CN116179608B - 一种dsRNA在免疫缺陷中华蜜蜂模型构建中的应用 - Google Patents
一种dsRNA在免疫缺陷中华蜜蜂模型构建中的应用 Download PDFInfo
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Abstract
本发明公开了一种dsRNA的新用途,即其在免疫缺陷中华蜜蜂模型构建中的应用,dsRNA的核酸序列如SEQ ID NO:1所示,本发明通过体外合成Spz4基因的部分片段的dsRNA,再给中华蜜蜂喂食dsRNA 72h后,即得免疫缺陷的中华蜜蜂模型,喂食dsRNA的免疫缺陷蜜蜂,Spz4基因的表达量降低70%,同时在致病菌侵染实验中7天的死亡率相比野生型蜜蜂增加了60%以上;且在粪肠球菌感染24小时后,4种抗菌肽的表达量显著降低。
Description
技术领域
本发明属于动物模型构建技术领域,具体的说,涉及一种dsRNA在免疫缺陷中华蜜蜂模型构建中的应用。
背景技术
免疫缺陷动物(immunodeficient animal)是指由于先天性基因突变或使用人工方法而导致免疫系统一种或多种成分缺陷的动物,可以利用免疫缺陷型的中华蜜蜂模型,对研究致病菌,寄生虫等致病机理有着极大的作用,从而可以达到有针对性的防治蜜蜂疾病的目的。
目前,在构建蜜蜂的免疫缺陷模型中,主要有两种技术方法,分别是CRISPR/Cas9和RNAi的方法。CRISPR/Cas9可使基因功能丧失更彻底,但是其操作复杂,耗时长,成本高。RNAi方法不能使基因的功能完全丧失,但可以大幅度降低目标基因的表达,且操作简单,耗时少,成本相对较低。RNAi是由双链RNA(dsRNA)诱导内源性特异性基因转录后沉默的一种机制。dsRNA进入生物体后,在细胞中被Dicer酶的RNaseⅢ分解为21-23bp的小干扰RNA(siRNA)。siRNA在沉默复合物(RISC)的作用下与靶mRNA结合,从而使其降解,达到降低基因表达水平的目的。
发明内容
本发明提供了一种dsRNA的新用途,即其在免疫缺陷中华蜜蜂模型构建中的应用,dsRNA的核酸序列如SEQ ID NO:1所示;本发明通过体外合成Spz4基因的部分片段的dsRNA,再给中华蜜蜂喂食dsRNA 72h后,即得到免疫缺陷的中华蜜蜂。
本发明采用RNAi的方法构建中华蜜蜂的免疫缺陷模型,RNAi的靶基因为Spaetzle4(Spz4,Gene ID: 108002210),Spz4蛋白是Toll信号转导途径所必需的胞外蛋白,与Toll受体结合并激活通路,产生抗菌肽以抵抗病原体,因此我们通过RNAi沉默Spz4基因从而构建免疫缺陷的蜜蜂模型。
本发明免疫缺陷中华蜜蜂模型的构建方法如下步:
(1)提取中华蜜蜂的总RNA,反转录获取cDNA;以cDNA作为模板,使用引物Spz4F:CAACGAATTCAGGGACGAGG,Spz4R:AGTAGTGCCGGGGAAATTCA进行PCR,获得Spz4的第426-930位的基因片段;将Spz4基因片段与质粒载体pCE2连接后转化入大肠杆菌DH5α中,筛选阳性克隆并提取质粒进行测序鉴定,将验证阳性的含有pCE2-Spz4RNAi质粒的大肠杆菌DH5α接种于含有卡纳霉素的LB液体培养基中进行增菌培养,抽提质粒,获得pCE2-Spz4RNAi质粒;
(2)以pCE2-Spz4RNAi质粒为模板,采用如下引物进行PCR扩增,获得用于扩增dsRNA序列的正向序列和反向序列,反应完成后进行琼脂糖凝胶电泳,后回收电泳产物,以回收的电泳产物为模板,体外转录合成dsRNA;
Spz4F:CAACGAATTCAGGGACGAGG
T7+Spz4R:TAATACGACTCACTATAGGGAGTAGTGCCGGGGAAATTCA;
Spz4R:AGTAGTGCCGGGGAAATTCA
T7+Spz4F:TAATACGACTCACTATAGGGCAACGAATTCAGGGACGAGG;
(3)每日给中华蜜蜂喂食20μg的dsRNA,喂食72小时后,即得免疫缺陷型中华蜜蜂。
本发明的有益效果在于:相较于CRISPR/Cas9系统,RNAi的方法更加高效,72小时就获得免疫缺陷型中华蜜蜂,且在蜜蜂各年龄段都可应用,相较于注射会导致蜜蜂引起免疫反应,喂食可以避免该问题,每只蜜蜂通过用单独的200μLEP管进行喂食,相较于传统灌喂,更加的快速方便。
附图说明
图1为实施例2中喂食PBS和dsSpz4 48h后感染粪肠球菌24h后中华蜜蜂Spz4基因的相对表达量图;
图2为实施例2中喂食PBS和dsSpz4 48h后感染粪肠球菌24h后中华蜜蜂抗菌肽基因的相对表达量图;
图3为实施例3中喂食PBS和dsSpz4 48h后感染粪肠球菌7天后中华蜜蜂的死亡情况。
具体实施方式
为了使本发明的目的、技术方案和有益技术效果更加清晰明白,以下结合附图和具体实施方式,对本发明进行进一步详细说明。应当理解的是,本说明书中描述的具体实施方式仅仅是为了解释本发明,并不是为了限定本发明。
实施例1:dsRNA的合成
