CN109136200A - 一种重组传染性造血器官坏死病毒及其构建方法与应用 - Google Patents
一种重组传染性造血器官坏死病毒及其构建方法与应用 Download PDFInfo
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- CN109136200A CN109136200A CN201811100029.1A CN201811100029A CN109136200A CN 109136200 A CN109136200 A CN 109136200A CN 201811100029 A CN201811100029 A CN 201811100029A CN 109136200 A CN109136200 A CN 109136200A
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Abstract
本发明公开了一种重组传染性造血器官坏死病毒及其构建方法与应用。本发明所提供的重组传染性造血器官坏死病毒与改造前的传染性造血器官坏死病毒相比，差别仅在于在所述改造前的传染性造血器官坏死病毒的基因组RNA中的P基因和M基因之间还含有目的蛋白的编码基因(如传染性胰脏坏死病毒的VP2基因)。本发明以IHNV为亲本病毒，利用反向遗传操作技术体外拯救成功获得了能够表达传染性胰脏坏死病毒VP2基因的重组病毒rIHNV‑VP2。本发明所构建得到的重组病毒rIHNV‑VP2，通过系列试验证明了该重组病毒能够起到同时防治IHNV和IPNV的作用，本发明为进一步开展IHNV和IPNV的防治研究奠定了坚实的基础。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种重组传染性造血器官坏死病毒及其构建方法与应用。
背景技术
传染性造血器官坏死病(Infectious hematopoietic necrosis,IHN)和传染性胰脏坏死病(Infectious pancreatic necrosis,IPN)是最常见的、严重危害鲑鳟鱼健康的病毒性传染病，是目前造成世界范围内鲑鳟鱼产业重大经济损失的最主要的两种疾病。传染性造血器官坏死病毒(Infectious haematopoietic necrosis virus，IHNV)，属于弹状病毒科(Rhabdoviridae)，诺拉弹状病毒属(Novirhabdovirus)，为单股负链RNA病毒，其病毒基因组全长约为11kb，包含六个基因，分别编码病毒核蛋白(N)、磷蛋白(P)、基质蛋白(M)、糖蛋白(G)、非结构蛋白(NV)和聚合酶蛋白(L)。根据G蛋白基因序列可将世界范围内的IHNV分为U、M、L、E和J五种基因型。其中，U、M、L基因型主要流行于北美，E基因型流行于欧洲，J基因型流行于亚洲。根据致病毒株、环境因素、鱼龄的不同，IHNV可造成鲑鳟鱼高达100％的死亡率，是目前严重阻碍鲑鳟鱼健康可持续发展的头号杀手。传染性胰脏坏死病毒(Infectious pancreatic necrosis virus,IPNV)属于双片段RNA病毒科的水生双RNA病毒属。IPNV基因组由两条双链RNA组成(A片段和B片段)，A片段全长3092bp，编码106kDa多聚蛋白(NH2-VP2-VP4-VP3-COOH)，其中VP2和VP3是该病毒的两种主要结构蛋白，VP2是诱导保护性中和抗体的主要免疫原。B片段全长2777bp，编码蛋白pVP1(94kDa)，是病毒粒子依赖的RNA聚合酶。由于毒株、宿主及环境因素的不同，IPNV可造成10％～90％死亡率。IHN和IPN主要危害鱼苗和幼鱼阶段的虹鳟(Onchorhyncus mykiss)、美洲红点鲑(Salvelinusfontinalis)、褐鳟(Salmo trutta)、大西洋鲑(Salmo salar)和大麻哈属(Oncorhynchusspp.)的鱼苗及幼鱼，是目前严重阻碍鲑鳟鱼健康可持续发展的主要病毒性传染病。
与经典的从表型改变到进行基因特征研究的思路相反，反向遗传操作技术(reverse genetics)是指通过构建RNA病毒的感染性分子克隆，在病毒cDNA分子水平上对其进行体外人工操作(如进行基因点突变、缺失、插入、颠换、转位和互补等改造)，改变病毒的某些特征(如减低病毒的毒力，提高病毒的侵染力等等)，将病毒的cDNA分子连入表达载体，转染细胞获得病毒的感染性克隆，也被称为“病毒拯救”(the rescue of virus)。通过反向遗传操作技术可以研究RNA病毒的基因复制与表达调控机理、RNA编辑和自发重组与诱导重组、病毒与宿主间的相互作用关系、抗病毒策略、基因治疗研究，以及构建新型病毒载体来表达外源基因和进行疫苗的研制等。
发明内容
本发明的目的是提供一种重组传染性造血器官坏死病毒及其构建方法与应用。
第一方面，本发明要求保护一种重组传染性造血器官坏死病毒。
本发明所提供的重组传染性造血器官坏死病毒与改造前的传染性造血器官坏死病毒相比，差别仅在于在所述改造前的传染性造血器官坏死病毒的基因组RNA中的P基因和M基因之间还含有目的蛋白的编码基因。
本发明所提供的重组传染性造血器官坏死病毒是在传染性造血器官坏死病毒(IHNV)的基因组RNA中的P基因和M基因之间插入目的蛋白的编码基因后得到的重组病毒。
其中，所述P基因编码传染性造血器官坏死病毒(IHNV)的磷蛋白(P)；所述M基因编码传染性造血器官坏死病毒(IHNV)的基质蛋白(M)。
