CN116463345A - 一种凡纳滨对虾钠钾ATPase alpha基因的dsRNA及其应用 - Google Patents
一种凡纳滨对虾钠钾ATPase alpha基因的dsRNA及其应用 Download PDFInfo
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Abstract
本发明涉及一种凡纳滨对虾钠钾ATPase alpha(PvATP1A)基因的dsRNA，序列如SEQ ID NO：1所示。首先获得凡纳滨对虾ATP1A基因，选择序列为SEQ ID NO：1的基因片段，用于dsRNA的合成。随后将合成的dsRNA注射到对虾体内，干扰成功后注射WSSV。与对照组相比，干扰组病毒拷贝数和病毒基因(IE1和VP28)表达下降以及对虾存活率上升，表明ATP1A有助于WSSV增殖。进一步通过免疫荧光分析ATP1A影响WSSV增殖的机制，发现ATP1A与WSSV共定位，并且干扰ATP1A后WSSV入胞减少。本发明的PvATP1A基因的dsRNA为WSSV的防治提供了一种潜在的药物，其可显著抑制WSSV入胞，进而高效抑制WSSV的复制。本发明的PvATP1A基因的dsRNA分子质量小易降解，不会破坏养殖过程中的生态平衡，是一种既环保又高效的病毒防御物质。
Description
技术领域
本发明涉及水产养殖的病毒性疾病防治领域，尤其涉及一种凡纳滨对虾钠钾ATPase alpha基因(简称PvATP1A)的dsRNA在抗白斑综合症病毒中的应用。
背景技术
凡纳滨对虾(又称南美白对虾)由于其营养要求低、生长快、适应性强，出肉率高等优点，目前已发展成为我国对虾养殖产量最高的虾类。据统计数据显示，2021年我国凡纳滨对虾养殖总产量高达197万吨，占全年对虾养殖总产量的80％以上。然而，长期以来我国对虾养殖业中的病害问题十分严重，导致对虾养殖成活率不高。其中，由白斑综合症病毒(white spot syndrome virus，WSSV)引起的白斑综合症对我国对虾养殖的影响最为严重。WSSV是一种具有囊膜、无包涵体的杆状双链环状DNA病毒，其是线形病毒科(Nimaviridae)、白斑病毒属(Whispovirus)的唯一成员，具有宿主范围广、传染性强和致死率高等特点。
病毒是一种严格的细胞内寄生的生物，其生命周期大致包括三个阶段：进入宿主细胞、基因组复制以及病毒粒子组装与释放。其中，病毒进入宿主细胞是决定其是否成功感染的关键环节之一。学者们认为，如果能够抑制病毒入胞，则可以从源头抑制病毒感染，从而有效提高宿主的存活率。然而，目前关于抑制/阻断WSSV入胞的药物报道较少。目前防治WSSV的主要措施是在养殖环境中添加广谱性的抗生素，此法在一定程度可以抑制病原菌的侵害，但是作用时间短，容易残留在在自然环境中，最终在人体内积累，损害人的身体健康。大部分已报道的WSSV防治药物如蛋白/多肽、miRNA以及中草药制剂等都是通过直接杀伤病毒、抑制病毒复制和提高对虾免疫能力等作用机制发挥作用，例如用香豆素类衍生物和中草药抗病免疫饲料添加剂等作为免疫增强剂，但这些免疫增强剂所需材料繁多，且制作过程繁琐。
因此，积极寻找和开发抑制/阻断病毒入胞的药物，能够大大降低防治WSSV的难度，达到高效环保的防治WSSV的目的，对解决我国对虾养殖中白斑综合症病害问题具有重要意义。
发明内容
本发明的目的在于提供一种凡纳滨对虾ATP1A基因的dsRNA及其在抗白斑综合症病毒中的应用。本发明PvATP1A基因的dsRNA可有效地抑制WSSV入胞从而抑制WSSV在凡纳滨对虾体内的增殖，提高对虾存活率，以解决凡纳滨对虾感染WSSV而致死的问题。
一种凡纳滨对虾钠钾ATPase alpha基因的dsRNA，序列如SEQ ID NO：1所示。
