CN114292879A - 一种用于在对虾中递送和表达外源基因的对虾病毒表达系统 - Google Patents
一种用于在对虾中递送和表达外源基因的对虾病毒表达系统 Download PDFInfo
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Abstract
本发明提供一种用于在对虾细胞中递送和表达外源基因的对虾病毒表达系统,其中包含有用于将外源基因在对虾细胞中进行表达的重组质粒,所述的重组质粒包含有对虾IHHNV病毒的基因组DNA、昆虫多角体蛋白基因的启动子PH、用于插入外源基因的多克隆酶切位点MCS和SV40多聚腺苷酸信号pA。所述的表达系统还包含有插入有对虾WSSV病毒囊膜蛋白VP28基因的昆虫杆状病毒重组表达质粒和昆虫Sf9包装细胞。本发明所提供的对虾病毒表达系统能够高效的在对虾细胞中表达外源基因,且表达系统中的所有质粒DNA遗传信息是游离于对虾细胞染色体之外,不会整合到对虾细胞的基因组中,提高了该系统的生物安全性。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种用于在对虾细胞中递送和表达外源基因的对虾病毒表达系统。
背景技术
对虾是一种重要的海水养殖经济品种,但随着养殖规模的不断扩大,养殖环境的水质不断恶化,导致对虾养殖中的病害问题愈发严重。尤其是频繁爆发的病毒性疾病,因缺乏有效的防治措施,而给对虾养殖业造成了巨大损失。为了满足对虾病毒的分离和纯化、研制诊断试剂与核酸疫苗以及对虾病毒病的致病机制研究的需要,亟需建立对虾永生性细胞系。
但是,由于缺乏有效的对虾细胞基因转移技术,导致人们无法将永生性转化相关基因高效导入体外培养的对虾细胞中,以诱导对虾细胞的永生性转化,从而大大阻碍了对虾永生性细胞系的成功建立。另外,由于针刺易导致对虾受精卵破裂和显微注射后的受精卵孵化率低等原因,导致对虾活体的基因转移与基因编辑的研究进展远滞后于鱼类。因此,对虾高效基因转移与表达系统的建立,也将大大推动转基因对虾的研制以及基因编辑技术在对虾活体中的应用。
目前,基于哺乳动物病毒的逆转录病毒和慢病毒基因转移和表达系统已经成功构建,并广泛应用于哺乳动物细胞的高效基因转移与表达,大大推动了相关研究领域的发展。但是,由于病毒具有严格的宿主专一性,上述哺乳动物病毒介导的基因转移与表达系统在对虾组织和细胞中的亲嗜性极低。
发明内容
本发明的目的是提供一种用于在对虾细胞中递送和表达外源基因的对虾病毒表达系统,从而弥补现有技术的不足。
本发明首先提供一种用于将外源基因在对虾细胞中进行表达的重组质粒,所述的重组质粒包含有对虾IHHNV病毒的基因组DNA、昆虫多角体蛋白基因的启动子PH、用于插入外源基因的多克隆酶切位点(MCS)和SV40多聚腺苷酸信号(pA);
作为本发明实施例的一种具体记载,所述的对虾IHHNV病毒的基因组DNA序列为SEQ ID NO:1;
作为实施例的具体记载,所述的PH启动子的核酸序列为SEQ ID NO:2,MCS的核酸序列为SEQ ID NO:3;SV40的pA的核酸序列为SEQ ID NO:4。
更进一步,所述启动子PH和多克隆酶切位点之间还插入有GUS报告基因;
所述的GUS报告基因的编码核酸和氨基酸序列分别为SEQ ID NO:5和SEQ ID NO:6;
所提供的重组质粒用于制备将外源基因在对虾细胞中进行表达的系统;
一种将外源基因在对虾细胞中进行表达的系统,包含有上述的重组质粒,以及插入有对虾WSSV病毒囊膜蛋白VP28基因的昆虫杆状病毒重组表达质粒Bacmid-VP28;
其中,VP28基因的编码核酸和氨基酸序列分别为SEQ ID NO:7和SEQ ID NO:8;
所述的表达系统还包含有昆虫Sf9包装细胞。
本发明还提供了一种使外源基因在对虾成体组织和体外培养细胞中稳定、高效表达的方法:将外源基因克隆到上述对虾病毒表达质粒中,然后通过上述述对虾病毒表达系统,制备混合包装病毒,然后通过肌肉注射和孵育的方式分别感染对虾成体组织及其细胞,检测外源基因的表达。
本发明所提供的对虾病毒表达系统能够高效的在对虾细胞中表达外源基因,且表达系统中的所有质粒DNA遗传信息是游离于对虾细胞染色体之外,不会整合到对虾细胞的基因组中,提高了该系统的生物安全性。
附图说明
图1:PCR扩增对虾IHHNV病毒全基因组前后两个片段的凝胶电泳结果图,其中,泳道1为IHHNV基因组前半部分的PCR扩增片段(1-1910bp),泳道2为IHHNV基因组后半部分的PCR扩增片段(1870-3833bp),M为DNA Marker。
图2:pUC19-IHHNV环化质粒的图谱;
图3:pUC19-IHHNV环化质粒的凝胶电泳结果图,其中泳道1为pUC19-IHHNV质粒(6.5kb),M为DNA Marker。
图4:pUC19-IHHNV环化质粒测序分析结果图,其中下划线代表酶切位点。
图5:pFastBacTM 1-GUS质粒的图谱;
图6:PCR扩增带有pUC19-IHHNV质粒线性化位点附近30bp同源臂的PH-GUS-MCS片段的凝胶电泳结果;其中泳道1为扩增产物PPH-GUS-MCS,M为DNA Marker。
图7:对虾IHHNV病毒表达质粒pUC19-IHHNV-PH-GUS的图谱。
图8:pUC19-IHHNV-PH-GUS表达质粒的测序分析结果;其中,下划线代表酶切位点,灰色阴影代表PH启动子,大框内代表GUS基因,小方框内为起始密码子和终止密码子。
图9:pUC19-IHHNV-PH-GUS与Bacmid分别单独转染昆虫Sf9细胞的GUS染色光镜结果图;其中,A为未转染质粒的昆虫Sf9细胞,B为pUC19-IHHNV-PH-GUS质粒在昆虫Sf9细胞单独转染120h的染色结果图,C为Bacmid质粒在昆虫Sf9细胞单独转染120h的染色结果图。比例尺为100μm。
