Endogenous long terminal repeat nucleotide sequence and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to an endogenous long-terminal repeat nucleotide sequence and application thereof, and the nucleotide sequence comprises a nucleotide sequence in SEQ ID No.1, a nucleotide sequence in SEQ ID No.2, a nucleotide sequence in SEQ ID No.3 and a nucleotide sequence in SEQ ID No.4. A nucleic acid sequence as shown in SEQ ID No.2; a nucleic acid sequence as shown in SEQ ID No. 4; a nucleic acid sequence as shown in SEQ ID No.5; the derived nucleic acids are obtained by replacing and / or adding and / or deleting one or more basic groups of the nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5; a nucleic acid sequence which can be hybridized with the nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5 under highly rigorous conditions; the immunogenic nucleic acid sequence has more than 90% of homology with the nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5, and the length of the immunogenic nucleic acid sequence is more than 20bp, or the nucleic acid sequence has the function of coding immunogenic polypeptide. The nucleic acid sequence provided by the invention has good practical application value through knowledge processing such as in-situ detection of spatial omics, continuous monitoring of in-vivo imaging, optimization analysis of intelligent calculation and the like.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine technology and transposon technology, and in particular to an endogenous long terminal repeat nucleic acid sequence and application thereof. Background Art

[0002] Systemic lupus erythematosus is a typical autoimmune disease that often affects multiple tissues such as the kidneys, heart, lungs, and skin. It has the characteristics of low early diagnosis rate and difficulty in radical cure. The serum anti-nucleic acid antibodies in the body are related to the evolution of lupus and are one of the commonly used indicators in important stages such as clinical diagnosis of lupus, monitoring of disease progression, and evaluation of drug efficacy.

[0003] Endogenous long terminal repeat nucleic acid sequences are endogenous transposons, accounting for more than 8% of the mammalian genome. The long terminal repeat sequences of the genome of adult individuals are in a silent state under physiological conditions, but under pathological conditions, their transcription products participate in immune responses such as macrophage phagocytosis, autoreactive B and T cell proliferation, antinuclear antibody formation, MHC molecule-T cell signaling, and are related to the complex process of lupus evolution. The physiological and pathological functions of such transposon nucleic acid sequences can be divided into two categories: 1) DNA sequences, transcription products, and complexes formed with protein molecules can induce adaptive immune responses; 2) regulate innate immune responses through the nucleic acid sensing pathway of Toll-like receptors. Such transposon nucleic acid sequences have highly overlapping fragments with the human genome chromosome 21 CH507 database, and this sequence is significantly upregulated in the Ncf1 mutant model animal with blocked reactive oxygen production, indicating that it is associated with autoimmune pathological manifestations under the environment of blocked reactive oxygen production. Summary of the invention

[0004] Based on this, the present invention proposes an endogenous long terminal repeat nucleic acid sequence and its application, which can be used as an early diagnostic target to assist in the early diagnosis of autoimmune diseases and has good practical application value.

[0005] According to a first aspect of the present invention, there is provided an endogenous long terminal repeat nucleic acid sequence, comprising:

[0006] The nucleic acid sequence in SEQ ID No. 1;

[0007] The nucleic acid sequence in SEQ ID No. 2;

[0008] The nucleic acid sequence in SEQ ID No. 4;

[0009] The nucleic acid sequence in SEQ ID No. 5; and

[0010] Derivative nucleic acids of the nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5, wherein one or more bases are substituted and / or added and / or deleted;

[0011] A nucleic acid sequence that can hybridize to the nucleic acid sequences of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 5 under highly stringent conditions;

[0012] An immunogenic nucleic acid sequence having more than 90% homology with the nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5, with a length of more than 20 bp or a nucleic acid sequence having the function of encoding an immunogenic polypeptide.

[0013] According to an embodiment of the present invention, the nucleic acid sequence is one of cDNA, recombinant DNA, ssDNA, ssRNA, mRNA, tRNA, siRNA, and hnRNA.

[0014] According to an embodiment of the present invention, the highly stringent conditions include:

[0015] 4×SSC concentration solution, constant temperature condition of 50℃.

[0016] According to an embodiment of the present invention, the immunogenic nucleic acid sequence refers to:

[0017] After the nucleic acid is delivered into the model animal, it can induce significantly upregulated autoantibody indicators within 4 weeks, such as dsDNA antibodies, Ro antibodies, P antibodies, cardiolipin antibodies, etc.

