WO2022120819A1 - Ires序列、ires序列的应用和多顺反子表达载体 - Google Patents

Ires序列、ires序列的应用和多顺反子表达载体 Download PDF

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WO2022120819A1
WO2022120819A1 PCT/CN2020/135845 CN2020135845W WO2022120819A1 WO 2022120819 A1 WO2022120819 A1 WO 2022120819A1 CN 2020135845 W CN2020135845 W CN 2020135845W WO 2022120819 A1 WO2022120819 A1 WO 2022120819A1
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ires sequence
expression vector
ires
sequence
cov
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French (fr)
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戴俊彪
卢俊南
杨炜钐
温栾
林鑫
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中国科学院深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the application belongs to the technical field of genetic engineering, and specifically relates to an internal ribosome entry site (Internal Ribosome Entry Sites, IRES) sequence, an application of the IRES sequence and a polycistronic expression vector.
  • IRES Internal Ribosome Entry Sites
  • the translation process of eukaryotes is an important stage of gene expression, which refers to the process of synthesizing proteins from genetic information on eukaryotic messenger ribonucleic acid (mRNA) transcribed from deoxyribonucleic acid (DNA).
  • mRNA messenger ribonucleic acid
  • DNA deoxyribonucleic acid
  • ribosomes can be recruited into the viral mRNA to initiate translation based on the cis-acting element IRES sequence that regulates the translation of the 5' untranslated region (UTR) of the RNA virus.
  • the IRES sequence is a cap-independent nucleotide sequence capable of initiating translation, capable of recruiting ribosomes into the translation initiation site of viral mRNA with the assistance of trans-acting factors. And some studies have shown that by fusing the IRES sequence with the exogenous cDNA, the IRES can initiate translation independently. Therefore, the study of IRES sequence is of great significance to gene expression.
  • the present application provides an IRES sequence, an application of the IRES sequence, and a polycistronic expression vector.
  • the IRES sequence has the ability to initiate translation for eukaryotes and can be applied to the expression process of a target gene.
  • the application provides an IRES sequence
  • the IRES sequence is obtained based on the ORF8 gene fragment in SARS-Cov-2
  • the ORF8 gene fragment is shown in SEQ ID NO.1.
  • the IRES sequence provided in this application has the ability to initiate translation to eukaryotes and can be applied to the expression process of target genes.
  • the IRES sequence is shown in SEQ ID NO.1.
  • the IRES sequence is a sequence obtained by elongation, trimming, recombination and/or mutation of the ORF8 gene fragment.
  • the IRES sequence includes: the ORF8 gene segment, and the upstream segment and/or the downstream segment of the ORF8 gene segment in the SARS-Cov-2.
  • the IRES sequence includes the ORF8 gene fragment, the upstream segment of 120 bp and the downstream segment of 14 bp, and the IRES sequence is shown in SEQ ID NO.2.
  • the IRES sequence is isolated from the SARS-Cov-2.
  • the present application provides an application of the IRES sequence described in the first aspect or any optional manner of the first aspect in the expression of a target gene.
  • the present application provides an application of the IRES sequence described in the first aspect or any optional manner of the first aspect in anti-SARS-Cov-2 drug screening.
  • the application provides a polycistronic expression vector, comprising the IRES sequence as described in the first aspect or any optional manner of the first aspect, wherein the IRES sequence is located in the polycistronic expression vector upstream of the initiation codon of the gene of interest.
  • the polycistronic expression vector provided in the present application can simultaneously express the target gene contained in the polycistronic expression vector.
  • the polycistronic expression vector is a dual fluorescent protein expression vector
  • the dual fluorescent protein expression vector includes the IRES sequence, GFP and mCherry, and the IRES sequence is located at the stop codon of the GFP and all between the initiation codons of mCherry.
  • Fig. 1 is the map of a kind of dual fluorescent protein expression vector with IRES sequence provided in the embodiment of this application;
  • Fig. 2 is the electrophoresis figure of a kind of purpose vector and purpose fragment provided in the embodiment of the present application;
  • FIG. 3 is a map of a dual fluorescent protein expression vector without an IRES sequence provided in the embodiment of the present application.
  • FIG. 4 is a fluorescence image of HEK-293T cells 48 hours after transfection provided in the examples of the present application.
  • references in this specification to "one embodiment” or “some embodiments” and the like mean that a particular feature, structure or characteristic described in connection with the embodiment is included in one or more embodiments of the present application.
  • appearances of the phrases “in one embodiment,” “in some embodiments,” “in other embodiments,” “in other embodiments,” etc. in various places in this specification are not necessarily All refer to the same embodiment, but mean “one or more but not all embodiments” unless specifically emphasized otherwise.
