CN116376795B - 一种表达hsp70的重组乳酸菌及其制备方法与应用 - Google Patents
一种表达hsp70的重组乳酸菌及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于基因工程及分子免疫学技术领域，具体涉及一种表达HSP70的重组乳酸菌及其制备方法与应用。该重组乳酸菌以乳酸菌作为活载体，表达HSP70蛋白，既能发挥乳酸菌的益生功能，又能发挥功能性蛋白(HSP70蛋白)在鱼类黏膜防御系统中关键调节作用，阻断小瓜虫感染，提高乳酸菌的抗小瓜虫感染的生态防病效果。
Description
技术领域
本发明属于基因工程及分子免疫学技术领域，具体涉及一种表达HSP70的重组乳酸菌及其制备方法与应用。
背景技术
多子小瓜虫(Ichthyophthirius multifiliis,Ich)属于纤毛虫纲、膜口目、凹口科、小瓜虫属，几乎能感染所有的淡水鱼类，分为掠食体、包囊前体、包囊和滋养体阶段，其中掠食体钻入鱼类皮肤表面后，分解鱼体表面黏液和上皮组织为食，可以造成鳞片基部和鳃盖处发生局灶性出血，并在真皮层中积聚血细胞和炎性结节。小瓜虫入侵鱼类表皮后，周围的白细胞和炎性细胞不仅没有诱导免疫保护，还被寄生虫摄取，导致炎症细胞的数量减少了，进而造成更加严重的继发性感染，也为小瓜虫病的防控带来巨大困难。因此，发病后常常引发鱼群大规模死亡并造成了巨大的经济损失。目前小瓜虫病的防治方法主要有物理方法和化学方法，但均不能对小瓜虫感染发挥有效控制作用。因此研制既能够确保水产品质量安全、环境友好，且操作简便、易于应用的绿色渔药，是促进我国水产养殖健康发展的当务之急。
发明内容
本发明第一方面的目的，在于提供一种重组乳酸菌。
本发明第二方面的目的，在于提供本发明第一方面的重组乳酸菌的制备方法。
本发明第三方面的目的，在于提供本发明第一方面的重组乳酸菌的应用。
本发明第四方面的目的，在于提供一种产品。
为了实现上述目的，本发明所采取的技术方案是：
本发明的第一个方面，提供一种重组乳酸菌，所述重组乳酸菌包含HSP70蛋白的编码基因，所述HSP70蛋白的氨基酸序列如SEQ ID NO.4所示的HSP70蛋白。
优选地，所述编码基因的序列如SEQ ID NO.3所示。
优选地，所述乳酸菌选自乳球菌亚种、链球菌亚种、乳杆菌亚种、明串珠菌亚种、片球菌亚种、短杆菌亚种以及丙酸杆菌亚种。
优选地，所述乳酸菌包含乳酸乳球菌；进一步包含乳酸乳球菌NZ9000。
优选地，所述重组乳酸菌的名称为pNZ8148-HSP70/L.lactis，保藏于位于中国武汉.武汉大学的中国典型培养物保藏中心，保藏时间为2023年2月27日；保藏编号是CCTCCNO：M2023213，分类命名为：Lactococcus lactis pNZ8148-HSP70(乳酸乳球菌pNZ8148-HSP70)。
本发明的第二个方面，提供本发明第一个方面的重组乳酸菌的制备方法，将HSP70蛋白的编码基因导入乳酸菌中。
优选地，所述HSP70蛋白的编码基因通过重组载体导入乳酸菌。
优选地，所述重组载体为将所述HSP70蛋白的编码基因插入表达载体的多克隆位点后得到的载体。
优选地，所述表达载体可为现有技术中常见的表达载体，例如：pNZ8148、pNZ8048、pNZ9530、pNZ8149、pLEISS、pNZ2013、pNZ2103、pLEB590、pNZ8112、pIAβ5、pW425e t、pW425t、pW425等。
优选地，所述表达载体为pNZ8148。
优选地，所述导入的方式为电转化。
本发明的第三个方面，提供本发明第一个方面的重组乳酸菌在制备产品中的应用；所述产品具有c1)～c2)中至少一种功能：
c1)预防小瓜虫感染；
c2)治疗和/或预防小瓜虫感染所引起的疾病。
优选地，所述小瓜虫为多子小瓜虫；进一步为淡水鱼多子小瓜虫。
