CN113278643B - 一种构建具有降低肝脏脂肪积累且保护肠道屏障功能的基因工程菌的方法及应用 - Google Patents
一种构建具有降低肝脏脂肪积累且保护肠道屏障功能的基因工程菌的方法及应用 Download PDFInfo
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Abstract
本发明公开了一种构建具有降低肝脏脂肪积累且保护肠道屏障功能的基因工程菌的方法及应用。本发明公开的重组菌的构建方法,包括将自溶素AcmA的编码基因和膜蛋白AM的编码基因导入受体菌中,得到重组菌;所述受体菌为乳酸乳球菌(Lactococcus lactis)。实验证明:利用本发明的方法构建的重组菌具有降低肝脏脂肪含量、减少肝损伤,保护鱼类肠道屏障的作用。表明利用本发明的方法构建的重组菌可以作为水产益生菌。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种构建具有降低肝脏脂肪积累且保护肠道功能的基因工程菌的方法及应用。
背景技术
水产动物脂肪肝问题是水产行业集约化养殖过程不可避免的难题之一,从微生物角度挖掘出有益的元件,实施脂代谢精准调控来缓解或解决鱼类脂肪肝问题是目前研究的热点。
益生菌是单一或复合菌株,它能够通过减少或去除病原菌,提高宿主原有的菌群特性,维持肠道微生物平衡,提高宿主的存活率,促进生长;从而被广泛的应用于水产养殖中。乳酸菌是一类可以分解糖类产生乳酸的无芽孢革兰氏阳性菌,在饲料添加剂中具有重要地位。农业农村部2013年批准允许鱼类饲用微生物30种,其中乳酸菌类达20种。而AcmA是乳酸菌细胞壁具有双向锚定功能的蛋白,其可以从细胞外环境中向细胞壁回向锚定,甚至是锚定到其它细菌种的细胞壁上。因此,AcmA成为乳酸菌中具有研究潜力的运载蛋白之一。
发明内容
本发明所要解决的技术问题是如何在高脂饲料喂养条件下抑制鱼类肝脏脂肪增加和/或降低鱼类肝脏脂肪含量和/或降低鱼类肝损伤。
为解决上述技术问题,本发明首先提供了重组菌的构建方法。
本发明所提供的重组菌的构建方法,包括将自溶素AcmA的编码基因和膜蛋白AM的编码基因导入受体菌中,得到重组菌。
上述方法中,所述受体菌为乳酸乳球菌(Lactococcus lactis);所述乳酸乳球菌为乳酸乳球菌(Lactococcus lactis)ZHY1,于2021年3月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC),其保藏编号为CGMCC No.22024,
上述方法中,所述重组菌与出发菌相比,具有抑制鱼类肝脏脂肪增加、降低鱼类肝脏脂肪含量、减少鱼类肝损伤、抑制鱼类肝脏产生炎症和/或改善鱼类肠道屏障的功能。
上述方法中,所述自溶素AcmA的编码基因可来源于乳酸乳球菌(Lactococcuslactis)ZHY1。
上述方法中,所述usp45信号肽的氨基酸序列如序列表中序列3第1-32位所示;所述自溶素AcmA的氨基酸序列如序列表中序列3第33-468位所示;所述膜蛋白AM的氨基酸序列如序列表中序列3第471-757所示;8×组氨酸标签的氨基酸序列为HHHHHHHH。
相应地,在基因水平上,所述usp45信号肽的编码基因为如下(a1)或(a2)或(a3):
(a1)核苷酸序列如序列表中序列2第9-104位所示的DNA分子;
(a2)在严格条件下与(a1)限定的DNA分子杂交且编码所述usp45信号肽的DNA分子;
(a3)与(a1)或(a2)所限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述usp45信号肽的DNA分子。
相应地,在基因水平上,所述自溶素AcmA的编码基因为如下(b1)或(b2)或(b3):
(b1)核苷酸序列如序列表中序列2第105-1412位所示的DNA分子;
(b2)在严格条件下与(a1)限定的DNA分子杂交且编码所述自溶素AcmA的DNA分子;
(b3)与(a1)或(a2)所限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述自溶素AcmA的DNA分子。
相应地,在基因水平上,膜蛋白AM的编码基因为如下(c1)或(c2)或(c3):
(c1)核苷酸序列如序列表中序列2第1419-2279位所示的DNA分子;
(c2)在严格条件下与(a1)限定的DNA分子杂交且编码所述膜蛋白AM的DNA分子;
(c3)与(a1)或(a2)所限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述膜蛋白AM的DNA分子。
上述方法中,所述自溶素AcmA的编码基因和膜蛋白AM的编码基因是通过重组载体导入所述受体菌中的,所述重组载体含有所述自溶素AcmA的编码基因和所述膜蛋白AM的编码基因的。
上述方法中,所述重组载体含有由usp45信号肽的编码基因、自溶素AcmA的编码基因、膜蛋白AM的编码基因和8×组氨酸标签编码基因连接而成的融合基因。
在本发明的一个实施例中,所述重组载体为重组载体pMG36e-usp45-AcmA-AM。所述重组载体pMG36e-usp45-AcmA-AM为将序列2所示的DNA分子替换质粒pMG36e上SacI和XbaI识别位点之间DNA序列(包括SacI和XbaI识别位点在内的小片段),且保持质粒pMG36e的其他序列不变,得到的重组载体。重组载体pMG36e-usp45-AcmA-AM表达带有HIS标签的AM-AcmA融合蛋白,带有HIS标签的AM-AcmA融合蛋白的氨基酸序列如序列表中序列3所示。
为了解决上述技术问题,本发明还提供了由上述重组菌的构建方法构建的重组菌。
所述重组菌具体可为乳酸乳球菌(Lactococcus lactis)AM-ZHY1,于2021年3月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC),其保藏编号为CGMCC No.22025。
为了解决上述技术问题,本发明还提供了一种具有降鱼类肝脏脂肪含量功能的饲料组合物,包括上述重组菌。所述饲料组合物还包括高脂饲料。
上述饲料组合物中,所述重组菌的含量为107-109CFUs/g;在本发明的一个实施例中具体为108CFUs/g。
为了解决上述技术问题,本发明还提供了上述重组菌或饲料组合物在如下1)-8)中的应用;
