CN114875036B - 黄河鲤抗菌肽hepcidin基因酵母表达产物及其应用 - Google Patents
黄河鲤抗菌肽hepcidin基因酵母表达产物及其应用 Download PDFInfo
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Abstract
本发明公开了一种黄河鲤抗菌肽hepcidin基因酵母表达产物及其应用，克隆黄河鲤抗菌肽hepcidin基因全长cDNA序列，扩增出基因片段，构建到毕赤酵母表达载体pPICZα A，得到重组表达载体，重组载体电转至毕赤酵母X‑33后，通过筛选获得重组毕赤酵母菌株，使用甲醇的培养基诱导表达，成功表达出重组Hepcidin蛋白，重组Hepcidin蛋白对嗜水气单胞菌、大肠杆菌、鳗弧菌、枯草芽孢杆菌均具有较好的抑菌活性。在黄河鲤基础饲料中添加重组Hepcidin蛋白，饲养周期为28d后，可以显著提高黄河鲤感染嗜水气单胞菌后的存活率。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种黄河鲤抗菌肽hepcidin基因酵母表达产物及其应用。
背景技术
鱼类抗菌肽Hepcidin，是一种具有抗菌活性的防御素类的小肽，还可参与调节铁代谢。在近20年，Hepcidin在硬骨鱼类中的基因序列、分子结构和生物学功能的研究已被广泛报道。Shike等研究了Hepcidin在几种不同物种中的序列和分子结构，发现Hepcidin的基因组结构在硬骨鱼中高度保守，包含三个由两个内含子分隔的外显子。这三个外显子编码高度保守的信号肽、前体肽和成熟肽组成的前肽。信号肽含有24个氨基酸，具有抗菌功能。不同物种的Hepcidin蛋白具有高度保守的基序，在前体肽含有典型的RX（K/R）R序列，用于招募前肽转化酶形成由19-31个氨基酸组成的成熟肽。从鱼类到哺乳动物的Hepcidin成熟肽序列显示，在成熟肽中发现的8个半胱氨酸残基具有高度保守性，这些半胱氨酸残基通常形成四个分子内二硫键，其中一个相邻的二硫键桥形成发卡结构有助于Hepcidin折叠成亲水和疏水侧链，是Hepcidin发挥抗菌活性的关键。
鱼类Hepcidin既是一种铁稳态的调节剂，又是一种抗菌肽，可以通过调节铁代谢来发挥其抗菌功能。黄颡鱼Hepcidin的表达上调，能降低血清铁水平来抵抗维氏气单胞菌的感染。草鱼Hepcidin可以通过细胞铁转运蛋白的内化和降解阻止肝细胞系的铁释放，并抑制细胞外细菌对铁的使用，以抑制细菌生长。另外，在网箱养殖条件下，饲粮中添加不同剂量的草鱼重组Hepcidin，能够控制鱼体内铁的分布，从而防止细菌感染，具有保护作用。
鱼类Hepcidin除了通过调节铁代谢抑制细菌生长外，还具有直接杀伤细菌的作用。鱼类Hepcidin在体外对病原菌具有广泛的杀菌活性，如源自斑马鱼的Hepcidin-2具有直接杀菌活性，能使大肠杆菌和金黄色葡萄球菌的细胞表面破裂和细胞质变薄、透明。Huang等观察到棕色鳟鱼Hepcidin处理的嗜水气单胞菌细胞起皱并被破坏，这表明Hepcidin能破坏病原菌的细胞膜。大黄鱼Hepcidin处理的谷氨酸棒状杆菌和美人鱼发光杆菌菌体表面变得粗糙，并在处理2小时后形成蜂窝状结构，表明Hepcidin对细菌具有杀菌作用。在海鲈中，已经证明Hepcidin通过与某些细胞内靶点结合或与细菌膜结合而对鳗鲡弧菌的复制产生影响。鳟鱼中，Hepcidin通过其N-末端的ATCUN金属结合基序促进细菌DNA水解。研究还证明鱼类Hepcidin在体内具有抗菌活性。与未经处理的受感染的对照鱼相比，事先在体内注射合成的大菱鲆Hepcidin蛋白，可以大大降低受感染鱼肾脏、脾脏和肝脏中的细菌数量。
