CN115197319A - 一种莱茵衣藻rab7蛋白的多克隆抗体、制备方法和应用 - Google Patents
一种莱茵衣藻rab7蛋白的多克隆抗体、制备方法和应用 Download PDFInfo
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- CN115197319A CN115197319A CN202210595918.XA CN202210595918A CN115197319A CN 115197319 A CN115197319 A CN 115197319A CN 202210595918 A CN202210595918 A CN 202210595918A CN 115197319 A CN115197319 A CN 115197319A
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- rab7
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- chlamydomonas reinhardtii
- polyclonal antibody
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Abstract
本发明公开一种莱茵衣藻RAB7蛋白的多克隆抗体,所述多克隆抗体能够特异性识别莱茵衣藻RAB7蛋白;所述莱茵衣藻RAB7蛋白的氨基酸序列如序列表SEQ ID NO.1所示,所述莱茵衣藻RAB7蛋白的核苷酸序列如SEO ID NO.2所示。本发明莱茵衣藻RAB7蛋白多克隆抗体能特异性识别莱茵衣藻RAB7蛋白,且具有效价高和特异性好的特点;该莱茵衣藻RAB7蛋白多克隆抗体可用于莱茵衣藻RAB7蛋白的检测以及后续以莱茵衣藻为模式生物的RAB7蛋白的功能研究。
Description
技术领域
本发明属于生物技术领域,尤其是一种莱茵衣藻RAB7蛋白的多克隆抗体、制备方法和应用。
背景技术
莱茵衣藻(Chlamydomonas reinhardtii)是一种细胞结构较为单一的藻类生物,细胞大小在10μm左右,胞体侧面长有一个眼点,胞内含有一个或多个液泡以及一个呈“U”型的半环状叶绿体,与细胞核、蛋白核、线粒体等藻细胞结构相比,叶绿体体积相对偏大,约占细胞总体积的40%。莱茵衣藻细胞结构虽简单却具有与动物细胞相似的特征,其中最典型的特点是在细胞顶部长有两根等长的鞭毛,使其能够自由运动。由于其运动鞭毛与哺乳动物的纤毛具有相同的结构和许多相同的组成蛋白质,并且关键蛋白的同源性达到90%以上,因此目前对哺乳动物纤毛的认识在很大程度上依赖于以莱茵衣藻为模式生物的研究。
纤毛是一种从基体(源自中心粒)顶端延伸而出,基于微管呈毛发状突出于细胞表面的细胞器,广泛分布于多种细胞表面,并已进化到在感知内外环境、驱动细胞运动和信号传导等方面发挥作用。纤毛主要由基体、纤毛膜及轴丝三部分组成,是进化上保守的细胞器。初级纤毛在信号转导、细胞增殖和分化以及胚胎发育等方面都具有重要功能,它的组装或者功能的缺陷,会直接引起很广泛的人类疾病,这类疾病现在被定义为纤毛类营养病,如视网膜变性、多囊肾、巴德-毕氏综合征等。
RAB蛋白家族是小G蛋白超家族中最大的亚家族,广泛存在于动物、植物和微生物等真核生物中,在进化过程中高度保守,是参与调控细胞内膜运输的关键因子,其中RAB8和RAB11参与了纤毛形成过程中的多个步骤,包括囊泡对接、纤毛膜形成以及鞭毛内运输。当纤毛进入细胞分裂期前,需要进行中心粒的复制及运动时,需要进行纤毛的解聚,其解聚异常会导致细胞周期进程受阻。2019年,Wang等鉴定了RAB7是一种新的纤毛解聚调节因子,通过调控纤毛内F-肌动蛋白聚合在纤毛外胞和纤毛解聚方面发挥作用。RAB7在纤毛形成中起负调控作用,RAB7基因的敲除可以增加初级纤毛的数量和长度来促进纤毛的形成,并且其缺失显著抑制了纤毛外胞现象。RAB7作为一种小G蛋白,可以在无活性(gdp结合)和活性(gtp结合)状态之间不断转换,而纤毛解聚和纤毛外胞都需要具有活性的RAB7。初级纤毛在细胞信号转导过程中,通过组装和解聚的动态转换发挥其功能,利用衣藻这种模式生物能够更加深入了解纤毛的形成与解聚的机理,为研究RAB7相关的发育缺陷和发病机制提供线索,进一步揭示纤毛病发生的遗传基础和分子机制,对纤毛病的预防、诊断和治疗提供依据。
本发明以实验室保存的莱茵衣藻cDNA为模板,通过设计的引物做PCR扩增,得到大小为621bp的rab7基因的目的片段;将PCR扩增得到的rab7片段和pET-28a(+)载体同时用BamHⅠ和HindⅢ双酶切,将目的基因与载体片段回收,以摩尔质量比为1:3室温用T4DNA连接酶连接1h;转化至E.coli XL1-blue,在含有相应抗性的平板上37℃过夜培养;挑取转化子至LB液体培养基中过夜培养,提取质粒后用BamHⅠ和HindⅢ进行双酶切验证,条带大小与预期一致则进行测序验证。
经检索,本发明在国内外未见公开报道,在国内外未见公开使用。
发明内容
本发明目的在于克服现有技术的不足之处,提供一种莱茵衣藻RAB7蛋白的多克隆抗体、制备方法和应用。
本发明解决其技术问题所采用的技术方案是:
一种莱茵衣藻RAB7蛋白的多克隆抗体,所述多克隆抗体能够特异性识别莱茵衣藻RAB7蛋白;
所述莱茵衣藻RAB7蛋白的氨基酸序列如序列表SEQ ID NO.1所示,所述莱茵衣藻RAB7蛋白的核苷酸序列如SEO ID NO.2所示。
