Enhanced pancreatic acellular cell specific promoter and application thereof

Abstract

The invention provides an enhanced pancreatic acinus cell specific promoter and application thereof, and belongs to the technical field of gene engineering. The enhanced pancreatic acinus cell specific promoter comprises a promoter core region, the nucleotide sequence of the promoter core region is shown as SEQ ID No: 1 or SEQ ID No: 2, the specific expression of an exogenous target gene in pancreatic acinus can be increased, the expression quantity of the exogenous target gene in other tissues and organs (such as liver) can be remarkably reduced, and the targeting of pancreas is improved. The enhanced pancreatic acinus cell specific promoter can further comprise an enhancer region, the nucleotide sequence of the enhancer region is shown as SEQ ID No: 5 or SEQ ID No: 6, and the effectiveness of exogenous target gene delivery can be further improved. Clinical application scenes are widened, and reference is provided for pancreatic disease mechanism exploration and gene therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to an enhanced pancreatic acinar cell-specific promoter and its application. Background Art

[0002] Pancreatitis is divided into acute and chronic pancreatitis, which are common clinical emergencies and refractory diseases. The recurrence and chronicity of pancreatitis are dynamic, progressive processes. Approximately 21% of acute pancreatitis (AP) will progress to recurrent pancreatitis, and 38% of the latter patients will progress to irreversible chronic pancreatitis (CP), presenting repeated abdominal pain, pancreatic exocrine and endocrine insufficiency, and a heavy disease burden; some CP will progress to pancreatic cancer with a poor prognosis. [1] Current treatment strategies mainly include endoscopic treatment, pain management, and complication control. [2] However, the treatment effect is limited, and none of them can achieve radical cure of the disease, seriously threatening human health. With the progress of genetic research, it has been found that some gene mutations are related to the occurrence and chronicity of pancreatitis. More than half of CP patients in China carry pathogenic mutations. Among them, the imbalance of trypsin activation-inhibition related genes in pancreatic acinar cells is the key process in the occurrence of AP and CP, and also participates in the occurrence and development of CP carcinogenesis. [3] Specifically regulating gene expression is of great significance for exploring the disease mechanism of pancreatitis, studying the "inflammation-cancer transformation" of pancreatitis, and gene therapy of pancreatitis.

[0003] In terms of exploring the disease mechanism and process, current research mainly relies on transgenic animals. However, organ-specific transgenic animals have disadvantages such as a long breeding cycle, high construction cost, high feeding cost, and inability to flexibly control gene expression. In addition, there is no international research on gene therapy for pancreatitis. Exogenous genes enter the pancreas mainly through non-viral or viral vectors. Non-viral vectors have problems such as low transduction efficiency, poor targeting, and short expression time of the mediated gene fragments. Adeno-associated virus (AAV) vectors in viral vectors have characteristics such as persistent gene expression and low immunogenicity, and are considered the most promising gene delivery vectors. [4] The inventor team of the present application previously targeted the loss-of-function mutation of the main pathogenic gene SPINK1 c.194+2T>C in China (accounting for about 40%). [3] It has been confirmed that the overexpression method of the normal human SPINK1 gene mediated by AAV under the control of the general promoter CMV can effectively relieve the CP phenotype of mice, but there are still deficiencies in the expression time and targeting. [5]。Currently, there is no highly targeted and specific gene vector for the pancreas. The non-specific expression of the carried target gene not only reduces the effective expression level in target cells but also increases the risk of adverse immune reactions. [6] 。

[0004] Using a specific promoter to mediate the expression of foreign genes is the most effective solution for precisely targeting tissues, regulating gene expression, and gene therapy. A promoter is a DNA sequence located upstream of the 5' end of a gene that can interact with RNA polymerase and transcription factors. The core promoter elements and upstream regulatory elements it contains can jointly affect the spatiotemporality of gene expression. [7] ,They can be mainly divided into two categories: general promoters and tissue-specific promoters. At present, the promoters for delivering foreign genes to the pancreas based on AAV are mainly general promoters (such as CMV, CAG, and CBA, etc.), which can achieve high-level expression of the target gene, but have low targeting specificity and are easily methylated by the host, resulting in transcriptional silencing. [8] 。

[0005] Tissue-specific promoters can direct gene expression in specific tissues. Even if the vector has poor targeting specificity, it can limit the expression of the target gene in non-target organs / tissues and reduce the immune response of antigen-presenting cells to the transgene. [6] 。The pancreas is divided into pancreatic acinar cells, ductal cells, and islet cells. The key site of pancreatitis is in the acinar. Previous scholars have constructed transgenic mice based on acinar cell-specific promoters such as Cela1, etc., to achieve specific expression of the transgene in pancreatic acinar cells. [9] 。However, in terms of delivering foreign target genes, there is no relevant research report on pancreatic acinar cell-specific promoters internationally at present. It should be noted that the packaging capacity of the AAV vector is ~4 kb, and the full-length promoter will exceed the packaging limit, so it is more necessary to seek short-fragment specific promoters.