图10:对虾病毒表达质粒pUC19-IHHNV-PH-GUS在昆虫Sf9细胞中的包装效果(透射电镜检测);其中,A为未感染病毒的昆虫Sf9细胞,B为pUC19-IHHNV-PH-GUS转染昆虫Sf9细胞后的上清液所感染的Sf9细胞,C为Bacmid转染昆虫Sf9细胞后的上清液所感染的Sf9细胞,D为pUC19-IHHNV-PH-GUS和Bacmid共转染昆虫Sf9细胞后的上清液所感染的Sf9细胞。比例尺为2μm。放大倍数为4×。
图11:病毒表达质粒pUC19-IHHNV-PH-GUS转染Sf9细胞后的上清二次侵染Sf9细胞后所收集的培养基上清液的负染电镜结果;其中比例尺为100nm。
图12:Bacmid与pUC19-IHHNV-PH-GUS的共转染实验中转染试剂Cellfectin II的最佳转染剂量的优化结果图;其中,A为未转染昆虫Sf9细胞光镜图,B为质粒与转染试剂比例为1:4(μg:μl),C为质粒与转染试剂比例为1:5(μg:μl),D为质粒与转染试剂比1:6(μg:μl)。比例尺为100μm。
图13:Bacmid与pUC19-IHHNV-PH-GUS的最佳共转染比例的优化结果;A:未转染昆虫Sf9细胞光镜图;B、C、D、E和F分别为pUC19-IHHNV-PH-GUS与Bacmid以1:1/3、1:1/2、1:1、1:2和1:3的比例共转染到昆虫Sf9细胞。比例尺为100μm。
图14:对虾病毒质粒pUC19-IHHNV-PH-GUS在昆虫Sf9细胞中的包装效果检测;其中,A为未感染病毒的昆虫Sf9细胞光镜图,B为Bacmid转染Sf9细胞后的培养基上清液所感染的昆虫Sf9细胞,C为Bacmid-GUS转染昆虫Sf9细胞后的培养基上清液所感染的Sf9细胞,D为pUC19-IHHNV-PH-GUS和Bacmid共转染Sf9细胞后的上清液所感染的Sf9细胞。比例尺为100μm。
图15:在1.5×L-15完全培养基中培养的对虾血淋巴细胞感染pUC19-IHHNV-PH-GUS/Bacmid-VP28混合病毒5天后的结果图;其中,A为未感染病毒,B为pUC19-IHHNV-PH-GUS/Bacmid-VP-28混合病毒感染。比例尺为100μm。
图16:pUC19-IHHNV-PH-GUS/Bacmid-VP28混合病毒感染刀额新对虾活体后不同组织中的GUS基因表达结果图,其中,I图为体视显微镜下结果,II图为1.5mL EP管内观察结果。I图中,A0-E0为对虾成体肌肉注射PBS后的心脏、鳃、Oka、肌肉与肠GUS染色结果。A1-E1为对虾成体肌肉注射7×104TU病毒后的心脏、鳃、Oka、肌肉与肠GUS染色结果。A2-E2为对虾成体肌肉注射7×105TU病毒后的心脏、鳃、Oka、肌肉与肠GUS染色结果。A3-E3为对虾成体肌肉注射7×106TU病毒后的心脏、鳃、Oka、肌肉与肠GUS染色结果。II图与I图结果对应。
具体实施方式
本发明所使用的对虾传染性皮下及造血组织坏死病毒(IHHNV)与哺乳动物腺相关病毒(AAV2)同属于细小病毒科,是一种单链DNA病毒,其基因组只有4kb左右,易于开展分子生物学操作。
在之前的研究中申请人发现Bac-to-Bac昆虫杆状病毒基因转移与表达系统不能有效侵染对虾活体及其体外培养细胞。在将对虾病毒WSSV的囊膜蛋白(VP28)引入上述昆虫杆状病毒的囊膜中,其在对虾体外培养细胞中的侵染效率也很低,不能应用于对虾细胞的永生性转化研究。
本发明首先构建了一种基于对虾IHHNV病毒全基因组的对虾高效基因转移与表达系统,首先扩增了对虾IHHNV病毒的基因组DNA全长,并构建其环化质粒pUC19-IHHNV,从而实现了在大肠杆菌中扩增对虾病毒基因组DNA的目的。然后将昆虫多角体蛋白基因的启动子PH、用于插入外源基因的多克隆酶切位点(MCS)和SV40多聚腺苷酸信号(pA)插入到对虾IHHNV病毒的基因组DNA之后,构建重组表达质粒pUC19-IHHNV-PH-GUS。
通过检测GUS报告基因的表达以及透射电镜和电镜负染分析,探究对虾病毒表达质粒pUC19-IHHNV-PH-GUS在昆虫Sf9细胞中的表达与包装能力;但结果表明重组表达质粒pUC19-IHHNV-PH-GUS单独转染到昆虫Sf9细胞中,检测不到报告基因GUS的表达。
而通过将对虾WSSV病毒囊膜蛋白VP28基因插入到昆虫杆状病毒表达质粒Bacmid中,构建成重组表达质粒Bacmid-VP28,然后将重组表达质粒Bacmid-VP28和pUC19-IHHNV-PH-GUS共转染昆虫Sf9包装细胞后,收集和制备pUC19-IHHNV-PH-GUS和Bacmid-VP28的混合病毒上清;将一代混合病毒感染sf9细胞,4天后收集细胞培养上清,离心,获得第二代混合病毒上清;超速离心,纯化和浓缩得到混合病毒,并根据GUS基因的表达,测定包装病毒(pUC19-IHHNV-PH-GUS)的滴度;最后,将混合病毒感染对虾活体及其细胞,检测GUS基因的表达。结果混合病毒Bacmid-VP28和pUC19-IHHNV-PH-GUS共感染对虾细胞后,GUS基因的表达量明显提高。
实现上述技术方案的方法,其一种具体的步骤如下:
扩增对虾IHHNV病毒的基因组DNA全长,将其与pUC19质粒连接环化,构建其环化质粒pUC19-IHHNV;然后将昆虫多角体蛋白基因启动子(PH)、报告基因GUS(β-葡萄糖苷酸酶)、多克隆酶切位点(MCS)和SV40多聚腺苷酸信号(pA)引入上述环化质粒中IHHNV序列之后。
上述环化质粒pUC19-IHHNV的构建方法为:分段扩增对虾IHHNV病毒的第1-1910bp和1870-3833bp的两个片段,然后采用大片段同源重组技术,将上述2个克隆片段与线性化的pUC19质粒连接。
病毒表达质粒Bacmid-VP28的构建方法为:将VP28基因克隆到供体质粒pFastBac1的多克隆酶切位点,构建重组质粒pFastBac1-VP28,然后将其转化到感受态细胞DH10Bac中,蓝白斑筛选阳性克隆,然后提取重组病毒表达质粒Bacmid-VP28。
