CN111484993A - Long-chain non-coding RNA I L21-AS 1 and application thereof - Google Patents

Abstract

Translated fromChinese

本发明提供了一种长链非编码RNA IL21‑AS1、能够表达长链非编码RNA IL21‑AS1的载体及其用途；还给出了将Naive T CD4+T细胞诱导成TFH细胞的方法，以及非编码RNA IL21‑AS1在制备用于诱导分泌IL‑21的物质中的用途。

The present invention provides a long-chain non-coding RNA IL21-AS1, a vector capable of expressing the long-chain non-coding RNA IL21-AS1 and uses thereof; also provides a method for inducing Naive T CD4+ T cells into TFH cells, and Use of non-coding RNA IL21-AS1 in the preparation of a substance for inducing secretion of IL-21.

Description

Translated fromChinese

长链非编码RNA IL21-AS1及其用途Long non-coding RNA IL21-AS1 and its use

技术领域technical field

本发明涉及本发明涉及生物医学领域，具体涉及长链非编码RNA及其用途。The present invention relates to the field of biomedicine, in particular to long-chain non-coding RNAs and uses thereof.

背景技术Background technique

长链非编码RNA(Long non-coding RNA,LncRNA)是长度大于200个核苷酸的非编码RNA，一般长度在200-100000nt之间，不编码蛋白质的转录本。根据在基因组的定位和背景，LncRNA分为反义长非编码、内含子非编码、基因间、非翻译区lncRNA等。LncRNA在生命科学研究领域具有重要作用，涉及DNA 复制、转录调控、细胞内物质运输、染色体重塑、表观遗传、参与细胞分化、细胞凋亡等。LncRNA参与在机体生长发育、应激反应和疾病包括癌症、自身免疫性疾病、心血管疾病的发生发展等进程。Long non-coding RNA (LncRNA) is a non-coding RNA with a length of more than 200 nucleotides, generally between 200-100000 nt, and does not encode a protein transcript. According to their location and background in the genome, lncRNAs are divided into antisense long non-coding, intron non-coding, intergenic, and untranslated lncRNAs. LncRNA plays an important role in the field of life science research, involving DNA replication, transcriptional regulation, intracellular material transport, chromosome remodeling, epigenetics, participation in cell differentiation, apoptosis, etc. LncRNA is involved in the process of growth and development, stress response and diseases including cancer, autoimmune diseases, cardiovascular diseases and other processes.

表达载体是在克隆载体基本骨架的基础上增加表达元件如启动子、RBS、终止子等，使目的基因能够表达的载体。构建过程中运用限制性核酸内切酶切割出与目的基因相合的末端，多为黏性末端，也有平末端，采用DNA连接酶连接，导入生物体实现克隆表达。An expression vector is a vector that adds expression elements such as promoter, RBS, terminator, etc. on the basis of the basic skeleton of the cloning vector, so that the target gene can be expressed. In the construction process, restriction endonucleases are used to cut out the ends that are compatible with the target gene. Most of them are sticky ends, and there are also blunt ends. DNA ligase is used to connect them and introduce them into organisms to realize cloning and expression.

滤泡辅助性T细胞(Follicular helper Tcell,TFH)，是重要的辅助性 (Thelpercell,Th)细胞亚群，主要来源于扁桃体组织，定位淋巴滤泡。TFH 不同于Th1、Th2、Th17等CD4+T细胞，其表型为CXCR5+ICOS+PD1+，胞内转录因子BCL-6和TOX2高表达，合成大量分泌的IL-21。TFH的主要功能是辅助 B细胞活化和成熟：TFH高表达CXCR5与CXCL3结合迁移至滤泡区参与生发中心(germinal center,GC)的形成，TFH分泌的IL-21，诱导B细胞分化成熟为浆细胞,分泌IgM、IgG和IgA，亦可调节Ig的同型转换。TFH数量过多或者功能过强导致自身免疫病,反之导致免疫缺陷病。TFH参与自身免疫性疾病如SLE、RA、Graves病和肿瘤的发生发展。Naive CD4+T细胞体外诱导分化 TFH的条件已经成熟，为研究TFH的发育、分化和功能提供细胞模型。Follicular helper T cells (TFH) are an important subset of helper (Thelpercell, Th) cells, which are mainly derived from tonsil tissue and locate lymphoid follicles. TFH is different from Th1, Th2, Th17 and other CD4+ T cells, its phenotype is CXCR5+ICOS+PD1+, the intracellular transcription factors BCL-6 and TOX2 are highly expressed, and a large amount of secreted IL-21 is synthesized. The main function of TFH is to assist in the activation and maturation of B cells: TFH highly expresses CXCR5 and binds to CXCL3 and migrates to the follicular area to participate in the formation of germinal centers (GC); IL-21 secreted by TFH induces B cells to differentiate and mature into plasma Cells, which secrete IgM, IgG and IgA, can also regulate Ig isotype switching. Excessive quantity or function of TFH leads to autoimmune diseases, and vice versa. TFH is involved in the occurrence and development of autoimmune diseases such as SLE, RA, Graves disease and tumors. The conditions for Naive CD4+ T cells to induce differentiation of TFH in vitro are mature, providing a cellular model for studying the development, differentiation and function of TFH.

长非编码RNA是否参与TFH的发育和分化，及其具体功能尚未知晓，本发明解决了这些问题中的至少一部分。Whether long non-coding RNA is involved in the development and differentiation of TFH, and its specific function is not yet known, and the present invention solves at least some of these problems.

发明内容SUMMARY OF THE INVENTION

本发明提供了长链非编码RNA IL21-AS1的全长序列(SEQ ID No：1)。The present invention provides the full-length sequence (SEQ ID No: 1) of the long non-coding RNA IL21-AS1.

本发明还提供了表达载体，其能够表达长链非编码RNA IL21-AS1；前述表达载体可以是质粒或者病毒；优选地，前述质粒是pcDNA3.1表达质粒。The present invention also provides an expression vector capable of expressing long-chain non-coding RNA IL21-AS1; the aforementioned expression vector can be a plasmid or a virus; preferably, the aforementioned plasmid is a pcDNA3.1 expression plasmid.

本发明提供了一种Naive CD4+T细胞，其含有上文所述的表达载体。本发明还提供了构建前述Naive CD4+T细胞的方法，所述方法包括将前述表达载体转染至Naive CD4+T细胞，优选地，使用含有长链非编码RNA IL21-AS1的 pcDNA3.1-IL21-As1表达载体转染至Naive CD4+T细胞。The present invention provides a Naive CD4+ T cell, which contains the above-mentioned expression vector. The present invention also provides a method for constructing the aforementioned Naive CD4+ T cells, the method comprising transfecting the aforementioned expression vector into the Naive CD4+ T cells, preferably, using pcDNA3.1- containing long-chain non-coding RNA IL21-AS1 The IL21-As1 expression vector was transfected into Naive CD4+ T cells.

