CN118141901A - 草鱼sPLA2蛋白在制备用于增强鱼类抗病力的产品中的应用 - Google Patents
草鱼sPLA2蛋白在制备用于增强鱼类抗病力的产品中的应用 Download PDFInfo
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Abstract
本发明提供了一种草鱼sPLA2蛋白在制备用于增强鱼类抗病力的产品中的应用。本发明通过研究发现，草鱼sPLA2蛋白能够显著提高鱼类肠道紧密连接分子ZO‑1和Occludin的表达，并降低血浆LPS和D‑乳酸的含量；显著提高鱼类肠道抗菌肽IL‑22、Hepcidin‑1、LEAP‑2和Lyzl的表达；显著提高鱼类血清中SOD、CAT、ACP和AKP的表达。因此，草鱼sPLA2蛋白通过强化鱼类肠黏膜屏障、提高鱼类抗菌能力和提高鱼类抗氧化能力，综合增强了鱼类抗病力，能够更好地抵抗细菌侵染，提高鱼类存活率，可作为新型的饲料添加剂或治疗剂用于制备鱼类饲料或药物。
Description
技术领域
本发明涉及分子生物技术领域，特别涉及一种草鱼sPLA2蛋白在制备用于增强鱼类抗病力的产品中的应用。
背景技术
草鱼是我国淡水鱼类养殖产量最高的一种经济鱼类。草鱼性温、味甘；具有平肝、祛风、活痹、截疟、暖胃的作用；还含有丰富的不饱和脂肪酸，对血液循环和心血管患者很有益处，是温中补虚的养生品。草鱼营养成分较丰富，使其具有较高的食用价值。但是近几年来，草鱼的细菌病害问题日趋严重，严重阻碍了水产养殖业的绿色、健康和可持续发展。目前，针对草鱼细菌疾病的预防和治疗，主要依靠抗生素等化学药物。然而抗生素的长期使用，不仅扰乱了水体的生态系统，而且可能导致出现抗生素耐药、药物残留和食品安全等严重问题。
因此，研制具有广谱、高效和安全的生物杀菌活性制剂，则显得尤为重要。利用分子生物学方法研制草鱼抗菌活性蛋白，可以避免抗生素带来的种种弊端。但是，目前可应用于鱼类增强抗病力的活性蛋白仍鲜有报道。
发明内容
基于此，有必要提供一种能够增强鱼类抗病力的活性蛋白的应用。
本发明的一个方面，提供了一种草鱼sPLA2蛋白在制备用于增强鱼类抗病力的产品中的应用。
在其中一个实施例中，上述增强鱼类抗病力包括强化鱼类肠黏膜屏障、提高鱼类抗菌能力和提高鱼类抗氧化能力。
在其中一个实施例中，上述产品为药物或饲料。
在其中一个实施例中，上述药物的剂型为片剂、胶囊剂、颗粒剂或注射剂，上述饲料为植物性饲料或动物性饲料。
在其中一个实施例中，上述鱼类为草鱼。
在其中一个实施例中，上述草鱼sPLA2蛋白的制备方法包括以下步骤：以草鱼肝脏cDNA为模板，使用引物进行PCR扩增，获得草鱼sPLA2核酸片段；将上述草鱼sPLA2核酸片段进行双酶切，并与经过相同酶切的质粒连接，获得重组质粒；将上述重组质粒转化到大肠杆菌中，加入诱导剂诱导表达，经蛋白纯化即获得草鱼sPLA2蛋白。
在其中一个实施例中，上述引物的核苷酸序列如SEQ ID NO：2和SEQ ID NO：3所示，上述质粒为pET28a，上述诱导剂为IPTG。
本发明的另一方面还提供了一种用于增强鱼类抗病力的药物，包括草鱼sPLA2蛋白以及药用辅料。
本发明的另一方面还提供了一种用于增强鱼类抗病力的饲料，其特征在于，包括草鱼sPLA2蛋白以及标准鱼饲料。
本发明的另一方面还提供了一种草鱼sPLA2蛋白作为鱼类肠道紧密连接分子、肠道抗菌肽或抗氧化酶的表达促进剂的应用，上述肠道紧密连接分子选自ZO-1和Occludin中的一种或多种，上述肠道抗菌肽选自IL-22、Hepcidin-1、LEAP-2和Lyzl中的一种或多种，上述抗氧化酶选自超氧化物歧化酶、过氧化氢酶、酸性磷酸酶和碱性磷酸酶中的一种或多种。
本发明的上述方案具有如下的有益效果：
