CN106801089A - 一种用于扩增鸟类ifna基因cds序列的引物及其方法 - Google Patents
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Abstract
本发明公开了一种用于扩增鸟类IFNA基因CDS序列的引物及其方法，所述引物包括序列号为SEQ ID NO.1的DNIFNA‑F和序列号为SEQ ID NO.2的DNIFNA‑R。所述方法包括：(1)提取待测样品的总基因组DNA；(2)采用所述引物对步骤(1)中提取的总基因组DNA进行PCR扩增；(3)对步骤(2)扩增后的产物进行琼脂糖凝胶电泳检测。本发明通过设计一对特异性PCR引物，对待测样品通过一次PCR扩增，经琼脂糖凝胶电泳检测是否有条带，从显示判定扩增效果，且所用的模板可从动物肌肉组织中提取，大大降低了取样的难度。
Description
技术领域
本发明属于生物检测技术领域，具体涉及一种用于扩增鸟类IFNA基因CDS序列的引物及其方法。
背景技术
IFNA基因(interferon alpha)CDS干扰素-α。干扰素(interferon，IFN)是一类在特定诱导剂(细菌、病毒等)作用下产生的一类具有抗病毒[1]、抗肿瘤[2]、免疫调节[3]等生物学功能的细胞因子，是由Isaacs等在利用鸡胚绒毛尿囊膜研究流感病毒的干扰现象时发现。根据氨基酸的序列、干扰素的功能及相应受体的结合，可将干扰素分为3种类型：Ⅰ型、Ⅱ型、Ⅲ型。Ⅰ型干扰素包括IFN-α、IFN-β、IFN-ω、IFN-ε、IFN-τ、IFN-δ、IFN-κ、IFN-ξ。Ⅱ型干扰素包括IFN-γ，Ⅲ型干扰素包括IL-28A、IL-28B、IL29。干扰素-α是由脊椎动物细胞受到病毒侵染时，由白细胞产生的一种调节蛋白，主要作用是抗病毒。在人和小鼠中，IFNA基因分别位于9号染色体的短臂和4号染色体的着丝粒近端区域。在原鸡中，IFNA位于Z染色体上。目前，NCBI上已释放鸟类IFNA序列较少，而诸多鸟类基因组注释尚不完整，从基因组中获取IFNA基因序列有限，所以亟需丰富鸟类IFNA基因的序列信息，以为研究鸟类干扰素抗病毒机制研究提供理论基础。
雁形目鸟类属于水禽，我国是水禽养殖大国，近年来受禽流感疫情等影响，各种禽类疾病成为阻碍水禽发展的重要因素。鉴于干扰素-α广谱的抗病毒作用，因此对雁形目鸟类IFNA抗病毒机制和应用的研究迫在眉睫。而目前关于雁形目鸟类IFNA，特别是CDS区的分子数据较少。
发明内容
根据以上现有技术的不足，本发明所要解决的技术问题是提出一种用于扩增鸟类IFNA基因CDS序列的引物及其方法，目的是快速、准确的获取鸟类的IFNA基因CDS序列。
为了解决上述技术问题，本发明采用的技术方案为：
一种用于扩增鸟类IFNA基因CDS序列的引物，所述引物包括序列号为SEQ ID NO.1的DNIFNA-F和序列号为SEQ ID NO.2的DNIFNA-R。
优选的，所述鸟类为雁形目鸟类。
一种利用所述引物扩增鸟类IFNA基因CDS序列的方法，包括如下步骤：
(1)提取待测样品的总基因组DNA；
(2)采用所述引物对步骤(1)中提取的总基因组DNA进行PCR扩增；
(3)对步骤(2)扩增后的产物进行琼脂糖凝胶电泳检测。
PCR扩增采用12.5uL体系:ddH2O 6.985uL，10×Ex-taq buffer 1.25uL，dNTPs(2.5mmol/L)1uL，Template 1uL，DNIFNA-F(5umol/L)1.1uL，DNIFNA-R(5umol/L)1.1uL，Ex-taq(5U/uL)0.065uL。
PCR反应体系为：95℃预变性5min。然后进行36个循环，该循环包括94℃变性30s，55℃退火30s，72℃延伸1min。最后72℃延伸10min。
