CN109438567B - 一种鲟鱼抗病免疫蛋白及其制备方法和应用 - Google Patents
一种鲟鱼抗病免疫蛋白及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种鲟鱼抗病免疫蛋白及其制备方法和应用,所述鲟鱼抗病免疫蛋白为鲟鱼白细胞介素‑6,其具有如SEQ ID NO.1所示的氨基酸序列。本发明提供了一种新的鲟鱼白细胞介素‑6,其表达受多种免疫刺激的诱导,具有促进免疫组织细胞增殖、调节免疫分子的表达和抑制细菌感染的功能。本发明提供的鲟鱼白细胞介素‑6可以作为免疫增强剂等抗病制剂提高鲟鱼的免疫力,有效防治鲟鱼的病害,促进鲟鱼养殖业的发展。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种鲟鱼抗病免疫蛋白及其制备方法和应用。
背景技术
鲟鱼(Sturgeon)属于软骨硬鳞类,是目前世界上最古老的原始鱼类,被称为“活化石”,素有“水中熊猫”之称。由于鲟鱼属于软骨硬鳞类,介于软骨鱼和硬骨鱼类之间,故其具有很高的学术研究价值。与此同时,鲟鱼肉味鲜美,营养价值极高,鲟鱼鱼子酱有“黑色黄金”之称(姜礼燔,1998;陈曾龙,1993)。鲟鱼养殖潜力巨大,市场前景广阔。我国鲟鱼养殖业起步较晚但发展迅速,经过20多年的发展,养殖规模不断扩大,养殖品种不断增多,目前包括杂交种在内养殖品种有十多种,养殖产量6万余吨,占世界鲟鱼养殖量的85%以上(潘鹏,2015)。然而随着养殖规模不断扩大,养殖环境也发生了巨大变化,鲟鱼养殖集约化程度提高,加之水流逐年减少、高温持续时间长、水质环境恶化及种质退化等因素,使得鲟鱼养殖病害呈逐年上升趋势,给鲟鱼养殖业带来重大的经济损失,已成为当前鲟鱼养殖业发展瓶颈(王静波,2013)。现有技术中关于鲟鱼病害防治的制剂的研究,主要有细菌疫苗(李圆圆,2008),和微生态制剂,但是对于免疫防治制品的研究仍然比较少,因此,亟需开展鲟鱼的免疫学研究工作,为鲟鱼的健康养殖和病害防控提供新思路。
在鱼类的免疫系统中,细胞因子参与控制鱼类免疫防御的启动,其在体内是可溶的,是重要的免疫感受器(Fletcher T C,1981),并且能够在外分泌、旁分泌、内分泌(激素)中调节多种细胞的增殖、分化和功能发挥。白细胞介素6(IL-6)是一种来源于T细胞的可溶性细胞因子,可以激活B细胞,促进B细胞产生免疫球蛋白,是一种多效性的细胞因子,参与免疫调节、造血、炎症等生理或病理过程(Belosevic M,2006)。白细胞介素6作为促炎因子,可参与炎症反应,召集免疫细胞,有利于抑制和清除病原微生物。关于鲟鱼白细胞介素6的基因克隆、表达分析、重组蛋白的制备及其促淋巴细胞增殖、抗菌活性的研究目前尚未见报道。
发明内容
为了解决现有技术中存在的问题,本发明的目的是提供一种鲟鱼抗病免疫蛋白及其制备方法和应用。
本发明提供一种鲟鱼白细胞介素-6,其具有如下氨基酸序列:
(1)如SEQ ID NO.1所示的氨基酸序列;
(2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、缺失或插入得到的具有相同功能蛋白的氨基酸序列。
此外,本发明还提供编码所述鲟鱼白细胞介素-6的基因。
优选地,本发明所述的编码鲟鱼白细胞介素-6的基因具有如下核苷酸序列:
(1)如SEQ ID NO.2所示的核苷酸序列;
(2)如SEQ ID NO.2所示的核苷酸序列经一个或多个碱基的替换、缺失或插入得到的编码具有相同功能蛋白的核苷酸序列;
(3)在严格条件下,能够与(1)或(2)所述的核苷酸序列杂交且编码具有相同功能蛋白的核苷酸序列。
进一步地,本发明还提供含有所述编码鲟鱼白细胞介素-6的基因的载体以及含有所述编码鲟鱼白细胞介素-6的基因或含有所述载体的宿主细胞。