  • the terms “including”, “including”, “having” and their variants mean “including but not limited to” unless specifically emphasized otherwise.
  • the content of the relevant components mentioned in the description of the examples of this application can not only refer to the specific content of each component, but also can represent the proportional relationship between the contents of each component. It is within the scope disclosed in the description of the embodiments of the present application that the content of the ingredients is scaled up or down.
  • the volume, the number of nucleotides, the rotational speed, and the time described in the description of the examples of the present application may be units of measurement known in the field of genetic engineering technology such as ⁇ L, mL, bp, kbp, rpm, s, min, and h.
  • the IRES sequence provided in this application is obtained based on the ORF8 gene fragment in SARS-Cov-2. Wherein, the ORF8 gene fragment in SARS-Cov-2 is shown in SEQ ID NO.1.
  • SARS-Cov-2 also known as novel coronavirus, is a positive-stranded single-stranded RNA virus with an outer membrane.
  • Coronaviruses belong to the genus Coronaviridae of the family Coronaviridae of the order Netoviridae, and the genus of coronaviruses can be subdivided into 4 genera, namely alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus.
  • SARS-Cov-2 belongs to the genus betacoronavirus.
  • SARS-Cov-2 is spherical or elliptical, and the diameter of virus particles is generally between 60 and 200 nm, with an average diameter of 100 nm.
  • SARS-Cov-2 The genetic material of SARS-Cov-2 is by far the most abundant of any RNA virus. SARS-Cov-2 can infect humans, mice, pigs, cats, dogs and avian vertebrates. It is the seventh type of coronavirus discovered so far that can infect humans. It has the characteristics of high transmissibility, long incubation period and high mutation rate.
  • the ORF8 gene fragment has the IRES function, that is, the IRES sequence can be obtained based on the ORF8 gene fragment, and the IRES sequence is applied to the expression process of the target gene.
  • the IRES sequence can be the original full-length sequence of the ORF8 gene fragment, that is, the IRES sequence is shown in SEQ ID NO.1. It can also be a sequence obtained after the ORF8 gene fragment is extended, trimmed, recombined and/or mutated.
  • the sequence of a part of the segment can be trimmed at the end of the ORF8 gene fragment to obtain the IRES sequence.
  • the ORF8 gene segment can be extended by the upstream segment and/or downstream segment of the ORF8 gene segment in SARS-Cov-2 to obtain the IRES sequence. That is, the IRES sequence includes: the ORF8 gene segment, and the upstream segment and/or the downstream segment of the ORF8 gene segment in SARS-Cov-2.
  • the IRES sequence provided in this application can include the ORF8 gene fragment, an upstream segment of 120 bp and a downstream segment of 14 bp.
  • the cut or extended length of the ORF8 gene fragment can be designed based on actual needs under the condition that the IRES function of the ORF8 gene fragment can be guaranteed, which is not limited in this application.
  • the ORF8 gene fragment can also be modified by means of recombination and/or mutation to obtain an IRES sequence.
  • the ORF8 gene fragment is recombined and/or mutated to obtain an IRES sequence.
  • the specific method of recombination and/or mutation of the ORF8 gene fragment can also be designed based on actual needs, which is not limited in this application.
  • the ORF8 gene fragment when modifying the ORF8 gene fragment, can also be cut or extended first, and then recombination and/or mutation can be performed.
  • SEQ ID NO. 2 is recombined and/or mutated to obtain the IRES sequence.
  • the IRES sequence provided in this application can be directly isolated from SARS-Cov-2, or can be synthesized artificially.
  • the IRES sequences provided in this application can be applied to the expression of target genes. Based on the IRES sequence, a polycistronic expression vector can be constructed, which can realize the synchronous expression of multi-purpose genes.
  • a polycistronic expression vector refers to a vector capable of synchronously expressing two or more target genes. Among them, a polycistronic expression vector containing two target genes is also commonly referred to as a bicistronic expression vector.
  • the application provides a polycistronic expression vector, comprising the IRES sequence provided in the application, and the IRES sequence is located upstream of the initiation codon of the target gene in the polycistronic expression vector.
  • the following takes the dual fluorescent protein expression vector as an example to illustrate the polycistronic expression vector provided in this application, the construction of the polycistronic expression vector, and the application of the IRES sequence in the expression of the target gene.
  • the dual fluorescent protein expression vector provided for this application includes human cytomegalovirus (Cytomegalovirus, CMV) promoter, green fluorescent protein (Green fluorescent protein, GFP), IRES sequence and red fluorescent protein (mCherry) And bovine growth hormone (Bovine growth hormone, BGH) tail signal.