1)抑制鱼类肝脏脂肪增加和/或降低鱼类肝脏脂肪含量;
2)制备具有抑制鱼类肝脏脂肪增加和/或降低鱼类肝脏脂肪含量的产品;
3)抑制鱼类肝脏产生炎症;
4)制备具有抑制鱼类肝脏产生炎症的产品;
5)抑制鱼类肝损伤;
6)制备具有鱼类肝损伤的产品;
7)改善鱼类肠道屏障功能;
8)制备具有鱼类改善肠道屏障功能的产品。
上述应用中,所述肝脏脂肪增加为高脂饲料诱导引起的肝脏脂肪增加;所述肝脏产生炎症为高脂饲料诱导引起的肝脏产生炎症;所述肝损伤为高脂饲料诱导引起的肝损伤。
为了解决上述技术问题,本发明还提供下述任一种方法:
M1、在高脂饲料饲喂过程中降低鱼类肝脏脂肪积累的方法,包括在高脂饲料中添加上述重组菌;
M2、在高脂饲料饲喂过程中降低鱼肝脏脂肪积累的方法,包括采用上述饲料组合物饲喂鱼;
M3、在高脂饲料饲喂过程中减少鱼肝脏损伤的方法,包括在高脂饲料中添加上述重组菌;
M4、在高脂饲料饲喂过程中减少鱼肝脏损伤的方法,包括采用上述饲料组合物饲喂鱼;
M5、在高脂饲料饲喂过程中改善鱼肠道屏障功能的方法,包括在高脂饲料中添加上述重组菌;
M6、在高脂饲料饲喂过程中改善鱼肠道屏障功能的方法,包括采用上述饲料组合物饲喂鱼。
上述应用或方法中,所述抑制鱼类体肝脏脂肪增加和/或降低鱼类肝脏脂肪含量主要体现为降低肝脏内甘油三酯的含量,和/或抑制鱼类肝脏脂肪合成相关基因SREBP-1c和/或PPARγ基因的表达,和/或降低鱼类肝脏脂肪吸收相关的基因CD36和/或FABP6基因的表达。
上述应用或方法中,所述抑制鱼类产生炎症体现在抑制鱼体促炎因子基因IL-6和/或TNF-α基因的表达。
上述应用或方法中,所述抑制鱼类肝损伤体现为降低肝脏中甘油三酯的含量,和/或降低血清谷丙转氨酶(ALT)和/或谷草转氨酶(AST)的含量。
上述应用或方法中,所述改善斑马鱼肠道屏障功能体现在提高肠道内紧密连接蛋白基因occludina、claudin7、claudin7b、claudin11a、claudin12和/或claudin15a的表达。
本发明中,所述鱼类可为鲤科鱼类,在本发明的实施例中以斑马鱼为例。
本发明通过在乳酸乳球菌ZHY1中表达的AM-AcmA融合蛋白,构建了一株乳酸乳球菌表面展示菌株AM-ZHY1。并通过实验证明,在高脂饲料中添加108CFUs/g的AM-ZHY1可以显著降低鱼类肝脏脂肪含量、减少肝损伤,保护鱼类肠道屏障,表明乳酸乳球菌AM-ZHY1具有降低肝脏脂肪含量、减少肝损伤,保护鱼类肠道屏障的作用。因此,乳酸乳球菌AM-ZHY1可以作为水产益生菌。
保藏说明
菌种名称:乳酸乳球菌ZHY1
拉丁名:Lactococcus lactis
菌株编号:ZHY1
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
地址:北京市朝阳区北辰西路1号院3号
保藏日期:2021年03月17日
保藏中心登记入册编号:CGMCC No.22024
菌种名称:乳酸乳球菌AM-ZHY1
拉丁名:Lactococcus lactis
菌株编号:AM-ZHY1
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
地址:北京市朝阳区北辰西路1号院3号
保藏日期:2021年03月17日
保藏中心登记入册编号:CGMCC No.22025
附图说明
图1为乳酸乳球菌ZHY1在GM17固体培养基上菌体形态图。
图2为重组质粒pMG36e-usp45-AcmA-AM的质粒图谱。
图3为乳酸乳球菌ZHY1、重组乳酸乳球菌AM-ZHY1与乳酸乳球菌工程菌MG1363的生长曲线的比较结果。
图4为重组乳酸乳球菌AM-ZHY1中融合蛋白AM-AcmA的表达检测结果。
图5为不同饲料组饲喂斑马鱼后肝脏脂肪的积累图;其中,A、B、C和D分别为低脂饲喂组、高脂饲喂组、AM-ZHY1饲喂组和ZHY1饲喂组斑马鱼肝脏内脂肪的积累情况。
图6为不同饲料组饲喂斑马鱼后肝脏中TAG的含量。
图7为不同饲料组饲喂斑马鱼后肝脏中脂代谢相关基因的相对表达量。
图8为不同饲料组饲喂斑马鱼后肝脏中炎症相关基因的相对表达量。
图9为不同饲料组饲喂斑马鱼后斑马鱼血清中ALT和AST的含量。
图10为不同饲料组饲喂斑马鱼后肠道屏障因子基因的相对表达量。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
Tu品系斑马鱼(记载于如下文献中:Loucks E,Carvan III M J.Strain-dependent effects of developmental ethanol exposure inzebrafish.Neurotoxicology and teratology,2004,26(6):745-755.)由北京大学斑马鱼水体实验室提供。
高脂饲料为在基础饲料基础上,通过添加猪油及豆油替代等量糊精构建而成,其中幼鱼的基础饲料和高脂饲料的原料成分、配比和营养水平见表1。
表1 基础饲料和高脂饲料的配方表
注:1.多维(各组分及含量,单位为mg/kg):硫胺素50;维生素E醋酸酯600;维生素C100;维生素D3 0.6;肌醇2000;烟酸200;叶酸15;核黄素50;维生素B6 40;维生素B12 0.1;维生素A醋酸酯8.6;维生素B12 0.1;维生素K3 40;生物素6。2.多矿(各组分及含量,单位为g/kg):K2SO4 13.10;(CH3CHOHCOO)2Ca·5H2O 37.90;NaCl 2.60;KCl 5.30;KI 0.002;CoCl2·6H2O(1%)0.02;CuSO4·5H2O 0.02;FeSO4·H2O 0.90;C6H5FeO7 3.1;ZnSO4·H2O0.04;MnSO4·H2O 0.03;MgSO4·7H2O 3.5;Ca(H2PO4)2·H2O 9.8;纤维素42。
荧光定量PCR分析采用SuperReal PreMix Plus试剂盒(天根公司的产品)进行。
MRS固体培养基:称取MRS琼脂(青岛海博生物技术公司产品,产品目录号为HB0384)66.2g,用去离子水溶解并定容至1L,pH=5.7,115℃高压蒸汽灭菌。GM17液体培养基:称取M17肉汤(青岛海博生物技术公司产品,产品目录号为HB0391)42.3g、葡萄糖5g,用去离子水溶解并定容至1L,pH=7.2,115℃高压蒸汽灭菌。