[0018] According to a second aspect of the present invention, there is provided a polypeptide derived from the above-mentioned endogenous long terminal repeat nucleic acid sequence, characterized in that it comprises:

[0019] The polypeptide sequence in SEQ ID No.6;

[0020] The polypeptide sequence in SEQ ID No.7;

[0021] A translated polypeptide sequence obtained by translating the above-mentioned endogenous long terminal repeat nucleic acid sequence through a nucleic acid sequence with one or more bases substituted and / or added and / or deleted;

[0022] The polypeptide sequences in SEQ ID No.6 and SEQ ID No.7 are obtained by translating the nucleic acid sequences in SEQ ID No.2 and SEQ ID No.4, respectively.

[0023] According to an embodiment of the present invention, it also includes a derived polypeptide sequence obtained by replacing and / or adding and / or deleting one or several amino acid residues from the SEQ ID No. 6, the SEQ ID No. 7 or the translated polypeptide sequence and having a homology of more than 90%.

[0024] According to a third aspect of the present invention, there is provided an application of an endogenous long terminal repeat nucleic acid sequence in the study of the mechanism of autoimmune diseases; comprising:

[0025] Delivery of endogenous long terminal repeat nucleic acid sequences via organ-targeted liposomes;

[0026] Construct a specific nucleic acid-induced autoimmune model;

[0027] Based on medical detection methods, the pathological mechanism of the autoimmune model is studied.

[0028] According to an embodiment of the present invention, the medical detection means include: immunohistochemistry, flow cytometry, and Elisa.

[0029] According to an embodiment of the present invention, the organ-targeting liposome comprises:

[0030] C12-200 liposomes, DOPE liposomes, Cholesterol, DMG-PEG liposomes, and 18-PA liposomes were mixed at a molar ratio of 24.5:11.2:32.6:1.8:30 to obtain specific delivery liposomes;

[0031] The endogenous long terminal repeat nucleic acid sequence is encapsulated in the specific delivery liposome to obtain the organ-targeted liposome.

[0032] According to an embodiment of the present invention, the construction of a specific nucleic acid-induced autoimmune model comprises:

[0033] The organ-targeted liposomes are injected intravenously into recipient mice to obtain a specific nucleic acid-induced autoimmune model.

[0034] According to a fourth aspect of the present invention, there is provided an application of an endogenous long terminal repeat nucleic acid sequence in the study of the spatial and cellular typing distribution of nucleic acid and protein molecules, specifically electrically enhanced spatial multi-omics in vivo detection; comprising:

[0035] preparing a test slide containing an endogenous long terminal repeat nucleic acid sequence;

[0036] Using the detection slide to perform electrically enhanced spatial multi-omics detection on biological tissues;

[0037] Determine the spatial and cellular distribution of affinity nucleic acids and protein molecules to obtain the spatial transcriptional map of the tissue in situ.

[0038] It can be seen from the above technical solution that an endogenous long terminal repeat nucleic acid sequence and its application provided by the present invention have the following beneficial effects:

[0039] The endogenous long terminal repeat nucleic acid sequence and derived polypeptide provided by the present invention can be used as a small molecule adjuvant for the construction of inflammatory-mediated disease models through knowledge processing such as in situ detection of spatial omics, continuous monitoring of in vivo imaging, and optimization analysis of intelligent computing; it can also be used as an early diagnostic target to assist in the early diagnosis of autoimmune diseases; it can also be used as a small molecule biological drug for precision medicine and personalized treatment.

[0040] The endogenous long terminal repeat nucleic acid sequence and the derived polypeptide provided by the invention have good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The images of kidney glomerular IgG and C3 antibody staining of mice injected with ssRNA-60 and ssRNA-60-scramble nucleic acid 14 days after the present invention (scale bar 50 micrometers);

[0042] Figure 2 The staining of IgG and C3 antibodies, indicators of glomerular inflammation, in mice injected with ssRNA-60 and ssRNA-60-scramble nucleic acid 14 days after injection in the example of the present invention;

[0043] Figure 3 Elisa was used to detect the expression of anti-nucleic acid antibodies in the serum of mice injected with ssRNA-60 and ssRNA-60-scramble nucleic acid 14 days after injection;

[0044] Figure 4 This is a schematic diagram of the process of an embodiment of in vivo detection of ssRNA-60 nucleic acid affinity molecules electrically enhanced spatial multi-omics.