1、Spz4基因特异引物的设计
根据Spz4基因序列信息,设计Spz4部分基因特异引物,引物序列如表1所示;
表1
引物 | 5’-3’ |
Spz4F | CAACGAATTCAGGGACGAGG |
Spz4R | AGTAGTGCCGGGGAAATTCA |
2、中华蜜蜂RNA的提取
取中华蜜蜂的肠道组织20mg,参照FastPure® Cell/Tissue TotalRNA IsolationKit V2试剂盒(购自Vazyme公司,中国)的操作说明,提取总RNA,具体操作步骤如下:
①匀浆处理:在20mg的肠道组织中添加500μL的Buffer RL,用电动匀浆器进行匀浆;
②将匀浆裂解后样本转移至FastPure gDNA-Filter Columns Ⅲ(FastPuregDNA-Filter Columns Ⅲ已放入收集管)中,12000rpm离心30s,弃掉FastPure gDNA-Filter Columns Ⅲ,收集滤液;
③向滤液中加入0.5倍滤液体积的无水乙醇并充分混匀;
④将步骤③混合液全部转移至FastPure RNA Columns Ⅲ(FastPure RNAColumns Ⅲ已放入收集管)中,12000rpm离心30s,弃滤液;
⑤向FastPure RNA Columns Ⅲ中加入700μL Buffer RW1,12000 rpm离心30s,弃滤液;
⑥向FastPure RNA Columns Ⅲ中加入700μL Buffer RW2(已加入无水乙醇),12000rpm离心30s,弃滤液;
⑦向FastPure RNA Columns Ⅲ中加入500μL Buffer RW2(已加入无水乙醇),12000 rpm离心2 min,将吸附柱从收集管中取;
⑧将吸附柱转移至新的RNase-free Collection Tubes 1.5 mL离心管中,向吸附柱中央悬空滴加50μL的RNase-free ddH2O,室温静置1min,12000 rpm离心1 min,洗脱获得RNA;
3、cDNA的合成
参照HiScript III RT SuperMix for qPCR (+gDNA wiper)试剂盒(购自Vazyme公司,中国)中说明书合成cDNA;具体如下,配制反转录体系:5μL步骤2提取的总RNA,4μL 4×gDNA wiper Mix,7μLNucleacen free-water,PCR仪中42℃,2min;之后添加4μL5 ×HiScript III qRTSuperMix;在PCR仪37℃,15min;85℃,5s;合成后将cDNA放于-20℃冰箱保存。
4、pCE2-Spz4RNAi质粒的制备
以步骤3中的cDNA为模板,使用步骤1引物扩增Spz4基因的第426-930位的基因片段,PCR反应体系(25μL):2×Phanta Max Master Mix (Dye Plus) 12.5μL、10μmol/L 上下游引物各1μL、模板1μL、无菌无酶水10.5μL;PCR反应程序95℃,3 min;95℃,15s;56℃,15s,72℃,30s,35个循环;72℃,5min;
参照5min TA/Blunt-Zero Cloning Kit试剂盒(购自Vazyme公司,中国)中的操作说明书,将Spz4基因片段(505bp)与质粒载体pCE2连接;具体如下:将1μLSpz4基因片段、1μL5×TA/Blunt-Zero Cloning Mix、3μL无菌无酶水混合,轻弹混匀,室温下反应5min,反应结束转化入大肠杆菌DH5α,用含有卡纳霉素(浓度50μg/mL)的LB平板筛选阳性克隆,提取质粒进行鉴定,获得了在质粒载体pCE2的TOPO位点之间插入了核苷酸序列如SEQ ID NO:1所示的Spz4基因片段的阳性克隆质粒pCE2-Spz4 RNAi;将含有pCE2-Spz4 RNAi的大肠杆菌DH5α接种于含有卡纳霉素的LB液体培养基中进行增菌培养,大量抽提质粒,用无菌无酶水溶解,即得pCE2-Spz4 RNAi质粒;pCE2-Spz4 RNAi质粒用M13通用引物测序进行验证,-20℃保存正确质粒。
5、dsRNA的合成
以pCE2-Spz4RNAi质粒为模板,采用如下引物进行PCR扩增;
Spz4F:CAACGAATTCAGGGACGAGG
T7+Spz4R:TAATACGACTCACTATAGGGAGTAGTGCCGGGGAAATTCA;
Spz4R:AGTAGTGCCGGGGAAATTCA
T7+Spz4F:TAATACGACTCACTATAGGGCAACGAATTCAGGGACGAGG;
PCR 反应体系(25μL):2×Phanta Max Master Mix(Dye Plus)12.5μL、10μmol/L上下游引物各1μL、模板1μL、无菌无酶水10.5μL;PCR反应程序95℃,3 min;95℃,15s;65℃,15s,72℃,30s,35个循环;72℃,5min;反应完成后分别进行琼脂糖凝胶电泳,然后回收电泳产物,获得用于扩增dsRNA序列的正向序列和反向序列;