GM17固体培养基:称取M17肉汤(青岛海博生物技术公司产品,产品目录号为HB0391)42.3g、葡萄糖5g、琼脂粉15g,用去离子水溶解并定容至1L,pH=7.2,115℃高压蒸汽灭菌。
实施例1 乳酸乳球菌ZHY1的获得与鉴定
1、乳酸乳球菌菌株ZHY1的分离纯化
超净工作台中取中华鲟肠道内容物,用浓度为10mM、pH值为7.4的PBS缓冲液稀释,利用MRS固体培养基培养24h,获得的单菌落ZHY1。
2、单菌落ZHY1的鉴定
1)形态鉴定
观察步骤1分离得到的单菌落ZHY1在MRS固体培养基上的生长形态。
结果如图1所示:菌落大小:2-6mm;圆形,湿润,不透明,浅黄色,边缘整齐圆润。
2)16s rRNA基因测序鉴定
将步骤1分离得到的单菌落ZHY1在GM17固体培养基上划线,28℃条件下培养24h左右。无菌条件下,挑取平板上的单菌落于GM17液体培养基中,28℃培养24h。离心收集菌体,提取基因组DNA。送生工生物工程(上海)股份有限公司进行16s rRNA基因测序鉴定。测序结果表明:单菌落ZHY1的16S rRNA基因具有如序列表中序列1所示的核苷酸片段。经比对,菌落ZHY1为乳酸乳球菌。
乳酸乳球菌(Lactococcus lactis)ZHY1于2021年3月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101),其保藏编号为CGMCC No.22024,分类命名为Lactococcus lactis,以下简称乳酸乳球菌ZHY1。
实施例2 乳酸乳球菌表面展示菌株的构建
一、重组质粒pMG36e-usp45-AcmA-AM的构建
人工合成序列表中序列2所示的DNA分子,其编码序列3所示的AM-AcmA融合蛋白。其中,序列2第9-104位为usp45信号肽的编码基因,编码氨基酸序列为序列表中序列3的第1-32位的usp45信号肽;序列2第105-1412位为自溶素AcmA的编码基因,编码氨基酸序列为序列表中序列3的第33-468位的自溶素AcmA;序列2第1419-2279位为膜蛋白AM的编码基因,编码氨基酸序列为序列表中序列3的第471-757位的膜蛋白AM;序列2第2280-2304位为组氨酸标签的编码基因。
将序列2所示的DNA分子替换质粒pMG36e(北京华越洋生物,货号SWGH695)上SacI和XbaI识别位点之间DNA序列(包括SacI和XbaI识别位点在内的小片段),且保持质粒pMG36e的其他序列不变,得到重组质粒pMG36e-usp45-AcmA-AM(质粒图谱参见图2)。
二、重组乳酸乳球菌AM-ZHY1的获得
1、乳酸乳球菌ZHY1感受态细胞的制备
GM17液体培养基中溶解添加甘氨酸和蔗糖,得到G-SGM17培养基。G-SGM17培养基中,甘氨酸的质量百分含量为2.5%,蔗糖的质量百分含量为17.1%。将过夜培养的乳酸乳球菌ZHY1以1%(v/v)接种量接种于50mL G-SGM17培养基中,置于30℃静置培养至OD600 nm吸光值达到0.5-0.7;将菌液转移至无菌的50mL离心管中,4℃,6000×g离心10min收集菌体;弃上清,加入50mL预冷的Washing buffer洗涤菌体,4℃,6000×g离心10min收集菌体,重复一次;弃上清,加入200μL预冷的Washing buffer重悬菌体,每管40μL分装到1.5ml离心管中,置于冰浴中备用或在液氮中速冻后置于-80℃冰箱中保存。
2、重组乳酸乳球菌AM-ZHY1的获得
取5μL质粒pMG36e-usp45-AcmA-AM通过电转化导入乳酸乳球菌ZHY1感受态细胞中。电转仪中设定的电击参数为:2.0kV,25μF,200Ω。电击后立即加入1mL预冷的复苏溶液(含有17.1%蔗糖、0.42%MgCl2和0.02%CaCl2的GM17液体培养基);将电转杯中的培养物转移至1.5mL无菌离心管中,置于30℃预培养2h;室温6000×g离心5min收集菌体,除去部分上清,将复苏菌液混匀后涂布于含有10μg/mL红霉素的GM17固态培养基上,倒置于30℃培养36-48h。筛选得到阳性转化子-将pMG36e-usp45-AcmA-AM转入乳酸乳球菌ZHY1得到的重组乳酸乳球菌,命名为AM-ZHY1。用天根质粒小提中量试剂盒提取重组乳酸乳球菌AM-ZHY1的质粒,送样测序鉴定。结果表明:序列表中序列2所示的DNA分子已导入乳酸乳球菌ZHY1中。
乳酸乳球菌AM-ZHY1(Lactococcus lactis)AM-ZHY1于2021年03月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101),其保藏编号为CGMCC No.22025,分类命名为Lactococcus lactis,以下简称重组乳酸乳球菌AM-ZHY1。
3、比较乳酸乳球菌ZHY1及重组乳酸乳球菌AM-ZHY1与乳酸菌工程菌MG1363的生长优势
将乳酸乳球菌ZHY1及重组乳酸乳球菌AM-ZHY1与乳酸菌工程菌MG1363按照OD600nm=1的接菌量分别接种到10ml的GM17培养基中连续32h测定生长曲线。
结果如图3所示,发现乳酸菌工程菌MG1363的生长速度低于乳酸乳球菌ZHY1和乳酸乳球菌AM-ZHY1。特别是在培养20h后,菌液浓度下降很多。因此,ZHY1在展示外源蛋白时得率较高,优于工程菌MG1363。
三、重组乳酸乳球菌AM-ZHY1中融合蛋白AM-AcmA的表达检测
1、重组乳酸乳球菌AM-ZHY1菌液的制备
将重组乳酸乳球菌AM-ZHY1接种于含有10μg/mL红霉素的GM17液体培养基中,30℃培养48h,得到重组乳酸乳球菌AM-ZHY1菌液。
2、融合蛋白AM-AcmA的表达检测
将重组乳酸乳球菌AM-ZHY1菌液室温6000×g离心5min,收集菌体。将菌体重悬于1ml的50mM Tris-HCl(pH 7.0)中,其中含有10mM MgCl2、4%SDS缓冲液中,37℃孵育2h。裂解后,沸水浴煮5min,3500×g离心15min,收集上清即为裂解后的细胞壁组分。加入5×SDSloading buffer,沸水浴煮5min,待冷却后上样SDS-PAGE,120V恒压电泳2h。