如上所述的莱茵衣藻RAB7蛋白的多克隆抗体在检测莱茵衣藻RAB7蛋白方面中的应用。
一种检测莱茵衣藻RAB7蛋白的试剂盒,其特征在于:所述试剂盒包括如上所述的多克隆抗体。
一种如上所述的莱茵衣藻RAB7蛋白的多克隆抗体的制备方法,所述方法包括如下步骤:
构建重组表达载体,所述重组表达载体的核苷酸序列包括如SEO ID NO.2所示的序列;
将所述重组表达载体转化至大肠杆菌感受态细胞BL21中,得到重组表达菌株;诱导重组表达菌株的蛋白表达,得到莱茵衣藻RAB7蛋白;
将莱茵衣藻RAB7蛋白经过亲和纯化后作为免疫原免疫动物,得到莱茵衣藻蛋白RAB7的多克隆抗体。
进一步地,构建重组表达载体的方法包括:
将莱茵衣藻cc-125cDNA作为模板,进行PCR扩增,获得莱茵衣藻rab7目的基因;
将莱茵衣藻rab7目的基因与原核表达载体pET-28a(+)连接,得到重组表达载体pET-28a(+)-rab7。
进一步地,在PCR扩增时采用上游引物和下游引物进行扩增,所述上游引物序列如SEQ ID NO.3所示,所述下游引物如SEQ ID NO.4所示;
扩增程序为:预变性95℃,5min;
(变性95℃,45s;退火58℃,30s;延伸72℃,1min)×35个循环;
延伸72℃,5min。
进一步地,所述诱导重组表达菌株的蛋白表达的具体方法包括如下步骤:
挑取重组表达菌株的单克隆到含有卡那抗性的LB液体培养基中,37℃过夜培养,以1:20的体积扩大培养,当600nm处的吸光值A值达到0.6~0.8时,加入1M异丙基-β-D-硫代半乳糖苷(isopropy-beta-D-thiogalactoside,IPTG)进行诱导,使其终浓度至0.2mM,且诱导的温度为25℃,诱导的时间为8h,将收集菌体高压破碎,全蛋白、上清和沉淀进行12%SDS-PAGE分析蛋白表达情况,在25kDa处能够看到RAB7蛋白大量表达并且表达在上清液中,由此得到莱茵衣藻RAB7蛋白。
进一步地,将得到的莱茵衣藻RAB7蛋白进行纯化,纯化的方法包括:将莱茵衣藻RAB7蛋白进行亲和纯化,挑取单克隆到含有卡那抗性的的LB液体培养基中,37℃过夜培养,以1:20的体积扩大培养,当600nm处的吸光值A值达到0.6~0.8时,加入1M异丙基-β-D-硫代半乳糖苷(isopropy-beta-D-thiogalactoside,IPTG)进行诱导,使其终浓度至0.2mM,且诱导的温度为25℃,诱导的时间为8h,8000rpm,3min离心收集RAB7菌体,得到菌体沉淀;
将菌体沉淀用PBS重悬,再次离心收集菌体沉淀;用含有20mmol/L咪唑的pH=8.0的Tris-HCl溶液溶解菌体沉淀并进行高压破碎,将破碎后的菌液离心8000rpm,10min后取上清液;
将上清液即莱茵衣藻RAB7蛋白用0.45μm滤膜过滤后加入预先平衡好的NiSepharoseTM6Fast Flow蛋白纯化柱中,室温条件下结合2h,用含20mmol/L咪唑的Tris盐溶液漂洗3次,最后用含500mmol/L咪唑的pH=8.0的Tris-HCl溶液洗脱;
将收集的全蛋白、沉淀、流穿液、漂洗液、洗脱液用12%SDS-PAGE检测,选择浓度>0.5mg/ml、纯度>85%的目标蛋白用于后续动物免疫实验;
其中,含20mmol/L咪唑的Tris盐溶液为:20mM咪唑,50mM pH 7.4Tris-HCl,0.5mol/L NaCl,溶剂为水;
含500mmol/L咪唑的pH=8.0的Tris-HCl溶液为:500mM咪唑、50mM pH 7.4Tris-HCl、0.5mol/LNaCl,溶剂为水。
较优地,所述免疫的方法包括:以莱茵衣藻RAB7蛋白为免疫原,取400ug或200ug免疫原,用生理盐水稀释到200-500ul,再加入等体积弗氏佐剂,初次免疫用弗氏完全佐剂,加强免疫用弗氏不完全佐剂;用混匀仪器将溶液和佐剂混匀,形成油包水,得到混合液;将混合液采用背部皮下注射法每次打8-10个点,形成初次免疫,初次免疫量为400μg,两周后进行加强免疫,免疫量分别为200μg,每次10-14天,一共3次,最后1次加强免疫后耳静脉取血,间接ELISA法测定免疫效价合格后,颈动脉取血,收集抗血清。
较优地,所述免疫动物为新西兰白兔。
进一步地,所述莱茵衣藻蛋白RAB7的多克隆抗体还经过如下处理:
将莱茵衣藻蛋白RAB7的多克隆抗体运用proteinA纯化珠进行IgG亚型抗体富集,以得到特异性更高的抗体;
将特异性更高的抗体利用MBP-RAB7融合蛋白作为抗原通过溴化氢进行抗原抗体纯化,得到特异性进一步提高的莱茵衣藻蛋白RAB7的多克隆抗体。
进一步地,进行IgG亚型抗体富集的步骤包括:按体积比为1:10将莱茵衣藻蛋白RAB7的多克隆抗体与结合缓冲液混合后,加入预先平衡好的含有ProteinA填料的结合柱中,室温结合2h后,用结合缓冲液漂洗至1×G-250不变色为止,最后用洗脱缓冲液洗脱进行洗脱,收集管中提前加入中和液,以得到纯度更高的RAB7抗体;