本发明通过研究发现，草鱼sPLA2蛋白能够显著提高鱼类肠道紧密连接分子ZO-1和Occludin的表达，并降低血浆LPS和D-乳酸的含量，从而强化肠道黏膜屏障，减少肠道黏膜的通透性；草鱼sPLA2蛋白能够显著提高鱼类肠道抗菌肽IL-22、Hepcidin-1、LEAP-2和Lyzl的表达，从而提高鱼类抗菌能力；同时，草鱼sPLA2蛋白还能够显著提高鱼类血清中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、酸性磷酸酶(ACP)和碱性磷酸酶(AKP)的表达，从而提高鱼类抗氧化能力。因此，草鱼sPLA2蛋白通过强化鱼类肠黏膜屏障、提高鱼类抗菌能力和提高鱼类抗氧化能力，综合增强了鱼类抗病力、抗感染能力，能够更好地抵抗细菌侵染，提高鱼类存活率，可作为新型的饲料添加剂或治疗剂用于制备鱼类药物或饲料。
附图说明
图1为本发明实施例1制备的草鱼sPLA2蛋白的SDS-PAGE电泳图；
图2为本发明实施例2中草鱼sPLA2蛋白对鱼类肠道紧密连接分子表达影响的测试结果；其中，A为ZO-1基因，B为Occludin基因；
图3为本发明实施例2中草鱼sPLA2蛋白对鱼类肠道抗菌肽表达影响的测试结果；其中，A为IL-22基因，B为Hepcidin-1基因，C为LEAP-2基因，D为Lyzl基因；
图4为本发明实施例2中草鱼sPLA2蛋白对鱼类血浆LPS和D-乳酸含量影响的测试结果；其中，A为血浆LPS，B为血浆D-乳酸；
图5为本发明实施例2中草鱼sPLA2蛋白对鱼类血清中抗氧化酶表达影响的测试结果；其中，A为超氧化物歧化酶，B为过氧化氢酶，C为酸性磷酸酶，D为碱性磷酸酶；
图6为本发明实施例3中草鱼sPLA2蛋白对鱼类抵抗细菌侵染能力影响的测试结果；
图7为本发明实施例4中草鱼sPLA2蛋白对鱼类存活率影响的测试结果。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
此外，下面所描述的本发明不同实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互结合。
本发明的一个方面，提供了一种草鱼sPLA2蛋白在制备用于增强鱼类抗病力的产品中的应用。
分泌型磷脂酶A2(sPLA2)是一种由宿主组织和细胞产生的Ca2+依赖型的内源性蛋白，分子量约为15kDa。有研究显示，人sPLA2可通过引起肿瘤坏死因子α生成，释放细胞色素c而发挥其凋亡活性；眼镜蛇毒sPLA2则具有影响血小板聚集、溶血活性、突触前神经毒性、肌毒性、心脏毒性、水肿和惊厥等多种活性。因此，不同物种中sPLA2蛋白的活性各不相同，功能上存在差异，而草鱼sPLA2蛋白的功能活性目前尚未有报道。
本发明通过研究发现，草鱼sPLA2蛋白能够显著提高鱼类肠道紧密连接分子ZO-1和Occludin的表达，并降低血浆LPS和D-乳酸的含量，从而强化肠道黏膜屏障，减少肠道黏膜的通透性；草鱼sPLA2蛋白能够显著提高鱼类肠道抗菌肽IL-22、Hepcidin-1、LEAP-2和Lyzl的表达，从而提高鱼类抗菌能力；同时，草鱼sPLA2蛋白还能够显著提高鱼类血清中超氧化物歧化酶、过氧化氢酶、酸性磷酸酶和碱性磷酸酶的表达，从而提高鱼类抗氧化能力。因此，草鱼sPLA2蛋白通过强化鱼类肠黏膜屏障、提高鱼类抗菌能力和提高鱼类抗氧化能力，综合增强了鱼类抗病力、抗感染能力，能够更好地抵抗细菌侵染，提高鱼类存活率，可作为新型的饲料添加剂或治疗剂用于制备鱼类药物或饲料。
在一个具体示例中，上述用于增强鱼类抗病力的产品为药物或饲料。可选的，药物的剂型为片剂、胶囊剂、颗粒剂或注射剂等，饲料为植物性饲料、动物性饲料或合成饲料等。
在一个具体示例中，上述鱼类为草鱼。
在一个具体示例中，草鱼sPLA2蛋白的制备方法包括以下步骤：以草鱼肝脏cDNA为模板，使用引物进行PCR扩增，获得草鱼sPLA2核酸片段；将草鱼sPLA2核酸片段进行双酶切，并与经过相同酶切的质粒连接，获得重组质粒；将重组质粒转化到大肠杆菌中，加入诱导剂诱导表达，经蛋白纯化即获得草鱼sPLA2蛋白。可以理解，制备方法不限于此，也可以采用本领域常用的其他蛋白表达技术制备该蛋白。
