CN114344453A - 一种罗非鱼cl-k1在制备预防和/或治疗罗非鱼病害的药物中的应用 - Google Patents
一种罗非鱼cl-k1在制备预防和/或治疗罗非鱼病害的药物中的应用 Download PDFInfo
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- CN114344453A CN114344453A CN202210052918.5A CN202210052918A CN114344453A CN 114344453 A CN114344453 A CN 114344453A CN 202210052918 A CN202210052918 A CN 202210052918A CN 114344453 A CN114344453 A CN 114344453A
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Abstract
本发明提供了罗非鱼CL‑K1或编码所述罗非鱼CL‑K1的基因在制备预防和/或治疗水产病害的药物中的应用，属于水产养殖技术领域。本发明利用真核表达系统构建、表达、纯化得到具有良好生物活性的真核蛋白罗非鱼CL‑K1，在体外采用牛津杯法测定罗非鱼CL‑K1具有抑制无乳链球菌增殖的能力，在体内试验证明敲降CL‑K1基因具有促进无乳链球菌感染引起的炎症反应，回补表达CL‑K1蛋白后可提高罗非鱼抗无乳链球菌的免疫保护，CL‑K1真核蛋白可以显著降低罗非鱼由于无乳链球菌感染而造成的死亡。罗非鱼CL‑K1作为水产动物先天免疫系统的重要防御机制从根本上防治鱼病，为实践中减少鱼病的爆发提供具有重要的指导意义。
Description
技术领域
本发明属于水产养殖技术领域，具体涉及一种罗非鱼CL-K1在制备预防和/或治疗罗非鱼病害的药物中的应用。
背景技术
罗非鱼，俗称：非洲鲫鱼，非鲫、越南鱼、南洋鲫等。它是世界水产业的重点科研培养的淡水养殖鱼类，且被誉为未来动物性蛋白质的主要来源之一。通常生活于淡水中，也能生活于不同盐度的咸水中，也可以存活于湖，河，池塘的浅水中。它有很强的适应能力，在面积狭小之水域中亦能繁殖，甚至在水稻田里能够生长，且对溶氧较少之水有极强之适应性。绝大部分罗非鱼是杂食性，常吃水中植物和碎物。
罗非鱼抗病力强，但在越冬期间，由于水温、水质等各种因素，加之密集饲养，鱼体体质差，病害较多。可在短期内引起大量死亡，要密切注意防治。无乳链球菌作为罗非鱼链球菌病的主要致病菌，造成了巨大的经济损失，已经对水产养殖行业形成巨大冲击。目前缺乏安全、高效的鱼病防治措施。虽然抗生素的使用能够一定程度上遏制病害的发生和发展，但抗生素滥用给水产品的质量和安全性受到极大威胁和质疑。因此，探究利用鱼类免疫系统的防御机制，挖掘先天免疫系统中重要分子潜能，从根本上防治鱼病，以适应长效、安全的可持续发展，是水产绿色养殖产业的迫切需求。
胶原凝集素(CL-K1)是一种功能多效且保守的分泌型蛋白质，作为先天免疫系统中重要的模式识别分子。现有技术报道，胶原凝集素家族成员在尿路感染、剂型肾损伤中发挥重要作用，然而这些是人来源的CL-K1的作用。关于硬骨鱼来源的CL-K1的报道较少，更未检索到关于罗非鱼来源的 CL-K1及其生物学功能的信息。
发明内容
有鉴于此，本发明的目的在于提供一种罗非鱼CL-K1制备预防和/或治疗罗非鱼病害的药物中的应用。
本发明提供了罗非鱼CL-K1或编码所述罗非鱼CL-K1的基因在制备预防和/或治疗水产病害的药物中的应用。
优选的，所述罗非鱼CL-K1的氨基酸序列如SEQ ID NO:1所示。
优选的，所述罗非鱼CL-K1是利用真核表达系统构建、表达、纯化的罗非鱼CL-K1。
优选的，所述编码所述罗非鱼CL-K1的基因的核苷酸序列如SEQ ID NO:2所示。
优选的，所述水产病害包括以下一种或几种：无乳链球菌感染导致的病害、败血症、脑膜炎、腹水症、嗜水气单胞菌病、烂尾病和赤皮病。
优选的，所述水产包括以下一种或几种：罗非鱼、大菱鲆、半滑舌鳎、金鲳、斑点叉尾鮰、草鱼和石斑鱼。
