CN111378689B - 一种用于对虾的假型昆虫杆状病毒基因转移系统、病毒及构建方法和应用 - Google Patents
一种用于对虾的假型昆虫杆状病毒基因转移系统、病毒及构建方法和应用 Download PDFInfo
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Abstract
本发明涉及一种用于对虾的假型昆虫杆状病毒基因转移系统、病毒及构建方法和应用，属于基因工程技术领域。本发明所述用于对虾的假型昆虫杆状病毒基因转移系统，包括Bac‑to‑Bac昆虫杆状病毒表达系统、对虾病毒囊膜蛋白基因表达质粒和昆虫包装细胞。本发明所述用于对虾的假型昆虫杆状病毒基因转移系统能够在对虾组织和细胞中稳定高效地转移与表达外源基因。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种用于对虾的假型昆虫杆状病毒基因转移系统、病毒及构建方法和应用，即一种嗜对虾的假型昆虫杆状病毒基因转移系统、嗜对虾的假型昆虫杆状病毒及构建方法和在对虾成体组织与对虾细胞(体外培养细胞)中的应用。
背景技术
Bac-to-Bac昆虫杆状病毒表达系统(Invitrogen产品)，已是一种很成熟的昆虫细胞基因转移技术。该系统由转移质粒、辅助质粒、杆状病毒Bacmid质粒以及感受态细胞DH10Bac组成，是基于大肠杆菌转座酶Tn7的转座原理，成功实现在大肠杆菌中快速将外源基因重组到杆状病毒Bacmid质粒中，并扩增重组杆状病毒的目的。其中，转移质粒(pFASTBac1)的特点：在其昆虫强启动子PPH(多角体蛋白基因启动子)及其之后的多克隆酶切位点的两翼各插入转座酶Tn7的左右识别位点Tn7L和Tn7R；杆状病毒质粒(Bacmid)的特点：插入了原核细胞低拷贝质粒复制子(mini-F)、卡那霉素抗性基因以及插入了转座酶识别位点attTn7的LacZ基因(不移码插入)；辅助质粒为转座酶表达质粒；宿主细胞DH10Bac是稳定转化有辅助质粒和杆状病毒质粒Bacmid的大肠杆菌感受态细胞。
该昆虫杆状病毒表达系统的技术流程为：首先将外源基因例如GUS(β-D-glucoronidase，β-D-葡萄糖苷酸酶)克隆到转移质粒pFastBac1的多克隆酶切位点，纯化出重组质粒pFastBac-GUS；然后将其转化到大肠杆菌感受态细胞DH10Bac，然后在辅助质粒所表达的转座酶的作用下，GUS基因被转座到Bacmid质粒中的attTn7位点；提取和纯化Bacmid-GUS重组质粒；将Bacmid-GUS重组质粒转染包装细胞sf9，包装与纯化Bacmid-GUS重组病毒；包装病毒感染靶细胞，从而实现外源基因在靶细胞中的转移与表达。
但已有研究表明，该昆虫杆状病毒表达系统不能有效侵染对虾细胞，表明其在对虾细胞中的亲嗜性极低，不能应用于对虾组织和细胞的基因转移研究。
目前，在对虾成体和体外培养细胞中尚缺乏一种高效的基因转移与表达技术，严重阻碍转基因对虾和对虾基因编辑研究的顺利开展。
发明内容
本发明的目的在于提供一种用于对虾的假型昆虫杆状病毒基因转移系统、病毒及构建方法和应用。本发明所述用于对虾的假型昆虫杆状病毒基因转移系统能够在对虾组织和细胞中稳定高效地转移与表达外源基因。
本发明提供了一种用于对虾的假型昆虫杆状病毒基因转移系统，所述基因转移系统包括Bac-to-Bac昆虫杆状病毒表达系统、对虾病毒囊膜蛋白基因表达质粒和昆虫包装细胞。
优选的是，所述对虾病毒囊膜蛋白基因表达质粒包括对虾白斑综合症病毒囊膜蛋白基因表达质粒。
优选的是，所述对虾病毒囊膜蛋白基因表达质粒中所述囊膜蛋白基因的核苷酸序列如SEQ ID NO.1所示。
优选的是，所述对虾病毒囊膜蛋白基因表达质粒的构建用骨架载体包括pcDNA3.1。
本发明还提供了基于上述技术方案所述基因转移系统的用于对虾的假型昆虫杆状病毒的构建方法，包括以下步骤：
1)利用Bac-to-Bac昆虫杆状病毒表达系统构建得到含外源基因的昆虫杆状病毒重组质粒；
2)将步骤1)得到的昆虫杆状病毒重组质粒和对虾病毒囊膜蛋白基因表达质粒共转染昆虫包装细胞，得到用于对虾的假型昆虫杆状病毒。
本发明还提供了基于上述技术方案所述构建方法构建得到的用于对虾的假型昆虫杆状病毒。
本发明还提供了基于上述技术方案所述基因转移系统或上述技术方案所述用于对虾的假型昆虫杆状病毒在对虾细胞中表达外源基因的应用。
优选的是，所述对虾细胞包括对虾体外培养细胞，所述对虾体外培养细胞包括对虾原代培养血淋巴细胞。
本发明还提供了上述技术方案所述基因转移系统或上述技术方案所述用于对虾的假型昆虫杆状病毒在对虾成体组织中表达外源基因的应用。
优选的是，所述对虾成体组织包括对虾成体的鳃、心脏和肠组织。
本发明提供了一种用于对虾的假型昆虫杆状病毒基因转移系统。本发明所述基因转移系统是对现有Bac-to-Bac昆虫杆状病毒表达系统的改进，通过将对虾病毒囊膜蛋白基因表达质粒与Bac-to-Bac昆虫杆状病毒表达系统得到的含外源基因的昆虫杆状病毒重组质粒共转染昆虫包装细胞，将对虾病毒囊膜蛋白引入杆状病毒囊膜中，可显著提高所获得的假型杆状病毒的包装效率和嗜对虾细胞性，可成功应用于对虾成体及对虾细胞的基因转移研究中，从而弥补现有技术的不足。