通过对雁形目四个属七种鸟类采用本发明的方法实验，通过琼脂糖凝胶电泳图可以看出，对应PCR产物泳道均有条带。在本发明中，出现的7条条带中，所含有的DNA片段的长度大致相等，在750bp-1000bp之间。
为了达到更好的PCR扩增效果，使得得到的琼脂糖凝胶电泳图更清晰可见，同时节约成本，以12.5ul的PCR扩增体系为基准，每条引物的浓度分别为1.1ul，DNA模板的用量为1ul。其中，用于PCR扩增的聚合酶以及缓冲液可以为本领域常规聚合酶和缓冲液，其用量具体的可以参照产品说明书中的详细说明。本发明在此不再详细赘述。
所述PCR扩增过程中的退火过程可以为本领域所常规使用的退火方式及退火参数，在本发明中，为了得到更好的扩增效果，所述PCR扩增过程的退火温度为55℃，退火时间为30s。另外，对于PCR扩增中的变性条件、延伸条件以及循环数等参数的设置均为本领域常规的设置，本发明在此不再详细赘述。
本发明有益效果是:本发明通过设计一对特异性PCR引物，对待测样品通过一次PCR扩增，经琼脂糖凝胶电泳检测是否有条带，从显示判定扩增效果，且所用的模板可从动物肌肉组织中提取，大大降低了取样的难度，而且该方法可简便快速获取雁形目七种鸟类IFNA基因序列，从而达到简便易行，经济实用的目的。为深入研究雁形目鸟类干扰素抗病毒机制研究提供理论基础。
附图说明
下面对本说明书附图所表达的内容及图中的标记作简要说明：
图1是本发明雁形目七种鸟类PCR扩增后琼脂凝胶电泳图。
其中，泳道1来自斑嘴鸭肌肉样品、泳道2来自白额雁的肌肉样品、泳道3来自翘鼻麻鸭的肌肉样品、泳道4来自鸿雁的肌肉样品、泳道5来自赤麻鸭的肌肉样品、泳道6来自大天鹅的肌肉样品、泳道7来自豆雁的肌肉样品，泳道0为分子量标记。
具体实施方式
下面通过对实施例的描述，对本发明作进一步详细的说明，以帮助本领域技术人员对本发明的发明构思、技术方案有更完整、准确和深入的理解。
需要阐明的是，所述引物均由通用生物系统(安徽)有限公司合成且引物浓度为5umol，本发明中使用的dNTPMix为Takara公司的市售品，其余所使用的化学试剂均为常规市售分析纯试剂。
样品取材：雁形目四个属七种鸟类：1只斑嘴鸭(鸭属)，1只赤麻鸭(麻鸭属)，1只翘鼻麻鸭(麻鸭属)，1只大天鹅(天鹅属)，1只鸿雁(雁属)、1只豆雁(雁属)、1只白额雁(雁属)。
实施例1
1)总基因组的提取：采用酚-氯仿抽提法，取约100mg肌肉组织样本，剪碎后置于一只已灭菌的2.0ml的EP管；
2)在EP管中加入1ml的DNA提取液及4ul 20mg/ml的RNaseA(核糖核酸酶)，摇匀后置于37℃水浴锅中水浴1h；
3)取出EP管，加入5ul 20mg/ml的蛋白酶K，摇匀后置于37℃水浴锅中水浴约2-3h，直至肌肉完全消化，期间每隔10min上下摇匀；
4)取出EP管，待冷却至室温后，加入等体积的饱和酚，温和上下摇匀约7分钟直至水相与酚相混合成乳状液；
5)10000rPm，10℃下离心15min，取出上层水相至另一20mlEP管中；
6)重复酚提取一二次；
7)加入等体积的CI(氯仿异戊醇)，温和上下摇匀约7min，10000rPm，10℃下离心15min，取出上层水相至另一2.0mlEP管中；
8)再次加入等体积的CI和上下摇匀约7min，10000rPm，10℃下离心15min，取出上层水相至另一1.5mlEP管中(分装到2管中，每管300ul)
9)加入1/5体积的3mol/l NaAC及2倍体积预冷的无水乙醇，室温轻摇EP管置于-20℃冰柜中冷冻3h；
10)从冰柜中取出EP管，12000rPm，4℃离心15min，并弃上清；
11)加入500ul 75％乙醇漂洗，12000rPm，4℃离心15min，并弃上清；