优选的，所述药物具有抑制无乳链球菌增殖、抑制病害细菌感染导致的炎症反应的作用。
本发明提供了一种罗非鱼CL-K1或编码罗非鱼CL-K1的基因在制备罗非鱼致病菌抑制剂中的应用。
本发明提供了一种罗非鱼CL-K1或编码罗非鱼CL-K1的基因在制备罗非鱼致病菌感染引起的炎症反应的抑制剂中的应用
优选的，所述罗非鱼致病菌包括以下一种或几种：无乳链球菌、嗜水气单胞菌、迟缓爱德华氏菌和荧光假单胞菌。
本发明提供了罗非鱼CL-K1或编码所述罗非鱼CL-K1的基因在制备预防和/或治疗水产病害的药物中的应用。本发明利用真核表达系统构建、表达、纯化得到具有良好生物活性的真核蛋白罗非鱼CL-K1，在体外试验中，采用牛津杯法测定罗非鱼CL-K1具有抑制无乳链球菌增殖的能力，在体内试验中，证明敲降CL-K1基因具有促进无乳链球菌感染引起的炎症反应，回补表达CL-K1蛋白后可提高罗非鱼抗无乳链球菌的免疫保护，CL-K1真核蛋白可以显著降低罗非鱼由于无乳链球菌感染而造成的死亡。因此，罗非鱼 CL-K1作为水产动物先天免疫系统的重要防御机制。可通过发挥罗非鱼 CL-K1的免疫防御作用从根本上防治鱼病，为实践中减少鱼病的爆发提供具有重要的指导意义。
附图说明
图1为本发明技术方案的技术路线图；
图2为尼罗罗非鱼CL-K1真核蛋白检测结果，其中图2A：SDS-PAGE 检测OnCL-K1真核蛋白的结果；图2B：OnCL-K1真核蛋白的质谱检测结果，氨基酸序列覆盖度(灰色部分)为95％，该蛋白全肽段质谱检测是样品送到国家蛋白质科学中心(北京)进行的检测)；
图3为尼罗罗非鱼CL-K1真核蛋白抑制无乳链球菌增殖能力的检测结果，图3A：牛津杯法检测OnCL-K1抑菌能力的结果(1.OnCL-K1；2.BSA； 3.Ampicillin)；图3B：各组抑菌圈面积的统计结果，n＝3。
图4为CL-K1基因的敲降促进无乳链球菌对尼罗罗非鱼的感染结果图，图4A：涂板检测OnCL-K1 RNA干扰对体内病原菌清除的结果；图4B：病理组织切片检测结果；图4C：荧光定量PCR检测炎症因子表达量动态变化；图4D：罗非鱼存活率的统计；
图5为CL-K1蛋白提高罗非鱼抗无乳链球菌的免疫保护结果图，图5A：重新注入OnCL-K1蛋白能够恢复对体内病原菌清除的涂板检测结果；图5B：病理组织切片检测结果；图5C：荧光定量PCR检测炎症因子表达量动态变化；图5D：罗非鱼存活率的统计结果。
具体实施方式
本发明提供了罗非鱼CL-K1或编码所述罗非鱼CL-K1的基因在制备预防和/或治疗水产病害的药物中的应用。
在本发明中，所述水产病害优选包括以下一种或几种：无乳链球菌感染导致的病害、败血症、脑膜炎、腹水症、嗜水气单胞菌病、烂尾病和赤皮病。在本发明实施例中，以无乳链球菌引发的水产病害为例具体说明罗非鱼 CL-K1通过发挥免疫防御作用来防治水产病害的效果。
在本发明中，所述水产优选包括以下一种或几种：罗非鱼、大菱鲆、半滑舌鳎、金鲳、斑点叉尾鮰、草鱼和石斑鱼。在本发明实施例中，以尼罗罗非鱼为水产动物代表，说明罗非鱼CL-K1对水产病害的防治效果。在本发明实施例中，通过体外实验证实，具有抑制无乳链球菌增殖的能力；通过体内基因敲除实验表明，罗非鱼CL-K1促进无乳链球菌感染罗非鱼导致的炎症反应，利用真核表达系统重新表达罗非鱼CL-K1蛋白或者体外注射重组罗非鱼CL-K1蛋白后，可提高罗非鱼的免疫保护作用，同时CL-K1真核蛋白可以显著降低罗非鱼由无乳链球菌感染而造成的死亡。
在本发明中，所述罗非鱼CL-K1的氨基酸序列优选如SEQ ID NO:1所示。由于CL-K1在鱼体内含量较少，可通过真核表达系统重组表达的方式获得罗非鱼CL-K1蛋白进行体外抑菌试验。鱼体内试验优选利用真核表达系统构建、体内重组表达罗非鱼CL-K1实现。在重组表达过程中，所述编码所述罗非鱼CL-K1的基因的核苷酸序列优选如SEQ ID NO:2所示。
本发明提供了一种罗非鱼CL-K1或编码罗非鱼CL-K1的基因在制备罗非鱼致病菌抑制剂中的应用。