dsRNA合成转录体系(20μL):NTP Mix8μL、10×Transcription Buffer2μL、T7Enzyme Mix2μL,正、反向模板各4μL(0.5μg);PCR 仪中 37℃,2h后;加入DNaseI 2μL、RNaseT1(10U/μL)2μL、无菌无酶水17μL,PCR 仪中37℃,30min,去除DNA模板及ssRNA,加入80μL的磁珠溶液,充分混匀后室温孵育8min,使RNA与磁珠充分结合,将PCR管置于磁力架上约5min移除上清液,保持PCR管处于磁力架上,加入200μL新配制的80%乙醇,室温孵育30s,移除上清,重复该步骤一次;开盖空气干燥磁珠5-10min,将PCR管从磁力架上取下,加入40μL的RNase-free H2O,充分混匀,室温孵育3min;将PCR管置于磁力架上,待溶液澄清后,将上清液转移至新的RNase-free EP管中,在A260处测产物的吸光值,确定其浓度为1765ng/μL,-20℃保存纯化产物;电泳检测上述扩增获得的dsRNA的质量及片段大小。经凝胶电泳验证,其条带较为单一无其他杂带,大小为500bp左右、浓度均符合要求,可用于RNAi试验。
实施例2:免疫缺陷型中华蜜蜂的构建以及用于病原体感染实验
1、每天给中华蜜蜂喂食20μg的dsRNA,72小时后,即得免疫缺陷的中华蜜蜂。
2、病原体感染实验
挑取1日龄的中华蜜蜂,将实施例1合成的dsRNA给中华蜜蜂喂食,同时以用PBS喂食为对照,每只蜜蜂每日的dsRNA的灌喂量均为20μg,本实验共设置6个生物学重复,每个生物学重复1只蜜蜂,共计6只;每只蜜蜂置于单个奶茶杯中饲养,每24h将20μg的dsRNA与糖水混合后装入200μL的EP管中,让蜜蜂自然进食(24h吃完),饲喂48h后感染粪肠球菌,感染24h后统计中华蜜蜂基因沉默效率,以及抗菌肽的表达情况。
提取中华蜜蜂肠道的总RNA,反转录合成cDNA,以cDNA为模板,以ATGCCGTGGTATTTTCAGCG、TTCGTTGCTTTTGAGTGCGA为引物,采用qRT-PCR法测定灌喂dsRNA、PBS后被粪肠球菌感染的中华蜜蜂的Spz4基因表达量,统计分析沉默效率,结果见图1,结果显示灌喂dsRNA的中华蜜蜂的Spz4基因表达量受到抑制,经计算沉默率约为70%(Spz4的表达量在喂食dsRNA后,降低到正常蜜蜂的30%,说明沉默该基因是成功的),而喂食PBS的中华蜜蜂的Spz4基因没有被沉默。
以cDNA为模板,采用如下引物,通过qRT-PCR法测定粪肠球菌感染后的中华蜜蜂的抗菌肽(abaecin、apidaecin、defensin1、defensin2、hymenoptaecin和lysozyme)的表达量,基因的相对表达量的获得是通过qRT-PCR得到结果后,验证溶解曲线后通过计算Ct值的方法进行数据分析。每一个所测基因设置三个重复,以actin作为内参基因,用Excel软件分析样本间差异显著性;
abaecin F:AGAGTTTGATCCTGGCTCAG
abaecin R:CTGCTGCCTCCCGTAGGAGT;
apidaecin F:CCAGATCCGCCTACTCAACC
apidaecin R:GGTTTAGCTTCACGGCGTAG;
defensin1 F:AGCCACTTGAGCATCCTGAG
defensin1 R:CCGTTCTTGCAATGACCTCC;
defensin2 F:TTTCGCGATTCTCGTCGCTA
defensin2R:TGTCGTAGCAGTAGCGGTTC;
hymenoptaecin F:CGTGTTGGTTGTCTTCTGCG
hymenoptaecin R:CACCATAGGCATCTCCCGTC;
lysozyme F:ACACGGTTGGTCACTGGTCC
lysozyme R:GTCCCACGCTTTGAATCCCT;
结果见图2:结果显示喂食dsRNA的中华蜜蜂的抗菌肽abaecin、apidaecin的表达量没有显著性,其中4个抗菌肽defensin1、defensin2、hymenoptaecin和lysozyme的表达量均显著降低,说明在Spz4基因被沉默后,中华蜜蜂的抗菌肽的表达量下降,说明蜜蜂的免疫病原体的能力降低。
实施例3:喂食dsRNA的中华蜜蜂的存活实验
挑取1日龄的中华蜜蜂,将实施例1合成的dsRNA给中华蜜蜂喂食,同时以用PBS喂食为对照,每只蜜蜂的dsRNA的喂食量均为20μg,本实验共设置8个生物学重,每只蜜蜂置于单个奶茶杯中饲养,每24h将20μg的dsRNA与糖水混合后装入200μL的EP管中,让蜜蜂自然进食,48h后,使其感染粪肠球菌,在感染后的7天,每天定点统计中华蜜蜂的存活情况;中华蜜蜂的死亡率的获取是在中华蜜蜂感染粪肠球菌后,每日定点统计中华蜜蜂的存活数量;用Excel软件分析死亡率差异显著性。
