CN113930527A - 一种非编码RNA lnc-ALVE-env1表达检测引物及试剂盒和应用 - Google Patents
一种非编码RNA lnc-ALVE-env1表达检测引物及试剂盒和应用 Download PDFInfo
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Abstract
本发明涉及一种非编码RNA lnc‑ALVE‑env1表达检测引物及试剂盒和应用，属于分子生物学技术领域。所述引物包括链特异性RT引物和荧光定量PCR检测引物；其中链特异性RT引物的序列如SEQ ID NO.3和SEQ ID NO.4所示；荧光定量PCR检测引物的序列如SEQ ID NO.5和SEQ ID NO.6所示。本研究发明还提供了lnc‑ALVE‑env1表达检测试剂盒，为lnc‑ALVE‑env1表达规律分析提供了方法和基础，在生产实践可用于检测鸡群的禽白血病天然免疫状态。本发明的非编码RNA lnc‑ALVE‑env1表达检测引物及试剂盒特异性好、方便便宜，可以快速检测出lnc‑ALVE‑env1的表达丰度；适用性广，可以有效筛选禽白血病病毒抗性品系鸡。
Description
技术领域
本发明涉及一种非编码RNA lnc-ALVE-env1表达检测引物及试剂盒和应用，属于分子生物学技术领域。
背景技术
目前，内源性反转录病毒(endogenous retroviruses)是基因组成分，被认为源于进化中感染生物体的反转录病毒。作为远古反转录病毒感染在宿主体内的残留，加之过去对它的功能不够了解，曾将其视为垃圾DNA(Junk DNA)。直至最近有研究表明，某些内源性反转录病毒不仅对早期胚胎发育和胚胎干细胞的多能性具有重要作用，还可能与细胞天然免疫有关。
最新研究发现，内源性反转录病毒转录物是非编码RNA(non-coding RNA，ncRNA)的重要来源，具有激活干扰素应答反应的潜能。事实上，非编码RNA在细胞生物学调控中的作用越来越受到重视，在抗病毒天然免疫中也具有重要调节作用。内源性反转录病毒衍生的非编码RNA在对抗外源性反转录病毒增殖的作用机制将逐渐被阐明，有望成为抵抗外源性病毒的新型疫苗或免疫增强剂。
禽内源性白血病病毒ALVE是最早发现的内源性反转录病毒，可能与宿主免疫应答和遗传抗性有关。特别是，来源于鸡内源性反转录病毒ALVE的env转录物的非编码RNA可能与禽白血病病毒的感染和增殖具有一定关联。
有鉴于上述的缺陷，本发明以期创设一种非编码RNA lnc-ALVE-env1表达检测引物及试剂盒，使其更具有产业上的利用价值。
发明内容
为解决上述技术问题，本发明的目的是提供一种非编码RNA lnc-ALVE-env1表达检测引物及试剂盒。本发明通过生物信息学分析在鸡基因组中筛选出一条禽内源性反转录病毒ALVE衍生的非编码RNA，命名为lnc-ALVE-env1，而lnc-ALVE-env1可以激活细胞抗病毒干扰素应答反应并抑制与其高度同源的外源性J亚群禽白血病病毒增殖，因此，可以通过进一步了解lnc-ALVE-env1在不同抗性品系鸡中的表达规律来制备检测用试剂盒。
本发明的一种用于非编码RNA lnc-ALVE-env1表达检测引物，
包括链特异性RT引物和荧光定量PCR检测引物；
所述链特异性RT引物包括链特异性引物lnc-ALVE-env1-RT1和lnc-ALVE-env1-RT2，所述链特异性引物lnc-ALVE-env1-RT1和lnc-ALVE-env1-RT2的核苷酸序列如下：
lnc-ALVE-env1-RT1(SEQ ID NO.3)：5’-TTGTGCCTATCCGCTGTC-3’
lnc-ALVE-env1-RT2(SEQ ID NO.4)：5’-CCGTAGACACCACTTGGA-3’；
所述荧光定量PCR检测引物包括特异性引物lnc-ALVE-env1-F和lnc-ALVE-env1-R，所用特异性引物lnc-ALVE-env1-F和lnc-ALVE-env1-R的核苷酸序列如下：
lnc-ALVE-env1-F(SEQ ID NO.5)：5’-AACGGATTTCTGCCTCTCT-3’
lnc-ALVE-env1-R(SEQ ID NO.6)：5’-GATAAGTGAGGGTGGTGTTGT-3’。
一种包括权利要求1所述用于非编码RNA lnc-ALVE-env1表达检测引物的试剂盒，包括用于非编码RNA lnc-ALVE-env1表达检测引物、逆转录试剂和定量PCR检测试剂。
一种用于非编码RNA lnc-ALVE-env1表达检测引物在制备lnc-ALVE-env1表达检测试剂盒中的应用。
借由上述方案，本发明至少具有以下优点：
