Enhanced pancreatic acinar cell-specific promoters and uses thereof

Abstract

The application provides an enhanced pancreatic acinar cell-specific promoter and application thereof, and belongs to the technical field of genetic engineering. The enhanced pancreatic acinar cell-specific promoter comprises a promoter core region, and the nucleotide sequence of the promoter core region is shown in SEQ ID No: 1 or SEQ ID No: 2. The enhanced pancreatic acinar cell-specific promoter can increase the specific expression of an exogenous target gene in pancreatic acinar cells, can significantly reduce the expression amount of the exogenous target gene in other tissues and organs (such as liver), and can improve the targeting of the pancreas. The enhanced pancreatic acinar cell-specific promoter can further comprise an enhancer region, and the nucleotide sequence of the enhancer region is shown in SEQ ID No: 5 or SEQ ID No: 6. The enhanced pancreatic acinar cell-specific promoter can further improve the delivery effectiveness of the exogenous target gene. The application widens the clinical application scene, and provides a reference for pancreatic disease mechanism exploration and gene therapy.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an enhanced pancreatic acinar cell-specific promoter and its application. Background Technology

[0002] Pancreatitis is classified into acute and chronic pancreatitis, and is a common acute and refractory disease in clinical practice. The recurrence and chronicity of pancreatitis is a dynamic and progressive process. Approximately 21% of acute pancreatitis (AP) will progress to recurrent pancreatitis, and 38% of patients with recurrent pancreatitis will progress to irreversible chronic pancreatitis (CP), experiencing recurrent abdominal pain, pancreatic exocrine and endocrine insufficiency, and a heavy disease burden. Some cases of CP will progress to pancreatic cancer, with a poor prognosis. [1] Current treatment strategies mainly include endoscopic treatment, pain management, and complication control. [2] Treatments for pancreatitis (CP) have limited effectiveness and cannot achieve a complete cure, posing a serious threat to human health. With advancements in genetic research, it has been discovered that some gene mutations are associated with the occurrence and chronicity of CP. More than half of CP patients in my country carry pathogenic mutations. Among these, the imbalance of trypsin activation-inhibition related genes in pancreatic acinar cells is a key process in the development of acute pancreatitis (AP) and CP, and also participates in the development and progression of CP carcinogenesis. [3] Specific regulation of gene expression is of great significance for exploring the disease mechanism of pancreatitis, studying the "inflammatory-cancer transformation" of pancreatitis, and gene therapy for pancreatitis.

[0003] In exploring disease mechanisms and processes, current research primarily relies on transgenic animals. However, organ-specific transgenic animals suffer from drawbacks such as long breeding cycles, high construction costs, high feeding costs, and the inability to flexibly control gene expression. Furthermore, there is currently no international research on gene therapy for pancreatitis. Exogenous genes enter the pancreas mainly through non-viral or viral vectors—non-viral vectors suffer from low transduction efficiency, poor targeting, and short-term gene fragment expression. Among viral vectors, adeno-associated virus (AAV) vectors are considered the most promising gene delivery vectors due to their persistent gene expression and low immunogenicity. [4] The inventors of this application previously targeted the loss-of-function mutation of SPINK1 c.194+2T>C, a major pathogenic gene in my country (accounting for approximately 40%). [3] It has been confirmed that AAV-mediated overexpression of the normal human SPINK1 gene under the universal promoter CMV can effectively alleviate the CP phenotype in mice, but there are still shortcomings such as poor expression time and poor targeting. [5]However, there is no specific gene vector targeting pancreas at present, and the non-specific expression of the carried target gene not only reduces the effective expression in the target cells, but also increases the risk of adverse immune response [6] .

[0004] Using specific promoters to mediate the expression of foreign genes is the most effective solution for precise targeting of tissues and regulating gene expression and gene therapy. Promoter is a DNA sequence located upstream of the 5' end of the gene, which can interact with RNA polymerase and transcription factors. The core promoter elements and upstream regulatory elements contained in the promoter can jointly affect the spatial and temporal expression of genes [7] , which can be divided into two categories: general promoters and tissue-specific promoters. At present, the promoters used 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 target genes, but have low targeting and are easily methylated by the host, leading to transcriptional silencing [8] .