优选地，所述小瓜虫感染所引起的疾病为小瓜虫病。
优选地，所述产品包含饲料、饲料添加剂、药物、试剂中的至少一种。
优选地，所述药物还包含药学上可接受的辅料。
优选地，所述产品为口服制剂。
优选地，所述产品的施用对象为水产动物；进一步为淡水鱼；更进一步为草鱼。
本发明的第四个方面，提供一种产品，包含本发明第一个方面的重组乳酸菌。
优选地，所述产品具有c1)～c2)中至少一种功能：
c1)预防小瓜虫感染；
c2)治疗和/或预防小瓜虫感染所引起的疾病。
优选地，所述小瓜虫为多子小瓜虫；进一步为淡水鱼多子小瓜虫。
优选地，所述小瓜虫感染所引起的疾病为小瓜虫病。
优选地，所述产品包含饲料、饲料添加剂、药物、试剂中的至少一种。
优选地，所述药物还包含药学上可接受的辅料。
优选地，所述产品为口服制剂。
优选地，所述产品的施用对象为水产动物；进一步为淡水鱼；更进一步为草鱼。
优选地，一种口服制剂，包含本发明第一个方面的重组乳酸菌。
本发明的有益效果是：
本发明以乳酸菌作为活载体，表达HSP70蛋白，既能发挥乳酸菌的益生功能，又能发挥功能性蛋白(HSP70蛋白)在鱼类黏膜防御系统中关键调节作用，阻断小瓜虫感染，提高乳酸菌的抗小瓜虫感染的生态防病效果，在抗小瓜虫感染中取得了意料不到的技术效果，21天相对保护率高达90.48％。
同时，乳酸菌具有调节免疫功能，抑制肠道致病菌生长，维持肠道微生态平衡，合成多种维生素、氨基酸和酶，促进消化吸收，提供饲料利用率等功能，并且，乳酸菌的稳定性好，耐消化道的酸性环境，可以长时间在鱼体肠道驻留。
本发明以乳酸菌为载体，递送具有黏膜防御功能的草鱼HSP70蛋白，鱼体口服后，重组乳酸菌在鱼体肠道黏膜中固定和繁殖，展示表达的HSP70蛋白，直接作用鱼类黏膜防御系统发挥自身的活性功能，使鱼体获得抗小瓜虫感染能力。该重组乳酸菌具有小瓜虫感染阻断功能，与现有的技术比较，从宿主本源益生菌防控小瓜虫感染，在兼具功能性蛋白的生物活性，所以本发明改造后具有预防小瓜虫病的重组乳酸菌不同于现有的微生物生态防病技术。
附图说明
图1是重组质粒pNZ8148-HSP70的PCR鉴定和酶切鉴定结果图：其中，M：DNA marker(DL5000)；泳道1：PCR鉴定；泳道2、3：单酶切鉴定；泳道4：双酶切鉴定。
图2是重组乳酸菌pNZ8148-HSP70/L.lactis表达产物的免疫印记(Western-blot)分析结果图：其中，M：蛋白Marker；泳道1：经诱导的pNZ8148-HSP70/L.lactis；泳道2：未经诱导的pNZ8148-HSP70/L.lactis。
图3是重组乳酸菌pNZ8148-HSP70/L.lactis表达产物的间接ELISA分析结果图。
图4是重组乳酸菌pNZ8148-HSP70/L.lactis在草鱼肠黏膜驻留评价结果图。
图5是草鱼口服重组乳酸菌pNZ8148-HSP70/L.lactis后免疫调节作用评价结果图：其中，(A)是草鱼口服重组乳酸菌后IL-1β表达量结果图；(B)是草鱼口服重组乳酸菌后IFN-γ表达量结果图；(C)是草鱼口服重组乳酸菌后IL-2表达量结果图；(D)是草鱼口服重组乳酸菌后IL-4表达量结果图；*表示与PBS组相比，P＜0.05；**表示与PBS组相比，P＜0.01。
图6是草鱼口服重组乳酸菌pNZ8148-HSP70/L.lactis后对小瓜虫感染的拮抗阻断效果评价结果图。
图7是草鱼口服重组乳酸菌pNZ8148-HSP70/L.lactis后感染小瓜虫的生存曲线图。
具体实施方式
以下通过具体的实施例对本发明的内容作进一步详细的说明。
应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。本实施例中所使用的材料、试剂等，如无特别说明，为从商业途径得到的试剂和材料。