进一步地，所述目的蛋白的编码基因可为靶标病毒的抗原基因或标记蛋白的编码基因。
更进一步地，所述靶标病毒可为传染性造血器官坏死病毒外的其他病毒，如传染性胰脏坏死病毒(IPNV)等；相应的，所述靶标病毒的抗原基因具体可为传染性胰脏坏死病毒(IPNV)的VP2基因等。所述标记蛋白可为绿色荧光蛋白等。
在本发明的具体实施方式中，所述改造前的传染性造血器官坏死病毒(IHNV)具体为传染性造血器官坏死病毒BLk94株(IHNV-BLk94)。所述传染性胰脏坏死病毒(IPNV)具体为传染性胰脏坏死病毒ChRtm213株(IPNV-ChRtm213)。
进一步地，所述改造前的传染性造血器官坏死病毒(IHNV)的基因组RNA中的所述P基因编码SEQ ID No.1所示的磷蛋白(P)；所述改造前的传染性造血器官坏死病毒(IHNV)的基因组RNA中的所述M基因编码SEQ ID No.2所示的基质蛋白(M)。
对应于基因水平，所述改造前的传染性造血器官坏死病毒(IHNV)的基因组RNA中的所述P基因的编码区核苷酸序列如SEQ ID No.4所示；所述改造前的传染性造血器官坏死病毒(IHNV)的基因组RNA中的所述M基因的编码区核苷酸序列如SEQ ID No.5所示。
进一步地，所述传染性胰脏坏死病毒(IPNV)的VP2基因编码SEQ ID No.3所示的VP2蛋白。
对应于基因水平，所述传染性胰脏坏死病毒(IPNV)的VP2基因的核苷酸序列如SEQID No.6所示。
更进一步地，所述重组传染性造血器官坏死病毒与改造前的传染性造血器官坏死病毒相比，差别仅在于将所述改造前的传染性造血器官坏死病毒的基因组RNA中的P基因的编码区和所述M基因的编码区之间的A片段(图3中A中的“P GE-M GS”)替换为了片段B(图3中B中的“P GE-M GS-VP2gene-P GE-M GS”)。其中，所述片段A的序列为SEQ ID No.7；所述片段B的序列为SEQ ID No.8(即SEQ ID No.7+SEQ ID No.6+SEQ ID No.7)。
更加具体地，所述重组传染性造血器官坏死病毒可按照如下第二方面中所述方法制备获得。
第二方面，本发明要求保护一种制备前文第一方面中所述的重组传染性造血器官坏死病毒的方法。
本发明所提供的制备前文第一方面中所述的重组传染性造血器官坏死病毒的方法，可包括如下步骤：将含有所述重组传染性造血器官坏死病毒的基因组RNA对应的cDNA序列的重组质粒和辅助质粒共转染EPC细胞，从而获得所述重组传染性造血器官坏死病毒。
其中，所述“含有所述重组传染性造血器官坏死病毒的基因组RNA对应的cDNA序列的重组质粒”中启动所述重组传染性造血器官坏死病毒的基因组RNA对应的cDNA序列表达的启动子为T7启动子，在所述重组传染性造血器官坏死病毒的基因组RNA对应的cDNA序列后还含有丁肝病毒核酶的编码序列和T7终止序列(记作pIHNV-VP2)。所述辅助质粒共有4个：含有所述改造前的传染性造血器官坏死病毒的基因组RNA中N基因(核蛋白N的编码基因)的编码区对应的cDNA序列的辅助质粒1(记作pHelp-N)；含有所述改造前的传染性造血器官坏死病毒的基因组RNA中P基因(磷蛋白P的编码基因)的编码区对应的cDNA序列的辅助质粒2(记作pHelp-P)；含有所述改造前的传染性造血器官坏死病毒的基因组RNA中NV基因(非结构蛋白NV的编码基因)的编码区对应的cDNA序列的辅助质粒3(记作pHelp-Nv)；含有所述改造前的传染性造血器官坏死病毒的基因组RNA中L基因(聚合酶蛋白L的编码基因)的编码区对应的cDNA序列的辅助质粒4(记作pHelp-L)。
在所述方法中，进行所述共转染时，所述pIHNV-VP2、所述pHelp-N、所述pHelp-P、所述pHelp-Nv和所述pHelp-L的质量配比为2.0：1.0：0.5：0.25：0.5。
在所述方法中，当60％以上的细胞出现CPE时，收获所述重组传染性造血器官坏死病毒。
第三方面，本发明要求保护如下(a1)或(a2)或(a3)的生物材料：
(a1)含有前文所述的重组传染性造血器官坏死病毒的离体动物细胞或重组菌；
(a2)含有前文所述的重组传染性造血器官坏死病毒的基因组RNA或cDNA的载体；
(a3)含有前文所述的重组传染性造血器官坏死病毒的疫苗。
由于IHNV-BLk94该病毒为弱毒株，直接注射免疫60d后即可进行免疫效果检测，无需灭活处理。
第四方面，本发明要求保护如下任一应用：
(b1)前文所述的重组传染性造血器官坏死病毒或所述的生物材料在制备用于预防和/或治疗由于传染性造血器官坏死病毒(IHNV)和/或前文所述的靶标病毒感染所致疾病的产品中的应用；
(b2)前文所述的重组传染性造血器官坏死病毒或所述的生物材料在制备用于抑制传染性造血器官坏死病毒(IHNV)和/或前文所述的靶标病毒感染的产品中的应用；
(b3)前文所述的重组传染性造血器官坏死病毒或所述的生物材料在预防和/或治疗由于传染性造血器官坏死病毒(IHNV)和/或前文所述的靶标病毒感染所致疾病中的应用；
(b4)前文所述的重组传染性造血器官坏死病毒或所述的生物材料在抑制传染性造血器官坏死病毒(IHNV)和/或前文所述的靶标病毒感染中的应用。
在本发明的具体实施方式中，所述靶标病毒具体为传染性胰脏坏死病毒(IPNV)。所述传染性造血器官坏死病毒(IHNV)和/或传染性胰脏坏死病毒(IPNV)感染为感染虹鳟鱼。