本发明系统具有以下优势：(1)相对于市场上已有的Bac-to-Bac昆虫杆状病毒表达系统不能侵染对虾(如成体组织及其体外培养细胞等)的现状，本发明系统在对虾成体组织中的侵染和表达能力得到极大提高，可实现100％的侵染与表达效率；(2)本发明系统中的囊膜蛋白基因独立于杆状病毒质粒Bacmid而存在，只提高杆状病毒的包装效率以及侵染与表达效率，其质粒DNA遗传信息不会进入对虾细胞，提高了该系统的生物安全性；(3)本发明系统完全克服了对虾成体和对虾细胞(体外培养细胞)的基因转移难的问题，任一外源基因都可在对虾细胞中得到高效转移与表达。
附图说明
图1为本发明所克隆的VP28开放阅读框的电泳结果以及pcDNA-VP28质粒的图谱；
图2为本发明提供的重组杆状病毒质粒Bacmid-GUS的制备图；
图3为本发明提供的Bacmid-GUS质粒在sf9细胞中的最佳转染条件的优化；
图4为本发明提供的假型杆状病毒Bacmid-GUS/VP28在包装细胞Sf9中的最适包装条件研究结果及其与杆状病毒Bacmid-GUS的包装效率比较；
图5为本发明提供的两种杆状病毒Bacmid-GUS与Bacmid-GUS/VP28在Sf9细胞中的P2代扩增结果；
图6为本发明提供的杆状病毒Bacmid-GUS感染Sf9细胞96h后的GUS染色结果；
图7为本发明提供的假型杆状病毒Bacmid-GUS/VP28感染Sf9细胞96h后的结果；
图8为本发明提供的假型杆状病毒Bacmid-GUS/VP28可感染对虾原代培养血淋巴细胞，可检测到GUS基因的蛋白表达，而Bacmid-GUS病毒则不能成功感染对虾原代培养血淋巴细胞；
图9为本发明提供的两种杆状病毒Bacmid-GUS/VP28和Bacmid-GUS都不能感染对虾原代培养胚胎细胞，不能检测到GUS表达；
图10为本发明提供的杆状病毒Bacmid-GUS不能感染对虾成体各个组织；
图11为本发明提供的假型杆状病毒Bacmid-GUS/VP28可高效感染对虾成体的鳃、心脏和肠组织；
图12为本发明提供的假型杆状病毒Bacmid-GUS/VP28感染对虾1-4天的GUS表达结果。
具体实施方式
本发明提供了一种用于对虾的假型昆虫杆状病毒基因转移系统，所述基因转移系统包括Bac-to-Bac昆虫杆状病毒表达系统、对虾病毒囊膜蛋白基因表达质粒和昆虫包装细胞。具体的，本发明所述用于对虾的假型昆虫杆状病毒基因转移系统是通过制备一种假型昆虫杆状病毒的方法，从而实现对昆虫杆状病毒表达系统的成功改造，大大提高该系统在对虾成体组织及其体外培养细胞中的亲嗜性以及侵染与表达的效率，在本发明具体实施例中，制备得到的是在囊膜中不仅含有昆虫杆状病毒来源的囊膜蛋白，还含有对虾病毒来源的囊膜蛋白VP28的病毒。在本发明中，所述Bac-to-Bac昆虫杆状病毒表达系统包括转移质粒(pFASTBac1)、辅助质粒、杆状病毒质粒Bacmid以及感受态细胞DH10Bac。所述杆状病毒质粒Bacmid中携带了原核细胞低拷贝质粒复制子(mini-F)、卡那霉素抗性基因以及插入了转座酶识别位点attTn7的LacZ基因(不移码插入)。外源基因可被重组到转座酶识别位点attTn7，形成携带外源基因的昆虫杆状病毒重组质粒。
在本发明中，所述对虾病毒囊膜蛋白基因表达质粒包括对虾白斑综合症病毒囊膜蛋白基因表达质粒。在本发明中，所述对虾白斑综合症病毒(WSSV)囊膜蛋白优选包括VP28和VP19等。在本发明具体实施例中，所述对虾白斑综合症病毒囊膜蛋白优选为VP28，更优选为含有细胞肥大病毒(CMV)启动子驱动的改造过的VP28(本发明下文所称的VP28，均指改造过的对虾病毒囊膜蛋白VP28)，在本发明中，所述对虾病毒囊膜蛋白基因表达质粒中所述囊膜蛋白基因的核苷酸序列如SEQ ID NO.1所示，对应的氨基酸序列如SEQ ID NO.2所示。本发明所述含SEQ ID NO.1所示核苷酸序列的质粒可在昆虫sf9细胞中高效表达对虾病毒的VP28蛋白。在本发明中，所述对虾病毒囊膜蛋白基因表达质粒的构建用骨架载体包括pcDNA3.1，当所述囊膜蛋白为VP28时，相应的质粒为真核表达载体pcDNA-VP28。本发明通过将pcDNA-VP28质粒与昆虫杆状病毒(Bacmid)重组质粒共转染包装细胞，可显著提高昆虫杆状病毒的包装效率，同时将其引入所包装病毒的囊膜中，大大提高其嗜对虾细胞性。
本发明还提供了基于上述技术方案所述基因转移系统的用于对虾的假型昆虫杆状病毒的构建方法，包括以下步骤：
1)利用Bac-to-Bac昆虫杆状病毒表达系统构建得到含外源基因的昆虫杆状病毒重组质粒；
2)将步骤1)得到的昆虫杆状病毒重组质粒和对虾病毒囊膜蛋白基因表达质粒共转染昆虫包装细胞，得到用于对虾的假型昆虫杆状病毒。
本发明利用Bac-to-Bac昆虫杆状病毒表达系统构建得到含外源基因的昆虫杆状病毒重组质粒。本发明对所述构建的方法没有特殊限定，采用本领域技术人员熟知的Bac-to-Bac昆虫杆状病毒表达系统使用方法即可。