一种凡纳滨对虾钠钾ATPase alpha基因的dsRNA及其在制备抗白斑综合症病毒制剂中的应用。首先获得凡纳滨对虾ATP1A基因，经研究发现，利用dsRNA在体内被酶分解成siRNA，可以达到敲降基因的目的。使用DNAMAN软件对ATP1A出现siRNA多的区域进行预测，发现数量最多的是SEQ ID NO：1的基因片段，因此选择序列为SEQ ID NO：1的基因片段用于dsRNA的合成。将合成的dsRNA注射到对虾体内，干扰成功的基础上注射WSSV。与对照组相比，干扰组病毒拷贝数及病毒基因(IE1和VP28)表达下降以及对虾存活率上升，表明ATP1A有助于WSSV增殖。进一步通过免疫荧光分析ATP1A影响WSSV增殖的机制，发现ATP1A与WSSV共定位，并且干扰ATP1A后WSSV入胞减少。本发明的PvATP1A基因的dsRNA为白斑综合症病毒的防治提供了一种潜在的药物，可显著抑制WSSV入胞，进而高效抑制WSSV的复制。本发明的PvATP1A基因的dsRNA分子质量小易降解，不会破坏海洋养殖过程中的生态平衡，是一种既环保又高效的病毒防御物质。
本发明还提供一种上述dsRNA的表达盒。
本发明还提供一种上述dsRNA的重组菌。
本发明还提供一种上述dsRNA的重组载体。
本发明还提供上述凡纳滨对虾钠钾ATPase alpha基因的dsRNA或重组载体在制备抑制WSSV的制剂中的用途。
本发明还提供一种含有上述凡纳滨对虾钠钾ATPase alpha基因的dsRNA、重组载体中的一种或者多种混合的制剂。
优选的，所述制剂包括生物抑制剂、试剂盒、饲料添加剂中的一种或多种。
优选的，所述制剂可用于凡纳滨对虾。
优选的，所述制剂可抑制WSSV增殖。
优选的，所述制剂可降低WSSV感染导致的凡纳滨对虾的死亡。
本发明的凡纳滨对虾钠钾ATPase alpha基因的dsRNA可以采用本领域技术人员已知的方法合成，并采用本领域技术人员已知的方法进行原核表达和纯化。
与现有技术相比，本发明采用RNA干扰技术对PvATP1A在WSSV感染中的功能与作用机制进行探究。干扰PvATP1A后WSSV攻毒，发现与对照组相比，血细胞中病毒拷贝数及病毒基因(IE1和VP28)表达下降，且对虾存活率上升，表明PvATP1A基因的dsRNA抑制WSSV增殖。进一步通过免疫荧光实验探索PvATP1A影响WSSV增殖的机制。结果发现，PvATP1A与WSSV共定位，并且干扰PvATP1A后WSSV内化受到抑制，表明PvATP1A参与WSSV入胞过程。因此PvATP1A基因的dsRNA可作为抗WSSV的新药物，应用于WSSV的防治工作中。
附图说明
图1为干扰PvATP1A对WSSV增殖的影响分析；其中：A为PvATP1A mRNA水平干扰效果分析；B为PvATP1A蛋白水平干扰效果分析；C为PvATP1A干扰前后IE1 mRNA水平表达分析；D为PvATP1A干扰前后VP28 mRNA水平表达分析；E为PvATP1A干扰前后WSSV拷贝数分析；F为PvATP1A干扰前后，IE1、VP28蛋白水平表达分析。
图2为干扰PvATP1A后WSSV攻毒，对虾存活率分析。
图3为PvATP1A与WSSV共定位分析；其中：A为荧光显微镜观察PvATP1A与WSSV共定位情况(箭头为WSSV与PvATP1A共定位区域)；B为共定位率统计。
图4为干扰PvATP1A对WSSV入胞影响分析；其中：A为荧光显微镜观察PvATP1A干扰前后WSSV内化情况分析(箭头为WSSV病毒粒子)；B为WSSV病毒粒子的内化率统计。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。但本领域技术人员了解，下述实施例不是对本发明保护范围的限制，任何在本发明基础上做出的改变和变化，都在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法