本发明提供了一种将Naive CD4+T细胞诱导成滤泡辅助性T细胞的方法，所述方法包括：将含有长链非编码RNA IL21-AS1的pcDNA3.1-IL21-As1表达载体电转染至Naive CD4+T细胞，然后所获得的经转染细胞继续培养3天。本发明的内容也包括了通过前述方法所获得的滤泡辅助性T细胞。The present invention provides a method for inducing Naive CD4+ T cells into follicular helper T cells, the method comprising: electroporating a pcDNA3.1-IL21-As1 expression vector containing long-chain non-coding RNA IL21-AS1 into Naive CD4+ T cells were transfected and the resulting transfected cells were cultured for 3 days. The content of the present invention also includes follicular helper T cells obtained by the aforementioned methods.

本发明提供了长链非编码RNA IL21-AS1以及含有长链非编码RNA IL21-AS1的表达载体在制备用于诱导Naive CD4+T细胞分化为滤泡辅助性T 细胞的物质的用途。进一步地，所获得的滤泡辅助性T细胞中IL-21表达增加，所述增加是指与自然生成的滤泡辅助性T细胞中的IL-21表达相比。The present invention provides the use of long-chain non-coding RNA IL21-AS1 and an expression vector containing long-chain non-coding RNA IL21-AS1 in preparing substances for inducing Naive CD4+ T cells to differentiate into follicular helper T cells. Further, IL-21 expression was increased in the obtained follicular helper T cells as compared to IL-21 expression in naturally occurring follicular helper T cells.

本发明提供了长链非编码RNA IL21-AS1以及能够表达长链非编码RNA IL21AS1的表达载体在制备用于诱导分泌IL-21的物质中的用途。The present invention provides the use of long-chain non-coding RNA IL21-AS1 and an expression vector capable of expressing long-chain non-coding RNA IL21AS1 in the preparation of substances for inducing secretion of IL-21.

本发明的优点在于：The advantages of the present invention are:

1、获得了长链非编码RNA IL21-As1的全长序列。1. Obtained the full-length sequence of long non-coding RNA IL21-As1.

2、构建了长链非编码RNA IL21-As1的表达载体。2. The expression vector of long-chain non-coding RNA IL21-As1 was constructed.

3、证明了长链非编码RNA IL21-As1的一部分功能和用途。3. Prove some functions and uses of long non-coding RNA IL21-As1.

4、验证了能够表达长链非编码RNA IL21-As1的表达载体促进Naive CD4+T 细胞向TFH细胞分化，并进一步促进TFH细胞分泌的IL-21增加。4. It was verified that the expression vector capable of expressing long-chain non-coding RNA IL21-As1 promoted the differentiation of Naive CD4+ T cells into TFH cells, and further promoted the increase of IL-21 secreted by TFH cells.

附图说明Description of drawings

附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。在附图中：The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification, and are used to explain the present invention together with the embodiments of the present invention, and do not constitute a limitation to the present invention. In the attached image:

图1示出了本发明的长链非编码RNA IL21-As1的序列；Fig. 1 shows the sequence of long-chain non-coding RNA IL21-As1 of the present invention;

图2示出了真核表达载体pcDNA3.1-IL21-AS1经双酶切后，1％琼脂糖电泳的结果验证，其中，泳道1:pcDNA3.1；泳道2:pcDNA3.1-IL21-AS1酶切产物泳道M:KB Ladder；Figure 2 shows the results of 1% agarose electrophoresis after double digestion of the eukaryotic expression vector pcDNA3.1-IL21-AS1, wherein, lane 1: pcDNA3.1; lane 2: pcDNA3.1-IL21-AS1 Enzyme digestion product lane M:KB Ladder;

图3为RT-qPCR验证TFH细胞中表达载体pcDNA3.1-IL21-AS1的过表达效果。Figure 3 shows the overexpression effect of the expression vector pcDNA3.1-IL21-AS1 in TFH cells verified by RT-qPCR.

图4示出了pcDNA3.1和pcDNA3.1-IL21-As1分别转染Naive CD4+T细胞，转染后培养3天，流式细胞仪分选结果以及TFH细胞的比例(*:P<001)。Figure 4 shows Naive CD4+ T cells transfected with pcDNA3.1 and pcDNA3.1-IL21-As1 respectively, cultured for 3 days after transfection, flow cytometry sorting results and the proportion of TFH cells (*:P<001 ).

图5示出了pcDNA3.1和pcDNA3.1-IL21-As1分别转染Naive CD4+T细胞，转染后培养3天，RT-qPCR验证IL-21的表达(*:P<001)。Figure 5 shows that pcDNA3.1 and pcDNA3.1-IL21-As1 were transfected into Naive CD4+ T cells, respectively, and the cells were cultured for 3 days after transfection, and the expression of IL-21 was verified by RT-qPCR (*: P<001).

图6示出了pcDNA3.1和pcDNA3.1-IL21-As1分别转染Naive CD4+T细胞，转染后培养3天，ELISA检测细胞培养基上清IL-21的表达。Figure 6 shows that pcDNA3.1 and pcDNA3.1-IL21-As1 were respectively transfected into Naive CD4+ T cells, and cultured for 3 days after transfection, and the expression of IL-21 in the supernatant of cell culture medium was detected by ELISA.

具体实施方式Detailed ways

以下结合附图对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

本发明的实施例中涉及到的实验试剂和仪器等如下：The experimental reagents and instruments involved in the embodiments of the present invention are as follows:

TRNzol Universal由TIANGEN提供，

RACE5’/3’Kit由Clontech 提供，Gel Extraction Kit由OMEGA提供，

Max DNA Polymerase 由Takara提供，pEASY-Blunt Cloning Kit由TransGen Biotech提供，Trans1-T1Phage ResistantChemically Competent Cell由TransGen Biotech 提供，Green Taq Mix由Vazyme提供；P3Primary Cell 4D-Nucleofector X Kit转染试剂和配套转染仪器，由LonZa提供；生长培养基为1640由Gibco 提供；FBS胎牛血清由Gibco提供；离心机生产厂家为美国Thermo公司、型号为FRESC017高速冷冻离心机；电泳仪生产厂家为美国BIO-RAD公司、型号为PowerPacTMand Mini-Sub cell GT；凝胶成像仪生产厂家为美国BIO-RAD 公司、型号为ChemiDocTMXRS+System；鼠抗人CD3抗体、鼠抗人CD28抗体、人-IL6、人-IL12和人-TGFβ由peprotech公司提供、鼠抗人CD4-FITC抗体、鼠抗人PD1-APC抗体、鼠抗人CXCR5-percp-cy5.5抗体；Naive CD4+T Cell Isolation Kit II由Miltenyi公司提供。Human-IL21ELISA检测试剂盒有 proteintech提供。TRNzol Universal is provided by TIANGEN,