得到昆虫杆状病毒重组质粒后，本发明将昆虫杆状病毒重组质粒和对虾病毒囊膜蛋白基因表达质粒共转染昆虫包装细胞，得到用于对虾的假型昆虫杆状病毒。本发明对所述共转染的条件没有特殊限定，具体实施例中，本发明Bacmid-GUS重组病毒质粒与pcDNA3.1-VP28共转染的比例优选为3：1。本发明在共转染后，优选还包括收集细胞培养上清，并从中纯化和浓缩的步骤。本发明构建方法得到的用于对虾的假型昆虫杆状病毒可高效感染对虾成体及对虾细胞。
具体的，本发明将含外源基因的昆虫杆状病毒重组质粒与表达质粒pcDNA-VP28共转染昆虫sf9细胞；4天后收集细胞培养上清，离心，获得第一代病毒上清；将一代病毒感染sf9细胞，4天后收集细胞培养上清，离心，获得第二代病毒上清；超速离心，纯化和浓缩，得到嗜对虾细胞的假型昆虫杆状病毒。本发明得到的病毒粒子的囊膜中，不仅含有昆虫杆状病毒来源的囊膜蛋白，还含有对虾病毒来源的囊膜蛋白VP28。本发明对虾病毒囊膜蛋白VP28的引入，可大大提高该假型昆虫杆状病毒的包装效率和嗜对虾细胞性。在本发明更具体实施例中，假型杆状病毒Bacmid-GUS/VP28的制备方法优选如下：通过转染试剂Cellfectin II(Invitrogen公司)，将Bacmid-GUS重组病毒质粒和pcDNA-VP28质粒以3：1比例，共转染到昆虫sf9细胞中，进行病毒的包装，并收集4天后的培养基上清，离心，获得P1代病毒；再次感染sf9细胞，扩增病毒，4天后收集培养基上清；经过0.45μm的滤膜过滤后，超速离心，分离纯化P2代病毒；分装储存在-80冰箱中。
本发明还提供了基于上述技术方案所述构建方法构建得到的用于对虾的假型昆虫杆状病毒。本发明所述病毒在对虾成体组织中的感染和表达具有组织特异性，在对虾的鳃、心脏和肠组织中的侵染和表达效率可高达100％，但是不能在Oka器官和肌肉组织中检测到外源基因的表达；本发明所述病毒在对虾细胞(体外培养细胞)中的侵染与表达也具有细胞特异性，能够成功感染对虾体外培养血淋巴细胞，但是不能感染对虾体外培养胚胎细胞。
本发明还提供了基于上述技术方案所述基因转移系统或上述技术方案所述用于对虾的假型昆虫杆状病毒在对虾细胞中表达外源基因的应用。
在本发明中，所述对虾细胞包括对虾体外培养细胞，所述对虾体外培养细胞包括对虾原代培养血淋巴细胞。
本发明还提供了上述技术方案所述基因转移系统或上述技术方案所述用于对虾的假型昆虫杆状病毒在对虾成体组织中表达外源基因的应用。
在本发明中，所述对虾成体组织包括对虾成体的鳃、心脏和肠组织。
在本发明具体实施例中，以报告基因GUS(β-D-glucoronidase，β-D-葡萄糖苷酸酶)为外源基因，对Invitrogen公司的Bac-to-Bac昆虫杆状病毒表达系统(Cat.No.10359-016)进行改造为例，进行阐述如下：首先将GUS报告基因克隆到转移质粒pFastBac1的多克隆酶切位点，纯化出重组质粒pFastBac-GUS；然后将其转化到大肠杆菌感受态细胞DH10Bac中，GUS将被转座到Bacmid质粒中的attTn7位点，得到重组病毒质粒Bacmid-GUS；提取和纯化重组病毒质粒Bacmid-GUS；将Bacmid-GUS重组病毒质粒DNA和pcDNA-VP28质粒共转染包装细胞sf9，包装得到假型杆状病毒Bacmid-GUS/VP28；纯化和浓缩后，测定病毒滴度；包装病毒感染对虾成体组织或体外培养对虾细胞，从而实现外源基因在靶组织或细胞中的转移与表达。在本发明中，所述GUS报告基因的核苷酸序列如SEQ ID NO.3所示，所述GUS报告基因对应的蛋白的氨基酸序列如SEQ ID NO.4所示。
本发明中所涉及的转移质粒pFastBac1、大肠杆菌感受态细胞DH10Bac和包装细胞sf9均来自于Bac-to-Bac昆虫杆状病毒表达系统(Cat.No.10359-016)。
其中，转移质粒pFASTBac1含有昆虫强启动子PPH(多角体蛋白基因启动子)、多克隆酶切位点以及侧翼的转座酶Tn7的识别位点Tn7R和Tn7L。
其中，宿主细胞DH10Bac为稳定转化有辅助质粒和杆状病毒Bacmid质粒的大肠杆菌感受态细胞。
其中，杆状病毒Bacmid质粒含有原核细胞低拷贝质粒复制子(mini-F)、卡那霉素抗性基因(Kanr)以及插入了转座酶识别位点attTn7的LacZ基因(不移码插入)；辅助质粒为转座酶表达质粒。
本发明中所涉及的包装病毒滴度的测定方法如下：将病毒浓缩液感染sf9细胞，X-Gluc(索莱宝生物科技有限公司)染色，统计染成蓝色的Sf9细胞的数目，计算病毒浓缩液的滴度。
本发明中所涉及的对虾成体组织的病毒感染方法如下：将病毒浓缩液注射到对虾成体肌肉组织中，4天后，取对虾不同组织进行X-Gluc染色，观察GUS报告基因的表达情况。
本发明中所涉及的对虾体外培养细胞的病毒感染方法如下：将体外培养对虾细胞的旧培养基弃除，更换为无抗生素无血清的培养基；病毒浓缩液以一定剂量加到体外培养对虾细胞的培养基上清中，4天后，进行X-Gluc染色，观察GUS报告基因的表达情况。
下面结合具体实施例对本发明所述的一种用于对虾的假型昆虫杆状病毒基因转移系统、病毒及构建方法和应用做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。
实施例1