实验动物：草鱼购于广州市京阳养殖场，体长(10±0.5)cm。
菌、毒株：淡水鱼多子小瓜虫由珠江水产研究所水产病害与免疫研究室分离保存，感染小瓜虫的鱼购自陕广东省广州市芳村花鸟鱼虫市场，其传代方式将感染鱼与健康金鱼一同置于若干50-L水族箱中，保持上述的饲养条件，用充氧泵进行增氧和虹吸法吸污，隔天换水1/3。收集小瓜虫的方法，首先将若干全身布满小瓜虫的金鱼置于装有若干蒸馏水的烧杯(100m L/尾)30分钟。由于金鱼不停地游动及轻微的人为刺激，成熟的小瓜虫从金鱼体表脱落，收集后用蒸馏水冲洗三次以去除其带有的宿主粘液。然后将其放入24孔板中并在22.0±0.5℃温度中培养，经过6h发育形成包囊，再经16-18小时后小瓜虫掠食体破膜而出，具体方法可见《厚朴酚抗多子小瓜虫活性及机制研究》【D】宋晨光，西北农林科技大学。
主要试剂：pNZ8148表达载体、草鱼IgM单克隆抗体由本实验室制备，具体方法参见Weiwei Zeng,et al.Development of a VP38 recombinant protein-based indirectELISA for detection of antibodies against grass carp reovirus genotype II(iELISA for detection of antibodies against GCRV II).JFD(2018)41:1811-1819.Chen JM,et al.Establishment of a rare minnow(Gobiocypris rarus)model forevaluation of experimental vaccines against a disease induced by grass carpreovirus genotype II.Fish Shellfish Immunol(2021)117:53-61.doi:10.1016/j.fsi.2021.07.014。Trizol Reagent、PrimeScriptTMRT reagent Kit、Takara Nco I、HindⅢ限制性内切酶、Takara Ex 基因组DNA提取试剂盒、Premix Ex TaqTM(ProbeqPCR)、Competent Cell Preparation Kit均购买于宝日医生物技术有限公司，SYBR GreenPro Taq HS预混型qPCR购买于艾科瑞生物科技有限公司，LB肉汤培养基购买于广东环凯微生物科技有限公司，GM17肉汤培养基购买于北京酷来搏科技有限公司，氯霉素购买于北京索莱宝科技有限公司，乳酸链球菌素Nisin购买于上海麦克林生化科技有限公司，Goatanti-Rabbit IgG(H&L)-FITC抗体购买于安诺伦(北京)生物科技有限公司，Goat Anti-Mouse IgG,(H+L)抗体购买于赛默飞世尔科技公司，TMB显色液、反应终止液、SDS-PAGE和Western blot相关试剂均购买于苏州新赛美生物科技有限公司。
本实施例中存活率及相对保护率的计算方法参考文献：陈佳明.Ⅱ型草鱼呼肠孤病毒稀有鮈鲫感染模型和疫苗评价模型的建立[D].上海海洋大学,2021.DOI:10.27314/d.cnki.gsscu.2021.000211.。
实施例1表达HSP70重组质粒的制备
重组表达载体pNZ8148-HSP70的合成和鉴定
GenBank上公布的草鱼类HSP70的基因序列，设计引物HSP70-F(AGATCTATGTCATCAGCAAAAGGAGT，SEQ ID NO.1)、HSP70-R(CTTAAGATGGTGATGGAGATGATGTTAATCCACCTCTTCAATAG，SEQ ID NO.2)，同时在引物中引入His标签和限制性酶切位点，提取各组织中的mRNA。以mRNA作为模板反转录获得cDNA，以HSP70-F/HSP70-R作为引物进行PCR扩增，将纯化的PCR产物连接pMD19T载体，转入E.coli JM109感受态细胞，挑选阳性克隆进行PCR和酶切鉴定，将鉴定正确的重组子测序。
