation of human lung adenocarcinoma epithelial cell line A549

[0183] A549 cells were routinely cultured in DMEM low-glucose medium (10% fetal bovine serum, 100 IU penicillin, 100 μg / mL streptomycin solution, 2 mM glutamine), 5% CO2, 37°C constant temperature incubator, and the medium was replaced every 48 hours.

[0184] When A549 cells grew to 70-80% confluence, they were digested with 2.5‰ trypsin and subcultured at a ratio of 1:3.

[0185] 7.2 Transfection of A549 cells

[0186] 1) One day before transfection, A549 cells were cultured in 24-well plates, with 0.5×10 cells per well. 5 -2×10 5 , the culture medium must be free of antibiotics;

[0187] When transfecting cells, ensure that the cell confluence reaches 90%-95%;

[0188] 2) Dilute 0.8 μg DNA (pSingle-tTS-shRNA-HMGB1-X / pSingle-tTS-shRNA:pCDNA-HMGB1=2:1) ​​in 50 μL serum-free medium and mix gently;

[0189] 3) Gently mix Lipfentamine TM 2000, dilute 1.6 μL of serum-free medium, total volume 50 μL, mix gently, and incubate at room temperature for 5 min (the next step must be performed within 25 min);

[0190] 4) Mix the mixture according to the ratio of DNA (μg): LipfentamineTM 2000 (μL) = 1:2, mix gently, and incubate at room temperature for 20 min;

[0191] 5) Add 100 μL of DNA and LipfentamineTM 2000 mixture to each cell culture well and mix the mixture by shaking the culture plate back and forth;

[0192] 6) Place the culture plate in a 37°C constant temperature incubator with 5% CO2 for 6-8 hours and replace the plate with a mixture of DNA and Lipfentamine TM 2000 culture medium;

[0193] The expression of the transgene can be detected after 18h-48h of culture.

[0194] 7.3 Extraction of total RNA from A549 cells

[0195] Use Invitrogen Reagent was used to extract RNA, and the steps were modified according to the instruction manual.

[0196] 1) Remove the cell culture medium and gently wash the cells 3 times with RNase-free PBS to remove as much PBS as possible;

[0197] 2) Add 0.2 mL of Trizol to each well and incubate at room temperature for 5 min to completely lyse the nuclear protein;

[0198] 3) Add 0.04 mL of chloroform (chloroform (v): Trizol (v) = 1:5) and mix well;

[0199] 4) Shake vigorously for 15 seconds;

[0200] 5) Incubate at room temperature for 2-3 minutes;

[0201] 6) 12000g, 15min, 4℃;

[0202] 7) Tilt 45° and transfer the supernatant to an RNase-free centrifuge tube;

[0203] 8) Add 0.1 mL of isopropanol (isopropanol (V): Trizol (V) = 1:2), mix well, and incubate at room temperature for 10 min;

[0204] 9) 12000g, 10min, 4℃;

[0205] 10) Remove the supernatant, add 0.2 mL of 75% alcohol (alcohol (V): Trizol (V) = 1:1), and vortex to mix;

[0206] 11) 7500 g, 5 min, 4 °C;

[0207] 12) Air-dry RNA and place at room temperature for 5-10 minutes;

[0208] 13) Add 20 μL RNase-free water and pipette several times to dissolve the RNA;

[0209] 14) 55-60℃ water bath for 10-15min;

[0210] 15) RNA was stored at -70°C until use.

[0211] 7.4 Synthesis of first-strand cDNA

[0212] RNA reverse transcription was performed according to the instructions of SuperScripTMIII Reverse Transcriptase and improved. The specific steps are as follows:

[0213] 1) Add the reagents shown in Table 6 to the Nuclease-free PCR tube:

[0214] Table 6 Reagents

[0215]

[0216] 2) Incubate in a water bath at 65°C for 5 min and place on ice for at least 1 min;

[0217] 3) Centrifuge briefly and add the reagents shown in Table 7 to the PCR tube:

[0218] Table 7 Reagents

[0219]

[0220] 4) Mix gently and centrifuge;

[0221] 5) Perform the following program on the PCR instrument: 50°C, 60 min;

[0222] 6) 70°C, 15 min, terminate the reaction;

[0223] The reaction product was stored at -20°C for future use.