对虾WSSV病毒囊膜蛋白基因(VP28)编码框核酸序列的克隆、改造及其真核表达载体pcDNA3.1-VP28的构建。
利用全式金公司的DNA提取试剂盒(Easypure MarineAnimal Genomic DNAkit，Cat.No.EE151-01)提取WSSV感染对虾的基因组DNA，具体操作方法根据试剂盒说明书来进行。对虾病毒VP28基因编码框核酸序列的改造通过扩增引物的方式引入，即分别设计含有BamHI和EcoRI酶切位点的VP28基因特异性引物：正向突变引物序列为5’-CGCGGATCCATTGCCACCATGGATCTTTCTTTCAC-3’(SEQ ID NO.5)，反向突变引物序列为5’-CCGGAATTCGTTACTCGGTCTCAGTGCC-3’(SEQ ID NO.6)。通过PCR技术扩增得到目的片段。PCR反应的体系以及程序如下：50μl的体系组成为：5.0μl 10×PCRbuffer(含Mg2+)，4.0μl2.5mmol/L dNTPs，2.5μL 10μmol/L上游和下游引物，0.5μL 5IU/μL Taq DNA聚合酶，2μL基因组DNA和33.5μL ddH2O。PCR反应程序为：95℃预变性10分钟，按照以下方式循环30次：95℃变性45秒，60℃退火45秒，72℃延伸50秒。最后72℃10分钟。PCR产物用1％的琼脂糖凝胶电泳分析，并送测序公司测序。(图1，左图，其中，M为DNA分子量2000Marker，A为VP28的扩增产物)。
利用BamHI和EcoRI核酸内切酶酶切质粒pcDNA3.1-V5/HisA(Invitrogen公司)，将带有相同粘性末端(BamHI/EcoRI)VP28序列(SEQ ID NO:1)插入其中从而获得pcDNA-VP28(图1，右图)。图1为本发明所克隆的VP28开放阅读框的电泳结果以及pcDNA-VP28质粒的图谱。
实施例2
将外源基因克隆到供体质粒pFastBac1中。
以pFastBac1-GUS重组质粒的构建为例(图2，重组杆状病毒质粒Bacmid-GUS的制备图；其中，i为pFastBac1-GUS质粒DNA的电泳结果，ii为重组杆状病毒质粒Bacmid-GUS的蓝白斑筛选结果，iii为重组杆状病毒质粒Bacmid-GUS的提取与电泳结果)。
分别设计含有BamHI和EcoRI酶切位点的GUS基因特异性引物：正向引物序列为5’-CGCGGATCCATGGTCCGTCCTGTAGAAAC-3’(SEQ ID NO.7)，反向引物序列为5’-CCGGAATTCTCATTGTTTGCCTCCCTGCT-3’(SEQ ID NO.8)。通过PCR技术扩增得到目的片段。PCR反应的体系以及程序如下：50μL的体系组成为：25μL2×Hieff PCRMasterMix(含Mg2+)，2.5μL 10μmol/L上游和下游引物，2μL基因组DNA和18μL ddH2O。PCR反应程序为：95℃预变性5分钟，按照以下方式循环30次：95℃变性45秒，55℃退火45秒，72℃延伸1分30秒。最后72℃10分钟。PCR产物用1％的琼脂糖凝胶电泳分析，并送测序公司测序。
利用BamHI和EcoRI核酸内切酶酶切质粒pFastBac1(Invitrogen公司)，将带有相同粘性末端(BamHI/EcoRI)GUS序列插入其中从而获得pFastBac1-GUS。
实施例3
将外源基因重组到病毒质粒Bacmid中。
以Bacmid-GUS的构建为例(图2)。
将pFastBac1-GUS质粒转化至DH10Bac感受态细胞。转化产物涂板并经过蓝白斑筛选与菌液PCR检测，纯化出含GUS基因的重组Bacmid质粒：Bacmid-GUS。
实施例4
重组杆状病毒的制备与浓缩纯化。
以Bacmid-GUS为例。
利用Cellfectin II转染试剂，将Bacmid-GUS重组杆状病毒质粒DNA转染昆虫Sf9细胞。Bacmid-GUS质粒在sf9细胞中的最佳转染条件的优化结果如图3所示；其中，A和A1，为对照细胞的GUS染色前后的结果，B和B1、C和C1、D和D1分别为Bacmid-GUS质粒与CellfectinII转染试剂，以1:4、1:5和1:6(μg:μL)的比例转染Sf9细胞的GUS染色前后的结果，结果表明，当质粒DNA与转染试剂比值为1:5(μg:μL)时可得到最佳转染结果。收集P1代病毒，再次感染sf9细胞，扩增昆虫杆状病毒，然后分离纯化P2代病毒(图5中的A-D)。
其中，GUS基因的蛋白表达检测采用X-gluc染色，具体方法：弃除培养孔中的旧培养基，PBS洗涤细胞单层，并加入含2％甲醛和0.05％戊二醛的细胞固定液，室温固定5分钟。弃除旧固定液，PBS洗涤细胞2次，加入20mg/mLX-Gluc染色，28℃孵育12h，光镜拍照记录结果。
实施例5
假型昆虫杆状病毒的制备与浓缩纯化。
以Bacmid-GUS/VP28为例。
