3 'UTR element and application thereof

Abstract

The invention relates to a 3 'UTR element and an application thereof. The nucleotide sequence of the 3 'UTR element contains a fragment sequence of an Rtn1 gene from a termination codon to a poly A tailing signal site of a positive-sense strand. According to the characteristics of 3 'UTR of mRNA, a fragment sequence from a termination codon to a poly A tailing signal site of a sense strand of an Rtn1 gene is selected, a 3' UTR element which is more specifically expressed in the brain relative to bGHpA is obtained, the 3 'UTR element is applied to the recombinant adeno-associated virus, exogenous genes can be better helped to translate and express in neurons, and the expression efficiency of the recombinant adeno-associated virus is improved. The expression efficiency of transgenes in the brain is improved, and the method has a wide application prospect in the fields of neural circuit tracing, gene therapy and transgenosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a 3'UTR element and an application thereof. Background Art

[0002] Ribonucleic Acid (RNA) is a long-chain molecule formed by phosphodiester bond condensation of ribonucleotides. It is widely present in various organisms and is one of the key molecules for the transmission of genetic information in organisms. It participates in important biological processes such as protein synthesis and gene expression. Organisms contain a variety of RNA molecules, which are divided into messenger RNA (mRNA) and non-coding RNA (nc-RNA) according to whether they can be translated into proteins. mRNA is a template for guiding protein synthesis and can transfer genetic information from DNA to protein. mRNA is a temporary copy with the characteristics of few copies, short lifespan, and few modified components. The life process in the cell requires the coordinated action of multiple proteins, so there are many types of mRNA, and they change with the constant changes in cell status and external environment.

[0003] During the transcription process, RNA synthase cannot predict the location of the termination codon in the transcript and can only complete the shearing and polyadenylic acid tailing of pre-mRNA by recognizing the polyadenylation signal (AAUAAA, AUUAAA) on the pre-mRNA (precursor RNA), which is referred to as polyadenylation. This is a process that mRNA must undergo during its maturation.

[0004] 3'UTR refers to an untranslated sequence downstream of the mature messenger ribonucleotide coding region, which contains a polyadenylic acid tailing signal and a GU-rich auxiliary sequence 10-30 bases downstream. In eukaryotes, 3'UTR can be recognized by possible post-transcriptional regulatory targets, thereby participating in maintaining the stability of mRNA and controlling its translation. In eukaryotes, the initiation of mRNA degradation mainly depends on polyadenylic acid tailing (poly A), which is mainly formed by cutting at the 3' end of mRNA, and then poly A polymerase introduces polyadenylic acid into the 3' end of mRNA through covalent linkage to form a poly A tail. This process can protect mRNA from the action of nuclease exonucleases and affect the nuclear export, transport, stability and translation of mRNA.

[0005] Therefore, solving the expression of heterologous genes in host cells depends on some elements in the expression vector, such as enhancers, promoters, introns, poly A signals, integration sites and regulatory sequences. Correct selection of these elements can improve the expression level and stability of recombinant proteins. However, most studies focus on the combined effects of enhancers, internal ribosome entry sites (IRES), introns, promoters and regulatory elements and promoters, and there are few studies on the effects of poly A elements and derived 3'UTRs on exogenous expression in brain neurons. In eukaryotes, 3'UTR can be recognized by post-transcriptional regulatory targets, thereby participating in maintaining the stability of mRNA and controlling its translation. Therefore, the sequence design and optimization of 3'UTR are very important.

[0006] The brain is mainly composed of two types of cells, namely neurons responsible for processing and storing information related to brain function and glial cells that provide structural support, nutrient delivery, information processing and transmission for neurons. For Alzheimer's disease, Parkinson's disease, and multiple sclerosis, it is often associated with neuronal damage or even death. 3'UTR can affect the stability, translation and protein synthesis of mRNA, and play a key role in the function, development, plasticity and regeneration of neurons. The currently commonly used 3'UTR is bGHpA (bovine growth factor poly A), but it is not very stable for transgenic expression and requires the auxiliary effect of enhancers. There are reports that WPRE (Woodchuck PosttranscriptionalRegulatory Element) enhancer may cause cancer. Summary of the invention

[0007] Based on this, it is necessary to provide a 3'UTR element and its application. The 3'UTR element can better help the translation and expression of exogenous genes in neurons, and improve the expression efficiency of transgenes in the brain.