本发明所述的含有所述编码鲟鱼白细胞介素-6的基因的载体可以为携带所述编码鲟鱼白细胞介素-6的基因的现有技术中公开的动物、植物或微生物的任意一种克隆载体、表达载体或非复制型载体。
本发明所述的含有所述载体的宿主细胞可以为携带含有所述编码鲟鱼白细胞介素-6的基因的载体的任意一种微生物细胞、动物细胞或植物细胞;包括但不限于大肠杆菌细胞、酵母细胞。
本领域技术人员应当理解,随着科技发展,所述载体和宿主细胞的选择也许会发生变化,但只要含有本发明所述基因或本发明所述的载体,均在本发明的保护范围之内。
本发明提供的鲟鱼白细胞介素-6具有促进免疫细胞增殖、促进多种免疫分子的表达、抑制微生物感染的功能,可以用于制备鲟鱼抗病制剂。
具体地,本发明提供一种鲟鱼抗病制剂,所述抗病制剂包含本发明所述的鲟鱼白细胞介素-6或所述的鲟鱼白细胞介素-6的重组蛋白。
优选地,所述抗病制剂为免疫增强剂。
本发明所述的抗病制剂还包括药物制剂领域允许的佐剂或辅料。
进一步地,本发明提供一种鲟鱼抗病制剂的制备方法,为表达本发明所述的编码鲟鱼白细胞介素-6的基因,纯化表达产物,得到鲟鱼白细胞介素-6或鲟鱼白细胞介素-6的重组蛋白。
具体地,所述制备方法包括如下步骤:
(1)构建白细胞介素-6基因的表达载体;
(2)将步骤(1)得到的表达载体转化至宿主细胞;
(3)诱导表达白细胞介素-6;
(4)纯化表达产物,得到白细胞介素-6。
作为本发明的优选实施方式,以pET30a(+)为出发载体构建白细胞介素-6基因的表达载体,所述宿主细胞为大肠杆菌BL21菌株;所述白细胞介素-6的表达为以携带His标签的重组蛋白的方式表达;所述纯化表达产物采用亲和层析。
此外,本发明提供所述的鲟鱼白细胞介素-6或所述编码鲟鱼白细胞介素-6的基因或所述的载体在制备转基因鲟鱼或制备鲟鱼病害防治制剂中的应用。
以及所述的鲟鱼白细胞介素-6或所述编码鲟鱼白细胞介素-6的基因或所述的载体或所述的抗病制剂在鲟鱼病害防治或提高鲟鱼免疫力中的应用。
本发明所述的鲟鱼白细胞介素-6或所述的鲟鱼白细胞介素-6重组蛋白或所述的抗病制剂可以采用体内注射的方式给药。
优选地,所述应用为将在鲟鱼体内提高白细胞介素-6的表达和/或注射所述鲟鱼抗病制剂。
所述鲟鱼病害防治包括但不限于微生物感染,优选为细菌、病毒或真菌感染。
本发明的有益效果在于:本发明提供了一种新的鲟鱼白细胞介素-6,通过鲟鱼白细胞介素-6的表达和纯化,获得了重组鲟鱼白细胞介素-6。鲟鱼白细胞介素-6基因的表达受细菌感染、LPS等多种免疫刺激的诱导,具有促进脾脏和头肾等免疫组织的细胞增殖、调节IL-1β、CXCI10、IgM和MHCII等免疫分子的表达和抑制微生物感染的功能,注射本发明提供的重组鲟鱼白细胞介素-6,脾脏的病原菌感染量最高下降了72%。本发明提供的鲟鱼白细胞介素-6可以作为免疫增强剂等抗病制剂提高鲟鱼的免疫力,有效防治鲟鱼的病害,促进鲟鱼养殖业的发展。
附图说明
图1为实施例2中西伯利亚鲟的白细胞介素-6的诱导表达SDS-PAGE电泳结果,其中,泳道1是pET30a(+)转化BL21、未加IPTG诱导剂的菌液包涵体样品(对照组),泳道2~7为pET30a(+)-AbIL6转化BL21后挑取不同单克隆诱导表达后的包涵体样品。
图2为实施例2中纯化的重组AbIL6蛋白的SDS-PAGE电泳结果和western blot检测结果,其中,A为SDS-PAGE电泳考马斯亮蓝染色结果,B为western blot检测结果;A和B中第一泳道均为纯化的AbIL6重组蛋白,第二泳道均为BSA蛋白。
图3为实施例3中西伯利亚鲟的鳃、眼、头肾、脾脏、心脏、肌肉、皮肤、肝脏、脑和肠组织中AbIL6基因的表达分析。
图4为实施例4中嗜水气单胞菌感染后不同时间西伯利亚鲟AbIL6的表达变化,其中Ah代表灭菌嗜水气单胞菌。