[0005] Tissue-specific promoters can guide gene expression in specific tissues. Even if the vector has poor targeting, it can limit the expression of the target gene in non-target organs / tissues and reduce the immune response of antigen-presenting cells to transgenes [6] . Pancreas is divided into pancreatic acinar cells, duct cells, and islet cells, and the key site of pancreatitis is acinar. Previous scholars have constructed transgenic mice based on acinar cell-specific promoters such as Cela1, etc., to achieve specific expression of transgenes in pancreatic acinar cells [9] . However, there is no related research on acinar cell-specific promoters for delivering exogenous target genes in the international community at present. It is worth noting that the packaging capacity of AAV vectors is ~4kb, and the full-length promoter will exceed the packaging limit, so it is necessary to seek short fragment-specific promoters.

[0006] In addition, although tissue-specific promoters have high specificity The drawback of a generally low start-up efficiency . Hybrid promoters are new promoters composed of multiple functional regions of different promoters, such as core promoters, upstream enhancers, and upstream silencers, which jointly regulate gene expression. This strategy has been widely used in the construction of muscle-specific promoters and islet β-cell-specific promoters [10,11] . Therefore, how to achieve high-specific targeting of pancreas while maintaining high-efficiency expression of target genes under the limited packaging capacity of AAV is also a key research issue.

[0007] Therefore, the defects of the prior art are summarized as follows: (1) there is no pancreatic acinar-specific promoter to mediate delivery of exogenous genes to pancreatic tissue at the present stage; (2) AAV as an ideal gene delivery vector has the problem of low packaging capacity, and it is necessary to screen a short fragment of pancreatic acinar-specific promoter with optimal starting efficiency; (3) the starting efficiency of pancreatic acinar-specific promoter is weak, and whether the combination of pancreatic acinar-specific enhancer and promoter can improve the starting efficiency is related.

[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.

[0012] 4. High KA, Roncarolo MG. Gene Therapy. N Engl J Med 2019; 381: 455-464.

[0013] 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.

[0014] 6. Pupo A, Fernandez A, Low SH, et al. AAV vectors: The Rubik’s cube of human gene therapy. Mol Ther 2022; 30: 3515-3541.

[0015] 7. Oudelaar AM, Higgs DR. The relationship between genome structure and function. Nat Rev Genet 2021; 22: 154-168.

[0016] 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.

[0017] 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.

[0018] 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

[0019] The present application is to solve the above problems, and aims to provide an enhanced pancreatic acinar cell-specific promoter and its application.

[0020] The present application provides an enhanced pancreatic acinar cell-specific promoter, which has the following characteristics: a promoter core region, the nucleotide sequence of which is shown in SEQ ID No: 1 or SEQ ID No: 2.

[0021] In the enhanced pancreatic acinar cell-specific promoter provided by the present application, it can also have the following characteristics: an enhancer region, the nucleotide sequence of which is shown in SEQ ID No: 5 or SEQ ID No: 6.

[0022] In the enhanced pancreatic acinar cell-specific promoter provided by the present application, it can also have the following characteristics: wherein the nucleotide sequence of the enhancer region is shown in SEQ ID No: 5.

[0023] In the enhanced pancreatic acinar cell-specific promoter provided by the present application, it can also have the following characteristics: wherein the enhanced pancreatic acinar cell-specific promoter can mediate high-targeted expression of an exogenous target gene in the pancreas.

[0024] The present application provides an enhanced pancreatic acinar cell-specific transgenic vector, which has the following characteristics: the above-mentioned enhanced pancreatic acinar cell-specific promoter.

[0025] In the enhanced pancreatic acinar cell-specific transgenic vector provided by the present application, it can also have the following characteristics: wherein the enhanced pancreatic acinar cell-specific transgenic vector uses a mutant scAAV8 to mediate a ZsGreen tag protein gene vector.

[0026] In the enhanced pancreatic acinar cell-specific transgenic vector provided by the present application, it can also have the following characteristics: wherein the mutant scAAV8 vector is a hybrid virus vector produced by the combination of AAV2 type genome and Y447+Y733F double point mutation of AAV8 capsid protein, and the vector contains ampicillin resistance gene.

[0027] The application also provides the use of the enhanced pancreatic acinar cell-specific transgenic vector in the preparation of a product for preventing and treating pancreatitis.

