CN112458128B - 一种海洋盐单胞菌胞外多糖在制备免疫增强剂中的应用 - Google Patents
一种海洋盐单胞菌胞外多糖在制备免疫增强剂中的应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体涉及到一种海洋盐单胞菌胞外多糖的制备方法及其在制备免疫增强剂中的应用。从一株海洋盐单胞菌Halomonas sp.2E1的发酵液中分离纯化得到了一种胞外多糖(EPS2E1),并对它进行了理化分析和体外免疫增强活性测定。结果表明,EPS2E1为一种甘露聚糖,能够通过激活MAPKs(ERK和JNK)和NF‑κB信号通路提高炎症因子(NO、IL‑6、IL‑1β和TNF‑α)和环氧化酶‑2(COX‑2)的表达,具有显著的免疫增强活性,且没有明显的细胞毒性。因此,本发明提供的海洋盐单胞菌胞外多糖EPS2E1可用于制备与免疫增强相关的药物或保健品,具有良好的开发利用价值。
Description
技术领域
本发明涉及生物技术领域,具体涉及到一种海洋盐单胞菌胞外多糖的制备方法及其在制备免疫增强剂中的用途。
背景技术
免疫***的主要功能是识别和消灭病原体,以维持机体的生理平衡和稳定,但过强或过低的免疫力可引起多种不良的免疫反应。免疫缺陷是免疫功能低下的表现,其是导致感染和恶性肿瘤的主要因素之一。目前,治疗免疫缺陷最常见的方法是使用免疫增强剂,如真菌多糖、胸腺肽、干扰素和卡介苗。在这些药物中,多糖作为免疫增强剂被证明是安全无毒的。多糖能与巨噬细胞表面受体结合,激活信号转导通路,促进白细胞介素(IL)、肿瘤坏死因子(TNF-α)、干扰素(IFN)和一氧化氮(NO)等细胞因子的分泌,这些细胞因子作为细胞间相互作用的内源性信号继续诱导其他细胞因子的产生,并在调节免疫应答中发挥重要作用。
海洋生物的多样性远远超过陆地生物,尤其海洋微生物种类繁多。由于低温、高盐、高压和寡营养的极端海洋环境,海洋微生物的代谢产物往往表现出新颖的遗传或生理特征和广泛的生物活性。在所有的代谢产物中,胞外多糖可以提高微生物生长和代谢过程中对环境的适应性。近年来,从海底热液喷口和冷泉区域发现的海洋细菌中分离出了大量具有多种特性的胞外多糖,其中一些表现出良好的免疫刺激活性,具有潜在的药物开发价值。因此,海洋微生物胞外多糖备受人们的关注。
本发明从一株海洋盐单胞菌(Halomonas sp.2E1)中分离纯化得到一种胞外多糖(EPS2E1),同时对其性质进行了分析。鉴于海洋多糖广泛的生物活性,我们利用RAW 264.7巨噬细胞模型评价了EPS2E1的体外免疫增强活性,并阐明了其活性的作用机制。本发明首次发现从海洋盐单胞菌发酵物中纯化获得的胞外多糖EPS2E1具有显著的免疫增强活性,为开发海洋多糖类免疫增强剂和保健品奠定了基础。
发明内容
本发明的目的是提供一种海洋盐单胞菌胞外多糖在制备免疫增强剂、或药物组合物、或保健品中的应用。
为实现上述目的,本发明采用以下技术方案:
一种海洋盐单胞菌来源于中国南海(119°17′04.956″E,22°06′58.384″N,水深1142米)盐单胞菌Halomonas sp.2E1,该菌种保藏于中国微生物菌种保藏管理委员会普通微生物中心,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),保藏日期:2020年10月29日,保藏编号:CGMCC NO.20969。