在本发明中，所述罗非鱼致病菌优选包括以下一种或几种：无乳链球菌、嗜水气单胞菌、迟缓爱德华氏菌和荧光假单胞菌。在本发明实施例中，体外实验证明，罗非鱼CL-K1显著抑制罗非鱼致病菌(无乳链球菌)的增殖，含有罗非鱼CL-K1的抑制剂可发挥抑制罗非鱼致病菌生长和增殖的作用。所述抑制剂还优选包括辅料。本发明对所述辅料的种类没有特殊限制，采用本领域所熟知的含蛋白活性成分的抑制剂的辅料即可，例如注射用水。本发明对所述抑制剂的制备方法没有特殊限制，采用本领域所熟知的微生物抑制剂的制备方法即可。本发明对抑制剂中罗非鱼CL-K1的浓度没有特殊限制，罗非鱼CL-K1的工作浓度不低于0.5mg/mL即可。
本发明提供了一种罗非鱼CL-K1或编码罗非鱼CL-K1的基因在制备罗非鱼致病菌感染引起的炎症反应的抑制剂中的应用。
在本发明中，所述罗非鱼致病菌优选包括以下一种或几种：无乳链球菌、嗜水气单胞菌、迟缓爱德华氏菌、荧光假单胞菌。在本发明实施例中，敲低罗非鱼CL-K1表达，促进罗非鱼致病菌(无乳链球菌)感染罗非鱼引起的炎症反应，通过提高罗非鱼CL-K1的体内含量，提高罗非鱼抗无乳链球菌的免疫保护作用。其中提高罗非鱼CL-K1的体内含量优选包括体外注射重组罗非鱼CL-K1和利用真核表达系统体内表达的罗非鱼CL-K1。因此，含有罗非鱼CL-K1的抑制剂可抑制罗非鱼致病菌感染引起的炎症反应。本发明对所述抑制剂的种类不做具体限定，采用本领域所熟知的炎症反应抑制剂的种类即可。
下面结合实施例对本发明提供的一种罗非鱼CL-K1在制备预防和/或治疗罗非鱼病害的药物中的应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
材料与试剂来源说明
1.OnCL-K1蛋白：根据已经克隆的尼罗罗非鱼OnCL-K1基因序列进行建真核表达载体的构建，真核表达载体为pcDNA3.1(-)表达载体(上海近岸科技有限公司)。然后经过阳性克隆菌株(pLVX-AcGFP1-N1-目的基因)的挑选、质粒提取、细胞转染(选择的哺乳动物表达系统为HEK293细胞)、表达、纯化，可以获得目的基因的真核蛋白。OnCL-K1真核蛋白的构建、表达和纯化是由上海近岸科技有限公司。最后，通过12％SDS-PAGE胶和 LC-MS/MS质谱(国家蛋白质科学中心(BPRC)，北京)对OnCL-K1真核蛋白进行纯化效果检测。结果见图2，获得纯度较高的重组OnCL-K1蛋白。 OnCL-K1蛋白于-80℃超低温冰箱中保存，备用。
2.dsOnCL-K1和dsGFP是合成的RNA。利用目的基因的序列和设计的引物，按照试剂盒合成RNA，进行相关基因的敲降。GFP是通用对照基因，dsGFP的合成引物见表1。尼罗罗非鱼CL-K1 dsRNA的合成是按照Promega公司的 T7 RiboMAXTMExpress RNAi System试剂盒的说明书进行。
3.BSA为商品化蛋白，购买至Sigma公司。
表1本专利所涉及的引物
实施例1
尼罗罗非鱼CL-K1真核蛋白抑制无乳链球菌增殖能力的检测(体外实验)
(1)培养基的配制：液体培养基是称取37g脑-心浸萃液态培养基粉末，用水定容至1L；固体培养基是称取37g脑-心浸萃液态培养基粉末，然后加入15g琼脂，用水定容至1L。通过高温蒸汽灭菌锅灭菌，其中固体培养基需要待温度降低到50℃左右时，在超净工作台中铺制平板。4℃保存备用。
(2)无乳链球菌的培养：将准备好的无乳链球菌(S.agalactiae)接种于脑心浸萃液体培养基中，37℃振荡培养，待OD值到0.6～1.0时取出进行下步操作。
(3)制备菌悬液：吸取一定量的无乳链球菌，3000×rpm离心。利用无菌的1×PBS(10mM磷酸盐，150mM NaCl，pH 7.4)清洗3次，然后用 1×PBS稀释菌的浓度为1×107CFU/mL。
(4)菌液涂布：在超净工作台，吸取调整好浓度的无乳链球菌200μL，加入含有脑心浸萃固体培养基的培养板中。用涂布器将菌液均匀涂布。
(5)摆放牛津杯；在培养基表面垂直摆放灭过菌的牛津杯，轻轻加压，使其与培养基接触无空隙。