[0045] Reference numerals:

[0046] 1-Indium tin oxide glass slide; 2-γ-glycidyloxypropyltrimethoxysilane coating; 3-phosphorylated ssRNA-60 nucleic acid; 4-Indium tin oxide glass slide (negative electrode); 5-conductive buffer; 6-tissue section; 7-switch-disconnect; 8-Indium tin oxide glass slide (positive electrode); 9-free negatively charged RNA, DNA and protein molecules in tissue; 10-switch; 11-under the action of electric field, negatively charged molecules are captured by ssRNA-60 nucleic acid; 12-immunofluorescence, detection of in situ proteins in tissue; 13-peeling tissue, spatial omics detection of in situ genes. DETAILED DESCRIPTION

[0047] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0048] According to a first aspect of the present invention, there is provided an endogenous long terminal repeat nucleic acid sequence, comprising:

[0049] The nucleic acid sequence in SEQ ID No. 1;

[0050] The nucleic acid sequence in SEQ ID No. 2;

[0051] The nucleic acid sequence in SEQ ID No. 4;

[0052] The nucleic acid sequence in SEQ ID No. 5; and

[0053] Derivative nucleic acids of the nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5 with one or more base substitutions and / or additions and / or deletions;

[0054] A nucleic acid sequence that can hybridize to the nucleic acid sequences of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 5 under highly stringent conditions;

[0055] An immunogenic nucleic acid sequence having more than 90% homology with the nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5, having a length of more than 20 bp or a nucleic acid sequence having the function of encoding an immunogenic polypeptide.

[0056] According to an embodiment of the present invention, the nucleic acid sequence is one of cDNA, recombinant DNA, ssDNA, ssRNA, mRNA, tRNA, siRNA, and hnRNA.

[0057] According to an embodiment of the present invention, high stringency conditions include:

[0058] 4×SSC concentration solution, constant temperature condition of 50℃.

[0059] According to an embodiment of the present invention, immunogenicity means that compared with a randomly scrambled control sequence, the targeted delivery into a model animal can induce significantly upregulated autoantibody indicators within 4 weeks, such as dsDNA antibodies, Ro antibodies, P antibodies, cardiolipin antibodies, etc.

[0060] According to an embodiment of the present invention, the nucleic acid sequence provided by the present invention can be artificially synthesized, or its upstream and downstream primers can be synthesized first, and then obtained by PCR amplification or / and reverse transcription. The nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQID No.5 are obtained by calculating gene interactions and screening based on key genes of autoimmune diseases. After experimental verification, the SEQ ID No.1 sequence is highly expressed in the autoimmune disease model and can activate inflammation. The sequence has application value in participating in the construction of inflammatory disease models, as a target to assist in the early diagnosis of autoimmune diseases, and as a small molecule biological drug for precision medicine and personalized treatment. Specifically, SEQ ID No. 1 is the endogenous LTR nucleic acid sequence of mice, and fluorescence in situ hybridization verifies that the sequence is highly expressed in the autoimmune disease model; SEQ ID No. 2 is the sequence of SEQ ID No. 1 obtained by TLR interaction screening and interception, and in vivo lipid delivery verifies that the sequence can activate inflammation; SEQ ID No. 4 is the sequence of SEQ ID No. 2 obtained by APOBEC3 cytosine deaminase base editing, which is suitable for studying the mechanism of autoimmune diseases related to gene polymorphism; SEQ ID No. 5 is the artificial thiolated sequence of SEQ ID No. 2, which has stronger RNA stability and is suitable for use as an in vitro detection antigen. The autoimmune disease model specifically refers to B6.Lyz2 luci / luci .Ncf1 * / * .Fas lpr / lpr Mouse, this model is composed of B6.Fas lpr / lpr In the classic model, natural gene polymorphism mutations are introduced. The DNA, RNA and polypeptide residues of mice and humans are highly similar, which is suitable for the study of the mechanism of autoimmune diseases.

[0061] According to a second aspect of the present invention, there is provided a polypeptide derived from the above-mentioned endogenous long terminal repeat nucleic acid sequence, characterized in that it comprises:

[0062] The polypeptide sequence in SEQ ID No.6;

[0063] The polypeptide sequence in SEQ ID No.7;

[0064] A translated polypeptide sequence obtained by translating the above-mentioned endogenous long terminal repeat nucleic acid sequence through a nucleic acid sequence with one or more bases substituted and / or added and / or deleted;

[0065] The polypeptide sequences in SEQ ID No.6 and SEQ ID No.7 are obtained by translating the nucleic acid sequences in SEQ ID No.2 and SEQ ID No.4, respectively.