[0224] 7.5 PCR reaction to screen effective shRNA target sequences

[0225] The PCR reaction was performed with reference to the PCR Amplification Kit and improved. The components shown in Table 8 were added to the PCR tube:

[0226] Table 8 Reagents

[0227]

[0228] Perform the reaction program shown in Table 9 on the PCR instrument:

[0229] Table 9 Reaction procedure

[0230]

[0231] After the reaction was completed, 3 μL of PCR product was taken for electrophoresis detection.

[0232] The recombinant plasmid pSingle-tTS-shRNA-HMGB1-X / pCDNA-HMGB1 was co-transfected into A549 cells, doxycycline was added to the culture medium (final concentration was 2 mg / mL), total cell RNA was extracted, and RT-PCR identification and screening were performed. The RT-PCR results are shown in Figure 5 From the figure, we can see that the four recombinant vectors pSingle-tTS-shRNA-HMGB1-1-4 basically showed no interference effect, while the recombinant vector pSingle-tTS-shRNA-HMGB1-5 showed a strong interference effect, and its PCR band brightness was significantly lower than that of the other four treatment groups. Therefore, pSingle-tTS-shRNA-HMGB1-5 was selected as an effective RNAi recombinant plasmid.

[0233] The recombinant plasmid that was detected to be able to significantly interfere with the expression of HMGB1 was stored in the form of glycerol bacteria at -80°C for future use.

[0234] 8. Preparation of shRNA-HMGB1 Transgenic Mice

[0235] The preparation of shRNA-HMGB1 transgenic mice was commissioned to the Model Animal Institute of Nanjing University. The main production process is as follows.

[0236] 8.1 Amplification of the recombinant vector pSingle-tTS-shRNA-HMGB1-5

[0237] The detection results of step 7 showed that pSingle-tTS-shRNA-HMGB1-5 had the best RNAi effect, and the pSingle-tTS-shRNA-HMGB1-5 recombinant vector was intended to be used as the insert DNA for preparing transgenic mice.

[0238] The amplification method of the pSingle-tTS-shRNA-HMGB1-5 recombinant vector is as follows:

[0239] 1) DH5α containing the pSingle-tTS-shRNA-HMGB1-5 recombinant vector was streaked and cultured on LB solid medium at 37°C overnight;

[0240] 2) Pick a single DH5α colony and transfer it to LB liquid medium for culture at 37°C with shaking overnight.

[0241] 8.2 Extraction of pSingle-tTS-shRNA-HMGB1-5 recombinant vector

[0242] The method is the same as step 6.2 double enzyme digestion identification of recombinant plasmid

[0243] 8.3 Triple enzyme digestion of pSingle-tTS-shRNA-HMGB1-5 recombinant vector

[0244] The sequence of the prokaryotic expression vector will seriously affect the expression of the transgene, so the backbone sequence of the recombinant vector was removed, and the expression of shRNA was not affected by the recombinant vector after enzyme digestion. Through the enzyme spectrum analysis of pSingle-tTS-shRNA-HMGB1-5, the three restriction enzyme sites of PvuII, ApaII and NaeI were the most suitable, and it was decided to use a three-enzyme digestion scheme. The length of the recovered fragment after enzyme digestion was 3659bp. Figure 6 shown.

[0245] The enzyme digestion reaction system is shown in Table 10:

[0246] Table 10 Enzyme digestion reaction system

[0247]

[0248] Incubate in a 37°C water bath for 6 hours. After the reaction is complete, 3 μL of the digestion product is taken for agarose electrophoresis detection. The results show that the fragments obtained by double digestion are basically the same size as the expected fragments, indicating that the pSingle-tTS-shRNA-HMGB1-5 recombinant vector was successfully constructed.

[0249] 8.4 Recovery of triple-enzyme digested vector

[0250] The recovery and purification steps are the same as 2.3.