(6)加入蛋白：在杯中分别加入200μL OnCL-K1(0.5mg/mL)、BSA (牛血清白蛋白购买于美国Sigma公司，0.5mg/mL，阴性对照)和Ampicillin (氨苄青霉素购买于广州蓝泽生物技术有限公司，0.5mg/mL，阳性对照) 实验重复3次。
(7)37℃静置培养12-15h。观察、统计抑菌圈直径和拍照。
结果见图3。结果表明，尼罗罗非鱼CL-K1真核蛋白具有抑制无乳链球菌增殖能力。
实施例2
CL-K1基因的敲降促进无乳链球菌对尼罗罗非鱼的感染(体内实验)
(1)尼罗罗非鱼的饲养：本实验所用的尼罗罗非鱼购买自广东省罗非鱼良种场。尼罗罗非鱼饲养于生物系半自动循环养殖系统内，保持水质、水温良好，每天投喂体重10％质量的浮性饲料。
(2)尼罗罗非鱼的免疫：从养殖系统内捕捞40尾健康的罗非鱼，随机分入4组并驯化若干天，使罗非鱼充分适应养殖桶环境。各组分别记为PBS 组、PBS+S.agalactiae组、dsGFP+S.agalactiae组、dsOnCL-K1+S.agalactiae 组。配制无菌10mM PBS。罗非鱼免疫当日以一定浓度的MS-2,2,2将其麻醉， dsOnCL-K1(20μg)、dsGFP(20μg)和PBS进行肌肉注射(鱼体中部侧线偏上部位)，每尾100μL。12h后，将提前准备的S.agalactiae(1×107CFU/mL)进行腹腔注射，每尾100μL。免疫后的12h和24h分别从各组中取罗非鱼3 尾，使用解剖工具取其脾脏和肝脏的组织样品。
(3)涂板检测：获取组织样品后，通过天平称重并记录每个样品的质量，以PBS溶液稀释组织匀浆液浓度，使每微升溶液中含有的组织质量相同。涂板检测的时间必须是取样当天，以确保组织样品中菌落的存活状态。在超净工作台中以电动匀浆器将组织样本充分匀浆。以500×g，4℃离心10min，并转移上清至新的离心管内，得到组织匀浆上清。将上一步得到的上清以无菌PBS进行稀释，分别稀释10倍和100倍。将肝脏、脾脏组织匀浆上清液、10倍稀释液、100倍稀释液分别涂布于脑心浸萃固体平板上。每个组别，每种组织，每种浓度各涂布3个平板作为平行。涂布后将平板置于37℃培养箱内培养过夜。次日将平板取出拍照、计数，并统计出每个组织样本中的菌落数量 (CFU)。
(4)病理切片：将“(3)尼罗罗非鱼的免疫”中获取的所述各组别罗非鱼的肝脏、脾脏组织样品转移至4％多聚甲醛溶液中，并于4℃固定过夜。此后，对样品编号并送往武汉市赛尔维生物科技有限公司，通过石蜡包埋、脱蜡至水、HE染色、脱水封片、显微拍摄等步骤，获得罗非鱼组织样本的组织病理学切片结果，以分析罗非鱼组织样本的病理变化。
(5)荧光定量PCR检测炎症因子表达量动态变化：将“(3)尼罗罗非鱼的免疫”中获取的组织样本取出置于冰面上，向每个样本加入200μL Trizol，以电动匀浆器充分研磨成组织匀浆。研磨结束后，继续加入800μL Trizol于室温静置10min。以12000rpm、4℃离心10min，上清转移到新的离心管中。向上清液中加入300μL氯仿，充分涡旋，使其混匀。静置10min后以12000 rpm、4℃离心15min。吸取上层水相转移到新的离心管中。然后加入500μL异丙醇，轻轻混匀，直到不出现分层状态后，静置10min。以12000rpm、4℃离心15min，弃去上清。并向沉淀中加入1mL无水乙醇，轻轻上下颠倒悬浮沉淀。重复上述步骤，洗涤沉淀2-3次。室温晾干沉淀，后加入适量DEPC 水，以溶解所得的RNA沉淀。通过NanoDrop 2000超微量紫外分光光度计检测所得RNA浓度与OD值，以1％琼脂糖凝胶电泳检测RNA的提取效果，是否发生明显的降解，后保存于-80℃备用。通过Hifair III反转录试剂盒 (YEASEN)以总RNA为模板合成cDNA，所有步骤按说明书执行。合成的cDNA以DEPC水稀释10倍后作为qPCR的模板。