在一个具体示例中，上述引物的核苷酸序列如SEQ ID NO：2和SEQ ID NO：3所示，该引物具有优异的扩增效果，特异性和扩增效率高。可选的，质粒为pET28a但不限于此，诱导剂为IPTG。
本发明另一方面提供了一种用于增强鱼类抗病力的药物，其包括草鱼sPLA2蛋白以及药用辅料。
可选的，药物的剂型为片剂、胶囊剂、颗粒剂或注射剂。在一个具体示例中，药物的剂型为注射剂，其使用时草鱼sPLA2蛋白的浓度为15～25ug/mL。
可选的，药用辅料包括稀释剂、防腐剂、缓冲剂、崩解剂、抗氧剂、助悬剂、着色剂和赋形剂中的一种或多种。
可选的，稀释剂选自聚乙二醇、丙二醇、植物油和矿物油中的一种或多种。在一个具体示例中，防腐剂选自山梨酸、山梨酸甲酯、对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯、对羟基苯甲酸丁酯、对羟基苯甲酸苄酯、对羟基苯甲酸甲酯钠、苯甲酸和苯甲醇中的一种或多种。在一个具体示例中，缓冲剂选自磷酸氢钠、磷酸二氢钠、枸橼酸钠、酒石酸钠和醋酸钠中的一种或多种。
可选的，崩解剂选自交联羧甲基纤维素钠、羧甲基淀粉钠、交联聚乙烯吡咯烷酮或低取代羟丙基纤维素中的一种或多种。在一个具体示例中，抗氧剂选自乙二胺四乙酸、乙二胺四乙酸二钠盐、二丁基羟基甲苯、甘氨酸、肌醇、抗坏血酸、抗坏血酸钠、卵磷脂、苹果酸、氢醌、枸橼酸、琥珀酸和焦亚硫酸钠中的一种或多种。在一个具体示例中，助悬剂选自蜂蜡、乙基羟乙基纤维素、甲壳素、甲壳糖、甲基纤维素、羧甲基纤维素、琼脂、羟丙基甲基纤维素和黄原胶中的一种或多种。在一个具体示例中，着色剂选自炭黑、铁黑、铁棕、铁红和二氧化钛中的一种或多种。在一个具体示例中，赋形剂选自甘露醇、葡萄糖、乳糖、葡聚糖、右旋糖苷和氯化钠中的一种或多种。
本发明另一方面提供了一种用于增强鱼类抗病力的饲料，包括草鱼sPLA2蛋白以及标准鱼饲料。
可选的，上述标准鱼饲料为动物性饲料或植物性饲料。在一个具体示例中，动物性饲料包含鱼粉、虾粉、贝壳粉、鸡肠粉、蚯蚓粉和复合维生素等，植物性饲料包含豆粕、菜粕、棉粕、花生粕、淀粉和复合维生素等。可选的，复合维生素包括维生素A、维生素D3、维生素E、维生素K3、维生素B1、维生素B2、烟酸、泛酸钙、维生素B6、维生素B12、叶酸、生物素和维生素C中的一种或多种。
本发明另一方面提供了一种草鱼sPLA2蛋白作为鱼类肠道紧密连接分子、肠道抗菌肽或抗氧化酶的表达促进剂的应用，其中肠道紧密连接分子选自ZO-1和Occludin中的一种或多种，肠道抗菌肽选自IL-22、Hepcidin-1、LEAP-2和Lyzl中的一种或多种，抗氧化酶选自超氧化物歧化酶、过氧化氢酶、酸性磷酸酶和碱性磷酸酶中的一种或多种。
可以理解，草鱼sPLA2蛋白能够用作促进鱼类肠道紧密连接分子、肠道抗菌肽或抗氧化酶表达的工具试剂，从而调节鱼类生理生化状态，以便鱼类相关研究工作的进行。
下面主要结合具体实施方式和附图对本发明作进一步详细的说明。
以下实施例中所涉及到的常规实验试剂购于如下公司：
(1)质粒提取试剂盒、DNA胶回收试剂盒、总RNA提取试剂盒、卡那霉素、LB培养基、蛋白裂解液、大肠杆菌BL21(DE3)、IPTG、His Trap HP Columns Kit和PBS购于“生工生物工程(上海)股份有限公司”。
(2)PCR mix、反转录试剂盒、T4连接酶、BamH和XhoI限制性内切酶、pMD18-T载体、qRT-PCR SYBR Mix购于“宝日医生物技术(北京)有限公司”。
(3)引物合成由“北京擎科生物科技股份有限公司”完成。
(4)脂多糖(LPS)、D-乳酸(D-lactate)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、酸性磷酸酶(ACP)及碱性磷酸酶(AKP)试剂盒购于“南京建成生物工程研究所有限公司”。