鱼类Hepcidin还可以作为有效的免疫调节剂，诱导免疫相关因子形成，从而调节炎症反应。里海鳟鱼Hepcidin1通过诱导原代细胞中促炎细胞因子IL-6、TNF-α和MHC基因的表达来发挥免疫调节功能。在创伤弧菌感染转基因斑马鱼后，Hepcidin诱导IL-10、IL-26、溶菌酶、TLR-4和Myd88的表达，从而有效抑制细菌生长。通过中华乌塘鳢对进行腹腔注射100μg合成的Hepcidin成熟肽，可以提高中华乌塘鳢感染副溶血性弧菌的存活率。
毕赤酵母真核表达系统能够进行许多真核生物翻译并修饰。Hepcidin含有4对二硫键，其特殊的结构使其很难以正确的折叠构型合成，采用毕赤酵母真核表达系统更有利于二硫键桥的形成，更容易获得大量具有活性的Hepcidin蛋白。由毕赤酵母真核表达的松江鲈Hepcidin蛋白具有模式识别功能和对多种细菌的凝集活性。毕赤酵母真核表达系统易于从摇瓶培养扩展到高密度发酵罐培养，因此更适合应用到工业上生产Hepcidin蛋白。因此我们选用了毕赤酵母表达系统来进行黄河鲤抗菌肽Hepcidin基因的重组表达，并研究了重组Hepcidin蛋白的抗菌活性以及作为饲料添加剂对鱼类抗病能力的增强效果，为Hepcidin重组蛋白应用于生产实践奠定基础。
发明内容
本发明的目的是提供了一种黄河鲤抗菌肽Hepcidin基因酵母表达产物及其应用，该黄河鲤抗菌肽Hepcidin基因酵母表达产物对嗜水气单胞菌、大肠杆菌、鳗弧菌、枯草芽孢杆菌有明显的抗菌功能，并且以120mg/kg的剂量添加到饲料中投喂，能显著提高黄河鲤感染嗜水气单胞菌后的存活率。
本发明为实现上述目的采用如下技术方案，黄河鲤抗菌肽Hepcidin基因，该黄河鲤抗菌肽Hepcidin基因的碱基序列如序列表SEQ ID NO.1所示。
本发明所述黄河鲤抗菌肽Hepcidin基因酵母表达产物，其特征在于：该黄河鲤抗菌肽Hepcidin基因酵母表达产物的氨基酸序列如序列表SEQ ID NO.2所示。
进一步限定，该黄河鲤抗菌肽Hepcidin基因酵母表达产物为分子量10kDa的黄河鲤抗菌肽Hepcidin重组蛋白。
本发明所述黄河鲤抗菌肽Hepcidin基因酵母表达产物的制备方法，其特征在于具体步骤为：
步骤S1：表达质粒pPICZα A-cchepcidin的构建，以黄河鲤hepcidin全长cDNA序列为模板，扩增其43位至第315位的共273bp的开放阅读框片段，并在片段两端引入酶切位点Xho I和Xba I，通过双酶切和T4连接酶将该片段构建到pPICZα A载体，重组载体经Sac Ⅰ酶线性化以后，电转化毕赤酵母X-33，用含100μg/mL Zeocin的YPD平板筛选转化成功的菌体即重组酵母菌株，并且提取菌体DNA，通过PCR鉴定，确定Hepcidin基因已成功构建到pPICZαA载体。pPICZα A载体单独转染毕赤酵母X-33作为对照菌株。
步骤S2：表达产物的诱导和浓度测定：重组酵母菌株接种于含100μg/mL ZeocinYPD液体培养基中活化，30℃震荡培养，而后接种到BMGY培养基中，培养至OD600至2.0左右，4℃、6000g离心7min，将菌体转入BMMY培养基中，30℃震荡培养；每隔24h添加甲醇诱导表达，且取样SDS-PAGE检测是否表达，直到其达到最大表达量，将诱导表达好的菌液12000g，4℃离心，将表达上清液过0.45μm滤膜后用切向回流超滤器（赛多利斯VIVAFLOW 200）浓缩培养液上清。用同样的方法浓缩pPICZα A空载体毕赤酵母菌表达上清。用BCA蛋白浓度试剂盒测定重组毕赤酵母X-33/pPICZα A-ccHepcidin发酵上清总蛋白浓度。将上述浓缩的表达上清取样进行SDS-PAGE分析，并通过Quantity one软件进行蛋白灰度分析定量目的蛋白Hepcidin占发酵上清中总蛋白的百分比。