通过ABI的QuantStudio 5qPCR仪检测各组别罗非鱼的肝脏、脾脏中促炎因子OnIL-1β、OnIL-6、OnTNF-α与抑炎因子OnIL-10表达量的动态变化， OnIL-1β、OnIL-6、OnTNF-α与抑炎因子OnIL-10的相关扩增引物见表1。 qPCR的体系包括Mix 10μL、浓度为2μM/L的上、下游引物各2μL、模板 3μL、ddH2O 3μL。引物由北京六合华大基因科技有限公司合成。qPCR以罗非鱼β-Actin作为内参基因，通过2-ΔΔCt的方法计算相对表达量。
(6)存活率检测：从养殖系统中随机选取约30g重的罗非鱼80尾，平均分成4组，分组情况同“(3)尼罗罗非鱼的免疫”。对上述罗非鱼按组别进行免疫，具体材料制备与免疫流程参照“(3)尼罗罗非鱼的免疫”(菌浓度调整为1×108CFU/mL)。免疫当天起，10天内每天统计各组罗非鱼的死亡数量。
结果见图4。CL-K1基因的敲降促进无乳链球菌对尼罗罗非鱼的感染。
实施例3
CL-K1蛋白提高罗非鱼抗无乳链球菌的免疫保护(体内实验)
(1)表型拯救实验：从养殖系统内捕捞20尾健康的罗非鱼，随机分入 2组并驯化若干天，使罗非鱼充分适应养殖桶环境。各组分别记为dsOnCL-K1 +Trx(空载蛋白)+S.agalactiae组、dsOnCL-K1+OnCL-K1+S.agalactiae 组。罗非鱼免疫当日以一定浓度的MS-2,2,2将其麻醉，dsOnCL-K1(20μg) 进行肌肉注射，每尾100μL。12h后，将提前准备的菌液进行腹腔注射，每尾100μL。实验组腹腔注射S.agalactiae(1×107CFU/mL)与OnCL-K1(500 μg/mL)或Trx(500μg/mL)共同孵育的菌液。菌应激后的第12h和24h 采集肝脏、脾脏和外周血样品。分别进行涂板计数、RNA提取和组织病理学分析，参照实施例2中步骤(4)～(6)。
(2)从养殖系统中随机选取约30g重的罗非鱼40尾，平均分成2组，分别为dsOnCL-K1+Trx+S.agalactiae组、dsOnCL-K1+OnCL-K1+S. agalactiae组。对上述罗非鱼按组别进行免疫，具体材料制备与免疫流程参照“(1)尼罗罗非鱼的免疫”(菌浓度调整为1×108CFU/mL)。免疫当天起，10天内每天统计各组罗非鱼的死亡数量。
结果见图5。重新注入OnCL-K1蛋白能够恢复对体内病原菌清除，显著抑制炎症反应，提高罗飞鱼的存活率。可见，CL-K1蛋白可显著提高罗非鱼抗无乳链球菌的免疫保护。
以上所述仅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，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 华南师范大学
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ctgtcggcag aaccgtgtcc 20
<210> 15
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ggatcctaat acgactcact atagcttaca ttgtttgtca gt 42
<210> 16
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aaactgacaa acaatgtaag ctatagtgag tcgtattagg atcc 44
<210> 17
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ggatcctaat acgactcact ataactgaca aacaatgtaa gc 42
<210> 18
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aagcttacat tgtttgtcag ttatagtgag tcgtattagg atcc 44
<210> 19
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ggatcctaat acgactcact atagccacaa cgtctatatc at 42