[0251] EZgeneTM Gel Extraction Kit was used to recover and purify the double-digested pSingle-tTS-shRNA vector. The recovery and purification steps were as follows according to the kit instructions and slightly optimized:

[0252] 1) Cut the agarose gel containing the target DNA fragment, and try to weigh as little gel as possible, and add Buffer GC at a ratio of gel: Buffer GC (m:g) = 1:1;

[0253] 2) Incubate at 55°C for 8 min, shake 2-3 times in a water bath, and after complete dissolution, cool to room temperature, add 15 μL of solution B, and mix thoroughly;

[0254] 3) Place the carrier / Buffer GC mixture in a centrifugal column, let stand for 2 min, and centrifuge at 10,000 g for 1 min;

[0255] 4) Discard the filtrate, add 500 μL Wash Buffer to the centrifuge column, centrifuge at 10,000 g for 30 seconds, and discard the filtrate;

[0256] 5) Repeat step 4) once;

[0257] 6) Centrifuge the empty centrifugal column at 13000 g for 2 min to remove residual alcohol;

[0258] 7) Place the centrifuge column in a new 1.5 mL sterile centrifuge tube and leave it for 10 minutes until the alcohol evaporates;

[0259] 8) Take 100 μL of preheated (60°C) nuclease-free ddH2O, place it in the center of the centrifuge column, and let it stand for 2 minutes;

[0260] 9) 15000g, 1 min, the eluted solution is the desired DNA solution, the DNA solution is transferred to the centrifugal column, centrifuged again, and eluted for the second time;

[0261] 10) The DNA solution was stored at -20°C for future use.

[0262] Note: The above experiment uses ethidium bromide (EB) as the dye for nucleic acid electrophoresis, but EB is highly toxic to cells, so EB contamination of nucleic acids should be avoided during nucleic acid electrophoresis. Take the following measures: Do not add EB when preparing agarose gel, and divide it into a marker lane, a small hole lane and a large hole lane during electrophoresis. After the end, cut the small hole lane and the marker lane, and stain the small hole lane and the marker lane with EB. After staining, place the small hole lane and the marker lane gel side by side with the large hole lane gel but not in contact. Cut the target DNA fragment of the large hole lane according to the position of the target DNA band in the small hole lane, and recover and purify the target DNA.

[0263] 8.5 Preparation of ligated male mice

[0264] The donors used in this example are all C57BL / 6 inbred mice, and the steps for preparing ligated male mice are as follows:

[0265] 1) 4-week-old C57BL / 6 male mice, weighed;

[0266] 2) Intraperitoneal injection of sodium pentobarbital (0.8 mg / kg) was performed for anesthesia, and the rat was placed on the operating table with the abdomen facing upward and the limbs fixed;

[0267] 3) Gently press the mouse abdomen to push the testicle into the scrotum, cut the fine hair on the scrotum with ophthalmic curved scissors, and disinfect the scrotal skin with an alcohol cotton ball;

[0268] 4) Make a small incision of about 0.5 cm in the middle and lower 1 / 3 of the right scrotum near the midline, cut open the testicular capsule, and a pale vas deferens can be seen on the inner side of the testicle. Use ophthalmic forceps to clamp it out, and use red-hot ophthalmic curved forceps to cauterize it. Then cauterize a small section of the vas deferens and return the broken end to the testicular capsule;

[0269] 5) Perform the same treatment on the left vas deferens;

[0270] 6) Use sutures to sew up the scrotal skin and disinfect the wound with an alcohol cotton ball;

[0271] 7) To prevent infection, a mixed antibiotic solution of penicillin and streptomycin can be injected intraperitoneally;

[0272] 8) Return the operated mice to their cages, wait for them to regain consciousness, and return them to the animal room for close observation for 2 days;

[0273] 9) When the mice grow to 8 weeks old, place them in a cage with sexually mature female mice and observe whether the female mice are pregnant to determine whether the operation is successful. Those with successful operation can be used to prepare pseudo-pregnant female mice.