将质粒Bacmid-GUS与pcDNA-VP28共转染昆虫Sf9细胞，可成功包装得到假型杆状病毒Bacmid-GUS/VP28。结果如图4(假型杆状病毒Bacmid-GUS/VP28在包装细胞Sf9中的最适包装条件研究结果及其与杆状病毒Bacmid-GUS的包装效率比较；其中，A为未转染对照细胞，B为转染Bacmid-GUS质粒的包装结果，C、D、E和F分别为质粒Bacmid-GUS与pcDNA-VP28以3:1、4:1、5:1和6:1的比例共转染sf9细胞的包装结果。所有细胞的DNA与转染试剂的转染比例均为1：5(μg:μL))所示，Bacmid-GUS与pcDNA-VP28二者共转染的最佳比例为3：1，而且质粒pcDNA-VP28的引入，大大提高了病毒Bacmid-GUS/VP28的包装效率。
收集P1代病毒，再次感染sf9细胞，扩增昆虫杆状病毒，然后分离纯化P2代病毒(图5中的E-H)。图5为两种杆状病毒Bacmid-GUS与Bacmid-GUS/VP28在Sf9细胞中的P2代扩增结果；其中，A、B、C和D分别为杆状病毒Bacmid-GUS感染sf9细胞后的第1、2、3和4天的结果图；E、F、G、H分别为假型杆状病毒Bacmid-GUS/VP28感染sf9细胞后的第1、2、3和4天的结果图；感染剂量为每个直径10cm的培养皿中接种400μl P1代病毒上清。
实施例6
昆虫杆状病毒的滴度测定。
病毒滴度的测定通过GUS报告基因的表达来实现。以杆状病毒Bacmid-GUS或Bacmid-GUS/VP28感染Sf9细胞为例。
利用无血清无双抗的SIM昆虫细胞培养基，将杆状病毒Bacmid-GUS或Bacmid-GUS/VP28梯度稀释10-1～10-7倍，然后向96孔细胞培养板中添加100μL病毒稀释液/孔，于细胞感染后第4天，加入X-gluc染色以检测GUS报告基因的表达，并计算病毒滴度。计算公式：病毒滴度TU/mL(transduction units permL)＝阳性细胞％×细胞总数×稀释倍数/病毒体积(mL)。
如图6(杆状病毒Bacmid-GUS感染Sf9细胞96h后的GUS染色结果，左图和右图分别为重组杆状病毒Bacmid-GUS感染Sf9细胞的GUS染色前后的结果，病毒滴度为5.3×108TU/ml)所示，Bacmid-GUS的病毒滴度可达5.3×108TU/mL。
如图7(假型杆状病毒Bacmid-GUS/VP28感染Sf9细胞96h后的结果；
其中，A1和A2分别为对照细胞GUS染色前后的结果。B1和B2分别为P2代Bacmid-GUS病毒浓缩液的病毒滴度测定结果，病毒滴度可达5.3×108TU/ml。、C1和C2分别为对照细胞GUS染色前后的结果。D1和D2分别为P1代Bacmid-GUS/VP28假型杆状病毒培养上清的病毒滴度测定结果，病毒滴度可达5×107TU/ml。E1和E2分别为P2代Bacmid-GUS/VP28假型杆状病毒浓缩液的病毒滴度测定结果，病毒滴度可达3×1010TU/ml)所示，Bacmid-GUS/VP28的病毒滴度可达3×1010TU/mL。
实施例7
昆虫杆状病毒感染对虾体外培养细胞。
以杆状病毒Bamcid-GUS和Bacmid-GUS/VP28感染对虾原代培养血淋巴细胞和胚胎细胞为例。
对虾原代培养血淋巴细胞的病毒感染方法：将对虾血淋巴细胞接种至48孔细胞培养板，待细胞贴壁5小时后，弃旧培养基，每孔添加100μL病毒浓缩液(用1.5×L-15培养基稀释)。病毒孵育4小时后，弃除旧培养基，更换为正常的1.5×L-15完全培养基，于病毒感染第五天，用X-Gluc染液，检测GUS报告基因的表达。结果如图8(假型杆状病毒Bacmid-GUS/VP28可感染对虾原代培养血淋巴细胞，可检测到GUS基因的蛋白表达，而Bacmid-GUS病毒则不能成功感染对虾原代培养血淋巴细胞；其中，左列图中A-G为GUS染色结果，中间一列图中A1-G1为细胞核DAPI荧光染色结果，而右列图中A2-G2为同一行中的前两张图片的合并照片。GUS表达的检测时间为病毒感染后120小时。A、A1和A2，无病毒上清处理的对照细胞。B、B1和B2，3.0×107TU/孔Bacmid-GUS病毒感染结果。C、C1和C2，1.5×108TU/孔Bacmid-GUS病毒感染结果。D1、D2和D3，3.0×108TU/孔Bacmid-GUS病毒感染结果。E1、E2和E3，3.0×107TU/孔Bacmid-GUS/VP28假型病毒感染结果。F1、F2和F3，1.5×108TU/孔Bacmid-GUS/VP28假型病毒感染结果。G1、G2和G3，3.0×108TU/孔Bacmid-GUS/VP28假型病毒感染结果。比例尺＝100μm)所示，杆状病毒Bacmid-GUS不能感染对虾原代培养血淋巴细胞，染色后未观察到明显的GUS表达信号(图8中的B-D)。这一结果还表明，对虾细胞中没有GUS基因的本底表达，可用于对虾转基因研究。而假型杆状病毒Bacmid-GUS/VP28能够有效感染对虾原代培养血淋巴细胞，在感染剂量为3×108TU/mL时，可观察到明显的GUS基因表达信号(图8中的G)。
上述结果还表明，未经改造的昆虫杆状病毒Bac-to-Bac基因转移与表达系统在对虾细胞中的亲嗜性极低，不能直接应用于对虾细胞的基因转移研究。而对虾病毒的VP28囊膜蛋白的引入，能够显著提高假型杆状病毒Bacmid-GUS/VP28在对虾血淋巴细胞中的亲嗜性，促进该假型杆状病毒对对虾血淋巴细胞的感染与表达。