[0066] According to an embodiment of the present invention, it also includes a derived polypeptide sequence obtained by replacing and / or adding and / or deleting one or more amino acid residues from SEQ ID No. 6, SEQ ID No. 7 or a translated polypeptide sequence and having a homology of more than 90%.

[0067] According to the embodiments of the present invention, the polypeptide sequence provided by the present invention can be artificially synthesized, or its encoding nucleic acid can be synthesized first and then expressed by an organism. The polypeptide sequence provided by the present invention has a potential immune mechanism of participating in MHC signal presentation as a small molecule target, and can be used as a target for the study of autoimmune diseases.

[0068] According to a third aspect of the present invention, there is provided an application of an endogenous long terminal repeat nucleic acid sequence in the study of the mechanism of autoimmune diseases, comprising:

[0069] Organ-targeted liposome delivery of endogenous long terminal repeat nucleic acid sequences;

[0070] Construct a specific nucleic acid-induced autoimmune model;

[0071] Based on medical detection methods, the pathological mechanism of the autoimmune model is studied.

[0072] According to an embodiment of the present invention, the organ-targeted liposome comprises:

[0073] C12-200 liposomes, DOPE liposomes, Cholesterol, DMG-PEG liposomes, and 18-PA liposomes were mixed at a molar ratio of 24.5:11.2:32.6:1.8:30 to obtain specific delivery liposomes;

[0074] The endogenous long terminal repeat nucleic acid sequence is encapsulated in a specific delivery liposome to obtain an organ-targeted nucleic acid delivery liposome.

[0075] According to an embodiment of the present invention, the organ-targeted liposome is an organ-specific delivery liposome that can be delivered to the spleen and kidney, and the nucleic acid sequence or the polypeptide derived therefrom of the present invention is encapsulated in the organ-targeted liposome.

[0076] According to an embodiment of the present invention, constructing a specific nucleic acid-induced autoimmune model includes:

[0077] Organ-targeted liposomes were injected intravenously into recipient mice to obtain a specific nucleic acid-induced autoimmune model.

[0078] According to an embodiment of the present invention, taking SEQ ID No.2 as an example, it specifically refers to the organ-targeted liposomes coated with the nucleic acid sequence provided by the present invention, and the organ-targeted liposomes coated with the control nucleic acid sequence, respectively, injected into two groups of 4-week-old mice by intravenous injection, to obtain a specific nucleic acid-induced autoimmune model and a control model. The nucleic acid sequence used for modeling is specifically SEQ ID No.2 in the sequence table, and its name is ssRNA-60. The control nucleic acid sequence, specifically SEQ ID No.3 in the sequence table, is named ssRNA-60-scramble, which is generated by random scrambling of the ssRNA-60 sequence, and its CG ratio is consistent with the ssRNA-60 sequence. The two groups of 4-week-old mice, specifically C57BL / 6 mice, are divided into a ssRNA-60 injection group and a control group (the control group is injected with ssRNA-60-scramble), and the number of mice in each group is greater than 3. The specific nucleic acid-induced autoimmune model can characterize autoimmune characteristics such as nephritis and serum antinuclear antibodies within 14 days. It is suitable for evaluating the immunogenicity of the nucleic acid sequence described in the present invention and its homologous sequence, and as an autoimmune disease model, and is applied to the study of the mechanism of autoimmune diseases.

[0079] According to a fourth aspect of the present invention, there is provided an application of an endogenous long terminal repeat nucleic acid sequence in the study of the spatial and cellular typing distribution of nucleic acid and protein molecules, specifically ssRNA-60 nucleic acid affinity molecule electrically enhanced spatial multi-omics in vivo detection; comprising:

[0080] preparing a test slide containing an endogenous long terminal repeat nucleic acid sequence;

[0081] The test slides were subjected to electrically enhanced spatial multi-omics detection to determine the spatial and cellular typing distribution of affinity nucleic acids and protein molecules, and to obtain the in situ spatial transcriptional map of the tissue.

[0082] According to an embodiment of the present invention, for example, the preparation of the detection slide specifically refers to modifying the phosphorylated ssRNA-60 nucleic acid (SEQ ID No. 2 sequence) or the sulfated thio-modified ssRNA-60 nucleic acid (SEQ ID No. 5 sequence) on an indium tin oxide conductive glass slide.