[0274] 9. Preparation of Donor Female Mice and Recipient Mice

[0275] 1) At 16:00 pm 3 days before microinjection of fertilized eggs, 10 3-week-old C57BL / 6 female mice were selected and intraperitoneally injected with PMSG (10 μL / mouse) to serve as fertilized egg donor mice;

[0276] 2) One day before microinjection of fertilized eggs, at 16:00 in the afternoon, the donor female mice were intraperitoneally injected with HCG, 10 IU / mouse, and were placed in cages with C57BL / 6 male mice. The mice were observed the next morning, and those with vaginal plugs could be used as fertilized egg donor mice.

[0277] 3) Select 10-20 10-week-old naturally estrus C57BL / 6 female mice and place them in cages with C57BL / 6 neutered male mice. Observe them the next morning. Those with vaginal plugs can be used as recipient mice for fertilized eggs.

[0278] 10. Collection and processing of fertilized eggs

[0279] 1) Ultraviolet disinfection of oral pipettes, culture dishes, clean benches, and operating rooms for 30 minutes;

[0280] 2) Drop the M16 solution on the culture dish, cover it with paraffin oil, and equilibrate it in a cell culture incubator for 1-2 hours, 5% CO2, 37°C;

[0281] 3) Sacrifice the fertilized egg donor mouse by cervical dislocation, open the abdominal cavity, remove the oviduct, and place it in M2 culture medium;

[0282] 4) Use a dissecting needle to puncture the ampulla of the fallopian tube and remove the fertilized egg;

[0283] 5) Digestion of granulosa cells with hyaluronidase (0.3%);

[0284] 6) Wash the digested fertilized eggs 4-8 times with fresh M2 to remove granulosa cells, residues and hyaluronidase;

[0285] 7) The washed fertilized eggs were transferred to M16 droplets covered with mineral oil and cultured in a 5% CO2, 37°C incubator.

[0286] 11. Gene Injection

[0287] 1) Take out the quantitative target DNA stored at -20℃, melt it at room temperature, then invert the injection needle into the EP tube containing the target DNA, absorb the target DNA into the top of the injection needle, and carefully install the injection needle on the right needle holder;

[0288] 2) Install the egg holding needle onto the left needle holder and apply pressure to fill the egg holding needle with paraffin oil;

[0289] 3) Place a drop of M2 on a petri dish, cover it with paraffin oil, and then place it on the stage of an inverted microscope;

[0290] 4) Transfer about 20 fertilized eggs from the M16 droplet to the M2 droplet, hold the egg needle and adjust the position of the fertilized egg so that the male pronucleus is located on the side close to the injection needle, fine-tune the focus of the microscope until the outline of the male pronucleus is clearly visible, and the injection needle pierces the zona pellucida and cell membrane into the nucleolar space of the pronucleus. Apply pressure, and after the pronucleus swells, immediately withdraw the needle, then replace another fertilized egg and continue to inject the gene;

[0291] 5) After all the fertilized eggs have been injected, they are transferred to M16 droplets and cultured in a 5% CO2, 37°C incubator.

[0292] 12. Embryo Transfer

[0293] 1) The prepared female recipient mice were intraperitoneally injected with 1% sodium pentobarbital solution to anesthetize the mice; the anesthetized female recipient mice were placed on the operating table, the fallopian tubes of the recipient female mice were surgically removed, and the fallopian tube transplantation was performed under a stereo microscope. After completion, the muscles and skin were sutured with silk thread, and the skin was disinfected once with 75% alcohol;

[0294] 2) After the transplant surgery, the mice were returned to the cages and the cages were heated on a hot plate at 55°C until the mice woke up and were then returned to the animal room.

[0295] 13. Identification of shRNA-HMGB1 transgenic mice

[0296] 13.1 Primer Design for Identification of shRNA-HMGB1 Transgenic Mice

[0297] Seven days after birth, the mice were numbered by clipping their toes and cutting their tails for genomic DNA extraction for gene identification. Figure 7 shown.