电泳后将SDS-PAGE的蛋白条带转移至PVDF膜上,15V恒压30min。电转完成后,于5%的脱脂奶粉中封闭2h;去除封闭液,加入一抗(按1:5000的比例,稀释于含有0.05%吐温20的封闭液),室温反应1h后置于4℃缓慢摇动过夜;去除一抗,加入HRP标记的二抗(按1:1000的比例,稀释于封闭液),室温下缓慢摇动2h;用Chemiluminescent HRP Substrate显色试剂盒显色。
结果如图4所示:蛋白条带的大小接近于理论分子量82.2kDa。这表明序列2所示的DNA分子能够在乳酸乳球菌ZHY1体内表达AM-AcmA融合蛋白。重组乳酸乳球菌AM-ZHY1为乳酸乳球菌表面展示菌株。
实施例3 在高脂饲料诱导下重组乳酸乳球菌AM-ZHY1在对鱼具有降低肝脏脂肪含量功能的研究
一、重组乳酸乳球菌AM-ZHY1在高脂饲料背景下饲喂斑马鱼
挑取重组乳酸乳球菌AM-ZHY1的单克隆接种于含有10ug/ml红霉素的GM17液体培养基中,同样挑取乳酸乳球菌ZHY1作为对照接种于GM17液体培养基中,培养48h,得到AM-ZHY1菌液(109CFUs/ml)和ZHY1菌液(109CFUs/ml)。
按照表1中的饲料配方依次制备四组饲料:1)基础饲料(为了与高脂饲料形成对比,后简称低脂饲料)、2)高脂饲料、3)高脂饲料+ZHY1组(简称ZHY1组饲料)、4)高脂饲料+AM-ZHY1组(简称AM-ZHY1组饲料)。其中,高脂饲料+ZHY1组为将乳酸乳球菌ZHY1菌液混入高脂饲料中,使乳酸乳球菌ZHY1的含量达到108CFUs/g得到的饲料;高脂饲料+AM-ZHY1组为将实施例2步骤三中培养的重组乳酸乳球菌AM-ZHY1菌液混入高脂饲料中,使重组乳酸乳球菌AM-ZHY1的含量达到108CFUs/g得到的饲料。
乳酸乳球菌ZHY1菌液的制备方法除将实施例2步骤三中重组乳酸乳球菌AM-ZHY1替换为乳酸乳球菌ZHY1外,其它操作均相同。
将1月龄的Tu品系斑马鱼分成四组,分别利用低脂饲料、高脂饲料、ZHY1组饲料和AM-ZHY1组饲料进行投喂。每天投喂饲料的量为鱼体重质量百分比6%(其中ZHY1组饲料和AM-ZHY1组饲料投喂量以高脂饲料的含量为标准进行计算),每天投喂2次,斑马鱼自由采食,养殖周期为4周,分别获得低脂饲喂组(后称低脂饲喂组)、高脂饲喂组(后称高脂饲喂组)、ZHY1饲喂组(后称ZHY1饲喂组)和AM-ZHY1饲喂组(后称AM-ZHY1饲喂组)斑马鱼。
二、重组乳酸乳球菌AM-ZHY1抑制高脂饲料诱导的斑马鱼肝脏脂肪的积累
1、表型观察
分别取低脂饲喂组、高脂饲喂组、ZHY1饲喂组和AM-ZHY1饲喂组斑马鱼的肝脏,使用4%的多聚甲醛固定,置于4℃冰箱中12h以上;依次使用二甲苯、95%乙醇、95%乙醇、80%乙醇以及蒸馏水清洗,再使用苏木精-伊红(hematoxylin-eosin staining,H&E)染色,然后依次用使用蒸馏水、80%乙醇、95%乙醇、95%乙醇、二甲苯进行清洗,中性树胶封固。显微镜(Leica DMIL-LED,Germany)下观察并拍照。肝脏中的甘油三酯(TAG)水平可以间接显示。
结果(如图5所示),在同是高脂饲料饲喂斑马鱼的背景下,与高脂饲喂组和ZHY1饲喂组相比,AM-ZHY1饲喂组的斑马鱼肝脏内脂肪积累含量显著下降。
2、斑马鱼肝脏TAG的检测
分别取低脂饲喂组、高脂饲喂组、ZHY1饲喂组和AM-ZHY1饲喂组斑马鱼的肝脏,每3条斑马鱼混合成一个样,每组6个重复,每个重复约5-10mg左右的组织,然后迅速加入有机溶剂混合液(氯仿甲醇=2:1),并同时震荡,1000rpm离心10min使之堆积。取出下层的含脂溶液在70℃加热下用氮气吹干,加入5%Triton X 100的氯仿1ml,轻轻震荡混匀,继续用70℃加热下用氮气吹干,然后加入200μL的超纯水,震荡,获得组织样品。组织TAG含量以Sigma公司的甘油标准品溶液(货号:G7793)为标准品,用Sigma公司的游离甘油试剂(货号:F6428)和甘油三酯试剂(货号:T2449)进行测定,测定方法按照说明书进行。以每mg组织样品的重量所含的TAG的量表示各组样品TAG的含量。
结果(如图6所示)表明,在同是高脂饲料饲喂斑马鱼的背景下,与高脂饲喂组和ZHY1饲喂组相比,AM-ZHY1饲喂组斑马鱼肝脏中的TAG含量明显降低,与低脂饲喂组斑马鱼肝脏中的TAG含量无显著差异。这说明AM-ZHY1组饲料可显著抑制高脂饲料诱导鱼肝脏TAG的积累。
3、脂代谢相关基因表达水平检测
分别取低脂饲喂组、高脂饲喂组、ZHY1饲喂组和AM-ZHY1饲喂组斑马鱼的肝脏,每3条斑马鱼混合成一个样,每组6个重复,提取RNA,反转录,得到各组斑马鱼的cDNA;以cDNA作为模板,采用荧光定量PCR检测斑马鱼cDNA中脂代谢相关基因PPARγ、SREBP-1c、UCP2、CD36和FABP6的表达量,以rps11作为内参基因。其中,PPARγ基因(GenBank:NM_131467.1,2021-02-28)、SREBP-1c基因(GenBank:NM_001105129.1)均为脂合成相关基因;UCP2基因(GenBank:XM_009305562.3,2017-06-15)为脂分解相关基因;CD36基因(GenBank:NM_001002363,2021-02-28)、FABP6基因(GenBank:EU665314,2008-06-18)均为脂吸收相关基因。
表2 荧光定量PCR检测脂代谢相关基因所用引物
结果如图7所示,在同是高脂饲料饲喂斑马鱼的背景下,与高脂饲喂组和ZHY1饲喂组相比,AM-ZHY1饲喂组斑马鱼肝脏内脂合成相关基因PPARγ、SREBP-1c与脂肪吸收相关基因CD36、FABP6的表达量均显著下降;而脂肪分解相关的基因UCP2表达量无显著性变化。这表明重组乳酸乳球菌AM-ZHY1能够显著抑制高脂饲料诱导鱼肝脏内脂肪合成相关基因PPARγ、SREBP-1c的表达,且降低了高脂饲料诱导鱼肝脏内脂肪吸收相关的基因CD36、FABP6的表达,降低鱼肝脏对饲料脂肪的吸收。但对脂肪分解相关基因UCP2的表达没有显著性影响。
由此可见,投喂高脂饲料时,重组乳酸乳球菌AM-ZHY1具有降低鱼类肝脏脂肪合成、降低肝脏脂肪吸收的作用。
实施例4 重组乳酸乳球菌AM-ZHY1降低高脂饲料诱导斑马鱼炎症