[0006] In addition, although tissue-specific promoters have high specificity, Generally has the disadvantage of low starting efficiency 。A hybrid promoter is a new promoter formed by fusing multiple functional regions of different promoters, such as the core promoter, upstream enhancer, and upstream silencer, etc., to jointly regulate gene expression. This strategy has now been widely used in the construction of muscle-specific promoters, islet β cell-specific promoters, etc. [10,11] 。Therefore, how to achieve high-specific targeting of the pancreas while maintaining high-efficiency expression of the target gene under the limited AAV packaging capacity is also the key to the research.

[0007] Therefore, the deficiencies of the existing technologies are summarized as follows: (1) At present, there is no pancreatic acinar-specific promoter to mediate the delivery of foreign genes to pancreatic tissues; (2) As an ideal gene delivery vector, AAV has the problem of low packaging capacity, and it is necessary to screen short fragments of pancreatic acinar-specific promoters with the optimal promoter efficiency; (3) The promoter efficiency of pancreatic acinar-specific promoters is weak, and there is no relevant research on whether the combination of pancreatic acinar-specific enhancers and promoters can improve the promoter efficiency.

[0008] References

[0009] 1. Petrov MS, Yadav D. Global epidemiology and holistic prevention of pancreatitis. Nat Rev Gastroenterol Hepatol 2019; 16: 175 - 184.

[0010] 2. Hines OJ, Pandol SJ. Management of chronic pancreatitis. Bmj 2024; 384: e070920.

[0011] 3. Zou WB, Tang XY, Zhou DZ, et al. SPINK1, PRSS1, CTRC, and CFTR Genotypes Influence Disease Onset and Clinical Outcomes in Chronic Pancreatitis. Clin Transl Gastroenterol 2018; 9: 204.

[0013] 4. High KA, Roncarolo MG. Gene Therapy. N Engl J Med 2019; 381: 455 - 464.

[0014] 5. Wang YC, Mao XT, Sun C, et al. Pancreas-directed AAV8-hSPINK1 gene therapy safely and effectively protects against pancreatitis in mice. Gut 2024; 73: 1142 - 1155.

[0015] 6. Pupo A, Fernández A, Low SH, et al. AAV vectors: The Rubik's cube of human gene therapy. Mol Ther 2022; 30: 3515-3541.

[0016] 7. Oudelaar AM, Higgs DR. The relationship between genome structure and function. Nat Rev Genet 2021; 22: 154-168.

[0017] 8. Duan B, Cheng L, Gao Y, et al. Silencing of fat-1 transgene expression in sheep may result from hypermethylation of its driven cytomegalovirus (CMV) promoter. Theriogenology 2012; 78: 793-802.

[0019] 9. Huang H, Swidnicka-Siergiejko AK, Daniluk J, et al. Transgenic expression of PRSS1 (R122H) sensitizes mice to pancreatitis. Gastroenterology 2020; 158: 1072-1082.e7. 10. Salva MZ, Himeda CL, Tai PW, et al. Design of tissue-specific regulatory cassettes for high-level rAAV-mediated expression in skeletal and cardiac muscle. Mol Ther 2007; 15: 320-9.

[0021] 11. Chai S, Kim Y, Galivo F, et al. Development of a Beta Cell-Specific Expression Control Element for Recombinant Adeno-Associated Virus. Hum Gene Ther 2022; 33: 789-800. Summary of the Invention

[0022] This invention is made to solve the above problems, and aims to provide an enhanced pancreatic acinar cell-specific promoter and its applications.

[0023] This invention provides an enhanced pancreatic acinar cell-specific promoter, which has the following characteristics: including a promoter core region, whose nucleotide sequence is as shown in SEQ ID No: 1 or as shown in SEQ ID No: 2.

[0024] In the enhanced pancreatic acinar cell-specific promoter provided by this invention, it may also have the following characteristics: further including an enhancer region, whose nucleotide sequence is as shown in SEQ ID No: 5 or as shown in SEQ ID No: 6.

