CN116762898B - 基于CpG寡核苷酸串联分子的对虾免疫增强剂及其应用 - Google Patents
基于CpG寡核苷酸串联分子的对虾免疫增强剂及其应用 Download PDFInfo
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Abstract
本发明提供一种基于CpG寡核苷酸串联分子的对虾免疫增强剂及其应用。本发明提供了8种CpG ODNs串联分子，研究结果表明它们在改善对虾肠道菌群结构的同时，显著上调对虾肝胰腺和肠道免疫基因表达，以及增强对虾肝胰腺和肠道抗氧化酶活性，从而增强对虾免疫力。该产品的开发对于降低对虾养殖过程中的病害率，提高经济效益，推动对虾养殖业的发展有重要意义。
Description
技术领域
本发明属于水产养殖技术领域，特别涉及基于CpG寡核苷酸串联分子的对虾免疫增强剂及其应用。
背景技术
凡纳滨对虾（Penaeus vannamei），又名南美白对虾，由于生长周期短、营养价值高、便于运输等优势，已经成为对虾养殖业中利润最高的品种之一。随着对虾养殖业在世界范围内的迅速发展，高密度、集约化的养殖模式增加了水体生态环境负担，不仅导致养殖环境严重恶化、苗种质量不断退化、养殖产量急剧下降，而且造成对虾机体抗逆性及抗病性下降，由细菌、病毒以及毒素引起的疫病频繁暴发。例如，由副溶血弧菌引起的对虾急性肝胰腺坏死病或早期死亡综合症，由桃拉病毒引起的对虾桃拉病，由黄头病毒引起的黄头病以及由微孢子虫引起的对虾肝肠孢虫病等，各种防治手段束手无策，养殖对虾产量持续下降，严重制约着对虾养殖业的健康可持续发展。因此，如何利用现代生物学技术，研究对虾免疫调控的分子机理，开发新型对虾抗病制剂，采取有效策略提高对虾自身的免疫力和抵抗力，对于提高对虾养殖经济收益，推动对虾养殖业的发展有重要意义。
传统上治疗和预防对虾各类疫病主要依赖使用抗生素和化学类药物等，然而药物的滥用又造成养殖水体中药物残留、耐药性病原微生物产生以及环境污染等严重问题，阻碍了对虾养殖业的健康发展。免疫增强剂是一类能够激活机体免疫力的物质，以其广谱、高效、环保等优点，在水产动物疫病防治中得到了广泛的关注和应用。对虾免疫增强剂主要是通过提高对虾非特异免疫能力，增强其抗病性，根据其成分可分为人工合成制剂、动植物制剂、微生物制剂及其衍生物和营养因子物质等。其中，CpG oligodeoxynucleotides（CpGODNs）为含有未甲基化的CG基序寡核苷酸单链，均能够有效的激活并调控机体的先天免疫系统，属于活性较强的免疫增强剂。CpG ODNs的功能受CpG二核苷酸侧翼序列、ODN骨架及特殊序列影响，它们具有种属特异性、细胞特异性，有的甚至作用完全相反。目前，关于CpGODNs在作为凡纳滨对虾免疫增强剂方面的相关报道并不多见。因此，开发一种基于CpGODNs串联分子、针对于凡纳滨对虾的免疫增强制剂，对于降低其养殖过程中的病害率，提高经济效益，推动对虾养殖业的发展有重要意义。
发明内容
本发明旨在提供一种基于CpG 寡核苷酸串联分子的对虾免疫增强剂，该免疫增强剂通过调控对虾肠道菌群结构、上调对虾肝胰腺和肠道免疫基因表达，以及增强对虾肝胰腺和肠道抗氧化酶活性，从而增强对虾免疫力。
本发明技术方案如下：
本发明的目的之一是提供一种基于CpG 寡核苷酸串联分子的对虾免疫增强剂，包括S1、S2、S3、S4、S5、S6、S7和S8 CpG ODNs 串联分子中的至少一种，其中，
S1 CpG ODNs 串联分子的核苷酸序列如SEQ ID NO.1所示；
S2 CpG ODNs 串联分子的核苷酸序列如SEQ ID NO.2所示；
S3 CpG ODNs 串联分子的核苷酸序列如SEQ ID NO.3所示；
S4 CpG ODNs 串联分子的核苷酸序列如SEQ ID NO.4所示；
S5 CpG ODNs 串联分子的核苷酸序列如SEQ ID NO.5所示；
S6 CpG ODNs 串联分子的核苷酸序列如SEQ ID NO.6所示；
S7 CpG ODNs 串联分子的核苷酸序列如SEQ ID NO.7所示；
S8 CpG ODNs 串联分子的核苷酸序列如SEQ ID NO.8所示。