或者,抗原抗体纯化的步骤包括:用BamHⅠ和HindⅢ同时将重组表达载体pET-28a(+)-rab7和表达载体pMAL-c2x进行双酶切,回收rab7目的基因和pMAL-c2x表达载体;将酶切后的莱茵衣藻rab7目的基因与原核表达载体pMAL-c2x连接,得到重组表达载体pMAL-c2x-rab7;将构建好的重组表达载体pMAL-c2x-rab7转入E.coli BL21(DE3)感受态细胞中,在含有氨苄抗性的LB平板上37℃培养12~14h,待平板长出转化子后,挑取单克隆菌落至含有氨苄抗性的液体LB培养基中扩大培养,当600nm处的吸光值A值达到0.6~0.8时,加入1M异丙基-β-D-硫代半乳糖苷(isopropy-beta-D-thiogalactoside,IPTG)至终浓度为0.2mM后进行诱导,且所述诱导的温度为25℃,所述诱导的时间为8h;离心菌液得到菌体,用PBS重悬后离心,重复两次以达到清洗目的;将菌体用pH 7.4的结合缓冲液重悬后进行高压破碎,取全蛋白、上清和沉淀进行12%SDS-PAGE凝胶电泳分析,得到莱茵衣藻MBP-RAB7融合蛋白;将破碎后的菌液离心后取上清液,用0.45μm滤膜过滤后加入预先平衡好的DextrinSepharoseTM High Performance蛋白纯化柱中,室温条件下结合2h,用pH 7.4结合缓冲液结合缓冲液漂洗去掉杂蛋白,而后用pH 7.4的洗脱缓冲液洗脱,得到纯度较高MBP-RAB7融合蛋白;称取CNBr-activated Sepharose 4B干粉溶胀于1mmol/LHCl,用pH 8.3的CouplingBuffer将溶胀后的CNBr-activated Sepharose 4B漂洗至pH为8.3;以MBP-RAB7融合蛋白作为抗原与CNBr-activated Sepharose 4B 4℃过夜结合,之后以pH 8.0的缓冲液即0.1mol/L Tris-HCl室温结合2~4h,最后用pH 8.5的缓冲液和pH 3.5的缓冲液交替洗脱8次;将经过ProteinA纯化后的IgG亚型抗体与以上处理好的CNBr-activated Sepharose 4B室温结合2h后,以甘氨酸洗脱,最终获得纯度进一步提高的抗体;
其中,所述结合缓冲液的配方为:20mM Tris、200mMNaCI、1mM EDTA,溶剂为水;所述洗脱缓冲液的配方为:20mM Tris、200mM NaCI、1mM EDTA、10mM麦芽糖,溶剂为水;所述中和液为:pH9.0的1M Tris;所述pH 8.3的Coupling Buffer的配方为:0.1mol/L NaHCO3、0.5mol/LNaCI,溶剂为水;
所述pH 8.5的缓冲液的配方为:100mmol/LTris-HCl、0.5mol/LNaC,溶剂为水;所述pH 3.5的缓冲液的配方为:50mmol/LGly、1mol/LNaCl,溶剂为水。
本发明取得的优点和积极效果为:
1、本发明莱茵衣藻RAB7蛋白多克隆抗体能特异性识别莱茵衣藻RAB7蛋白,且具有效价高和特异性好的特点;该莱茵衣藻RAB7蛋白多克隆抗体可用于莱茵衣藻RAB7蛋白的检测以及后续以莱茵衣藻为模式生物的RAB7蛋白的功能研究。
2、本发明莱茵衣藻RAB7蛋白多克隆抗体的特异性较好,为进一步研究RAB7在相关疾病发生机制中的作用奠定了基础。
3、本发明选择MBP标签的RAB7抗原,将其结合到CNBr-activated Sepharose 4B介质上,利用抗原抗体纯化,不仅能得到特异性高的抗体,提高了抗体制备的效率和得率;而且结合了抗原的CNBr-activated Sepharose 4B介质可以反复利用,节约了制备成本。
4、本发明多克隆抗体可以识别同一抗原的多个表位,因此在免疫检测中,可以识别更多的抗原,也比较少受到抗原构象变化的影响,从而可以提高检测的灵敏度和稳定性,对一些丰度偏低的蛋白更容易检出。其亲和性更高,可放大低表达水平靶蛋白的信号,这有利于后续在免疫沉淀(IP)和染色质免疫沉淀(ChIP)实验中的应用。
5、多克隆抗体制备的主要流程包括制备抗原、免疫动物、测定效价合格后收集血清并纯化、鉴定抗体,本发明在实验室完成,过程简单,成本低廉且速度较快,抗体的价格与商业抗体的价格相比更加低廉,极大地节约了成本。单克隆抗体在免疫动物后,还需要花费较长时间制备、筛选出能生产高效价抗体的单克隆杂交瘤细胞株,因技术要求较高、制备周期较长,通常成本也较高。
附图说明
图1为本发明中实施例提供的PCR扩增与载体的双酶切验证图(RAB7的CDS序列PCR扩增及重组质粒双酶切验证);其中,图A中,M为100bp的DNALadder;1为PCR扩增rab7目的片段;图B中,M为1kb的DNA Ladder;1为BamHⅠ和HindⅢ双酶切质粒pET-28a(+)-rab7;
图2为发明中实施例提供的融合蛋白成功表达图(重组PET-28a(+)-rab7融合蛋白表达及亲和纯化的检测结果);其中,横坐标中M为蛋白质标志物;横坐标1为异丙基-β-D-硫代半乳糖苷(IPTG)诱导前全蛋白;2为IPTG诱导后全蛋白;3为IPTG诱导后上清;4为IPTG诱导后沉淀;5为流穿液;6为漂洗液;7为洗脱液1;8为洗脱液2;9为洗脱液3;10为洗脱液4;11为洗脱液5;12为洗脱液6;
图3为本发明中实施案例提供的ELISA法评定抗体效价图(抗血清效价的ELISA检测结果),图中横坐标为稀释倍数,纵坐标为酶标仪测定450nm处的A值;
图4为本发明中实施案例提供的莱茵衣藻RAB7蛋白多克隆抗体与莱茵衣藻CC-125样品分离鞭毛所得鞭毛蛋白的Westernblot测定结果图(RAB7抗体特异性检测结果);其中,左侧纵坐标标注为蛋白质标准分子量,Pre-immuune为免疫前血清检测莱茵衣藻野生型藻种CC-125中RAB7蛋白,ProteinApurified为ProteinA纯化后血清检测莱茵衣藻野生型藻种CC-125中RAB7蛋白;
图5为本发明中实施例提供的pMAL-c2x-rab7重组表达载体的双酶切验证图;其中,M为1kb的DNALadder;1为BamHⅠ和HindⅢ双酶切质粒pMAL-c2x-rab7;