[0025] In the enhanced pancreatic acinar cell-specific promoter provided by this invention, it may also have the following characteristics: wherein, the nucleotide sequence of the enhancer region is as shown in SEQ ID No: 5.

[0026] In the enhanced pancreatic acinar cell-specific promoter provided by this invention, it may also have the following characteristics: wherein, the enhanced pancreatic acinar cell-specific promoter can mediate the highly targeted expression of exogenous target genes in the pancreas.

[0027] This invention provides an enhanced pancreatic acinar cell-specific transgenic vector, which has the following characteristics: including the above enhanced pancreatic acinar cell-specific promoter.

[0028] In the enhanced pancreatic acinar cell-specific transgenic vector provided by this invention, it may also have the following characteristics: wherein, the enhanced pancreatic acinar cell-specific transgenic vector uses a mutant scAAV8-mediated vector of the ZsGreen tag protein gene.

[0029] In the enhanced pancreatic acinar cell-specific transgenic vector provided by this invention, it may also have the following characteristics: wherein, the mutant scAAV8 vector is a hybrid virus vector produced by the combination of the AAV2 genome and the AAV8 capsid protein with double-point mutations of Y447+Y733F, and this vector contains an ampicillin resistance gene.

[0030] The present invention also provides the use of an enhanced pancreatic acinar cell-specific transgenic vector in the preparation of a product for preventing and treating pancreatitis.

[0031] In the use of the enhanced pancreatic acinar cell-specific transgenic vector provided by the present invention in the preparation of a product for preventing and treating pancreatitis, it may further have the following characteristics: among them, in the product, the optimal infection titer of the enhanced pancreatic acinar cell-specific transgenic vector is 2×10 11 vg / animal, and the optimal expression time is the 5th day after injection.

[0032] Functions and effects of the invention

[0033] According to the enhanced pancreatic acinar cell-specific promoter and its application involved in the present invention, since the enhanced pancreatic acinar cell-specific promoter includes a promoter core region, the nucleotide sequence of which is as shown in SEQ ID No: 1 or as shown in SEQ ID No: 2, therefore, this enhanced pancreatic acinar cell-specific promoter can increase the specific expression of the exogenous target gene in pancreatic acinar cells, and can significantly reduce the expression level of the exogenous target gene in other tissues and organs (such as the liver), and improve the targeting of the pancreas.

[0034] Furthermore, since this enhanced pancreatic acinar cell-specific promoter also includes an enhancer region, the nucleotide sequence of which is as shown in SEQ ID No: 5 or as shown in SEQ ID No: 6, it can further improve the effectiveness of the delivery of the exogenous target gene.

[0035] The present invention broadens the clinical application scenarios and provides a reference for the exploration of pancreatic disease mechanisms and gene therapy at the same time. Brief description of the drawings

[0036] Figure 1 is a schematic diagram for screening pancreatic acinar cell-specific candidate genes in the examples of the present invention;

[0037] Figure 2 is a diagram of a dual-luciferase backbone plasmid in the examples of the present invention, including the pGL4.10 plasmid (upper) and the pRL-CMV plasmid (lower);

[0038] Figure 3 is a plasmid map of different promoters in the examples of the present invention, where A. pGL4.10 empty vector; B. pGL4.10-CMV promoter; C. pGL4.10-hPRSS1 promoter; D. pGL4.10-Cela1(-500 / +8) promoter; E. pGL4.10-Cela1(-205 / +8) promoter; F. pGL4.10-hSPINK1 promoter.Figure 4 It is a result graph of the in vitro activities of different promoter sequences in the embodiments of the present invention.

[0039] Figure 5 It is a graph of scAAV8-ZsGreen vectors driven by different promoters in the embodiments of the present invention, where A. scAAV8-CMV-ZsGreen; B. scAAV8-hPRSS1-ZsGreen; C. scAAV8-Cela1(-500 / +8)-ZsGreen; D. scAAV8-Cela1(-205 / +8)-ZsGreen; E. scAAV8-hSPINK1-ZsGreen.

[0040] Figure 6 It is a result graph for evaluating the efficiency of ZsGreen gene activation and pancreatic tissue specificity by different promoters in the embodiments of the present invention. Among them, A. Representative pictures of spontaneous green fluorescence of ZsGreen gene activated by different promoters shown in frozen sections; B. Quantitative analysis of the proportion of spontaneous green fluorescence in the head and tail of the pancreas by different promoters; C. The pancreas / liver ratio of ZsGreen gene mRNA by different promoters, and a larger value represents higher specificity in pancreatic tissue.