结果见图3,经计算在感染粪肠球菌三日后,免疫缺陷组的中华蜜蜂的死亡率相比对照组高了60%以上,在基因沉默之后,中华蜜蜂在被病原体侵染后的生存时间大幅度降低。
Claims (1)
1.一种dsRNA在免疫缺陷型中华蜜蜂模型构建中的应用,所述dsRNA的核酸序列如SEQID NO:1所示;
给中华蜜蜂喂食dsRNA,喂食72小时后,即得免疫缺陷型中华蜜蜂。
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Citations (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995002684A1 (en) * | 1993-07-16 | 1995-01-26 | The Board Of Trustees Of The Leland Stanford Junior University | Regulated apoptosis |
WO2003009812A2 (en) * | 2001-07-25 | 2003-02-06 | New York University | Use of glycosylceramides as adjuvants for vaccines against infections and cancer |
WO2005116220A1 (en) * | 2004-05-28 | 2005-12-08 | Cambridge University Technical Services Limited | Production of recombinant protein using heterologous prosequences |
CN101139603A (zh) * | 2007-08-07 | 2008-03-12 | 重庆拓桑生物科技有限公司 | 家蚕bmspz-like基因 |
EP3017045A2 (en) * | 2013-07-02 | 2016-05-11 | University Court Of The University Of Aberdeen | Control of varroa mite infestation |
CN109393254A (zh) * | 2017-08-18 | 2019-03-01 | 彦臣生技药品股份有限公司 | 用于提升昆虫免疫力的组合物及方法 |
CN111088281A (zh) * | 2020-01-09 | 2020-05-01 | 山东农业大学 | 中华蜜蜂耐热相关基因DnaJ1及其应用 |
CN113265350A (zh) * | 2021-05-11 | 2021-08-17 | 昆明理工大学 | 一种双歧杆菌w8118及其应用 |
CN113265351A (zh) * | 2021-05-11 | 2021-08-17 | 昆明理工大学 | 一株乳杆菌w8172及其应用 |
CN113621620A (zh) * | 2021-08-17 | 2021-11-09 | 安徽农业大学 | 具有调控小菜蛾免疫功能的基因及其制备方法和应用 |
CN114437113A (zh) * | 2022-03-18 | 2022-05-06 | 西安交通大学 | 一种噻唑并吡啶环联三氮唑类化合物及其制备方法和应用 |
CN115181745A (zh) * | 2022-04-01 | 2022-10-14 | 中国农业科学院蜜蜂研究所 | 抑制蜜蜂Rho1基因的siRNA及其应用 |
CN116589551A (zh) * | 2023-01-10 | 2023-08-15 | 中国农业科学院植物保护研究所 | 蝗虫免疫相关蛋白LmEaster基因及其应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8097712B2 (en) * | 2007-11-07 | 2012-01-17 | Beelogics Inc. | Compositions for conferring tolerance to viral disease in social insects, and the use thereof |
US20230272395A1 (en) * | 2022-02-28 | 2023-08-31 | The United States Of America, As Represented By The Secretary Of Agriculture | Engineered microalgae feed to improve honey bee pathogen resistance and nutrition |
-
2023
- 2023-01-05 CN CN202310012287.9A patent/CN116179608B/zh active Active