所述海洋盐单胞菌胞外多糖优选以下方法制备:将海洋盐单胞菌Halomonassp.2E1按0.1~1%接种于2216E培养基(0.1~1%蛋白胨、0.1~0.2%酵母浸粉、0.5~2%蔗糖和陈海水)中,置于摇床上震荡培养(25~40℃、100~200rpm、24~48h);发酵完毕后将发酵液离心(4000~8000rpm、5~30min)去除菌体,收集上清液,加入2~4倍体积的95%乙醇溶液,离心收集沉淀,加适量蒸馏水复溶后透析,冻干(-60~-80℃,24~48h)得到粗胞外多糖;采用DEAE Fast Flow离子交换柱层析对粗多糖进行纯化(先用蒸馏水洗脱1~3个柱体积,再用0~2mol/L氯化钠溶液洗脱8~12个柱体积),得到的纯化组份进行透析、冻干,继续采用Sephadex G75凝胶柱层析进行纯化(流动相为蒸馏水或0.1~1mol/L碳酸氢铵溶液),最终得到纯化的胞外多糖,即为EPS2E1。
所述海洋盐单胞菌Halomonas sp.2E1的胞外多糖(EPS2E1)在制备免疫增强剂、或药物组合物、或保健品中的应用。
所述EPS2E1为一种甘露聚糖,单糖组成为甘露糖和葡萄糖(摩尔比为5:1~2:1),总糖含量范围为80%~100%,分子量范围为30~60kD。
所述盐单胞菌胞外多糖能够激活MAPKs和NF-κB信号通路,提高ERK、JNK、IκB和P65等蛋白的磷酸化水平。
所述盐单胞菌胞外多糖能显著下调环氧化酶-2(COX-2)的表达量,同时抑制NO、IL-6、IL-1β和TNF-α等炎症因子的产生。
所述盐单胞菌胞外多糖在免疫增强剂中的浓度为1~1000μg/mL。所述盐单胞菌胞外多糖在1~1000μg/mL浓度下对RAW264.7巨噬细胞无毒性。
以所述盐单胞菌胞外多糖为活性成分,与药学或食品上可接受的辅料或辅助添加剂成分混合后,可以用来制备具有抗炎作用的药物或药物组合物。
本发明所具有的优点:
本发明首次提供了从盐单胞菌Halomonas sp.2E1中制备胞外多糖(EPS2E1)的方法,并提供了EPS2E1在制备免疫增强剂、或药物组合物、或保健品中的应用。EPS2E1能够上调RAW264.7巨噬细胞MAPKs和NF-κB信号通路中ERK、JNK、IκB和P65蛋白的磷酸化水平,增加巨噬细胞COX-2的表达量,同时促进NO、IL-6、IL-1β和TNF-α等炎症因子的产生,从而提高机体的免疫功能。同时,EPS2E1没有明显细胞毒性,具有潜在的开发利用价值。
附图说明
图1A为本发明实施例提供的海洋盐单胞菌胞外多糖在DEAE Fast Flow离子交换柱层析上的洗脱色谱图;
图1B为本发明实施例提供的海洋盐单胞菌胞外多糖在Sephadex G75凝胶柱层析上的洗脱色谱图;
图2为本发明实施例提供的海洋盐单胞菌胞外多糖的单糖组成测定色谱图;
图3为本发明实施例提供的海洋盐单胞菌胞外多糖的相对分子量测定色谱图;
图4为本发明实施例提供的海洋盐单胞菌胞外多糖体外给药对RAW264.7细胞增殖活性的影响;
图5A为本发明实施例提供的海洋盐单胞菌胞外多糖体外给药对RAW264.7细胞NO生成量的影响;
图5B为本发明实施例提供的海洋盐单胞菌胞外多糖与多粘菌素B共孵育后体外给药对RAW264.7细胞中NO生成量的影响;
图6为本发明实施例提供的海洋盐单胞菌胞外多糖体外给药对RAW264.7细胞中IL-1β、IL-6、TNF-α和COX-2表达量的影响;
图7为本发明实施例提供的海洋盐单胞菌胞外多糖对RAW264.7细胞MAPKs信号通路中磷酸化ERK、JNK和P38表达量的影响;
图8为本发明实施例提供的海洋盐单胞菌胞外多糖对RAW264.7细胞NF-κB信号通路中磷酸化IκB和P65表达量的影响。
具体实施方式
下面结合附图和实施例对本发明进行详细描述。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。此外应理解,在阅读了本发明的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本发明所限定的范围。
实施例1
海洋盐单胞菌胞外多糖EPS2E1的制备方法,具体步骤如下:
1)粗多糖的提取