本发明以IHNV毒株BLk94为亲本病毒，利用反向遗传操作技术体外拯救成功获得了rIHNV-BLk94。本发明将IPNV的结构蛋白VP2基因插入到rIHNV-BLk94基因组的P与M基因之间，构建得到了重组病毒rIHNV-VP2。通过系列试验证明了该重组病毒能够起到同时防治IHNV和IPNV的作用，本发明为进一步开展IHNV和IPNV的防治研究奠定了坚实的基础。
附图说明
图1为rIHNV-BLk94毒株全长cDNA序列质粒构建策略图。
图2为重组质粒pIHNV-GFP-N/P的构建策略图。
图3为本发明将IPNV毒株的VP2基因插入到rIHNV-BLk94基因组的P与M基因之间插入位置示意图。A为rIHNV-BLk94基因组相关区域示意图；B为重组病毒rIHNV-VP2基因组相关区域示意图。
图4为rIHNV-VP2毒株全长cDNA序列质粒构建策略图。
图5为IHNV-BLk94全基因组序列的分段克隆。H1、H2、H3为IHNV-BLk94全基因组序列分段克隆产物；M为DL15000DNA分子量标准；pFLC-LaSota真核表达载体骨架扩增。
图6为表达GFP的各重组质粒PCR鉴定结果。M为DL2000DNA分子量标准；pIHNV-GFP-N/P、pIHNV-GFP-P/M、pIHNV-GFP-M/G、pIHNV-GFP-G/NV、pIHNV-GFP-NV/L分别为各重组质粒PCR鉴定结果。
图7为IHNV-BLk94辅助质粒的克隆。N、P、Nv和L为IHNV-BLk94基因片段；M1为DL15000DNA分子量标准；M2为DL2000DNA分子量标准。
图8为各重组病毒的TCID50检测。
图9为各重组病毒的生长曲线。
图10为各重组病毒的GFP mRNA表达水平。
图11为各重组病毒的GFP蛋白表达水平。
图12为重组病毒rIHNV-VP2的细胞病变。
图13为IHNV攻毒后保护率检测。
图14为IPNV攻毒后虹鳟体内病毒载量检测。
具体实施方式
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。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所涉及的传染性造血器官坏死病毒BLk94株(IHNV-BLk94)的基因组RNA中的P基因的编码区核苷酸序列如SEQ ID No.4所示，其编码SEQ ID No.1所示的磷蛋白(P)。IHNV-BLk94的基因组RNA中的M基因的编码区的核苷酸序列如SEQ ID No.5所示，其编码SEQ ID No.2所示的基质蛋白(M)。在IHNV-BLk94的基因组RNA中位于所述P基因的编码区和所述M基因的编码区之间的RNA片段(对应图3中A中的“P GE-M GS”)的序列如SEQ IDNo.7所示。
下述实施例中所涉及的传染性胰脏坏死病毒ChRtm213株(IPNV-ChRtm213)的VP2基因的编码区的核苷酸序列如SEQ ID No.6所示，其编码SEQ ID No.3所示的VP2蛋白。
实施例1、重组病毒rIHNV-VP2的构建及应用
一、材料与方法BLk94
(一)实验材料与试剂
Trizol为Invitrogen公司(10296028)产品；传染性造血器官坏死病毒BLk94株(简称IHNV-BLk94)(Genbank登录号：DQ164100)和传染性胰脏坏死病毒ChRtm213株(简称IPNV-ChRtm213)(Genbank登录号：KX234591)由本实验室保存；EPC细胞ATCC CRL-2872；含有丁肝病毒核酶的pBluescript II SK改造载体pFLC-LaSota，辅助质粒载体pTM-NP由本实验室保存(参考文献“张振宇.新型重组新城疫病毒(NDV)表达系统的建立及最佳外源基因表达位置的确定[D].黑龙江:东北农业大学，2015:16-19.”)。细胞培养液MEM、胰蛋白酶、Hank’s液为Hyclone生物公司产品。胎牛血清为Gibco生物公司产品。RT-PCR一步法试剂盒(12574035)、脂质体2000为Invitrogen生物公司产品。In-Fusion PCR Cloning Kit试剂盒为Clontech公司(600670)产品。抗IHNV抗体由本实验室制备保存，该抗体为表达的IHNV病毒的表面糖蛋白为免疫原，免疫新西兰大白兔得到的多克隆抗体(参考文献为“赵景壮,徐黎明,刘淼,等.鱼类传染性造血器官坏死病病毒糖蛋白的截短表达及免疫原性检测[J].细胞与分子免疫学杂志,2014,30(12):1238-1242.”)，荧光标记二抗为Ebioscience生物公司。
(二)引物设计与合成
根据IHNV-BLk94全基因组序列及pFLC-LaSota真核表达载体骨架序列，利用Primeprimer 5.0软件设计特异性引物，用于构建重组病毒rIHNV-BLk94全基因组cDNA序列的真核转录质粒，引物序列如表1所示。
表1 rIHNV-BLk94全基因组cDNA序列克隆引物
| 引物 | 序列(5’→3’) |
| IH1-F | 5’-CGACTCACTATAGGGGTATAAAAAAAGTAACTTGACTA-3’ |
| IH1-R | 5’-AATCCAATCATACAGGCCCGATGCAGT-3’ |
| IH2-F | 5’-CTGTATGATTGGATTCTGTGGGGGGCAGTGGATAC-3’ |
| IH2-R | 5’-ACTTTGTTGTTGACGCGCTCT-3’ |
| IH3-F | 5’-TGTCAACAACAAAGTCGGGGTGCATCTCTTT-3’ |
| IH3-R | 5’-GGGACCATGCCGGCCGTATAAAAAAAGTAACAGAGAGA-3’ |