实施例1制备草鱼sPLA2蛋白
草鱼sPLA2重组蛋白的制备具体包括如下步骤：
步骤(1)：克隆草鱼sPLA2的核苷酸序列
利用总RNA提取试剂盒提取健康草鱼(重约50g)的肝脏总RNA，利用反转录试剂盒将总RNA反转为cDNA。以合成的cDNA为模板，采用F1引物(5’端带BamH酶切位点)和R1引物(5’端带XhoI酶切位点)，PCR法扩增得到带有酶切位点的草鱼sPLA2的核苷酸序列(如SEQID NO：1所示)。PCR法扩增的反应条件为：94℃预变性2分钟，1个循环；94℃变性30秒，60℃退火30秒，72℃延伸30秒，共35个循环；72℃延伸8分钟，1个循环。通过胶回收试剂盒回收目的片段，经DNA测序证实该片段为草鱼sPLA2的核苷酸序列。
草鱼sPLA2的核苷酸序列(SEQ ID NO：1)：CTAGACTACCGTGCGGTGTGGCAGTTCAGGTCCATGATCATCTGTACCGTCCCTGGTAGCTGGCCTGCACTGGATTACGCAGACTACGGATGCTACTGCGGCCTAGGAGGCTCAGGCACCCCGGTGGATGCACTGGACAGGTGCTGTCAGGTGCATGATCAGTGTTATTCAGACTCACGGCAGCATGATGACTGCCGGGGTATCTTTGACAACCCCTACACTGAAATCTATTCATTCTCATGTGACAAACCGGCAAAGAAAGTCACCTGCAATGCTGACAAGAATCGCCCCTGTGAGATGTTCATCTGTGAATGTGACAGAAAAGCAGCTGAATGCTTTGCAAAAGCGGGTTACAACCCAGAGTACAAGTACTATCCTAGTCAAAACTGCAAA
上述引物F1为：GGATCCCTAGACTACCGTGCGGTGTG(SEQ ID NO：2)，R1为CTCGAGTTTGCAGTTTTGACTAGGAT(SEQ ID NO：3)，其中下划线为酶切位点。
步骤(2)：制备草鱼sPLA2重组蛋白
使用BamH和XhoI限制性内切酶分别对上述草鱼sPLA2的核苷酸序列和pET28a载体(购于天根生化科技(北京)有限公司)进行双酶切，通过胶回收试剂盒回收草鱼sPLA2的核苷酸序列和pET28a载体，利用T4连接酶进行连接反应。经过转化、菌落筛选，得到重组质粒pET-GCsPLA2。将pET-GCsPLA2转化入大肠杆菌BL21(DE3)，活化菌株在含有卡那霉素(50ug/ml)和0.4mM的IPTG的LB液体培养基中16℃转速120rpm震荡培养6小时。之后，低温离心收集菌体，加入3ml蛋白裂解液。2小时后低温离心收集上清；将上清加入到His Trap HPColumns中，纯化上清中的蛋白，即为制备成功的草鱼sPLA2重组蛋白。经质谱分析证实纯化的蛋白具有草鱼sPLA2的氨基酸序列(如SEQ ID NO：4所示)。将纯化的蛋白经SDS-PAGE电泳(80V电压下电泳1.5小时)测定其分子量大小，如图1所示。
草鱼sPLA2的氨基酸序列(SEQ ID NO：4)：LDYRAVWQFRSMIICTVPGSWPALDYADYGCYCGLGGSGTPVDALDRCCQVHDQCYSDSRQHDDCRGIFDNPYTEIYSFSCDKPAKKVTCNADKNRPCEMFICECDRKAAECFAKAGYNPEYKYYPSQNCK
实施例2草鱼sPLA2蛋白保护肠黏膜屏障的应用
草鱼sPLA2重组蛋白保护肠黏膜屏障的应用测试，具体包括以下步骤：
步骤(1)：草鱼sPLA2重组蛋白的注射
使用PBS将实施例1中制备的草鱼sPLA2重组蛋白稀释至20ug/ml，即为制备成功的草鱼sPLA2重组蛋白稀释液。将30条草鱼(重约15g)随机分为2组，每组15条，并分别命名为A组和B组。将A组的每条鱼分别腹腔注射50ul的草鱼sPLA2重组蛋白稀释液，将B组的每条鱼分别腹腔注射50ul的PBS。