1、本发明的非编码RNA lnc-ALVE-env1表达检测引物及试剂盒特异性好、方便便宜，可以快速检测出lnc-ALVE-env1的表达丰度；
2、本发明的非编码RNA lnc-ALVE-env1表达检测引物及试剂盒适用性广，可以有效筛选禽白血病病毒抗性品系鸡。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某个实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1是本发明lnc-ALVE-env1抑制J亚群禽白血病病毒增殖图；
其中：
A：过表达lnc-ALVE-env1抑制J亚群禽白血病病毒env基因mRNA表达；
B：过表达lnc-ALVE-env1抑制J亚群禽白血病p27蛋白表达；
C：过表达lnc-ALVE-env1抑制J亚群禽白血病病毒滴度；
图2是本发明lnc-ALVE-env1荧光定量PCR检测结果图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1 lnc-ALVE-env1过表达载体构建
本实施例1中，根据NCBI公布的序列(XXX)扩增lnc-ALVE-env1全长序列，构建lnc-ALVE-env1过表达质粒。
具体包括如下步骤：
(1)从AZA处理96小时的鸡胚成纤维细胞中，使用
Reagent提取总RNA，然后用RNase-free DNase I去除基因组。使用PrimeScript RT reagent Kit试剂盒将其逆转录成cDNA产物。
(2)以步骤(1)中所获得的cDNA产物为模板，使用高保真酶扩增lnc-ALVE-env1全长序列，其中，所用引物lnc-ALVE-env1-HindIII-F和lnc-ALVE-env1-XbaI-R的核苷酸序列如下：
lnc-ALVE-env1-HindIII-F:
5′-TGCTCTAGACAATCACTTTGAGCGTGG-3′(SEQ ID NO.1)
lnc-ALVE-env1-XbaI-R:
5′-CGCGGATCCATTCCTTGCCATGCGCGAT′(SEQ ID NO.2)
其反应体系包括：100ng步骤(1)所获得的cDNA产物作为模板，1μL(10μM)上游引物lnc-ALVE-env1-HindIII-F与1μL(10μM)lnc-ALVE-env1-XbaI-R分别作为扩增引物，1μLDNA Polymerase，10μL 5x SF Buffer，1μL(10μM)dNTP Mix和32μL ddH2O。
其反应条件为：95℃3min；95℃15s，58℃90s，72℃1min，35x循环；72℃7min；4℃维持。
(3)以步骤(2)中PCR扩增产物进行琼脂糖凝胶电泳，然后切胶回收产物并连接至T载体进行克隆测序。测序正确的阳性克隆质粒与pcDNA3.1(+)(Invitrogen)过表达质粒载体分别用HindIII和XbaI酶切。最后，酶切回收的lnc-ALVE-env1目的片段DNA和pcDNA3.1(+)载体DNA用T4 DNA ligase进行连接，连接产物直接转化大肠杆菌。重组子经PCR、酶切和克隆测序鉴定正确后，命名为pcDNA3.1-lnc-ALVE-env1。
实施例2 lnc-ALVE-env1抗J亚群禽白血病病毒增殖能力分析
本实施例2中，使用从实施例1中所获得的pcDNA3.1-lnc-ALVE-env1过表达质粒，通过RT-qPCR、Western Blot、ELISA和TCID50方法评估了在鸡巨噬细胞系HD11中lnc-ALVE-env1抗J亚群禽白血病病毒增殖能力。
具体包括如下步骤：
(1)将鸡巨噬细胞系HD11接种至6孔细胞培养板，然后感染J亚群禽白血病病毒(毒株JS09GY3，GenBank accession number GU982308)。
(2)病毒感染后4小时，转染质粒pcDNA3.1-lnc-ALVE-env1；同时转染GFP过表达质粒作为载体对照。
(3)转染后48小时，收集细胞，使用RT-qPCR测定J亚群禽白血病病毒囊膜基因env的mRNA表达水平。
(4)转染后48小时，收集上清，使用IDEXX禽白血病抗原检测试剂盒检测J亚群禽白血病病毒p27蛋白表达水平。
(5)转染后48小时，收集细胞和上清，使用TCID50方法测定J亚群禽白血病病毒滴度。
通过RT-qPCR、ELISA和TCID50实验，我们观察到在鸡巨噬细胞系HD11中过表达lnc-ALVE-env1可以显著激活抑制J亚群禽白血病病毒增殖(图1)。
实施例3 lnc-ALVE-env1 RT-qPCR检测
(1)分别从2日龄不同品系鸡脾脏组织中，使用
Reagent提取总RNA，测定浓度。