本发明的目的之二是提供所述基于CpG 寡核苷酸串联分子的对虾免疫增强剂的制备方法，该方法包括以下步骤：
（1）将合成的CpG ODNs串联分子克隆到pUC57质粒上并进行扩增；
（2）用鸡蛋清将CpG ODNs串联分子包裹，添加到对虾饲料原料中充分搅拌均匀，制成对虾饲料。
优选的，所述CpG ODNs串联分子在饲料中的含量为30~60 mg/kg，所述鸡蛋清在饲料中的含量为3~6 mg/kg且被证明不会影响使用效果。
更优选的，所述CpG ODNs串联分子在饲料中的含量为50 mg/kg，所述鸡蛋清在饲料中的含量为5 mg/kg。
进一步的，本发明的目的之三是提供所述基于CpG 寡核苷酸串联分子的对虾免疫增强剂的应用，所述应用包括：在制备用于改善对虾肠道菌群结构的产品中的应用。
肠道是对虾消化吸收和营养代谢的主要器官，同时也是机体抵御病原入侵的第一道防线，肠道内栖居的菌群与肠道黏膜结合和黏附，形成具有一定规律的膜菌群，与宿主的微空间结构组成的相互依赖、相互作用的微生态系统。肠道菌群在调节肠道有益菌和有害菌的比例、调节机体免疫以及营养消化和吸收等方面发挥着重要作用。
本发明将制备的含有50 mg/kg CpG ODNs串联分子的饲料喂养对虾，饲喂过程持续14天，每天饲喂3次，根据本发明的实施例，饲喂量为对虾体重的5%-8%。收集饲喂第0、7和14天的对虾完整肠道，通过对肠道菌群进行16S rDNA测序，发现饲喂后对虾肠道菌群结构发生显著变化，物种丰富度显著增加，致病菌如弧菌相对丰度明显降低。对不同肠道菌群进行功能注释发现，与免疫相关的通路如抗原加工呈递途径得到加强。说明CpG ODNs串联分子能够通过饲喂的方式进入凡纳滨对虾体内，显著改善对虾肠道菌群结构，调节肠道有益菌和有害菌的比例，进而增强对虾免疫力，从而可进一步开发作为一种新型对虾免疫增强剂，应用于对虾养殖产业中，增强对虾抗病能力，提高对虾养殖产量。
本发明的目的之四是提供所述基于CpG 寡核苷酸串联分子在制备用于改善对虾免疫力和抗氧化活性的产品中的应用。
优选的，所述对虾为凡纳滨对虾。
与现有技术相比，本发明的有益成果在于：
1.本发明提供了一种对虾免疫增强剂，其通过CpG ODNs串联分子改善对虾肠道菌群结构，增强其免疫力。
2.本发明提供了一种将CpG ODNs串联分子添加到对虾饲料中的方法，具体为将CpG ODNs串联分子用少量鸡蛋清包裹后，添加到对虾饲料原料中混匀进行制备。
3.本发明中的对虾免疫增强剂被对虾取食后，随着饲料的被消化，CpG ODNs串联分子被肠道吸收，并随着对虾的开放式循环系统运输至其它组织器官，在改善对虾肠道菌群结构的同时，显著上调对虾肝胰腺和肠道免疫基因表达，以及增强对虾肝胰腺和肠道抗氧化酶活性，从而增强对虾免疫力。
附图说明
图1-图4：饲喂CpG ODNs串联分子后对虾肝胰腺和肠道免疫相关基因表达量变化情况。图1：第7天肝胰腺；图2：第7天肠道；图3：第14天肝胰腺；图4： 第14天肠道。
图5-图6：饲喂CpG ODNs串联分子后对虾肝胰腺和肠道抗氧化酶活性变化情况。图5：第14天肝胰腺；图6：第14天肠道。
具体实施方式
为使本领域技术人员更好的理解本发明技术内容，下面结合具体实施例和附图对本发明做进一步的说明。
实施例1对虾免疫增强剂的制备
8个由4种不同CpG ODNs单体分子组合而成的串联分子序列如表1所示，合成后将其克隆到pUC-57质粒上。将重组质粒转化到DH5α感受态细胞中，扩大培养后采用抽提法大量提取重组质粒。将重组质粒用微量鸡蛋清包裹后，添加到对虾饲料原料中充分搅拌均匀，制备成与对虾商业饲料营养成分相同的饲料，饲料原料及营养成分如表2所示，其中CpGODNs串联分子含量可以为30~60 mg/kg（本实施例中选择的用量为50 mg/kg），鸡蛋清用量可以为3~6 mg/kg（本实施例中选择的用量为5 mg/kg）。
表1本发明所用CpG ODNs串联分子序列组成及长度。
表2 本发明所制备饲料原料及营养成分 (干重， g/kg)。
实施例2 一种改善对虾肠道菌群结构的方法
将本发明实施例1制备的含CpG ODNs串联分子饲料饲喂对虾，对虾肠道菌群结构得到有效改善。