对虾原代培养胚胎细胞的病毒感染方法：将对虾胚胎细胞接种至48孔细胞培养板，待细胞贴壁2天后，弃旧培养基，每孔添加100μL病毒浓缩液(用1.5×L-15培养基稀释)。病毒孵育4小时后，弃除旧培养基，更换为正常的1.5×L-15完全培养基，于病毒感染第五天，用X-Gluc染液，检测GUS报告基因的表达。结果如图9(两种杆状病毒Bacmid-GUS/VP28和Bacmid-GUS都不能感染对虾原代培养胚胎细胞，不能检测到GUS表达；其中，左列图中A1-G1为GUS染色前的结果，而右列图中A2-G2为同一行中的GUS染色后的结果。A1和A2，无病毒上清处理的对照细胞。GUS表达的检测时间为病毒感染后120小时。B1和B2，3.0×107TU/孔Bacmid-GUS病毒感染结果。C1和C2，1.5×108TU/孔Bacmid-GUS病毒感染结果。D1和D2，3.0×108TU/孔Bacmid-GUS病毒感染结果。E1和E2，3.0×107TU/孔Bacmid-GUS/VP28假型杆状病毒感染结果。F1和F2，1.5×108TU/孔Bacmid-GUS/VP28假型杆状病毒感染结果。G1和G2，3.0×108TU/孔Bacmid-GUS/VP28假型杆状病毒感染结果。比例尺＝100μm)所示，两种杆状病毒Bacmid-GUS和Bacmid-GUS/VP28都不能有效感染对虾原代培养胚胎细胞，不能检测到GUS基因的表达。这表明，本发明所构建的假型杆状病毒在对虾细胞中的感染是具有细胞特异性的，这可能与本发明所使用的对虾病毒囊膜蛋白VP28有关。
对虾细胞的X-Gluc染色方法同昆虫Sf9细胞。
实施例8
昆虫杆状病毒病毒感染对虾成体组织。
以Bacmid-GUS和Bacmid-GUS/VP28为例。
上述两种杆状病毒感染对虾成体的方法采用肌肉注射的方式。具体操作：用微量注射器于对虾第一对游泳足中间腹线偏右约1mm处注射杆状病毒，注射后轻按注射处，并将对虾放至海水中；于病毒注射第5天，分别取对虾鳃、Oka器官、心脏、肌肉和肠组织，置于1.5mL离心管中，加入组织专用的GUS染液，避光置于28℃培养箱中孵育12小时，PBS清洗组织除去染液，用体视显微镜拍照并记录各个组织的染色结果。
结果如图10(杆状病毒Bacmid-GUS不能感染对虾成体各个组织；其中，图中A0-E0分别为肌肉注射PBS后的对照对虾的鳃、Oka器官、心脏、肌肉和肠组织的GUS染色结果，图中A1-E1分别为肌肉注射3×107TU Bacmid-GUS病毒后的对虾鳃、Oka器官、心脏、肌肉和肠组织的GUS染色结果。图中A2-E2分别为肌肉注射1.5×108TU Bacmid-GUS病毒后的对虾鳃、Oka器官、心脏、肌肉和肠组织的GUS染色结果。感染方式为肌肉注射，GUS表达的检测时间为病毒感染后120小时。比例尺：500μm)所示，所有感染剂量的杆状病毒Bacmid-GUS均未在感染对虾的鳃、Oka器官、心脏、肌肉和肠组织中检测到GUS基因的表达，这表明未改造的杆状病毒Bacmid-GUS不能有效侵染对虾上述五种组织。
但是，如图11(假型杆状病毒Bacmid-GUS/VP28可高效感染对虾成体的鳃、心脏和肠组织；其中，图中A0-A8分别为每只注射0(PBS)、1.5×104、1.5×105、1.5×106、1.5×107、3.0×107、1.5×108、3.0×108和6.0×108TU Bacmid-GUS/VP28假型杆状病毒后的对虾鳃组织GUS染色的显微观察结果。而图中B0-B8、C0-C8、D0-D8和E0-E8分别为Oka器官、心脏、肌肉与肠组织的相对应的GUS染色的显微观察结果。结果。感染方式为肌肉注射，GUS表达的检测时间为病毒感染后120小时。比例尺＝500μm)所示，所有感染剂量的假型杆状病毒Bacmid-GUS/VP28都能在所有感染对虾的鳃和心脏组织中检测到GUS基因的表达，而且当感染剂量达到1.5×107TU时，也能够在所有感染对虾的肠组织中检测到GUS基因的表达信号。这表明，对虾病毒VP28囊膜蛋白的引入，也大大促进了假型杆状病毒Bacmid-GUS/VP28对对虾成体组织的亲嗜性，且该假型杆状病毒侵染的靶器官为鳃、心脏与肠组织，其侵染具有组织特异性。而在肌肉注射PBS的对照对虾的所有5种组织中均未检测到GUS基因的表达，表明GUS基因在对虾成体组织中也没有明显的本底表达，可被应用于对虾成体的基因转移研究。
如图12(假型杆状病毒Bacmid-GUS/VP28感染对虾1-4天的GUS表达结果；其中，A1-A4分别为感染病毒1、2、3和4天后的对虾鳃组织的GUS染色结果，而B1-B4、C1-C4、D1-D4和E1-E4分别为对虾Oka器官、心脏、肌肉与肠组织的相对应的GUS染色结果。感染方式为肌肉注射，感染剂量为每只对虾注射3×108TU病毒)所示，假型杆状病毒Bacmid-GUS/VP28在对虾成体组织中的侵染和表达具有时间依赖性，GUS表达信号随感染时间的增加而加强。