RACE5'/3'Kit is provided by Clontech, Gel Extraction Kit is provided by OMEGA,

Max DNA Polymerase was provided by Takara, pEASY-Blunt Cloning Kit was provided by TransGen Biotech, Trans1-T1Phage ResistantChemically Competent Cell was provided by TransGen Biotech, Green Taq Mix was provided by Vazyme; P3Primary Cell 4D-Nucleofector X Kit transfection reagent and supporting transfection instrument , provided by LonZa; growth medium is 1640 provided by Gibco; FBS fetal bovine serum is provided by Gibco; centrifuge manufacturer is American Thermo Company, model FRESC017 high-speed refrigerated centrifuge; electrophoresis instrument manufacturer is American BIO-RAD Company, The model is PowerPacTM and Mini-Sub cell GT; the manufacturer of the gel imager is BIO-RAD, USA, the model is ChemiDocTMXRS+System; mouse anti-human CD3 antibody, mouse anti-human CD28 antibody, human-IL6, human-IL12 and human- TGFβ was provided by peprotech company, mouse anti-human CD4-FITC antibody, mouse anti-human PD1-APC antibody, mouse anti-human CXCR5-percp-cy5.5 antibody; Naive CD4+T Cell Isolation Kit II was provided by Miltenyi company. Human-IL21ELISA detection kit is provided by proteintech.

实施例1:RACE实验确定长链非编码RNA IL21-As1基因全长Embodiment 1: RACE experiment determines the full length of long-chain non-coding RNA IL21-As1 gene

1.1RNA提取(TIANGEN说明书操作，其内容通过引用并入本文)1.1 RNA extraction (TIANGEN manual operation, the content of which is incorporated herein by reference)

1.2RACE-Ready cDNA合成1.2 RACE-Ready cDNA synthesis

按照SMARTerRACE5’/3’Kit说明书进行RACE-Ready cDNA合成。RACE-Ready cDNA synthesis was performed according to the SMARTerRACE 5'/3'Kit instructions.

设计3’和5’RACE特异性扩增引物如下：Design 3' and 5' RACE specific amplification primers as follows:

Smart-3’RACE内引物：GCCTATGACCCTGGTGTCGTTT(SEQ ID No：2)Smart-3' RACE inner primer: GCCTATGACCCTGGTGTCGTTT (SEQ ID No: 2)

Smart-3’RACE外引物：TTTTGCTCTGTGAGTGTTGGGC(SEQ ID No：3)Smart-3' RACE outer primer: TTTTGCTCTGTGAGTGTTGGGC (SEQ ID No: 3)

Smart-5’RACE内引物：CACCACCGGTTGAGAACCACT(SEQ ID No：4)Smart-5'RACE inner primer: CACCACCGGTTGAGAACCACT (SEQ ID No: 4)

Smart-5’RACE外引物：ACCTCACGGAAGGCCAAAGACAAGA(SEQ ID No：5)Smart-5' RACE outer primer: ACCTCACGGAAGGCCAAAGACAAGA (SEQ ID No: 5)

1.3PCR扩增，产物回收和连接反应(常规操作，参照分子克隆)1.3 PCR amplification, product recovery and ligation reaction (routine operation, refer to molecular cloning)

1.4转化，PCR菌落和测序验证(常规操作，参照分子克隆)1.4 Transformation, PCR colony and sequencing verification (routine operation, reference molecular cloning)

长链非编码RNA IL21-As1进行DNA测序验证(由上海吉凯生物科技公司提供)，如图1所示。The long non-coding RNA IL21-As1 was verified by DNA sequencing (provided by Shanghai Jikai Biotechnology Co., Ltd.), as shown in Figure 1.

实施例2：长链非编码RNA IL21-AS1表达质粒载体的构建。Example 2: Construction of long-chain non-coding RNA IL21-AS1 expression plasmid vector.

先合成长链非编码RNA IL21-AS1(图1所示)基因(由南京金斯瑞生物科技公司提供)，接着将其连接到BamH I和XhoI酶切pcDNA3.1过得载体质粒，得到pcDNA3.1-IL21-AS1表达质粒。具体构建过程如下：First synthesize the long-chain non-coding RNA IL21-AS1 (shown in Figure 1) gene (provided by Nanjing GenScript Biotechnology Co., Ltd.), and then connect it to BamH I and XhoI to digest pcDNA3.1 to obtain a vector plasmid to obtain pcDNA3 .1-IL21-AS1 expression plasmid. The specific construction process is as follows:

(1)pcDNA3.1双酶切:取4μg pcDNA3.1载体到EP管，用BamH I和 XhoI双酶切酶切，体系如下(1) Double digestion of pcDNA3.1: Take 4 μg of pcDNA3.1 vector into EP tube, and cut with BamH I and XhoI double digestion, the system is as follows

加无酶水补充到总体积为50μL,37℃酶切反应20分钟(min)。Add enzyme-free water to make up to a total volume of 50 μL, and digest at 37°C for 20 minutes (min).

(2)长链非编码RNA IL21-AS1基因与(1)连接：体系如下(2) The long non-coding RNA IL21-AS1 gene is linked to (1): the system is as follows

加无酶水补充到总体积为20μL，37℃连接30分钟(min)，冰浴5min。Add enzyme-free water to make up to a total volume of 20 μL, connect at 37° C. for 30 minutes (min), and ice bath for 5 minutes.

(3)将步骤(2)所得转化至感受态扩增和抽提质粒：冰上解冻100uL DH5 ɑ感受态细菌，再加入4ul pcDNA3.1-IL21-AS1，混匀，再冰浴30min后，立刻42℃水浴45秒(s)，立刻再冰浴2min，转移到1ml LB培养基，37℃、225r/min 振荡培养1h，将得到的细菌涂布到LB培养板(Amp浓度100μg/mL)，37℃培养箱中培养12h，挑取单菌落放入LB培养基(Amp浓度100μg/mL)，37℃、 300r/min震荡培养15h，抽提质粒pcDNA3.1-IL21-AS1，即可。(4)对步骤(3)所得质粒进行酶切和测序鉴定：酶切体系如下，(3) Transform the obtained in step (2) into competent amplification and extraction plasmid: thaw 100uL DH5ɑ competent bacteria on ice, add 4ul pcDNA3.1-IL21-AS1, mix well, and then ice bath for 30min, Immediately take a 42°C water bath for 45 seconds (s), then immediately ice bath for 2 minutes, transfer to 1ml LB medium, shake at 37°C, 225r/min for 1h, and spread the obtained bacteria onto LB plates (Amp concentration 100μg/mL) , cultured in a 37°C incubator for 12h, pick a single colony into LB medium (Amp concentration 100μg/mL), 37°C, 300r/min shaking culture for 15h, and extract plasmid pcDNA3.1-IL21-AS1. (4) The plasmid obtained in step (3) is subjected to enzyme digestion and sequencing identification: the enzyme digestion system is as follows,

加无酶水补充到总体积为50μ,37℃酶切反应20min。Add enzyme-free water to make up the total volume to 50μ, and react at 37℃ for 20min.