  • CMV human cytomegalovirus
  • Green fluorescent protein, GFP green fluorescent protein
  • IRES sequence IRES sequence and red fluorescent protein (mCherry) And bovine growth hormone (Bovine growth hormone, BGH) tail signal.
  • the IRES sequence is located between the stop codon of GFP and the start codon of mCherry.
  • the stop codon of GFP is TAA codon
  • the start codon of GFP is ATG codon.
  • the IRES sequence provided in this application is the sequence shown in SEQ ID NO.2, and the destination vector is the pcDNA3.1-EGFP vector constructed on the basis of the pcDNA3.1 (+/-) vector.
  • the construction process of the dual fluorescent protein expression vector can be as follows:
  • Step 1 prepare the target vector.
  • the pcDNA3.1-EGFP vector plasmid was single digested with restriction endonuclease (Xba I) and incubated at 37°C for 3h. Then, the digestion product is detected by agarose gel electrophoresis, and the linearized vector frame is recovered to obtain the target vector.
  • Xba I restriction endonuclease
  • the electropherograms of the pcDNA3.1-EGFP before and after restriction digestion can be shown in (b) of FIG. 2 , and the size of the target vector obtained after restriction digestion is about 6 kb.
  • Step 2 prepare the target fragment.
  • the target fragments include IRES sequence fragments and target gene mCherry fragments.
  • the plasmid carrying the sequence shown in SEQ ID NO.2 is used as a template to carry out polymerase chain reaction (Polymerase Chain Reaction, PCR) amplification.
  • the PCR program is: firstly preheat at 95°C for 30s. Then, 30 amplification cycles were performed, and the reaction conditions of each cycle were: denaturation at 95°C for 15s, then annealing at 50°C for 15s, and finally extension at 72°C for 1 min. After the cycle, the cells were extended at 72°C for 5 min to obtain the amplification product of the IRES sequence.
  • the 5' ends of the forward primer and the reverse primer of the amplified product of the IRES sequence were added with the homologous sequence of the pcDNA3.1-EGFP vector and the downstream mCherry fragment to obtain the IRES sequence fragment.
  • the electropherogram of the IRES sequence fragment can be shown in (a) of FIG. 2 , and the size of the IRES sequence fragment is about 541 bp.
  • PCR amplification was performed using the plasmid carrying the mCherry gene as a template.
  • the PCR program is: firstly preheat at 95°C for 30s. Then, 33 amplification cycles were performed. The reaction conditions of each cycle were: denaturation at 95°C for 15s, annealing at 50°C for 15s, and extension at 72°C for 1 min. After the cycle, the cells were extended at 72°C for 5 min to obtain the amplification product of mCherry.
  • a homologous sequence of the pcDNA3.1-EGFP vector was added to the 5' end of the reverse primer of the mCherry amplification product to obtain an mCherry fragment.
  • the electropherogram of the mCherry fragment can be shown in (a) of FIG. 2 , and the size of the mCherry fragment is about 732 bp.
  • Step 3 ligation of the target fragment and the target vector.
  • the target fragment and the target vector were incubated at 50°C for 15 minutes using the recombination kit of Novozymes Biotechnology (Cat. No.: C115), and homologous recombination was performed to obtain a homologous recombination product.
  • the homologous recombination product was then transformed into E. coli DH5 ⁇ competent cells.
  • the conversion steps can look like this:
  • the mixed product was ice-bathed for 30 min, and then heat-shocked at 42 °C for 90 s. After the heat shock, ice bath for 5 min. Then, 1 mL of anti-LB (Luria-Bertani) medium was added, and the culture was recovered for 1 h at 37° C. and 220 rpm to obtain a recovered culture. During the incubation period, LB plates containing antibiotics were kept at room temperature for use.
  • anti-LB Lia-Bertani
  • step 4 a plasmid is prepared to obtain a double fluorescent protein expression vector.
  • plasmid preparation can be carried out using an endotoxin-free plasmid extraction kit to obtain a dual fluorescent protein expression vector containing the IRES sequence.
  • an endotoxin-free plasmid extraction kit for example, the Midi Low Endotoxin Plasmid Body Medium Kit (Cat. No.: P1112-02) can be selected to prepare plasmids, and the specific operations can be carried out according to the instructions of the kit.
  • the purpose vector, endonuclease, recombination kit and plasmid extraction kit used are all exemplary enumeration schemes.
  • the purpose vector, endonuclease, recombination kit and plasmid extraction kit used are all exemplary enumeration schemes.
  • the gene expression function of the dual fluorescent protein expression vector can be further verified.
  • three experimental groups can be set, including blank control group (Mock), negative control group (Vector) and positive control group (IRES).
  • the blank control group (Mock) is the control group without the expression vector transfected
  • the negative control group is the control group transfected with the double fluorescent protein expression vector without IRES sequence
  • the positive control group is the double fluorescent protein expression vector transfected with the IRES sequence.