分别取实施例2中低脂饲喂组、高脂饲喂组、ZHY1饲喂组和AM-ZHY1饲喂组斑马鱼的肝脏,每3条斑马鱼混合成一个样,每组6个重复,提取RNA,反转录,得到各组斑马鱼的cDNA;以cDNA作为模板,采用荧光定量PCR分析斑马鱼cDNA中炎症相关因子IL-6(GenBank:NM_001261449.1,2019-12-21)和TNF-α(GenBank:NM_212859.2,2019-12-21)的表达量,以rps11作为内参基因(检测引物见表2)。
表3 荧光定量PCR检测炎症相关基因所用引物
结果如图8所示,在同是高脂饲料饲喂斑马鱼的背景下,与高脂饲喂组和ZHY1饲喂组相比,AM-ZHY1饲喂组斑马鱼肝脏内炎症相关基因IL-6和TNF-α的表达量均显著下降。这表明重组乳酸乳球菌AM-ZHY1能够显著抑制了高脂饲料诱导鱼体内炎症相关基因IL-6和TNF-α的表达。由此可见,投喂高脂饲料时,重组乳酸乳球菌AM-ZHY1菌可以降低高脂饲料诱导斑马鱼肝脏的炎症。
实施例5 重组乳酸乳球菌AM-ZHY1减少高脂饲料诱导的肝损伤
分别取实施例2中低脂饲喂组、高脂饲喂组、ZHY1饲喂组和AM-ZHY1饲喂组斑马鱼的血清,检测血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)的含量。检测方法按照南京建成AST、ALT说明书进行(货号:C010-2-1,C009-2-1)。
结果如图9所示,在同是高脂饲料饲喂斑马鱼的背景下,与高脂饲喂组和ZHY1饲喂组相比,AM-ZHY1饲喂组斑马鱼血清中ALT、AST的含量均显著下降。这表明重组乳酸乳球菌AM-ZHY1能够显著降低高脂饲料诱导鱼血清中ALT、AST的含量。由此可见,投喂高脂饲料时,重组乳酸乳球菌AM-ZHY1菌可以降低高脂饲料诱导的斑马鱼肝脏损伤。
实施例6 在高脂饲料诱导下AM-ZHY1在对鱼具有保护肠道屏障功能的研究
分别取实施例2中低脂饲喂组、高脂饲喂组、ZHY1饲喂组和AM-ZHY1饲喂组斑马鱼的肠道,每3条斑马鱼混合成一个样,每组6个重复,提取RNA,反转录,得到各组斑马鱼的肠道cDNA;以肠道cDNA作为模板,采用荧光定量PCR检测斑马鱼肠道内紧密连接蛋白基因occludina(GenBank:NM_212832,2021.02.20)、claudin7(GenBank:AF260240.1,2002.06.21)、claudin7b(GenBank:NM_001002340.1,2017.08.23)、claudin11a(GenBank:NM_001002624.1,2020.04.09)、claudin12(GenBank:NM_131773.2,2020.10.12)和claudin15a(GenBank:NM_200404.1,2021.06.17)的表达量,以rps11作为内参基因(检测引物见表2)。
表4 荧光定量PCR检测紧密连接蛋白基因所用引物
结果如图10所示,在同是高脂饲料饲喂斑马鱼的背景下,与ZHY1饲喂组相比,AM-ZHY1饲喂组斑马鱼肠道内紧密连接蛋白基因occludina、claudin7、claudin7b、claudin11a、claudin12和claudin15a的表达量显著升高。这表明重组乳酸乳球菌AM-ZHY1能够显著提高鱼体肠道内紧密连接蛋白基因的表达。由此可见,投喂高脂饲料时,重组乳酸乳球菌AM-ZHY1可以保护肠道屏障。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
序列表
<110> 中国农业科学院饲料研究所
<120> 一种构建具有降低肝脏脂肪积累且保护肠道屏障功能的基因工程菌的方法及应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1418
<212> DNA
<213> 乳酸乳球菌(Lactococcus lactis)
<400> 1
agttgagcgc tgaaggttgg tacttgtacc gactggatga gcagcgaacg ggtgagtaac 60
gcgtggggaa tctgcctttg agcgggggac aacatttgga aacgaatgct aataccgcat 120
aacaacttta aacacaagtt ttaagtttga aagatgcaat tgcatcactc aaagatgatc 180
ccgcgttgta ttagctagtt ggtgaggtaa aggctnacca aggcgatgat acatagccga 240
cctgagaggg tgatcggcca cattgggact gagacacggc ccaaactcct acgggaggca 300
gcagtaggga atcttcggca atggacgaaa gtctgaccga gcaacgccgc gtgagtgaag 360
aaggttttcg gatcgtaaaa ctctgttggt agagaagaac gttggtgaga gtggaaagct 420
catcaagtga cggtaactac ccagaaaggg acggctaact acgtgccagc agccgcggta 480
atacgtaggt cccgagcgtt gtccggattt attgggcgta aagcgagcgc aggtggttta 540
ttaagtctgg tgtaaaaggc agtggctcaa ccattgtatg cattggaaac tggtagactt 600
gagtgcagga gaggagagtg gaattccatg tgtagcggtg aaatgcgtag atatatggag 660
gaacaccggt ggcgaaagcg gctctctggc ctgtaactga cactgaggct cgaaagcgtg 720
gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag tgctagatgt 780
agggagctat aagttctctg tatcgcagct aacgcaataa gcactccgcc tggggagtac 840
gaccgcaagg ttgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 900
gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatactcgt gctattccta 960