| pB-F | 5’-GGCCGGCATGGTCCCAGCCTCCTCG-3’ |
| pB-R | 5’-CCCTATAGTGAGTCGTATTAGCGGC-3’ |
根据IHNV-BLk94全基因组序列及辅助质粒载体pTM-NP，利用Prime primer 5.0软件设计特异性引物，分别用于构建重组病毒rIHNV-BLk94核蛋白(N)、磷蛋白(P)、非结构蛋白(NV)和聚合酶蛋白(L)cDNA序列的真核辅助质粒，引物序列如表2所示。
表2 rIHNV-BLk94辅助质粒cDNA序列克隆引物
根据IHNV-BLk94全基因组序列及GFP基因序列，利用Prime primer 5.0软件设计特异性引物，用于构建rIHNV-BLk94载体表达GFP重组质粒，引物序列如表3所示。
表3构建rIHNV-BLk94载体表达GFP质粒克隆引物
根据IPNV毒株的VP2基因序列，利用Prime primer 5.0软件设计特异性引物用于构建rIHNV-VP2载体表达VP2重组质粒引物序列如表4所示。
表4构建rIHNV-VP2重组质粒克隆引物
| 引物 | 序列(5’→3’) |
| ZT-F | 5’-ATGTCCATTTTCAAGAGAGCAAAGA-3’ |
| ZT-R | 5’-GCTCTCGTTTGAACTGACTCTTGGACTT-3’ |
| VP2-F | 5’-AGTTCAAACGAGAGCATGAACACATCCAAGGCAACCGCAA-3’ |
| VP2-R | 5’-CTTGAAAATGGACATTCATGCCTTTGAGGTTGGTAGGTCA-3’ |
(三)病毒的扩增与RNA提取
将IHNV-BLk94和IPNV-ChRtm213的病毒悬液分别用细胞维持液(含有2％FBS的MEM培养基)稀释105倍，接种于单层汇合的EPC细胞，于15℃培养。当80％以上细胞出现细胞病变(cytopathic effect，CPE)时，收集细胞培养液(即病毒悬液)，取250μL病毒悬液(调整至含有105病毒粒子)于无RNA酶的离心管中。12 000g离心5min，去除沉淀。加入750μL Trizol裂解液震荡混匀，加入200μL酚氯仿混匀，10min后12 000g离心15min，吸取上清液加入到新的离心管中，加入等体积的异丙醇，反复颠倒混匀，12 000g离心15min，弃尽上清，加入75％的乙醇1mL，颠倒洗涤，12 000g离心10min，弃尽上清，4000离心10s，吸干管底的液体，室温干燥3min；加入100μL的无RNA酶水，将RNA沉淀完全溶解，分装于-80℃备用。
(四)rIHNV-BLk94毒株全长cDNA序列质粒及真核辅助质粒的构建
1、rIHNV-BLk94毒株全长cDNA序列质粒的构建
rIHNV-BLk94毒株全长cDNA序列质粒的构建策略如图1所示。
以步骤(三)提取的IHNV-BLk94RNA为模板、表1的序列为引物，利用RT-PCR一步反应试剂盒扩增涵盖IHNV-BLk94毒株全长基因组的IH1、IH2、IH3片段。扩增IH1片段采用引物IH1-F和IH1-R；扩增IH2片段采用引物IH2-F和IH2-R；扩增IH3片段采用引物IH3-F和IH3-R。然后，按照Pfu UltraTMII Fusion HS DNA聚合酶说明书，以实验室保存的pFLC-LaSota真核表达载体为模板，利用表1的序列(pB-F和pB-R)为引物，扩增pFLC-LaSota真核表达载体骨架片段。利用胶回收试剂盒对以上目的基因片段回收，根据In-Fusion PCR Cloning Kit说明书连接IH1、IH2、IH3及pFLC-LaSota真核表达载体骨架片段，构建出含有rIHNV-BLk94毒株全长cDNA序列的质粒pIHNV-BLk94，并经测序验证正确。
2、真核辅助质粒的构建
以步骤(三)提取的IHNV-BLk94RNA为模板、表2的序列为引物，利用RT-PCR一步反应试剂盒分别扩增IHNV-BLk94毒株N、P、NV和L基因片段。扩增N基因片段采用引物IHN-F和IHN-R；扩增P基因片段采用引物IHP-F和IHP-R；扩增NV基因片段采用引物IHNv-F和IHNv-R；扩增L基因片段采用引物IHL-F和IHL-R。按照Pfu UltraTMII Fusion HS DNA聚合酶说明书，以实验室保存的辅助质粒载体pTM-NP为模板，利用表2的序列(phelp-F和phelp-R)为引物，扩增辅助质粒载体pTM-NP骨架片段。利用胶回收试剂盒对以上目的基因片段回收，根据In-Fusion PCR Cloning Kit说明书分别将N、P、NV和L基因片段与辅助质粒载体pTM-NP骨架片段连接，构建出拯救病毒用辅助质粒pHelp-N、pHelp-P、pHelp-Nv和pHelp-L。并经测序验证正确。
(五)GFP亚克隆的构建
以步骤四1中构建的pIHNV-BLk94全长cDNA为模板，表3中的NF和NR为引物，将IHNV的N基因与M基因之间的序列克隆到pcDNA3.1载体中，得到IHNV P基因亚克隆pcDNA3.1-NM/P+。
将利用p519gfp质粒(ATCC：87452)为模板，表3中的GFP insert F和GFP insert R为引物，扩增得到GFP片段；利用pcDNA3.1-NM/P+为模板，表3中的GFP Vet F和GFP Vet R为引物，扩增得到去除P基因开放阅读框的载体片段pcDNA3.1-NM/P-；将GFP片段与pcDNA3.1-NM/P-载体连接后，得到包含有Gene end-Gene start-GFP的GFP亚克隆(GFP template)，并经测序验证正确，方便于后期重组质粒的构建。