将海洋盐单胞菌Halomonas sp.2E1接种于2216E(含1%蔗糖)培养基中,置于摇床上28℃、150rpm震荡培养48h。发酵完毕,将盐单胞菌发酵液离心(4000rpm)15min去除菌体,收集上清液,向上清液中边搅拌边加入三倍体积的95%乙醇溶液,搅拌均匀后于冰箱中4℃静置过夜,随后离心(4000rpm,15min)后收集沉淀,加水复溶后透析(截留分子量为3.5kDa),冻干得到粗多糖。
2)胞外多糖的纯化
采用DEAE Fast Flow离子交换柱层析对粗多糖进行纯化,上样后,先用纯水洗脱2个柱体积(CV),随后采用0~2.0mol/L NaCl溶液线性梯度洗脱10CV,根据硫酸苯酚法检测多糖,收集含糖组分,透析(截留分子量为3.5kDa)、冻干(图1A)。将离子交换柱纯化组分再采用Sephadex G75凝胶柱层析进行纯化,采用纯水作为流动相,同样采用硫酸苯酚法检测多糖,收集含糖组分,得到的纯化多糖,即为EPS2E1(图1B)。
3)总糖含量分析
采用硫酸-苯酚法,以D-甘露糖(Man)为对照品测定总糖含量。精密准确称取适量D-甘露糖对照品,加纯水配制成0.1mg/mL溶液。依次取50、100、150、200、250、300μL Man对照品,放置于10mL的EP管中,用纯水补至500μL,空白对照为500μL纯水。移取300μL 0.1mg/mL的EPS2E1,用纯水补至500μL。再向各EP管中加入5%苯酚和浓硫酸,加入量分别为300μL和1.5mL。摇匀,放置于100℃沸水浴中,反应10min。待放冷后,借助紫外可见分光光度计测定492nm处的光密度值。每组设定3个平行样,以Man对照品的质量数和492nm处的光密度值分别作为横、纵坐标,绘制标准曲线。将样品的光密度值得平均值带入标准曲线中,计算EPS2E1的总糖含量。由结果可知,EPS2E1的总糖含量范围为83%。
4)单糖组成分析
采用PMP柱前衍生高效液相色谱法对多糖进行单糖组成分析。精密称取5mg样品,置于安瓿瓶中,加入1mL 2mol/L三氟乙酸,封口,于105℃反应6h。反应完毕,反复加甲醇,减压旋蒸除去TFA。最后,用纯水将样品溶出,使样品浓度为10mg/mL。取全水解液100μL,分别加入100μL的氢氧化钠溶液(0.3mol/L)和120μL PMP溶液(0.5mol/L),震荡混匀,于70℃水浴中反应1h。衍生完毕,加入110μL盐酸溶液(0.3mol/L),终止反应。加入500μL的三氯甲烷萃取3次,除掉多余的PMP。单糖标准品和样品均采用上述方法衍生,处理完毕,经0.22μm微孔过滤膜过滤,运用高效液相进行分析。Zorbax KP-C18色谱柱(4.6mm×150mm,5μm);检测波长:245nm;流动相:磷酸盐缓冲液(pH=6.8)/乙腈(体积分数,83:17);流速:0.8ml/mL;柱温:30℃;上样量:10μL。由结果可知,EPS2E1单糖组成为甘露糖和葡萄糖(摩尔比为4:1)(图2)。
5)相对分子量分析
采用高效凝胶渗透色谱法来分析多糖样品相对分子质量。色谱柱:TSK Gel-G3000PWXL凝胶色谱柱(300mm×7.8mm);流动相:0.1mol/L硫酸钠溶液;流速:0.5mL/min;柱温:35℃;检测器:RID;进样量:20μL。系列右旋糖酐葡聚糖相对分子质量对照品,用0.1mol/L硫酸钠溶液配成浓度为5mg/mL,经0.22μm微孔过滤膜过滤,进样,记录保留时间(tR)。以右旋糖酐葡聚糖对照品的相对分子质量的对数(logMw)对保留时间(tR)作图,绘制标准曲线。精密称取5mg样品,用0.1mol/L硫酸钠溶液配成5mg/mL,经0.22μm微孔过滤膜过滤,进样,记录保留时间(tR)。将保留时间(tR)带入标准曲线中,计算样品对应的分子量。由结果可知,EPS2E1的重均分子量范围为47.0kDa。(图3)。
实施例2
EPS2E1体外给药对RAW264.7细胞增殖活性的影响
实验方法:
取生长对数期的RAW264.7细胞,吹打制成单细胞悬液,经细胞计数后,将RAW264.7细胞按照5×105个/mL细胞密度接于96孔板中培养,待细胞生长至50%左右时,用100μL不含FBS培养基代替旧培养基,饥饿处理12h。然后用100μL空白或含不同浓度EPS2E1的培养基代替旧培养基,继续培养24h。向每孔中加入20μL浓度为5mg/mL的MTT溶液,继续孵育4h,弃去上清,每孔加入150μL DMSO,室温轻轻震摇10min使其结晶充分溶解,在490nm波长下测定吸光度。
细胞存活率=(实验组OD值/空白对照组OD值)×100%。
结果如图4所示,EPS2E1在浓度为6.25~200μg/mL时对RAW264.7巨噬细胞无明显毒性。
实施例3
EPS2E1体外给药抑制RAW264.7细胞分泌NO的分泌。
实验方法:
将RAW264.7细胞按照5×105个/mL细胞密度接于96孔板置于恒温培养箱中培养(37℃,5%CO2),待细胞生长至50%左右时,用100μL不含FBS培养基代替旧培养基,饥饿处理12h。按照以下分组加样:(1)空白对照组;(2)LPS处理组(LPS终浓度1μg/mL);(3)EPS2E1处理组(终浓度6.25~200μg/mL);每组6复孔,置于培养箱中培养24h。收集上清,采用Griess法测定NO浓度:取上清液50μL依次各加入50μL Griess试剂Ⅰ、Ⅱ液,混匀后室温放置10min,使其充分反应,在540nm波长下测定吸光度,以亚硝酸钠溶液为标准溶液建立NO标准曲线,计算各实验组细胞培养上清中的NO浓度。
NO抑制率=(LPS处理组-实验组)/LPS处理组×100%
为了排除内毒素(内源性LPS)对实验结果的影响,采用多粘菌素B与EPS2E1共孵育后给药,其它操作同上。
结果如图5所示,图5A表明EPS2E1单独作用于RAW264.7细胞时,在6.25μg/mL~200μg/mL浓度时能有效地刺激NO产生,并且呈浓度依懒性;图5B表明EPS2E1与多粘菌素B共孵育后同样对RAW264.7细胞有刺激作用,能有效地促进其NO的产生。NO作为炎症的主要标志因子,其产生量增加说明EPS2E1具有较强的增强免疫作用。
实施例4
EPS2E1体外给药对环氧化酶-2(COX-2)以及IL-6、IL-1β和TNF-α等炎症因子产生的影响。
将RAW264.7细胞按照5×105个/mL细胞密度接于12孔板置于恒温培养箱中培养(37℃,5%CO2),待细胞生长至50%左右时,用不含FBS培养基代替旧培养基,饥饿处理12h后分为四组:(1)空白对照组;(2)LPS组(LPS终浓度1μg/mL);(3)实验组:EPS2E1(6.25μg/mL~200μg/mL)。培养24h后,吸去培养板内的培养基,用预冷的PBS润洗3次,加入适量细胞裂解液,于冰上震荡裂解10min。裂解完后用枪吹打细胞,将裂解液转移至离心管中,于4℃下12000rpm离心5min。离心后的上清采用ELISA试剂盒进行检测,具体按试剂盒说明书操作。
结果如图6所示,结果表明,各浓度的EPS2E1均能刺激RAW264.7细胞产生COX-2、IL-6、IL-1β和TNF-α,说明EPS2E1能有效促进炎症介质的表达,具有免疫增强作用。
实施例5
采用western blotting检测EPS2E1体外给药对MAPKs和NF-κB信号通路中关键蛋白质表达的影响。
按照实施例4中方法培养及药物处理RAW 264.7巨噬细胞。培养24h后,收集巨噬细胞并加入100μL RIPA裂解缓冲液,提取总蛋白,用BCA蛋白检测试剂盒测定各处理组的蛋白含量。将各组的蛋白质调整一致。然后加入Loading buffer,100℃加热5min使蛋白质变性。,使用10%SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)上分离(上样量为30μg/孔),并转移至硝酸纤维素(NC)膜上。立即用5%封闭剂封闭膜2h,与一抗杂交4℃过夜。用TBST漂洗4次5min后,将膜与二抗在室温下孵育1h,应用Vazyme增强型ECL化学发光检测试剂盒显影,采用ChemiDoc MP***检测蛋白的印迹。