水气单胞菌、鳗弧菌、枯草芽孢杆菌和大肠杆菌培养至OD600为0.5，分别取50μL涂布于LB固体平板上，放入牛津杯于LB固体平板上，将浓缩20倍的pPICZα A-ccHepcidin毕赤酵母培养液上清100μL加入牛津杯，过夜培养，对水气单胞菌、鳗弧菌、枯草芽孢杆菌和大肠杆菌具有抑制作用。
本发明所述的黄河鲤抗菌肽Hepcidin基因酵母表达产物的应用，其特征在于在基础饲料中添加 120mg/kg黄河鲤抗菌肽Hepcidin重组蛋白，饲养周期为28d后，可以显著提高黄河鲤感染嗜水气单胞菌后的存活率，有效提高了黄河鲤抗细菌感染的能力，从而可以用于预防水产养殖中因细菌感染引起的黄河鲤疾病。
本发明具有以下优点和有益效果：
1、本发明的酵母表达产物来自于黄河鲤抗菌肽Hepcidin基因，该重组蛋白具有抗菌活性，与传统使用的抗生素和人工合成的化学抗菌药相比，无环境污染性，且不会使细菌产生耐药性；
2、本发明所使用的毕赤酵母真核表达系统更有利于蛋白质中二硫键桥的形成，产生出来的Hepcidin重组蛋白质活性高；
3、本发明产生Hepcidin重组蛋白在酵母培养上清中得到了分泌型表达，无需进行蛋白纯化，经浓缩后即可以添加到饲料中，生产成本低，使用方便，可实现规模化生产应用。
附图说明
图1为黄河鲤Hepcidin基因ORF片段PCR 扩增图，M：Marker，泳道1：Hepcidin PCR产物。
图2为pPICZα A-ccHepcidin重组毕赤酵母菌株的表达产物，M：超低分子量蛋白Maker；1-2：pPICZα A-ccHepcidin毕赤酵母转化子诱导表达产物。
图3为重组Hepcidin对嗜水气单胞菌、大肠杆菌、枯草芽孢杆菌、鳗弧菌的抑菌活性鉴定；（A）嗜水气单胞菌，（B）大肠杆菌，（C）枯草芽孢杆菌，（D）鳗弧菌；1：氨苄青霉素10μg；2：毕赤酵母X-33/pPICZα 诱导表达产物；3：毕赤酵母X-33/pPICZα A-ccHepcidin诱导表达产物。
图4为肝脏、脾脏、肾脏和肠道中的细菌载量，结果以平均值±标准差(n=3)表示，用*标记时，有显著差异（P<0.05）；用**标记时，有极显著差异（P<0.01）。
图5为 黄河鲤不同预处理组感染嗜水气单胞菌的存活率，各组重组蛋白添加量为，H1组：120mg/kg Hepcidin重组蛋白，H2组：60mg/kg Hepcidin重组蛋白，H3组：30mg/kgHepcidin重组蛋白，CT组：120mg/kg对照菌株表达蛋白。通过Log-rank检验分析各预处理组的存活率。“*”表示差异显著（P <0.05）。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明，此处所描述的具体方式仅用于解释相关内容，并非对本发明的限定。
实施例
黄河鲤抗菌肽Hepcidin基因酵母表达产物的制备
1、表达质粒pPICZα A-ccHepcidin的构建
黄河鲤购买自河南省新乡市一养殖基地。从肝脏组织中提取RNA，用Oligo dT引物逆转录为cDNA。以cDNA为模板，用引物Hepcidin-F：5’- CCGCTCGAGAAAAGAATGAAGTTCTCACGTGGG-3’和Hepcidin-R：5’-GCTCTAGATCAATGATGATGATGATGATGGAATTTGCAGCAGTA-3’进行PCR扩增。PCR反应条件：95℃预变性5min；95℃ 30s，55℃ 30s，72℃ 60s，共35个循环；72℃终延伸10min。扩增得到一约270bp的片段（图1），与预期相符。PCR产物纯化后用Xho I和Xba I双酶切，pPICZα A载体也同时用这两种酶双酶切，PCR产物和载体双酶切后的片段进行回收，用T4 DNA连接酶连接，连接产物转化大肠杆菌DH5α，涂布于含25μg/mL Zeocin低盐LB平板，过夜培养后挑取单克隆菌落扩大培养后，PCR检测，筛选阳性克隆，阳性克隆送测序验证。阳性克隆菌液用内毒素质粒小提试剂盒按说明书提取重组表达质粒pPICZα A-ccHepcidin。
