CN107287216B - CvBV5-3基因在降低果蝇免疫力和制备免疫低下型果蝇模型中的应用 - Google Patents
CvBV5-3基因在降低果蝇免疫力和制备免疫低下型果蝇模型中的应用 Download PDFInfo
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Abstract
本发明公开了CvBV5‑3基因在降低果蝇免疫力和制备免疫低下型果蝇模型中的应用，该CvBV5‑3基因是菜蛾盘绒茧蜂多分DNA病毒的35个基因组片段中的第5个环上的第3个基因，其碱基序列如SEQ ID NO.1所示。本发明利用转基因技术，将菜蛾盘绒茧蜂多分DNA病毒基因CvBV5‑3转入果蝇基因组，获得了稳定遗传纯合体的转基因果蝇品系；该转基因品系表达多分DNA病毒基因CvBV5‑3后，存在血细胞凋亡的现象，并且在受到病原物侵染时，具有明显的免疫力低的性状，在对研究昆虫免疫相关机制、增强人类免疫类药剂的筛选等方面有重要意义。
Description
技术领域
本发明涉及分子生物学和基因工程技术领域，尤其涉及CvBV5-3基因在降低果蝇免疫力和制备免疫低下型果蝇模型中的应用。
背景技术
昆虫是目前地球陆地上最繁盛的物种类群，是人类取之不尽的资源宝库。近年来，昆虫的免疫在其基础和应用研究方面受到极大关注，通过实验来研究与昆虫免疫相关的机制、信号等问题，对害虫防治、益虫防病、开发利用抗菌物、研究人类免疫机制等有着非常重要的现实意义。
果蝇属于双翅目、果蝇属、果蝇科昆虫，约1000种。其中，果蝇具有生活史短、易饲养、繁殖快、染色体少、突变型多、易于观察等特点，被广泛地用作遗传和演化的室内外研究材料，是一种重要的模式生物。在生命科学发展的历史长河中，果蝇扮演了十分重要的角色，是十分活跃的模型生物。随着转基因技术的发展，各种转基因果蝇模型的应用也越来越广泛。
目前，转基因果蝇模型主要用于遗传学的研究、发育的基因调控的研究、各类神经疾病的研究、帕金森氏病、老年痴呆症、药物成瘾和酒精中毒、衰老与长寿、学习记忆与某些认知行为等研究中。
低免疫力果蝇模型在对研究昆虫免疫相关机制、增强人类免疫类药剂的筛选等方面有重要意义。但是，毒性较强的基因通常容易造成转基因果蝇的死亡；因此，寻找合适的毒性基因是建立免疫低下型果蝇的重要环节。
而Polydnaviruses(多分DNA病毒，PDV)是一类寄生蜂专性共生的特殊病毒，主要包括两个属：分别是存在于膜翅目茧蜂科(Braconidae)的茧蜂病毒(Bracovirus,BV)与姬蜂科(Ichneumonidae)昆虫体内的姬蜂病毒属(Ichnovirus,IV)(Turnbull,M.and B.Webb(2002).Perspectives on polydnavirus origins and evolution.Advances in Virus Research,Academic Press.58:203-254)。PDV粒子随雌蜂产卵时注入到寄生蜂的寄主体体腔内，首先侵染血细胞，随后侵染所有组织。PDV作为寄生蜂的“强力武器”(Bai,S.,X.Chen,J.Cheng,W.Fu and J.He(2005)."Effects of wasp-associated factors of Cotesiaplutellae on growth and development of Plutella xylostella larvae."Acta Phytophylacica Sinica 32(3):235-240)，其主要生理功能是抑制寄主的免疫(Dupuy,C.,E.Huguet and J.M.Drezen(2006)."Unfolding