(2)使用PrimeScriptTM RT reagent Kit with gDNA Eraser试剂盒中提供的试剂去除基因组。
其反应体系包括：1μg步骤(1)所获得的总RNA作为模板，2μL5×gDNA EraserBuffer，1μL gDNA Eraser，RNase Free dH2O补齐至10μL。
其反应条件为：42℃2min；4℃维持。
(3)使用PrimeScriptTM RT reagent Kit with gDNA Eraser试剂盒中提供的试剂进行链特异性逆转录反应，将总RNA逆转录成cDNA产物。其中，所用链特异性引物lnc-ALVE-env1-RT1和lnc-ALVE-env1-RT2的核苷酸序列如下：
lnc-ALVE-env1-RT1(SEQ ID NO.3)：5’-TTGTGCCTATCCGCTGTC-3’
lnc-ALVE-env1-RT2(SEQ ID NO.4)：5’-CCGTAGACACCACTTGGA-3’
其反应体系包括：步骤(2)所获得的10μL反应产物作为模板，5μLlnc-ALVE-env1-RT1和lnc-ALVE-env1-RT2作为链特异性RT混合引物，1μL PrimeScript RT Enzyme Mix I，4μL 5×PrimeScript Buffer 2和4μL RNase Free dH2O。其反应条件为：42℃15min；85℃5s；4℃维持。
(4)使用
Premix Ex TaqTM试剂盒中提供的试剂进行链特异性荧光定量PCR反应。其中，所用特异性引物lnc-ALVE-env1-F和lnc-ALVE-env1-R的核苷酸序列如下：
lnc-ALVE-env1-F(SEQ ID NO.5)：5’-AACGGATTTCTGCCTCTCT-3’
lnc-ALVE-env1-R(SEQ ID NO.6)：5’-GATAAGTGAGGGTGGTGTTGT-3’
其反应体系包括：1μL步骤(3)所获得的cDNA产物作为模板，0.4μL(10μM)上游引物lnc-ALVE-env1-F2与0.4μL(10μM)lnc-ALVE-env1-R2分别作为扩增引物，10μL
Premix Ex TaqTM(2×)和8.2μL ddH2O。
其反应条件为：95℃5min；95℃5s；60℃34s；40个循环。
链特异性荧光定量PCR检测结果如图2所示：非编码RNA lnc-ALVE-env1在2日龄不同品系鸡脾脏组织中均表达；与其他品系鸡相比，lnc-ALVE-env1在A品系鸡中异常高表达。因此，此试剂盒可用于lnc-ALVE-env1表达检测。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Claims (3)
1.一种用于非编码RNA lnc-ALVE-env1表达检测引物，其特征在于，
包括链特异性RT引物和荧光定量PCR检测引物；
所述链特异性RT引物包括链特异性引物lnc-ALVE-env1-RT1和lnc-ALVE-env1-RT2，所述链特异性引物lnc-ALVE-env1-RT1和lnc-ALVE-env1-RT2的核苷酸序列如下：
lnc-ALVE-env1-RT1(SEQ ID NO.3)：5’-TTGTGCCTATCCGCTGTC-3’
lnc-ALVE-env1-RT2(SEQ ID NO.4)：5’-CCGTAGACACCACTTGGA-3’；
所述荧光定量PCR检测引物包括特异性引物lnc-ALVE-env1-F和lnc-ALVE-env1-R，所用特异性引物lnc-ALVE-env1-F和lnc-ALVE-env1-R的核苷酸序列如下：
lnc-ALVE-env1-F(SEQ ID NO.5)：5’-AACGGATTTCTGCCTCTCT-3
lnc-ALVE-env1-R(SEQ ID NO.6)：5’-GATAAGTGAGGGTGGTGTTGT-3’。
2.一种包括权利要求1所述用于非编码RNA lnc-ALVE-env1表达检测引物的试剂盒，其特征在于，包括用于非编码RNA lnc-ALVE-env1表达检测引物、逆转录试剂和定量PCR检测试剂。
3.一种权利要求1所述的用于非编码RNA lnc-ALVE-env1表达检测引物在制备lnc-ALVE-env1表达检测试剂盒中的应用。
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| CN110885823A (zh) * | 2019-12-05 | 2020-03-17 | 湖北省农业科学院畜牧兽医研究所 | 一种长链非编码RNA猪Lnc-000649及其应用 |
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