图6为本发明中实施例提供的融合蛋白成功表达图(重组pMAL-c2x-rab7融合蛋白表达及亲和纯化的检测结果);其中,横坐标中M为蛋白质标志物;横坐标1为异丙基-β-D-硫代半乳糖苷(IPTG)诱导前全蛋白;2为IPTG诱导后全蛋白;3为IPTG诱导后上清;4为IPTG诱导后沉淀;5为流穿液;6为第一次漂洗液;7为最后一次漂洗液;8为洗脱液1;9为洗脱液2;
图7为本发明中实施案例提供的莱茵衣藻RAB7蛋白多克隆抗体与莱茵衣藻CC-125样品分离鞭毛所得鞭毛蛋白的Westernblot测定结果图(RAB7抗体特异性检测结果);其中,左侧纵坐标标注为蛋白质标准分子量,Pre-immuune为免疫前血清检测莱茵衣藻野生型藻种CC-125中RAB7蛋白,Affinity purified为抗原抗体纯化后血清检测莱茵衣藻野生型藻种CC-125中RAB7蛋白。
具体实施方式
下面详细叙述本发明的实施例,需要说明的是,本实施例是叙述性的,不是限定性的,不能以此限定本发明的保护范围。
本发明中所使用的原料,如无特殊说明,均为常规的市售产品;本发明中所使用的方法,如无特殊说明,均为本领域的常规方法。
为探讨RAB7在纤毛形成和解聚过程中的更多作用,本发明特研究制备了莱茵衣藻RAB7蛋白多克隆抗体。首先PCR扩增获得莱茵衣藻rab7的目的基因,通过分子克隆技术构建带有6×His标签的原核表达载体pET-28a(+)-rab7;将重组表达载体转化至大肠杆菌BL21(DE3)中,经过IPTG诱导后,从而获得融合蛋白6×His-RAB7。融合蛋白经亲和纯化后达到免疫原纯度要求,免疫新西兰大白兔,分离血清用间接ELISA方法测得效价值为1:102400。运用proteinA纯化珠对所得RAB7抗血清进行IgG亚型抗体富集,接着利用MBP-RAB7融合蛋白对IgG亚型抗体进行抗原抗体纯化。最后,利用纯化后得的RAB7多克隆抗体通过WesternBlot检测莱茵衣藻野生型藻种CC-125中的RAB7蛋白,结果表明RAB7多克隆抗体的特异性较好,为进一步研究RAB7在相关疾病发生机制中的作用奠定了基础。
下面简单介绍一下该莱茵衣藻RAB7蛋白多克隆抗体的制备方法,该方法包括:
构建重组表达载体,以实验室保存的莱茵衣藻cDNA为模板,进行PCR扩增,获得莱茵衣藻rab7目的基因,引物序列如序列表中SEQ ID NO:3和SEQ ID NO:4所示的序列;
将重组表达的载体转化至感受态细胞中,得到重组表达菌株;
诱导所述重组表达菌株的蛋白表达,得到莱茵衣藻RAB7蛋白;
将所述莱茵衣藻Rab7蛋白作为抗原,并进行免疫处理,得到所述莱茵衣藻RAB7蛋白多克隆抗体。
具体地,构建重组表达载体的方法包括:以实验室保存的莱茵衣藻cDNA为模板,通过设计的引物做PCR扩增,得到莱茵衣藻rab7目的基因。
将所述莱茵衣藻rab7目的基因与原核表达载体链接,得到重组表达载体。
具体地,所述原核表达载体为双酶切的大肠杆菌表达载体pET-28a(+)-rab7。
具体地,所述诱导表重组表达菌株的蛋白表达的方法包括:将所述重组表达菌株37℃摇床中培养14~16h,质粒提取后进行双酶切验证,正确则进行测序。挑取单克隆接种至种子培养基中,37℃培养过夜,以1:20的比例接种至发酵培养基中,当OD600为0.6~0.8时,加入0.2mM的IPTG,25℃,220rpm/min,培养8h。8000rpm、3min离心收集菌体,PBS清洗3次后,经超声破碎后,分离上清及沉淀,进行12%SDS-PAGE,对RAB7蛋白在诱导前后及上清,沉淀的表达情况进行分析,从而获得最优表达条件。
具体地,感受态细胞为大肠杆菌感受态细胞。
进一步地,感受态细胞为大肠杆菌BL 21感受态细胞。
具体地,所述方法还包括:将所述莱茵衣藻RAB7蛋白进行纯化;所述纯化的方法包括:将所述得到莱茵衣藻RAB7融合蛋白进行亲和纯化,将扩大培养后的6×His-RAB7菌体经过第一次离心,得到菌体沉淀;
将菌体沉淀用PBS重悬,再次离心收集菌体沉淀;用含有20mmol/L咪唑的Tris-HCl溶液(pH8.0)溶解菌体沉淀并进行高压破碎,将破碎后的菌液离心后取上清液;
将所述上清液用0.45μm滤膜过滤后加入预先平衡好的Ni SepharoseTM 6FastFlow蛋白纯化柱中,室温条件下结合2h,用含20mmol/L咪唑的Tris盐溶液漂洗3次,最后用含500mmol/L咪唑的Tris-HCl溶液(pH8.0)洗脱;
将上述收集的全蛋白,沉淀、流穿、漂洗,洗脱液用12%SDS-PAGE检测,选择浓度>0.5mg/ml,纯度>85%的目标蛋白用于后续动物免疫实验;
具体地,免疫处理的方法包括:以6×His-RAB7融合蛋白为免疫原,与完全弗氏佐剂混合,形成油包水。得到第一混合液,将所述第一混合液采用背部皮下注射法每次打8-10个点,形成3次加强免疫,每次10-14天,最后1次加强免疫后耳静脉取血,间接ELISA测定免疫效价合格后,颈动脉取血。收集抗血清。
进一步地,免疫动物为新西兰白兔。
该莱茵衣藻RAB7蛋白多克隆抗体的应用,该应用包括将莱茵衣藻RAB7蛋白的多克隆抗体用于莱茵衣藻RAB7蛋白的检测和功能鉴定。
本实施例中,构建重组表达载体的方法包括:
通过PCR扩增获得621bp的rab7目的基因,连接至载体后,转化挑取单克隆提取质粒并酶切验证。以实验室保存的莱茵衣藻cDNA为模板,通过设计的上、下游引物进行PCR扩增,PCR扩增时采用上游引物和下游引物进行扩增,扩增程序为预变性95℃,5min;(变性95℃,45s;退火58℃,30s;延伸72℃,1min)×35个循环;延伸72℃,5min。上游引物为5’-CGGGATCCATGTCCACCAAGAAGCGG-3’,下游引物为5’-CCCAAGCTT TCAGCAGCAG CCAGCC-3’。用BamHⅠ和HindⅢ同时将目的基因、表达载体pET-28a(+)进行双酶切,将回收得到大小为621bp的rab7目的基因和载体片段分别按照摩尔质量1:3的比例室温连接1h,转化至E.coliXL1-blue中后涂板至相应抗性平板上,挑取阳性转化子至LB液体培养基中,37℃摇床中培养14~16h,质粒提取后进行双酶切验证,正确则进行测序。结果如图1所示,1A显示PCR扩增产物,条带单一且大小正确;图1B显示重组表达质粒pET-28a(+)-rab7阳性克隆双酶切验证结果,双酶切后两条带大小与预期果一致,经测序验证结果正确,说明表达载体pET-28a(+)-rab7构建成功。
将构建成功的重组表达载体pET-28a(+)-rab7转化至E.coli BL21中,挑取单克隆接种至种子培养基中,37℃培养过夜,以1:20的比例接种至发酵培养基中,当OD600为0.6~0.8时,加入0.2mM的IPTG,25℃,220rpm/min,培养8h。8000rpm、3min离心收集菌体,PBS清洗3次后,经超声破碎后,分离上清及沉淀,进行12%SDS-PAGE,对RAB7蛋白在诱导前后及上清,沉淀的表达情况进行分析,从而获得最优表达条件。
将诱导表达后的菌体经超声破碎后离心后取上清,上清液用0.45μm滤膜过滤后,加入预先平衡好的Ni SepharoseTM 6Fast Flow填料柱中,室温结合2小时,收集流穿液,用漂洗液(20mM咪唑,50mM pH 7.4Tris-HCl,0.5mol/LNaCl)漂洗3次后收集漂洗液。最后用含500mM咪唑的洗脱液(500mM咪唑、50mM pH 7.4Tris-HCl、0.5mol/LNaCl)洗脱目的蛋白,静置5~10min后收集洗脱液,将收集的全蛋白、流穿液、漂洗液、洗脱液进行SDS-PAGE检测,并做灰度分析,实验选取纯度大于85%,浓度大于0.5mg/mL的6×His-RAB7融合蛋白用于后续动物免疫实验。结果如图2所示,纯化后经SDS-PAGE分析纯化结果,并利用Image J软件进行灰度分析,融合蛋白经纯化后,纯度为85%以上,浓度可达到0.5mg/ml,符合免疫标准,将足够纯度和浓度的6×His-RAB7抗原用于后续动物免疫实验。
采用间接ELISA法检测抗血清效价。以包被液稀释6×His-RAB7抗原,至终浓度为2μg/mL,每孔加入100μL,4℃过夜。PBS洗涤2次后,封闭液封闭,每孔加200μL,37℃静置2h,后用PBS洗涤1次。抗血清从200倍开始以PBS溶液逐次做2倍梯度稀释,空白对照为PBS,阴性对照为稀释后的抗血清,均为100μL/well,37℃条件下静置1h,PBS洗涤3次,加入PBS稀释20000倍的二抗,100μL/well,37℃静置1h,再用PBS洗涤3次。每孔加100μL显色液,5-15min后,每孔加50μL终止液。最后,双波长(450nm,630nm)处测吸光值,做好数据的记录,并做折线图进行分析。效价值为1/2最大OD值所对应的稀释倍数。间接ELISA法测抗血清效价,以免疫前血清做阴性对照组,以免疫后抗血清为实验组,检测结果如图3所示,由图3可看出,抗血清效价达到1:102400。
将离心分离后的抗血清首先经过ProteinA Sepharose TM CL-4B填料进行IgG亚型抗体富集,专一性的吸附IgG,从而去除IgG之外的其他抗体分子。纯化方法为:首先取1mL抗血清与10mL结合缓冲液混合,加入预先平衡好的含ProteinA填料的柱子中,室温结合1h后,用结合缓冲液漂洗至G-250不变色,最后用洗脱缓冲液洗脱,从而得到的纯度更高的抗体。结果如图4所示。
用BamHⅠ和HindⅢ同时将重组表达载体pET-28a(+)-rab7和表达载体pMAL-c2x进行双酶切,回收rab7目的基因和pMAL-c2x表达载体。将酶切后的莱茵衣藻rab7目的基因与原核表达载体pMAL-c2x连接,得到重组表达载体pMAL-c2x-rab7。结果如图5所示。将构建好的重组表达载体pMAL-c2x-rab7转入E.coli BL21(DE3)感受态细胞中,在含有氨苄抗性的LB平板上37℃培养12~14h,待平板长出转化子后,挑取单克隆菌落至含有氨苄抗性的液体LB培养基中扩大培养,当600nm处的吸光值(A)值达到0.6~0.8时,加入1M异丙基-β-D-硫代半乳糖苷(isopropy-beta-D-thiogalactoside,IPTG)至终浓度为0.2mM后进行诱导,且所述诱导的温度为25℃,所述诱导的时间为8h。离心菌液得到菌体,用结合缓冲液重悬后离心,重复两次以达到清洗目的。将菌体用pH 7.4结合缓冲液(20mM Tris、200mM NaCI、1mMEDTA)重悬后进行高压破碎,取全蛋白、上清和沉淀进行12%SDS-PAGE凝胶电泳分析,得到所述莱茵衣藻MBP-RAB7融合蛋白。结果如图6所示。将破碎后的菌液离心后取上清液,用0.45μm滤膜过滤后加入预先平衡好的Dextrin SepharoseTM High Performance蛋白纯化柱中,室温条件下结合2h,用pH 7.4结合缓冲液(20mM Tris、200mM NaCI、1mM EDTA)结合缓冲液漂洗去掉杂蛋白,而后用pH 7.4的洗脱缓冲液(20mM Tris、200mM NaCI、1mM EDTA、10mM麦芽糖)洗脱,得到纯度较高MBP-RAB7融合蛋白。