图5为实施例4中原代脾脏细胞在不同的免疫刺激下AbIL6基因的表达变化,其中,A为LPS和灭菌嗜水气单胞菌(Ah),B为PolyI:C和Glucan。
图6为实施例5中重组AbIL6蛋白对西伯利亚鲟脾脏和头肾细胞增殖活性的影响,其中,A为脾脏细胞,B为头肾细胞。
图7为实施例6中体内注射重组AbIL6蛋白后脾脏和头肾细胞中免疫基因的表达情况分析,其中,A为促炎因子IL-1β,B为趋化因子CXCL10,C为IgM,D为MHCII。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
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。
实施例1鲟鱼白细胞介素-6基因的获得和表达载体的构建
1、鲟鱼白细胞介素-6基因的获得
为研究免疫原刺激或高温等条件胁迫是否影响鲟鱼免疫功能,发明人所在团队进行了不同条件下鲟鱼的肝脏转录组测序,根据测序结果,从中找到一段免疫功能发生变化的不同条件下差异表达的基因,其cDNA的核苷酸序列如SEQ ID NO.2所示,其编码蛋白的氨基酸序列如SEQ ID NO.1所示,根据同源序列比对和蛋白结构模拟分析,推测该基因编码的蛋白质为白细胞介素-6。
根据上述靶定的鲟鱼的白细胞介素-6基因的mRNA序列设计分别携带NcoI和XhoI酶切位点的扩增引物F:(SEQ ID NO.3:CCCATGGCTCCTATTGTAGAATTTTC)和R(SEQ ID NO.4:CCGCTCGAGTCATACAAATATTATCTTT)。以西伯利亚鲟的脾脏cDNA为模板,以F和R为引物进行PCR扩增,得到西伯利亚鲟IL-6的成熟肽(如SEQ ID NO.1所示的27~218位氨基酸)的cDNA片段(AbIL6)。PCR扩增反应体系(25μL)含有2.5μL 10×Ex buffer,2μLdNTP,F和R引物各0.3μmol,1U Ex taq酶(Takara),500ng cDNA模板;PCR反应程序为:94℃10min;94℃25s,52℃25s,72℃30s(10个循环);94℃25s,60℃25s,72℃30s(25个循环);72℃7min。扩增产物经1%琼脂糖凝胶电泳分析后,用DNA凝胶回收试剂盒(Takara)进行回收纯化。
2、表达载体pET30a(+)-AbIL6的构建
(1)pET30a(+)质粒与AbIL6片段的双酶切及胶回收
采用Thermo公司的限制性内切酶进行双酶切处理,20μL双酶切反应体系为:4μL10×Tango Buffer,1μLNcoI,1μLXhoI,1μg pET30a(+)质粒(Merck millipore公司,货号69909)与AbIL6片段,以ddH2O补足20μL。37℃,反应6h。双酶切产物经电泳鉴定后,利用Takara试剂盒切胶回收纯化双酶切的质粒pET30a(+)与目的片段AbIL6。
(2)连接反应
将经过双酶切后纯化的质粒和目的基因片段以1:3摩尔比进行连接(采用Thermo公司的T4连接酶)。连接体系(20μL)为:2μL 10×T4Buffer,1μL T4连接酶,3μL目的DNA,14μL pET30a(+)质粒。
(3)转化感受态细胞
取50μL大肠杆菌DH5α感受态细胞,冰上融化,加入上述连接体系,轻轻混匀后在冰上放置30min;在42℃水浴中热激90s,再冰浴3min;加入500μL LB培养基,混匀后置于37℃,180rpm震荡培养1h,使菌复苏抗性。吸取50μL菌液涂布在含卡那霉素(50μg/mL)的LB平板上,37℃恒温培养16h。平板上挑取单菌落,利用通用引物T7和T7t进行菌落PCR,筛选阳性转化子。提取阳性转化子的质粒,经测序验证后,得到pET30a(+)-AbIL6重组质粒。将质粒转化至大肠杆菌BL21感受态细胞中,在含有卡那霉素(50μg/mL)的LB固体培养基上,37℃培养18h,挑取一个单菌落,经验证可成功表达重组IL-6蛋白后,将其命名为pET30a(+)-AbIL6-BL21。