[0028] In the use of the enhanced pancreatic acinar cell-specific transgenic vector in the preparation of a product for preventing and treating pancreatitis provided by the application, the following features can also be present: in the product, the optimal infection titer of the enhanced pancreatic acinar cell-specific transgenic vector is 2 x 10 11 vg / animal, and the optimal expression time is the 5th day after injection.

[0029] Effects of the application

[0030] According to the enhanced pancreatic acinar cell-specific promoter and the use thereof provided by the application, because the enhanced pancreatic acinar cell-specific promoter comprises a promoter core region, the nucleotide sequence of which is shown in SEQ ID No: 1 or SEQ ID No: 2, the enhanced pancreatic acinar cell-specific promoter can increase the specific expression of an exogenous target gene in pancreatic acinar cells, significantly reduce the expression amount of the exogenous target gene in other tissues and organs (such as the liver), and improve the targeting of the pancreas.

[0031] Further, because the enhanced pancreatic acinar cell-specific promoter further comprises an enhancer region, the nucleotide sequence of which is shown in SEQ ID No: 5 or SEQ ID No: 6, the delivery effectiveness of the exogenous target gene can be further improved.

[0032] The application broadens the clinical application scenarios and provides a reference for pancreas disease mechanism exploration and gene therapy. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is a schematic diagram of screening of a pancreatic acinar cell-specific candidate gene in an embodiment of the application;

[0034] Figure 2 is a diagram of a dual-luciferase backbone plasmid in an embodiment of the application, in which a pGL4.10 plasmid (top) and a pRL-CMV plasmid (bottom) are shown;

[0035] Figure 3 is a diagram of different promoter plasmids in an embodiment of the application, in which 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; and F. pGL4.10-hSPINK1 promoter.Figure 4 Figure 1 is a result chart of in vitro activity of different promoter sequences in an embodiment of the present application.

[0036] Figure 5 Figure 2 is a chart of scAAV8-ZsGreen vectors driven by different promoters in an embodiment of the present application, wherein A. scAAV8-CMV-ZsGreen; B. scAAV8-hPRSS1-ZsGreen; C. scAAV8-Cela1(-500 / +8)-ZsGreen; D. scAAV8-Cela1(-205 / +8)-ZsGreen; E. scAAV8-hSPINK1-ZsGreen.

[0037] Figure 6 Figure 3 is a chart of efficiency and pancreatic tissue specificity evaluation of ZsGreen gene driven by different promoters in an embodiment of the present application, wherein A. Representative pictures of frozen sections showing spontaneous green fluorescence of ZsGreen gene driven by different promoters; B. Quantification of spontaneous green fluorescence ratio of head and tail of pancreas of different promoters; C. Pancreas / liver ratio of ZsGreen gene mRNA of different promoters, the greater the value, the higher the specificity in pancreatic tissue.

[0038] Figure 7 Figure 4 is a chart of plasmid maps of different hybrid promoters in an embodiment of the present application, wherein 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.

[0039] Figure 8 Figure 5 is a chart of in vitro experimental results of AMY2(human) and Amy2(rat) enhancing the activity of hPRSS1 and Cela1(-500 / +8) promoter sequences.

[0040] Figure 9Figure is a scAAV8-ZsGreen vector map driven by different promoter combination enhancer in the embodiments of the present application, wherein 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.

[0041] Figure 10 Figure is a result map of hybrid promoter significantly improving ZsGreen gene efficiency in the embodiments of the present application. DETAILED DESCRIPTION

[0042] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the following embodiments will be specifically described in combination with the drawings.

[0043] EMBODIMENT

[0044] The present embodiment provides an enhanced pancreatic acinar cell-specific promoter and its application.

[0045] I. Screening of Promoter Sequences

[0046] 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 the genes were ranked from high to low according to enrichment scores; through gene description, genes playing a function in pancreatic islet cells were excluded. Then, the expression of each gene in different tissues was checked through the Human Protein Atlas database (https: / / www.proteinatlas.org / ), and genes with poor specificity were excluded. A total of 200 pancreatic acinar expression genes were obtained through the human single-cell RNA sequencing database (http: / / bis.zju.edu.cn / HCL / ), ranked from high to low according to the scores, and the top 30 genes were screened. Based on the mouse RNA-seq data of the team [5] , the top 20 genes were screened. According to the screening results of the above databases and the original data of the team, it was determined to use the key regulatory sequences upstream of the transcription start site of PRSS1, AMY2A and CELA genes for subsequent research (see Figure 1 ).