(六)重组质粒pIHNV-GFP-N/P、pIHNV-GFP-P/M、pIHNV-GFP-M/G、pIHNV-GFP-G/NV、pIHNV-GFP-NV/L的构建及病毒的拯救
为了确定IHNV表达外源基因的最佳位置，本发明先后将指示蛋白GFP插入到了IHNV基因组的N和P基因之间、P和M之间、M和G之间、G和NV之间以及NV和L之间，构建了一系列插入位置不同的pIHNV-GFP质粒。
下面以外源GFP基因插入到N和P基因之间为例，详细描述系列重组质粒的构建与拯救策略，具体策略如图2所示。
1、重组质粒pIHNV-GFP-N/P的构建
分别以步骤五得到的GFP template及步骤四1中得到的pIHNV-BLk94质粒为模板，利用表3中的引物扩增GFP N/P片段及pIHNV-GFP-N/P Vet片段。扩增GFP N/P片段时采用的引物为N/P insert F和N/P insert R；扩增pIHNV-GFP-N/P Vet片段时采用的引物为N/PVet F和N/P Vet R。将PCR得到的片段分别胶回收后进行连接，连接产物转化大肠杆菌DH5α感受态细胞，挑取单个菌落鉴定正确的阳性质粒命名为pIHNV-GFP-N/P质粒(并经测序验证正确)。
2、重组病毒rIHNV-GFP-N/P的拯救
取pIHNV-GFP-N/P质粒2.0μg，辅助质粒pHelp-N 1.0μg、pHelp-P 0.5μg、pHelp-Nv0.25μg和pHelp-L 0.5μg共转染EPC细胞，具体操作方法按照Invitrogen公司的Lipofectamine 2000转染操作说明书进行。将转染后的细胞置于15℃培养箱培养，每天观察细胞状态，当60％以上的细胞出现CPE时，收获病毒悬液置于-80℃分装保存备用。
3、重组质粒pIHNV-GFP-P/M、pIHNV-GFP-M/G、pIHNV-GFP-G/NV、pIHNV-GFP-NV/L的构建及病毒的拯救
重组质粒pIHNV-GFP-P/M、pIHNV-GFP-M/G、pIHNV-GFP-G/NV、pIHNV-GFP-NV/L的构建与病毒的拯救除了GFP插入片段及载体扩增时所用引物有所不同，其他步骤均与pIHNV-GFP-N/P的构建及病毒拯救相同。扩增GFP P/M片段时采用的引物为表3中的P/M insert F和P/M insert R，扩增pIHNV-GFP-P/M Vet片段时采用的引物为表3中的P/M Vet F和P/MVet R；扩增GFP M/G片段时采用的引物为表3中的M/G insert F和M/G insert R，扩增pIHNV-GFP-M/G Vet片段时采用的引物为表3中的M/G Vet F和M/G Vet R；扩增GFP G/NV片段时采用的引物为表3中的G/NV insert F和G/NV insert R，扩增pIHNV-GFP-G/NV Vet片段时采用的引物为表3中的G/NV Vet F和G/NV Vet R；扩增GFP NV/L片段时采用的引物为表3中的NV/L insert F和NV/L insert R，扩增pIHNV-GFP-NV/L Vet片段时采用的引物为表3中的NV/L Vet F和NV/L Vet R。
(七)拯救病毒rIHNV-GFP-N/P、rIHNV-GFP-P/M、rIHNV-GFP-M/G、rIHNV-GFP-G/NV及rIHNV-GFP-NV/L的TCID50测定
取生长状态良好的EPC细胞，消化后按照2×104个/100μL分装于96孔板，接种各分离株不同稀释度的病毒悬液，每个稀释度8孔，每孔100μL同时设立空白对照组，15℃培养，观察7天后，利用Reed-Muench法计算TCID50。
(八)拯救病毒rIHNV-GFP-N/P、rIHNV-GFP-P/M、rIHNV-GFP-M/G、rIHNV-GFP-G/NV及rIHNV-GFP-NV/L的生长曲线
取生长状态良好的EPC细胞，消化后按照2×104个/100μL分装于96孔板，接种各分离株不同稀释度的病毒悬液，每个稀释度8孔，每孔100μL同时设立空白对照组，15℃培养，每12h收取一次病毒，利用Reed-Muench法计算TCID50绘制病毒生长曲线。
(九)转录水平检测拯救病毒rIHNV-GFP-N/P、rIHNV-GFP-P/M、rIHNV-GFP-M/G、rIHNV-GFP-G/NV及rIHNV-GFP-NV/L GFP mRNA的表达量
接种EPC单层细胞于6孔细胞培养板内，接种病毒前更换为含有10％血清的MEM培养基。以0.1MOI(Multiplicity of infection)分别接种各拯救病毒于6孔板内，15℃培养1h后更换为2％血清的维持液。15℃，0.5％CO2条件下培养48h后提取病毒RNA，利用OneStep SYBR PrimeScript PLUS RT-PCR Kit(RR096A,Takara,Japan)对重组病毒GFP mRNA的表达量进行检测，采用的检测引物为RT-GFP F：5′-CGAGGTGGTGTACATGAACGA-3′，RT-GFPR：5′-GCTGTAGAACTTGCCGCTGTT-3′，内参基因为病毒的N蛋白基因，内参引物为RT-N F：5′-AGGAGAGGGAACGAGAAGG-3′，RT-N R：5′-TGTTGGGATCTGCGAAAGTG-3′。