[0041] Figure 7 It is a plasmid map of different hybrid promoters in the embodiments of the present invention, where A. pGL4.10-AMY2(human)-hPRSS1 promoter; B. pGL4.10-AMY2(human)-Cela1(-500 / +8)promoter; C. pGL4.10-Amy2(rat)-hPRSS1 promoter; D. pGL4.10-Amy2(rat)-Cela1(-500 / +8)promoter.

[0042] Figure 8 It is a result graph of in vitro experiments on the enhancement of the promoter activities of hPRSS1 and Cela1(-500 / +8) by AMY2(human) and Amy2(rat) in the embodiments of the present invention.

[0043] Figure 9It is a diagram of the scAAV8-ZsGreen vector driven by different promoters combined with enhancers in the embodiments of the present invention. Among them, A. scAAV8-AMY2(human)-hPRSS1-ZsGreen; B. scAAV8-AMY2(human)-Cela1(-500 / +8)-ZsGreen; C. scAAV8-Amy2(rat)-hPRSS1-ZsGreen; D. scAAV8-AMY2(rat)-Cela1(-500 / +8)-ZsGreen.

[0044] Figure 10 It is a result diagram showing that the hybrid promoter significantly improves the efficiency of the ZsGreen gene in the embodiments of the present invention. Detailed implementation manners

[0045] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the following embodiments will specifically elaborate on the enhanced pancreatic acinar cell-specific promoter of the present invention and its application in conjunction with the accompanying drawings.

[0046] Embodiment

[0047] This embodiment provides an enhanced pancreatic acinar cell-specific promoter and its application.

[0048] I. Screening of promoter sequences

[0049] Based on the TiGER database (http: / / bioinfo.wilmer.jhu.edu / tiger / ) of human genome data, 205 genes highly expressed in pancreatic tissue were screened and sorted according to the enrichment scores from high to low; through gene descriptions, genes that function in islet cells were excluded. Subsequently, the expression of each gene in different tissues was checked through the The Human Protein Atlas database (https: / / www.proteinatlas.org / ), and genes with poor specificity were excluded. A total of 200 pancreatic acinar-expressed genes were obtained through the human single-cell RNAsequencing database (http: / / bis.zju.edu.cn / HCL / ), sorted according to the scores from high to low, and the top 30 with the highest scores were screened. Then, based on the mouse RNA-seq data of our team [5] , the top 20 with the highest scores were screened. According to the screening results of the above databases and the original data of our team, it was planned to determine to use the key regulatory sequences upstream of the transcription start sites of the PRSS1, AMY2A, and CELA genes for subsequent research (see Figure 1 ).

[0050] Sequences around the transcriptional start site (TSS) are highly conserved during evolution. It is inferred that the short sequence around the TSS is the core region of the promoter. Based on the transcriptional active sites and the rat Cela1 gene reported in previous transgenic research [9] (which Figure 1 is the homologous gene of human CELA3A and CELA3B genes), this patent finally incorporates the -395 to +13 fragment (408 bp) of the human PRSS1 promoter (NM_002769.5), the -500 to +8 fragment (508 bp) and -205 to +8 fragment (213 bp) of the rat Cela1 promoter (NM_012552.4), and the -67 to +53 (120 bp) of the human SPINK1 promoter (NM_003122.5). The AMY2 gene is mainly an enhancer region, and this patent finally incorporates the enhancer fragments of human AMY2 (34 bp) and rat Amy2 (40 bp).

[0051] The following are the specific sequence information of each promoter and enhancer involved in this patent:

[0052] (1) The -395 to +13 fragment (408 bp) of the human PRSS1 promoter (NM_002769.5), denoted as human PRSS1 promoter, has the nucleotide sequence as shown in SEQ ID No: 1, specifically as follows: tgcttgcccttccttaagtacattaaatacattcccaataggtggaaagatttgtaaacagctaggccattctccctccttctaaccctcatctccagggcctaagaaagcccagctctgttatctggggcttgactttccctgtcttccccatcccagtatccttgcaggaaacagccggtctcgctctctgctctcagaaggaaagtttccttatcacctgtgaatcacaaacccagagagtggccaaacatagccaggctgatgcaagaccctgggaagaggaaagctgcaggtgtgtttgtgctgggaggagtggtgaccctcacctcacagtcacctcctctctggatcctcgtgaggtataaagacgagtcctc caccaccagtcaggcACACTCTACCACC