[0047] The sequence around the transcriptional start site (TSS) is highly conserved during evolution, and the short sequence around the TSS is inferred to be the core region of the promoter. According to the transcriptionally active sites, and the reported Cela1 gene in the previous transgenic studies [9] (are Figure 1 The homologous genes of human CELA3A and CELA3B), the patent finally includes the-395 to +13 fragment (408 bp) of human PRSS1 promoter (NM_002769.5), the-500 to +8 fragment (508 bp) and the-205 to +8 fragment (213 bp) of rat Cela1 promoter (NM_012552.4), and the-67 to +53 (120 bp) of human SPINK1 promoter (NM_003122.5). The AMY2 gene is mainly an enhancer region, and the patent finally includes the enhancer fragments of human AMY2 (34 bp) and rat Amy2 (40 bp).

[0048] The following are the specific sequence information of each promoter and enhancer involved in the patent:

[0049] (1) The-395 to +13 fragment (408 bp) of human PRSS1 promoter (NM_002769.5) is recorded as human PRSS1 promoter, and the nucleotide sequence is shown in SEQ ID No: 1, which is as follows: tgcttgcccttccttaagtacattaaatacattcccaataggtggaaagatttgtaaacagctaggccattctccctccttctaaccctcatctccagggcctaagaaagcccagctctgttatctggggcttgactttccctgtcttccccatcccagtatccttgcaggaaacagccggtctcgctctctgctctcagaaggaaagtttccttatcacctgtgaatcacaaacccagagagtggccaaacatagccaggctgatgcaagaccctgggaagaggaaagctgcaggtgtgtttgtgctgggaggagtggtgaccctcacctcacagtcacctcctctctggatcctcgtgaggtataaagacgagtcctccaccaccagtcaggcACACTCTACCACC

[0050] (2) rat Cela1 promoter (NM_012552.4) -500 to +8 fragment (508bp), denoted rat Cela1 500 promoter, having the nucleotide sequence as shown in SEQ ID No: 2, in particular as follows: tatgaaaaaaaaagcaatcctcactcttcatcttggatttttattaccgtgtttacctatctatgcactcatggtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcatgagcacatgctcgttgtggtcaggggacaacttgcaggaacccatccactctttctgccatgtcagttccaggggattgaatgtgggtggtcgcctagcggccaatggcttcagccatcttgaagaccctcttaggatccgctggatgcaactcagctggggtcagctcagtcgacttgggttaactgagtgccggccttgttctgtctttgaatatcagataaatgagttgacttaaaatttgttcatttgtactttcatgtcacctgtgcttttccctgccttctacccacagccctgcccagctggcggaggaaggtcagcagagctgctgataagagccgtataaagagggttccgctcatggcaaggggcAGTGGTCT

[0051] (3) rat Cela1 promoter (NM_012552.4) -205 to +8 fragment (213bp), denoted rat Cela1 205 promoter, having the nucleotide sequence as shown in SEQ ID No: 3, in particular as follows: gtcgacttgggttaactgagtgccggccttgttctgtctttgaatatcagataaatgagttgacttaaaatttgttcatttgtactttcatgtcacctgtgcttttccctgccttctacccacagccctgcccagctggcggaggaaggtcagcagagctgctgataagagccgtataaagagggttccgctcatggcaaggggcAGTGGTCT

[0052] (4) human SPINK1 promoter (NM_003122.5) from -67 to +53 (120bp), denoted as human SPINK1 promoter, the nucleotide sequence of which is shown as SEQ ID No: 4, and specifically as follows:

[0053] atgacacagagtcaatcaataaccagggagatctgtgatatagcccagtaggtggggccttgctgccATCTGCCATATGACCCT TCCAGTCCCAGGCTTCTGAAGAGACGTGGTAAGTGC

[0054] (5) Enhancer fragment of human AMY2 (34bp), denoted as human AMY2 enhancer, the nucleotide sequence of which is shown as SEQ ID No: 5, and specifically as follows:

[0055] ttccatgagagactttttgatgttctttacctgt

[0056] (6) Enhancer fragment of rat Amy2 (40bp), denoted as rat Amy2 enhancer, the nucleotide sequence of which is shown as SEQ ID No: 6, and specifically as follows:

[0057] ttccatgagagtttctgaagaaccttcagctgtgcacatc.