[0008] The present application provides a 3'UTR element, wherein the nucleotide sequence of the 3'UTR element comprises: a fragment sequence from the stop codon to the poly A tailing signal site of the positive strand of the Rtn1 gene.

[0009] In the present application, according to the characteristics of the 3'UTR of mRNA, a fragment sequence from the stop codon to the poly A tailing signal site of the positive chain of the Rtn1 gene is selected and modified to obtain a 3'UTR element that is better than bGHpA in promoting the expression of exogenous genes in the brain, and the 3'UTR element of the present application is applied to a recombinant adeno-associated virus, which can better help the translation and expression of exogenous genes in neurons and improve the expression efficiency of transgenes in the brain. It has broad application prospects in the fields of neural circuit tracing, gene therapy and transgenics.

[0010] In some embodiments, the nucleotide sequence of the 3'UTR element contains the nucleotide sequence of the Rtn1 gene.

[0011] The second aspect of the present application provides a recombinant vector, characterized in that the recombinant vector carries the above-mentioned 3'UTR element.

[0012] In some embodiments, the recombinant vector is a recombinant adeno-associated virus vector packaged with the 3'UTR element;

[0013] Furthermore, the recombinant vector also carries a fluorescent protein reporter gene.

[0014] The third aspect of the present application provides a method for constructing the above-mentioned recombinant vector, characterized in that it comprises the following steps:

[0015] Constructing a backbone vector;

[0016] Amplifying Rtn1 using amplification primers to obtain a gene fragment of the 3'UTR element;

[0017] The backbone vector is connected to the gene fragment of the 3'UTR element to obtain the recombinant vector.

[0018] In some embodiments, the backbone vector is an adeno-associated virus backbone vector and contains a fluorescent protein reporter gene;

[0019] The amplification primer pair contains a sequence that recognizes the 3'UTR element and complements the fluorescent protein reporter gene;

[0020] Furthermore, the amplification primer pair is used to amplify the nucleotide sequence of the Rtn1 gene.

[0021] A fourth aspect of the present application provides a recombinant virus, characterized in that the recombinant virus is packaged with the recombinant vector described above.

[0022] In some embodiments, the recombinant virus is a recombinant adeno-associated virus.

[0023] The fifth aspect of the present application provides a method for preparing the above-mentioned recombinant virus, characterized in that it comprises the following steps:

[0024] The recombinant vector, packaging vector and auxiliary vector are co-transfected into 293T cells, and the virus is separated to obtain the recombinant virus.

[0025] In a sixth aspect, the present application provides the use of the above-mentioned 3'UTR element, or the above-mentioned recombinant vector, or the above-mentioned recombinant virus in neural circuit tracing, gene therapy and / or transgenic related reagents and / or drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the construction of the pAAV-pCALM1-EYFP-Rtn1 vector in Example 1;

[0027] Figure 2 It is a comparison chart of the cell transfection results and fluorescence values ​​of pAAV-pCALM1-EYFP-Rtn1 and pAAV-pCALM1-EYFP-bGHpA in Example 1;

[0028] Figure 3 This is a comparison of the WB results after transfection of pAAV-pCALM1-EYFP-Rtn1 and pAAV-pCALM1-EYFP-bGHpA cells in Example 1;

[0029] Figure 4 This is a comparison of the M1 brain region of mice infected with the pAAV-pCALM1-EYFP-Rtn1 and pAAV-pCALM1-EYFP-bGHpA viruses in Example 1. DETAILED DESCRIPTION

[0030] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described in the application, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0031] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used in this application and in the specification of this application are only for the purpose of describing the implementation methods and embodiments and are not intended to limit this application.

[0032] Unless otherwise specified or there is a contradiction, the terms and phrases used in this application have the following meanings:

[0033] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0034] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0035] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0036] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0037] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0038] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0039] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In the present application, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in the present application should be understood to include any and all sub-ranges included therein.

[0040] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0041] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0042] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0043] Adeno-associated virus (AAV) is a tiny, non-enveloped virus with an icosahedral structure that contains a linear single-stranded DNA genome. In most cases, AAV can only produce toxic infections when it relies on other viruses, such as adenovirus, herpes virus, etc. AAV has highly specific infection, long-term stable expression ability and low immunogenicity. Recombinant adeno-associated virus (rAAV) is a new gene vector modified on the basis of non-pathogenic wild-type AAV. It is a tool with good safety, a wide range of host cells, low immunogenicity, and can efficiently deliver exogenous genes for long-term expression in vivo.