其中，HSP70的核苷酸序列为：ATGTCATCAGCAAAAGGAGTAGCTATTGGAATTGA CCTGGGCACCACCTACTCCTGTGTGGGGGTGTTTCAGCATGGAAAGGTGGAGATCATCGCCAATGACCAGGGGAACAGAACAACACCCAGCTATGTTGCCTTCACAGACACAGAGAGGCTCATTGGGGATGCAGCTAAAAACCAGGTGGCCATGAACCCCAACAACACTGTGTTTGATGCTAAGAGGCTGATTGGCAGGAAGTTTGATGACCCAGTTGTGCAGTCTGACATGAAGCACTGGTCCTTCAAAGTGGTCAGTGATGGAGGAAAACCAAAGGTTCAAGTCGAATACAAAGGAGAAAACAAGACATTTTATCCTGAAGAAATTTCCTCTATGGTCCTGGTGAAGATGAAGGAGATTGCTGAAGCTTATCTGGGGCAGAAGGTGACAAATGCAGTTATCACAGTTCCAGCCTATTTCAATGACTCCCAGAGGCAAGCCACTAAAGACGCCGGAGTAATCGCCGGGCTCAATGTCCTCAGAATCATCAACGAGCCCACAGCTGCAGCCATCGCCTACGGCCTTGACAAAGGCAAAGCAGCAGAACGCAACGTCCTGATCTTTGACCTGGGTGGAGGCACCTTTGACGTGTCCATCCTGACCATTGAAGATGGCATCTTTGAGGTGAAGGCCACAGCCGGAGACACTCATCTGGGTGGCGAGGACTTTGACAACCGCATGGTGAATCACTTTGTAGAAGAATTCAAGAGGAAGCACAAGAAGGACATCAGTCAGAACAAGAGGGCACTGAGGAGGCTGCGTACAGCGTGTGAGCGAGCCAAGAGAACCCTCTCCTCCAGCTCTCAGGCCAGCCTTGAGATCGACTCGCTGTACGAGGGCATCGACTTCTACACGTCCATCACCAGAGCACGCTTTGAAGAGATGTGCTCAGACCTCTTCAGGGGAACGCTTGAGCCTGTGGAGAAAGCCCTGAGAGACGCCAAGATGGACAAGTCTCAGATCCATGACATCGTTCTGGTTGGTGGATCAACAAGAATCCCAAAGATCCAGAAGCTTCTGCAGGATTTCTTCAACGGCAGGGACTTGAACAAGAGCATCAACCCAGATGAGGCAGTGGCTTATGGTGCAGCGGTGCAAGCCGCCATCCTCATGGGTGACACATCTGGAAATGTCCAGGACCTGCTGCTGCTGGATGTGGCTCCTCTGTCCCTGGGTATTGAAACCGCAGGTGGAGTCATGACGGCCCTCATCAAACGCAACACCACCATCCCCACCAAACAGACCCAGACCTTCACCACCTACTCTGACAACCAGCCCGGTGTCCTGATCCAGGTGTATGAGGGAGAGAGGGCCATGACAAAAGACAACAACCTGCTGGGTAAATTTGAGCTCACAGGAATTCCACCTGCACCACGTGGAGTCCCGCAGATTGAAGTGACCTTTGACATCGACGCCAACGGAATCCTAAATGTGTCCGCGGTGGACAAAAGCACTGGAAAAGAGAACAAGATCACCATCACCAATGACAAGGGCAGACTGAGCAAAGAGGAGATTGAGAGAATGGTGCAGGAAGCAGATAAGTACAAAGCTGAAGATGATCTGCAAAGAGAGAAGATTGCTGCCAAAAACTCCCTGGAGTCTTACGCCTTCAACATGAAGAACAGTGTGGAAGATGAGAACCTGAAAGGCAAGATCAGCGAGGATGACAAGAAGAAAGTCATAGAGAAGTGCAACCAGACTATCAGCTGGCTAGAGAACAACCAGCTGGCTGATAAGGAGGAGTATGAACATCAGCTGAAGGAGCTGGAGAAAGTCTGCAACCCAATCATCACTAAGCTTTATCAGGGAGGGATGCCAGCTGGAGGCTGTGGAGCTCAGGCACGTGGAGGATCAGGGGCCGCTTCCCAGGGACCAACTATTGAAGAGGTGGATTAA(SEQ IDNO.3)；
HSP70的氨基酸序列为：MSSAKGVAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPS YVAFTDTERLIGDAAKNQVAMNPNNTVFDAKRLIGRKFDDPVVQSDMKHWSFKVVSDGGKPKVQVEYKGENKTFYPEEISSMVLVKMKEIAEAYLGQKVTNAVITVPAYFNDSQRQATKDAGVIAGLNVLRIINEPTAAAIAYGLDKGKAAERNVLIFDLGGGTFDVSILTIEDGIFEVKATAGDTHLGGEDFDNRMVNHFVEEFKRKHKKDISQNKRALRRLRTACERAKRTLSSSSQASLEIDSLYEGIDFYTSITRARFEEMCSDLFRGTLEPVEKALRDAKMDKSQIHDIVLVGGSTRIPKIQKLLQDFFNGRDLNKSINPDEAVAYGAAVQAAILMGDTSGNVQDLLLLDVAPLSLGIETAGGVMTALIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERAMTKDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGILNVSAVDKSTGKENKITITNDKGRLSKEEIERMVQEADKYKAEDDLQREKIAAKNSLESYAFNMKNSVEDENLKGKISEDDKKKVIEKCNQTISWLENNQLADKEEYEHQLKELEKVCNPIITKLYQGGMPAGGCGAQARGGSGAASQGPTIEEVD(SEQ IDNO.4)。
分别将pMD19T-HSP70和乳酸菌表达载体pNZ8148以限制性内切酶Nco I、Xba I酶切，回收纯化酶切产物后T4连接酶连接。转入E.coli MC1061感受态细胞，挑取pNZ8148-HSP70/MC1061单克隆，接种抗性LB液体培养基，37℃、200r/min震荡培养12h，提取质粒后进行酶切验证和PCR鉴定，并送至测序公司进行测序。PCR和酶切鉴定结果表明成功构建重组质粒pNZ8148-HSP70(图1)。
实施例2表达HSP70的重组乳酸菌的制备
