CN113717943A - St irf3/irf7 ko细胞系及其构建方法和应用 - Google Patents
St irf3/irf7 ko细胞系及其构建方法和应用 Download PDFInfo
- Publication number
- CN113717943A CN113717943A CN202110937887.7A CN202110937887A CN113717943A CN 113717943 A CN113717943 A CN 113717943A CN 202110937887 A CN202110937887 A CN 202110937887A CN 113717943 A CN113717943 A CN 113717943A
- Authority
- CN
- China
- Prior art keywords
- irf3
- cell line
- irf7ko
- cells
- lenticrispr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0683—Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Toxicology (AREA)
- Reproductive Health (AREA)
- Urology & Nephrology (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Virology (AREA)
- Dermatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于分子生物学领域,涉及ST IRF3/IRF7KO细胞系及其构建方法和应用,本发明针对猪源IRF3、IRF7基因序列的外显子设计特异性sgRNA,插入载体lentiCRISPR v2上,得到慢病毒载体lentiCRISPR v2‑IRF3和lentiCRISPR v2‑IRF7。将重组质粒转染HEK293T细胞后,收集慢病毒感染ST细胞。通过抗生素筛选出成功被感染的细胞,并进行测序、Western Blot验证。本发明所述的ST IRF3/IRF7KO细胞系可运用于猪源病毒的基础和应用研究,也为病毒疫苗生产中细胞系的选用提供了有价值的新材料。
Description
技术领域
本发明属于分子生物学领域,涉及ST IRF3/IRF7 KO细胞系及其构建方法和应用。
背景技术
CRISPR/Cas9系统是古细菌和细菌在长期发展过程中形成的适应性免疫防御系统,可用来对抗入侵的病毒及外源DNA。CRISPR/Cas9基因编辑技术是一种高效的核酸切割工具。近年来,该技术因其操作性强、效率高、适用范围广等优点,已经成为定点编辑的重要手段,广泛应用于猪、家兔、鼠等的基因定点修饰中。目前,以CRISPR/Cas9为基础的基因编辑技术在一系列疾病的基因治疗的应用领域都展现出极大的应用前景。
干扰素调节因子家族(Interferon regulatory factors,IRFs)参与机体细胞凋亡、肿瘤发生、宿主防御、病毒潜伏和免疫反应等多种生物学功能的调节。其中IRF3和IRF7在I型IFN介导的先天免疫中起着关键作用。IRF3在多种类型的细胞中表达,负责诱导早期IFN-β的转录。 IRF7主要存在于B细胞、浆细胞样树突状细胞(PDC)和单核细胞中,可诱导产生IFN-α和 IFN-β。产生的I型IFN可以与细胞表面的干扰素受体结合,激活下游信号通路,诱导细胞产生抗病毒蛋白(AVP)而发挥广谱抗病毒作用。
虽然研究人员已经建立了敲除IRF3基因的PK15细胞系,但双敲除IRF3、IRF7基因的猪源细胞系仍处于空白状态。因此,本研究利用CRISPR/Cas9基因组编辑技术,将I型IFN通路中的关键因子IRF3、IRF7进行双敲除,得到一株ST IRF3/IRF7 KO细胞,该细胞系的成功构建有助于揭示病毒-宿主细胞的相互作用、解析病毒的致病机理。
发明内容
本发明的目的是构建一株ST IRF3/IRF7 KO细胞系,可应用于多种猪源病毒感染的相关研究。根据猪源IRF3、IRF7基因序列的外显子设计sgRNA,并引入相应的酶切位点。通过退火扩增出目的片段后,分别插入载体lentiCRISPR v2上,并转化至感受态细胞Top10中进行克隆,得到lentiCRISPR v2-IRF3,lentiCRISPR v2-IRF7慢病毒载体。经测序鉴定正确后,将其转染HEK293T细胞,分别在转染后48h、72h收集慢病毒,随后感染ST细胞。慢病毒感染48h后,利用潮霉素B或嘌呤霉素筛选出成功感染的细胞,经测序及WB鉴定正确后扩大培养,进行后续实验。
随后,对ST IRF3/IRF7 KO细胞进行生长特性研究,并且与ST细胞进行比对。另外,我们还运用ST IRF3/IRF7 KO细胞系测定了SVA(塞内卡病毒)、PSV(萨佩罗病毒)及GETV(盖他病毒)的病毒生长曲线,结果表明,敲除IRF3及IRF7后,病毒的滴度均明显提升。我们还运用该细胞系测定了SVA、PSV、GETV感染后,部分基因的表达变化。结果显示,敲除IRF3及IRF7后,下游基因的变化幅度显著减小。
一株ST IRF3/IRF7 KO细胞系,分类命名为猪睾丸上皮细胞系ST IRF3/IRF7 KO,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:C2021178,保藏地点为中国武汉,保藏日期为2021年7月12日。
一种ST IRF3/IRF7 KO细胞系的构建方法,包括如下步骤:
采用IRF3、IRF7基因序列的外显子设计对应的sgRNA,随后在得到的sgRNA两端分别加上BsmbI酶切位点;
退火扩增出目的sgRNA后,分别插入至载体lentiCRISPR v2上,并转化至感受态细胞 Top10中,分离单菌落,提取质粒;得到慢病毒表达载体lentiCRISPR v2-IRF3和lentiCRISPR v2-IRF7;
慢病毒表达载体共同转染HEK293T细胞;将慢病毒加入靶细胞中,接毒后,添加嘌呤霉素或潮霉素B,筛选得到目的细胞系ST IRF3/IRF7 KO。
一种ST IRF3/IRF7 KO细胞系的构建方法,包括如下步骤:
采用IRF3、IRF7基因序列的外显子设计对应的sgRNA,随后在得到的sgRNA两端分别加上BsmbI酶切位点;
退火扩增出目的sgRNA后,分别插入至载体lentiCRISPR v2上,并转化至感受态细胞 Top10中,分离单菌落,提取质粒;得到慢病毒表达载体lentiCRISPR v2-IRF3和lentiCRISPR v2-IRF7;
慢病毒表达载体共同转染HEK293T细胞;将慢病毒加入靶细胞中,接毒后,添加嘌呤霉素或潮霉素B,筛选得到目的细胞系ST IRF3/IRF7 KO。ST IRF3/IRF7 KO细胞系的构建方法,其特征在于,包括如下步骤:
sgRNA的设计;
慢病毒表达载体的构建;
ST IRF3/IRF7 KO细胞系的构建。
进一步的,采用下列引物:
进一步的,所述慢病毒表达载体的检测引物序列为:
U6F:5′-TACGATACAAGGCTTAGA-3′;
U6R:5′-ACATCACTTTCCCAGTTTAC-3′。
进一步的,目的sgRNA插入至载体lentiCRISPR v2上的酶切体系为:
进一步的,反应体系、条件如下表所示:
一种ST IRF3/IRF7 KO细胞系的检测方法,检测引物序列为:
进一步的,采用所述ST IRF3/IRF7 KO细胞系接种SVA、PSV及GETV,并采用如下引物进行RT-qPCR荧光检测:
进一步的,以管家基因β-actin为内参,根据公式2-ΔΔCT,计算出DEGs的倍数变化:
ΔΔCT=(CQ目的基因-CQβ-actin)病毒组-(CQ目的基因-CQβ-actin)阴性对照。进一步的, sgRNA的设计方法为:采用猪源IRF3、IRF7基因序列的外显子设计对应的sgRNA。随后在得到的sgRNA两端分别加上BsmbI酶切位点。
进一步的,慢病毒表达载体的构建包括如下步骤:通过退火扩增出目的sgRNA后,分别插入至载体lentiCRISPR v2上,并转化至感受态细胞Top10中,分离单菌落,提取质粒;得到慢病毒表达载体lentiCRISPR v2-IRF3,lentiCRISPR v2-IRF7。
进一步的,将慢病毒表达载体7μg,包装质粒psPAX2 5μg,pMD2.G 3μg,共同转染HEK293T细胞。转染后2-3天收集慢病毒。
进一步的,将慢病毒加入靶细胞中,接毒48h后,添加嘌呤霉素或潮霉素B,筛选得到目的细胞。
本发明还提供ST IRF3/IRF7 KO细胞系在制备病毒疫苗活药物筛选中的应用。
有益效果
本发明针对猪源IRF3、IRF7基因序列的外显子设计特异性sgRNA,插入载体lentiCRISPR v2上,得到慢病毒载体lentiCRISPR v2-IRF3和lentiCRISPR v2-IRF7。将重组质粒转染HEK293T细胞后,收集慢病毒感染ST细胞。通过抗生素筛选出成功被感染的细胞,并进行测序、WB验证。
本研究利用CRISPR/Cas9技术构建了ST IRF3/IRF7 KO细胞系,弥补了现阶段猪源双缺失细胞系的不足。图5显示,在细胞水平上,IRF3、IRF7基因能显著降低SVA,PSV及GETV的病毒毒价,在抑制病毒的生长复制方面具有重要作用。同时,RT-qPCR结果显示(图6),对比正常ST细胞,ST IRF3/IRF7 KO细胞中处于IRF3及IRF7基因下游的相关因子的表达量显著降低。该细胞系还可以作为有价值的细胞材料,应用于多种猪源病毒感染的相关基础和应用研究,有助于揭示病毒-宿主细胞的相互作用、解析病毒的致病机理。
附图说明
图1为lentiCRISPR v2-IRF3和lentiCRISPR v2-IRF7慢病毒表达载体测序鉴定结果。
图2为ST IRF3/IRF7 KO细胞系测序验证结果。
图3为通过Western Blot检测ST IRF3/IRF7 KO细胞中IRF3和IRF7的表达水平结果。
图4为ST IRF3/IRF7 KO细胞生长曲线测定结果。
图5为SVA(a)、PSV(b)、GETV(b)在ST IRF3/IRF7 KO细胞上的毒价测定结果。
图6为SVA(a)、PSV(b)、GETV(c)感染后,通过RT-qPCR测定下游相关基因的表达变化水平。
具体实施方式
1.生物材料与试剂:
1.1 生物材料
ST、HEK293T细胞;PSV、GETV、SVA病毒;质粒lentiCRISPR v2、psPAX2、pMD2.G。
1.2 实验试剂与耗材
DMEM培养基,胎牛血清,青链霉素,胰酶,DNA提取试剂盒,Takara PrimeSTAR MaxDNAPolymerase,限制性核酸内切酶BsmBI,T4 DNA连接酶,50×TAE电泳缓冲液,琼脂糖,PCR产物回收试剂盒,胶回收试剂盒,质粒提取试剂盒,质粒转染试剂盒。
2.实验方法
2.1 基因敲除位点选择和sgRNA设计
选择基因敲除位点有以下三个原则:第一,靶位点应位于基因组外显子上。第二,选择特异性高的位点以降低脱靶概率。第三,尽量避开GC含量较高的区域,防止甲基化造成干扰。
根据上述原则,我们采用猪源IRF3、IRF7基因序列的外显子,设计对应的sgRNA。根据评分从高到低,每个基因筛选出2条sgRNA用于后续实验。随后在得到的sgRNA两端分别加上BsmbI酶切位点,最终设计的sgRNA序列如表1所示。
表1 基因sgRNA引物序列
2.2 慢病毒表达载体的构建
2.2.1 sgRNA双链DNA片段的合成
(1)将两条合成好的sgRNA溶于TE Buffer中,终浓度为100μM;
(2)在PCR管中分别配置25μL的反应体系;
(3)将反应体系混匀;
(4)PCR管放入PCR仪中,95℃孵育5min;
(5)PCR仪梯度降温,逐渐从95℃降至37℃,具体反应程序如下:
(6)PCR产物-20℃保存。
2.2.2 载体线性化
使用限制性核酸内切酶Bsmb I酶切载体lentiCRISPR v2,酶切体系,如下所示:
反应体系混匀后,55℃酶切3h;
酶切产物经1%浓度的琼脂糖凝胶电泳分析后,回收酶切产物,具体操作如下:
(1)待DNA条带分离后,用一把干净、锋利的手术刀小心地切下目的条带,并去除周围多余的琼脂糖以减小凝胶体积;
(2)将切下的目的条带置于清洁的1.5mL微量离心管中,并称量凝胶质量来确定适当的体积。假设凝胶密度为1g/mL,则1g凝胶的体积为1mL;
(3)根据凝胶质量,加入同等体积的Binding Buffer;
(4)置于50-60℃金属浴上进行溶胶,每2-3min涡旋一次直到凝胶完全融化;
(6)10000×g,室温离心1min后,弃去滤液并将吸附柱重新置于收集管中;
(7)在吸附柱内加入300μL Binding Buffer,13000×g,室温离心1min,弃去滤液并将吸附柱重新置于收集管中;