[0044] At present, recombinant adeno-associated viruses are widely used in transgenic research and gene therapy, and the key point lies in the selection of vectors. For example, in the treatment of neurological diseases, due to the existence of the blood-brain barrier, peripheral gene expression products are difficult to be transported to the brain to achieve therapeutic effects. Therefore, in order to overcome this problem, researchers have selected methods such as stereotactic brain injection through the blood-brain barrier and intramuscular injection to enter the nervous system in reverse transport to transfer exogenous genes into the nervous system. Most neurological diseases have varying degrees of neuronal cell death and nervous system function degeneration. Therefore, neurotrophic factors play an important role in the treatment of neurological diseases. However, how to make these exogenous neurotrophic factors better enter the body and express more stably.

[0045] The expression of heterologous genes in host cells depends on some elements in the expression vector, such as enhancers, promoters, introns, poly A signals, integration sites and regulatory sequences. Correct selection of these elements can improve the expression level and stability of recombinant proteins. In eukaryotes, 3'UTR can be recognized by post-transcriptional regulatory targets, thereby participating in maintaining the stability of mRNA and controlling its translation. Therefore, the sequence design and optimization of 3'UTR are very important. The brain is mainly composed of two types of cells, namely, neurons responsible for processing and storing information related to brain function and glial cells that provide structural support, nutrient delivery, information processing and transmission for neurons. For Alzheimer's disease, Parkinson's disease, and multiple sclerosis, it is often associated with neuronal damage or even death. 3'UTR can affect the stability, translation and protein synthesis of mRNA, and play a key role in the function, development, plasticity and regeneration of neurons. The commonly used 3'UTR is bGHpA (bovine growth factor poly A), but it is not very stable for transgenic expression and requires the assistance of an enhancer. There are reports that the WPRE (Woodchuck Posttranscriptional Regulatory Element) enhancer may cause cancer.

[0046] Based on this, in a first aspect of the embodiments of the present application, a 3'UTR element is provided, wherein the nucleotide sequence of the 3'UTR element comprises: a fragment sequence from the stop codon to the poly A tailing signal site of the positive strand of the Rtn1 gene.

[0047] Among them, the Rtn1 gene is the reticular family 1 gene. This gene belongs to the reticular encoding gene family. Reticular cells are related to the endoplasmic reticulum and are involved in neuroendocrine secretion or membrane transport of neuroendocrine cells. This gene is considered to be a specific marker for neurological diseases and cancers and a potential molecular target for treatment.

[0048] In the present application, according to the characteristics of the 3'UTR of mRNA, a fragment sequence from the stop codon to the poly A tailing signal site of the positive chain of the Rtn1 gene is selected to obtain a 3'UTR element that is more specifically expressed in the brain than bGHpA, and the 3'UTR element of the present application is applied to a recombinant adeno-associated virus, which can better help the translation and expression of exogenous genes in neurons and improve the expression efficiency of transgenes in the brain. It has broad application prospects in the fields of neural circuit tracing, gene therapy and transgenics.

[0049] The nucleotide sequence of the 3'UTR element contains: a 578 bp sequence from the Rtn1 termination codon to the positive strand polyA tailing signal site (AATAAA).

[0050] In some embodiments, the nucleotide sequence of the 3'UTR element contains the nucleotide sequence of the Rtn1 gene.

[0051] Specifically, the 3'UTR element is pAAV-pCALM1-EYFP-Rtn1.

[0052] The above 3'UTR element can better translate exogenous genes in neurons and increase expression, improve the efficiency of transgenics, and can be applied to the fields of neural circuit tracing, gene therapy and transgenics, such as neural circuit tracing, gene therapy and / or transgenic related reagents and / or drugs.

[0053] A second aspect of the embodiments of the present application provides a recombinant vector, characterized in that the recombinant vector carries the 3'UTR element described in the first aspect.

[0054] In some embodiments, the recombinant vector is a recombinant adeno-associated virus vector packaged with the 3'UTR element.

[0055] Furthermore, the recombinant vector also carries a fluorescent protein reporter gene.

[0056] By constructing a recombinant vector carrying the above 3'UTR element, it is beneficial to the application and research of the element in the fields of neural circuit tracing, gene therapy and transgenic fields, such as neural circuit tracing, gene therapy and / or transgenic related reagents and / or drugs.