Patent Citations (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995002684A1 (en) * | 1993-07-16 | 1995-01-26 | The Board Of Trustees Of The Leland Stanford Junior University | Regulated apoptosis |
WO2003009812A2 (en) * | 2001-07-25 | 2003-02-06 | New York University | Use of glycosylceramides as adjuvants for vaccines against infections and cancer |
WO2005116220A1 (en) * | 2004-05-28 | 2005-12-08 | Cambridge University Technical Services Limited | Production of recombinant protein using heterologous prosequences |
CN101139603A (zh) * | 2007-08-07 | 2008-03-12 | 重庆拓桑生物科技有限公司 | 家蚕bmspz-like基因 |
EP3017045A2 (en) * | 2013-07-02 | 2016-05-11 | University Court Of The University Of Aberdeen | Control of varroa mite infestation |
CN109393254A (zh) * | 2017-08-18 | 2019-03-01 | 彦臣生技药品股份有限公司 | 用于提升昆虫免疫力的组合物及方法 |
CN111088281A (zh) * | 2020-01-09 | 2020-05-01 | 山东农业大学 | 中华蜜蜂耐热相关基因DnaJ1及其应用 |
CN113265350A (zh) * | 2021-05-11 | 2021-08-17 | 昆明理工大学 | 一种双歧杆菌w8118及其应用 |
CN113265351A (zh) * | 2021-05-11 | 2021-08-17 | 昆明理工大学 | 一株乳杆菌w8172及其应用 |
CN113621620A (zh) * | 2021-08-17 | 2021-11-09 | 安徽农业大学 | 具有调控小菜蛾免疫功能的基因及其制备方法和应用 |
CN114437113A (zh) * | 2022-03-18 | 2022-05-06 | 西安交通大学 | 一种噻唑并吡啶环联三氮唑类化合物及其制备方法和应用 |
CN115181745A (zh) * | 2022-04-01 | 2022-10-14 | 中国农业科学院蜜蜂研究所 | 抑制蜜蜂Rho1基因的siRNA及其应用 |
CN116589551A (zh) * | 2023-01-10 | 2023-08-15 | 中国农业科学院植物保护研究所 | 蝗虫免疫相关蛋白LmEaster基因及其应用 |
Non-Patent Citations (6)
Title |
---|
PREDICTED: Apis cerana protein spaetzle 4 (LOC108002210), transcript variant X1, mRNA;NCBI;Genbank Database;Accession No:XM_028668966.1 * |
Stably transmitted defined microbial community in honeybees preserves Hafnia alvei inhibition by regulating the immune system;Jieni Wang 等;Frontiers in Microbiology;摘要,第5页右栏第2-3段,图3 * |
Wenhao Zhang等.Toll receptor ligand Spa¨tzle 4 responses to the highly pathogenic Enterococcus faecalis from Varroa mites in honeybees.PLOS Pathogens.2023,第1-19页. * |
北方山溪鲵精巢中性类固醇激素的免疫细胞化学研究;李亚琳;;西北农业学报(第01期);第35-40页 * |
家蚕Bmspatzle4新剪接异构体的发现及其在体壁中对不同微生物的免疫响应;黄心怡;中国优秀硕士学位论文全文数据库农业科技辑;摘要,正文第35-50页 * |
郭丽娜 ; 赵慧婷 ; 任有蛇 ; 徐兵 ; 姜玉锁 ; .中华蜜蜂气味受体AcerOr2基因最佳干扰片段的筛选.山西农业科学.2020,(第06期),第1-8页. * |
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