实施例2西伯利亚鲟IL-6重组蛋白的诱导表达及纯化
1、西伯利亚鲟IL-6重组蛋白的诱导表达
将pET30a(+)-AbIL6-BL21接种于含有卡那霉素(50ng/μL)的LB培养基中,在37℃震荡培养至OD600约为0.6,加入1mM异丙基-β-D-硫代半乳糖苷,继续培养6h,离心收集菌体。以培养基1/10体积的PBS悬起菌体,并加入PMSF至终浓度1mM,在冰浴条件下,经100W超声破碎裂解细胞,离心分别收集裂解液上清和沉淀,进行SDS-PAGE电泳分析,结果表明,西伯利亚鲟Il6重组蛋白存在于包涵体(图1)。
2、西伯利亚鲟IL-6重组蛋白的纯化
将收集的经超声破碎的pET30a(+)-AbIL6-BL21的菌体沉淀重悬于BufferA(6M盐酸胍,137mMNaCl,8mM Na2HPO4,2.7mMKCl,1.5mM KH2PO4充分溶解于800mL双蒸水,定容至1L,调节pH至7.4)中,利用TALON Superflow Resin Co2+亲和层析柱纯化带有6×His标签的AbIL6蛋白。
Co2+亲和层析纯化蛋白的步骤如下:
(1)收集超声破碎的菌体沉淀,向沉淀中加入适量的洗涤Buffer(50mM Tris-HCl(pH8.0),1mM EDTA(pH8.0),50mMNaCl,2M尿素,0.5%Triton x-100),重悬沉淀,洗涤包涵体,洗涤完毕后,4℃,12000g离心10min,弃去上清。反复操作3次;
(2)将洗涤后的包涵体重悬于BufferA中,振荡摇匀使蛋白充分溶解。溶解后,4℃,12000g离心10min收集上清;将收集的上清(即溶解于BufferA的包涵体溶解液)用0.45μm孔径的滤膜过滤,去除杂质;
(3)将Co2+树脂装入层析柱中,用经0.45μm孔径的滤膜过滤后的5倍柱体积的清水洗柱;然后加5倍柱体积的Buffer A,平衡柱子,至紫外分析仪的数值稳定后调为0.000。
(4)将过滤好的包涵体溶解液加入层析柱中,流速控制在1mL/min;待柱体饱和,停止加上清,改用BufferA洗柱,至紫外吸收峰接近0.000;
(5)加入含150mM咪唑的洗脱液BufferC(5.4M盐酸胍,150mM咪唑,137mMNaCl,8mMNa2HPO4,2.7MmKCl,1.5mM KH2PO4,充分溶解于800mL双蒸水,定容至1L,调节pH至7.4)洗脱蛋白,收集洗脱组分;
(6)待收集完,依次用10倍柱体积ddH2O、5倍柱体积MES、10倍柱体积ddH2O洗柱,然后用20%的乙醇保存柱子以备下次使用;
(7)将收集的洗脱组分置于透析袋中,将透析袋的两头用夹子夹好,放于复性缓冲液(50mM Tris-HCl,pH8.0;6M尿素)中,于4℃透析,每24h更换尿素浓度逐级降低的复性缓冲液,使蛋白复性。将复性后的蛋白用10KDa的超滤管(Millipore)超滤浓缩,然后用SDS-PAGE检测目的蛋白AbIL6的纯化结果。
3、西伯利亚鲟IL-6重组蛋白6×His标签的westernblot检测
取上述纯化的IL-6重组蛋白3μg上样,并以BSA作对照,进行SDS-PAGE,电泳结果如图2的A所示,其中泳道1为IL-6重组蛋白,泳道2为BSA蛋白对照。将电泳结束后凝胶上的蛋白转移至PVDF膜上。PBST漂洗3次,每次10min,在含1%BSA的PBST溶液中,4℃下封闭过夜,PBST漂洗后,加入单抗Anti-His-HRP(1:10000),在37℃下孵育2h,PBST漂洗3次,每次10min,然后用DAB显色。结果如图2的B所示,泳道1对应的条带大小处目的条带发生显色,而泳道2的BSA蛋白对照无显色条带。结果表明,经诱导表达和纯化成功获得了纯度较高的IL-6重组蛋白。