[0298] Primer 5 was used to design primers for identifying shRNA-HMGB1 transgenic mice. The primer sequences are shown in SEQ ID NOs. 22-23, respectively, as follows:

[0299] HMGB1-checkF:5'-TAGGCGTGTACGGTCATATGCTTACC-3';

[0300] HMGB1-checkR:5'-TCTCGCCAAGCTCATTAATGCAGAC-3';

[0301] The length of the amplified fragment of the positive mouse was 230 bp, while there was no amplified fragment in the wild type (WT).

[0302] 13.2 Mouse tail genome preparation

[0303] 1) Ophthalmic scissors, forceps, and 1.5 mL centrifuge tubes are sterilized by high pressure;

[0304] 2) Cut tail tissue of 3-week-old mice, about 0.5 cm long, and place it in a 1.5 mL sterile centrifuge tube. Label the tube wall and cut the mouse toes and number them. Place the centrifuge tube containing the specimen on ice to keep it low.

[0305] After collecting a mouse specimen, the scissors should be treated with high temperature and wiped with alcohol to avoid contamination of the next mouse specimen;

[0306] 3) Add 100 μL of mouse tail tissue lysis buffer A, and the tail tissue should be completely immersed in the lysis buffer;

[0307] 4) Incubate at 95°C for 20 min. Centrifuge after incubation to allow the lysate on the tube wall to flow back to the bottom of the tube;

[0308] 5) Add 100 μL of mouse tail tissue lysis buffer B and vortex for 30 seconds;

[0309] 6) 10000g, 10min, 4°C;

[0310] 7) The supernatant is the desired mouse tail tissue DNA solution, and it is transferred to a new sterile 1.5 mL centrifuge tube;

[0311] 8) Store at -20℃ for future use.

[0312] 13.3 PCR Identification

[0313] Add the components shown in Table 11 to the PCR tube:

[0314] Table 11 PCR reaction system

[0315]

[0316] Execute the reaction program shown in Table 12 on the PCR instrument:

[0317] Table 12 PCR reaction system

[0318]

[0319] After the reaction is completed, 3 μL of PCR product is taken for electrophoresis detection. The PCR results are as follows Figure 8 As shown, those marked with numbers are positive results. It can be seen from the figure that 32 mice out of 118 chimeric mice are transgenic mice, and the positive rate is 27%.

[0320] 14. Identification of HMGB1 gene expression in shRNA-HMGB1 transgenic mice

[0321] 14.1 DOX feeding

[0322] Add DOX to the drinking water of mice to a final concentration of 2 mg / mL. To mask the bitter taste of DOX, add sucrose to a final concentration of 5%. Protect from light. Change the water every 1 day. Feed continuously for 14 days.

[0323] 14.2 Lung tissue

[0324] 2% sodium pentobarbital was injected intraperitoneally, and the eyeballs were removed and blood was drawn after anesthesia. The neck was dehaired and disinfected with alcohol, and the chest was opened to remove the lungs, which were quickly stored in liquid nitrogen.

[0325] 14.3 Lung total protein extraction

[0326] 1) Remove the preserved lung tissue from liquid nitrogen and place it on ice;

[0327] 2) Add RIPA lysis buffer at a ratio of 1:100 (mL / mg) and liquid nitrogen at the same time, and grind until the tissue is completely broken;

[0328] 3) 4°C, 10,000 g, 10 min;

[0329] 4) Take the supernatant and store it at -80℃ for later use.

[0330] 14.4 Western Blot Detection of HMGB1 Expression

[0331] 1) Prepare separation gel and stacking gel;

[0332] 2) Sample application: Mix 5× loading buffer with sample, mix well, and incubate at 95°C for 5 min.