按上述实施例1的流程制备好包被有对虾免疫增强剂的对虾饲料后，按每尾虾每天5％-8%体重的饲料量饲喂对虾，以未包被对虾免疫增强剂的饲料为对照。连续饲喂14d。每组随机选择9尾对虾，分别采集饲喂第0、7、14天对虾完成肠道，提取肠道微生物总DNA，扩增其V3-V4可变区，扩增引物为：F：5'-CCTACGGGNGGCWGCAG-3'（SEQ ID NO.9），R：5'-GACTACHVGGGTATCTAATCC-3'（SEQ ID NO.10），PCR反应体系：上下游引物各2.5 μL、PhusionHot start flex 2X Master Mix 12.5 μL、模板DNA 2 μL、ddH2O 5.5 μL。反应程序如下：98℃, 30 s; 98℃, 10 s; 54℃, 30 s；72℃, 45 s; 循环30次; 72℃, 10min。PCR扩增产物通过2%琼脂糖凝胶电泳进行检测，回收采用AMPure XT beads回收试剂盒。对纯化后的PCR产物使用Agilent 2100生物分析仪和Illumina的文库定量试剂盒进行评估，合格的文库浓度应在2nM以上。将合格的各上机测序文库（Index序列不可重复）梯度稀释后，根据所需测序量按相应比例混合，并经NaOH变性为单链进行上机测序；使用NovaSeq 6000测序仪进行2×250bp的双端测序。
对测序获得的双端数据，首先需要根据barcode(条形码标签)信息对样品进行数据拆分，并去除接头和barcode序列。对数据进行拼接和过滤，进一步进行多样性分析、物种注释、差异分析后发现，对虾肠道菌群结构发生显著变化，物种丰富度显著增加，致病菌（例如弧菌）相对丰度明显降低，与免疫相关的通路（例如抗原加工呈递途径）得到加强。说明CpG ODNs串联分子能够通过饲喂的方式进入凡纳滨对虾体内，显著改善对虾肠道菌群结构，调节肠道有益菌和有害菌的比例，进而增强对虾免疫力。其中CpG ODNs串联分子S1效果最佳。
实施例3 一种上调对虾免疫相关基因以及提高抗氧化酶活性的方法
饲喂CpG ODNs串联分子后，评估对虾非特异性免疫相关指标。
将上述实施例1制备的含CpG ODNs串联分子饲料，按每尾虾每天5%-8%体重的饲料量饲喂对虾14天，以未包被对虾免疫增强剂的饲料为对照。每组随机选取9尾虾，收集饲喂第0、7、14天对虾肝胰腺与肠道，分别用于提取总RNA测定免疫相关基因表达量和抗氧化酶活性。测定的免疫相关基因分别是TNF-α（登录号：HQ696609）、IL-8（登录号：KY218792.1）、IL-10（登录号：HQ388294）、IL-1β（登录号：JQ692172）、TOR（登录号：XM_027365549.1）、ALF（登录号：XM_027372864.1）、Toll（登录号：DQ923424）、proPO（登录号：EF115296.1）。抗氧化酶活性测定指标分别是MDA（丙二醛）、AKP（碱性磷酸酯酶）、GST（谷胱甘肽S-转移酶）、LZM（溶菌酶）、CAT（过氧化氢酶）、T-AOC（总抗氧化能力）、GPX（谷胱甘肽过氧化物酶）、ACP（酸性磷酸酶）、LPS（脂肪酶）、SOD（超氧化物歧化酶），所用试剂盒均为南京建成生物科技有限公司生产，具体步骤均按试剂盒操作说明书进行。
评估结果发现，经过含CpG ODNs串联分子饲料持续饲喂后，相较于对照组，对虾肠道和肝胰腺免疫相关基因出现不同程度上调，其中肠道TNF-α表达量上调15.73倍，ALF上调达18.42倍，肝胰腺TOR、IL-8、IL-8、Toll表达量上调6.16-14.75倍。肠道和肝胰腺抗氧化酶活性也出现较大程度增强，相较于对照组增强3-6倍。以上结果说明对虾先天免疫力得到加强。其中CpG ODNs串联分子S1效果最佳。
以上所述仅为本发明的部分实施例而已，并不用限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (7)
1.一种基于CpG 寡核苷酸串联分子的对虾免疫增强剂，包括S1 CpG ODNs 串联分子，其特征在于，
S1 CpG ODNs 串联分子的核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述基于CpG 寡核苷酸串联分子的对虾免疫增强剂的制备方法，其特征在于，包括以下步骤：