  • Fluorescent protein expression vector that is, the above-mentioned dual fluorescent protein expression vector provided in this application
  • the double fluorescent protein expression vector without IRES sequence can be shown in Figure 3, including GFP, IRES sequence, mCherry and BGH tail signal. There is no IRES sequence inserted between the stop codon of GFP and the start codon of mCherry.
  • the dual fluorescent protein expression vector without IRES sequence does not need to prepare IRES sequence fragments during the construction process, and when preparing mCherry fragments, it is necessary to increase the identity of the target vector at the 5' ends of the reverse primer and forward primer of the mCherry amplification product.
  • the source sequence is convenient for the homologous recombination of the mCherry fragment and the destination vector. The rest of the process is similar to that of the dual fluorescent protein expression vector containing the IRES sequence.
  • the negative control group and the positive control group need to be transfected first, so that the expression of GFP and mCherry can be observed by fluorescence phenomenon.
  • electroporation can be used for cell transfection
  • commercial transfection reagents can also be used for cell transfection.
  • Celetrix electroporator can be used for electroporation
  • Lipo8000 TM reagent Cat. No.: C0533FT
  • Lipofectamine 3000 reagent Cat. No.: L3000015) from Thermo Fisher, etc.
  • the amount of the reagent can refer to the reagent dosage for 12-well plates described in the instructions of the commercial transfection reagent.
  • the 3'UTR provided by the CMV promoter will guide the ribosome to enter and initiate translation, so that GFP is expressed and green fluorescence can be observed.
  • the ribosome is translated to the stop codon of GFP and separated from the mRNA, so that the downstream gene mCherry of GFP cannot be translated, and the red fluorescence cannot be observed.
  • green fluorescence can finally be observed.
  • the IRES sequence can recruit ribosomes into the IRES sequence again.
  • the downstream gene mCherry starts translation, so that mCherry is expressed and red fluorescence can be observed.
  • the mixed light of green fluorescence and red fluorescence can finally be observed.