[0057] The third aspect of the embodiment of the present application provides a method for constructing the recombinant vector of the second aspect, comprising the following steps S110-S130:

[0058] S110, constructing a backbone vector;

[0059] S120, amplifying Rtn1 using an amplification primer to obtain a gene fragment of the 3'UTR element;

[0060] S130, connecting the backbone vector and the gene fragment of the 3'UTR element to obtain the recombinant vector.

[0061] In some embodiments, the backbone vector is an adeno-associated virus backbone vector and contains a fluorescent protein reporter gene;

[0062] The amplification primer pair contains a sequence that recognizes the 3'UTR element and complements the fluorescent protein reporter gene;

[0063] It should be noted that the backbone vector is not limited to an adeno-associated virus backbone vector, and may also be other backbone vectors capable of cloning or expressing the above-mentioned 3'UTR element.

[0064] In some embodiments, the backbone vector is connected to the gene fragment of the 3'UTR element by homologous recombination. It should be noted that the connection method is not limited to homologous recombination, and other gene fragment connection methods may also be used.

[0065] In a fourth aspect of the embodiment of the present application, a recombinant virus is provided, wherein the recombinant virus is packaged with the recombinant vector described in the third aspect. By constructing the recombinant virus, it is beneficial to the application and research of 3'UTR elements in the fields of neural circuit tracing, gene therapy and transgenic fields, such as neural circuit tracing, gene therapy and / or transgenic related reagents and / or drugs.

[0066] In some embodiments, the recombinant virus is a recombinant adeno-associated virus. It should be noted that the recombinant virus is not limited to a recombinant adeno-associated virus, and may also be other viruses that can package the above 3'UTR element.

[0067] In a fifth aspect of the embodiments of the present application, a method for preparing the recombinant virus of the fourth aspect is provided, comprising the following steps:

[0068] The recombinant vector, packaging vector and auxiliary vector are co-transfected into 293T cells, and the virus is separated to obtain the recombinant virus.

[0069] The 3'UTR element provided in the present application can improve the stability of transgenic expression and the translation level in the brain, and can be used for the transgenic efficiency of nerve cells and other tissue cells.

[0070] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0071] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0072] Example 1

[0073] The 3'UTR element of this example is pAAV-pCALM1-EYFP-Rtn1.

[0074] First, the Rtn1 sequence was packaged into a recombinant adeno-associated virus vector containing a fluorescent protein. In this example, the fluorescent protein EYFP was used as the reporter gene of the 3'UTR element to obtain the recombinant adeno-associated virus vector pAAV-pCALM1-EYFP-Rtn1. The Rtn1 virtual plasmid was constructed using Snapgene software (see Figure 1 As stated, Figure 1 Schematic diagram for the construction of pAAV-pCALM1-EYFP-Rtn1 vector), using pAAV-pCALM1-EYFP-bGHpA as the backbone and control for replacing bGHpA with Rtn1 and its transformation and deletion models. During the construction process, pAAV-pCALM1-EYFP-bGHpA was treated with restriction endonucleases BsrGI and AgeI, and digested at 37°C for 2h to obtain the connection skeleton of pAAV-pCALM1-EYFP-Rtn1. The enzyme digestion system is shown in Table 1. At the same time, DNA extracted from human embryonic kidney cells 293T was used as a template to amplify the Rtn1 fragment. Since the recognition site of BsrGI is located on the fluorescent protein EYFP sequence, when designing primers to amplify Rtn1, it is necessary to supplement the EYFP sequence that is digested by enzymes so that the EYFP amino acid sequence is complete and no frameshift mutation occurs. In the process of designing primers, in order to reduce the loss of individual bases caused by enzyme digestion, the homologous recombination method is used for connection. Therefore, in the process of designing primers, it is necessary to make the primers contain sequences that recognize the Rtn1 sequence, supplement the EYFP sequence, and overlap with the vector.

[0075] The fragment amplification reaction system and reaction steps are shown in Tables 2 and 3. The backbone and fragments were homologously recombined at 50°C for 30 min. The ligation system is shown in Table 4.

[0076] Table 1 pAAV-pCALM1-EYFP-bGHpA vector restriction enzyme digestion system

[0077] Reagents Dosage Enzyme digestion buffer 10×rCutSmart Buffer 5μL BsrGI-HF 1μL AgeI-HF 1μL pAAV-pCALM1-EYFP-bGHpA 1 μg <![CDATA[ddH2O]]> up to 50μL

[0078] Table 2 Rtn1 fragment amplification system

[0079]

[0080] Table 3 Fragment amplification program

[0081]

[0082] Table 4 Connection system

[0083]

[0084]

[0085] After the plasmid is constructed, cell transfection is performed for cell verification. The main steps include:

[0086] 1) Cell preparation: 293T cells were plated in a 24-well plate containing complete culture medium (10% fetal bovine serum, 1% penicillin-streptomycin mixture, 89% DMEM high-glucose culture medium) at a density of 60% and cultured in a cell culture incubator containing 5% CO2.