（1）将合成的CpG ODNs串联分子克隆到pUC57质粒上并进行扩增；
（2）用鸡蛋清将CpG ODNs串联分子包裹，添加到对虾饲料原料中充分搅拌均匀，制成对虾饲料。
3.根据权利要求2所述的制备方法，其特征在于，所述CpG ODNs串联分子在饲料中的含量为30~60 mg/kg，所述鸡蛋清在饲料中的含量为3~6 mg/kg。
4.根据权利要求2所述的制备方法，其特征在于，所述CpG ODNs串联分子在饲料中的含量为50 mg/kg，所述鸡蛋清在饲料中的含量为5 mg/kg。
5.权利要求1所述基于CpG 寡核苷酸串联分子的对虾免疫增强剂的应用，其特征在于，所述应用为：在制备用于改善对虾肠道菌群结构的产品中的应用。
6.权利要求1所述基于CpG 寡核苷酸串联分子的对虾免疫增强剂的应用，其特征在于，所述应用为：在制备用于改善对虾免疫力和抗氧化活性的产品中的应用。
7.根据权利要求5或权利要求6所述的应用，其特征在于，所述对虾为凡纳滨对虾。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970772A (zh) * | 2006-12-01 | 2007-05-30 | 浙江大学 | 用于水产动物病害防治的序列、质粒、工程菌及其应用 |
| CN108949802A (zh) * | 2018-06-25 | 2018-12-07 | 浙江皇冠科技有限公司 | 一种高产CpG ISS工程菌构建方法与用途 |
| CN113209126A (zh) * | 2021-05-31 | 2021-08-06 | 中山大学 | 一种基于rna干扰技术的对虾免疫增强剂及其制备方法与应用 |
| CN113841813A (zh) * | 2021-08-19 | 2021-12-28 | 广东海洋大学 | CpG ODN在促进凡纳滨对虾对虾生长和改善肠道菌群中的应用 |
-
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- 2023-08-16 CN CN202311027946.2A patent/CN116762898B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1970772A (zh) * | 2006-12-01 | 2007-05-30 | 浙江大学 | 用于水产动物病害防治的序列、质粒、工程菌及其应用 |
| CN108949802A (zh) * | 2018-06-25 | 2018-12-07 | 浙江皇冠科技有限公司 | 一种高产CpG ISS工程菌构建方法与用途 |
| CN113209126A (zh) * | 2021-05-31 | 2021-08-06 | 中山大学 | 一种基于rna干扰技术的对虾免疫增强剂及其制备方法与应用 |
| CN113841813A (zh) * | 2021-08-19 | 2021-12-28 | 广东海洋大学 | CpG ODN在促进凡纳滨对虾对虾生长和改善肠道菌群中的应用 |
Non-Patent Citations (1)
| Title |
|---|
| Comparative study of the impact of dietary supplementation with different types of CpG oligodeoxynucleotides (CpG ODNs) on enhancing intestinal microbiota diversity, antioxidant capacity, and immune-related gene expression profiles in Pacific white shrimp ( Litopenaeus vannamei);Feng Hu等;《Front Immunol》;第1页摘要、第12页左栏第2段、表S1-S2 * |
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