  • the IRES sequence provided by the application has the high-efficiency non-cap structure-dependent initiation translation ability, can be applied to the expression process of the target gene, and improves the expression efficiency of the target gene.
  • the IRES sequence can also be used as a drug target to screen for anti-SARS-Cov-2 drugs.
  • the IRES sequence can also be used as a drug target to screen for anti-SARS-Cov-2 drugs.
  • the dual fluorescent protein expression vector shown in Figure 1 transfecting it into cells, adding the drug molecules to be screened, and then analyzing the ratio of the red fluorescence intensity to the green fluorescence intensity, you can quickly understand the corresponding drug molecules on the IRES function inhibitory effect.
  • this IRES is responsible for the translation of nucleocapsid protein, which is one of the core structural proteins of SARS-Cov-2, at least at the initial stage of virus invasion. In this way, an effective IRES inhibitor is expected to be screened, which inhibits the function of SARS-Cov-2 by inhibiting the translation of the nucleocapsid protein of SARS-Cov-2.

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Abstract

提供了一种IRES序列、IRES序列的应用和多顺反子表达载体。所述IRES序列是基于SARS-Cov-2中的ORF8基因片段得到的。该IRES序列可以直接从SARS-Cov-2中分离得到，也可以人工合成。该IRES序列具备对真核生物的起始翻译能力，可以应用于目标基因的表达过程，也可以应用于抗SARS-Cov-2药物的筛选。

Description

IRES序列、IRES序列的应用和多顺反子表达载体 技术领域
本申请属于基因工程技术领域，具体涉及一种内部核糖体进入位点(Internal Ribosome Entry Sites，IRES)序列、IRES序列的应用和多顺反子表达载体。
背景技术
真核生物的翻译(Translation)过程是基因表达的重要阶段，是指将从脱氧核糖核酸(DNA)转录得到的真核生物的信使核糖核酸(mRNA)上的遗传信息合成蛋白质的过程。真核生物的翻译过程中，一般需要依赖mRNA的5’端帽子结构与核糖体结合，起始翻译。但对于缺乏帽子构的RNA病毒，可以基于RNA病毒的5’端非翻译区(untranslated region,UTR)翻译调控的顺式作用元件IRES序列，招募核糖体进入病毒的mRNA起始翻译。
IRES序列是一种具有非帽子结构依赖性的起始翻译能力的核苷酸序列，能够在反式作用因子的辅助下，招募核糖体进入病毒mRNA的翻译起始位点。并且有研究表明，将IRES序列与外源cDNA融合，IRES能独立的起始翻译。因此对IRES序列的研究对基因表达有着重要的意义。
发明内容
有鉴于此，本申请提供一种IRES序列、IRES序列的应用和多顺反子表达载体，该IRES序列具备对真核生物的起始翻译能力，能够应用于目标基因的表达过程。
第一方面，本申请提供一种IRES序列，所述IRES序列是基于SARS-Cov-2中的ORF8基因片段得到，所述ORF8基因片段如SEQ ID NO.1所示。
本申请提供的IRES序列具备对真核生物的起始翻译能力，能够应用于目 标基因的表达过程。
可选的，所述IRES序列如SEQ ID NO.1所示。
可选的，所述IRES序列是所述ORF8基因片段经过延长、裁切、重组和/或突变得到的序列。
可选的，所述IRES序列包括：所述ORF8基因片段，以及，所述ORF8基因片段在所述SARS-Cov-2中的上游区段和/或下游区段。
可选的，所述IRES序列包括所述ORF8基因片段、120bp的所述上游区段和14bp的所述下游区段，所述IRES序列如SEQ ID NO.2所示。
可选的，所述IRES序列从所述SARS-Cov-2中分离得到。
第二方面，本申请提供一种如第一方面或第一方面的任一可选方式所述的IRES序列在目的基因表达上的应用。
第三方面，本申请提供一种如第一方面或第一方面的任一可选方式所述的IRES序列在抗SARS-Cov-2药物筛选中的应用。