[0053] (2) The -500 to +8 fragment (508 bp) of the rat Cela1 promoter (NM_012552.4), denoted as rat Cela1500 promoter, has the nucleotide sequence shown in SEQ ID No: 2 as follows: tatgaaaaaaaaagcaatcctcactcttcatcttggatttttattaccgtgtttacctatctatgcactcatggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcatgagcacatgctcgttgtggtcaggggacaacttgcaggaacccatccactctttctgccatgtcagttccaggggattgaatgtgggtggtcgcctagcggccaatggcttcagccatcttgaagaccctcttaggatccgctggatgcaactcagctggggtcagctcagtcgacttgggttaactgagtgccggccttgttctgtctttgaatatcagataaatgagttgacttaaaatttgttcatttgtactttcatgtcacctgtgcttttccctgccttctacccacagccctgcccagctggcggaggaaggtcagcagagctgctgataagagccgtataaagagggttccgctcatggcaaggggcAGTGGTCT

[0054] (3) The -205 to +8 fragment (213 bp) of the rat Cela1 promoter (NM_012552.4), denoted as rat Cela1205 promoter, has the nucleotide sequence shown in SEQ ID No: 3 as follows: gtcgacttgggttaactgagtgccggccttgttctgtctttgaatatcagataaatgagttgacttaaaatttgttcatttgtactttcatgtcacctgtgcttttccctgccttctacccacagccctgcccagctggcggaggaaggtcagcagagctgctgataagagccgtataaagagggttccgctcatggcaaggggcAGTGGTCT

[0055] (4) The -67 to +53 (120 bp) of the human SPINK1 promoter (NM_003122.5), denoted as human SPINK1 promoter, and its nucleotide sequence is as shown in SEQ ID No: 4, specifically as follows:

[0056] atgacacagagtcaatcaataaccagggagatctgtgatatagcccagtaggtggggccttgctgccATCTGCCATATGACCCT TCCAGTCCCAGGCTTCTGAAGAGACGTGGTAAGTGC

[0057] (5) The enhancer fragment of human AMY2 (34 bp), denoted as human AMY2 enhancer, and its nucleotide sequence is as shown in SEQ ID No: 5, specifically as follows:

[0058] ttccatgagagactttttgatgttctttacctgt

[0059] (6) The enhancer fragment of rat Amy2 (40 bp), denoted as rat Amy2 enhancer, and its nucleotide sequence is as shown in SEQ ID No: 6, specifically as follows:

[0060] ttccatgagagtttctgaagaaccttcagctgtgcacatc.

[0061] II. Construction of Dual-Luciferase Reporter Gene Plasmids

[0062] To achieve the above object, in the first aspect of the present invention, different promoters are introduced, and the promoter activity is preliminarily detected in vitro by using dual-luciferase reporter gene plasmids.

[0063] 1. Construction of Dual-Luciferase Backbone Plasmids

[0064] The dual-luciferase reporter system usually requires two plasmids: one is the firefly luciferase reporter gene plasmid containing the candidate promoter sequence (the plasmid backbone used in the present invention is pGL4.1), and the other is the Renilla luciferase plasmid containing the broad-spectrum promoter CMV (the plasmid backbone used in the present invention is pRL-CMV), which is used as an internal reference to correct the deviation in the experiment (see Figure 2 ).

[0065] 2. Construction of Linearized Expression Vectors

[0066] Using the pGL4.10 vector system as a template, analyze the target sequence and vector sequence, and digest the vector with restriction endonucleases BglII and HindIII. Detect the digestion effect of the digestion products by agarose gel electrophoresis, and recover the target vector from the gel.

[0067] 3. Introduce the target sequence into the linearized expression vector

[0068] Use a seamless cloning kit to add the target gene fragment and the linearized vector to a centrifuge tube at a molar ratio of 2:1 for recombination reaction and transformation. Finally, construct six plasmids including pGL4.10 empty vector, pGL4.10-CMV promoter, pGL4.10-hPRSS1 promoter, pGL4.10-Cela1(-500 / +8) promoter, pGL4.10-Cela1(-205 / +8) promoter, and pGL4.10-hSPINK1 promoter (see Figure 3 ).

[0069] Subsequently, prepare competent cells for the above plasmids, transform them, identify positive transformants, and send positive clones for sequencing verification. These series of steps are all mature steps and will not be elaborated here.