the evolutionary story ofpolydnaviruses."Virus Research 117(1):81-89,Strand,M.R.and G.R.Burke(2012)."Polydnaviruses as symbionts and gene delivery systems."Plos Pathogens 8(7))，却不造成寄主的死亡，以保证寄生蜂的正常生长发育(Ye,X.,M.Shi and X.Chen(2014)."Origin and character-istics of polydnaviruses carried by parasitoid wasps."Scientia Sinica Vitae 44(4):342)。菜蛾盘绒茧蜂多分DNA病毒(CvBV)是国内研究较多的一种多分DNA病毒，共有35个环状DNA组成，编码一百多个毒性基因。因此，有必要对CvBV基因做更进一步地深入研究。
发明内容
本发明发现了CvBV5-3基因在降低果蝇免疫力和制备免疫低下型果蝇中的新用途。
CvBV5-3基因是菜蛾盘绒茧蜂多分DNA病毒的35个基因组片段中的第5个环上的第3个基因，碱基序列如SEQ ID NO.1所示；其能够编码127个氨基酸，其氨基酸序列如SEQ IDNO.2所示。
本发明提供了CvBV5-3基因在降低果蝇免疫力中的应用，所述CvBV5-3基因的碱基序列如SEQ ID NO.1所示。
经实验发现，CvBV5-3基因在促进果蝇血细胞凋亡中的用途，所述CvBV5-3基因的碱基序列如SEQ ID NO.1所示。
本发明还提供了CvBV5-3基因在制备免疫低下型果蝇模型中的应用，所述CvBV5-3基因的碱基序列如SEQ ID NO.1所示。
本发明提供了一种免疫低下型果蝇模型的制备方法，包括：
(1)制备包含有CvBV5-3基因的重组质粒，将重组质粒注入白眼野生型果蝇胚胎中，胚胎发育至成虫后，获得红眼转基因果蝇；所述CvBV5-3基因的碱基序列如SEQ ID NO.1所示；
(2)将红眼转基因果蝇与平衡系进行两轮杂交，依据后代表型，挑选纯合体转基因果蝇；
(3)利用UAS/GAL4系统，将步骤(2)制得的纯合体转基因果蝇与BS8700果蝇品系进行杂交，使得CvBV5-3基因在后代果蝇血细胞中特异性表达，从而获得免疫低下型果蝇。
其中，所述白眼野生型果蝇为W1118；平衡系的基因型为w-/w-；Sp/Cyo；TM2/TM6B。
所述纯合体转基因果蝇的基因型为w-/w-；CvBV5-3/CvBV5-3；TM2/TM6B。
本发明还提供了一种免疫低下型果蝇模型，其染色体中包含有CvBV5-3基因，所述CvBV5-3基因的碱基序列如SEQ ID NO.1所示。
本发明还提供了一种采用上述制备方法制备的免疫低下型果蝇模型。
与现有技术相比，本发明具有以下有益效果：
(1)本发明利用转基因技术，将菜蛾盘绒茧蜂多分DNA病毒基因CvBV5-3转入果蝇基因组，获得了稳定遗传、纯合体的转基因果蝇品系；利用UAS/GAL4系统，纯合体的CvBV5-3转基因果蝇与BS8700果蝇品系进行杂交，得到的品系表达多分DNA病毒基因CvBV5-3后，存在血细胞凋亡的现象，并且在受到病原物侵染时，具有明显的免疫力低的性状，在对研究昆虫免疫相关机制、增强人类免疫类药剂的筛选等方面有重要意义。
(2)本发明构建的CvBV5-3转基因果蝇借助UAS/GAL4系统的调控以及杂交试验，实现了CvBV5-3基因在果蝇特定组织的高表达。
附图说明
图1为实施例1中构建CvBV5-3转基因果蝇的PCR检测结果。
图2为实施例1中半定量PCR检测CvBV5-3在转基因果蝇中的表达结果。