称取一定量的CNBr-activated Sepharose4B干粉溶胀于1mmol/LHCl,用pH 8.3的Coupling Buffer(0.1mol/L NaHCO3、0.5mol/LNaCI)将溶胀后的CNBr-activated Sepharose 4B漂洗至pH为8.3。以MBP-RAB7融合蛋白作为抗原与CNBr-activated Sepharose 4B 4℃过夜结合,之后以0.1mol/L Tris-HCl(pH8.0)缓冲液室温结合2~4h,最后用pH 8.5的缓冲液(100mmol/LTris-HCl、0.5mol/LNaCl)和pH 3.5的缓冲液(50mmol/L Gly、1mol/LNaCl)交替洗脱8次。将经过ProteinA纯化后的抗血清与以上处理好的CNBr-activated Sepharose 4B室温结合2h后,以甘氨酸洗脱,最终获得纯度较高的抗体。
以莱茵衣藻CC-125野生型藻种检测抗体特异性。先取20μg微藻做SDS-PAGE电泳,转膜,封闭后,以1:1000的比例孵育RAB7多克隆抗体,二抗采用1:10000倍稀释HRP标记的羊抗兔IgG,最后进行ECL显色。Western印迹法检测抗体特异性,结果显示纯化后的抗血清在1:1000的稀释比下,可与莱茵衣藻CC-125样品于相对分子质量约2.3×104kDa处形成特异性结合条带,以标签蛋白抗体进行Western印迹法检测作为对照,可见同样大小的特异性结合条带,同时以免疫前血清做阴性对照。由此可知抗血清可与莱茵衣藻RAB7蛋白发生特异性结合(图7),表明所制备的RAB7多克隆抗体特异性良好。
核苷酸序列:
>C.reinhardtii v5.6|Cre06.g311900.t1.2 CDS
ATGTCCACCAAGAAGCGGAGGCTGCTGAAGGTCATCATTCTGGGCGATAGCGGCGTCGGGAAGACGTCGCTGATGAACCAGTATGTTCAGAAGAAGTTCACCAAGGAGTACAAGGCCACAATCGGTGCCGACTTCCTCACCAAGGAGATTGAGGTGGACGACAAAAAAGTCACCATGCAGATCTGGGACACGGCCGGCCAGGAGCGCTTCCAGAGCTTGGGCTCCGCATTCTACCGCGGTGCAGACTGCTGTGTGCTTGTGTTTGACGTAAACAATGCCAAGAGCTTTGACGACCTAGACAACTGGCGGGATGAGTTCATCATACAGGCTGGGCCGCCCGACCCAGACAACTTCCCGTTCATGGTGCTGGGGAACAAGATTGATGAGAACGGGGGCAGCAGCCGCCAGGTGTCGGAGAAGAAGGCCAAGGCCTGGTGCGCCTCCAAGGGCAGCATACCCTACTTCGAGACGTCGGCCAAGGAGGACATCAACGTGGAGGCGGCCTTCACATGCATCACACGCAACGCACTGCGCAACGAGAAGGAGGAGGAGCTGTTTATGCCGGACGCTGTGGACATGAACACCACTGCCACGCAGCGGAAGCGGGCTGGCTGCTGCTGA
氨基酸序列:
>C.reinhardtii v5.6|Cre06.g311900.t1.2
MSTKKRRLLKVIILGDSGVGKTSLMNQYVQKKFTKEYKATIGADFLTKEIEVDDKKVTMQIWDTAGQERFQSLGSAFYRGADCCVLVFDVNNAKSFDDLDNWRDEFIIQAGPPDPDNFPFMVLGNKIDENGGSSRQVSEKKAKAWCASKGSIPYFETSAKEDINVEAAFTCITRNALRNEKEEELFMPDAVDMNTTATQRKRAGCC*
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例所公开的内容。
序列表
<110> 天津科技大学
<120> 一种莱茵衣藻RAB7蛋白的多克隆抗体、制备方法和应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 206
<212> PRT
<213> 莱茵衣藻RAB7蛋白的氨基酸序列(Unknown)
<400> 1
Met Ser Thr Lys Lys Arg Arg Leu Leu Lys Val Ile Ile Leu Gly Asp
1 5 10 15
Ser Gly Val Gly Lys Thr Ser Leu Met Asn Gln Tyr Val Gln Lys Lys
20 25 30
Phe Thr Lys Glu Tyr Lys Ala Thr Ile Gly Ala Asp Phe Leu Thr Lys
35 40 45
Glu Ile Glu Val Asp Asp Lys Lys Val Thr Met Gln Ile Trp Asp Thr
50 55 60
Ala Gly Gln Glu Arg Phe Gln Ser Leu Gly Ser Ala Phe Tyr Arg Gly
65 70 75 80
Ala Asp Cys Cys Val Leu Val Phe Asp Val Asn Asn Ala Lys Ser Phe