2、黄河鲤Hepcidin基因在毕赤酵母中的表达
重组表达质粒pPICZα A-ccHepcidin经内切酶Sac I线性化后电转化毕赤酵母X-33感受态细胞，用含100μg/mL Zeocin的YPD平板筛选重组子，30°C培养48h。挑取长出的单菌落到YPD培养基中，30°C培养48h后回收菌体，提取酵母菌基因组DNA，PCR鉴定阳性克隆。
含重组表达质粒pPICZα A-ccHepcidin的毕赤酵母阳性菌接种于20mL BMGY培养基中，30℃震荡培养，待OD600至2.0左右，4℃、6000g离心7min，将菌体转入500mL BMMY 培养基中，30℃震荡培养，每隔24h添加甲醇至终浓度1%诱导表达，且取培养上清 SDS-PAGE 检测重组蛋白是否表达，直到其达到最大表达量（图2）。
将诱导表达好的菌液12000g，4℃离心，将表达上清液过0.45μm滤膜后用切向回流超滤器（赛多利斯VIVAFLOW 200）浓缩培养液上清。用同样的方法浓缩pPICZα A空载体毕赤酵母菌表达上清。用BCA蛋白浓度试剂盒测定重组毕赤酵母X-33/pPICZα A-ccHepcidin发酵上清总蛋白浓度。将上述浓缩的表达上清取样进行SDS-PAGE分析，并通过Quantity one软件进行蛋白灰度分析定量目的蛋白Hepcidin占发酵上清中总蛋白的百分比。
实施例
黄河鲤抗菌肽Hepcidin基因酵母表达产物的应用
1.黄河鲤Hepcidin基因酵母表达产物对病原菌的抑制作用
将嗜水气单胞菌、鳗弧菌、枯草芽孢杆菌和大肠杆菌分别分离到的单菌落培养至OD600为0.5-1，分别取50μL涂布与LB固体平板上，放入3个灭过菌的牛津杯，分别加入100μL氨苄青霉素（0.1mg/mL）作为阳性对照、浓缩20倍的pPICZα A空载体毕赤酵母表达上清为阴性对照和浓缩20倍的pPICZα A-ccHepcidin毕赤酵母培养液上清100μL（含Hepcidin重组蛋白300μg），培养过夜。重组毕赤酵母表达的重组蛋白对嗜水气单胞菌、大肠杆菌、鳗弧菌和枯草芽孢杆菌均产生了较大的抑菌圈，抑菌圈直径分别为36.9cm、36.2cm、34.7cm和32.7cm，且转化pPICZα A空载体的重组毕赤酵母，经甲醇诱导表达并浓缩的上清无抑菌圈产生（图3）。证明通过毕赤酵母诱导表达的重组蛋白对嗜水气单胞菌、大肠杆菌、鳗弧菌和枯草芽孢杆菌都有较好的抑菌活性。
2.饲料中添加黄河鲤Hepcidin基因酵母表达产物对鱼体抗病能力的增强作用
选择通威鱼饲料作为基础饲料。将所添加的制剂均匀喷洒于膨化饲料表面并混匀，阴凉处晾干，4℃保存备用。健康黄河鲤随机分为4组，对照组（CT组）投喂基础饲料添加毕赤酵母表达pPICZα A空载培养液上清，实验组（H-1组、H-2组、H-3组）投喂基础饲料添加不同剂量黄河鲤Hepcidin基因毕赤酵母表达产物，H-1组添加量为120mg/kg，H-2组添加量为60mg/kg，H-3组添加量为30mg/kg。每组90尾鱼，设3个平行，每个平行30尾。每天投喂3次，投喂量为鱼体重的1%，每天换一半经曝气后的水，实验持续28d。饲料中添加Hepcidin重组蛋白（H-1组）可显著降低嗜水气单胞菌感染后24h肝脏、脾脏、肾脏和肠道中的细菌载量（图4）。
投喂28d后，各组每组随机选取20尾腹腔注射100μL 5×106 CFU/mL（约5×105CFU/30 g鱼体重）嗜水气单胞菌，在注射后24h后，每组随机选取3条鱼，用MS-222麻醉后取肝脏、脾脏和肾脏大约0.1g新鲜组织于1mL 生理盐水中匀浆，然后将匀浆液稀释10倍后取100μL均匀涂布于LB琼脂平板上，孵育16h，计数平板上的菌落数，实验分为3个重复。