gagataggaa gttccttcgg gacacgggat acaggtggtg catggttgtc gtcagctcgt 1020
gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cctattgtta gttgccatca 1080
ttaagttggg cactctaacg agactgccgg tgataaaccg gaggaaggtg gggatgacgt 1140
caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga 1200
gtcgcgagac agtgatgttt agctaatctc ttaaaaccat tctcagttcg gattgtaggc 1260
tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcac gccgcggtga 1320
atacgttccc gggccttgta cacaccgccc gtcacaccac gggagttggg agtacccgaa 1380
gtaggttgcc taaccgcaag gagggcgctc ctaagtag 1418
<210> 2
<211> 2312
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gagctcatat gaaaaaaaag attatctcag ctattttaat gtctacagtg atactttctg 60
ctgcagcccc gttgtcaggt gtttacgctg acacaaactc agatccagta tcacgtgtta 120
aagttaaaaa tagacattta aaaaagaaaa ctaaaaaacc actcgctttt tataaaccaa 180
ccacaaaatt tgttggcgct gttcttattg ccggaacatt gacaaccaca catgaacttc 240
ttctgcaaca gacaagtcca atggttcaag cagcaactaa ctcatcagag gcttttattg 300
aaagtattgc cgcatcagca aaacctgtgg cagatgctaa tggcttatat ccttcggtca 360
tgattgccca agctattttg gaaagcaact ggggctcaag tcagctttca cgagcacctt 420
attataattt atttggtatt caaggcactt atcaaggaaa gagtgtcgta tttaaaactc 480
aagagtatct caatggtaaa tgggtgacta aagatatgcc ctttagggtc tatccttcct 540
ttaatcaaag ttttcaagac aatacttatg tcctaaaaac aacaaacttt gggaatggtc 600
cctattacgc taaggcttgg cgggctaatg ctgccaccta tcaagacgct actgctgctt 660
tgacgggcaa atatgctacc gacccaagtt atggcgcttc actaaatcgc attatttctc 720
aatataattt gactcgtttt gacggagctt cttcagctgg aaatactaat tctggtggct 780
cgacaaccac aaatacgaat aataattctg gaaccaatag cagttcaact acttataccg 840
tcaaatctgg tgatactctt tggggaattt cacaaagata tggaattagt gtcgctcaaa 900
ttcaaagtgc gaataatctt aaaagtacca ttatctacat tggtcaaaaa cttttactga 960
caggttcagc ttcttctaca aattcaggtg gttcaaacaa ttccgcaagc actactccaa 1020
ccacttctgt gacacctgca aagccagctt cacaaacatc tgttaaggtt aagtccggag 1080
ataccctttg ggcgctatca gtaaaatata aaactagtat tgctcaattg aaaagttgga 1140
atcatttaag ttctgatacc atttatattg gtcaaaatct tattgtttca caatctgctg 1200
ctacttcaaa tccttcgaca ggttcaggct caactgctac caataactca aactcgactt 1260
cttctaactc aaatgcctca attcataagg tcgttaaagg agatactctc tggggacttt 1320
cgcaaaaatc tggcagccca attgcttcaa tcaaggcttg gaatcattta tctagcgata 1380
ctattttaat tggtcagtat ctgcgaataa aactcgagat cgtcaattcc aaacgcagtg 1440
aactggacaa aaaaatcagc atcgccgcca aggaaatcaa gtccgccaat gctgcggaaa 1500
tcactccgag ccgatcatcc aacgaagagc tggaaaaaga actgaaccgc tatgccaagg 1560
ccgtgggcag cctggaaacg gcctacaagc ccttccttgc ctcctccgcg ctggtcccca 1620
ccacgcccac ggcattccag aatgaactga aaacattcag ggattccctg atctcctcct 1680
gcaagaaaaa gaacattctc ataacggaca catcctcctg gctcggtttc caggtttaca 1740