[0058] II. Construction of dual-luciferase reporter plasmid

[0059] To achieve the above-mentioned purpose, in the first aspect of the present application, different promoters are introduced, and the activity of the promoters is preliminarily detected in vitro by using a dual-luciferase reporter plasmid.

[0060] 1. Construction of dual-luciferase backbone plasmid

[0061] The dual-luciferase reporter system generally requires two plasmids: one is a firefly luciferase reporter plasmid containing a candidate promoter sequence (the plasmid backbone used in the present application is pGL4.1), and the other is a Renilla luciferase plasmid containing a broad-spectrum promoter CMV (the plasmid backbone used in the present application is pRL-CMV), which is used as an internal reference to correct the bias in the experiment (see Figure 2 ).

[0062] 2. Construction of linearized expression vector

[0063] With pGL4.10 vector system as template, analyze the sequence of interest and vector sequence, and use restriction endonuclease BglII and HindIII to cut the vector. Perform agarose gel electrophoresis on the enzyme cutting product to detect the enzyme cutting effect, and perform gel recovery on the vector of interest.

[0064] 3. Introduce the sequence of interest into the linearized expression vector

[0065] Using seamless cloning kit, add the gene fragment of interest and linearized vector to the centrifuge tube at a molar ratio of 2:1 for recombination reaction and transformation. Finally, construct 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, a total of 6 kinds of plasmids (see Figure 3 ).

[0066] Subsequently, prepare competent cells and transform the above plasmids, identify positive transformants, and send the positive clones for sequencing verification. The series of steps are mature steps, which will not be described here.

[0067] III. In vitro expression of firefly luciferase driven by different promoters

[0068] 1. Cell transfection:

[0069] (1) Plate mouse pancreatic acinar carcinoma 266-6 cells, 12-well plate, cell amount 4x10 5 / well.

[0070] (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 concentration of the plasmid, take 3ug of the plasmid (the ratio of firefly luciferase plasmid and sea panicle luciferase plasmid is 20:1), and dilute it to 50ul with Opti-MEM; take 3ul of Lipofectamine TM 2000, and dilute it to 50ul with Opti-MEM. Add the diluted plasmid to the diluted Lipofectamine TM 2000, and incubate at room temperature for 5min. Finally, add the plasmid-Lipofectamine TM 2000 complex to the cells, 100ul / well. Incubate at 37℃ for 3d, and perform reporter gene expression detection.

[0071] 2. Reporter gene expression detection:

[0072] (1) Lysis of cells

[0073] Discard the culture medium, wash the residual medium with PBS, and then aspirate all the liquid. Add an appropriate amount of 1x Cell Lysis Buffer to each well, and stand or shake at room temperature for 15 min. Blow and aspirate all the cell lysates into a 1.5 mL centrifuge tube, and centrifuge at 16000 g for 5 min. Take the supernatant for subsequent detection.

[0074] (2) Firefly Luciferase reaction detection

[0075] Aspirate 20 uL of cell lysate supernatant into the enzyme-labeled plate, and then add 100 uL of Firefly Luciferase Reaction Buffer containing substrate equilibrated to room temperature. Mix quickly and immediately detect the Firefly Luciferase reporter gene activity (wavelength 560 nm) in the enzyme-labeled instrument.

[0076] (3) Renilla Luciferase reaction detection

[0077] Add 100 uL of Renilla detection working solution to the above reaction solution, mix quickly, and immediately detect the Renilla Luciferase reporter gene activity (wavelength 465 nm) in the enzyme-labeled instrument. Divide the data of Firefly Luciferase by the data of Renilla Luciferase, and analyze the normalized average value.

[0078] As can be seen from the results, the hPRSS1 promoter and Cela1 (-500 / +8) promoter have promoter activity, which is significantly improved compared with the empty vector; the promoter activity of Cela1 (-205 / +8) and hSPINK1 is not significantly improved compared with the empty vector (see Figure 4 ).