85 90 95
Asp Asp Leu Asp Asn Trp Arg Asp Glu Phe Ile Ile Gln Ala Gly Pro
100 105 110
Pro Asp Pro Asp Asn Phe Pro Phe Met Val Leu Gly Asn Lys Ile Asp
115 120 125
Glu Asn Gly Gly Ser Ser Arg Gln Val Ser Glu Lys Lys Ala Lys Ala
130 135 140
Trp Cys Ala Ser Lys Gly Ser Ile Pro Tyr Phe Glu Thr Ser Ala Lys
145 150 155 160
Glu Asp Ile Asn Val Glu Ala Ala Phe Thr Cys Ile Thr Arg Asn Ala
165 170 175
Leu Arg Asn Glu Lys Glu Glu Glu Leu Phe Met Pro Asp Ala Val Asp
180 185 190
Met Asn Thr Thr Ala Thr Gln Arg Lys Arg Ala Gly Cys Cys
195 200 205
<210> 2
<211> 621
<212> DNA
<213> 莱茵衣藻RAB7蛋白的核苷酸序列(Unknown)
<400> 2
atgtccacca agaagcggag gctgctgaag gtcatcattc tgggcgatag cggcgtcggg 60
aagacgtcgc tgatgaacca gtatgttcag aagaagttca ccaaggagta caaggccaca 120
atcggtgccg acttcctcac caaggagatt gaggtggacg acaaaaaagt caccatgcag 180
atctgggaca cggccggcca ggagcgcttc cagagcttgg gctccgcatt ctaccgcggt 240
gcagactgct gtgtgcttgt gtttgacgta aacaatgcca agagctttga cgacctagac 300
aactggcggg atgagttcat catacaggct gggccgcccg acccagacaa cttcccgttc 360
atggtgctgg ggaacaagat tgatgagaac gggggcagca gccgccaggt gtcggagaag 420
aaggccaagg cctggtgcgc ctccaagggc agcataccct acttcgagac gtcggccaag 480
gaggacatca acgtggaggc ggccttcaca tgcatcacac gcaacgcact gcgcaacgag 540
aaggaggagg agctgtttat gccggacgct gtggacatga acaccactgc cacgcagcgg 600
aagcgggctg gctgctgctg a 621
<210> 3
<211> 26
<212> DNA
<213> PCR扩增上游引物(Unknown)
<400> 3
cgggatccat gtccaccaag aagcgg 26
<210> 4
<211> 25
<212> DNA
<213> PCR扩增下游引物(Unknown)
<400> 4
cccaagcttt cagcagcagc cagcc 25
Claims (10)
1.一种莱茵衣藻RAB7蛋白的多克隆抗体,其特征在于:所述多克隆抗体能够特异性识别莱茵衣藻RAB7蛋白;
所述莱茵衣藻RAB7蛋白的氨基酸序列如序列表SEQ ID NO.1所示,所述莱茵衣藻RAB7蛋白的核苷酸序列如SEO ID NO.2所示。
2.如权利要求1所述的莱茵衣藻RAB7蛋白的多克隆抗体在检测莱茵衣藻RAB7蛋白方面中的应用。
3.一种检测莱茵衣藻RAB7蛋白的试剂盒,其特征在于:所述试剂盒包括如权利要求1所述的多克隆抗体。
4.一种如权利要求1所述的莱茵衣藻RAB7蛋白的多克隆抗体的制备方法,其特征在于:所述方法包括如下步骤:
构建重组表达载体,所述重组表达载体的核苷酸序列包括如SEO ID NO.2所示的序列;
将所述重组表达载体转化至大肠杆菌感受态细胞BL21中,得到重组表达菌株;诱导重组表达菌株的蛋白表达,得到莱茵衣藻RAB7蛋白;
将莱茵衣藻RAB7蛋白经过亲和纯化后作为免疫原免疫动物,得到莱茵衣藻蛋白RAB7的多克隆抗体。
5.根据权利要求4所述的莱茵衣藻蛋白RAB7的多克隆抗体的制备方法,其特征在于:构建重组表达载体的方法包括:
将莱茵衣藻cc-125cDNA作为模板,进行PCR扩增,获得莱茵衣藻rab7目的基因;
将莱茵衣藻rab7目的基因与原核表达载体pET-28a(+)连接,得到重组表达载体pET-28a(+)-rab7。
6.根据权利要求5所述的莱茵衣藻蛋白RAB7的多克隆抗体的制备方法,其特征在于:在PCR扩增时采用上游引物和下游引物进行扩增,所述上游引物序列如SEQ ID NO.3所示,所述下游引物如SEQ ID NO.4所示;
扩增程序为:预变性95℃,5min;
(变性95℃,45s;退火58℃,30s;延伸72℃,1min)×35个循环;
延伸72℃,5min。