[0083] According to an embodiment of the present invention, electrically enhanced spatial multi-omics detection specifically refers to attaching the target tissue slice to the ssRNA-60 nucleic acid coating of an indium tin oxide conductive glass slide, applying an electric field up and down, migrating electronegative molecules such as RNA and protein in the tissue to the ssRNA-60 nucleic acid coating, digesting and eluting the molecules not bound to the ssRNA-60 nucleic acid coating, detecting genes and proteins targeted and bound to the ssRNA-60 nucleic acid sequence through spatial omics technology, constructing an in situ ssRNA-60 interaction map of the tissue, and assisting in the early diagnosis of ssRNA-60 related immune inflammation.

[0084] According to an embodiment of the present invention, the spatial omics technology may be selected as 10x Visium technology, STARmap technology, smFISH technology, etc., and the lower limit of detection is that the spatial resolution is higher than 200 μm and the number of detected genes is greater than 10.

[0085] The technical solution of the present invention is described in detail below through preferred embodiments. It should be noted that the specific embodiments below are only used for illustration and are not used to limit the present invention.

[0086] Functional verification of ssRNA-60 nucleic acid sequence and autoimmune model induced by ssRNA-60

[0087] The specific delivery liposomes are prepared, including: dissolving C12-200 liposomes, DOPE liposomes, Cholesterol, and DMG-PEG liposomes in anhydrous ethanol respectively, dissolving 18-PA liposomes in tetrahydrofuran, and mixing the above five liposomes at a molecular weight ratio of 24.5:11.2:32.6:1.8:30, and mixing them on an oscillator to form specific delivery liposomes.

[0088] Nucleic acid coating, including: dissolving a commercially synthesized ssRNA-60 nucleic acid sequence, preferably RNA, at a concentration of 65ng / μl in a 10mmol / L, pH=4.0 citrate buffer. Mix the nucleic acid solution with the specific delivery liposome at a nucleic acid solution / lipid solution volume ratio of 3 / 1 and a nucleic acid / lipid mass ratio of 1 / 40, and oscillate to obtain liposome-coated ssRNA-60 nucleic acid. Replace the nucleic acid sequence with SEQ ID No. 3 in the sequence table, named ssRNA-60-scramble, which is generated by randomly scrambling the ssRNA-60 sequence, and its CG ratio is consistent with the ssRNA-60 sequence, and prepare a liposome-coated nonsense nucleic acid control in the same steps.

[0089] The liposome-encapsulated nucleic acid is intravenously injected into mice, specifically comprising: taking 10 3-5 week-old mice, preferably 4-week-old B6.lpr female mice, and dividing them into an ssRNA-60 experimental group and an ssRNA-60-scramble control group. The experimental group is injected with liposome-encapsulated ssRNA-60 nucleic acid through the ocular vein, and the control group is injected with liposome-encapsulated ssRNA-60-scramble control nucleic acid through the ocular vein, and the injection volume is based on the weight of the mouse, specifically 0.5 μl per gram of mouse.

[0090] In vivo monitoring of the disease progression and renal function level of autoimmune inflammation includes: weekly autofluorescence in vivo imaging of the nucleic acid-induced inflammation model and the control to detect the progression of inflammation in the joints and skin on the body surface; urine protein testing every 3 days using a commercial urine protein kit; and antinuclear antibody testing every 7 days using a serum Elisa kit to obtain renal function indicators and blood antinuclear antibody indicators.

[0091] At the end of inflammation, multi-organ detection is performed on the nucleic acid-induced inflammation model and the control to quantitatively evaluate the progression of inflammation, specifically including: extracting serum from the nucleic acid-induced inflammation model and the control mice in the 2nd to 4th week after intravenous injection, preferably in the third week, performing cardiac perfusion, and obtaining tissues such as kidneys, spleens, and lymph nodes. Immunohistochemistry staining is used to detect the progression of glomerular inflammation and the activation level of ssRNA-60 nucleic acid targeted delivery between the two groups, preferably IgG and C3 antibodies; flow cytometry is used to detect the differences in spleen and lymph node immune cell clustering between the two groups, preferably CD3, CD4, CD11b, and CD68 antibodies; enzyme-linked immunosorbent assay is used to detect the expression levels of key autoimmune antibodies between the two groups, preferably Ro, P0, cardiolipin, and ds-DNA antibodies; RNA sequencing is used to detect the activation level of inflammatory genes between the two groups.