养殖实验结束后，饥饿处理24h，各组黄河鲤随机选取30尾腹腔注射100μL 1×107CFU/mL（约106CFU/30g鱼体重）嗜水气单胞菌，每天监测各组鱼7天期间的疾病症状和生存率。H-1组感染嗜水气单胞菌7d后存活率（40%）显著高于对照组（16.67%）。H-2组、H-3组的存活率与对照组相似。H-1组的相对免疫保护率为23.33%（图5）。
以上结果表明饲料中添加120 mg/kg黄河鲤Hepcidin基因酵母表达产物，有助于增强鱼体免疫力，有助于鱼体清除体内感染的嗜水气单胞菌，并能提高鱼体感染后的存活率。
序列表
<110> 河南师范大学
<120> 黄河鲤抗菌肽hepcidin基因酵母表达产物及其应用
<130> 2022
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<212> DNA
<213> Cyprinus carpio
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gctctcgctg ctgcagtcat catcgcatgc gtctgcatcc tccagaccgc agccgttccc 120
ttcacacagc agactgaaga tgagcatcat gtggagagtg aagcacaaca ggagaaccag 180
catctgacag aaacttcaca ggaacaaacc aatcccctgg cattcttcag ggtgaaacgt 240
caaagccatc tgtccctgtg cagatactgc tgcaactgct gccgcaacaa aggctgtgga 300
tactgctgca aattctgaag gctgtggata ctgctgcaac tactaatgat tttctctgat 360
taatgattaa tcagtatcgt ataatgacat atgctatttg aactttttca gacatcgctt 420
tgtcaacatg taatttcagg caataaaaca gttaagcatt taagagaaaa aaaaaaaaaa 480
ggatccggta cctctagatc agaatcgtcg acctgcaggc atgcaagctt ggcgtaatca 540
tggtcatagc tgtt 554
<210> 2
<211> 91
<212> PRT
<213> Cyprinus carpio
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Met Lys Phe Ser Arg Gly Ala Leu Ala Ala Ala Val Ile Ile Ala Cys
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Val Cys Ile Leu Gln Thr Ala Ala Val Pro Phe Thr Gln Gln Thr Glu
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Asp Glu His His Val Glu Ser Glu Ala Gln Gln Glu Asn Gln His Leu
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Thr Glu Thr Ser Gln Glu Gln Thr Asn Pro Leu Ala Phe Phe Arg Val