制备L.lactis NZ9000感受态细胞。将L.lactis NZ9000划线在0.5wt％葡萄糖的GM17固体培养基上，30℃静置培养过夜。挑取新鲜的乳酸乳球菌单菌落于5mL含0.5wt％葡萄糖GM17肉汤培养基，30℃静置培养6h，得到培养物。按1∶10(v/v)比例取上述培养物于50mL含0.5wt％葡萄糖+1wt％甘氨酸的GM17肉汤培养基，30℃静置培养过夜，得到培养物。按1∶10(v/v)比例取上述培养物于400mL含0.5wt％葡萄糖+0.5mol·L-1蔗糖+2wt％甘氨酸的GM17肉汤培养基，继续静置培养至光密度(OD600)＝0.5，分装置于50mL离心管中，4℃5000g离心15min弃上清。加入10mL预冷溶液(0.5mol·L-1蔗糖+10wt％甘油)重悬，4℃5 000g离心15min弃上清。加入5mL预冷溶液[2.5mL(0.5mol·L-1蔗糖+10wt％甘油)+2.5mL0.05mol·L-1Na-EDTA(pH7.5)]重悬，4℃5 000g离心15min弃上清。加入200uL预冷溶液(0.5mol·L-1蔗糖+10wt％甘油)重悬，分装，-80℃保存备用。
pNZ8148-HSP70质粒电转化L.lactis NZ9000感受态细胞。试验前将电转杯、恢复培养基(0.5M蔗糖+0.02M MgCL2+0.002M CaCl2的GM17肉汤培养基)预冷。将100μL的L.lactis NZ9000感受态细胞冰上融化，与10μL质粒混匀，冰中静置10min。将混合物转入预冷电转杯中，置于Eppendorf Eporator电转化仪电击(2500V，5ms)，迅速加入900μL预冷恢复培养基，均匀混合后置于冰上3min，30℃厌氧培养2h，3500r/min离心1min，弃掉900μL上清。将剩余菌液吹悬后涂布至抗性GM17固体平板，30℃厌氧静置过夜培养。挑取单菌落接种抗性GM17肉汤培养基中，30℃静置培养。将鉴定正确的重组乳酸菌命名为pNZ8148-HSP70/L.lactis，保藏于位于中国武汉.武汉大学的中国典型培养物保藏中心，保藏时间为2023年2月27日；保藏编号是CCTCC NO：M2023213，分类命名为：Lactococcus lactis pNZ8148-HSP70(乳酸乳球菌pNZ8148-HSP70)。
重组乳酸菌的表达。将挑取pNZ8148-HSP70/L.lactis菌落接种于10mL抗性GM17肉汤培养基，30℃厌氧静置培养过夜。取培养菌液按1：50(v/v)比例接种于300mL抗性GM17肉汤培养基中，30℃静置培养4h，分别加入终浓度为10ng/mL、50ng/mL、200ng/mL、500ng/mL、1000ng/mL和1000ng/mL诱导剂乳链球菌肽Nisin，以未经诱导(未加入乳链球菌肽Nisin)的重组乳酸菌作为对照，30℃诱导4h。
表达产物的免疫印记(Western-blot)分析。将诱导和未经诱导后重组乳酸菌4℃，6000r/min离心5min，弃上清。用3mLPBS溶液悬浮，超声破碎(功率120w，工作2s，休息5s)，菌液破碎效果以澄清为宜。取20μL破碎菌液加入5μL5×SDS凝胶上样缓冲液，混匀后置于沸水中10min，在12％ SDS-PAGE上进行蛋白电泳(120V，60min)。电泳后将凝胶上的蛋白转移至PVDF膜(400mA，20min)，于5％脱脂奶粉中封闭，37℃2h。加入PBST清洗3次，每次5min。以鼠抗His单克隆抗体作为一抗，4℃孵育过夜。加入PBST清洗3次，每次5min。加入HRP标记的羊抗鼠IgG作为二抗，室温孵育1h。加入PBST清洗3次，每次5min。按照高敏ECL化学发光试剂盒说明书进行显色，凝胶成像仪观察结果。重组乳酸菌pNZ8148-HSP70/L.lactis经诱导后，在表达蛋白带的预期位置出现明显的反应，未加入诱导剂对照组没有(图2)。