5ul DNA Marker和5μL酶切产物进行1.5％琼脂糖凝胶电泳检测,如图2 所示。5ul DNA Marker and 5μL digestion products were detected by 1.5% agarose gel electrophoresis, as shown in Figure 2.

实施例3：使用含有长链非编码RNA IL21-AS1表达质粒载体转染Naive CD4+T细胞，进行培养并对表达结果进行验证Example 3: Naive CD4+ T cells were transfected with the expression plasmid vector containing long-chain non-coding RNA IL21-AS1, cultured and the expression results were verified

3.1全血中单个核细胞(PBMC)的分选，要求全过程无菌操作，主要步骤如下：3.1 The sorting of mononuclear cells (PBMC) in whole blood requires aseptic operation in the whole process. The main steps are as follows:

(1)采集肝素抗凝血100ml，Ficoll分选PBMC：室温将全血用PBS稀释稀释三倍，备用；(1) Collect 100ml of heparin for anticoagulation, and sort PBMCs with Ficoll: dilute whole blood three times with PBS at room temperature for use;

(2)50ml离心管，室温加15ml Ficoll，备用；(2) 50ml centrifuge tube, add 15ml Ficoll at room temperature, spare;

(3)将步骤(2)中的每个离心管加入35ml步骤(1)所得稀释后全血，要求操作动作温柔，保证液面分层明显，备用；(3) adding 35ml of the diluted whole blood obtained in step (1) to each centrifuge tube in step (2), requiring gentle operation, and ensuring that the liquid level is clearly stratified, for subsequent use;

(4)水平离心机调整到离心力为750g，温度20℃，加速度为8，减速度为0，将步骤(3)的样本配平后离心20min，备用；(4) The horizontal centrifuge is adjusted to a centrifugal force of 750 g, a temperature of 20° C., an acceleration of 8, and a deceleration of 0, and the sample in step (3) is balanced and centrifuged for 20 min, for use;

(5)巴氏吸管吸取步骤(4)中的云雾状富集有PBMC细胞液体到50ml细心管，每管存放15ml，剩余体积补加35mlPBS，混匀，备用；(5) suck the cloudy enriched PBMC cell liquid in step (4) into a 50ml thin tube, store 15ml in each tube, add 35ml PBS to the remaining volume, mix well, and set aside;

(6)水平离心机调整到离心力为350g，温度4℃，加速度为8，减速度为9，将(5)的样本配平后离心10min，备用；(6) The horizontal centrifuge is adjusted to a centrifugal force of 350 g, a temperature of 4 °C, an acceleration of 8, and a deceleration of 9, and the sample in (5) is balanced and centrifuged for 10 min for use;

(7)弃掉(6)上清液，用15ml Buffer(PBS+0.5％BSA)重悬细胞沉淀，转移到15ml离心管，备用；(7) Discard the supernatant of (6), resuspend the cell pellet with 15ml Buffer (PBS+0.5%BSA), and transfer it to a 15ml centrifuge tube for later use;

(8)水平离心机调整到离心力为300g，温度4℃，加速度为8，减速度为 9，将(7)的样本配平后离心10min，备用；(8) Adjust the horizontal centrifuge to a centrifugal force of 300g, a temperature of 4°C, an acceleration of 8, and a deceleration of 9, and the sample in (7) is balanced and centrifuged for 10min for use;

(9)弃掉(8)上清液，用5mlBuffer(PBS+0.5％BSA)重悬细胞沉淀，取适量细胞样本，0.45％的台盼蓝计数活细胞的数量，备用。(9) Discard the supernatant of (8), resuspend the cell pellet with 5ml Buffer (PBS+0.5%BSA), take an appropriate amount of cell sample, count the number of viable cells with 0.45% trypan blue, and set aside.

3.2磁珠分选Naive CD4+T细胞3.2 Magnetic bead sorting of Naive CD4+ T cells

严格按照Naive CD4+T Cell Isolation Kit II说明书操作(Miltenyi 公司)，该说明书通过引用并入本文，全过程无菌操作，得到Naive CD4+T 细胞，备用。Strictly follow the instructions of Naive CD4+T Cell Isolation Kit II (Miltenyi Company), which is incorporated herein by reference, and the whole process is aseptically operated to obtain Naive CD4+T cells for future use.

3.3使用电转染的方式转染Naive CD4+T细胞(4D-Nucleofector^TM X Unit，Lonza)，步骤如下：3.3 Transfect Naive CD4+ T cells (4D-Nucleofector^TM X Unit, Lonza) by electrotransfection, the steps are as follows:

(1)每个反应需要Naive CD4+T细胞量大于等于5*10⁶，细胞计数确认后备用；(1) Each reaction requires the amount of Naive CD4+ T cells greater than or equal to 5*10⁶ , and the cell count is confirmed for use;

(2)取pcDNA3.1-IL21-AS1和pcDNA3.1质粒4ug，放入EP管备用；(2) Take 4ug of pcDNA3.1-IL21-AS1 and pcDNA3.1 plasmids and put them into EP tubes for later use;

(3)转染反应液200ul加入1*10⁷Naive CD4+T细胞，混匀，取100ul 分别加入到(2)，混匀，备用；(3) Add 1*10⁷ Naive CD4+ T cells to 200ul of the transfection reaction solution, mix well, add 100ul to (2), mix well, and set aside;

(4)将(3)的细胞悬液转移到转染杯中，确保不能有气泡，并且细胞悬液覆盖电转杯底部，备用；(4) Transfer the cell suspension of (3) into the transfection cup, make sure that there are no air bubbles, and the cell suspension covers the bottom of the electroporation cup for use;

(5)调试电转仪器4D-Nucleofector^TM Core Unit参数：human UnstimulatedTcell-high functionality，备用；(5) Debugging the 4D-Nucleofector^TM Core Unit parameters: human UnstimulatedTcell-high functionality, spare;

(6)将(4)的反应杯正确对号放入到(5)的程序，进行电转染。(6) Put the cuvette of (4) into the procedure of (5) correctly, and perform electrotransfection.

3.4电转后的细胞培养与RNA提取，如下：3.4 Cell culture and RNA extraction after electroporation, as follows:

(1)将电转完毕的细胞，使用微量吸管(Lonza试剂盒提供)，吸取细胞悬液到预先备好1640培养基六孔板中，37℃，5％CO₂的培养箱培养6h，使细胞恢复状态。(1) Use a micropipette (provided by Lonza kit) to transfer the cells after electroporation, pipette the cell suspension into a six-well plate of 1640 medium prepared in advance, and incubate for 6 hours at 37°C in a 5% CO₂ incubator to make the cells restore state.