实施例3 AbIL6基因在西伯利亚鲟的不同组织中的表达分析
采用实时荧光定量PCR方法检测IL-6基因在健康西伯利亚鲟体内不同组织的相对表达量,研究AbIL6mRNA的组织分布。
具体操作方法:随机选取健康西伯利亚鲟(体重约150g)的10个组织,分别为:鳃、眼、头肾、脾脏、心脏、肌肉、皮肤、肝脏、脑和肠。用Trizol法提取组织总RNA,用Takara反转录试剂盒反转录为cDNA。
根据AbIL6的cDNA序列设计用于荧光定量PCR的引物,引物序列如下:
SEQ ID NO.5:Abil6F:5’-CACTGTCAGCGTTGCTGGTTC-3’;
SEQ ID NO.6:Abil6R:5’-GCCACTCGGCTAAAGATTCCC-3’;
内参基因18S rRNA的引物序列如下:
SEQ ID NO.7:18sF:5’-TGCCCTATCAACTTTCGATGG-3’;
SEQ ID NO.8:18sR:5’-CTGCCTTCCTTGGATGTGGT-3’。
采用ABI7500实时荧光定量PCR仪,检测上述10个组织中AbIL6的相对表达量。反应条件为:95℃,15s;95℃5s,59.4℃30s,72℃30s,40个循环。
结果表明(如图3所示),AbIL6基因在上述10个组织中均有表达,其中,在脾脏中的表达量最高,其次是肝和脑,这三种组织的表达量之间存在显著性差异(P<0.05),且分别与其余7个组织中的表达量存在显著性差异(P<0.05)。
实施例4免疫刺激对西伯利亚鲟AbIL6基因的表达的影响
分别利用在体和离体实验分析不同免疫刺激对西伯利亚鲟AbIL6基因的表达的影响。
1、细菌感染对西伯利亚鲟AbIL6基因的表达的影响
随机选取健康的西伯利亚鲟(体重约100g),分为对照组和感染组。给感染组的鲟鱼腹腔注射0.2mL嗜水气单胞菌NX830(保藏于中国农业部水生动物病原库,保藏编号:BYK20130805)(108CFU/mL),对照组的鲟鱼注射等量的PBS。在注射后6h,12h,24h,72h和7d,分别取各组的脾脏。实时荧光定量PCR的方法检测AbIL6基因的相对表达量。
结果表明(如图4所示),AbIL6mRNA表达量在嗜水气单胞菌感染6h迅速升高,达到峰值,之后逐渐下降,直至72h后基本和对照组保持相近的水平。
2、不同免疫刺激对原代脾细胞中西伯利亚鲟IL-6基因表达的影响
用0.01%(W/V)高锰酸钾溶液对西伯利亚鲟鱼体浸泡消毒30min,然后再用70%的酒精对鱼体体表进行消毒。将其脾脏取出后放置到无菌培养皿中,在含青霉素(500U/mL)和链霉素(500U/mL)的1×PBS溶液中洗涤3~4次。将脾脏组织过100目筛网,用2mL注射器内胆研磨,用不含血清的培养基清洗筛网。将细胞于1000rpm离心5min,并用无血清培养基清洗细胞一次。将离心的细胞沉淀用细胞培养基重悬、计数,并调整浓度为1×106个/ml,加1mL细胞悬液到24孔细胞培养板中。在26℃培养箱中进行原代培养。第二天吸出培养基,每孔添加1mL新鲜培养基,并分别添加不同的免疫刺激物LPS(100μg/mL)、灭活嗜水气单胞菌(2×106CFU/ml)(嗜水气单胞菌NX830于70℃高温孵育2h,涂LB平板,确认无菌落生长)、Glucan(100μg/mL)和PolyI:C(50μg/ml)。分别于免疫刺激的6h、24h、48h时收取细胞,提取RNA,并反转录得到cDNA。采用实时荧光定量PCR的方法检测不同免疫刺激物作用下,不同刺激时间,AbIL6mRNA的相对表达量。
结果表明(如图5所示),LPS、灭活嗜水气单胞菌和Poly I:C均能刺激西伯利亚鲟脾脏原代细胞上调AbIL6mRNA的表达。LPS、灭活嗜水气单胞菌刺激后12h,脾脏原代细胞AbIL6mRNA表达量迅速升高,显著高于PBS对照组(P<0.05)。在Poly I:C刺激后6h,脾脏细胞AbIL6mRNA的表达量迅速升高,显著高于PBS对照组(P<0.05)。