第四方面，本申请提供一种多顺反子表达载体，包括如第一方面或第一方面的任一可选方式所述的IRES序列，所述IRES序列位于所述多顺反子表达载体中目的基因的起始密码子上游。
本申请提供的多顺反子表达载体能够同步表达多顺反子表达载体中包含的目的基因。
可选的，所述多顺反子表达载体为双荧光蛋白表达载体，所述双荧光蛋白表达载体包括所述IRES序列、GFP和mCherry，所述IRES序列位于所述GFP的终止密码子和所述mCherry的起始密码子之间。
附图说明
图1是本申请实施例提供的一种有IRES序列的双荧光蛋白表达载体的图谱；
图2是本申请实施例提供的一种目的载体和目的片段的电泳图；
图3是本申请实施例提供的一种无IRES序列的双荧光蛋白表达载体的图谱；
图4是本申请实施例提供的一种HEK-293T细胞转染后48小时的荧光图。
具体实施方式
本申请中，术语“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B的情况。其中A，B可以是单数或者复数。字符“/”一般表示前后关联对象是一种“或”的关系。
在本申请实施例中使用的术语是仅仅出于描述特定实施例的目的，而非旨在限制本申请。在本申请实施例和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式，除非上下文清楚地表示其他含义。
在本申请说明书中描述的参考“一个实施例”或“一些实施例”等意味着在本申请的一个或多个实施例中包括结合该实施例描述的特定特征、结构或特点。由此，在本说明书中的不同之处出现的语句“在一个实施例中”、“在一些实施例中”、“在其他一些实施例中”、“在另外一些实施例中”等不是必然都参考相同的实施例，而是意味着“一个或多个但不是所有的实施例”，除非是以其他方式另外特别强调。术语“包括”、“包含”、“具有”及它们的变形都意味着“包括但不限于”，除非是以其他方式另外特别强调。
本申请实施例说明书中所提到的相关成分的含量不仅仅可以指代各组分的具体含量，也可以表示各组分间含量的比例关系，因此，只要是按照本申请实施例说明书相关组分的含量按比例放大或缩小均在本申请实施例说明书公开的范围之内。具体地，本申请实施例说明书中所述的体积、核苷酸的数 量、转速、时间可以是μL、mL、bp、kbp、rpm、s、min、h等基因工程技术领域公知的计量单位。
下面结合具体实施例，对本申请提供的IRES序列、IRES序列的应用以及多顺反子表达载体进行示例性的说明。应当理解，此处所描述的具体实施例仅仅用以解释本申请的方案，并不用于限定本申请。
本申请提供的IRES序列是基于SARS-Cov-2中的ORF8基因片段得到的。其中，SARS-Cov-2中的ORF8基因片段如SEQ ID NO.1所示。
SEQ ID NO.1：
Figure PCTCN2020135845-appb-000001
SARS-Cov-2也可以称为新型冠状病毒，是一种具有外膜的正链单股RNA病毒。冠状病毒隶属于网巢病毒目冠状病毒科冠状病毒属，而冠状病毒属又可细分为4个属，分别为α冠状病毒属、β冠状病毒属、γ冠状病毒属和δ冠状病毒属。其中，SARS-Cov-2属于β冠状病毒属。SARS-Cov-2呈球形或椭圆形，病毒颗粒的直径一般在60～200nm之间，平均直径为100nm。SARS-Cov-2的遗传物质是目前为止所有RNA病毒中含量最大的。SARS-Cov-2可以感染人、鼠、猪、猫、犬和禽类脊椎动物，是目前发现的第7种可以感染人类的冠状病毒，具有高传播力、潜伏期长，突变率高等特点。
通过对SARS-Cov-2的ORF8基因片段的克隆和实验，可以确定ORF8基因片段具备IRES功能，即能够基于ORF8基因片段来获得IRES序列，并将 该IRES序列应用于目的基因的表达过程。
在本申请实施例中，IRES序列可以是ORF8基因片段原本的全长序列，即IRES序列如SEQ ID NO.1所示。也可以是ORF8基因片段经过延长、裁切、重组和/或突变之后得到的序列。
示例性的，在对ORF8基因片段进行裁切时，可以在ORF8基因片段末端裁切一部分区段的序列，进而得到IRES序列。
在对ORF8基因片段进行延长时，可以在ORF8基因片段末端拼接一部分序列。例如，可以通过ORF8基因片段在SARS-Cov-2中的上游区段和/或下游区段来延长ORF8基因片段，得到IRES序列。即IRES序列包括：ORF8基因片段，以及，ORF8基因片段在SARS-Cov-2中的上游区段和/或下游区段。
例如，如SEQ ID NO.2所示，本申请提供的IRES序列可以包括ORF8基因片段、120bp的上游区段和14bp的下游区段。
SEQ ID NO.2：
Figure PCTCN2020135845-appb-000002
值得说明的是，对ORF8基因片段裁切或者延长的长度，在能够保证ORF8基因片段的IRES功能的情况下，可以基于实际需求进行设计，本申请不做限制。
可选的，除上述裁切和延长的改造方式外，还可以通过重组和/或突变的方式对ORF8基因片段进行改造，得到IRES序列。例如，以增强ORF8基因片段的IRES功能为目的，对ORF8基因片段进行重组和/或突变，得到得到IRES序列。当然，在能够保证ORF8基因片段的IRES功能的情况下，对ORF8基因片段的重组和/或突变的具体方式也可以基于实际需求来设计，对此本申请不做限制。
可以理解的是，在对ORF8基因片段进行改造时，也可以先裁切或者延长该ORF8基因片段，在进行重组和/或突变。例如，对SEQ ID NO.2进行重组和/或突变，得到IRES序列。
基于实际需求，本申请提供的IRES序列可以直接从SARS-Cov-2中分离得到，也可以人工合成。