(2)收集(1)的细胞到15ml离心管，离心力为300g，样本配平后离心 10min，留取细胞，使用6ml的1640培养重悬(1640+10％FBS)，备用。(2) Collect the cells of (1) into a 15ml centrifuge tube with a centrifugal force of 300g, centrifuge the sample for 10min after balancing, and collect the cells, and use 6ml of 1640 culture to resuspend (1640+10% FBS) for use.

(3)将(2)的细胞悬液，平均分为三份，培养到预先包被有鼠抗人CD3 抗体的(2ug/ml)12孔板中，37℃，5％CO₂的培养箱培养，48h和96h，备用。(3) Divide the cell suspension of (2) into three equal parts, and culture them in a 12-well plate (2ug/ml) pre-coated with mouse anti-human CD3 antibody in an incubator at 37°C, 5% CO₂ Culture, 48h and 96h, set aside.

(4)收集培养48h的细胞，利用Trizol方法抽提总RNA。(4) The cells cultured for 48 hours were collected, and total RNA was extracted by Trizol method.

3.5转染结果的检测3.5 Detection of transfection results

3.5.1经转染后的细胞培养三天，进行表达检测：3.5.1 The transfected cells were cultured for three days for expression detection:

(1)RNA为模板合成cDNA(takara公司的逆转录试剂盒)；(1) RNA is a template to synthesize cDNA (reverse transcription kit from takara company);

(2)RT-qPCR(罗氏)检测长链非编码RNAIL21-AS1的过表达效果，RT-PCR 检测长链非编码RNAIL21-AS1，图3所示：实验组pcDNA3.1-IL21-AS1的长链非编码RNAIL21-AS1表达高出pcDNA3.1对照组100倍，说明pcDNA3.1-IL21-AS1转染成功。(2) RT-qPCR (Roche) to detect the overexpression effect of long-chain non-coding RNAIL21-AS1, RT-PCR to detect long-chain non-coding RNAIL21-AS1, as shown in Figure 3: the length of pcDNA3.1-IL21-AS1 in the experimental group The expression of strand non-coding RNA IL21-AS1 was 100 times higher than that of the pcDNA3.1 control group, indicating that pcDNA3.1-IL21-AS1 was successfully transfected.

3.5.2收集经转染后培养3天的细胞，流式细胞术检测TFH细胞的比例，并分别通过RT-qPCR和ELISA检测IL-21的表达，步骤如下：3.5.2 Collect the cells cultured for 3 days after transfection, detect the proportion of TFH cells by flow cytometry, and detect the expression of IL-21 by RT-qPCR and ELISA respectively. The steps are as follows:

流式细胞术检测TFH细胞的比例：分别收取实验组pcDNA3.1-IL21-As1、对照组pcDNA3.1、阴性对照和补偿对照CD4、PD1、CXCR5，每管细胞数量1X10⁶，每个标记管中加入标记荧光单克隆抗体。采用流式细胞仪BD FACScantoII检测TFH细胞的比例(上述步骤中所使用的抗体和仪器均来自BD Bioscience)，流式细胞术分析结果见图4所示：转染三天后，实验组pcDNA3.1-IL21-AS1的 PD1+CXCR5+双阳性的细胞(分化的TFH细胞)占比高出pcDNA3.1对照组20％，说明pcDNA3.1-IL21-AS1转染促进TFH分化。Flow cytometry to detect the proportion of TFH cells: collect the experimental group pcDNA3.1-IL21-As1, the control group pcDNA3.1, the negative control and the compensation control CD4, PD1, CXCR5, the number of cells in each tube is 1X10⁶ , each labeled tube Add labeled fluorescent monoclonal antibody. Flow cytometry BD FACScantoII was used to detect the proportion of TFH cells (both antibodies and instruments used in the above steps were from BD Bioscience). The flow cytometry analysis results are shown in Figure 4: three days after transfection, the experimental group pcDNA3.1 The proportion of PD1+CXCR5+ double positive cells (differentiated TFH cells) of -IL21-AS1 was 20% higher than that of the pcDNA3.1 control group, indicating that pcDNA3.1-IL21-AS1 transfection promoted TFH differentiation.

收集实验组pcDNA3.1-IL21-As1和对照组pcDNA3.1经转染后3天的细胞，取细胞5*10⁵，检测RT-qPCR检测IL21的表达，实验结果见图5所示：转染三天后，实验组pcDNA3.1-IL21-AS1的IL21 mRNA的表达水平是pcDNA3.1对照组2.2倍，说明pcDNA3.1-IL21-AS1转染促进IL21 mRNA的表达。其中，RT-qPCR 使用如下引物：Collect the cells of the experimental group pcDNA3.1-IL21-As1 and the control group pcDNA3.1 3 days after transfection, take 5*10⁵ cells, and detect the expression of IL21 by RT-qPCR. The experimental results are shown in Figure 5: Transfection Three days after transfection, the expression level of IL21 mRNA in the experimental group pcDNA3.1-IL21-AS1 was 2.2 times that of the pcDNA3.1 control group, indicating that pcDNA3.1-IL21-AS1 transfection promoted the expression of IL21 mRNA. Among them, RT-qPCR uses the following primers:

IL21：IL21:

F：TGGTCCCTGAATTTCTGCCAG(SEQ ID No：6)F: TGGTCCCTGAATTTCTGCCAG (SEQ ID No: 6)

R：TTAGTTGGGCCTTCTGAAAGCA(SEQ ID No：7)R: TTAGTTGGGCCTTCTGAAAGCA (SEQ ID No: 7)

β-actinβ-actin

F：GAGCTACGAGCTGCCTGACG(SEQ ID No：8)F: GAGCTACGAGCTGCCTGACG (SEQ ID No: 8)

R：GTAGTTTCGTGGATGCCACAG(SEQ ID No：9)R: GTAGTTTCGTGGATGCCAAG (SEQ ID No: 9)

LncRNA-IL21AS1：LncRNA-IL21AS1:

F：AACTACATGCCAGGCCTCTT(SEQ ID No：10)F: AACTACATGCCAGGCCTCTT (SEQ ID No: 10)

R：AGGTCAAGATCGCCACATGA(SEQ ID No：11)R: AGGTCAAGATCGCCACATGA (SEQ ID No: 11)

收集实验组pcDNA3.1-IL21-As1和对照组pcDNA3.1经转染后3天的细胞培养液上清，ELISA检测IL-21的表达，实验结果见图6所示：转染三天后，实验组pcDNA3.1-IL21-AS1的IL21 mRNA的表达水平是pcDNA3.1对照组1.5 倍，说明pcDNA3.1-IL21-AS1转染促进IL21蛋白的表达。The cell culture supernatants of the experimental group pcDNA3.1-IL21-As1 and the control group pcDNA3.1 were collected 3 days after transfection, and the expression of IL-21 was detected by ELISA. The experimental results are shown in Figure 6: three days after transfection, The expression level of IL21 mRNA in the experimental group pcDNA3.1-IL21-AS1 was 1.5 times higher than that in the pcDNA3.1 control group, indicating that pcDNA3.1-IL21-AS1 transfection promoted the expression of IL21 protein.