(8)在吸附柱内加入700μL SPW Wash Buffer(确认已加入适当比例的酒精),10000×g,室温离心1min,弃去滤液并将吸附柱重新置于收集管中;
(9)重复步骤(8);
(10)将吸附柱置于空的收集管内,以最大速度离心2min以彻底去除吸附柱内的乙醇。注意:残留的乙醇可能会影响后续实验;
(11)将吸附柱重新置于洁净的1.5mL微量离心管中,并向吸附柱的膜中央中加入15-30 μL Elution Buffer溶液(溶液可提前预热),室温静置2-3min;
(12)13000×g,室温离心2min以洗脱DNA,纯化的DNA置于-20℃保存备用。
2.2.3 退火片段与酶切载体连接
将步骤1.7.1中的PCR产物40倍稀释,将退火片段与酶切载体进行连接,反应体系、条件如下表所示:
将所有组分混匀,PCR仪16℃连接2h后,4℃连接过夜。
2.2.4 连接产物转化
(1)取出Top10感受态细胞,冰上融化。将体积≤10μL的连接产物加入100μL感受态细胞中,轻轻混匀后冰上放置30min;
(2)将离心管置于42℃金属浴上,热应激90s(须严格控制热应激时间),热应激完成后迅速取出置于冰上冷却2-3min;
(3)离心管中加入900μL无污染的液体LB培养基,置于37℃,160rpm/min的摇床上振荡培养50min;
(4)5000rpm,室温离心3min,仅保留80μL左右上清,用移液枪吹打混匀后,吸取所有菌液均匀涂布于含有氨苄霉素抗性的固体LB平板上;
(5)倒置培养皿于37℃恒温培养箱中,过夜培养;
(6)待单菌落长出后,挑取单菌落于含氨苄霉素抗性的液体LB培养基中,置于37℃, 220rpm/min的摇床上振荡培养12-16h。
2.2.5 重组质粒提取
提取质粒操作如下:
(1)取3-4mL过夜培养的菌液于2mL微量离心管中,10000×g,室温离心1min,完全弃去上清;
(2)加入250μL Solution 1,旋涡或上下颠倒至沉淀完全悬浮(使用前确认Solution 1 中已添加RNase A);
(3)将步骤(2)中的溶液转移至新的1.5mL微量离心管中,加入250μL Solution 2,轻柔地上下颠倒数次直至溶液变得澄清透亮。注意:剧烈混合会引起染色体DNA剪切,降低质粒的纯度;
(4)加入350μL Solution 3,轻柔地上下颠倒数次,直至形成白色絮状沉淀。以最大速度室温离心10min,迅速进入下一步;
(5)将制备管放入2mL收集管中,小心吸取步骤(4)中的上清,13000×g,室温离心1min,弃尽滤液;
(6)将制备管重新放入收集管中,加入500μL HBC Buffer,13000×g,室温离心1min,弃尽滤液;
(7)将制备管重新放入收集管中,加入700μL DNA Wash Buffer,13000×g,室温离心 1min,弃尽滤液;
(8)重复步骤(7)
(9)将制备管重新放入收集管中,13000×g,室温离心2min;
(10)将制备管放入新的1.5mL微量离心管中,在制备管的膜中央滴加30-100μLElution Buffer(Elution Buffer在金属浴上提前预热至65℃),室温静置2-3min,13000×g,室温离心1min。用分光光度仪检测重组质粒浓度,并保存于-20℃备用。
2.2.6 慢病毒表达载体测序鉴定
慢病毒表达载体序列lentiCRISPR v2-108 IRF3如SEQ ID NO:1所示:
慢病毒表达载体序列lentiCRISPR v2-112 IRF3如SEQ ID NO:2所示:
慢病毒表达载体序列lentiCRISPR v2-65 IRF7如SEQ ID NO:3所示:
慢病毒表达载体序列lentiCRISPR v2-133 IRF7如SEQ ID NO:4所示。
将2.2.3中提取的重组质粒适当稀释,以U6 promoter序列: U6F:5′-TACGATACAAGGCTTAGA-3′,U6R:5′-ACATCACTTTCCCAGTTTAC-3′为引物送至南京擎科生物科技有限公司进行测序鉴定。测序结果鉴定成功后,进行后续实验。测序结果如下:
2.3 细胞培养与慢病毒制备
2.3.1 ST、HEK293T细胞复苏
(1)将先前冻存的细胞从液氮罐中取出并迅速置于37℃恒温水浴锅中,每2-3min轻轻摇晃,直至细胞完全融化;
(2)将完全融化的细胞移至含有5mL细胞生长液的10mL无菌离心管中;
(3)1000rpm,室温离心8min,完全弃去上清。加入2mL细胞生长液吹打均匀,使细胞完全悬浮;
(4)将步骤(3)中的所有液体吸到T25细胞培养瓶中,并加入3-4mL细胞生长液,左右轻轻摇动使细胞分布均匀;
(5)标记好复苏日期、细胞种类、培养人姓名等关键信息,随后置于37℃,5%CO2的细胞培养箱中进行培养。
2.3.2 细胞传代
当培养瓶中细胞的覆盖率达到90%且细胞状态良好时,需进行细胞传代。
(1)用真空泵吸去原细胞培养瓶中的旧培养基;
(2)用高压灭菌后的1×PBS洗涤细胞1-2次;
(3)向细胞瓶内加入适量浓度为0.25%的胰酶(含EDTA),以刚好能覆盖细胞培养瓶底部为宜;
(4)将细胞培养瓶置于恒温培养箱中进行消化,时间为2-3min;
(5)用倒置显微镜观察细胞形态,当细胞胞质回缩、细胞间隙增大时消化完成;
(6)迅速加入2mL细胞生长液终止消化,用巴氏滴管吸取瓶内生长液,反复温柔地吹打贴壁细胞,使之从瓶壁脱离形成单细胞悬液。注意吹打时动作要轻柔,防止用力过猛造成细胞损伤;
(7)取出步骤(6)中1/2的细胞悬液放入新的细胞培养瓶中,加入5mL细胞生长液,置于恒温细胞培养箱培养。
2.3.3 慢病毒制备
当六孔板中的HEK293T细胞的汇合度达到60%时,进行质粒转染。将冻存于-20℃的目的质粒提前取出,室温融化。取适量无血清和抗生素的空培养基分别稀释目的质粒和转染试剂H4000,使目的质粒(μg)∶转染试剂(μL)为1∶3。随后,按照目的质粒lentiCRISPRv2-IRF3、 lentiCRISPR v2-IRF7 7μg,包装质粒psPAX2 5μg,pMD2.G 3μg的量与稀释好的转染试剂混匀,室温静置20min。随后,将混合物加入六孔板中,摇晃均匀。将六孔板置于细胞培养箱中继续培养,在转染后4-6h,更换细胞培养基。转染后2-3天收集慢病毒,并保存于-80℃备用。
2.4 ST IRF3/IRF7 KO细胞系的构建
2.4.1 慢病毒感染与抗生素筛选
首先需要确认后续抗生素筛选的浓度,实验步骤如下:提前一天将ST细胞均匀铺于48 孔板中,待细胞密度达到100%时,梯度添加潮霉素B或嘌呤霉素,观察细胞全部死亡所需时日并记录。
随后,将保存的慢病毒与细胞生长液按照1∶1的比例混合均匀,加入汇合度为60%-70%的靶细胞中,同时以未加入慢病毒的靶细胞作为阴性对照。接毒24h后,更换培养基。接毒 48h后,根据目的质粒携带的抗性,在细胞生长液中添加1-1.5μg/mL的嘌呤霉素或800-100 μg/mL的潮霉素B。连续筛选7-10天,直至阴性对照细胞全部死亡。ST IRF3 KO细胞系构建完成后,将携带IRF7sgRNA的慢病毒与细胞生长液1∶1混匀加入细胞,完成ST IRF3/IRF7 KO双缺失细胞系的构建。
2.4.3 单克隆细胞挑取
将2.4.1 中携带抗性的细胞消化为单细胞,并进行细胞计数。以适宜的密度(50-100个/ 皿)均匀铺在100mm的细胞培养皿中,待单细胞形成肉眼可见的单细胞克隆时,进行挑取。
(1)提前将克隆环高压灭菌,并置于75%无水乙醇中备用;
(2)在倒置显微镜下观察单细胞克隆,并将细胞状态较好的单细胞克隆做上标记;
(3)将100mm培养皿中的培养基弃去约3/4,仅保留刚好能覆盖整个底面的培养基;
(4)用镊子小心夹取克隆环并在酒精灯外焰上灼烧5-10s,确保克隆环无菌;
(5)克隆环蘸取适量克隆胶,使胶水刚好能够覆盖到整个克隆环的边缘;
(6)将克隆环扣在先前做标记的单细胞克隆上,注意尽量使克隆位于环的正中间,以免造成细胞损伤;
(7)用10μL移液枪将克隆环内的培养基吸尽;
(8)向每个克隆环内小心滴加100μL PBS,漂洗1-2次,随后弃去。注意枪头不要碰到细胞;
(9)向每个克隆环内小心滴加约50μL胰酶,随后将培养皿置于细胞培养箱内,消化6-10min;
(10)确认细胞完全消化后,小心轻柔地吹打单细胞克隆直至形成单细胞悬液;
(11)将克隆环内所有液体转移至96孔细胞培养板中,并添加适量的细胞生长液;
(12)96孔细胞培养板置于恒温细胞培养箱中继续培养;
(13)用镊子将细胞培养皿中的克隆环取下,将原有的细胞生长液弃去,加入10mL全新培养基,置于37℃、5%CO2的细胞培养箱中继续培养。
2.4.4 细胞扩培
待96孔细胞培养板中细胞的密度达到80%至100%时,可进行细胞扩培。将原始96孔板中的细胞完全消化,转移至1个全新的48孔板中,并补加适量的细胞生长液,置于细胞培养箱中继续培养。待48孔板中细胞的密度达到80%-100%时,将细胞完全消化并移至1个全新24孔板中继续培养。重复此过程,直至细胞总量达到6孔板的90%至100%。此时,将细胞完全消化,取1/2细胞提取基因组DNA,剩余的细胞进行冻存。
细胞冻存步骤如下:
(1)用0.25%的胰酶将生长状态良好的细胞完全消化后,移至10mL无菌离心管中;
(2)离心管1000rpm,室温离心10min,弃去上清;
(3)配置细胞冻存液:60%DMEM,30%FBS和10%DMSO,混合均匀;
(4)用1mL细胞冻存液重悬细胞,并将其移至冻存管中,封上封口膜;
(5)冻存管标记好细胞名称、冻存日期、细胞抗性、培养人姓名等重要信息;
(6)将冻存管放入已平衡至室温的冻存盒中,置于超低温冰箱;
(7)24h后,将冻存的细胞从超低温冰箱转移至液氮罐中,并在本上详细记录相关信息。
2.5 ST IRF3/IRF7 KO细胞系鉴定
猪基因组DNA中IRF3突变基因的序列如SEQ ID NO:5所示;
猪基因组DNA中IRF7突变基因的序列如SEQ ID NO:6所示。
2.5.1 基因组DNA提取
提取细胞基因组DNA,操作步骤如下:
(1)贴壁细胞加入适量胰酶,待其消化为单细胞悬液后,10000rpm,室温离心1min,完全弃去上清;
(3)加入200μL缓冲液GA,涡旋震荡直至细胞彻底悬浮;
(2)加入20μL Proteinase K溶液,混合均匀;
(3)加入200μL缓冲液GB,充分地上下颠倒,使之完全混匀。金属浴70℃,10min。待溶液变得澄清透亮时,简短离心以去除内盖水珠。注意:加入缓冲液GB后可能会出现白色絮状沉淀,一般70℃加热时会消失,不影响后续实验;
(4)加入200μL无水乙醇,涡旋振荡15s,待溶液充分混匀后简短离心以去除内盖水珠;
(5)将步骤(4)中所得溶液与白色沉淀共同加入吸附柱CB3中,吸附柱放入收集管中,12000rpm,室温离心30s,弃掉废液;
(6)将吸附柱放回收集管中,加入500μL缓冲液GD(需确认缓冲液中已添加无水乙醇),12000rpm,室温离心1min,弃掉废液;
(7)将吸附柱放回收集管中,加入600μL漂洗液PW(需确认漂洗液PW中已添加无水乙醇),12000rpm,室温离心1min,弃掉废液;
(8)重复步骤(7);
(9)将吸附柱放回收集管中,13000rpm,室温离心3min,弃掉废液。吸附柱置于室温3-5min,直至吸附柱中残留的漂洗液彻底晾干;
(10)将吸附柱CB3移至一个全新的1.5mL离心管中,向吸附膜的中央滴加60-100μL洗脱缓冲液TE,室温静置3-5min,随后12000rpm,室温离心1min。DNA产物置于-20℃保存备用。
2.5.2 PCR扩增目的片段
根据设计的sgRNA所在位置,利用snapgene软件设计一条高特异性的引物用于后续PCR 验证。为防止测序前200-300bp结果不准确,选取sgRNA 5’端上游300bp左右的位置设为上游引物,扩增片段全长以700-900bp为佳。引物长度大概18-22bp,GC含量≤60%,上下游引物的TM值相差≤5℃。引物序列如下:
表2 验证引物序列
PCR反应体系如下:
PCR反应条件如下:
PCR反应结束后,取3-5μL PCR产物通过琼脂糖凝胶电泳检测结果。
2.5.3 目的片段连接T载体
首先,将获得的PCR产物PCR纯化:
(1)将PCR产物转移到一个全新的1.5mL微量离心管中;
(2)加入4倍体积(约200μL)的CP Buffer,用移液器吹打混匀;
(3)将混匀后的液体转移到吸附柱内,12000rpm,室温离心1min,弃去滤液;
(4)加入700μL的DNA Wash Buffer,12000rpm,室温离心1min,弃去滤液;
(5)重复步骤(4)
(6)将吸附柱重新放回接液管内,12000rpm,室温离心2min;
(7)将吸附柱转移至一个全新的的1.5mL微量离心管中,向吸附柱的膜中央滴加30μL 预热的Elution Buffer,室温静置2min后,12000rpm,室温离心1min,洗脱目的DNA片段。
随后,纯化的PCR片段进行加A,具体步骤如下:
PCR反应条件:72℃,30min。