(十)重组病毒rIHNV-GFP-N/P、rIHNV-GFP-P/M、rIHNV-GFP-M/G、rIHNV-GFP-G/NV及rIHNV-GFP-NV/L GFP蛋白表达量的检测
接种EPC单层细胞于6孔细胞培养板内，接种病毒前更换为含有10％血清的MEM培养基。以0.1MOI(Multiplicity of infection)分别接种各拯救病毒于6孔板内，15℃培养1h后更换为2％血清的维持液。15℃，0.5％CO2条件下培养48h后于利用胰酶将细胞消化下来，PBS洗涤2遍后利用流式细胞仪检测各重组病毒绿色荧光蛋白GFP的表达情况。
(十一)rIHNV-VP2毒株全长cDNA序列质粒构建
前期研究结果证明了IHNV mRNA转录量按照基因组3’至5’的顺序依次递减，并且与其他插入位点相比，当GFP插入到rIHNV-BLk94载体的P与M基因之间时，GFP蛋白的表达量最高。这说明，综合考虑IHNV基因组mRNA的转录特性与外源基因对病毒复制所产生的影响，rIHNV-BLk94基因组P与M基因之间的非编码区是表达外源基因的最佳位置。因此本发明将IPNV毒株的VP2基因插入到rIHNV-BLk94基因组的P与M基因之间，构建出含有IPNV毒株VP2基因的重组病毒rIHNV-VP2(图3)。
以步骤(三)提取的IPNV-ChRtm213的RNA为模板、表4的VP2-F和VP2-R为引物，利用RT-PCR一步反应试剂盒扩增涵盖IPNV毒株VP2基因的目的片段。以步骤六3中构建的pIHNV-GFP-P/M重组质粒为模板，利用表4的ZT-F和ZT-R为引物，扩增去除GFP基因的载体序列。利用Pfu UltraTMII Fusion HS DNA聚合酶，连接VP2基因目的片段和去除GFP基因的载体序列，构建出含有VP2 cDNA序列的重组质粒pIHNV-VP2，构建策略如图4所示。
(十二)重组病毒rIHNV-VP2毒株的拯救
取pIHNV-VP2质粒2.0μg，辅助质粒pHelp-N 1.0μg、pHelp-P 0.5μg、pHelp-Nv0.25μg和pHelp-L 0.5μg共转染EPC细胞，具体操作方法按照Invitrogen公司的Lipofectamine 2000转染操作说明书进行。将转染后的细胞置于15℃培养箱培养，每天观察细胞状态，当60％以上的细胞出现CPE时，收获病毒悬液置于-80℃分装保存备用。
(十三)重组病毒rIHNV-VP2的应用
将收获的重组病毒rIHNV-VP2注射免疫SPF虹鳟(5±1g)，每组免疫50尾，免疫剂量为每尾50pfu/尾，对照组采用PBS进行免疫注射。在免疫后的60d利用IHNV(102pfu/尾)及IPNV(106pfu/尾)病毒分别感染，进行免疫保护检测。
二、结果与分析
1、rIHNV-BLk94毒株全长cDNA质粒构建
IHNV-BLk94全基因组序列长11132bp，本发明将其分为IH1、IH2、IH3三段基因片段进行克隆，其大小依次为3756bp、3680bp、3726bp，经RT-PCR一步反应试剂盒扩增后的产物电泳分析结果显示，3段基因片段大小与预期结果相符(见图5)。应用特异性引物对pFLC-LaSota真核表达载体骨架序列进行扩增，电泳结果显示载体大小与预期结果相符3140bp(见图5)。根据In-Fusion PCR Cloning Kit说明书连接IH1、IH2、IH3及pFLC-LaSota真核表达载体骨架片段，构建出含有IHNV-BLk94毒株全长cDNA序列的质粒pIHNV-BLk94。
2、重组质粒pIHNV-GFP-N/P、pIHNV-GFP-P/M、pIHNV-GFP-M/G、pIHNV-GFP-G/NV、pIHNV-GFP-NV/L的构建
利用特异性的引物分别扩增GFP N/P片段、GFP P/M片段、GFP M/G片段、GFP G/NV片段、GFP NV/L片段以及pIHNV-GFP-N/P Vet载体片段、pIHNV-GFP-P/M Vet载体片段、pIHNV-GFP-M/G Vet载体片段、pIHNV-GFP-G/NV Vet载体片段、pIHNV-GFP-NV/L Vet载体片段，分别将目的片段与载体连接得到重组质粒pIHNV-GFP-N/P、pIHNV-GFP-P/M、pIHNV-GFP-M/G、pIHNV-GFP-G/NV和pIHNV-GFP-NV/L，构建成功的重组质粒PCR鉴定结果如图6所示。
3、rIHNV-GFP毒株真核辅助质粒的构建
以IHNV-BLk94毒株的RNA为模板，利用特异性引物分别扩增IHNV-BLk94毒株N、P、Nv和L基因片段，其大小分别为1176bp、693bp、336bp和5961bp(见图7)。同时用特异性引物扩增pFLC-LaSota载体骨架片段，根据In-Fusion PCR Cloning Kit说明书分别将N、P、Nv和L基因片段与pFLC-LaSota载体骨架片段连接，构建出拯救rIHNV-GFP毒株辅助质粒phelp-N、phelp-P、phelp-NV和phelp-L。
4、拯救病毒rIHNV-GFP-N/P、rIHNV-GFP-P/M、rIHNV-GFP-M/G、rIHNV-GFP-G/NV及rIHNV-GFP-NV/L的TCID50测定
为了确定不同位点插入GFP基因后，重组病毒感染能力的变化情况，本发明分别对各重组病毒的TCID50进行了检测。结果表明，各拯救病毒的TCID50滴度与亲本病毒rIHNV-BLk94相似，说明拯救病毒rIHNV-GFP-N/P、rIHNV-GFP-P/M、rIHNV-GFP-M/G、rIHNV-GFP-G/NV及rIHNV-GFP-NV/L保持了其亲本病毒的感染能力(图8)。