实施例1
干扰PvATP1A对WSSV增殖的影响分析
(1)根据NCBI中的PvATP1A核酸序列(Genbank ID:KF765670.1)和对照基因EGFP核酸序列(Genbank ID:20473140)分别设计并合成PvATP1A和EGFP的dsRNA引物，引物序列如下：
dsPvATP1A-F：5’-AAGCATCGCCGTTTACTTC-3’
dsPvATP1A-R：5’-CAATGTGCCTGAGGGTCTG-3’
dsPvATP1A-T7F：5’-GGATCCTAATACGACTCACTATAGGAAGCATCGCCGTTTACTTC-3’
dsPvATP1A-T7R：5’-GGATCCTAATACGACTCACTATAGGCAATGTGCCTGAGGGTCTG-3’
dsEGFP-F：5’-CGTAAACGGCCACAAGTT-3’
dsEGFP-R：5’-TTCACCTTGATGCCGTTC-3’
dsEGFP-T7F：5’-GGATCCTAATACGACTCACTATAGGCGTAAACGGCCACAAGTT-3’
dsEGFP-T7R：5’-GGATCCTAATACGACTCACTATAGGTTCACCTTGATGCCGTTC-3’
(2)以凡纳滨对虾血细胞cDNA和pEGFP-N1质粒为模板进行PCR扩增，以PCR产物为模板体外转录合成dsPvATP1A和dsEGFP；
(3)合成的dsPvATP1A与dsEGFP分别用DEPC水稀释为150ng/μL。每组各取15只对虾，每只对虾注射100μL dsPvATP1A或100μL dsEGFP；
(4)在注射dsRNA 48h后，实验组和对照组每只对虾注射100μL WSSV(1×105copies/对虾)。WSSV感染24h、48h后，每组随机抽取3只对虾血细胞，用于提取总RNA、基因组DNA和总蛋白；
(5)分别用海洋动物组织基因组DNA提取试剂盒(天根生化科技有限公司)和总RNA抽提试剂盒(上海飞捷生物有限公司)提取对虾基因组DNA和总RNA，然后用反转录试剂盒(全式金生物科技有限公司)将mRNA反转录成cDNA；
(6)干扰效果检测：采用qPCR和Western blot分析PvATP1A在mRNA水平和蛋白水平变化情况；
(7)WSSV拷贝数的计算：以基因组DNA为模板，VP28的qPCR引物检测对虾血细胞基因组中的WSSV拷贝数；
(8)WSSV极早期基因IE1和晚期基因VP28转录及蛋白水平的检测：用IE1、VP28和内参基因EF-1α进行qPCR分析，检测其转录情况。Western blot分析IE1和VP28蛋白水平的变化；
结果如图1所示，与对照组(dsEGFP)相比，干扰组(dsPvATP1A)血细胞PvATP1A在mRNA水平和蛋白水平的表达量明显下降，干扰效率在80％以上，说明RNA干扰成功(图1A-B)。接着采用qPCR和Western blot对病毒拷贝数和病毒基因表达进行检测。与对照组(dsEGFP)相比，干扰组(dsPvATP1A)血细胞中病毒拷贝数显著下降，存在极显著性差异(p<0.01)，病毒基因VP28和IE1在mRNA水平和蛋白水平都显著下降(图1C-F)，表明PvATP1A基因的dsRNA抑制WSSV增殖。
实施例2
干扰PvATP1A后WSSV刺激下对虾的存活率分析
(1)根据上述注射dsRNA的剂量，进行RNA干扰实验；
(2)实验组(dsPvATP1A组)和对照组(dsEGFP组)每只对虾注射100μL WSSV(1×105copies/对虾)；
(3)注射WSSV后每隔12h统计对虾的存活数量并分析其存活率；