[0070] III. In vitro expression of firefly luciferase driven by different promoters

[0071] 1. Cell transfection:

[0072] (1) Plate mouse pancreatic acinar carcinoma 266-6 cells in a 12-well plate at a cell density of 4×10 5 / well.

[0073] (2) After 24 hours, use Lipofectamine TM 2000 reagent to transfer the dual-luciferase plasmid into 266-6 cells. The specific process is as follows: According to the plasmid concentration, aspirate 3 μg of plasmid (the ratio of firefly luciferase plasmid to Renilla luciferase plasmid is 20:1), and dilute it to 50 μl with Opti-MEM; aspirate 3 μl of Lipofectamine TM 2000, and dilute it to 50 μl with Opti-MEM. Add the diluted plasmid to the diluted Lipofectamine TM 2000, and incubate at room temperature for 5 min. Finally, add the plasmid-Lipofectamine TM 2000 complex to the cells, 100 μl / well. Incubate at 37 °C for 3 d for reporter gene expression detection.

[0074] 2. Detection of reporter gene expression:

[0075] (1) Cell lysis

[0076] Discard the culture medium, wash away the residual culture medium with PBS, and then aspirate all the liquid. Add an appropriate amount of 1×CellLysis Buffer to each well, let it stand at room temperature or shake it gently for 15 min to lyse the cells, pipette and transfer all the cell lysates to a 1.5 mL centrifuge tube, centrifuge at 16000 g for 5 min, and take the supernatant for subsequent detection.

[0077] (2) Firefly Luciferase reaction detection

[0078] Aspirate 20 μL of the cell lysate supernatant and add it to the microplate. Then add 100 μL of Firefly Luciferase Reaction Buffer containing the substrate that has been equilibrated to room temperature. Mix quickly and immediately detect the activity of the Firefly Luciferase reporter gene (wavelength 560 nm) using a microplate reader.

[0079] (3) Renilla Luciferase reaction detection

[0080] Add 100 μL of the Renilla detection working solution to the above reaction solution, mix quickly, and immediately detect the activity of the Renilla Luciferase reporter gene (wavelength 465 nm) using a microplate reader. Divide the data of Firefly Luciferase by the data of Renilla Luciferase, and analyze using the normalized average value.

[0081] The results showed that the hPRSS1 promoter and the Cela1(-500 / +8) promoter had promoter activity, which was significantly increased compared with the empty vector; the promoter activities of the Cela1(-205 / +8) promoter and the hSPINK1 promoter were not significant compared with the empty vector (see Figure 4 ).

[0082] IV. Construction and identification of mutant scAAV8-ZsGreen vectors containing different promoters

[0083] The in vitro dual-luciferase assay only preliminarily explored the promoter activity. Whether it can drive the in vivo expression of the target gene needs to be further verified in vivo. This patent uses a vector (hereinafter referred to as scAAV8-ZsGreen) that mediates the ZsGreen-tagged protein gene with mutant scAAV8.

[0084] The mutant scAAV8 vector used is a hybrid viral vector produced by the combination of the AAV2 genome and the AAV8 capsid protein with the double-point mutations Y447+Y733F. This vector contains an ampicillin resistance gene, which is convenient for screening positive plasmids; tWPA is an optimized WPRE element that contains a polyA sequence, which can increase the available space of the vector and enhance the transgenic expression efficiency. Our team has confirmed the safety of this vector.

[0085] The target gene used is the 696bp zoanthus green fluorescent protein gene (ZsGreen). ZsGreen has the highest fluorescence signal-to-noise ratio, and extremely strong green fluorescence can be observed with the naked eye under white light. The fluorescence intensity is 8.6 times that of enhanced GFP.

[0086] 1. Construction and identification of scAAV8-promoter-ZsGreen vector

[0087] In the control group, the broad-spectrum promoter CMV was used to drive the expression of the target gene ZsGreen, which was later called "scAAV8-CMV-ZsGreen"; in the experimental groups, the hPRSS1, Cela1(-500 / +8), Cela1(-205 / +8), and hSPINK1 promoters were used to drive the expression of the target gene ZsGreen, which were called "scAAV8-hPRSS1-ZsGreen", "scAAV8-Cela1(-500 / +8)-ZsGreen", "scAAV8-Cela1(-205 / +8)-ZsGreen", and "scAAV8-hSPINK1-ZsGreen" respectively (as Figure 5 shown).