根据以上实验结果，本领域技术人员可以判定长链非编码RNA IL21-AS1 的过表达促进了Naive CD4+T细胞向TFH细胞的分化，并提高了IL-21的表达；TFH细胞在多种自身免疫疾病的发生中具有重要作用，因此可以推断长链非编码RNA IL21-AS1以及能够长链非编码RNA IL21-AS1的表达载体可以用于制备用于促进Naive CD4+T细胞向TFH细胞分化的物质，以及用于制备促进 TFH细胞中IL-21表达的物质。According to the above experimental results, those skilled in the art can determine that the overexpression of long-chain non-coding RNA IL21-AS1 promotes the differentiation of Naive CD4+ T cells into TFH cells, and increases the expression of IL-21; It plays an important role in the occurrence of immune diseases, so it can be inferred that the long-chain non-coding RNA IL21-AS1 and the expression vector capable of long-chain non-coding RNA IL21-AS1 can be used to prepare the expression vector for promoting the differentiation of Naive CD4+ T cells into TFH cells. Substances, and for the preparation of substances that promote the expression of IL-21 in TFH cells.

显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. Thus, provided that these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

序列表sequence listing

<110> 中南大学湘雅二医院<110> The Second Xiangya Hospital of Central South University

<120> 长链非编码RNA IL21-AS1及其用途<120> Long non-coding RNA IL21-AS1 and its use

<130> CP20122<130> CP20122

<141> 2020-04-30<141> 2020-04-30

<160> 11<160> 11

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 4082<211> 4082

<212> RNA<212> RNA

<213> 人(human species)<213> Human species

<400> 1<400> 1

gacccaagct ggctagcgtt taaacttaag cttggtaccg agctcggatc cggagaatcg 60gacccaagct ggctagcgtt taaacttaag cttggtaccg agctcggatc cggagaatcg 60

cttgaacctg ggaggcagag gttgcagtga gctgagatcg caccactgca ctccagccta 120cttgaacctg ggaggcagag gttgcagtga gctgagatcg caccactgca ctccagccta 120

gcaacatatc gagactccgt ctcaaaaaaa aaaaaaaaaa aaaaaaaaaa ggattacctt 180gcaacatatc gagactccgt ctcaaaaaaa aaaaaaaaaa aaaaaaaaaa ggattacctt 180

tgtcctacac acaaactagg tttatatcag ggtttctcaa cggtgatacc actgacattt 240tgtcctacac acaaactagg tttatatcag ggtttctcaa cggtgatacc actgacattt 240

ggggctagat aattctttgt tgtcagggct gtcttgtgca ttctagaatg tttagtacct 300ggggctagat aattctttgt tgtcagggct gtcttgtgca ttctagaatg tttagtacct 300

ggcttctacc tactagatgc cagtagcacc cccattctac ctccaagtgt gacaaccaga 360ggcttctacc tactagatgc cagtagcacc cccattctac ctccaagtgt gacaaccaga 360

aatttttcca gccaatgcca aatgttttct ggggcacaaa aatcaccacc ggttgagaac 420aatttttcca gccaatgcca aatgttttct ggggcacaaa aatcaccacc ggttgagaac 420

cactggttat atgattagac ggaaagaaaa agagaaaaag aaagatgcct ggctaggcca 480cactggttat atgattagac ggaaagaaaa agagaaaaag aaagatgcct ggctaggcca 480

attagagatc ttagataatt actatatgca tgcaacatta gatagtttta ttttcattaa 540attagagatc ttagataatt actatatgca tgcaacatta gatagtttta ttttcattaa 540

acttttaaat cttttaaaat ctcttacaga ccctactttg aagctagctt gatataatgc 600acttttaaat cttttaaaat ctcttacaga ccctactttg aagctagctt gatataatgc 600

taatgtgacc ttgaacgatt tctttcttcc tgagtttgcc tactagtata gcttaaagat 660taatgtgacc ttgaacgatt tctttcttcc tgagtttgcc tactagtata gcttaaagat 660

ccaagttaca ctattactca cttatatcat agccagcatt gcttgatcag attatcagat 720ccaagttaca ctattactca cttatatcat agccagcatt gcttgatcag attatcagat 720

agatacagta tcacggagac aaaatccttt cattatgatg taagataaaa aatatctttc 780agatacagta tcacggagac aaaatccttt cattatgatg taagataaaa aatatctttc 780

attaacaaca cacgtaaagg aagtacccac tggaccaact caggttagaa gggtaccaga 840attaacaaca cacgtaaagg aagtacccac tggaccaact caggttagaa gggtaccaga 840

ccagaagatc agatcgtaga agttactgga gaaaaagatc agcggacagc actcttgaac 900ccagaagatc agatcgtaga agttactgga gaaaaagatc agcggacagc actcttgaac 900

tacatgccag gcctcttctt cctggaactc taatggattt aaatggctta agagacggga 960tacatgccag gcctcttctt cctggaactc taatggattt aaatggctta agagacggga 960

tgaattgttg aaaatggttc tttcaaaaga cctcacggaa ggccaaagac aagactcact 1020tgaattgttg aaaatggttc tttcaaaaga cctcacggaa ggccaaagac aagactcact 1020

gaggccaagc tgatcaacaa tatctataag ttgacgcatt ctaatcatgt ggcgatcttg 1080gaggccaagc tgatcaacaa tatctataag ttgacgcatt ctaatcatgt ggcgatcttg 1080

accttgggag cttgatttgt ggaccagtgt ccccaagaag atgaccatca gacagatgac 1140accttgggag cttgatttgt ggaccagtgt ccccaagaag atgaccatca gacagatgac 1140

aatcctctcc atgttgccag gactggatct cataagtacc aacagtagag ctagaccttg 1200aatcctctcc atgttgccag gactggatct cataagtacc aacagtagag ctagaccttg 1200

gtctcgtttt cacttcagct tgactgagaa caggtatacg tgtgcatgtg ctaatgtgtg 1260gtctcgtttt cacttcagct tgactgagaa caggtatacg tgtgcatgtg ctaatgtgtg 1260

ggggcaggga ttgatggagt caacgaaatg tgccccatct gcatctttga caggaaaaag 1320ggggcaggga ttgatggagt caacgaaatg tgccccatct gcatctttga caggaaaaag 1320

agagaatggg tgaattccaa ttttcagcat gaatcaagaa tgtcaccgat gaagtttttg 1380agagaatggg tgaattccaa ttttcagcat gaatcaagaa tgtcaccgat gaagttttttg 1380

aatgctgtgc tcctaatctc attttcctcc tccagagact tcggtgaagg atgatgcaaa 1440aatgctgtgc tcctaatctc attttcctcc tccagagact tcggtgaagg atgatgcaaa 1440

ctgtgtttct tcagcatgtg agcaccatca gccatgagtg tggcagctcc agaatgtcta 1500ctgtgtttct tcagcatgtg agcaccatca gccatgagtg tggcagctcc agaatgtcta 1500