结果如图2所示，与对照组(dsEGFP+WSSV)相比，干扰组(dsPvATP1A+WSSV)对虾存活率显著上升，两者间均存在显著性差异(p<0.05)，再次说明PvATP1A基因的dsRNA抑制WSSV增殖。
实施例3
PvATP1A与WSSV共定位分析
(1)挑取10只健康对虾，每只对虾注射100μL WSSV(1×106copies/对虾)。在WSSV感染0h、2h、4h后，每组随机抽取3只对虾血细胞，混匀后加入Insect-XPRESS昆虫培养基重悬细胞，并稀释成1×106个/mL；
(2)各组取100μL稀释后的血细胞悬液铺在共聚焦皿中，放入28℃细胞培养箱培养2h，使细胞贴壁；
(3)加入4％多聚甲醛固定15min，PBS洗涤3次，每次3min；
(4)加入0.5％ Triton X-100通透20min，PBS洗涤3次，每次3min；
(5)加入3％ BSA(PBS配制)，室温封闭1h；
(6)加入兔抗ATP1A抗体(1：200)和鼠抗VP28抗体(1：200)，4℃过夜孵育，PBS洗涤3次，每次15min；
(7)加入Alexa Flour 555标记驴抗兔IgG(H+L)(1：400)和Alexa Flour488标记山羊抗小鼠IgG(H+L)(1：400)，室温避光孵育1h，PBS洗涤3次，每次10min；
(8)加入Hoechst 33342避光孵育15min，PBS洗涤3次，每次2min；
(9)使用ZEISS LSM 800共聚焦显微镜观察PvATP1A与WSSV病毒粒子的共定位情况，并统计其共定位率；
结果如图3所示，WSSV刺激2h后，PvATP1A与WSSV检测到共定位，且感染4h后共定位增多(图3A)。接着对两者共定位率进行统计，发现WSSV刺激2h后，PvATP1A与WSSV共定位率为1.5％，而刺激4h时的共定位率为7.5％(图3B)。由此说明WSSV与PvATP1A存在共定位。
实施例4
干扰PvATP1A对WSSV入胞影响分析
(1)根据上述注射dsRNA的剂量，进行RNA干扰实验；
(2)干扰48h后，各组对虾注射100μL WSSV(1×106copies/对虾)；
(3)在WSSV感染4h后，每组随机抽取3只对虾血细胞，采用免疫荧光实验观察WSSV内化情况，并对内化率进行统计；
结果如图4所示，对照组WSSV内化率为12％，而干扰组WSSV内化率为5％(图4A-B)，表明WSSV入胞需要PvATP1A的参与。
以上所揭露的仅为本发明的较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。
Claims (10)
1.一种凡纳滨对虾钠钾ATPase alpha基因的dsRNA，其特征在于，序列如SEQ ID NO：1所示。
2.一种含有根据权利要求1所述dsRNA的表达盒。
3.一种含有根据权利要求1所述dsRNA的重组菌。
4.一种含有根据权利要求1所述dsRNA的重组载体。
5.根据权利要求1所述凡纳滨对虾钠钾ATPase alpha基因的dsRNA、或权利要求4所述重组载体在制备抑制WSSV的制剂中的用途。
6.一种含有根据权利要求1所述凡纳滨对虾ATPase alpha基因的dsRNA、权利要求4所述重组载体中的一种或者多种混合的制剂。
7.根据权利要求6所述制剂，其特征在于，所述制剂包括生物抑制剂、试剂盒、饲料添加剂中的一种或多种。
8.根据权利要求7所述制剂，其特征在于，所述制剂用于凡纳滨对虾。
9.根据权利要求6所述制剂，其特征在于，所述制剂可抑制WSSV增殖。
10.根据权利要求6所述制剂，其特征在于，所述制剂可降低WSSV感染导致的凡纳滨对虾的死亡。
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