[0088] After the construction of the linearized expression vector, the target gene was inserted into the linearized expression vector, and the competent cells were prepared and transformed (the steps were the same as the corresponding parts in "II. Construction of Dual-Luciferase Reporter Gene Plasmids" above). The transformants grown on the plate were picked and resuspended in 10 μl of LB culture medium, and 1 μl was taken as a template for colony PCR identification. The reaction system and PCR cycling conditions were the same as the corresponding parts in "II. Construction of Dual-Luciferase Reporter Gene Plasmids" above. The positive clones were sent for sequencing for further verification and identification.

[0089] 2. Evaluation of ZsGreen expression efficiency and specificity in the pancreas

[0090] Eight-week-old wild-type C57BL / 6 mice were randomly divided into the "scAAV8-CMV-ZsGreen" group, "scAAV8-hPRSS1-ZsGreen" group, "scAAV8-Cela1(-500 / +8)-ZsGreen" group, "scAAV8-Cela1(-205 / +8)-ZsGreen" group, "scAAV8-hSPINK1-ZsGreen" group, and the untreated group. Except for the untreated group, each mouse in each group was intraperitoneally injected with 2×10 11 virus titer (vg / animal). Five days after virus infection, the mice were sacrificed by cervical dislocation, and the liver and pancreas were quickly dissected, separated and removed, washed with 1×PBS and placed in a culture dish.

[0091] Take a part of the mouse pancreas tissue and put it into a 1.5 ml EP tube, quickly store it in liquid nitrogen, embed it in OCT, cut it on ice, incubate with antibodies, and observe the fluorescence of the pancreatic frozen sections.

[0092] Take a part of the mouse pancreas and liver tissues and put them into a 1.5 ml EP tube. Extract RNA according to the column method FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme, RC112-01), use the HiScript IV All-in-Onne Ultra RT SuperMix for qPCR reverse transcription kit (Vazyme, R433), and use quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to detect the expression level of ZsGreen mRNA initiated by different promoters mediated by scAAV8, with the mouse endogenous Gapdh gene as the internal reference (see Table 1).

[0093] Table 1 qRT-PCR primers

[0094]

[0095] The results of immunofluorescence of frozen sections showed that five days after injection in vivo, the pancreas of the mice in the scAAV8-CMV-ZsGreen group showed strong green autofluorescence signals, the scAAV8-hPRSS1-ZsGreen group and the scAAV8-Cela1(-500 / +8)-ZsGreen group showed relatively strong green autofluorescence signals, while the scAAV8-Cela1(-205 / +8)-ZsGreen group and the scAAV8-hSPINK1-ZsGreen group did not show green autofluorescence signals (see Figure 6A); The detailed fluorescence quantitative results are shown in Figure 6 B.

[0096] Although the promoter activities of hPRSS1 and Cela1(-500 / +8) are not as good as that of CMV, they significantly enhance the enrichment of ZsGreen in pancreatic tissues and avoid the expression of the target gene in the liver (see Figure 6 C).

[0097] V. Evaluation of the Expression Efficiency by Combining Enhancers and Promoters

[0098] As confirmed above in this patent, the promoters of hPRSS1 and Cela1(-500 / +8) can initiate the expression of the target gene, with strong pancreatic specificity, but the promoter efficiency needs to be improved. Subsequently, in this patent, the (5) human AMY2 and (6) rat Amy2 enhancers in Method 1 are introduced to evaluate whether they can enhance the promoter efficiency of the above two promoters. The two enhancers are inserted upstream of the promoter to construct (1) the "AMY2(human)-hPRSS1" hybrid promoter, (2) the "AMY2(human)-Cela1(-500 / +8)" hybrid promoter, (3) the "Amy2(rat)-hPRSS1" hybrid promoter, and (4) the "Amy2(rat)-Cela1(-500 / +8)" hybrid promoter.

[0099] 1. Dual-Luciferase Reporter Gene Plasmids of Hybrid Promoters and In Vitro Promoter Efficiency Evaluation

[0100] First, verify the promoter efficiency of the above hybrid promoters in in vitro experiments. Using the pGL4.10 vector as the backbone, construct pGL4.10-AMY2(human)-hPRSS1 promoter, pGL4.10-AMY2(human)-Cela1(-500 / +8)promoter, pGL4.10-Amy2(rat)-hPRSS1 promoter, pGL4.10-Amy2(rat)-Cela1(-500 / +8)promoter (see Figure 7 ). The steps are the same as those above.