[0079] Four, construction and identification of mutant scAAV8-ZsGreen vectors containing different promoters

[0080] The in vitro dual luciferase experiment only preliminarily explores the promoter activity, and whether it can drive the expression of the target gene in vivo still needs to be further verified in vivo. The present patent uses a mutant scAAV8-mediated ZsGreen tag protein gene vector (hereinafter referred to as scAAV8-ZsGreen).

[0081] The applied mutant scAAV8 vector is a hybrid virus vector produced by combining the AAV2 type genome with the Y447+Y733F double point mutation of AAV8 capsid protein, which contains an ampicillin resistance gene for positive plasmid screening; tWPA is an optimized WPRE element containing a polyA sequence, which can increase the available space of the vector and enhance the transgene expression efficiency. Our team has confirmed the safety of this vector.

[0082] The target gene used is a 696 bp coral reef green fluorescent protein gene (zoanthus green fluorescent protein, ZsGreen). ZsGreen has the highest fluorescence signal-to-noise ratio, and the extremely strong green fluorescence can be observed with the naked eye under white light, with a fluorescence intensity 8.6 times that of enhanced GFP.

[0083] 1. Construction and identification of scAAV8-promoter-ZsGreen vector

[0084] The control group uses the broad-spectrum promoter CMV to drive the expression of the target gene ZsGreen, which is referred to as "scAAV8-CMV-ZsGreen" hereinafter; the experimental group uses the hPRSS1, Cela1(-500 / +8), Cela1(-205 / +8), and hSPINK1 promoters to drive the expression of the target gene ZsGreen, respectively, which are referred to as "scAAV8-hPRSS1-ZsGreen", "scAAV8-Cela1(-500 / +8)-ZsGreen", "scAAV8-Cela1(-205 / +8)-ZsGreen", and "scAAV8-hSPINK1-ZsGreen" (as shown in Figure 5 ).

[0085] After linearizing the expression vector, constructing the target gene into the linearized expression vector, preparing and transforming the competent cells (the steps are the same as the corresponding part in the above "II. Construction of dual luciferase reporter plasmid"), the transformants growing on the plate were resuspended in 10 μl of LB culture solution, and 1 μl was used as a template for colony PCR identification. The reaction system and PCR cycle conditions are the same as the corresponding part in the above "II. Construction of dual luciferase reporter plasmid". Positive clones were sent for sequencing for further verification and identification.

[0086] 2. Evaluation of ZsGreen expression efficiency and specificity in pancreas

[0087] Eight-week-old wild-type C57BL / 6 mice were randomly divided into "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 untreated group. Except for the untreated group, each mouse in each group was injected intraperitoneally with 2×10 11 Five days after viral infection, the mice were sacrificed by cervical dislocation, quickly dissected and the liver and pancreas were removed, washed with 1×PBS and placed in a culture dish.

[0088] Part of the mouse pancreas tissue was taken into a 1.5ml EP tube and quickly stored in liquid nitrogen, and after OCT embedding, ice cutting and antibody incubation, the fluorescence of the pancreas frozen section was observed.

[0089] Part of the mouse pancreas and liver tissue was taken into a 1.5ml EP tube, and RNA was extracted according to the column method FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme, RC112-01), and HiScript IV All-in-One Ultra RT SuperMix for qPCR reverse transcription kit (Vazyme, R433) was used, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to detect the expression of ZsGreen mRNA mediated by different promoters of scAAV8, and the mouse background Gapdh gene was used as an endogenous reference (see Table 1).

[0090] Table 1 qRT-PCR primers

[0091]

[0092] The results of immunofluorescence of frozen sections showed that 5 days after injection in vivo, the pancreas of mice in the scAAV8-CMV-ZsGreen group showed strong green autofluorescence signal, the scAAV8-hPRSS1-ZsGreen group and the scAAV8-Cela1(-500 / +8)-ZsGreen group showed strong green autofluorescence signal, while the scAAV8-Cela1(-205 / +8)-ZsGreen group and the scAAV8-hSPINK1-ZsGreen group did not show green autofluorescence signal (see Figure 6A); the results of the fluorescence quantification are shown in Figure 6 B.

[0093] Although the hPRSS1 and Cela1(-500 / +8) promoters are less potent than CMV, they significantly enhance the enrichment of ZsGreen in the pancreas and avoid the expression of the gene of interest in the liver (see Figure 6 C).