5、拯救病毒rIHNV-GFP-N/P、rIHNV-GFP-P/M、rIHNV-GFP-M/G、rIHNV-GFP-G/NV及rIHNV-GFP-NV/L的生长曲线
为了研究拯救病毒的复制特性及其生长动力学特性，本发明将病毒稀释10-5倍后分别感染EPC细胞，之后每12h收获一次病毒，并对不同感染时间点病毒的TCID50进行检测，绘制感染病毒的生长曲线(图9)。如图所示，表达GFP蛋白的各重组病毒在同一时间点上均匀rIHNV-BLk94亲本病毒保持了相似的地图，表现出了与rIHNV-BLk94亲本病毒相似的复制特性及生长动力学特性。但是与其他表达GFP的重组病毒相比rIHNV-GFP-N/P的生长速度稍显滞后，大约落后其他病毒0.5-1个滴度。虽然rIHNV-GFP-P/M病毒在同一时间点上略高于rIHNV-GFP-N/P，但是仍明显低于其他拯救病毒。同一时间点病毒的滴度由高到低依次为：rIHNV-GFP-N/P、rIHNV-GFP-P/M、rIHNV-GFP-M/G、rIHNV-GFP-G/NV、rIHNV-GFP-NV/L。这说明，外源蛋白GFP的插入对于病毒的复制有很大的影响，并且插入基因越接近基因组的3’端对病毒的复制效率影响越大。
6、转录水平检测拯救病毒rIHNV-GFP-N/P、rIHNV-GFP-P/M、rIHNV-GFP-M/G、rIHNV-GFP-G/NV及rIHNV-GFP-NV/L的表达量
利用特异性Real-time PCR引物，以病毒的N蛋白基因作为内参，在转录水平对各拯救病毒表达外源基因GFP的表达量进行了检测(图10)。结果显示，各拯救病毒GFP mRNA的表达量呈现rIHNV-GFP-N/P>rIHNV-GFP-P/M>rIHNV-GFP-M/G>rIHNV-GFP-G/NV>rIHNV-GFP-NV/L。
7、重组病毒rIHNV-GFP-N/P、rIHNV-GFP-P/M、rIHNV-GFP-M/G、rIHNV-GFP-G/NV及rIHNV-GFP-NV/L GFP蛋白表达量的检测
Real-time PCR结果显示，外源蛋白约靠近病毒基因组的3’端其mRNA的转录量越多；但是病毒的生长曲线却表明，外源基因越接近基因组的3’端对病毒的复制效率影响越大。为了确定外源蛋白的最佳表达位置，本研究对各拯救病毒的GFP蛋白表达量进行了流式细胞仪检测(图11)。由结果可以看出，拯救病毒rIHNV-GFP-P/M的GFP表达量最大，具体顺序为rIHNV-GFP-P/M>rIHNV-GFP-M/G>rIHNV-GFP-N/P>rIHNV-GFP-G/NV>rIHNV-GFP-NV/L。由以上结果可以看出，综合考虑IHNV基因组mRNA的转录特性与外源基因对病毒复制所产生的影响，将外源基因插入到IHNV-BLk94基因组的P基因与M基因之间可以获得外源基因的最高表达量。
8、rIHNV-VP2毒株全长cDNA质粒构建及病毒拯救
以IPNV-ChRtm213毒株的RNA为模板，利用特异性引物扩增IPNV毒株VP2基因片段，其大小为1329bp。应用特异性引物根据In-Fusion PCR Cloning Kit说明书将VP2基因替换掉pIHNV-GFP-P/M质粒的GFP基因，构建出含有IPNV毒株VP2基因片段的pIHNV-VP2重组质粒。将pIHNV-VP2、pHelp-N 1.0μg、pHelp-P、pHelp-Nv、和pHelp-L共转染EPC细胞后，拯救出重组病毒rIHNV-VP2，细胞病变结果如图12所示。
另外，对重组病毒rIHNV-VP2进行基因组序列测定，结果显示，重组病毒rIHNV-VP2与IHNV-BLk94相比，差别仅在于将IHNV-BLk94基因组RNA中位于P基因编码区(图3中A中的“P cds”)和M基因编码区(图3中A中的“M CDS”)之间的A片段(图3中A中的“P GE-M GS”)替换为了片段B(图3中B中的“P GE-M GS-VP2gene-P GE-M GS”)。其中，所述片段A的序列为SEQ ID No.7；所述片段B的序列为SEQ ID No.8(即SEQ ID No.7+SEQ ID No.6+SEQ IDNo.7)。
9、重组病毒rIHNV-VP2的应用
将重组病毒rIHNV-VP2稀释后，免疫SPF虹鳟，在免疫后的60d利用IHNV及IPNV病毒感染，分别对IHNV的免疫保护率及组织中IPNV病毒载量进行检测。由结果可以看出，经重组病毒rIHNV-VP2免疫的虹鳟鱼，在IHNV病毒感染时，与对照组(以PBS替代重组病毒rIHNV-VP2)相比重组病毒的免疫保护率约为85％(图13)；在IPNV病毒感染时，与对照组相比重组病毒的免疫保护组IPNV的病毒载量明显降低(图14)。以上结果说明重组病毒rIHNV-VP2对预防虹鳟IHNV及IPNV病毒感染具有良好的免疫保护效果，这一结果为我国虹鳟养殖业的健康发展提供了技术保障。
<110> 中国水产科学研究院黑龙江水产研究所
<120> 一种重组传染性造血器官坏死病毒及其构建方法与应用
<130> GNCLN181321
<160> 8
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Claims (10)
1.重组传染性造血器官坏死病毒，其特征在于：所述重组传染性造血器官坏死病毒与改造前的传染性造血器官坏死病毒相比，差别仅在于在所述改造前的传染性造血器官坏死病毒的基因组RNA中的P基因和M基因之间还含有目的蛋白的编码基因。