[0087] 2) Transfected cells: In this example, jetOPTIMUS transfection reagent was used for transfection, and then added dropwise to 293T cells grown to 80% to 90% density. Cell solution was replaced after 12 hours, and blue excitation light was used for photography after 72 hours. The experimental results are shown in Figure 2 As shown, the cells were lysed and the proteins were extracted. Figure 2 This is a comparison chart of the cell transfection results and fluorescence values ​​of pAAV-pCALM1-EYFP-Rtn1 and pAAV-pCALM1-EYFP-bGHpA.

[0088] 3) Western Blotting: Add 50 μL of lysis buffer to the cells after centrifugation, place on ice for 30 minutes and vortex every 10 minutes until fully lysed; mix the lysed sample and loading buffer at a ratio of 1:5, heat in a 100°C water bath for 10 minutes to denature the protein, cool to room temperature, centrifuge at 10,000 r / min for 5 minutes, and store at -80°C. Use 10% high-resolution precast gel from Yisheng Biotechnology (Shanghai) Co., Ltd., and perform constant voltage electrophoresis at 85V for 30 minutes after sample addition, and 110V for 90 minutes to make the protein bands run away. Cut the PVDF transfer membrane to cover the entire gel, activate it in methanol for 3 minutes, make a "sandwich" with sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the order, and place it in the transfer tank. The transfer tank is placed in a basin with ice packs, and the membrane is transferred at 400mA constant current for 30 minutes; take out the transferred VDF membrane, add blocking solution and shake for 60 minutes at 70r / min; pour out the blocking solution, add the diluted primary antibody (GFP antibody: primary antibody and secondary antibody diluent = 1:500; Tubulin antibody: primary antibody and secondary antibody diluent = 1:500), shake at 4°C overnight; the next day, wash 3 times with TBST, 10 minutes each time, add the diluted secondary antibody, incubate at room temperature for 1 hour, shake at 70r / min; wash 3 times with TBST, 10 minutes each time; use developer to color and expose, scan the protein blot bands, and the experimental results are as follows Figure 3 shown. Figure 3 This is a comparison of WB results after cell transfection with pAAV-pCALM1-EYFP-Rtn1 and pAAV-pCALM1-EYFP-bGHpA.

[0089] like Figure 3 As shown, through the verification of cell fluorescence value and WB protein detection, it was determined that the fluorescent protein expression of pAAV-pCALM1-EYFP-Rtn1 was better than that of pAAV-pCALM1-EYFP-bGHpA.

[0090] Therefore, the three-plasmid co-transfection method was used to prepare the virus. Before virus preparation, the plasmids required for packaging need to be extracted, including the recombinant adeno-associated virus vector pAAV-pCALM1-EYFP-bGHpA, the recombinant adeno-associated virus vector pAAV-pCALM1-EYFP-Rtn1, the packaging plasmid PHPeB and the auxiliary plasmid. The specific steps are as follows:

[0091] 1) Cell preparation: 293T cells were plated in a culture dish containing complete culture medium ((10% fetal bovine serum, 1% penicillin-streptomycin mixture, 89% DMEM high-glucose culture medium) and cultured in a cell culture incubator containing 5% CO2.

[0092] 2) Prepare transfection reagent: Pipette 15 μg of auxiliary plasmid, 15 μg of packaging plasmid, 15 μg of recombinant plasmid, 5.25 mL of ultrapure water and 0.8 mL of 2M calcium chloride solution, mix gently, add an equal volume of 2×HBS, vortex and incubate at room temperature for 30 min.

[0093] 3) Transfect cells: Add 20 μL of chloroquine solution to each tube of transfection reagent and add dropwise to the 293T cell culture dish.

[0094] 4) Collecting the virus: After 72 hours, collect the cells and put them into a centrifuge tube, centrifuge at 3000 rpm / min for 30 minutes, discard the supernatant, add cell lysis buffer, and freeze-thaw repeatedly to obtain a suspension containing virus and cell fragments.