其次,回收加A片段,具体步骤同2.2.2。
最后,将目的片段连接T载体进行连接,连接体系如下:
连接条件:4℃连接过夜。
2.5.4 连接产物转化
同2.2.4。
2.5.5 质粒提取
同2.2.5。
2.5.6 测序鉴定
测序验证步骤如下:将2.5.5中提取的重组质粒进行适当稀释,送至南京擎科生物科技有限公司进行测序鉴定。结果显示,IRF3和IRF7基因均有不同数量的核苷酸插入,造成了移码突变,重组质粒构建成功。
1.5.7 Western Blot鉴定
(1)ST、ST IRF3/IRF7 KO细胞弃上清,分别用PBS润洗三次;
(2)每孔加入100μL RIPA裂解缓冲液(含1%蛋白酶抑制剂),冰上静置20min后, 4℃,12000rpm离心3min收集上清;
(3)样品加入5×SDS-PAGE上样缓冲液并混匀,沸水浴10min,分装备用;
(4)制备分离胶浓度为12%,浓缩胶浓度为5%的10孔蛋白胶;
(5)按照蛋白Marker 26616、ST IRF3/IRF7 KO细胞、ST细胞的顺序,依次上样。80V恒定电压跑至分离胶后,将电压调为120V,待溴酚蓝指示线跑至蛋白胶底部时,关闭电源。电泳时间约2.5h;
(6)电泳结束后,根据蛋白胶的大小,裁剪出大小合适的NC膜,并将蛋白胶、滤纸、海绵浸泡在膜转移缓冲液中平衡5min;
(7)在转膜夹黑板面依次放入海绵、两层滤纸、蛋白胶、NC膜、两层滤纸、海绵,并轻轻赶走气泡;
(8)安装完毕后,将转膜夹放入装有冰块的电泳槽中,恒流200mA,45min进行转膜;
(9)转膜完成后,将NC膜置于5%的脱脂奶粉中,室温封闭2h;
(10)1∶1000稀释一抗,随后将NC膜与稀释后的一抗放入WB杂交袋中,4℃孵育过夜;
(11)回收一抗后,加入适量的1×TBST缓冲液,室温下洗涤10min。此过程重复三次;
(12)1∶8000稀释HRP标记的对应二抗,室温孵育2h;
(13)回收二抗后,加入适量的1×TBST缓冲液,室温下洗涤10min。此过程重复三次;
(14)配置ECL显影液,均匀滴加在NC膜上进行曝光拍照。
实验案例一:细胞生长曲线测定
为比较缺失细胞与正常细胞生长速度的差异,取代数相同的ST IRF3/IRF7 KO细胞及ST 细胞,通过MTT法测定细胞生长曲线。具体方法如下:
(1)用0.25%的胰酶将生长状态良好的细胞消化后,用DMEM进行稀释。
(2)以每孔2000个细胞接种至96孔板,每孔体积200μL。每组设置4个复孔,同时以DMEM为阴性对照。
(3)分别在培养0.5d,1d,1.5d,2d,3d,4d,5d,6d后,加入20μL MTT溶液 (5mg/mL),继续培养4h。
(4)小心吸去孔内的上清液,每孔加入150μL DMSO溶液,摇床震荡10min。
(5)选择490nm波长,在酶标仪上测定每孔的OD值。
(6)记录数据,并绘制出细胞生长曲线。
结果显示:ST及ST IRF3/IRF7 KO细胞均呈现出典型的“S”形生长曲线,且生长速率无明显差异。
实验案例二:病毒生长特性研究
为研究在细胞水平上,IRF3、IRF7基因对于病毒生长复制能力的影响,将SVA、PSV、GETV分别感染ST、ST IRF3/IRF7 KO,测定病毒滴度。具体方法如下:
(1)提前一天将ST、ST IRF3/IRF7 KO细胞铺于6孔板中,待细胞汇合度达到80%-90%时,弃去部分上清,仅保留能覆盖孔底面的即可;
(2)SVA、PSV、GETV分别以MOI=0.01感染ST IRF3/IRF7 KO细胞,同时将ST细胞作为阴性对照;
(3)培养板置于37℃,5%CO2的病毒培养箱中孵育2h后,完全弃去上清,用1×PBS清洗2-3遍;
(4)加入2mL含2%FBS的不完全培养基,置于恒温病毒培养箱中继续培养;
(5)分别在病毒感染后6h,12h,24h,36h,48h,60h收集病毒液200μL,再补加 200μL细胞不完全培养基;
(6)将收集的病毒液置于-80℃保存备用。
随后,进行病毒效价测定:
(1)提前1天将ST细胞铺于96孔板中,待细胞汇合度达到80%时,弃去上清;
(2)将冻存的病毒液室温融化,并用含2%FBS的细胞不完全培养基进行梯度稀释(10-2-10-8):
(3)用稀释好的病毒液感染ST细胞,每孔100μL,且每个梯度设置4个重复;
(4)96孔板置于37℃,5%CO2的病毒培养箱孵育2h;
(5)每孔补加50μL含2%FBS的细胞不完全培养基,继续恒温培养;
(6)观察细胞病变情况并及时做好标记,按照Karber法计算出病毒效价。
滴度结果显示:对比ST细胞,病毒滴度在ST IRF3/IRF7 KO细胞上明显提升。
实验案例一:下游基因表达测定
ST、ST IRF3/IRF7 KO细胞分别以MOI=0.1接种SVA、PSV及GETV。分别在病毒感染后24h时收集细胞,制备样品。选取7个重要基因,并根据Genbank上的基因序列设计对应引物,如表所示。
荧光定量PCR引物
将所得DNA产物稀释,进行RT-qPCR实验,反应体系如下:
在罗氏荧光定量PCR仪上进行扩增,反应程序如下:
在延伸阶段检测荧光。以管家基因β-actin为内参,根据公式2-ΔΔCT,计算出DEGs的倍数变化。
ΔΔCT=(CQ目的基因-CQβ-actin)病毒组-(CQ目的基因-CQβ-actin)阴性对照组
CQ目的基因为病毒感染组的CQ值;CQβ-actin为内参的CQ值;ΔΔCT为目的基因相对于内参的RNA的量。
结果显示:病毒感染后,ST IRF3/IRF7 KO细胞中相关基因的上调幅度明显低于ST细胞。
序列表
<110> 扬州大学
<120> ST IRF3/IRF7 KO细胞系及其构建方法和应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ccgcaagccg tgcttccaag ttttagagct agaaatagca agttaaaata 300
aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgaattcgct 360
agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca 420
tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg tgcctagaga 480
aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag 540
ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg 600
tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa gaagtacagc 660
atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag 720
gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac 780
ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga 840
accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc 900
agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg 960
gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg 1020
gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc 1080
gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc 1140
cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc 1200
cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg 1260
gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc 1320
gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg 1380
ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg 1440
agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 1500
gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg 1560
agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac 1620
gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag 1680
tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga 1740
gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc 1800
gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac 1860
aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag 1920
gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc 1980
cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc 2040
agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct 2100
tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag 2160
gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa 2220
gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag 2280
gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag 2340
gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg 2400
ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc 2460
ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt 2520
gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa 2580
gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg 2640
atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac 2700
ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag 2760
gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat 2820
ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag 2880
ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag 2940
aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag 3000
ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg 3060
cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag 3120
gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt 3180
ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag 3240
agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg 3300
ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc 3360
ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag 3420
atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat 3480
gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc 3540
cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac 3600
gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc 3660
gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag 3720
caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc 3780
aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac 3840
ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg 3900
ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc 3960
aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg 4020
gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg 4080
gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc 4140
accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc 4200
tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg 4260
gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg 4320
gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag 4380
ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg 4440
gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat 4500
ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc 4560
gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac 4620
tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc 4680
ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga 4740
ggcgac 4746
<210> 2
<211> 4742
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg acacgcgctt ccgcagtttt agagctagaa atagcaagtt aaaataaggc 300
tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgaa ttcgctagct 360
aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc 420
ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt 480
ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg 540
ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg 600
ccgccagaac acaggaccgg ttctagagcg ctgccaccat ggacaagaag tacagcatcg 660
gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc 720
ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga 780
tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg 840
ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca 900
acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg 960
aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct 1020
accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca 1080
aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact 1140
tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc 1200
tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg 1260
ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc 1320
agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc 1380
tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca 1440
aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg 1500
acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag 1560
tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc 1620
accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca 1680
aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca 1740
gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg 1800
aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg 1860
gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag 1920
atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca 1980
tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa 2040
agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg 2100
cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc 2160
tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga 2220
aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca 2280
tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact 2340
acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca 2400
acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg 2460
acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg 2520
acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga 2580
tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca 2640
acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct 2700
tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca 2760
tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg 2820
ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg 2880
tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc 2940
agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca 3000
tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga 3060
acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac 3120
tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga 3180
aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg 3240
acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga 3300
acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc 3360
tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca 3420
caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca 3480
agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga 3540
aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct 3600
acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt 3660
tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg 3720
aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga 3780
ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg 3840
aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga 3900
gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag 3960
agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc 4020
ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca 4080
aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca 4140
tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca 4200
aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa 4260
acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc 4320
tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct 4380
cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg 4440
agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg 4500
acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga 4560
atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg 4620
acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga 4680
tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg 4740
ac 4742
<210> 3
<211> 4747
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg tcagcagcgg ccgttacgca gttttagagc tagaaatagc aagttaaaat 300
aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcgc 360
tagctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac 420
atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg gtgcctagag 480
aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga 540
gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg 600
gtttgccgcc agaacacagg accggttcta gagcgctgcc accatggaca agaagtacag 660
catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa 720
ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa 780
cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag 840
aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt 900
cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag agtccttcct 960
ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt 1020
ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac 1080
cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg 1140
ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat 1200
ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt 1260
ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat 1320
cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct 1380
gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct 1440
gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta 1500
cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct 1560
gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga 1620
cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa 1680
gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg 1740
agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac 1800
cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga 1860
caacggcagc atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca 1920
ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt 1980
ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac 2040
cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc 2100
ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa 2160
ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa 2220
agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa 2280
ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga 2340
ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg 2400
gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt 2460
cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt 2520
tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa 2580
agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct 2640
gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga 2700
cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga 2760
ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa 2820
tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga 2880
gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga 2940
gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga 3000
gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct 3060
gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca 3120
ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt 3180
tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa 3240
gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct 3300
gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg 3360
cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca 3420
gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa 3480
tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt 3540
ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga 3600
cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag 3660
cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga 3720
gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt 3780
caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa 3840
cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt 3900
gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag 3960
caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg 4020
ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt 4080
ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat 4140
caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg 4200
ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct 4260
ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact 4320
ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa 4380
gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct 4440
ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa 4500
tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc 4560
cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta 4620
ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac 4680
cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg 4740
aggcgac 4747
<210> 4
<211> 4746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg tgcgcgagtc ggactcgccg ttttagagct agaaatagca agttaaaata 300
aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgaattcgct 360
agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca 420
tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg tgcctagaga 480
aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag 540
ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg 600
tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa gaagtacagc 660
atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag 720
gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac 780
ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga 840
accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc 900
agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg 960
gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg 1020
gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc 1080
gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc 1140
cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc 1200
cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg 1260
gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc 1320
gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg 1380
ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg 1440
agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 1500
gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg 1560
agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac 1620
gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag 1680
tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga 1740
gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc 1800
gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac 1860
aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag 1920
gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc 1980
cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc 2040
agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct 2100
tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag 2160
gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa 2220
gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag 2280
gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag 2340
gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg 2400
ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc 2460
ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt 2520
gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa 2580
gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg 2640
atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac 2700
ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag 2760
gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat 2820
ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag 2880
ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag 2940
aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag 3000
ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg 3060
cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag 3120
gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt 3180
ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag 3240
agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg 3300
ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc 3360
ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag 3420
atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat 3480
gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc 3540
cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac 3600
gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc 3660
gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag 3720
caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc 3780
aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac 3840
ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg 3900
ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc 3960
aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg 4020
gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg 4080
gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc 4140
accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc 4200
tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg 4260
gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg 4320
gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag 4380
ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg 4440
gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat 4500
ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc 4560
gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac 4620
tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc 4680
ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga 4740
ggcgac 4746
<210> 5
<211> 4401
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgggaactc agaagcctcg gatcctgccc tggctgatat ctcagctgaa ccaggggcaa 60
ctggagggcg tggcctggct ggatgagggc cacacgcgct tccgcatccc ttaggccaca 120
cgcgcttcca cacccattag gccacacgcg cttccacacc cattaggcca cacgcgcttc 180
cacacccatt aggccacacg cgcttccaca cccttccagg tgcgcgcgcg ccgggcccgg 240
ggactccaag gggacggggc cgagggatgg gatcagctcg accgcgcggc ccacgcttat 300
tctggcaggt gggggtggat ggagttagaa ctgggagggg cgaagttagt acgacgcgca 360
gatgggcgtc tgacagccgt gaggattgga gccgccaggt cccaggaata gaaccgggca 420
agtgaaggaa atgcatcctg gggtgggagg ggagtggtgg tggtagggaa ccccctgggg 480
tggggctttt ggttgtagaa ttctgggggc ggaccttgga atattagagg caagactcga 540
ccctgaagcc gaggttgcca cggaagtagg atatgccggt ggtgttaagc ctaatgacga 600
agagtgtcct ggggcagcgc actggcagtt agacacccat taggaccagg aggctggctt 660
agaaagcaaa tgtttttgaa gggtggaggt aagggggatt tggggcacag agaattccag 720
agcttgggga tgagaactaa gttagaacgt gcaaagggaa ttggcacaat attgtaaatc 780
aactatggtt tcaaaaacaa aaacaaaaac aaaacctgcc aggggagggc aaggtggtgt 840