[0101] Dual-luciferase assay showed that Amy2(rat)-hPRSS1 promoter significantly up-regulated the expression of hPRSS1 promoter by nearly 8-fold, but there was no significant difference in the promoter efficiency between AMY2(human)-hPRSS1 promoter and hPRSS1 promoter; both AMY2(human)-Cela1(-500 / +8)promoter and Amy2(rat)-Cela1(-500 / +8)promoter significantly up-regulated the promoter efficiency of Cela1(-500 / +8)promoter, about 3-fold and 4-fold respectively (see Figure 8 ).

[0102] 2. Construction and in vivo promoter efficiency evaluation of scAAV8-heterologous promoter-ZsGreen

[0103] Subsequently, the promoter efficiency of the above heterologous promoters was further verified in vivo. The above different heterologous promoters were inserted into the mutant scAAV8-ZsGreen vector to drive the expression of the target gene ZsGreen, and they were named "scAAV8-AMY2(human)-hPRSS1-ZsGreen", "scAAV8-AMY2(human)-Cela1(-500 / +8)-ZsGreen", "scAAV8-Amy2(rat)-hPRSS1-ZsGreen", "scAAV8-Amy2(rat)-Cela1(-500 / +8)-ZsGreen" ( Figure 9 ).

[0104] The mutant scAAV8-ZsGreen vectors containing the above different heterologous promoters were injected into mice at a dose of 2×10 11 vg / mouse. Samples of pancreatic tissue and liver tissue were collected on the fifth day after injection, and the expression was detected by frozen section. The results are shown in Figure 10 . It can be seen that the introduction of human AMY2 and rat Amy2 enhancers both significantly enhanced the in vivo promoter strength of hPRSS1 and Cela1(-500 / +8) promoters, and the ZsGreen fluorescence intensity increased.

[0105] In summary, the finally selected hybrid promoters in this patent, namely (1) the "AMY2(human)-hPRSS1" hybrid promoter, (2) the "AMY2(human)-Cela1(-500 / +8)" hybrid promoter, (3) the "Amy2(rat)-hPRSS1" hybrid promoter, and (4) the "Amy2(rat)-Cela1(-500 / +8)" hybrid promoter, can all be applied to the precise pancreatic delivery and expression of foreign genes. Among them, the "AMY2(human)-hPRSS1" hybrid promoter and the "AMY2(human)-Cela1(-500 / +8)" hybrid promoter have the best efficacy in both in vitro and in vivo experiments. The hybrid promoters screened in this patent will effectively avoid ectopic expression in non-target organs such as the liver, and improve the specificity and safety of foreign gene delivery.

[0106] Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. An enhanced pancreatic acinar cell-specific promoter, characterized in that: include: The promoter core region has a nucleotide sequence as shown in SEQ ID No: 1 or SEQ ID No:

2.

2. The enhanced pancreatic acinar cell-specific promoter according to claim 1, characterized in that Also includes: The enhancer region, whose nucleotide sequence is shown as SEQ ID No: 5 or SEQ ID No:

6.

3. The enhanced pancreatic acinar cell-specific promoter according to claim 2, characterized in that: in, The nucleotide sequence of the enhancer region is shown in SEQ ID No:

5.

4. The enhanced pancreatic acinar cell-specific promoter according to claim 2, characterized in that: in, The enhanced pancreatic acinar cell-specific promoter can mediate highly targeted expression of exogenous target genes in the pancreas.

5. An enhanced pancreatic acinar cell-specific transgenic vector, characterized in that: The method comprises the enhanced pancreatic acinar cell-specific promoter according to any one of claims 1 to 4.

6. The enhanced pancreatic acinar cell-specific transgenic vector according to claim 5, characterized in that: in, The enhanced pancreatic acinar cell-specific transgenic vector uses a mutant scAAV8-mediated ZsGreen tag protein gene vector.

7. The transgenic vector according to claim 6, characterized in that: in, The mutant scAAV8 vector is a hybrid viral vector produced by combining the AAV2 genome with the AAV8 capsid protein with double point mutations of Y447+Y733F, and the vector contains an ampicillin resistance gene.

8. Use of the enhanced pancreatic acinar cell-specific transgenic vector according to any one of claims 5 to 7 in the preparation of a product for preventing and treating pancreatitis.

9. Use of the enhanced pancreatic acinar cell-specific transgenic vector according to claim 8 in the preparation of a product for preventing and treating pancreatitis, characterized in that: in, The optimal infection titer of the enhanced pancreatic acinar cell-specific transgenic vector in the product is 2×10 11 vg / animal, the optimal expression time is the 5th day after injection.

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