[0094] V. Evaluation of the expression efficiency of enhancer-promoter combinations

[0095] The hPRSS1 and Cela1(-500 / +8) promoters identified above can initiate the expression of the gene of interest with a strong pancreas specificity, but their efficiency needs to be improved. The (5) human AMY2 and (6) rat Amy2 enhancers introduced in Method I (5) and (6) were then inserted upstream of the promoters to evaluate whether they can enhance the efficiency of the two promoters. The two enhancers were inserted upstream of the promoters 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.

[0096] 1. Hybrid promoter dual luciferase reporter plasmids and evaluation of the in vitro promoter efficiency

[0097] The efficiency of the hybrid promoters described above was first evaluated in vitro using the pGL4.10 vector as a backbone to construct pGL4.10-AMY2(human)-hPRSS1 promoter, pGL4.10-AMY2(human)-Cela1(-500 / +8) promoter, pGL4.10-Amy2(rat)-hPRSS1 promoter, and pGL4.10-Amy2(rat)-Cela1(-500 / +8) promoter (see Figure 7 ), following the same procedure as above.

[0098] The dual-luciferase assay found that Amy2(rat)-hPRSS1 promoter significantly up-regulated the expression of hPRSS1 promoter by nearly 8 times, but the starting efficiency of AMY2(human)-hPRSS1 promoter had no significant difference with hPRSS1 promoter; AMY2(human)-Cela1(-500 / +8) promoter and Amy2(rat)-Cela1(-500 / +8) promoter could significantly up-regulate the starting efficiency of Cela1(-500 / +8) promoter, about 3 times and 4 times respectively (see Figure 8 ).

[0099] 2. Construction of scAAV8-hybrid promoter-ZsGreen and evaluation of the starting efficiency in vivo

[0100] Subsequently, the starting efficiency of the above hybrid promoters was further verified in vivo. The above different hybrid promoters were inserted into the mutant scAAV8-ZsGreen vector to drive the expression of the target gene ZsGreen, which were respectively called "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 ).

[0101] The above mutant scAAV8-ZsGreen vectors containing different hybrid promoters were injected into mice at a dose of 2x10 11 vg / each, and the pancreatic tissue and liver tissue samples were collected on the fifth day after injection. The expression was detected by frozen section, and the results are shown in Figure 10 . It can be seen that the introduction of human AMY2 and rat Amy2 enhancers significantly enhances the in vivo starting strength of hPRSS1 and Cela1(-500 / +8) promoters, and the ZsGreen fluorescence intensity is increased.

[0102] In summary, the final screened hybrid promoters in the present patent, (1) "AMY2(human)-hPRSS1", (2) "AMY2(human)-Cela1(-500 / +8)", (3) "Amy2(rat)-hPRSS1", and (4) "Amy2(rat)-Cela1(-500 / +8)", can be applied to the precise delivery and expression of exogenous genes in pancreas, among which "AMY2(human)-hPRSS1" and "AMY2(human)-Cela1(-500 / +8)" have the best performance in in vivo and in vitro experiments. The hybrid promoters screened in the present patent can effectively avoid ectopic expression in non-target organs such as liver, and improve the specificity and safety of exogenous gene delivery.

[0103] Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. An enhanced pancreatic acinar cell-specific promoter, characterized in that, It consists of the following two parts composition: The promoter core region, whose nucleotide sequence is shown in SEQ ID No: 2; and The enhancer region has a nucleotide sequence as shown in SEQ ID No: 5 or SEQ ID No:

6.

2. The enhanced pancreatic acinar cell-specific promoter according to claim 1, characterized in that: in, The enhanced pancreatic acinar cell-specific promoter can mediate highly targeted expression of exogenous target genes in pancreatic acinar cells.

3. An enhanced pancreatic acinar cell-specific transgenic vector, characterized in that, Includes the enhanced pancreatic acinar cell-specific promoter as described in claim 1 or 2.

4. The enhanced pancreatic acinar cell-specific transgenic vector according to claim 3, characterized in that: in, The enhanced pancreatic acinar cell-specific transgenic vector was mediated using mutant scAAV8. ZsGreen Vectors for tag protein genes.

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