taccagagtg gctgagagtc agcaacttgg ctggaagcta cacttgaata gcaaacaaac 1560taccagagtg gctgagagtc agcaacttgg ctggaagcta cacttgaata gcaaacaaac 1560

cacttttact tagtttccat gtctatttgg taccttggag tacagtgatg agttttacta 1620cacttttact tagtttccat gtctatttgg taccttggag tacagtgatg agttttacta 1620

gatgtggact ttccaggaat atagccaagg tcgtcaccat gaaagtatga aactttagtc 1680gatgtggact ttccaggaat atagccaagg tcgtcaccat gaaagtatga aactttagtc 1680

atttacccaa ggggattcat ggacatatat taagaatttt ctttaatgga ctgtgtttat 1740atttacccaa ggggattcat ggacatatat taagaatttt ctttaatgga ctgtgtttat 1740

agaacacctg gtcaggacgg aaatgaggcc agggaactaa aggggtagaa gtctgaagag 1800agaacacctg gtcaggacgg aaatgaggcc agggaactaa aggggtagaa gtctgaagag 1800

gaatggtttt gcatgggaaa actgtgccag gtctaaaact gggctgaaca ctatttgtcc 1860gaatggtttt gcatgggaaa actgtgccag gtctaaaact gggctgaaca ctatttgtcc 1860

ccaactctct ggcccggtcc tcatagttct ttctgtgatc cctgcgtggt gaatactcct 1920ccaactctct ggcccggtcc tcatagttct ttctgtgatc cctgcgtggt gaatactcct 1920

tttctgcttt cactggcttt ggcccctcat ggatagaatt agatgttctg catggcaaac 1980tttctgcttt cactggcttt ggcccctcat ggatagaatt agatgttctg catggcaaac 1980

tgtgggaaga gaggcctcca gtgtttagag tgatattatc atgtgtacca ctactattat 2040tgtgggaaga gaggcctcca gtgtttagag tgatattatc atgtgtacca ctactattat 2040

acatactaaa ggtattcaga caggtggctt gtctctgggc tttatataga tctctgtcaa 2100acatactaaa ggtattcaga caggtggctt gtctctgggc tttatataga tctctgtcaa 2100

gctagaagaa aaatgtcact aaaataattc aagacaattt ttgtactttc caacgatgtt 2160gctagaagaa aaatgtcact aaaataattc aagacaattt ttgtactttc caacgatgtt 2160

caggtaacag ctgaaaatat tctcacttat ttgacttgag gaagaaaatt cgaacgagga 2220caggtaacag ctgaaaatat tctcacttat ttgacttgag gaagaaaatt cgaacgagga 2220

aaatcatcaa ggatttgcta aagtcccttc tgtaaaatct tccttaagga agtttaaaca 2280aaatcatcaa ggatttgcta aagtcccttc tgtaaaatct tccttaagga agtttaaaca 2280

ctcctattct ctcttctctc attcttttga actcactgca tgtattgata tcactgactt 2340ctcctattct ctcttctctc attcttttga actcactgca tgtattgata tcactgactt 2340

ggtttgtttt ctagaatata tgtaaaagta agagtgtgta tatataaccc attatgtaca 2400ggtttgtttt ctagaatata tgtaaaagta agagtgtgta tatataaccc attatgtaca 2400

taacaagaac agttccttcc aatattcaaa tttcatgact ctagatcact actgtgcatt 2460taacaagaac agttccttcc aatattcaaa tttcatgact ctagatcact actgtgcatt 2460

ctaagaaggt cagggactca tggagaccaa agggtcaatc ctggtcattg ttgtcttacg 2520ctaagaaggt cagggactca tggagaccaa agggtcaatc ctggtcattg ttgtcttacg 2520

agagaaaaac aagagccttc tcgggccccg tgttctctag ctcttgatgt ggcctcttcg 2580agagaaaaac aagagccttc tcgggccccg tgttctctag ctcttgatgt ggcctcttcg 2580

gtatcactgg cagccccaca gtctggtggt cagtgtgtcg cagcagaggg tcattcaccg 2640gtatcactgg cagccccaca gtctggtggt cagtgtgtcg cagcagaggg tcattcaccg 2640

atgctgctta tccacagcac cttttctgtg gggaatgggg gcctctattc tgctagtaga 2700atgctgctta tccacagcac cttttctgtg gggaatgggg gcctctattc tgctagtaga 2700

ctcaggtgtg tcactctgtg atactcttaa gactgggttc attgaggaca ggtctctttg 2760ctcaggtgtg tcactctgtg atactcttaa gactgggttc attgaggaca ggtctctttg 2760

ctaacattca cacaaagtca ttgtttggaa gcacagtcct gcttctgaaa tgctaggtct 2820ctaacattca cacaaagtca ttgtttggaa gcacagtcct gcttctgaaa tgctaggtct 2820

ggaggagttc cagcagcagc cacatgggcc ctctcctcag atcttggctc caacatggga 2880ggaggagttc cagcagcagc cacatgggcc ctctcctcag atcttggctc caacatggga 2880

tactcagcag tgaggctaga gatcagatgt ccttcattcc ttgaccacta agcctagagg 2940tactcagcag tgaggctaga gatcagatgt ccttcattcc ttgaccacta agcctagagg 2940

atggagagaa agaagtggaa catctgcact cttcaacctg agtccactcc aactccatcc 3000atggagagaa agaagtggaa catctgcact cttcaacctg agtccactcc aactccatcc 3000

cacaagggaa ctgcctccat tgctgatgtt ggattaaaca gaacagcctg tgcagttagt 3060cacaagggaa ctgcctccat tgctgatgtt ggattaaaca gaacagcctg tgcagttagt 3060

tcatggattc cataaaggga ctggacctta atgaagacgt tcctgacatg ggtttgggca 3120tcatggattc cataaaggga ctggacctta atgaagacgt tcctgacatg ggtttgggca 3120

agaagatatt ataattttgc aaatgaagaa actgagatac agaaatgaca tccagcaccc 3180agaagatatt ataattttgc aaatgaagaa actgagatac agaaatgaca tccagcaccc 3180

tcagtgtcac acagagagta aatggactgg cctgaaccag gctgcctcct ggaactggtt 3240tcagtgtcac acagagagta aatggactgg cctgaaccag gctgcctcct ggaactggtt 3240

tctttggttg gttgaatcca tggatggaga actcgtggat acaagggctg actataatga 3300tctttggttg gttgaatcca tggatggaga actcgtggat acaagggctg actataatga 3300

gaagatatga aagctgaaaa gacaaacatt actacatatt tacatatgtc caaagaagaa 3360gaagatatga aagctgaaaa gacaaacatt actacatatt tacatatgtc caaagaagaa 3360