[0092] The results of kidney immunohistochemistry were shown in Figure 1 and Figure 2 It can be seen that in the ssRNA-60 group, the expression of IgG and C3 in the glomerulus increased, indicating that ssRNA-60 induced renal inflammation; the results of serum enzyme-linked adsorbent assay are shown in Figure 3 It can be seen that in the ssRNA-60 group, antinuclear antibodies and cardiolipin antibodies were significantly increased, indicating that ssRNA-60 significantly activated autoimmune inflammatory manifestations compared with the control sequence. Therefore, the ssRNA-60 experimental group model mice obtained by this application can be used as a supplement to the research model of autoimmune disease mechanism.

[0093] ssRNA-60 nucleic acid affinity molecule electrically enhanced spatial multi-omics in vivo detection

[0094] like Figure 4As shown, the preparation of the ssRNA-60 detection slide includes: immersing a commercially available indium tin oxide conductive slide, preferably with a slide size of 75×25×0.2 mm, in acetone for ultrasonic cleaning at room temperature for 20 minutes, immersing it in isopropanol for ultrasonic cleaning at room temperature for 20 minutes, immersing it in ultrapure water for ultrasonic cleaning at room temperature for 20 minutes, immersing it in ultrapure water at room temperature and protected from light for at least 48 hours, immersing it in an acetone solution containing 2% γ-glycidyloxypropyltrimethoxysilane reagent for 2 hours, and rinsing it with acetone for 5 minutes. On the conductive side of the indium tin oxide, a 50 μl volume of a 1 μM concentration of a commercially synthesized 5' phosphorylated modified ssRNA-60 corresponding nucleic acid sequence (optionally SEQ ID No. 2 or SEQ ID No. 5) is added to the center of the surface, incubated in a nitrogen environment at room temperature for 1 hour, and rinsed with ultrapure water for 5 minutes to obtain a detection slide.

[0095] Tissue slice patch, electrically enhanced capture of negatively charged molecules, including: preparing 10 μm thick frozen slices of kidney, spleen, lung and other tissues of model animals or patients, and patching the tissues on the coated ssRNA-60 nucleic acid area, such as Figure 4 As shown, a tissue-attached indium tin oxide conductive glass slide is placed at the bottom as the positive electrode, and an untreated indium tin oxide conductive glass slide is placed at the top as the negative electrode. The upper and lower gaps are kept at 1 mm, and a conductive buffer (preferably 1×TAE buffer) is filled in between. A voltage of 1V is applied for 20 minutes, and negatively charged RNA and proteins are attracted by the electric field and attached to the positive electrode below, thereby achieving electrically enhanced capture of negatively charged molecules.

[0096] In situ tissue multi-omics testing includes: immunofluorescence antibody imaging of tissues on slides, preferably CD68 antibodies, Ly6g antibodies and CD3 antibodies, to determine their cellular spatial location and key immune cell typing.

[0097] The multi-omics detection of ssRNA-60 captured molecules includes: adding proteinase K buffer to the glass slide tissue and incubating at 37 degrees for 12 hours to digest the residual tissue, and performing protein molecule detection and nucleic acid spatial omics detection on the digested glass slide. The protein molecule detection specifically refers to immunofluorescence imaging of nucleic acid affinity protein molecules captured by the ssRNA-60 coating layer on the glass slide (NRLP6 antibody and TLR4 antibody are preferred in kidney tissue detection). The nucleic acid spatial omics detection specifically refers to spatial omics detection of nucleic acid molecules captured by the ssRNA-60 coating layer on the glass slide. The detection lower limit is a spatial resolution higher than 200μm and a number of detected genes greater than 10. Optional spatial omics technologies include 10xVisium technology, STARmap technology, smFISH technology, etc. Taking STARmap technology as an example, a detection gene library was established (preferably Pcdh7, Cd68, Ly6g, Cd31, Cd4, Cd8, Tlr2, Tlr4, Tlr7, C3 genes, etc. in mouse kidney tissue), STARmap mixed coding probes were commercially synthesized, and 50 μl of 5 nanomolar mixed coding probes were added to the nucleic acid area captured by the ssRNA-60 coating layer on the glass slide. The mixture was incubated at 40 degrees for 36 hours, washed with PBS after incubation, and subjected to rolling circle amplification at room temperature. Multiple rounds of multi-channel imaging were performed (preferably 5 rounds within 100 genes, 3-channel fluorescence Cy5 / Cy3 / Af488) to decode the gene spatial genomics information.