本申请提供的IRES序列可以应用于目的基因的表达。基于该IRES序列，可以构建多顺反子表达载体，能够实现多目的基因的同步表达。
多顺反子表达载体是指能够同步表达两个或两个以上目的基因的载体。其中，包含两个目的基因的多顺反子表达载体通常也称为双顺反子表达载体。
本申请提供一种多顺反子表达载体，包括本申请提供的IRES序列，该IRES序列位于该多顺反子表达载体中目的基因的起始密码子上游。
下面以双荧光蛋白表达载体为例，对本申请提供的多顺反子表达载体、多顺反子表达载体的构建以及IRES序列在目的基因表达上的应用进行示例性的说明。
如图1所示，为本申请提供的双荧光蛋白表达载体，包括人巨细胞病毒(Cytomegalovirus,CMV)启动子、绿色荧光蛋白(Green fluorescent protein，GFP)、IRES序列和红色荧光蛋白(mCherry)和牛生长激素(Bovine growth hormone，BGH)加尾信号。其中，IRES序列位于GFP的终止密码子和mCherry的起始密码子之间。GFP的终止密码子为TAA密码子，的起始密码子为ATG 密码子。
示例性的，假设本申请提供的IRES序列为SEQ ID NO.2所示序列，目的载体为以pcDNA3.1(+/-)载体为基础构建的pcDNA3.1-EGFP载体。该双荧光蛋白表达载体的构建过程可以如下：
步骤1，制备目的载体。
采用限制性核酸内切酶(Xba I)对pcDNA3.1-EGFP载体质粒进行单酶切，在37℃的环境下孵育3h。然后对酶切产物进行琼脂糖凝胶电泳检测，回收线性化的载体框架，得到目的载体。示例性的，该pcDNA3.1-EGFP在酶切前后的电泳图可以如图2中的(b)所示，酶切后得到的目的载体的大小约为6kb。
步骤2，制备目的片段。
目的片段包括IRES序列片段和目的基因mCherry片段。
其中，制备IRES序列片段时，以携带SEQ ID NO.2所示序列的质粒为模板进行聚合酶链式反应(Polymerase Chain Reaction，PCR)扩增。PCR程序为：首先在95℃条件下预热30s。然后执行30个扩增循环，每个循环的反应条件是：先在95℃条件下将变性15s，然后在50℃条件下退火15s，最后在72℃条件下延伸1min。循环结束后，在72℃条件下延伸5min，得到IRES序列的扩增产物。IRES序列的扩增产物的正向引物和反向引物的5’端增加有pcDNA3.1-EGFP载体和下游mCherry片段的同源序列，得到IRES序列片段。示例性的，IRES序列片段的电泳图可以如图2中的(a)所示，IRES序列片段的大小约为541bp。
制备mCherry片段时，以携带mCherry基因的质粒为模板进行PCR扩增。PCR程序为：首先在95℃条件下预热30s。然后执行33个扩增循环，每个循环的反应条件是：先在95℃条件下将变性15s，然后在50℃条件下退火15s，最后在72℃条件下延伸1min。循环结束后，在72℃条件下延伸5min，得到mCherry的扩增产物。mCherry的扩增产物的反向引物的5’端增加有pcDNA3.1-EGFP载体的同源序列，得到mCherry片段。示例性的，mCherry 片段的电泳图可以如图2中的(a)所示，mCherry片段的大小约为732bp。
步骤3，目的片段和目的载体的连接。
在一个示例中，采用诺唯赞生物科技的重组试剂盒(货号：C115)将目的片段和目的载体在50℃的条件下孵育15min，进行同源重组连接，得到同源重组产物。然后将同源重组产物转化到大肠杆菌DH5α感受态细胞上。转化步骤可以如下所示：
S1，冰上解冻50μL的DH5α感受态细胞。
S2，在解冻后的DH5α感受态细胞加入5μL的同源重组产物，混匀后得到混合产物。
S3，将混合产物冰浴30min，然后在42℃条件下热激90s。热激结束后再冰浴5min。然后加入1mL无抗LB(Luria-Bertani)培养基，在37℃，220rpm的条件下复苏培养1h，得到复苏培养物。培养期间，将含有抗生素的LB平板放置室温条件下备用。
S4，取200μL的复苏培养物涂于含有抗生素的LB平板上，在37℃条件下倒置培养约12h。然后从培养出来的菌落中挑选样本进行测序，获得测序结果正确的克隆序列。其中，挑选出来的样本可以基于实验需要选择送往基因测序公司，由基因测序公司来验证培养的克隆序列的正确性。
步骤4，制备质粒，获得双荧光蛋白表达载体。
获得正确的克隆序列后，可以采用无内毒素的质粒提取试剂盒进行质粒制备，得到含有IRES序列的双荧光蛋白表达载体。例如，可以选择美基生物低内毒素质粒小体中量试剂盒(货号：P1112-02)制备质粒，具体操作可以按照该试剂盒的说明书进行。
值得说明的是，在上述构建双荧光蛋白表达载体的过程中，所使用的目的载体、内切酶、重组试剂盒以及质粒提取试剂盒均为示例性的列举方案。目前市面上可供选择的产品较多，具体可以基于实际实验需求进行选择，对此，本申请不做限制。
构建得到双荧光蛋白表达载体后，即可进一步对双荧光蛋白表达载体的基因表达功能进行验证。
在一个实施例中，可以设置三个实验组，包括空白对照组(Mock)、阴性对照组(Vector)和阳性对照组(IRES)。
其中，空白对照组(Mock)为未转染表达载体的对照组，阴性对照组为转染了无IRES序列的双荧光蛋白表达载体的对照组，阳性对照组为转染了含有IRES序列的双荧光蛋白表达载体(即上述本申请提供的双荧光蛋白表达载体)实验组。