12)再加入500ul 75％乙醇漂洗，12000rPm，4℃离心15min，并弃上清，后风干沉淀；
13)加入100ul 1×TE溶解DNA；
14)取2ulDNA溶液进行琼脂糖凝胶电泳；
15)总DNA置于-40℃冰柜中储存。
以七种雁形目鸟类的总DNA为模板，进行PCR扩增。
PCR扩增采用12.5uL体系:ddH2O 6.985uL，10×Ex-taq buffer 1.25uL，dNTPs(2.5mmol/L)1uL，Template 1uL，DNIFNA-F(5umol/L)1.1uL，DNIFNA-R(5umol/L)1.1uL，Ex-taq(5U/uL)0.065uL。用于PCR扩增和测序的引物如下表1所示。
PCR反应体系为：95℃预变性5min。然后进行36个循环，该循环包括94℃变性30s，55℃退火30s，72℃延伸1min。最后72℃延伸10min。
表1.用于PCR扩增和测序的引物
将扩增后的产物进行琼脂糖凝胶电泳，电泳结果如图1所示。
每个物种重新扩增50uL体积PCR产物，将所得PCR产物送至通用生物系统(安徽)有限公司进行测序。
对测序后序列进行校正及整理，得到七个物种的IFNA基因的CDS全序列(CDS序列)，长度为576bp或579bp。如表2所示。
表2.所扩增物种的编号、拉丁名及CDS序列长度
上面对本发明进行了示例性描述，显然本发明具体实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种非实质性的改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围之内。本发明的保护范围应该以权利要求书所限定的保护范围为准。
SEQUENCE LISTING
<110> 安徽师范大学
<120> 一种用于扩增鸟类IFNA基因CDS序列的引物及其方法
<130> 1
<160> 2
<170> PatentIn version 3.3
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<213> DNIFNA-F
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ccatgacctg aaagcgacga 20
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gggctccggt cagttcttg 19
Claims (7)
1.一种用于扩增鸟类IFNA基因CDS序列的引物，其特征在于，所述引物包括序列号为SEQ ID NO.1的DNIFNA-F和序列号为SEQ ID NO.2的DNIFNA-R。
2.根据权利要求1所述用于扩增鸟类IFNA基因CDS序列的引物，其特征在于，所述鸟类为雁形目鸟类。
3.采用权利要求1或2所述引物在鸟类IFNA基因CDS序列测序中的应用。
4.一种利用权利要求1或2所述引物扩增鸟类IFNA基因CDS序列的方法，其特征在于，包括如下步骤：
(1)提取待测样品的总基因组DNA；
(2)采用所述引物对步骤(1)中提取的总基因组DNA进行PCR扩增；
(3)对步骤(2)扩增后的产物进行琼脂糖凝胶电泳检测。
5.根据权利要求4所述方法，其特征在于，步骤(3)所述扩增后的产物的条带所含有的DNA片段在750bp-1000bp之间。
6.根据权利要求4所述方法，其特征在于，所述PCR扩增是以12.5μl的PCR扩增体系为基准，每条引物的浓度分别为1.1μl，模板的用量均为1μl。
7.根据权利要求4所述方法，其特征在于，所述PCR扩增的过程中退火温度为55℃，退火时间为30s。
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