gcacccaggc tccctctgtt caggcggcct ccacgctggg ttttgaattg aaagccatca 1800
acagcctggt caacaaactg gcggaatgcg gcctgtccaa attcatcaag gtgtaccgcc 1860
cccagctccc cattgaaacc ccggcgaaca atccggaaga atcggacgaa gccgaccagg 1920
ccccatggac tcccatgcct ctggaaatag ccttccaggg cgaccgggaa agtgtattga 1980
aagccatgaa cgccataacc ggcatgcagg actatctgtt cacggtcaac tccatccgta 2040
tccgcaacga acggatgatg ccccctccca tcgccaatcc ggcagccgcc aaacctgccg 2100
cggcccaacc cgccacgggt gcggcttccc tgactccggc ggatgaggcg gctgcacctg 2160
cagccccggc catccagcaa gtcatcaagc cttacatggg caaggagcag gtctttgtcc 2220
aggtctccct gaatctggtc cacttcaacc agcccaaggc tcaggaaccg tctgaagatc 2280
atcatcacca tcaccatcac cattaatcta ga 2312
<210> 3
<211> 765
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Lys Lys Lys Ile Ile Ser Ala Ile Leu Met Ser Thr Val Ile Leu
1 5 10 15
Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala Asp Thr Asn Ser Asp
20 25 30
Pro Val Ser Arg Val Lys Val Lys Asn Arg His Leu Lys Lys Lys Thr
35 40 45
Lys Lys Pro Leu Ala Phe Tyr Lys Pro Thr Thr Lys Phe Val Gly Ala
50 55 60
Val Leu Ile Ala Gly Thr Leu Thr Thr Thr His Glu Leu Leu Leu Gln
65 70 75 80
Gln Thr Ser Pro Met Val Gln Ala Ala Thr Asn Ser Ser Glu Ala Phe
85 90 95
Ile Glu Ser Ile Ala Ala Ser Ala Lys Pro Val Ala Asp Ala Asn Gly
100 105 110
Leu Tyr Pro Ser Val Met Ile Ala Gln Ala Ile Leu Glu Ser Asn Trp
115 120 125
Gly Ser Ser Gln Leu Ser Arg Ala Pro Tyr Tyr Asn Leu Phe Gly Ile
130 135 140
Gln Gly Thr Tyr Gln Gly Lys Ser Val Val Phe Lys Thr Gln Glu Tyr
145 150 155 160
Leu Asn Gly Lys Trp Val Thr Lys Asp Met Pro Phe Arg Val Tyr Pro
165 170 175
Ser Phe Asn Gln Ser Phe Gln Asp Asn Thr Tyr Val Leu Lys Thr Thr
180 185 190
Asn Phe Gly Asn Gly Pro Tyr Tyr Ala Lys Ala Trp Arg Ala Asn Ala
195 200 205
Ala Thr Tyr Gln Asp Ala Thr Ala Ala Leu Thr Gly Lys Tyr Ala Thr
210 215 220
Asp Pro Ser Tyr Gly Ala Ser Leu Asn Arg Ile Ile Ser Gln Tyr Asn
225 230 235 240
Leu Thr Arg Phe Asp Gly Ala Ser Ser Ala Gly Asn Thr Asn Ser Gly
245 250 255
Gly Ser Thr Thr Thr Asn Thr Asn Asn Asn Ser Gly Thr Asn Ser Ser
260 265 270
Ser Thr Thr Tyr Thr Val Lys Ser Gly Asp Thr Leu Trp Gly Ile Ser
275 280 285
Gln Arg Tyr Gly Ile Ser Val Ala Gln Ile Gln Ser Ala Asn Asn Leu
290 295 300
Lys Ser Thr Ile Ile Tyr Ile Gly Gln Lys Leu Leu Leu Thr Gly Ser
305 310 315 320
Ala Ser Ser Thr Asn Ser Gly Gly Ser Asn Asn Ser Ala Ser Thr Thr
325 330 335
Pro Thr Thr Ser Val Thr Pro Ala Lys Pro Ala Ser Gln Thr Ser Val
340 345 350
Lys Val Lys Ser Gly Asp Thr Leu Trp Ala Leu Ser Val Lys Tyr Lys
355 360 365
Thr Ser Ile Ala Gln Leu Lys Ser Trp Asn His Leu Ser Ser Asp Thr
370 375 380
Ile Tyr Ile Gly Gln Asn Leu Ile Val Ser Gln Ser Ala Ala Thr Ser
385 390 395 400
Asn Pro Ser Thr Gly Ser Gly Ser Thr Ala Thr Asn Asn Ser Asn Ser
405 410 415