[0098] Through in situ protein imaging and RNA capture molecule multi-omics imaging, key immune cell populations such as macrophages, neutrophils, T cells and their adjacent ssRNA-60 affinity gene distributions were obtained, realizing multi-omics screening of ssRNA-60 sequences.

[0099] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An endogenous long terminal repeat nucleic acid sequence, characterized in that: include: The nucleic acid sequence in SEQ ID No. 1; The nucleic acid sequence in SEQ ID No. 2; The nucleic acid sequence in SEQ ID No. 4; The nucleic acid sequence in SEQ ID No. 5; and Derivative nucleic acids of the nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5, wherein one or more bases are substituted and / or added and / or deleted; A nucleic acid sequence that can hybridize to the nucleic acid sequences of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 5 under highly stringent conditions; An immunogenic nucleic acid sequence having more than 90% homology with the nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5, with a length of more than 20 bp or a nucleic acid sequence having the function of encoding an immunogenic polypeptide.

2. The endogenous long terminal repeat nucleic acid sequence according to claim 1, characterized in that The nucleic acid sequence is one of cDNA, recombinant DNA, ssDNA, ssRNA, mRNA, tRNA, siRNA, and hnRNA; The immunogenic nucleic acid sequence refers to an autoantibody index that can induce significant upregulation within 4 weeks after the nucleic acid is delivered into the model animal, including: dsDNA antibody, Ro antibody, P antibody, and cardiolipin antibody.

3. The endogenous long terminal repeat nucleic acid sequence according to claim 1, characterized in that: The highly stringent conditions include: 4×SSC concentration solution and 50° C. constant temperature conditions.

4. A polypeptide derived from the endogenous long terminal repeat nucleic acid sequence of claims 1-3, characterized in that: include: The polypeptide sequence in SEQ ID No.6; The polypeptide sequence in SEQ ID No.7; A translated polypeptide sequence obtained by translating the endogenous long terminal repeat nucleic acid sequence according to claims 1 to 3 through a nucleic acid sequence in which one or more bases are replaced and / or added and / or deleted; The polypeptide sequences in SEQ ID No.6 and SEQ ID No.7 are obtained by translating the nucleic acid sequences of SEQ ID No.2 and SEQ ID No.4, respectively.

5. The polypeptide derived from the endogenous long terminal repeat nucleic acid sequence according to claim 4, characterized in that: It also includes a derived polypeptide sequence obtained by replacing and / or adding and / or deleting one or more amino acid residues from the SEQ ID No. 6, the SEQ ID No. 7 or the translated polypeptide sequence and having a homology of more than 90%.

6. A method for using the endogenous long terminal repeat nucleic acid sequence according to claims 1 to 3 in the study of the mechanism of autoimmune diseases; comprising: Delivering the endogenous long terminal repeat nucleic acid sequence of claims 1-3 via organ-targeted liposomes; Construct a specific nucleic acid-induced autoimmune model; Based on medical detection methods, the pathological mechanism of the autoimmune model is studied.

7. The preparation method according to claim 6, characterized in that: The organ-targeting liposomes include: C12-200 liposomes, DOPE liposomes, Cholesterol, DMG-PEG liposomes, and 18-PA liposomes were mixed at a molar ratio of 24.5:11.2:32.6:1.8:30 to obtain specific delivery liposomes; The endogenous long terminal repeat nucleic acid sequence described in claims 1 to 3 is encapsulated in the specific delivery liposome to obtain the organ-targeted liposome.

8. The preparation method according to claim 7, characterized in that: The construction of the specific nucleic acid-induced autoimmune model comprises: The organ-targeted liposomes are injected intravenously into recipient mice to obtain a specific nucleic acid-induced autoimmune model.

9. An application of the endogenous long terminal repeat nucleic acid sequence according to claims 1 to 3 in the study of the spatial and cellular typing distribution of nucleic acid and protein molecules; comprising: Preparing a test slide comprising the endogenous long terminal repeat nucleic acid sequence of claims 1-3; performing electrically enhanced spatial multi-omics detection on the detection slide; Determine the spatial and cellular distribution of affinity nucleic acids and protein molecules to obtain the spatial transcriptional map of the tissue in situ.

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