对虾成体组织的GUS染色方法：避光条件下，现用现配GUS组织染色液：每1mL组织染色液中添加830μL无核酸酶灭菌水、100μL 1M磷酸钠溶液、20μL 0.5M EDTA-2Na、10μL10％TritonX-100,、20μL 50mM铁氰化钾溶液和20μL 0.1M X-Gluc溶液(50mg/mL)；向离心管中加入GUS组织染液，使其没过组织块，于28℃培养箱中孵育过夜；从培养箱中取出离心管，小心弃除染色液，用小镊子取出组织，置于盛有PBS缓冲液的小皿中，于体视显微镜下观察并拍照记录。
总之，本发明所构建的假型杆状病毒基因转移系统在对虾成体组织中的感染和表达效率可高达100％，显著优于未改造的杆状病毒基因转移与表达系统。
同时，本发明的构建方法也适用于对虾白斑综合征病毒的其它囊膜蛋白如VP19，以及其它对虾病毒的囊膜蛋白。将不同的对虾病毒囊膜蛋白引入昆虫杆状病毒的结果，将改变所制备的假型杆状病毒的组织和细胞的特异性。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国海洋大学
<120> 一种用于对虾的假型昆虫杆状病毒基因转移系统、病毒及构建方法和应用
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gaaacccaca caggcaatat cgagacaaac atggatgaaa acctccgcat tcctgtgact 180
gctgaggttg gatcaggcta cttcaagatg actgatgtgt cctttgacag cgacaccttg 240
ggcaaaatca agatccgcaa tggaaagtct gatgcacaga tgaaggaaga agatgcggat 300
cttgtcatca ctcccgtgga gggccgagca ctcgaagtga ctgtggggca gaatctcacc 360
tttgagggaa cattcaaggt gtggaacaac acatcaagaa agatcaacat cactggtatg 420
cagatggtgc caaagattaa cccatcaaag gcctttgtcg gtagctccaa cacctcctcc 480
ttcacccccg tctctattga tgaggatgaa gttggcacct ttgtgtgtgg taccaccttt 540
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Val Ser Ile Asp Glu Asp Glu Val Gly Thr Phe Val Cys Gly Thr Thr
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atggtccgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca 60
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cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa aggttgggca 240
ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat 300
aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga tgtcacgccg 360
tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga actgaactgg 420
cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa gcagtcttat 480
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ctgttgactg gcaggtggtg gccaatggtg atgtcagcgt tgaactgcgt gatgcggatc 660
aacaggtggt tgcaactgga caaggcacta gcgggacttt gcaagtggtg aatccgcacc 720
tctggcaacc gggtgaaggt tatctctatg aactgtgcgt cacagccaaa agccagacag 780
agtgtgatat ctacccgctt cgcgtcggca tccggtcagt ggcagtgaag ggcgaacagt 840
tcctgattaa ccacaaaccg ttctacttta ctggctttgg tcgtcatgaa gatgcggact 900
tacgtggcaa aggattcgat aacgtgctga tggtgcacga ccacgcatta atggactgga 960
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cagatgaaca tggcatcgtg gtgattgatg aaactgctgc tgtcggcttt aacctctctt 1080
taggcattgg tttcgaagcg ggcaacaagc cgaaagaact gtacagcgaa gaggcagtca 1140
acggggaaac tcagcaagcg cacttacagg cgattaaaga gctgatagcg cgtgacaaaa 1200
accacccaag cgtggtgatg tggagtattg ccaacgaacc ggatacccgt ccgcaaggtg 1260
cacgggaata tttcgcgcca ctggcggaag caacgcgtaa actcgacccg acgcgtccga 1320
tcacctgcgt caatgtaatg ttctgcgacg ctcacaccga taccatcagc gatctctttg 1380
atgtgctgtg cctgaaccgt tattacggat ggtatgtcca aagcggcgat ttggaaacgg 1440
cagagaaggt actggaaaaa gaacttctgg cctggcagga gaaactgcat cagccgatta 1500
tcatcaccga atacggcgtg gatacgttag ccgggctgca ctcaatgtac accgacatgt 1560
ggagtgaaga gtatcagtgt gcatggctgg atatgtatca ccgcgtcttt atcgcgtcag 1620
cgccgtcgtc ggtgaacagg tatggaattt cgccgatttt gcgacctcgc aaggcatatt 1680
gcgcgttggc ggtacaagaa agggatcttc actcgcgacc gcaaaccgaa gtcggcggct 1740
tttctgctgc aaaaacgctg gactggcatg aacttcggtg aaaaaccgca gcagggaggc 1800
aaacaatga 1809
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Ala Leu Val Ser Lys Arg Ala Thr Ser Arg Lys Asn Cys Thr Ala Lys
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Claims (7)
1.一种用于对虾的假型昆虫杆状病毒基因转移系统，其特征在于，所述基因转移系统包括Bac-to-Bac昆虫杆状病毒表达系统、对虾病毒囊膜蛋白基因表达质粒和昆虫包装细胞；
所述对虾病毒囊膜蛋白基因表达质粒包括对虾白斑综合症病毒囊膜蛋白基因表达质粒；
所述对虾病毒囊膜蛋白基因表达质粒中所述囊膜蛋白基因的核苷酸序列如SEQ IDNO.1所示；
所述对虾病毒囊膜蛋白基因表达质粒的构建用骨架载体为pcDNA3.1。
2.基于权利要求1所述基因转移系统的用于对虾的假型昆虫杆状病毒的构建方法，包括以下步骤：
1)利用Bac-to-Bac昆虫杆状病毒表达系统构建得到含外源基因的昆虫杆状病毒重组质粒；
2)将步骤1)得到的昆虫杆状病毒重组质粒和对虾病毒囊膜蛋白基因表达质粒共转染昆虫包装细胞，得到用于对虾的假型昆虫杆状病毒。
3.权利要求2所述构建方法构建得到的用于对虾的假型昆虫杆状病毒。
4.权利要求1所述基因转移系统或权利要求3所述用于对虾的假型昆虫杆状病毒在对虾细胞中表达外源基因的应用。
5.根据权利要求4所述的应用，其特征在于，所述对虾细胞包括对虾体外培养细胞，所述对虾体外培养细胞包括对虾原代培养血淋巴细胞。
6.权利要求1所述基因转移系统或权利要求3所述用于对虾的假型昆虫杆状病毒在对虾成体组织中表达外源基因的应用。
7.根据权利要求6所述的应用，其特征在于，所述对虾成体组织包括对虾成体的鳃、心脏和肠组织。
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Non-Patent Citations (4)
| Title |
|---|
| Recombinant baculovirus mediates dsRNA specific to rr2 delivery and its protective efficacy against WSSV infection;Triwit Rattanarojpong等;《J Biotechnol》;20160731;44-52 * |
| 以凡纳滨对虾β-actin基因启动子为元件的重组昆虫杆状病毒表达系统的建立;史英力;《中国博士学位论文全文数据库》;20160815;A006-191 * |
| 对虾白斑综合症病毒基因组同源重复区结合蛋白的筛选;朱艳冰等;《台湾海峡》;20061231;318-323 * |
| 对虾细胞基因转移技术研究以及对虾早期胚胎microRNAs的挖掘;濮龙军;《中国优秀硕士学位论文全文数据库》;20160715;A006-107 * |
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