<210> 20
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aaatgatata gacgttgtgg ctatagtgag tcgtattagg atcc 44
<210> 21
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ggatcctaat acgactcact ataatgatat agacgttgtg gc 42
<210> 22
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
aagccacaac gtctatatca ttatagtgag tcgtattagg atcc 44
Claims (10)
1.罗非鱼CL-K1或编码所述罗非鱼CL-K1的基因在制备预防和/或治疗水产病害的药物中的应用。
2.根据权利要求1所述应用，其特征在于，所述罗非鱼CL-K1的氨基酸序列如SEQ IDNO:1所示。
3.根据权利要求2所述应用，其特征在于，所述罗非鱼CL-K1是利用真核表达系统构建、表达、纯化的罗非鱼CL-K1。
4.根据权利要求1所述应用，其特征在于，所述编码所述罗非鱼CL-K1的基因的核苷酸序列如SEQ ID NO:2所示。
5.根据权利要求1所述应用，其特征在于，所述水产病害包括以下一种或几种：无乳链球菌感染导致的病害、败血症、脑膜炎、腹水症、嗜水气单胞菌病、烂尾病和赤皮病。
6.根据权利要求1所述应用，其特征在于，所述水产包括以下一种或几种：罗非鱼、大菱鲆、半滑舌鳎、金鲳、斑点叉尾鮰、草鱼和石斑鱼。
7.根据权利要求1～6任意一项所述应用，其特征在于，所述药物具有抑制无乳链球菌增殖、抑制病害细菌感染导致的炎症反应的作用。
8.一种罗非鱼CL-K1或编码罗非鱼CL-K1的基因在制备罗非鱼致病菌抑制剂中的应用。
9.一种罗非鱼CL-K1或编码罗非鱼CL-K1的基因在制备罗非鱼致病菌感染引起的炎症反应的抑制剂中的应用。
10.根据权利要求8或9所述应用，其特征在于，所述罗非鱼致病菌包括以下一种或几种：无乳链球菌、嗜水气单胞菌、迟缓爱德华氏菌和荧光假单胞菌。
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| CN1694961A (zh) * | 2002-09-10 | 2005-11-09 | 内蒂穆恩公司 | 胶原凝集素－补体激活蛋白嵌合体 |
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| US20170056481A1 (en) * | 2014-02-19 | 2017-03-02 | University Of Southampton | Treating Infection |
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| CN113058029A (zh) * | 2021-03-18 | 2021-07-02 | 西安交通大学 | 胶原凝集素-11在制备防治泌尿系统感染的药物中的应用 |
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| CN1694961A (zh) * | 2002-09-10 | 2005-11-09 | 内蒂穆恩公司 | 胶原凝集素－补体激活蛋白嵌合体 |
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| CN110734486A (zh) * | 2019-10-30 | 2020-01-31 | 青岛大学 | 一种半滑舌鳎凝集素家族collectin抗病基因 |
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