taggacaaga ggagccaagg ttaggacccc tgggggcaag atttaagtca tcctcttcac 900
tatcttggta ccactggtcc ccccaggcct gggccgaggc cagtggtgcc tacactcctg 960
ggaaggataa gcccgacctg cccacctgga agaggaattt ccggtctgcc ctgaaccgga 1020
aagaagcatt gcgtttagca gaggaccaca gcaaggaccc ccacgaccca cacaagatct 1080
atgagtttgt gacctcaggt gtgctgaagt gaggctttgc ttggagggat gggatttcgg 1140
gtgtccccag agcccctact ccactcacca cctttctcga caggagttgg ggactttcct 1200
gagccagaca cctctctaga cctcagtggc agatacagta cctctgatac ccacgtaaga 1260
atgcgccccg accgtcctgc gctcagcccc tgcctcatgc atcctcaggg gcctagcgag 1320
gccaccatca ctccaggctc cactctctcc ctgcagtctg ccccctacct gacccatggc 1380
tgaccctgtc cctcccctgc tcacaggcct cccactggcc tcccagcatc ctcaggagaa 1440
aatgtggagc tttcaccagg catttaagac cctcacccac ttttctagga cttcacagct 1500
tccctccctg cacagggtac ctcagcctct tggcaaccga gaccctccct acccttgatc 1560
ttgctgacgt ctgtgccagg gcaggtccct tctgaaatcc tacccaggct tctctgcccg 1620
ggtcttgctc gcactgaccc agatcctcgg ggtggaatta gccgctcccc tcgggctacg 1680
gtagtgccca gcctgtgtac aagctgtgta tattccaccg ccttggctgg tggctgggtc 1740
tccctcagac tagaatcccc ttaagggcaa agctcaggtc tgtgtcctct ttggaggcac 1800
ggctccacac cgctgagcac aaggcctggt ccccgtgaag tctccagaaa aggccgtgga 1860
aatacctggc cgcttttttt tttcccctct ccaggaagac agtctggata agttactaag 1920
tggcatggac ttggcctcgg atgcagggcc cccgagcctg accctggccc tcgagcagcc 1980
ccctcagctc tccctgagcc ccagtgtaga cgcccccgct tcttgcccca acctgggagt 2040
ccgtgaaaac ccactgaagc agctgctggc gaatgacgac ggtgagtgcc cgctgtgggc 2100
tgggcggggc tggcacggcc cgcggcccct gacactccct cctctctctc cgctcatgtc 2160
cctgctgccg ctgctgctca gagtgggagt tccaggtgac cgtcttctac cggggctgcc 2220
aagtcttcca gcagactgtc tgcagcccgg gggcctgcgg ctggtgggat ccgaggccga 2280
ggacgggacg ctggccgggc agccagtgag actgccagac cccgcagcct ccctgacgga 2340
caggggtgtg gcggactacg tgcgccgggt gctgagctgc ctgggcgggg ggctggctct 2400
gtggagggcg gggcagtggc tctgggccca gaggctgggg cactgccacg tgtactgggc 2460
catgggcgag gaactcatcc cggacagcgg tcacaagcct gacggtgagg tccccaagga 2520
cagggaagga ggcgtgttcg acctggggcc cttcatcgaa ggtgagtgcg gcggccttgg 2580
ccctgccgtg gaagcggcag gctgcggggc tccagacctc cggggtcagg gggtggatca 2640
aattcagagc ctctgctctg cttgtggttg agcctgggaa ggcaaggact gcttggaacc 2700
tgtaaagtct cttctctctc cttacctgct gggagctggc agggggcagg gggcccagtg 2760
cacagcaggt gctcaggcag ttcgctgctt tcgttgttgt cgtcatcacc accaccagtt 2820
atggtggaga gacagacgca ggagaaaaac tcctgcgggg ctttgttatc caactagaac 2880
tttctgccat gatggaaatg cactgcatgc actgcccagc gtggcagccg ccagctgcag 2940
agtggctact gaggacgtag ataaagggca tgcaaaggaa gaaacgagct ttaagtttta 3000
gttaactttt tttttttgcc attttagagc tgcacctgag acatatggag gttcccaggc 3060
taggggtcca actggagctg tcaccacagc cacagccacg ccagatccga gccacgtctg 3120
cgacctacac cacagctcac ggcaacgccg gatccataac ccactgagca gggccaggga 3180
tggaactagc gtcctcaagg atactagttg ggttcgttgc cgccaagcca caatggtaac 3240
tcccagtttt cgttgatttt aattagatac aattaaaact taactagcca tcatggctca 3300
tggttatata ttggaccagc ctgaacttga ggggttcaag agcaggaagc accaaccaca 3360
tgtcctccag actcactcag catccttggg ggagcaggct ccccttctgg gcctgtatcc 3420
catccaaatg ctgaggcggg gagcgggggg acggcagcaa agcagcccct acagccctac 3480
cctgatggaa gggagagagg gggaacacta aagttggtgc cagaactgca gggacttttc 3540
ccaggtcaac agcccctggt catggtggca gaggtaggtt gggctttgaa ccaggggaga 3600
aggtaaagac gttactcggt ttgggggagc aggtagaggg ccttcttcca tccctgacta 3660
gttcctgggc ccccagatct gattgccttc atcgaaggaa gcagacgctc accacgctac 3720
accctctggt tctgcatggg gcagtcgtgg cctcaggacg agccgtgggt caagaggctg 3780
gtgatggtca aggtacggca gcccctggct cctggagtgg agggacgatc ccaaggggtg 3840
gggggttgct cccaggaact tcagcgcccg gggccccata gagaaactgc ggctgccagg 3900
agtctctgct gctccttacc ctcgggggcc tctgcgtgtc cctgccatgg cctcaccccg 3960
agcctctttg gagctctccc agccacacag aaggtggcgg accccaagtc ctggcaggct 4020
cagctcccag gagtcctgac cacaagccgc ttgggccacg cttgggggac ccctctgcag 4080
cctcagcccc gaggacctca ctggcctccc cggctgttaa catggggaca accatttcac 4140
tccgagcagc cctgggcttc gtggttcatc ctaggggaca gtgggggcgg ctggtgccgg 4200
cgtttcccca actcctcctc ccacacccca caggttgtcc ccatgtgtct ccgggccctg 4260
gtagacatgg cgcgggacgg gggcgcctcc tccctggaga acacggtgga cctgcacatt 4320
tccaacagcc acccgctctc cctcacctcc gaccagtaca aggcctgcct ccgggacctg 4380
gtggaggaca tggatttcta g 4401
<210> 6
<211> 2465
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggccgcgg ctcctgacag gtgacagccc gcagccctac ccggtgccaa ccccaagcac 60
ctcgcggtcc gcgcgtgccc tgacgcccgc tgctctgccc gcagggggtg cccacgcgtg 120
ctcttcggag actggcttct gggcgaagtc agcagcggcc gttaccgcag ggctgcgatg 180
gctggatgaa gccagaacac ggttccgcgt gccctggaag cacttcgccc gaaaggacct 240
gggcgagtcc gactcgcgca tctttaaggt gggaccccag ccccgcctgg agtgcggggg 300
gaccggccca ggtccaggcc cgtactccat tccatccctc cccaggcctg ggccgtggcc 360
cgcggcaggt ggccgccccg cagcagcgaa ggtgcgcagc cgatcccgga gggtgccctc 420
cgcgcctcct ggaaaaccaa cttccgctgt gccctgcaca gcacgcggcg cttcgtgatg 480
ttgcaggaca actcagggga ccccactgac cctcataagg tgtatatgat caaccccgag 540
ctggggtgcg gaggtgagga aggccagggg agggctgcct gcagggaggg ctgtgccgcg 600
gagcaagccg ctgctgggtt agatttcccc ttttagaaac tggcgtggga gaggctgggg 660
ccagactgtt gggggaaggt ggggtagggg atggccggga ggccccagac gggcctctgg 720
ggactggtga aggggaggag ctgcccaggg gttcacccct gggtgaccca gtccagtctc 780
agatccccat gtccccttaa acctaattct ctcccagaag gcccaggctt caacccgggg 840
gaagaggagg ccctcgagga tgccccaccc ccaagggtaa ggccactcag ccctccttgc 900
tcccgggttg gggctgccac ttctcactgg ggctggggag ctgctcactg atgccggcac 960
actgtcttct ccgcagggtg agcttcccgg gccgcgcctg gcagcacata ctggtgagag 1020
gctgaggcac gagctggcca ggccgagccc tgaaccctgc accccaagcc cagctggaga 1080