实施例5重组AbIL6蛋白对西伯利亚鲟细胞增殖活性的影响
取西伯利亚鲟的脾脏细胞和头肾细胞,向96孔板的每个孔中加入150μL(1×105个/mL)细胞培养液。将重组AbIL6加入至细胞培养液中对细胞进行刺激,设置终浓度分别为10ng/mL,100ng/mL,500ng/mL,以添加等量体积的PBS作为对照,于刺激三天后,向每个孔中加入20μL的3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮哇嗅盐(MTT,5mg/mL),22℃孵育4h,小心吸出孔内培养液,然后加入150μLDMSO,震荡10min,将结晶物溶解。酶标仪检测各孔OD值(波长490nm),记录结果。
结果如图6所示,添加不同浓度的AbIL6蛋白对于脾脏细胞增殖的促进作用未表现出明显的剂量依赖性,其中,添加10ng/mL重组AbIL6蛋白的脾脏细胞的实验组的OD490为0.207(平均值),较添加PBS的对照组的OD490(平均值为0.087)提高了1.38倍,具有统计学意义;添加100ng/mL和500ng/mL重组蛋白组比对照组OD490分别提高了1倍和82.7%。添加不同浓度的AbIL6蛋白对于头肾细胞增殖的促进作用也未表现出明显的剂量依赖性,其中,添加100ng/mL重组AbIL6蛋白的头肾细胞OD490的平均值为0.166,添加PBS的对照组OD490的平均值为0.105,细胞增殖活性提高了58%,具有统计学意义。结果表明,AbIL6蛋白具有促进脾脏细胞和头肾细胞增殖的作用。
实施例6体内注射重组蛋白AbIL6后免疫基因的表达分析
随机选取9尾西伯利亚鲟(体重约为100g),注射0.2mg/mL纯化的重组蛋白AbIL6,并于注射后6h和24h,采集免疫组织脾脏和头肾,提取总RNA,反转录为cDNA。采用实时荧光定量PCR方法检测AbIL6mRNA相对表达量。
结果表明(如图7所示),注射重组AbIL6蛋白6h后可以迅速上调西伯利亚鲟的脾脏和头肾的促炎因子IL1β的表达(分别是PBS对照组的3.55倍和2.41倍)、脾脏趋化因子CXCL10的表达(是PBS对照组的1.72倍)及头肾IgM的表达(是PBS对照组的5.6倍);在注射重组蛋白AbIL6后24h,西伯利亚鲟脾脏IgM和MHCII的表达显著上调(分别是PBS对照组的13.37倍和2.1倍)。结果显示,重组AbIL6蛋白对脾脏IgM和MHCII基因的诱导作用较强,表明重组ABIL6具有促进炎症反应的作用,并可诱导MHCII参与的抗原呈递等免疫反应,诱导免疫球蛋白IgM参与的体液免疫反应。
实施例7两种规格的西伯利亚鲟体内注射AbIL6的抑菌功能分析
将重组AbIL6蛋白用生理盐水稀释至0.2mg/mL,即为重组AbIL6蛋白稀释液。将8尾西伯利亚鲟(体重约85g)随机分为2组,每组4尾鱼,一组为注射200μL生理盐水的对照组,一组为注射200μL重组AbIL6蛋白稀释液的实验组。
细菌悬液制备:LB培养基培养嗜水气单胞菌NX830至OD600为0.6-0.8,离心(8000g,2min),倾倒上清,将菌体用生理盐水悬浮,调至终浓度为5×107CFU/mL。
攻毒感染:对试验组和对照组分别注射重组AbIL6和生理盐水5h后,在试验组和对照组的鱼体腹腔注射200μL嗜水气单胞菌悬液。在注射24h后,麻醉鲟鱼,解剖,取出脾脏,称重,并根据重量加入不同量(1mL/100mg)的生理盐水,用无菌研磨棒研磨后,取50μL组织匀浆液均匀得涂在LB平板上,28℃培养16-24h,进行菌落计数,并进行统计分析。