2.根据权利要求1所述的重组传染性造血器官坏死病毒，其特征在于：所述目的蛋白的编码基因为靶标病毒的抗原基因或标记蛋白的编码基因；
进一步地，所述靶标病毒的抗原基因为传染性胰脏坏死病毒的VP2基因；所述标记蛋白为绿色荧光蛋白；
更进一步地，所述传染性胰脏坏死病毒为传染性胰脏坏死病毒ChRtm213株。
3.根据权利要求1或2所述的重组传染性造血器官坏死病毒，其特征在于：所述改造前的传染性造血器官坏死病毒的基因组RNA中的所述P基因编码SEQ ID No.1所示的磷蛋白；和/或
所述改造前的传染性造血器官坏死病毒的基因组RNA中的所述M基因编码SEQ ID No.2所示的基质蛋白；和/或
所述传染性胰脏坏死病毒的VP2基因编码SEQ ID No.3所示的VP2蛋白。
4.根据权利要求3所述的重组传染性造血器官坏死病毒，其特征在于：所述改造前的传染性造血器官坏死病毒的基因组RNA中的所述P基因的编码区的核苷酸序列如SEQ ID No.4所示；和/或
所述改造前的传染性造血器官坏死病毒的基因组RNA中的所述M基因的编码区的核苷酸序列如SEQ ID No.5所示；和/或
所述传染性胰脏坏死病毒的VP2基因的编码区的核苷酸序列如SEQ ID No.6所示。
5.根据权利要求1-4中任一所述的重组传染性造血器官坏死病毒，其特征在于：所述重组传染性造血器官坏死病毒与所述改造前的传染性造血器官坏死病毒相比，差别仅在于将所述改造前的传染性造血器官坏死病毒的基因组RNA中位于所述P基因的编码区和所述M基因的编码区之间的A片段替换为了片段B；所述片段A的序列为SEQ ID No.7；所述片段B的序列为SEQ ID No.8。
6.根据权利要求1-5中任一所述的重组传染性造血器官坏死病毒，其特征在于：所述重组传染性造血器官坏死病毒按照权利要求7或8所述方法制备获得。
7.一种制备权利要求1-5中任一所述的重组传染性造血器官坏死病毒的方法，包括如下步骤：将含有所述重组传染性造血器官坏死病毒的基因组RNA对应的cDNA序列的重组质粒和辅助质粒共转染EPC细胞，从而获得所述重组传染性造血器官坏死病毒。
8.根据权利要求7所述的方法，其特征在于：所述“含有所述重组传染性造血器官坏死病毒的基因组RNA对应的cDNA序列的重组质粒”中启动所述重组传染性造血器官坏死病毒的基因组RNA对应的cDNA序列表达的启动子为T7启动子；
和/或
所述辅助质粒共有4个：含有所述改造前的传染性造血器官坏死病毒的基因组RNA中N基因的编码区对应的cDNA序列的辅助质粒1；含有所述改造前的传染性造血器官坏死病毒的基因组RNA中P基因的编码区对应的cDNA序列的辅助质粒2；含有所述改造前的传染性造血器官坏死病毒的基因组RNA中NV基因的编码区对应的cDNA序列的辅助质粒3；含有所述改造前的传染性造血器官坏死病毒的基因组RNA中L基因的编码区对应的cDNA序列的辅助质粒4。
9.如下(a1)或(a2)或(a3)的生物材料：
(a1)含有权利要求1-6中任一所述的重组传染性造血器官坏死病毒的离体动物细胞或重组菌；
(a2)含有权利要求1-6中任一所述的重组传染性造血器官坏死病毒的基因组RNA或cDNA的载体；
(a3)含有权利要求1-6中任一所述的重组传染性造血器官坏死病毒的疫苗。
10.如下任一应用：
(b1)权利要求1-6中任一所述的重组传染性造血器官坏死病毒或权利要求9所述的生物材料在制备用于预防和/或治疗由于传染性造血器官坏死病毒和/或权利要求2中所述的靶标病毒感染所致疾病的产品中的应用；
(b2)权利要求1-6中任一所述的重组传染性造血器官坏死病毒或权利要求9所述的生物材料在制备用于抑制传染性造血器官坏死病毒和/或权利要求2中所述的靶标病毒感染的产品中的应用；
(b3)权利要求1-6中任一所述的重组传染性造血器官坏死病毒或权利要求9所述的生物材料在预防和/或治疗由于传染性造血器官坏死病毒和/或权利要求2中所述的靶标病毒感染所致疾病中的应用；
(b4)权利要求1-6中任一所述的重组传染性造血器官坏死病毒或权利要求9所述的生物材料在抑制传染性造血器官坏死病毒和/或权利要求2中所述的靶标病毒感染中的应用；
进一步地，所述靶标病毒为传染性胰脏坏死病毒。
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| CN115887638A (zh) * | 2022-06-16 | 2023-04-04 | 中国水产科学研究院东海水产研究所 | 鲑科鱼类传染性造血器官坏死病及传染性胰腺坏死病重组腺病毒疫苗的加工方法 |
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| CN112852873A (zh) * | 2021-02-04 | 2021-05-28 | 华中农业大学 | 一种猪δ冠状病毒感染性克隆质粒的构建方法 |
| CN113122510A (zh) * | 2021-04-12 | 2021-07-16 | 中国水产科学研究院黑龙江水产研究所 | 一种传染性造血器官坏死病疫苗及其病毒在胖头鱥肌肉细胞上扩增的方法 |
| CN115887638A (zh) * | 2022-06-16 | 2023-04-04 | 中国水产科学研究院东海水产研究所 | 鲑科鱼类传染性造血器官坏死病及传染性胰腺坏死病重组腺病毒疫苗的加工方法 |
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