ggttcatctc cacgtggacc tccctataga gcttcttgaa tggcctcaca acatggcggc 3420ggttcatctc cacgtggacc tccctataga gcttcttgaa tggcctcaca acatggcggc 3420

tggcttccca cagagtgagc aatccaagat ttccttgatt cagagaaaga agataaaaac 3480tggcttccca cagagtgagc aatccaagat ttccttgatt cagagaaaga agataaaaac 3480

acatacaaag agacggcacc agctcctttc ccagttgtga ccagattgca cgatacccaa 3540acatacaaag agacggcacc agctcctttc ccagttgtga ccagattgca cgatacccaa 3540

gccatggctc caaagagtgc gtgtactgaa gcctggccat tcatgacaga ctcatttcca 3600gccatggctc caaagagtgc gtgtactgaa gcctggccat tcatgacaga ctcatttcca 3600

cacctgagtc cggttttgct ctgtgagtgt tgggctttct ccaagggaat ggttggagct 3660cacctgagtc cggttttgct ctgtgagtgt tgggctttct ccaagggaat ggttggagct 3660

gttgcgagcg aacttcctgg ctgccacaga gttgattgga ttccttggaa atgcacactc 3720gttgcgagcg aacttcctgg ctgccacaga gttgattgga ttccttggaa atgcacactc 3720

acaggctgct gctctttgaa gctgtctcct tcactggcca tgattctcct gtctcattag 3780acaggctgct gctctttgaa gctgtctcct tcactggcca tgattctcct gtctcattag 3780

ctgttgccta tgaccctggt gtcgttttct ccttttactc atcaacaccc aaatccacag 3840ctgttgccta tgaccctggt gtcgttttct ccttttactc atcaacaccc aaatccacag 3840

ggttgtgatg gattagcagc ctttcttatt agcaagaagc cctgtggcct caagaggcaa 3900ggttgtgatg gattagcagc ctttcttatt agcaagaagc cctgtggcct caagaggcaa 3900

aatgaatcgt ggttagaaat ctccatctcc tccctccctt ggtataatgc catcatttaa 3960aatgaatcgt ggttagaaat ctccatctcc tccctccctt ggtataatgc catcatttaa 3960

ctatctgaaa atagagaaga ctccgttcca attgtaaagc cctaaggaga aaaactgagt 4020ctatctgaaa atagagaaga ctccgttcca attgtaaagc cctaaggaga aaaactgagt 4020

ttgaaacttt ccttaattat gctgatattt aacaataaac tttgtagtat gaaaatctca 4080ttgaaacttt ccttaattat gctgatattt aacaataaac tttgtagtat gaaaatctca 4080

aa 4082aa 4082

<210> 2<210> 2

<211> 22<211> 22

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 2<400> 2

gcctatgacc ctggtgtcgt tt 22gcctatgacc ctggtgtcgt tt 22

<210> 3<210> 3

<211> 22<211> 22

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 3<400> 3

ttttgctctg tgagtgttgg gc 22ttttgctctg tgagtgttgg gc 22

<210> 4<210> 4

<211> 21<211> 21

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 4<400> 4

caccaccggt tgagaaccac t 21caccaccggt tgagaaccac t 21

<210> 5<210> 5

<211> 25<211> 25

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 5<400> 5

acctcacgga aggccaaaga caaga 25acctcacgga aggccaaaga caaga 25

<210> 6<210> 6

<211> 21<211> 21

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 6<400> 6

tggtccctga atttctgcca g 21tggtccctga atttctgcca g 21

<210> 7<210> 7

<211> 22<211> 22

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 7<400> 7

ttagttgggc cttctgaaag ca 22ttagttgggc cttctgaaag ca 22

<210> 8<210> 8

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 8<400> 8

gagctacgag ctgcctgacg 20gagctacgag ctgcctgacg 20

<210> 9<210> 9

<211> 21<211> 21

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 9<400> 9

gtagtttcgt ggatgccaca g 21gtagtttcgt ggatgccaca g 21

<210> 10<210> 10

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 10<400> 10

aactacatgc caggcctctt 20aactacatgc caggcctctt 20

<210> 11<210> 11

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 11<400> 11

aggtcaagat cgccacatga 20aggtcaagat cgccacatga 20

Claims (10)

Translated fromChinese

1.一种长链非编码RNA IL21-AS1，其特征在于，其核苷酸序列如SEQ ID No：1所示。1. A long-chain non-coding RNA IL21-AS1, characterized in that its nucleotide sequence is shown in SEQ ID No: 1.2.表达载体，其特征在于，所述表达载体能够表达权利要求1所述的长链非编码RNAIL21-AS1。2 . An expression vector, wherein the expression vector is capable of expressing the long-chain non-coding RNAIL21-AS1 of claim 1 .3.根据权利要求2所述的表达载体，所述载体为真核表达质粒。3. The expression vector according to claim 2, which is a eukaryotic expression plasmid.4.根据权利要求3所述的表达载体，其特征在于，所述质粒为pcDNA3.1表达质粒pcDNA3.1-IL21-AS1。4. The expression vector according to claim 3, wherein the plasmid is pcDNA3.1 expression plasmid pcDNA3.1-IL21-AS1.5.一种Naive CD4+T细胞，其特征在于，所述Naive CD4+T细胞含有权利要求2所述的表达载体。5 . A Naive CD4+ T cell, wherein the Naive CD4+ T cell contains the expression vector of claim 2 . 6 .6.构建权利要求5所述的Naive CD4+T细胞的方法，其特征在于，将权利要求4的真核表达载体通过电转染至Naive CD4+T细胞。6. The method for constructing the Naive CD4+ T cell of claim 5, wherein the eukaryotic expression vector of claim 4 is electrotransfected into the Naive CD4+ T cell.7.一种将Naive CD4+T细胞诱导成滤泡辅助性T细胞的方法，其特征在于，将按照权利要求6所获得的经转染细胞继续培养3天。7. A method for inducing Naive CD4+ T cells into follicular helper T cells, characterized in that the transfected cells obtained according to claim 6 are further cultured for 3 days.8.权利要求1所述的长链非编码RNA IL21-AS1或权利要求2所述的表达载体在制备用于诱导Naive CD4+T细胞分化为滤泡辅助性T细胞的物质的用途。8. Use of the long-chain non-coding RNA IL21-AS1 of claim 1 or the expression vector of claim 2 in preparing a substance for inducing Naive CD4+ T cells to differentiate into follicular helper T cells.9.根据权利要求8所述的用途，其特征在于，所述滤泡辅助性T细胞表达的IL-21增加。9. The use of claim 8, wherein the expression of IL-21 by the follicular helper T cells is increased.10.权利要求1所述的长链非编码RNA IL21-AS1或权利要求2所述的表达载体在制备用于诱导分泌IL-21的物质中的用途。10. Use of the long-chain non-coding RNA IL21-AS1 of claim 1 or the expression vector of claim 2 in the preparation of a substance for inducing secretion of IL-21.

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