结果如表1所示,在体重为85g的鲟鱼的注射试验中,注射生理盐水的对照组鲟鱼脾脏细菌感染量显著大于注射重组AbIL6蛋白的试验组的鲟鱼脾脏的细菌感染量;注射重组AbIL6蛋白使得脾脏细菌感染量降低了72%。可见,注射重组AbIL6蛋白对鲟鱼感染病原菌具有显著的清除和抑制作用。
表185g鲟鱼的脾脏细菌感染量(104CFU/g)
表中a和b代表数据具有显著性差异(P<0.05)
将重组AbIL6蛋白用生理盐水稀释至0.2mg/mL,即为重组AbIL6蛋白稀释液。将8尾西伯利亚鲟(体重约30g)随机分为2组,每组4尾鱼,一组为注射200μL生理盐水的对照组,一组为注射200μL重组AbIL6蛋白稀释液的实验组。
细菌悬液制备:LB培养基培养嗜水气单胞菌NX830至OD600为0.6-0.8,离心(8000g,2min),倾倒上清,将菌体用生理盐水悬浮,调至终浓度为5×106CFU/mL。
攻毒感染:对试验组和对照组分别注射重组AbIL6蛋白和生理盐水5h后,在试验组和对照组的鱼体腹腔注射200μL嗜水气单胞菌悬液。在注射24h后,麻醉鲟鱼,解剖,取出脾脏,称重,并根据重量加入不同量(1.4mL/100mg)的生理盐水,用无菌研磨棒研磨后,取50μL组织匀浆液均匀得涂在LB平板上,28℃培养16-24h,进行菌落计数,并进行统计分析。
结果如表2显示,在体重为30g的鲟鱼试验中,注射生理盐水的对照组的鲟鱼脾脏细菌感染量显著大于注射重组AbIL6蛋白的试验组鲟鱼脾脏的细菌感染量;注射重组AbIL6蛋白使得脾脏的细菌感染量降低了63.5%。可见,注射重组AbIL6蛋白对鲟鱼感染病原菌具有显著的清除和抑制作用。
表230g鲟鱼的脾脏细菌感染量(104CFU/g)
表中a和b代表数据具有显著性差异(P<0.05)
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,在不脱离本发明技术原理的前提下,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> 北京市水产科学研究所
<120> 一种鲟鱼抗病免疫蛋白及其制备方法和应用
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Claims (9)
1.一种鲟鱼白细胞介素-6,其特征在于,其氨基酸序列如SEQ ID NO.1所示。
2.编码权利要求1所述的鲟鱼白细胞介素-6的基因。
3.根据权利要求2所述的基因,其特征在于,其核苷酸序列如SEQ ID NO.2所示。
4.含有权利要求2或3所述的基因的载体。
5.含有权利要求2或3所述的基因或含有权利要求4所述的载体的宿主细胞。
6.一种鲟鱼抗病制剂,其特征在于,所述抗病制剂包含权利要求1所述的鲟鱼白细胞介素-6或权利要求1所述的鲟鱼白细胞介素-6的重组蛋白。
7.一种鲟鱼抗病制剂的制备方法,其特征在于,表达权利要求2或3所述的基因,纯化表达产物,得到鲟鱼白细胞介素-6或鲟鱼白细胞介素-6的重组蛋白。
8.权利要求2或3所述的基因或权利要求4所述的载体在制备转基因鲟鱼中的应用。
9.权利要求1所述的鲟鱼白细胞介素-6或权利要求2或3所述的基因或权利要求4所述的载体在制备鲟鱼病害防治制剂或提高鲟鱼免疫力的制剂中的应用。
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Molecular cloning and characterisation of the flounder (Paralichthys olivaceus) interleukin-6 gene;Bo-Hye Nam et al.;《Fish & Shellfish Immunology》;20061011;第23卷;第231-236页 * |
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