HSP70蛋白间接ELISA分析。将诱导后超声破壁的pNZ8148-HSP70/L.lactis(pNZ8148-HSP70/L.lactis破碎)以及完整菌体(pNZ8148-HSP70/L.lactis全菌)分别作为抗原包被96孔ELISA反应板(酶标板)，以pNZ8148/L.lactis空载(L.lactis NZ9000)为阴性对照，每孔100μL，4℃包被过夜。PBST清洗3次，每次5min。每孔加入200μL含有5％脱脂奶粉37℃，封闭2h。PBST清洗3次，每次5min。每孔加入100μL His蛋白的单克隆抗体(1：3000稀释)，4℃孵育过夜，PBST清洗3次，每次5min。每孔加入100μL HRP标记羊抗鼠IgG(1:5000稀释)，37℃，孵育1h，PBST清洗3次，每次5min。按照说明书，加入150μL NcmTMB One显色液，37℃避光显色20min，加50μL反应终止液(2M H2SO4)终止反应，酶标测定仪测定OD450nm值。所有数据表示为平均值±标准误差。数据经过Student's T检验分析，P<0.05被认为具有统计学意义。pNZ8148-HSP70/L.lactis破碎组和pNZ8148-HSP70/L.lactis全菌组的OD450nm值与pNZ81480/L.lactis(L.lactis NZ9000)组相比均具有极显著性差异(p＜0.01)，表明重组蛋白HSP70主要展示在菌体表面(图3)。
实施例3草鱼口服重组乳酸菌后免疫调节作用评价
草鱼口服黏膜接种。将300尾健康的草鱼暂养实验动物房2周后，随机分为3组，每组100尾，分别为PBS空白对照组(PBS)，pNZ8148/L.lactis空载对照组(L.lactis NZ9000)，pNZ8148-HSP70/L.lactis实验组(pNZ8148-HSP70/L.lactis)。黏膜接种方式为灌胃口服接种，连续灌胃3d，免疫剂量为10μL 2×109CFU/mL/鱼/d。
草鱼肠黏膜驻留评价：以pNZ8148-HSP70/L.lactis，按照106CFU/尾剂量口服灌胃进行黏膜接种，在接种后72h内，取草鱼前肠，去除肠内容物后以无菌PBS反复冲洗，取肠粘膜上皮细胞，通过菌落计数对单位面积肠粘膜上重组乳酸菌的驻留情况通过肠黏膜菌落计数进行持续监测，结果如图4所示：在接种后3h即可在草鱼肠粘膜内监测到重组乳酸菌pNZ8148-HSP70/L.lactis，随后肠黏膜粘附菌落数量升高，在接种后6h达到峰值，且在接种后72小时内黏膜乳酸菌数量持续保持较高水平，表明pNZ8148-HSP70/L.lactis重组乳酸菌可以在鱼类的肠道中较长时间驻留，并发挥生物学功能。
样品采集。免疫后的第7d、14d、21d从各组中随机取3尾草鱼收集脾脏、肾脏和肠道后端组织进行免疫相关基因，提取各组织的总RNA后进行反转录，以β-actin作为内参基因，以获得的cDNA作为模板通过qRT-PCR测定各组织中IL-1β、IFN-γ、IL-2和IL-4的相对表达量。各基因引物序列信息如表1所示，引物序列由测序公司合成。实验进行三次生物学重复。qRT-PCR反应体系如下：10μL 2×SYBR Green Taq HS Premix，引物各0.4μL，0.4μL ROX，2μL cDNA模板以及加ddH2O补齐至20μL，95℃预变性5min后进入循环：95℃，15s变性；60℃，45s退火；共循环35次。测定结果使用2-ΔΔCt算法进行分析。所有数据为平均值±标准误差。数据经过Student's t检验分析，P<0.05被认为具有统计学意义。结果如图5所示：黏膜接种重组乳酸菌后草鱼中枢免疫器官、外周免疫器官和黏膜免疫组织中的IL-1β、IFN-γ、IL-2和IL-4的相对表达水平均发生显著提高，表明重组乳酸菌定植后既可以调节黏膜免疫，又可以调节系统免疫，对鱼体先天免疫、体液免疫和细胞免疫均有较好的调节作用；口服免疫重组乳酸菌(pNZ8148-HSP70/L.lactis)能够有效增强草鱼黏膜免疫组织、中枢免疫器官和外周免疫器官的炎症反应、体液免疫和细胞免疫。
表1荧光定量PCR中的引物序列
实施例4重组乳酸菌口服免疫保护评价