结果如图7-8所示,图7表明,EPS2E1能显著提高RAW264.7细胞MAPKs通路中JNK和ERK蛋白的磷酸化;图8表明,EPS2E1同样能显著提高RAW264.7细胞NF-κB通路中IκB和P65蛋白的磷酸化,说明EPS2E1能激活MAPKs和NF-κB信号通路,转导炎症相关信号,促进炎症因子的表达,从而达到免疫增强的效果。
Claims (5)
1.一种海洋盐单胞菌胞外多糖,其特征在于:海洋盐单胞菌来源于中国南海盐单胞菌Halomonassp.2E1,该菌种保藏于中国微生物菌种保藏管理委员会普通微生物中心,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,保藏日期:2020年10月29日,保藏编号:CGMCC NO. 20969;海洋盐单胞菌胞外多糖以下方法制备:将海洋盐单胞菌Halomonas sp. 2E1按0.1~1%接种于2216E培养基中,置于摇床上25~40℃、100~200 rpm震荡培养24~48 h;发酵完毕后将发酵液4000~8000 rpm下离心5~30 min去除菌体,收集上清液,加入2~4倍体积的95%乙醇溶液,离心收集沉淀,加适量蒸馏水复溶后透析,-60~-80℃冻干24~48 h得到粗胞外多糖;采用DEAE Fast Flow离子交换柱层析对粗多糖进行纯化,先用蒸馏水洗脱1~3个柱体积,再用0~2mol/L氯化钠溶液洗脱8~12个柱体积,得到的纯化组份进行透析、冻干,继续采用Sephadex G75凝胶柱层析进行纯化,流动相为蒸馏水或0.1~1 mol/L碳酸氢铵溶液,最终得到纯化的胞外多糖,即为EPS2E1,2216E培养基为0.1~1%蛋白胨、0.1~0.2%酵母浸粉、0.5~2%蔗糖和陈海水。
2.权利要求1所述的海洋盐单胞菌胞外多糖的制备方法,其特征在于:海洋盐单胞菌胞外多糖以下方法制备:将海洋盐单胞菌Halomonas sp. 2E1按0.1~1%接种于2216E培养基中,置于摇床上25~40℃、100~200 rpm震荡培养24~48 h;发酵完毕后将发酵液4000~8000rpm下离心5~30 min去除菌体,收集上清液,加入2~4倍体积的95%乙醇溶液,离心收集沉淀,加适量蒸馏水复溶后透析,-60~-80℃冻干24~48 h得到粗胞外多糖;采用DEAE Fast Flow离子交换柱层析对粗多糖进行纯化,先用蒸馏水洗脱1~3个柱体积,再用0~2mol/L氯化钠溶液洗脱8~12个柱体积,得到的纯化组份进行透析、冻干,继续采用Sephadex G75凝胶柱层析进行纯化,流动相为蒸馏水或0.1~1 mol/L碳酸氢铵溶液,最终得到纯化的胞外多糖,即为EPS2E1,2216E培养基为0.1~1%蛋白胨、0.1~0.2%酵母浸粉、0.5~2%蔗糖和陈海水。
3.一种权利要求1所述的海洋盐单胞菌胞外多糖的应用,其特征在于:海洋盐单胞菌Halomonas sp. 2E1的胞外多糖即为EPS2E1在制备药物组合物中的应用。
4.根据权利要求3所述的海洋盐单胞菌胞外多糖的应用,其特征在于:EPS2E1为一种甘露聚糖,分子量范围为30~60 kD,单糖组成为甘露糖和葡萄糖,摩尔比为5:1~2:1。
5.根据权利要求3所述的海洋盐单胞菌胞外多糖的应用,其特征在于:以EPS2E1为活性成分,与药学上可接受的辅料或辅助添加剂成分混合后,用来制备具有免疫增强作用的药物或药物组合物。
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