7.根据权利要求4所述的莱茵衣藻蛋白RAB7的多克隆抗体的制备方法,其特征在于:所述诱导重组表达菌株的蛋白表达的具体方法包括如下步骤:
挑取重组表达菌株的单克隆到含有卡那抗性的LB液体培养基中,37℃过夜培养,以1:20的体积扩大培养,当600nm处的吸光值A值达到0.6~0.8时,加入1M异丙基-β-D-硫代半乳糖苷进行诱导,使其终浓度至0.2mM,且诱导的温度为25℃,诱导的时间为8h,将收集菌体高压破碎,全蛋白、上清和沉淀进行12%SDS-PAGE分析蛋白表达情况,在25kDa处能够看到RAB7蛋白大量表达并且表达在上清液中,由此得到莱茵衣藻RAB7蛋白。
8.根据权利要求4所述的莱茵衣藻蛋白RAB7的多克隆抗体的制备方法,其特征在于:将得到的莱茵衣藻RAB7蛋白进行纯化,纯化的方法包括:
将莱茵衣藻RAB7蛋白进行亲和纯化,将上清液即莱茵衣藻RAB7蛋白用0.45μm滤膜过滤后加入预先平衡好的Ni SepharoseTM6Fast Flow蛋白纯化柱中,室温条件下结合2h,用含20mmol/L咪唑的Tris盐溶液漂洗3次,最后用含500mmol/L咪唑的pH=8.0的Tris-HCl溶液洗脱;
将收集的全蛋白、沉淀、流穿液、漂洗液、洗脱液用12%SDS-PAGE检测,选择浓度>0.5mg/ml、纯度>85%的目标蛋白用于后续动物免疫实验;
其中,含20mmol/L咪唑的Tris盐溶液为:20mM咪唑,50mM pH 7.4Tris-HCl,0.5mol/LNaCl,溶剂为水;
含500mmol/L咪唑的pH=8.0的Tris-HCl溶液为:500mM咪唑、50mM pH 7.4Tris-HCl、0.5mol/LNaCl,溶剂为水。
9.根据权利要求4至8任一项所述的莱茵衣藻蛋白RAB7的多克隆抗体的制备方法,其特征在于:所述莱茵衣藻蛋白RAB7的多克隆抗体还经过如下处理:
将莱茵衣藻蛋白RAB7的多克隆抗体运用proteinA纯化珠进行IgG亚型抗体富集,以得到特异性更高的抗体;
将特异性更高的抗体利用MBP-RAB7融合蛋白作为抗原通过溴化氢进行抗原抗体纯化,得到特异性进一步提高的莱茵衣藻蛋白RAB7的多克隆抗体。
10.根据权利要求9所述的莱茵衣藻蛋白RAB7的多克隆抗体的制备方法,其特征在于:进行IgG亚型抗体富集的步骤包括:按体积比为1:10将莱茵衣藻蛋白RAB7的多克隆抗体与结合缓冲液混合后,加入预先平衡好的含有ProteinA填料的结合柱中,室温结合2h后,用结合缓冲液漂洗至1×G-250不变色为止,最后用洗脱缓冲液洗脱进行洗脱,收集管中提前加入中和液,以得到纯度更高的RAB7抗体;
或者,抗原抗体纯化的步骤包括:用BamHⅠ和HindⅢ同时将重组表达载体pET-28a(+)-rab7和表达载体pMAL-c2x进行双酶切,回收rab7目的基因和pMAL-c2x表达载体;将酶切后的莱茵衣藻rab7目的基因与原核表达载体pMAL-c2x连接,得到重组表达载体pMAL-c2x-rab7;将构建好的重组表达载体pMAL-c2x-rab7转入E.coli BL21(DE3)感受态细胞中,在含有氨苄抗性的LB平板上37℃培养12~14h,待平板长出转化子后,挑取单克隆菌落至含有氨苄抗性的液体LB培养基中扩大培养,当600nm处的吸光值A值达到0.6~0.8时,加入1M异丙基-β-D-硫代半乳糖苷至终浓度为0.2mM后进行诱导,且所述诱导的温度为25℃,所述诱导的时间为8h;离心菌液得到菌体,用PBS重悬后离心,重复两次以达到清洗目的;将菌体用pH7.4的结合缓冲液重悬后进行高压破碎,取全蛋白、上清和沉淀进行12%SDS-PAGE凝胶电泳分析,得到莱茵衣藻MBP-RAB7融合蛋白;将破碎后的菌液离心后取上清液,用0.45μm滤膜过滤后加入预先平衡好的Dextrin SepharoseTM High Performance蛋白纯化柱中,室温条件下结合2h,用pH 7.4结合缓冲液结合缓冲液漂洗去掉杂蛋白,而后用pH 7.4的洗脱缓冲液洗脱,得到纯度较高MBP-RAB7融合蛋白;称取CNBr-activated Sepharose 4B干粉溶胀于1mmol/LHCl,用pH 8.3的Coupling Buffer将溶胀后的CNBr-activated Sepharose 4B漂洗至pH为8.3;以MBP-RAB7融合蛋白作为抗原与CNBr-activated Sepharose 4B 4℃过夜结合,之后以pH 8.0的缓冲液即0.1mol/LTris-HCl室温结合2~4h,最后用pH 8.5的缓冲液和pH 3.5的缓冲液交替洗脱8次;将经过ProteinA纯化后的IgG亚型抗体与以上处理好的CNBr-activated Sepharose 4B室温结合2h后,以甘氨酸洗脱,最终获得纯度进一步提高的抗体;
其中,所述结合缓冲液的配方为:20mM Tris、200mM NaCI、1mM EDTA,溶剂为水;所述洗脱缓冲液的配方为:20mM Tris、200mM NaCI、1mM EDTA、10mM麦芽糖,溶剂为水;所述中和液为:pH9.0的1M Tris;所述pH 8.3的Coupling Buffer的配方为:0.1mol/L NaHCO3、0.5mol/LNaCI,溶剂为水;
所述pH 8.5的缓冲液的配方为:100mmol/L Tris-HCl、0.5mol/L NaC,溶剂为水;所述pH 3.5的缓冲液的配方为:50mmol/L Gly、1mol/L NaCl,溶剂为水。
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