在实施例3的草鱼免疫后第21天，对草鱼进行人工感染小瓜虫。从已感染小瓜虫的鱼体表刮取小瓜虫，18℃清水中孵育18-20h，每条鱼约5000个掠食体的浓度进行浸泡感染草鱼(每组50尾草鱼)。观察各组草鱼状态，病记录统计感染小瓜虫后死亡草鱼数。在感染后的第7、14、21天，从每组中随机抽取3尾鱼剪取鳃部，在镜下进行多子小瓜虫体计数。结果如图6所示：PBS空白对照组在感染后粘附小瓜虫数量持续持续上升，口服黏膜接种空载乳酸菌(L.lactis NZ9000)/重组乳酸菌(pNZ8148-HSP70/L.lactis)在感染后粘附小瓜虫数量持续下降，在感染后第7、14d，空载乳酸菌(L.lactis NZ9000)的粘附小瓜虫数量显著低于PBS空白对照组(PBS，P＜0.05)，重组乳酸菌(pNZ8148-HSP70/L.lactis)的黏附小瓜虫数量极显著低于对照组(P＜0.01)，在感染后第21d，空载乳酸菌(L.lactis NZ9000)、重组乳酸菌(pNZ8148-HSP70/L.lactis)的粘附小瓜虫数量显著低于PBS空白对照组(P＜0.001)，并且在感染14d重组乳酸菌(pNZ8148-HSP70/L.lactis)组养殖草鱼鳃部无附着小瓜虫检出；与对照组相比较，黏膜接种空载乳酸菌(L.lactis NZ9000)虽然也对小瓜虫感染具有一定阻断作用，但在感染初期仍然虫体数量仍较多。结果表明口服黏膜接种重组乳酸菌pNZ8148-HSP70/L.lactis可以提前显著降低小瓜虫的黏附能力。
攻毒实验评价结果如图7所示：pNZ8148-HSP70/L.lactis实验组(pNZ8148-HSP70/L.lactis)草鱼在感染后鱼体体色、体态正常，游动、采食等行为无异常，未出现持续大规模死亡，感染21天后存活率为78.95％；pNZ8148/L.lactis空载对照组(L.lactis NZ9000)感染初期症状并不明显，在感染后第7天后出现了小瓜虫感染的典型症状，并伴有持续死亡发生，感染后21天存活率为28.57％；PBS空白对照组(PBS)草鱼在感染后发生与体色发黑、体表粘液减少、鱼体消瘦、聚团游泳等典型症状，并发生持续死亡，在感染后第17天全部死亡；与对照组相比，重组乳酸菌和空载乳酸菌口服黏膜接种后都具有生抗小瓜虫感染效果，相对保护率分别为90.48％和28.57％。结果表明乳酸菌(pNZ8148/L.lactis空载)黏膜接种对小瓜虫感染具有一定保护效果，HSP70蛋白(pNZ8148-HSP70/L.lactis)进一步提高了小瓜虫感染黏膜保护效果。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (9)
1.一种重组乳酸菌，所述重组乳酸菌的名称为Lactococcus lactis pNZ8148-HSP70，保藏于位于中国武汉.武汉大学的中国典型培养物保藏中心，保藏时间为2023年2月27日；保藏编号是CCTCC NO：M2023213。
2.权利要求1所述的重组乳酸菌在制备产品中的应用；所述产品具有c1)～c2)中至少一种功能：
c1)预防小瓜虫感染；
c2)治疗和/或预防小瓜虫感染所引起的疾病。
3.根据权利要求2所述的应用，其特征在于：所述产品包含饲料、饲料添加剂、药物、试剂中的至少一种。
4.根据权利要求3所述的应用，其特征在于：所述药物还包含药学上可接受的辅料。
5.根据权利要求3所述的应用，其特征在于：所述产品为口服制剂。
6.一种产品，包含权利要求1所述的重组乳酸菌。
7.根据权利要求6所述的产品，其特征在于：所述产品包含饲料、饲料添加剂、药物、试剂中的至少一种。
8.根据权利要求7所述的产品，其特征在于：所述药物还包含药学上可接受的辅料。
9.一种口服制剂，包含权利要求1所述的重组乳酸菌。
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