[0095] 5) Virus purification: centrifuge the suspension at 12000rpm / min for 30min, discard the supernatant, add 200μL HBS to mix, add 200μL chloroform and centrifuge at 12000rpm / min for 5min; aspirate the supernatant, add 100μL 2.5mM sodium chloride and 100μL 40% PEG8000, vortex to mix, and store in a 4°C refrigerator overnight; centrifuge at 12000rpm / min for 35min at 4°C, discard the supernatant, add 30μL HBS and 0.5μL nuclease, digest at room temperature for 30min, add 30μL chloroform, centrifuge at 12000rpm / min for 5min, and store at -80°C.

[0096] 6) Titer determination: Real-time fluorescence quantitative PCR (qPCR) was used for determination. In this example, a fluorescence quantitative kit (TAKARA) was used to determine the AAV titer. The test results are as follows:

[0097] The titer of pAAV-pCALM1-EYFP-bGHpA was 8.51E+12VG / mL.

[0098] The titer of pAAV-pCALM1-EYFP-Rtn1 was 6.81E+12VG / mL.

[0099] The purified recombinant adeno-associated virus was injected into the primary motor cortex (M1) of the mouse unilaterally (Bregma 2.0mm; Lamdga 2.1mm; Z-1.70mm) by stereotaxic injection. The promoter used in the recombinant adeno-associated virus is the promoter of pCALM1 that can mark neurons. Three weeks later, the mouse brain was perfused and frozen sectioned, nuclear stained (DAPI), and mounted, and the brain slice results were observed. The experimental results are as follows Figure 4 shown. Figure 4 This is a comparison of the M1 brain region of mice infected with pAAV-pCALM1-EYFP-Rtn1 and pAAV-pCALM1-EYFP-bGHpA viruses.

[0100] like Figure 4 As shown, the experimental results showed that pAAV-pCALM1-EYFP-Rtn1 was verified in mice and its effect was better than pAAV-pCALM1-EYFP-bGHpA, proving that the 3'UTR Rtn1 element has a higher function in maintaining the stability of mRNA and controlling its translation in the cortex.

[0101] The 3'UTR element provided in the present application can better help the translation and expression of exogenous genes in neurons. Experiments of in vitro transfection of 293T cells and in vivo stereotaxic injection into mouse brains have been carried out. The results show that it has better effects than bGHpA and can be applied to the fields of neural circuit tracing, gene therapy and transgenic fields, such as neural circuit tracing, gene therapy and / or transgenic related reagents and / or drugs.

[0102] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0103] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A 3'UTR element, characterized in that The nucleotide sequence of the 3'UTR element contains: a fragment sequence from the stop codon to the poly A tailing signal site of the positive chain of the Rtn1 gene.

2. The 3'UTR element according to claim 1, characterized in that The nucleotide sequence of the 3'UTR element contains the nucleotide sequence of the Rtn1 gene.

3. A recombinant vector, characterized in that: The recombinant vector carries the 3'UTR element according to any one of claims 1-2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector is a recombinant adeno-associated virus vector packaged with the 3'UTR element; Furthermore, the recombinant vector also carries a fluorescent protein reporter gene.

5. The method for constructing a recombinant vector according to any one of claims 3 to 4, characterized in that: The steps include: Constructing a backbone vector; Amplifying Rtn1 using amplification primers to obtain a gene fragment of the 3'UTR element; The backbone vector is connected to the gene fragment of the 3'UTR element to obtain the recombinant vector.

6. The construction method according to claim 5, characterized in that: The backbone vector is an adeno-associated virus backbone vector and contains a fluorescent protein reporter gene; The amplification primer pair contains a sequence that recognizes the 3'UTR element and complements the fluorescent protein reporter gene; Furthermore, the amplification primer pair is used to amplify the nucleotide sequence of the Rtn1 gene.

7. A recombinant virus, characterized in that The recombinant virus is packaged with the recombinant vector according to any one of claims 3-4.

8. The recombinant virus according to claim 7, characterized in that The recombinant virus is a recombinant adeno-associated virus.

9. The method for preparing a recombinant virus according to any one of claims 7 to 8, characterized in that: The steps include: The recombinant vector, packaging vector and auxiliary vector are co-transfected into 293T cells, and the virus is separated to obtain the recombinant virus.

10. Use of the 3'UTR element according to any one of claims 1 to 2, or the recombinant vector according to any one of claims 3 to 4, or the recombinant virus according to any one of claims 7 to 9 in the preparation of neural circuit tracing, gene therapy and / or transgenic related reagents and / or drugs.

Images