本发明另一个方面提供了一种基于对虾IHHNV病毒全基因组的高效基因转移与表达系统。该系统的组成为:对虾病毒表达质粒(pUC19-IHHNV-PH-GUS)、昆虫杆状病毒表达质粒(Bacmid-VP28)和昆虫Sf9包装细胞。
其中,上述对虾病毒介导的基因转移与表达系统的技术流程为:将对虾病毒表达质粒(pUC19-IHHNV-PH-GUS)和昆虫杆状病毒表达质粒(Bacmid-VP28)共转染昆虫Sf9包装细胞;4天后收集细胞培养上清,离心,获得第一代pUC19-IHHNV-PH-GUS和Bacmid-VP28的混合病毒上清;将一代混合病毒感染sf9细胞,4天后收集细胞培养上清,离心,获得第二代混合病毒上清;超速离心,纯化和浓缩得到混合病毒,并根据GUS基因的表达测定包装病毒(pUC19-IHHNV-PH-GUS)的滴度;将混合病毒感染对虾活体及其细胞,检测GUS基因的表达。
本发明还提供了一种使外源基因在对虾成体组织和体外培养细胞中稳定、高效表达的方法:将外源基因克隆到上述对虾病毒表达质粒中,然后通过上述述对虾病毒表达系统,制备混合包装病毒,然后通过肌肉注射和孵育的方式分别感染对虾成体组织及其细胞,检测外源基因的表达。
下面结合实施例和附图对本发明进行详细的描述。
实施例1:对虾IHHNV病毒基因组DNA全长的克隆及其环化质粒pUC19-IHHNV的构建。
设计特异性引物,PCR技术分段扩增对虾IHHNV病毒的第1-1910bp和1870-3833bp的两个片段(图1),测序后可拼接获得了对虾IHHNV病毒的基因组DNA全长,为3833bp。然后采用大片段同源重组技术(GibsonUltra Kit),将上述2个克隆片段与线性化的pUC19质粒连接,构建了对虾IHHNV基因组DNA的pUC19环化质粒:pUC19-IHHNV(图2-图4),从而实现了对虾病毒IHHNV基因组DNA在细菌中的扩增与纯化。
实施例2:基于对虾IHHNV病毒全基因组的对虾病毒表达质粒pUC19-IHHNV-PH-GUS的构建。
以质粒pFastBacTM1-GUS为模板(图5),PCR扩增其中的PH-GUS-MCS-SV40 pA片段(2383bp)。PCR扩增得到的PH-GUS-MCS片段的左右两端分别带有15bp的同源臂(2413bp)(图6)。然后利用abm公司无缝克隆试剂盒,将PH-GUS-MCS片段插入双酶酶切线性化的pUC19-IHHNV质粒中,成功构建了以PH作为启动子、GUS作为报告基因的对虾病毒表达质粒:pUC19-IHHNV-PH-GUS(图7-图8)。
实施例3:利用Sf9细胞成功包装得到对虾IHHNV病毒粒子(pUC19-IHHNV-PH-GUS)。
将病毒表达质粒pUC19-IHHNV-PH-GUS单独转染到昆虫Sf9细胞中,发现检测不到GUS基因的表达(图9),但是转染后Sf9细胞的培养基上清可成功感染Sf9细胞,并在感染后的Sf9细胞内和培养基上清液中观察到二十面体的类似于对虾IHHNV的病毒颗粒,其大小约为50-80nm(图10-图11)。
上述结果表明基于对虾IHHNV病毒的重组表达质粒pUC19-IHHNV-PH-GUS单独转染Sf9细胞,能够包装出pUC19-IHHNV-PH-GUS的病毒粒子。关于在转染和感染后的sf9细胞中检测不到GUS基因的表达的问题,我们认为,是由于PH启动子为昆虫杆状病毒的晚期启动子,可能需要昆虫杆状病毒的早期基因表达产物的激活。
进一步优化质粒DNA与转染试剂Cellfectin II在sf9细胞的最佳转染条件(图12-图13),并进一步将对虾病毒表达质粒pUC19-IHHNV-PH-GUS与昆虫杆状病毒表达质粒Bacmid共转染到昆虫Sf9细胞中,成功检测到了GUS报告基因的表达。将共转染后的培养基上清再次感染Sf9细胞,可在二次感染后的Sf9细胞中检测到GUS基因的表达(图14)。
透射电镜检测发现,在感染后的昆虫Sf9细胞中,既可以观察到杆状的Bacmid病毒粒子,也可以观察到二十面体的pUC19-IHHNV-PH-GUS病毒粒子(图10)。这表明,Sf9细胞中同时包装出了完整的pUC19-IHHNV-PH-GUS病毒粒子和Bacmid病毒粒子。上述结果表明,利用Sf9细胞成功包装得到了对虾病毒粒子,建立了基于对虾IHHNV病毒基因组全长的GUS报告基因转移与表达系统。
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/mL X-Gluc染色,28℃孵育12h,光镜拍照记录结果。
实施例4:对虾WSSV病毒囊膜蛋白VP28基因的昆虫杆状病毒重组表达质粒Bacmid-VP28的构建。
设计特异性引物,PCR扩增对虾WSSV病毒的VP28基因,然后定向克隆到供体质粒pFastBac1的多克隆酶切位点,构建重组质粒pFastBac1-VP28,然后将其转化到感受态细胞DH10Bac中,转化产物涂板并经过蓝白斑筛选与菌液PCR检测,提取和纯化出重组病毒表达质粒Bacmid-VP28。
实施例5:构建基于对虾IHHNV病毒全基因组的GUS报告基因转移与表达系统。
该系统由3种组分组成:基于对虾IHHNV病毒全基因组的对虾病毒表达质粒(pUC19-IHHNV-PH-GUS)、昆虫杆状病毒表达质粒(Bacmid-VP28)和昆虫Sf9包装细胞。
基于对虾IHHNV病毒的GUS报告基因转移与表达系统可应用于在对虾活体及其体外培养对虾细胞中表达外源基因。
首先利用sf9细胞,包装、纯化并浓缩得到pUC19-IHHNV-PH-GUS和Bacmid-VP28的混合病毒,然后将上述混合病毒以8×106TU/孔的感染剂量感染96孔板中对虾体外培养细胞,5天后可以在感染后的对虾细胞中观察到明显的细胞病变效应,表明pUC19-IHHNV-PH-GUS病毒可以感染对虾细胞(图15)。
如图16所示,将pUC19-IHHNV-PH-GUS和Bacmid-VP28混合病毒按照7×106TU/只、7×105TU/只和7×104TU/只的剂量注射到对虾体内,5天后,取注射后对虾的心脏、鳃、Oka器官、肌肉和肠组织检测GUS基因的表达。发现在心脏、鳃和Oka器官中均有GUS基因的表达,且GUS基因的表达随病毒注射剂量的增加而升高。这表明,pUC19-IHHNV-PH-GUS和Bacmid-VP28混合病毒可以感染对虾活体,并且具有组织特异性。
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。
序列表
<110> 中国海洋大学
<120> 一种用于在对虾中递送和表达外源基因的对虾病毒表达系统
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3833
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tcggagcgct tcgcaggaaa ccgttacaac ctatgacgtc ataggtccta tataagagat 60
gacggactca ccggtctccc agtttctaac tgacgagtga agaggctatt ccaagtgact 120
aaggacaatt ttggaacatg gaagatacga acgaccaccc atggcaatca atacctagtc 180
cgtcattatt tggatcatca agtaacagtg aaccaacaga agtctttcaa aacgtcttcg 240
gagaaagaca aacccaagga tacaaatgta agtacaagtg actgactaag tgacgatcca 300
ttaaattcct aattgacgca agtgacgacg tcatatgcgt cacttacaaa agacgtaacc 360
gctttcgtcc atcactcaca tatatctttc tctacctttc agacgacata ccccaacaaa 420
tatcgctgcg ctactgccca gatcacattc taccgtggtg cttcataggg aacagacccg 480
ttctctactg cctctgcaac gagtgtttta tagacaatct caatgtcaac ggacagtgtc 540
aacactgtca tcccgacgac gaagaatgga cagaaaatat ggccaaggac atactgcata 600
cacgtcaggg cgaaccagaa tcacttagtg aattgcttcg aaaacgcacg aacgaaactc 660
actccagcca gggaatttct ccaagccttc tcaccccagg tccaaatcaa gaccctaaac 720
ccactaccga acaacttctt aatatgtctg aagaactgtt ccagttttca gacgaggaag 780
acaactctca aactcctcca agaacttcaa caccagaaca aactgatcct aaggtctgcg 840
tggataacct gggaattcga gagggaacag gaaacggaac aattcaactt ggaagtgaat 900
cagaaacctc ccttggaagt gttggaaata gtaatgacag gggtaaaaag agacagagag 960
gaattactta catcagtgac acatcagatt cctccggatc agatgacgaa aatctgggta 1020
caccacatcg aaataaaaga accagaaact ccaacaccac aaaagagaca ggcggaggag 1080
acaaccgacg acatcaggaa agcgatcatg gaagcaatgg aaatcgactg gaacctaccg 1140
atggaggaga gagctgtagt agcggaacac aacccgactt tattgaaggg actcccaacg 1200
gaccggacga aatggacgga aggcgactgg aagagagtga gattgataaa caagtggaaa 1260
gtacaacatg gtacaccttc gtcatcagag aaaaaccaca accaagaaga ctctccggaa 1320
gaacaccaaa cttcaccatt acaaatcatg gtgaccactg gcacatcaca tactccggac 1380
acccaaccaa taagaccaga catagagcta caatcctcgc ctatttggga gttacctttg 1440
ctgccagagc cgaagctgaa gcgactacgg tacttgttag aaatatcaag agatggatac 1500
tctatcttat cagatacggt attgaacggc tttcgtattt tggtcttggc cacgccattt 1560
ttaaacgaat catcaaatac ttccaacaat acagaagaga cgaagacgca gtagacggac 1620
catgtccata tatgactact acaagagaag accgcgctga agaaaaacct aaagaaaaca 1680
gtgcagaata tgactacctc caacacttag tcaaaaccaa gtcagcaaga acagtacaag 1740
aacttgtcaa taaacttgac gatgaggaat ataaacaact atggacccgt accagaggac 1800
aatataaaga caaactcaga ggaatattaa catactacaa caacaagaaa aagtcaaacc 1860
aaagccaact gtcactaatt acaaacctgc agaatatatc aaaaagaaaa ccagactacg 1920
acaatatgca gtggataaaa tacatgttag ccaacaacaa catccgtgta ccagaaatct 1980
tagcttggat aattatcgta gcagacaaaa aactggacaa aataaacact cttgtactcc 2040
aaggaccaac aggaacagga aaatctctta ccatcggtgc actactcgga aaactgaaca 2100
ctggcctagt aacaagaaca ggagactcaa acaccttcca tctacaaaac ctcatcggaa 2160
agtcctacgc tctcttcgaa gaacccagaa tcagtcaaat aacagtggac gacttcaaac 2220
tccttttcga gggatcagac ttagaagtaa acataaaaca ccaagagtca gaaattatgg 2280
gacgaatacc aatcttcata tcaacaaaca aggatataga ctactgggta cctccagctg 2340
atggtaaagc tctacaaaca agaacaaaaa ccttccacct gacaagacaa ataaaaggcc 2400
tctcagacag gatgaacagc cagtacgaca tcaaccctcc accagacaag atcaccagcg 2460
acgacttcct aggactcttc caagaatacg aaaaggaaat cgacgacatc atagacagcc 2520
atgtgcgccg attcaacaag agcaagccca aggaaaagat ccaggaggga tgcacataat 2580
gaagacgaag aacacgccga aggatcaagt ggaccagacc cacacagatg tctacaattc 2640
aatactggag actcaataca tattactttc caaacaagaa gatacttcga attcgacgct 2700
gccaatgatg gaaacttcga cggaaaaaat ttatactgcc tcccactaca ttggatgaac 2760
ttatatctct atggtctaaa gagcagcgac agttcagcga cagaaacaca acgatataag 2820
atggtaaaat cactgatgaa gacctacgga tggaaagtac acaaagcagg cgtagtgatg 2880
cactcaatgg taccccttat gaaagactta aaagtatcag gaggcacatc atttgagact 2940
ctcacattta cagacacccc atatttagaa atatttaagg atactactgg actacataat 3000
caactatcaa ctaaggaagc cgacgtaaca ttggcaaaat ggatacaaaa tccccaactt 3060
gtgaccgtac aatcaacagc agcaaactat gaagacccaa tccaacaatt tggattcatg 3120
gaacaaatgc gaaccggtga cagaaaagcc tatacaatcc atggtgacac tagaaattgg 3180
tatggcggag aaataccaac aaccggaccc accttcatcc caaaatgggg tggtcaatta 3240
aaatgggaca aaccatccct tggaaaccta gtctacccag cagaccacca tacaaacgac 3300
tggcaacaga tcttcatgag aatgtcacca atcaaaggac caaatggaga cgaacttaaa 3360
cttggctgca gagtacaagc cgacttcttc ctacacctag aagtacgact cccaccacaa 3420
ggatgtaccg caagtttggg gatgttacaa tatcttcacg caccatgtac tggacaactt 3480
aacaaatgtt atattatgca tactaactaa atatattcga tgtgcaatat atacccgatt 3540
atatccagct tttaccaata aacatgtata gcttattatc atctatacct accctatata 3600
cataccagct acccaggcaa ggtgggactc cggctaccca ggcatggtgg gacacttttc 3660
ttctattgac gacatatttc ggcacttacg tcacttacaa aagactaaaa tccctatcgt 3720
cagtcagtca tttagagtca gggatattgt ccgccgtcac ttagagcgcg aagcgcgagt 3780
atccatcatt taaattagtg gtatgaagtc acatattaag ttaacggttt ctg 3833
<210> 2
<211> 92
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc 60
gtaacagttt tgtaataaaa aaacctataa at 92
<210> 3
<211> 89
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gaattcaaag gcctacgtcg acgagctcac tagtcgcggc cgctttcgaa tctagagcct 60
gcagtctcga ggcatgcggt accaagctt 89
<210> 4
<211> 135
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60
aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120
tatcatgtct ggatc 135
<210> 5
<211> 1812
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggtccgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca 60
ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag cgcgttacaa 120
gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga tgcagatatt 180
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 gcagtcttac 480
ttccatgatt tctttaacta tgccggaatc catcgcagcg taatgctcta caccacgccg 540
aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg taaccacgcg 600
tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg tgatgcggat 660
caacaggtgg ttgcaactgg acaaggcact agcgggactt tgcaagtggt gaatccgcac 720
ctctggcaac cgggtgaagg ttatctctat gaactgtgcg tcacagccaa aagccagaca 780
gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa gggcgaacag 840
ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga agatgcggac 900
ttacgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt aatggactgg 960
attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat gctcgactgg 1020
gcagatgaac atggcatcgt ggtgattgat gaaactgctg ctgtcggctt taacctctct 1080
ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga agaggcagtc 1140
aacggggaaa ctcagcaagc gcacttacag gcgattaaag agctgatagc gcgtgacaaa 1200
aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg tccgcaaggt 1260
gcacgggaat atttcgcgcc actggcggaa gcaacgcgta aactcgaccc gacgcgtccg 1320
atcacctgcg tcaatgtaat gttctgcgac gctcacaccg ataccatcag cgatctcttt 1380
gatgtgctgt gcctgaaccg ttattacgga tggtatgtcc aaagcggcga tttggaaacg 1440
gcagagaagg tactggaaaa agaacttctg gcctggcagg agaaactgca tcagccgatt 1500
atcatcaccg aatacggcgt ggatacgtta gccgggctgc actcaatgta caccgacatg 1560
tggagtgaag agtatcagtg tgcatggctg gatatgtatc accgcgtctt tgatcgcgtc 1620
agcgccgtcg tcggtgaaca ggtatggaat ttcgccgatt ttgcgacctc gcaaggcata 1680
ttgcgcgttg gcggtaacaa gaaagggatc ttcactcgcg accgcaaacc gaagtcggcg 1740
gcttttctgc tgcaaaaacg ctggactggc atgaacttcg gtgaaaaacc gcagcaggga 1800
ggcaaacaat ga 1812
<210> 6
<211> 603
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Val Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Asp
1 5 10 15
Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Ile Asp Gln
20 25 30
Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg Ala Ile Ala Val Pro
35 40 45
Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Asp Ile Arg Asn Tyr Ala
50 55 60
Gly Asn Val Trp Tyr Gln Arg Glu Val Phe Ile Pro Lys Gly Trp Ala
65 70 75 80
Gly Gln Arg Ile Val Leu Arg Phe Asp Ala Val Thr His Tyr Gly Lys
85 90 95
Val Trp Val Asn Asn Gln Glu Val Met Glu His Gln Gly Gly Tyr Thr
100 105 110
Pro Phe Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val
115 120 125
Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr Ile Pro
130 135 140
Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Lys Gln Ser Tyr
145 150 155 160
Phe His Asp Phe Phe Asn Tyr Ala Gly Ile His Arg Ser Val Met Leu
165 170 175
Tyr Thr Thr Pro Asn Thr Trp Val Asp Asp Ile Thr Val Val Thr His
180 185 190
Val Ala Gln Asp Cys Asn His Ala Ser Val Asp Trp Gln Val Val Ala
195 200 205
Asn Gly Asp Val Ser Val Glu Leu Arg Asp Ala Asp Gln Gln Val Val
210 215 220
Ala Thr Gly Gln Gly Thr Ser Gly Thr Leu Gln Val Val Asn Pro His
225 230 235 240
Leu Trp Gln Pro Gly Glu Gly Tyr Leu Tyr Glu Leu Cys Val Thr Ala
245 250 255
Lys Ser Gln Thr Glu Cys Asp Ile Tyr Pro Leu Arg Val Gly Ile Arg
260 265 270
Ser Val Ala Val Lys Gly Glu Gln Phe Leu Ile Asn His Lys Pro Phe
275 280 285
Tyr Phe Thr Gly Phe Gly Arg His Glu Asp Ala Asp Leu Arg Gly Lys
290 295 300
Gly Phe Asp Asn Val Leu Met Val His Asp His Ala Leu Met Asp Trp
305 310 315 320
Ile Gly Ala Asn Ser Tyr Arg Thr Ser His Tyr Pro Tyr Ala Glu Glu
325 330 335
Met Leu Asp Trp Ala Asp Glu His Gly Ile Val Val Ile Asp Glu Thr
340 345 350
Ala Ala Val Gly Phe Asn Leu Ser Leu Gly Ile Gly Phe Glu Ala Gly
355 360 365
Asn Lys Pro Lys Glu Leu Tyr Ser Glu Glu Ala Val Asn Gly Glu Thr
370 375 380
Gln Gln Ala His Leu Gln Ala Ile Lys Glu Leu Ile Ala Arg Asp Lys
385 390 395 400
Asn His Pro Ser Val Val Met Trp Ser Ile Ala Asn Glu Pro Asp Thr
405 410 415
Arg Pro Gln Gly Ala Arg Glu Tyr Phe Ala Pro Leu Ala Glu Ala Thr
420 425 430
Arg Lys Leu Asp Pro Thr Arg Pro Ile Thr Cys Val Asn Val Met Phe
435 440 445
Cys Asp Ala His Thr Asp Thr Ile Ser Asp Leu Phe Asp Val Leu Cys
450 455 460
Leu Asn Arg Tyr Tyr Gly Trp Tyr Val Gln Ser Gly Asp Leu Glu Thr
465 470 475 480
Ala Glu Lys Val Leu Glu Lys Glu Leu Leu Ala Trp Gln Glu Lys Leu
485 490 495
His Gln Pro Ile Ile Ile Thr Glu Tyr Gly Val Asp Thr Leu Ala Gly
500 505 510
Leu His Ser Met Tyr Thr Asp Met Trp Ser Glu Glu Tyr Gln Cys Ala
515 520 525
Trp Leu Asp Met Tyr His Arg Val Phe Asp Arg Val Ser Ala Val Val
530 535 540
Gly Glu Gln Val Trp Asn Phe Ala Asp Phe Ala Thr Ser Gln Gly Ile
545 550 555 560
Leu Arg Val Gly Gly Asn Lys Lys Gly Ile Phe Thr Arg Asp Arg Lys
565 570 575
Pro Lys Ser Ala Ala Phe Leu Leu Gln Lys Arg Trp Thr Gly Met Asn
580 585 590
Phe Gly Glu Lys Pro Gln Gln Gly Gly Lys Gln
595 600
<210> 7
<211> 615
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggatcttt ctttcactct ttcggtcgtg tcggccatcc tcgccatcac tgctgtgatt 60
gctgtattta ttgtgatttt taggtatcac aacactgtga ccaagaccat cgaaacccgc 120
acagacaata tcgagacaaa catggatgaa aacctccgca ttcctgtgac tgctgaggtt 180
ggatcaggct acttcaagat gactgatgtg tcctttgaca gcgacacctt gggcaaaatc 240
aagatccgca atggaaagtc tgatgcacag atgaaggaag aagatgcgga tcttgtcatc 300
actcccgtgg agggccgagc actcgaagtg actgtggggc agaatctcac ctttgaggga 360
acattcaagg tgtggaacaa cacatcaaga aagatcaaca tcactggtat gcagatggtg 420
ccaaagatta acccatcaaa ggcctttgtc ggtagctcca acacctcctc cttcaccccc 480
gtctctattg atgaggatga agttggcacc tttgtgtgtg gtaccacctt tggcgcacca 540
attgcagcta ccgccggtgg aaatcttttc gacatgtacg tgcacgtcac ctactctggc 600
actgagaccg agtaa 615
<210> 8
<211> 204
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Asp Leu Ser Phe Thr Leu Ser Val Val Ser Ala Ile Leu Ala Ile
1 5 10 15
Thr Ala Val Ile Ala Val Phe Ile Val Ile Phe Arg Tyr His Asn Thr
20 25 30
Val Thr Lys Thr Ile Glu Thr Arg Thr Asp Asn Ile Glu Thr Asn Met
35 40 45
Asp Glu Asn Leu Arg Ile Pro Val Thr Ala Glu Val Gly Ser Gly Tyr
50 55 60
Phe Lys Met Thr Asp Val Ser Phe Asp Ser Asp Thr Leu Gly Lys Ile
65 70 75 80
Lys Ile Arg Asn Gly Lys Ser Asp Ala Gln Met Lys Glu Glu Asp Ala
85 90 95
Asp Leu Val Ile Thr Pro Val Glu Gly Arg Ala Leu Glu Val Thr Val
100 105 110
Gly Gln Asn Leu Thr Phe Glu Gly Thr Phe Lys Val Trp Asn Asn Thr
115 120 125
Ser Arg Lys Ile Asn Ile Thr Gly Met Gln Met Val Pro Lys Ile Asn
130 135 140
Pro Ser Lys Ala Phe Val Gly Ser Ser Asn Thr Ser Ser Phe Thr Pro
145 150 155 160
Val Ser Ile Asp Glu Asp Glu Val Gly Thr Phe Val Cys Gly Thr Thr
165 170 175
Phe Gly Ala Pro Ile Ala Ala Thr Ala Gly Gly Asn Leu Phe Asp Met
180 185 190
Tyr Val His Val Thr Tyr Ser Gly Thr Glu Thr Glu
195 200
Claims (10)
1.一种用于将外源基因在对虾细胞中进行表达的重组质粒,其特征在于,所述的重组质粒包含有对虾IHHNV病毒的基因组DNA、昆虫多角体蛋白基因的启动子PH、用于插入外源基因的多克隆酶切位点(MCS)和SV40多聚腺苷酸信号pA。
2.如权利要求1所述的重组质粒,其特征在于,所述的启动子PH和多克隆酶切位点之间还插入有GUS报告基因。
3.如权利要求1所述的重组质粒,其特征在于,所述的对虾IHHNV病毒的基因组DNA序列为SEQ ID NO:1。
4.如权利要求1所述的重组质粒,其特征在于,所述的PH启动子的核酸序列为SEQ IDNO:2,MCS的核酸序列为SEQ ID NO:3;SV40多聚腺苷酸信号pA的核酸序列为SEQ ID NO:4。
5.如权利要求1所述的重组质粒,其特征在于,所述的GUS报告基因的编码核酸和氨基酸序列分别为SEQ ID NO:5和SEQ ID NO:6。
6.权利要求1-5任一项所述的重组质粒在制备表达系统中的的应用,所述的表达系统是将外源基因在对虾细胞中进行表达。
7.一种对虾病毒表达系统,其特征在于,所述的对虾病毒表达系统中包含有权利要求1-5任一项所述的重组质粒。
8.如权利要求7所述的对虾病毒表达系统,其特征在于,所述的表达系统还包含有插入有对虾WSSV病毒囊膜蛋白VP28基因的昆虫杆状病毒重组表达质粒。
9.如权利要求7或8所述的对虾病毒表达系统,其特征在于,所述的表达系统还包含有昆虫Sf9包装细胞。
10.一种使外源基因在对虾成体组织和体外培养细胞中表达的方法,其特征在于,所述的方法是使用权利要求7-9任一项所述的对虾病毒表达系统来进行外源基因的表达。
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