[0333] 3) Electrophoresis: 90V, until the sample completely enters the separation gel, then adjust the voltage to 110V until the bromophenol blue in the loading buffer reaches the bottom of the gel;

[0334] 4) Transfer: Soak the PVDF membrane in 80% formaldehyde solution for 15 minutes; after the electrophoresis is completed, place the gel in the transfer plate in the following order: anode plate, filter paper, PVDF transfer membrane, gel, filter paper, cathode plate; when stacking, drive out the bubbles between each layer, especially the bubbles between the gel and PVDF, and place it on a semi-dry transfer instrument, with the anode and cathode placed correctly, the current is 380mA, and the time is 2h;

[0335] 5) After the transfer is completed, take out the PVDF membrane and wash the membrane with TBST for 5 min × 3 times;

[0336] 6) 5% skimmed milk powder for 2 hours;

[0337] 7) Incubate with primary antibody: dilute primary antibody 1000:1 with 5% skim milk powder, incubate at 4°C overnight with gentle shaking;

[0338] 8) Wash the membrane with TBST for 5 min × 3 times;

[0339] 9) Incubate with secondary antibody, dilute the secondary antibody in TBST 1000:1, room temperature, 2h, gently shake;

[0340] 10) Wash the membrane with TBST for 5 min × 3 times;

[0341] 11) Exposure: Join West Dura Extended Duration Substrate, color development for 5 minutes, and imager to record data.

[0342] The experimental results show that the Wsetern Blot results are as follows Fig. 9 As shown in (1,3,4,6: TG mice; 2,5,7,8: WT mice), after feeding with DOX for 14 days, shRNA-HMGB1 can be expressed normally and produce an effective RNAi effect, and the expression of HMGB1 is downregulated, indicating that the prepared shRNA-HMGB1 transgenic mice meet the experimental requirements.

Claims

1. A shRNA target targeting HMGB1 gene, characterized in that: The shRNA target sequences are shown in SEQ ID NO.1-5.

2. A shRNA targeting the HMGB1 gene, characterized in that: It is a synthetic shRNA based on the shRNA target of claim 1, which has two complementary sense strand and antisense strand sequences respectively; The structure of the sense chain sequence is: 5' end-XhoI restriction enzyme cleavage site+target sequence sense chain+hairpin loop+target sequence antisense chain+terminator+MluI restriction enzyme cleavage site+residue-3' end.

3. The shRNA according to claim 2, characterized in that The antisense strand has a HindIII restriction enzyme site added to the 5' end, which then forms a sequence that is reverse complementary to the sense strand sequence; The sense strand sequence is reverse complementary to the antisense strand sequence.

4. The shRNA according to claim 2, characterized in that The sense sequence of the hairpin loop is TTCAAGAGA, and the antisense sequence is TCTCTTGAA.

5. The shRNA according to claim 2, characterized in that The sense strand and antisense strand sequences of the shRNA corresponding to the shRNA target site of claim 1 are shown in SEQ ID NO.6 to SEQ ID NO.15, respectively.

6. A recombinant plasmid targeting HMGB1 gene, characterized in that: The recombinant plasmid is a recombinant plasmid obtained by constructing the shRNA sequence described in claim 2 into a plasmid vector; The plasmid vector includes a pSingle-tTS-shRNA vector.

7. A method for constructing a recombinant plasmid targeting the HMGB1 gene according to claim 6, characterized in that: The steps include: 1) synthesizing the shRNA targeting the HMGB1 gene according to claim 2; 2) annealing the shRNA oligonucleotides from step 1) to form double-stranded DNA fragments; 3) Double enzyme digestion and recovery and purification of the expression vector; 4) connecting the double-stranded DNA fragment to the vector purified in step 3) to obtain a recombinant plasmid; 5) Transforming and expressing the recombinant plasmid through competent cells; The expression vector includes a pSingle-tTS-shRNA vector.

8. Use of the shRNA target site of claim 1 or the shRNA of claim 2 or the recombinant plasmid of claim 6 targeting the HMGB1 gene in the preparation of an HMGB1 gene expression inhibitor.

9. A method for constructing a transgenic mouse model with low HMGB1 expression, characterized in that: The steps include: 1) amplifying the recombinant plasmid according to claim 6; The primer sequences for the amplification are shown in SEQ ID NOs. 16 to 21; 2) recovering the vector by three enzyme digestion of the recombinant plasmid and extracting the target gene according to claim 3; 3) The target gene is transferred into recipient female mice by injection to obtain a transgenic mouse model expressing shRNA-HMGB1.

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