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Lys Arg Gln Ser His Leu Ser Leu Cys Arg Tyr Cys Cys Asn Cys Cys
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Arg Asn Lys Gly Cys Gly Tyr Cys Cys Lys Phe
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<211> 33
<212> DNA
<213> Cyprinus carpio
<400> 3
ccgctcgaga aaagaatgaa gttctcacgt ggg 33
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<212> DNA
<213> Cyprinus carpio
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gctctagatc aatgatgatg atgatgatgg aatttgcagc agta 44
Claims (6)
1.黄河鲤抗菌肽Hepcidin基因酵母表达产物，其特征在于：该黄河鲤抗菌肽Hepcidin基因酵母表达产物的氨基酸序列如序列表SEQ ID NO.2所示。
2.根据权利要求1所述的黄河鲤抗菌肽Hepcidin基因酵母表达产物，其特征在于：该黄河鲤抗菌肽Hepcidin基因酵母表达产物为分子量10kDa的黄河鲤抗菌肽Hepcidin重组蛋白。
3.一种权利要求1所述的黄河鲤抗菌肽Hepcidin基因酵母表达产物的制备方法，其特征在于具体步骤为：
步骤S1：表达质粒pPICZα A-cchepcidin的构建，以黄河鲤hepcidin全长cDNA序列为模板，扩增其43位至第315位的共273bp的开放阅读框片段，并在片段两端引入酶切位点Xho I和Xba I，通过双酶切和T4连接酶将该片段构建到pPICZα A载体，重组载体经Sac Ⅰ酶线性化以后，电转化毕赤酵母X-33，用含100μg/mL Zeocin的YPD平板筛选转化成功的菌体即重组酵母菌株，并且提取菌体DNA，通过PCR鉴定，确定Hepcidin基因已成功构建到pPICZα A载体；
步骤S2：表达产物的诱导，将重组酵母菌株接种于含100μg/mL Zeocin YPD液体培养基中活化，30℃震荡培养，而后接种到BMGY培养基中，培养至OD600至2.0，4℃、6000g离心7min，将菌体转入BMMY培养基中，30℃震荡培养；每隔24h添加甲醇诱导表达，且取样SDS-PAGE检测是否表达，直到其达到最大表达量，将诱导表达好的菌液12000g，4℃离心，将表达上清液过0.45μm滤膜后用切向回流超滤器浓缩培养液上清。
4.权利要求1所述黄河鲤抗菌肽hepcidin基因酵母表达产物在制备黄河鲤抗细菌感染制剂中的应用，其中细菌为嗜水气单胞菌、大肠杆菌、鳗弧菌或枯草芽孢杆菌中的一种或多种。
5.权利要求1所述黄河鲤抗菌肽hepcidin基因酵母表达产物在制备饲料添加剂中的应用，其特征在于：在基础饲料中添加黄河鲤抗菌肽hepcidin基因的酵母表达产物，用于显著提高黄河鲤感染嗜水气单胞菌后的存活率，有效提高了黄河鲤抗细菌感染的能力，从而能够用于预防水产养殖中因细菌感染引起的黄河鲤疾病。
6.根据权利要求5所述的应用，其特征在于：所述黄河鲤抗菌肽hepcidin基因的酵母表达产物在饲料中的添加量为120mg/kg，该黄河鲤抗菌肽hepcidin基因的酵母表达产物均匀喷洒于膨化饲料表面并混合均匀。
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