步骤(2)：攻毒感染
利用LB液体培养基培养嗜水气单胞菌培养至OD600为0.5，离心收集菌体，用PBS悬浮至终浓度为1×106cfu/ml，即为制备成功的嗜水气单胞菌悬液。在上述步骤(1)注射2小时后，将A组和B组的每条鱼分别腹腔注射嗜水气单胞菌悬液100ul。
所述嗜水气单胞菌来源已被文献报道(Zeng Q,Qiang J,Yang Y,Li Z,Li P,HuN,Zhou Z.WbuB,a glycosyltransferase family 4protein,regulates the activationof NLRP3 inflammasome and contributes to the virulence of Aeromonashydrophila CCL1,Reproduction and Breeding,2024,4(1):38-45,DOI:10.1016/j.repbre.2023.12.006)。
步骤(3)：分析草鱼肠黏膜的通透性、抗菌肽和抗氧化能力变化
在上述步骤(2)24小时后，采集草鱼的肠道组织。利用总RNA提取试剂盒提取肠道总RNA，利用反转录试剂盒将总RNA反转为cDNA。以合成的cDNA为模板，采用表1中引物，利用qRT-PCR法在QuantStudio 5实时荧光定量PCR系统(购于美国ABI公司)检测肠道紧密连接分子(ZO-1和Occludin)和肠道抗菌肽(IL-22、Hepcidin-1、LEAP-2和Lyzl)的表达，利用2-△△t法分析数据。qRT-PCR程序为：95℃，预变性30秒；95℃变性10秒，60℃反应30秒，共40个循环。实验结果表明，A组鱼的紧密连接(ZO-1和Occludin)和肠道抗菌肽(IL-22、Hepcidin-1、LEAP-2和Lyzl)的表达(分别为1.8、2.6、2.4、1.8、3.0和2.6)显著(Mann-Whitney test，P﹤0.01)高于B组鱼的紧密连接(ZO-1和Occludin)和肠道抗菌肽(IL-22、Hepcidin-1、LEAP-2和Lyzl)的表达(分别为1.0、1.0、1.0、1.0、1.0和1.0)，如图2和图3所示。
在上述步骤(2)24小时后，采集草鱼的血液，分离获得血浆和血清。利用LPS、D-乳酸、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、酸性磷酸酶(ACP)及碱性磷酸酶(AKP)试剂盒分析血浆LPS和D-乳酸的含量变化，以及血清中SOD、CAT、ACP和AKP的含量变化。实验结果表明，A组鱼的血浆LPS和D-乳酸的含量(分别为17.4pg/ml和142.3nmol/ml)显著(Mann-Whitney test，P﹤0.01)低于B组鱼的血浆LPS和D-乳酸的含量(分别为30.4pg/ml和259.7nmol/ml)，如图4所示。A组鱼的血清SOD、CAT、ACP和AKP的含量(分别为157.5U/ml、1.8U/ml、31.0金氏单位/100ml和9.5金氏单位/100ml)显著(Mann-Whitney test，P﹤0.01)高于B组鱼的血清中SOD、CAT、ACP和AKP的含量(分别为103.1U/ml、1.3U/ml、18.9金氏单位/100ml和6.4金氏单位/100ml)，如图5所示。
上述结果表明，草鱼sPLA2蛋白能够显著提高鱼类肠道紧密连接分子表达，减少血浆LPS含量和D-乳酸含量，从而强化肠道黏膜屏障，减少肠道黏膜的通透性，并且也能够显著提高鱼类肠道抗菌肽和抗氧化酶的表达，从而提高鱼类抗菌能力和抗氧化能力。
表1本实施例2使用到的引物
实施例3草鱼sPLA2蛋白抗菌功能的应用
草鱼sPLA2重组蛋白抗菌功能的应用测试，具体包括以下步骤：
步骤(1)：草鱼sPLA2重组蛋白的注射和细菌感染