图3为实施例1中CvBV5-3基因在果蝇血细胞中特异表达对血细胞影响的检测结果。
图4为实施例1中CvBV5-3基因引起果蝇幼虫血细胞凋亡的检测结果。
图5为实施例1中CvBV5-3基因影响果蝇免疫的检测结果。
具体实施方式
实施例1
1、转基因果蝇的构建
根据CvBV5-3基因开放阅读框(ORF,SEQ ID NO.1所示)两端序列以及pUASttb载体上多克隆位点序列，结合同源重组酶的作用方式，设计特异性引物，引物序列如下：
F：5’-TTCGTTAACAGATCTGCGGCCGCATGGTGTTCAACAAAACCTTAAT-3’
R：5’-TCCTCTAGAGGTACCCTCGAGTTAATCTTTGATAATAATTGGTCC-3’；
采用Trizol(Invitrogen,Carlsbad,CA,USA)提取寄生后小菜蛾血细胞的总RNA，并利用试剂盒SMART RACE cDNA Amplification kit(Clontech,USA)构建cDNA文库。然后，利用特异性引物F、R从构建的cDNA文库中PCR扩增出带有酶切位点的CvBV5-3基因片段，利用同源重组酶(ClonExpress II One Step Cloning Kit，诺唯赞，南京)将片段重组克隆到载体上。重组后的载体转到大肠杆菌进行扩繁，并对重组质粒进行测序验证序列的正确性。
将验证正确的重组质粒显微注射入白眼野生型黑腹果蝇W1118的胚胎中，胚胎发育至成虫即为G0代，G0代果蝇中红眼果蝇即为成功的转基因品系，以此为依据进行筛选。
利用PCR检测，提取CvBV5-3转基因果蝇和野生型W1118黑腹果蝇的基因组DNA，分别用特异性引物F、R对这些基因组DNA进行PCR检测，从CvBV5-3转基因果蝇扩增出与CvBV5-3大小一致的片段，而在野生型W1118黑腹果蝇的基因组DNA中没有扩增出任何片段。证明菜蛾盘绒茧蜂多分DNA病毒CvBV5-3基因成功插入到转基因果蝇的基因组中，转基因果蝇品系构建成功(如图1)。
2、CvBV5-3转基因果蝇纯合体品系的构建
本实施例中CvBV5-3插入到二号染色体上，故将上述步骤1构建成功的转基因品系与平衡系(w-/w-；Sp/Cyo；TM2/TM6B)进行杂交，依据后代表现型，挑选出子代具有卷翅、大平衡棒的处女果蝇(w-/w-；CvBV5-3/Cyo；TM2/+)以及具有卷翅、肩部多毛的雄果蝇(w-/w-；CvBV5-3/Cyo；TM6B/+)，并将挑选出的果蝇进行杂交；或者挑选出子代具有卷翅、大平衡棒的雄果蝇(w-/w-；CvBV5-3/Cyo；TM2/+)以及具有卷翅、肩部多毛的处女果蝇(w-/w-；CvBV5-3/Cyo；TM6B/+)，并将挑选出的果蝇进行杂交；杂交后代挑选直翅、大平衡棒、肩部多毛的纯合体的转CvBV5-3基因的果蝇(w-/w-；CvBV5-3/CvBV5-3；TM2/TM6B)进行保种。
3、CvBV5-3转基因果蝇在体内的表达
利用UAS/GAL4系统，将步骤2中纯合体的CvBV5-3转基因果蝇与BS8700(FlyBaseID:FBti0064641)果蝇品系进行杂交，使得CvBV5-3在果蝇血细胞中特异表达，并以CvBV5-3转基因品系与野生型W1118杂交作为对照。提取子代血细胞总RNA反转录为cDNA(具体方法同(1)中)，以管家基因Actin做内参，用定量特异性引物进行半定量检测。
定量特异性引物如下：
CvBV5-3-qPCR-F：5’-GAAAGGATTCGGAACGTGTG-3’；
CvBV5-3-qPCR-F：5’-CTGCTTGCTGGTCGAGAACG-3’；
Dro-actin-qPCR-F：5’-GCTGAGCGTGAAATCGTCCG-3’；