tggtggggac ctcctgctcc aggctctgca gcagagctgc atggaggacc atctgctgga 1140
agctgcatgg ggcgtggatc tgaccccccc cgaggttcca tgtgagagac ggctgctctg 1200
ccctgcaggg ttctgggcca aggggaggga caggcaggac agcaggaccc ctgggggggg 1260
gttgccccca gtagggatgg gctgaccaca ggtgggcccg aatagccgag cctcctcagg 1320
tccagagctc cctgcttaga aactgtacct gccactggca gggaaggtgg cccccagcct 1380
tgggccacag ccccaggccc agatgacaag gtgaggcgag ccaagtgaca cgtggaaggg 1440
agaggagctg ggggaccagg agccaactca ggccttcctc tccagatgca ggcccactcc 1500
cagccccggt gccctggaag ccccaagagg cggagccctg caccacactc tacccccgtg 1560
tctgcacgcg gatggcaggc gagcggggcg cggccagtcc cagctgctca cagctcggtc 1620
tgcacgcagg gcccagcttg gaggctctgg acttgaccat catgtacaaa ggtcgaacgg 1680
tgctgcagga gatggtgggg ggccccagct gtgtgctcct gtacgggtct ccgggcccgg 1740
ctggcgcggc tgcagagccc cagcaggtag ccttccccag ccccgctgag ctgcctgacc 1800
agaagcagct ccactacaca gagaagctgc tgcagcacgt ggcccccggc ctgcagctgg 1860
agttccgggg acccaagctg tgggcccggc gcctgggcaa gtgcaaagtc tactgggagg 1920
tgggagggcc cctgggctct ggcagcccct ccacccccgc ctgcctgctg ccccgagact 1980
gcgacacccc catcttcgac ttcggcacct tcttccaagg tcagtgacgc acctgcacct 2040
gccccggggc tgcggggggg gggggggggg cgctcctggc ccctgcccct cctcccgctc 2100
accagagccc tgtcctgcag agttgatgga gttccgggcc cgccggcgcc agggctcccc 2160
acactacacc atctacctgg gcttcgggca ggacctctcc gtggggaggc ccaaggagaa 2220
gagcctcgtc ctggtgaagg tgaggggcag ggaccctggg gcgaggctgc aggtagccct 2280
cgcccggcct gacagcgccc tacccgcagt tggagccatg gctgtgtcgg acacagctgg 2340
agagcgtgca gcgtgaaggc gtgtcctccc tggacagcgg cagcctcagc ctctgcctgt 2400
ccccctccaa cagcctctat gacgagctgg agcgcttcct gatggaggtg gagcagccgg 2460
cctag 2465
Claims (10)
1.一株ST IRF3/IRF7 KO细胞系,其特征在于,分类命名为猪睾丸上皮细胞系ST IRF3/IRF7 KO,保藏编号为CCTCC NO:C2021178。
2.根据权利要求1所述的ST IRF3/IRF7 KO细胞系的构建方法,其特征在于,包括如下步骤:
采用IRF3、IRF7基因序列的外显子设计对应的sgRNA,随后在得到的sgRNA两端分别加上BsmbI酶切位点;
退火扩增出目的sgRNA后,分别插入至载体lentiCRISPR v2上,并转化至感受态细胞Top10中,分离单菌落,提取质粒;得到慢病毒表达载体lentiCRISPR v2-IRF3和lentiCRISPR v2-IRF7;
慢病毒表达载体共同转染HEK293T细胞;将慢病毒加入靶细胞中,接毒后,添加嘌呤霉素或潮霉素B,筛选得到目的细胞系ST IRF3/IRF7 KO。
4.根据权利要求2所述的ST IRF3/IRF7 KO细胞系的构建方法,所述慢病毒表达载体的检测引物序列为:
U6F:5'-TACGATACAAGGCTTAGA-3';
U6R:5'-ACATCACTTTCCCAGTTTAC-3'。
9.根据权利要求8所述的ST IRF3/IRF7 KO细胞系的检测方法,以管家基因β-actin为内参,根据公式2-ΔΔCT,计算出DEGs的倍数变化:ΔΔCT=(CQ目的基因-CQβ-actin)病毒组-(CQ目的基因-CQβ-actin)阴性对照。
10.权利要求1所述的ST IRF3/IRF7 KO细胞系在制备病毒疫苗或药物筛选中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110937887.7A CN113717943B (zh) | 2021-08-16 | 2021-08-16 | St irf3/irf7 ko细胞系及其构建方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110937887.7A CN113717943B (zh) | 2021-08-16 | 2021-08-16 | St irf3/irf7 ko细胞系及其构建方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113717943A true CN113717943A (zh) | 2021-11-30 |
CN113717943B CN113717943B (zh) | 2023-04-04 |
Family
ID=78675974
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110937887.7A Active CN113717943B (zh) | 2021-08-16 | 2021-08-16 | St irf3/irf7 ko细胞系及其构建方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113717943B (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114107303A (zh) * | 2021-12-08 | 2022-03-01 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | sgRNA、质粒、IRF7功能缺失的细胞系及其构建方法和应用 |
CN114350615A (zh) * | 2021-12-20 | 2022-04-15 | 北京镁伽科技有限公司 | Stat2基因缺失细胞株及其制备方法和应用 |
-
2021
- 2021-08-16 CN CN202110937887.7A patent/CN113717943B/zh active Active
Non-Patent Citations (3)
Title |
---|
BASTIAN HATESUER: "Deletion of Irf3 and Irf7 Genes in Mice Results in Altered Interferon Pathway Activation and Granulocyte-Dominated Inflammatory Responses to Influenza A Infection", 《J INNATE IMMUN》 * |
CHING-LING LIN等: "HDAC1 and HDAC2 Double Knockout Triggers Cell Apoptosis in Advanced Thyroid Cancer", 《INT J MOL SCI》 * |
张爽: "IRF3基因敲除猪肾上皮细胞系的构建", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114107303A (zh) * | 2021-12-08 | 2022-03-01 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | sgRNA、质粒、IRF7功能缺失的细胞系及其构建方法和应用 |
CN114350615A (zh) * | 2021-12-20 | 2022-04-15 | 北京镁伽科技有限公司 | Stat2基因缺失细胞株及其制备方法和应用 |
CN114350615B (zh) * | 2021-12-20 | 2024-04-16 | 北京镁伽科技有限公司 | Stat2基因缺失细胞株及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN113717943B (zh) | 2023-04-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108486108B (zh) | 一种敲除人hmgb1基因的细胞株及其应用 | |
CN113717943B (zh) | St irf3/irf7 ko细胞系及其构建方法和应用 | |
AU2022275537A1 (en) | Nuclease systems for genetic engineering | |
CN109750035B (zh) | 靶向并引导Cas9蛋白高效切割TCR及B2M基因座的sgRNA | |
CN109825464B (zh) | 敲除t6ss-1基因簇的杀香鱼假单胞菌鱼用减毒疫苗 | |
CN111172114B (zh) | 一种人源化肠癌前病变永生化上皮细胞系、构建方法及其应用 | |
CN108148866B (zh) | 一种hcbp6基因敲除细胞系及其构建方法 | |
Seeger et al. | Why are hepadnaviruses DNA and not RNA viruses? | |
CN114836418A (zh) | 用于敲降猪流行性腹泻病毒的CRISPR-Cas13d系统 | |
CN114934066B (zh) | 石骨症的基因编辑体系及其应用 | |
CN111925449B (zh) | 一种表达鸡vp2和鸡gal-1融合蛋白的重组cho细胞株及其构建方法和应用 | |
CN114107303A (zh) | sgRNA、质粒、IRF7功能缺失的细胞系及其构建方法和应用 | |
WO2022006745A1 (en) | Guide rna for hsv-1 gene editing and method thereof | |
CN111378621B (zh) | Eb病毒潜伏期膜蛋白1稳定转染的b淋巴瘤细胞株、其构建方法和应用 | |
KR101677231B1 (ko) | 무혈청배지(serum free medium)를 이용한 메갈로사이티바이러스의 효율적 제조방법 | |
CN114107304A (zh) | 一种表达α毒素蛋白和荧光标签蛋白的重组球虫载体及其检测方法 | |
AU785214B2 (en) | Mutant bank | |
CN112553200A (zh) | 一种用于敲除猪ugt2c1基因的打靶载体制备方法及其应用 | |
CN113564165B (zh) | 一种胞内编辑伪狂犬病毒关键基因的细胞株及其构建方法和应用 | |
CN115725724B (zh) | Pop基因或蛋白为靶点在筛选抑制小rna病毒科病毒复制药物中的应用 | |
CN114606201B (zh) | 重组杆状病毒基因组及其改造方法、应用以及表达外源蛋白的方法 | |
CN112342244B (zh) | 一种表达Furin蛋白的细胞株及其在禽传染性支气管炎病毒培养中的应用 | |
CN108220217B (zh) | 一种用于递送及表达外源抗原的减毒单核细胞增生李斯特氏菌及其应用 | |
CN115261390B (zh) | 一种过表达pigr蛋白的细胞系及其制备方法和应用 | |
CN108624592B (zh) | 针对DJ-1基因编辑的sgRNA筛选及其载体与应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |