CN113274510B - 抑制j亚群禽白血病病毒复制的组合物及其应用 - Google Patents
抑制j亚群禽白血病病毒复制的组合物及其应用 Download PDFInfo
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Abstract
本发明公开了一种抑制J亚群禽白血病病毒复制的组合物及其应用，属于兽用生物制品技术领域。所述抑制J亚群禽白血病病毒复制的组合物包括：shRNA表达质粒和AZT；所述shRNA表达质粒是由shRNA序列连接入质粒载体中构建而成。应用shRNA表达载体及逆转录酶抑制剂AZT在清除细胞内的ALV的作用上具有协同效果，两者联合应用能够显著降低DF‑1细胞内禽白血病病毒的病毒量以及p27抗原水平，能够显著抑制ALV复制，在细胞水平上具有较好的抗禽白血病病毒效果。
Description
技术领域
本发明涉及兽用生物制品技术领域，具体涉及一种抑制J亚群禽白血病病毒复制的组合物及其应用。
背景技术
禽白血病(Avian leukosis，AL)是由禽白血病病毒(Avianleukosis virus，ALV)引起的、禽类多种肿瘤性疾病的统称，主要表现为鸡的良性、恶性肿瘤和亚临床感染，导致鸡只生长迟缓、产蛋下降和免疫抑制。自AL被发现以来，该病呈现出世界性流行趋势，近年来在中国鸡群中广泛传播，对鸡群的安全构成了严重威胁，给我国养殖业的发展带来了巨大困扰，并给地方品种鸡保种和选育工作带来很大的困难。禽白血病病毒属于反转录病毒科、正反录病毒亚科、甲型反转录病毒属。该科病毒的基因组为RNA，以具有反转录酶为特征。目前，根据gp85基因的特性，将ALV分为A、B、C、D、E、F、G、H、I、J和K共11个亚群。目前，ALV-J在很多种类鸡群中均发现感染且病毒呈现出极大的基因多样性。鉴于该病的重大危害，《国家中长期动物疫病防治规划(2012-2020年)》将AL列为我国需要优先净化的禽病。ALV主要通过种蛋垂直传播，另外，使用了被ALV污染的弱毒疫苗也是该病传播的一个重要方式。目前，尚无针对禽白血病的商品化疫苗与特效药物，主要通过对现有种鸡群净化来控制该病。
齐多夫定(Zidovudine，AZT)是一种核苷类逆转录酶抑制剂具有抗逆转录酶的活性，能够在组织培养中抑制HIV复制，临床试验证明它能延缓艾滋病程，推迟机会性感染。先前，已有研究证明了药物能够在一定程度上抑制J亚群禽白血病病毒的复制。由于AZT与脱氧胸腺嘧啶核苷酸(T)很相似，逆转录酶也错将其利用，但因其脱氧核糖的第3位缺少羟基，不能与另一核糖核苷酸上的第5’端的羟基与磷酸酯化相连，因此不能够形成3,5-磷酸二酯键，DNA链的编码被中断了，因此阻碍了逆转录病毒的复制。
RNA干扰(RNA interference，RNAi)是通过内源性或外源性双链RNA(doublestrand RNA，dsRNA)的介导，特异性降解相应序列的mRNA，导致靶基因的表达沉默，产生相应的功能型缺失，属于转录后水平的基因沉默(post-transcript gene silencing，PTGS)现象。作为古老的、进化保守的基因沉默机制，RNAi对细胞防御病毒的感染、修复遗传损伤及调节正常的基因功能具有重要作用。现在，RNAi作为一种简单有效的分子生物学工具，研究人员通常采用化学合成，或质粒载体、病毒载体表达所需要的dsRNA，进而诱导特异有效的RNAi进行基因功能研究和疾病治疗。前期研究表明，通过设计针对不同靶位点的siRNA(如有研究针对gag基因、env基因或LTR区域)，可以有效干扰ALV-J的复制。
但是，AZT虽然可以在细胞水平上抑制ALV-J的复制，具有一定的效果，但高剂量AZT会对细胞造成损伤；而且，高剂量AZT的长期使用可能会导致耐药毒株的出现。RNAi是一种强有效的抑制基因功能的工具，但RNAi技术的难点在于如何选择靶标区域设计shRNA表达质粒，针对ALV-J病毒基因组不同靶基因的RNAi在抑制禽白血病病毒复制的效果各不相同；而且，RNAi技术一般是单独使用，目前还很少见有基于RNAi技术设计的shRNA表达质粒与其他药物联合应用治疗J亚群禽白血病的报道。
发明内容
针对上述现有技术，本发明的目的是提供一种抑制J亚群禽白血病病毒复制的组合物及其应用。本发明发现，应用shRNA表达载体及逆转录酶抑制剂AZT在清除细胞内的ALV的作用上具有协同效果，两者联合应用能够显著降低DF-1细胞内禽白血病病毒的病毒量以及p27抗原水平，能够显著抑制ALV复制，在细胞水平上具有较好的抗禽白血病病毒效果。
为实现上述目的，本发明采用如下技术方案：
本发明的第一方面，提供一种抑制J亚群禽白血病病毒复制的组合物，所述组合物包括：shRNA表达质粒和AZT；
所述shRNA表达质粒是由shRNA序列连接入质粒载体中构建而成。
优选的，所述shRNA序列为如下(1)-(4)任一项所示：
(1)sh-1-1，其正义链序列如SEQ ID NO.6所示，反义链序列如SEQ ID NO.7所示；
(2)sh-1-2，其正义链序列如SEQ ID NO.8所示，反义链序列如SEQ ID NO.9所示；
(1)sh-2-1，其正义链序列如SEQ ID NO.10所示，反义链序列如SEQ ID NO.11所示；
(1)sh-2-2，其正义链序列如SEQ ID NO.12所示，反义链序列如SEQ ID NO.13所示。
优选的，所述质粒载体为载体pBAsi-hU6。
优选的，所述shRNA表达质粒的转染剂量为0.5-1.0μg；所述AZT的使用浓度为0.5-1.0μg/ml。
本发明的第二方面，提供上述组合物在制备抑制J亚群禽白血病病毒复制的药物中的应用。
本发明的第三方面，提供一种抑制J亚群禽白血病病毒复制的药物，所述药物以上述组合物为有效成分。
本发明的有益效果：
(1)对ALV-J抑制效果好：本发明采用shRNA与AZT联用的方式。其中，本发明设计的shRNA其基因靶标为ALV-J pol基因的高变区。这一区域是病毒反转录酶发挥活性的重要区域，通过shRNA干扰，可以有效抑制病毒反转录酶的活性。
(2)对细胞损伤程度极小：虽然AZT对ALV-J抑制作用良好，但高浓度的AZT对体外培养的细胞具有较高的毒性，限制了其在细胞层面的使用。为此，本发明利用了shRNA与AZT联用所发挥的联合效应，从而在保证效果的前提下，降低了AZT的使用浓度，避免了细胞损伤。
附图说明
图1：本发明设计shRNA的靶标区域。
图2：shRNA寡核苷酸序列形成示意图。
图3：pBAsi载体图谱。
图4：表达质粒的测序结果。
图5：不同浓度的AZT以及shRNA和AZT组合后对细胞损伤的影响结果。
图6：病毒RNA相对表达量。
图7：细胞培养上清S/P值；图中，横线代表是所使用的ELISA试剂盒的CUT OFF值(S/P＝0.2)。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案，以下将结合具体的实施例详细说明本申请的技术方案。
本发明实施例中所用的未进行具体说明试验材料均为本领域常规的试验材料，均可通过商业渠道购买得到。本发明实施例中未注明具体实验条件和方法的，通常按照常规条件，如J.萨姆布鲁克等主编，科学出版社，2002，分子克隆实验指南(第三版)；D.L.斯佩克特等主编，科学出版社，2001，细胞实验指南；或者按照制造厂商建议的条件。
实施例1：shRNA的设计与质粒构建
本实施例针对ALV-J pol基因高变区即为pol基因第426-698位核苷酸及pol基因第1098-1364位核苷酸(上述位置是以ALV-J的SDAU1005株的pol蛋白Genbank accessNo.AMP18890.1为基准)作为靶标设计shRNA，如图1所示。设计合成4条siRNA及一条对照序列(sh-NC)，其中sh-1-1和sh-1-2室针对第一段高变区所设计，而sh-2-1和sh-2-2为针对第二段高变区所设计。与ALV-J基因其他位置相比，这两段高变区为逆转录酶关键区域，第一段高变区与逆转录酶活性(RT)有关，第二段高变区与RNase H活性有关，通过shRNA干扰，可以有效抑制病毒反转录酶的活性。
4条siRNA及sh-NC的正义链(sense)序列如表1所示(对应序列表中的SEQ IDNO.1-SEQ ID NO.5)。表1中的siRNA是shRNA经Dicer酶加工产生RNAi的作用形式，依据表1的序列在中间加入发夹序列，前端加入BamH I序列，后端加入终止信号序列和Hind III序列(图2)，得到shRNA寡核苷酸序列，具体如表2所示。
表1：siRNA序列
表2：合成的shRNA寡核苷酸序列
将人工合成的shRNA寡核苷酸(表2)连接至pBAsi-hU6表达载体构建shRNA表达质粒。具体如下：
(1)首先将合成的寡核苷酸用双蒸水稀释至0.01OD/μL。
(2)配制10μL退火体系：Top strand 1μL，Bottom strand 1μL，TE Buffer 1μl，ddH2O 7μl，其中TE Buffer成分为：10mM Tris pH8.0，50mM NaCl，1mM EDTA。于95℃变性5min之后自然冷却至室温。
(3)pBAsi-hU6载体的线性化：将pBAsi-hU6载体(购自TaKaRa公司，载体图谱如图3所示)用限制性内切酶BamH I和Hind III进行双酶切，酶切反应体系为：BamH I 1μL，HindIII 1μL，10×K Buffer 1μL，dd H2O 7μl，于37℃酶切3h之后使用琼脂糖凝胶电泳分离线性化载体并用琼脂糖凝胶回收试剂盒回收纯化。
(4)将退火后的片段与回收纯化后的线性化载体使用T4 DNA连接试剂盒按说明书进行连接，配制10μL连接体系为：退火后片段5μL，线性化载体1μL，T4 DNA Ligase1μL，Ligase Buffer 1μL，dd H2O 2μl，于16℃连接12h。
(5)将连接片段热转化至JM109感受态细胞中并挑取单克隆菌落，提取无内毒素质粒进行PCR验证并送生物公司进行测序，测序结果如图4所示，确保序列准确无误。最终构建得到sh-1-1表达质粒、sh-1-2表达质粒、sh-2-1表达质粒和sh-2-2表达质粒。
实施例2：不同浓度的AZT以及shRNA和AZT组合后对细胞损伤的影响
将DF-1细胞接种至24孔细胞板中，待细胞长至80％时进行试验处理，具体为：
将含有不同剂量(0μg/mL、0.1μg/mL、0.5μg/mL、1μg/mL、2μg/mL、5μg/mL、10μg/mL)的AZT与DF-1细胞共同孵育48h，通过CCK-8法检测测试细胞存活率。
以及，将1μg/mL的AZT分别与0.8μg的sh-1-1表达质粒、sh-1-2表达质粒、sh-2-1表达质粒、sh-2-2表达质粒组合后与DF-1细胞共同孵育48h，通过CCK-8法检测测试细胞存活率。
结果如图5所示，从图中可以看出，当药物浓度在5μg/mL以下时，细胞活性在90％以上，而当使用药物浓度为1μg/mL时单独使用药物或者shRNA与药物组合细胞活性均在95％以上，表明shRNA与药物组合细胞损伤程度较小。
实施例3：shRNA与AZT联合使用抑制ALV-J的复制
将接种密度为1×105个/孔的DF-1细胞至24孔细胞板中，待细胞长至80％时，以LipofectamineTM 2000作转染试剂通过转染将0.8μg shRNA表达质粒转入细胞，在细胞表达并产生RNAi作用。在转染8h后每孔接150TCID50的ALV-J病毒株SDAU1005(Genbank accessNo.KT156668)，同时使用药物浓度为1μg/mL的AZT培养细胞，使AZT与靶向逆转录酶的干扰RNA共同作用于细胞，在48hpi收集细胞提取核酸进行反转录，以β-actin为内参检测病毒基因组RNA相对表达水平，同时收集细胞上清培养液利用禽白血病抗原检测试剂盒检测病毒p27抗原水平。
通过对单独使用AZT或shRNA表达载体(sh-1-1表达质粒、sh-1-2表达质粒、sh-2-1表达质粒和sh-2-2表达质粒)以及shRNA联合AZT的不同处理组DF-1细胞中ALV病毒的相对含量以及细胞培养上清中p27抗原水平进行定量以衡量shRNA联合AZT对ALV的抑制效果。
Real-Time PCR结果表明，与对照组(sh-NC)相比，单独使用AZT或者shRNA表达载体就能够使ALV基因组RNA的相对表达量显著降低；而AZT联合shRNA共同使用时使得ALV基因组RNA的相对表达量进一步降低(图6)。与单独使用AZT(sh-NC+AZT)相比，表明shRNA能够协同AZT抑制ALV基因组RNA的相对表达量。
用ELISA试剂盒检测细胞上清中的ALV-p27抗原水平，在48hpi时，与对照组(sh-NC)相比，单独使用AZT或者shRNA表达载体能够使细胞培养液上清p27-S/P值显著降低(图7)，而表达载体sh-1-1、sh-1-2、sh-2-1、sh-2-2分别联合AZT使用能够使S/P值进一步降低至0.188±0.0164、0.185±0.0319、0.254±0.0435、0.224±0.0128，处于ELISA试剂盒CUTOFF值(S/P＝0.2)附近，从细胞培养上清中p27抗原水平来看，shRNA表达载体能够协同AZT进一步抑制ALV的复制。
综上，应用shRNA表达载体及逆转录酶抑制剂AZT在清除细胞内的ALV的作用上具有协同效果，两者联合应用能够显著降低DF-1细胞内禽白血病病毒的病毒量以及p27抗原水平，能够显著抑制ALV复制，表明此方法在细胞水平上具有较好的抗禽白血病病毒效果。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
SEQUENCE LISTING
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Claims (4)
1.一种抑制J亚群禽白血病病毒复制的组合物，其特征在于，所述组合物包括：shRNA表达质粒和AZT；
所述shRNA表达质粒是由shRNA序列连接入质粒载体中构建而成；
所述shRNA序列为如下（1）-（4）任一项所示：
（1）sh-1-1，其正义链序列如SEQ ID NO.6所示，反义链序列如SEQ ID NO.7所示；
（2）sh-1-2，其正义链序列如SEQ ID NO.8所示，反义链序列如SEQ ID NO.9所示；
（1）sh-2-1，其正义链序列如SEQ ID NO.10所示，反义链序列如SEQ ID NO.11所示；
（1）sh-2-2，其正义链序列如SEQ ID NO.12所示，反义链序列如SEQ ID NO.13所示；
所述质粒载体为含有pol-III启动子的载体pBAsi-hU6。
2.根据权利要求1所述的组合物，其特征在于，所述shRNA表达质粒的转染剂量为0.5-1.0μg；所述AZT的使用浓度为0.5-1.0μg/ml。
3.权利要求1或2所述的组合物在制备抑制J亚群禽白血病病毒复制的药物中的应用。
4.一种抑制J亚群禽白血病病毒复制的药物，其特征在于，所述药物以权利要求1或2所述的组合物为有效成分。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2362909A2 (en) * | 2008-09-30 | 2011-09-07 | Zirus, Inc. | Mammalian genes involved in infection |
| CN103623426A (zh) * | 2012-08-29 | 2014-03-12 | 上海吉凯基因化学技术有限公司 | 人ppp5c基因的用途及其相关药物 |
| CN104262477A (zh) * | 2014-09-25 | 2015-01-07 | 山东农业大学 | 一种禽CCCH型锌指蛋白chZAP及其应用 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2362909A2 (en) * | 2008-09-30 | 2011-09-07 | Zirus, Inc. | Mammalian genes involved in infection |
| CN103623426A (zh) * | 2012-08-29 | 2014-03-12 | 上海吉凯基因化学技术有限公司 | 人ppp5c基因的用途及其相关药物 |
| CN104262477A (zh) * | 2014-09-25 | 2015-01-07 | 山东农业大学 | 一种禽CCCH型锌指蛋白chZAP及其应用 |
Non-Patent Citations (1)
| Title |
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| 病毒性疾病的免疫问题;王用揖;《微生物学报》;19751231;第15卷(第2期);全文 * |
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