Dro-actin-qPCR-R：5’-GGAGTTGTAGGTGGTCTCGTGGA-3’。
图2结果表明，在mRNA水平上，CvBV5-3转基因品系和W1118的杂交后代无CvBV5-3的表达，而CvBV5-3转基因品系和BS8700的杂交后代表达量很高。因此，说明得到了在血细胞中特异表达CvBV5-3的转基因果蝇。
4、CvBV5-3基因在果蝇血细胞中特异表达对果蝇的影响
a)CvBV5-3在果蝇血细胞中特异表达对血细胞影响的检测结果
利用CvBV5-3转基因品系与野生型W1118和BS8700的处女果蝇分别杂交，提取子代血细胞，对子代幼虫血细胞进行原代培养，并置于光学显微镜下进行形态学观察。
图3结果表明，对照组果蝇幼虫血细胞生长状态正常，而有CvBV5-3特异表达的果蝇幼虫血细胞，出现部分凋亡的现象，并伴随有凋亡小体的产生(如图中白色箭头所指)。
b)CvBV5-3引起果蝇幼虫血细胞凋亡的检测结果
利用CvBV5-3转基因品系与野生型W1118和BS8700的处女果蝇分别杂交，提取子代血细胞，对子代幼虫血细胞进行原代培养，并按照TUNEL BrightRed Apoptosis DetectionKit(诺唯赞生物科技有限公司)说明书所述方法进行检测。
图4结果表明，对照组血细胞无凋亡信号(白色斑点)；而CvBV5-3在果蝇幼虫血细胞中特异表达能引起果蝇幼虫血细胞的凋亡(如白色箭头所示)。
c)CvBV5-3影响果蝇免疫的检测结果
利用CvBV5-3转基因品系与野生型W1118和BS8700的处女果蝇分别杂交，向后代果蝇雄成虫(约60头)体内注射33nl PBS和金黄色葡萄球菌(OD＝0.4)。注射后每隔12h，统计果蝇生存情况，绘制生存曲线。
图5结果表明，注射PBS不影响果蝇的生存率；而注射金黄色葡萄球菌后，CvBV5-3转基因品系与BS8700的杂交后代的存活率显著(p＝0.0457)低于对照组(CvBV5-3转基因品系与野生型W1118的杂交后代)，表明CvBV5-3能降低果蝇的免疫。
SEQUENCE LISTING
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Claims (6)
1.CvBV5-3基因在降低果蝇免疫力中的应用，其特征在于，所述CvBV5-3基因的碱基序列如SEQ ID NO.1所示。
2.CvBV5-3基因在促进果蝇血细胞凋亡中的应用，其特征在于，所述CvBV5-3基因的碱基序列如SEQ ID NO.1所示。
3.CvBV5-3基因在制备免疫低下型果蝇模型中的应用，其特征在于，所述CvBV5-3基因的碱基序列如SEQ ID NO.1所示。
4.一种免疫低下型果蝇模型的制备方法，其特征在于，包括：
(1)制备包含有CvBV5-3基因的重组质粒，将重组质粒注入白眼野生型果蝇胚胎中，胚胎发育至成虫后，获得红眼转基因果蝇；所述CvBV5-3基因的碱基序列如SEQ ID NO.1所示；
(2)将红眼转基因果蝇与平衡系进行两轮杂交，依据后代表型，挑选纯合体转基因果蝇；
(3)利用UAS/GAL4系统，将步骤(2)制得的纯合体转基因果蝇与BS8700果蝇品系进行杂交，使得CvBV5-3基因在后代果蝇血细胞中特异性表达，从而获得免疫低下型果蝇。
5.如权利要求4所述的制备方法，其特征在于，所述白眼野生型果蝇为W1118；平衡系的基因型为w-/w-；Sp/Cyo；TM2/TM6B。
6.如权利要求4所述的制备方法，其特征在于，所述纯合体转基因果蝇的基因型为w-/w-；CvBV5-3/CvBV5-3；TM2/TM6B。
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