将20条草鱼(重约15g)随机分为2组，每组10条，分别命名为C组和D组。将C组的每条鱼分别腹腔注射50ul的实施例1中制备的草鱼sPLA2重组蛋白稀释液(20ug/ml)，将D组的每条鱼分别腹腔注射50ul的PBS。注射2小时后，将C组和D组的每条鱼分别腹腔注射实施例2中制备的嗜水气单胞菌悬液100ul。
步骤(2)：抵抗细菌侵染能力分析
在上述步骤(1)24小时后，取C组和D组鱼的肾脏和脾脏组织，经称重、组织研磨，将组织匀浆液用PBS稀释100倍后，涂布于LB固定培养基平板。将LB固定培养基平板放置在28℃培养箱中倒置培养24小时，统计每个平板出现的菌落数。实验结果表明，C组鱼的肾脏和脾脏组织的细菌数(分别为145和175CFU/mg)显著(Mann-Whitney test，P﹤0.01)低于D组鱼的肾脏和脾脏组织的细菌数(分别为357和418CFU/mg)，如图6所示。可见，草鱼sPLA2蛋白能够显著提高鱼类抵抗细菌侵染的能力。
实施例4草鱼sPLA2蛋白增强存活率的应用
草鱼sPLA2重组蛋白增强存活率的应用测试，具体包括以下步骤：
步骤(1)：草鱼sPLA2重组蛋白的注射和细菌感染
将40条草鱼(重约15g)随机分为2组，每组20条，分别命名为E组和F组。将E组的每条鱼分别腹腔注射50ul的实施例1中制备的草鱼sPLA2重组蛋白稀释液(20ug/ml)，将F组的每条鱼分别腹腔注射50ul的PBS。注射2小时后，将E组和F组的每条鱼分别腹腔注射实施例2中制备的嗜水气单胞菌悬液100ul。
步骤(2)：分析存活率
在上述步骤(1)后，持续观察15天，统计每天的草鱼死亡数量，分析草鱼感染嗜水气单胞菌后的存活率。实验结果表明，15天后E组鱼的生存率(35％)显著(Log-rank test，P﹤0.01)高于F组鱼的生存率(0％)，如图7所示。可见，草鱼sPLA2蛋白能够显著提高草鱼感染嗜水气单胞菌后的存活率。
以上所述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，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.草鱼sPLA2蛋白在制备用于增强鱼类抗病力的产品中的应用。
2.根据权利要求1所述的应用，其特征在于，所述增强鱼类抗病力包括强化鱼类肠黏膜屏障、提高鱼类抗菌能力和提高鱼类抗氧化能力。
3.根据权利要求1所述的应用，其特征在于，所述产品为药物或饲料。
4.根据权利要求3所述的应用，其特征在于，所述药物的剂型为片剂、胶囊剂、颗粒剂或注射剂，所述饲料为植物性饲料或动物性饲料。
5.根据权利要求1所述的应用，其特征在于，所述鱼类为草鱼。
6.根据权利要求1所述的应用，其特征在于，所述草鱼sPLA2蛋白的制备方法包括以下步骤：以草鱼肝脏cDNA为模板，使用引物进行PCR扩增，获得草鱼sPLA2核酸片段；将所述草鱼sPLA2核酸片段进行双酶切，并与经过相同酶切的质粒连接，获得重组质粒；将所述重组质粒转化到大肠杆菌中，加入诱导剂诱导表达，经蛋白纯化即获得所述草鱼sPLA2蛋白。
7.根据权利要求6所述的应用，其特征在于，所述引物的核苷酸序列如SEQ ID NO：2和SEQ ID NO：3所示，所述质粒为pET28a，所述诱导剂为IPTG。
8.一种用于增强鱼类抗病力的药物，其特征在于，包括草鱼sPLA2蛋白以及药用辅料。
9.一种用于增强鱼类抗病力的饲料，其特征在于，包括草鱼sPLA2蛋白以及标准鱼饲料。
10.草鱼sPLA2蛋白作为鱼类肠道紧密连接分子、肠道抗菌肽或抗氧化酶的表达促进剂的应用，其特征在于，所述肠道紧密连接分子选自ZO-1和Occludin中的一种或多种，所述肠道抗菌肽选自IL-22、Hepcidin-1、LEAP-2和Lyzl中的一种或多种，所述抗氧化酶选自超氧化物歧化酶、过氧化氢酶、酸性磷酸酶和碱性磷酸酶中的一种或多种。
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