Thr Ser Ser Asn Ser Asn Ala Ser Ile His Lys Val Val Lys Gly Asp
420 425 430
Thr Leu Trp Gly Leu Ser Gln Lys Ser Gly Ser Pro Ile Ala Ser Ile
435 440 445
Lys Ala Trp Asn His Leu Ser Ser Asp Thr Ile Leu Ile Gly Gln Tyr
450 455 460
Leu Arg Ile Lys Leu Glu Ile Val Asn Ser Lys Arg Ser Glu Leu Asp
465 470 475 480
Lys Lys Ile Ser Ile Ala Ala Lys Glu Ile Lys Ser Ala Asn Ala Ala
485 490 495
Glu Ile Thr Pro Ser Arg Ser Ser Asn Glu Glu Leu Glu Lys Glu Leu
500 505 510
Asn Arg Tyr Ala Lys Ala Val Gly Ser Leu Glu Thr Ala Tyr Lys Pro
515 520 525
Phe Leu Ala Ser Ser Ala Leu Val Pro Thr Thr Pro Thr Ala Phe Gln
530 535 540
Asn Glu Leu Lys Thr Phe Arg Asp Ser Leu Ile Ser Ser Cys Lys Lys
545 550 555 560
Lys Asn Ile Leu Ile Thr Asp Thr Ser Ser Trp Leu Gly Phe Gln Val
565 570 575
Tyr Ser Thr Gln Ala Pro Ser Val Gln Ala Ala Ser Thr Leu Gly Phe
580 585 590
Glu Leu Lys Ala Ile Asn Ser Leu Val Asn Lys Leu Ala Glu Cys Gly
595 600 605
Leu Ser Lys Phe Ile Lys Val Tyr Arg Pro Gln Leu Pro Ile Glu Thr
610 615 620
Pro Ala Asn Asn Pro Glu Glu Ser Asp Glu Ala Asp Gln Ala Pro Trp
625 630 635 640
Thr Pro Met Pro Leu Glu Ile Ala Phe Gln Gly Asp Arg Glu Ser Val
645 650 655
Leu Lys Ala Met Asn Ala Ile Thr Gly Met Gln Asp Tyr Leu Phe Thr
660 665 670
Val Asn Ser Ile Arg Ile Arg Asn Glu Arg Met Met Pro Pro Pro Ile
675 680 685
Ala Asn Pro Ala Ala Ala Lys Pro Ala Ala Ala Gln Pro Ala Thr Gly
690 695 700
Ala Ala Ser Leu Thr Pro Ala Asp Glu Ala Ala Ala Pro Ala Ala Pro
705 710 715 720
Ala Ile Gln Gln Val Ile Lys Pro Tyr Met Gly Lys Glu Gln Val Phe
725 730 735
Val Gln Val Ser Leu Asn Leu Val His Phe Asn Gln Pro Lys Ala Gln
740 745 750
Glu Pro Ser Glu Asp His His His His His His His His
755 760 765
Claims (9)
1.重组菌的构建方法,包括将自溶素AcmA的编码基因和膜蛋白AM的编码基因导入受体菌中,得到重组菌;所述受体菌为乳酸乳球菌(Lactococcus lactis);
所述乳酸乳球菌为乳酸乳球菌(Lactococcus lactis)ZHY1,其保藏编号为CGMCCNo.22024。
2.根据权利要求1所述的构建方法,其特征在于:所述自溶素AcmA的编码基因和膜蛋白AM的编码基因是通过重组载体导入所述受体菌中的,所述重组载体含有所述自溶素AcmA的编码基因和所述膜蛋白AM的编码基因的。
3.根据权利要求1或2所述的构建方法,其特征在于:所述重组载体含有由usp45信号肽的编码基因、自溶素AcmA的编码基因、膜蛋白AM的编码基因和8×组氨酸标签编码基因连接而成的融合基因。
4.根据权利要求3所述的构建方法,其特征在于:所述usp45信号肽的氨基酸序列如序列表中序列3第1-32位所示;
所述自溶素AcmA的氨基酸序列如序列表中序列3第33-468位所示;
所述膜蛋白AM的氨基酸序列如序列表中序列3第471-757所示。
5.由权利要求1-4任一所述的构建方法构建的重组菌。
6.一种重组菌,为重组乳酸乳球菌AM-ZHY1,其保藏编号为CGMCC No.22025。
7.一种具有降低肝脏脂肪含量功能的饲料组合物,其特征在于:所述饲料组合物包括权利要求5或6所述的重组菌。
8.权利要求5或6所述的重组菌或者权利要求7所述的饲料组合物在如下1)-4)中的应用;
1)制备具有抑制鱼类肝脏脂肪增加和/或降低鱼类肝脏脂肪含量的产品;
2)制备具有抑制鱼类肝脏产生炎症的产品;
3)制备具有抑制鱼类肝损伤的产品;
4)制备具有改善鱼类肠道屏障功能的产品。
9.方法,其特征在于:所述方法为下述任一种:
M1、在高脂饲料饲喂过程中降低鱼类肝脏脂肪积累的方法,包括在高脂饲料中添加权利要求5或6所述的重组菌;
M2、在高脂饲料饲喂过程中降低鱼类肝脏脂肪积累的方法,包括采用要求7所述的饲料组合物饲喂鱼;
M3、在高脂饲料饲喂过程中改善鱼类肠道屏障功能的方法,包括在高脂饲料中添加权利要求5或6所述的重组菌;
M4、在高脂饲料饲喂过程中改善鱼类肠道屏障功能的方法,包括采用要求7所述的饲料组合物饲喂鱼;
所述方法为非疾病诊断和治疗目的的方法。
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