其中，无IRES序列的双荧光蛋白表达载体可以如图3所示，包括GFP、IRES序列、mCherry和BGH加尾信号。其中，GFP的终止密码子和mCherry的起始密码子之间没有插入IRES序列。无IRES序列的双荧光蛋白表达载体在构过程中无需制备IRES序列片段，且在制备mCherry片段时，需在mCherry的扩增产物的反向引物和正向引物的5’端均增加目的载体的同源序列，便于mCherry片段与目的载体进行同源重组连接。其余过程与含有IRES序列的双荧光蛋白表达载体过程类似。
在进行功能验证时，首先需要对阴性对照组和阳性对照组进行细胞转染，以使得GFP和mCherry的表达可通过荧光现象被观测到。在对表达载体进行进行细胞转染的过程中，首先选择合适的孔板、培养瓶或者培养皿提前24h接种细胞。例如，选择12孔的HEK-293T细胞铺板。当接合率到达约80％时进行细胞转染。
示例性的，可以使用电转化进行细胞转染，也可以采用商业化转染试剂进行细胞转染。例如，电转化可以采用Celetrix电转仪，商业化转染试剂可以选择碧云天的Lipo8000 TM试剂(货号：C0533FT)，或者赛默飞世尔的Lipofectamine 3000试剂(货号：L3000015)等。如果采用商业化转染试剂进行细胞转染，试剂用量可以参考商业化转染试剂的说明书中描述的针对12孔板的试剂用量进行。
对阴性对照组和阳性对照组进行细胞转染后，经过48小时即可进行荧光观察。如图4所示，正常情况下，空白对照组中由于没有转染包含GFP和mCherry的载体，因此，空白对照组中无法观测到绿色荧光和红色荧光。
阴性对照组中，由于GFP与CMV启动子连接，CMV启动子提供的3’UTR会引导核糖体进入启动翻译，从而使得GFP表达，可以观测到绿色荧光。之后核糖体翻译到GFP的终止密码子，并脱离mRNA，使得GFP的下游基因mCherry无法翻译，进而无法观测到红色荧光。那么，正常情况下，阴性对照组中GFP和mCherry的表达结果叠加(merged)后，最终可以观测到绿色荧光。
阳性对照组中，由于GFP的终止密码子与mCherry的起始密码子之间存在IRES序列，即使核糖体经过GFP的终止密码子后脱离mRNA，IRES序列也能在再次招募核糖体进入IRES序列的下游基因mCherry，并启动翻译，从而使得mCherry表达，可以观测到红色荧光。那么，正常情况下，阳性对照组中GFP和mCherry的表达结果叠加(merged)后，最终可以观测到绿色荧光和红色荧光的混合光。
经过观测，显微镜标尺在50μm的情况下，三组实验组的荧光表达情况如图5中merged列所示，均为正常显示结果，即如下表1所示的实验结果：
表1
Figure PCTCN2020135845-appb-000003
经过实验验证，本申请提供的IRES序列具备高效的非帽子结构依赖性的起始翻译能力，能够应用于目的基因的表达过程，并提高目的基因的表达效 率。
此外，由于该IRES序列是基于SARS-Cov-2中的ORF8基因片段得到的，因此，该IRES序列还可以作为药物靶点来筛选抗SARS-Cov-2的药物。比如，采用如图1所示的双荧光蛋白表达载体，将其转染细胞后加入待筛选的药物分子，然后分析红色荧光强度与绿色荧光强度的比值，即可快速了解相应药物分子对IRES功能的抑制作用。该IRES在SARS-Cov-2中至少在病毒入侵初期负责核衣壳蛋白(Nucleocapsid protein)的翻译，而核衣壳蛋白是SARS-Cov-2的核心结构蛋白之一。通过这种方式有望筛选到有效的IRES抑制剂，其通过抑制SARS-Cov-2的核衣壳蛋白的翻译进而抑制SARS-Cov-2的功能。
以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内所作的任何修改、等同替换和改进等，均应包括在本申请的保护范围之内。

Claims (10)

  1. 一种内部核糖体进入位点IRES序列，其特征在于，所述IRES序列基于SARS-Cov-2中的ORF8基因片段得到，所述ORF8基因片段如SEQ ID NO.1所示。
  2. 如权利要求1所述的IRES序列，所述IRES序列如SEQ ID NO.1所示。
  3. 如权利要求1所述的IRES序列，所述IRES序列是所述ORF8基因片段经过延长、裁切、重组和/或突变得到的序列。
  4. 如权利要求1所述的IRES序列，其特征在于，所述IRES序列包括：
    所述ORF8基因片段，以及，所述ORF8基因片段在所述SARS-Cov-2中的上游区段和/或下游区段。
  5. 如权利要求4所述的IRES序列，其特征在于，所述IRES序列包括所述ORF8基因片段、120bp的所述上游区段和14bp的所述下游区段，所述IRES序列如SEQ ID NO.2所示。
  6. 根据权利要求2、4或5所述的IRES序列，其特征在于，所述IRES序列从所述SARS-Cov-2中分离得到。
  7. 如权利要求1-6任一项所述的IRES序列在目的基因表达上的应用。
  8. 如权利要求1-6任一项所述的IRES序列在抗SARS-Cov-2药物筛选中的应用。
  9. 一种多顺反子表达载体，其特征在于，包括如权利要求1-6任一所述的IRES序列，所述IRES序列位于所述多顺反子表达载体中目的基因的起始密码子上游。
  10. 根据权利要求9所述的多顺反子表达载体，其特征在于，所述多顺反子表达载体为双荧光蛋白表达载体，所述双荧光蛋白表达载体包括所述IRES序列、GFP和mCherry，所述IRES序列位于所述GFP的终止密码子和所述mCherry的起始密码子之间。
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