CN108802357B - 一种用于免疫印迹分析的生物发光探针及其应用 - Google Patents
一种用于免疫印迹分析的生物发光探针及其应用 Download PDFInfo
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Abstract
本发明提供了一种用于免疫印迹分析的生物发光探针,所述探针的氨基酸序列如SEQ ID NO.1所示,包括:可结合一级抗体的串联Z结构域;具有生物发光功能的萤光素酶结构域;上述两个结构域的连接区。本发明同时还提供了上述探针的制备方法以及应用。该探针可通过大肠杆菌表达获得,无需通过动物活体或哺乳动物细胞制备,因此具有生产成本低廉的特点;此外,该探针可识别不同来源的一级抗体,并针对待检测的目的蛋白质产生高灵敏度的生物发光信号,因此可替代二级抗体用于免疫印迹分析。
Description
技术领域
本发明涉及一种用于免疫印迹分析的新型生物发光探针,属于免疫分析领域。
背景技术
基于抗原-抗体特异性识别的蛋白质免疫印迹分析不仅是生物化学与分子生物学研究中的必要工具,同时也在疾病诊断、食品安全和法医鉴定中具有重要的应用价值。免疫印迹技术的关键在于将抗体对蛋白质的识别转化为可检测的信号。上述信号转化的方式可分为直接法和间接法两类。其中,直接法是在能够识别目的蛋白质的一级抗体上标记信号分子,从而实现目的蛋白质的直接检测。而间接法则先使用未标记的一级抗体对目的蛋白进行识别,然后再通过标记了信号分子的二级抗体对一级抗体进行识别,从而实现目的蛋白质的检测。相对于直接法,间接法具有灵敏度高的优点,因此已经成为了免疫印迹分析中应用最为广泛的方法。
然而,利用二级抗体进行免疫印迹分析仍存在诸多不足,主要包括:1)通过化学方法对抗体进行标记不仅复杂、繁琐,而且有时会导致抗体的稳定性和亲和力下降;2)二级抗体的生产大多依赖于动物活体或哺乳动物细胞,导致生产成本相对高昂;3)二级抗体的来源必须与一级抗体不同,因此通用性较差。
发明内容
为克服现有技术中的不足,本发明提供了一种用于免疫印迹分析的新型生物发光探针,该探针可通过大肠杆菌表达获得,无需通过动物活体或哺乳动物细胞制备,因此具有生产成本低廉的特点;此外,该探针可识别不同来源的一级抗体,并针对待检测的目的蛋白质产生高灵敏度的生物发光信号,因此可替代二级抗体用于免疫印迹分析。
实现本发明上述目的所采用的技术方案为:
一种用于免疫印迹分析的生物发光探针,所述探针的氨基酸序列如SEQ ID NO.1所示,包括:可结合一级抗体的串联Z结构域;具有生物发光功能的萤光素酶结构域;上述两个结构域的连接区。
一种编码权利要求1所述氨基酸序列的基因片段,所述基因片段的核苷酸序列如SEQ ID NO.2所示,包括:串联Z结构域的编码序列;萤光素酶结构域的编码序列;上述两个结构域连接区的编码序列。
本发明提供了上述用于免疫印迹分析的生物发光探针的制备方法,包括以下步骤:
(1)编码基因片段的设计与制备:对Z结构域和萤光素酶结构域的编码序列和连接区编码序列进行拼接,然后根据大肠杆菌密码子偏好性,对拼接序列进行序列优化,从而使其在大肠杆菌中具有更高的表达效率,优化后的基因片段通过全基因合成制备得到,并连入pUC57载体。
(2)编码基因片段的扩增和纯化:以上述pUC57载体为模板,通过特异性引物P1和P2扩增所述探针的编码基因片段,并在其两端添加NdeI和SalI酶切位点,所得扩增产物通过琼脂糖凝胶电泳回收,即制得编码基因片段;所述特异性引物P1和P2的核苷酸序列分别如SEQ ID NO.3和SEQ ID NO.4所示;
(3)酶切、连接及转化:第(2)步所得编码基因片段和pET-26b(+)载体分别通过限制性核酸内切酶NdeI和SalI进行双酶切,将酶切后的基因片段和质粒载体混合后,通过T4DNA连接酶进行连接,所得连接产物转化至大肠杆菌DH5α感受态细胞,并挑取转化子进行测序,测序结果正确的表达载体保存、待用;
(4)生物发光探针的表达与纯化:挑取含有所述表达载体的大肠杆菌到含有卡那霉素的LB液体培养基中,震荡培养,加入IPTG并继续震荡培养,所得菌液离心收集菌体沉淀,并用细菌裂解缓冲液重悬菌体沉淀,通过超声波对上述菌体进行破碎,所得裂解液离心,最后通过镍亲和层析对上清液中含有的生物发光探针进行纯化,即制得所述用于免疫印迹分析的生物发光探针。
本发明的另一目的是提供上述生物发光探针进行蛋白质免疫印迹分析的方法,包括以下步骤:
(1)待检测蛋白质的分离和转印:通过SDS聚丙烯酰胺凝胶电泳分离待检测蛋白质,并通过半干法将凝胶中分离的蛋白质转印到聚偏氟乙烯(PVDF)膜上。
(2)免疫印迹分析:用脱脂奶粉封闭上述转印有待检测蛋白质的PVDF膜,然后使用一级抗体对膜上的待检测蛋白质进行杂交,再通过所述生物发光探针对一级抗体进行杂交,最后在膜表面添加萤光素酶底物缓冲液,并通过成像仪拍摄结果。
与现有技术相比本发明的有益效果为:(1)本发明所述生物发光探针可替代二级抗体实现待检测蛋白质的免疫印迹分析,并且无需复杂、繁琐的化学标记即可产生高灵敏度的检测信号;(2)所述生物发光探针为单体蛋白质,可通过廉价的大肠杆菌制备,避免了昂贵的动物免疫或哺乳动物细胞培养;(3)所述生物发光探针可识别不同种类的一级抗体,因此具有良好的通用性。
附图说明
图1为本发明中所提供的生物发光探针的结构示意图;
图2为本发明中所提供的生物发光探针的生物发光光谱示意图;
图3为本发明中所提供的生物发光探针的免疫印迹分析原理示意图;
图4为本发明中所提供的生物发光探针在人流感病毒血凝素抗原(HA)免疫印迹分析中应用结果的示意图;
图5为本发明中所提供的生物发光探针在小鼠β-微管蛋白免疫印迹分析中应用结果的示意图。
具体实施方式
下面结合具体实施例对本发明做详细具体的说明,但是本发明的保护范围并不局限于以下实施例。
实施例1:一种用于免疫印迹分析的新型生物发光探针表达载体的构建
(1)编码基因片段的设计与制备:在GeneBank中查找Z结构域和萤光素酶结构域的编码序列(序列编号分别为:DQ172901.1和KY776554.1),通过Primer 5.0软件对上述序列和连接区编码序列进行拼接,然后根据大肠杆菌密码子偏好性,对拼接序列进行序列优化,从而使其在大肠杆菌中具有更高的表达效率,优化后的基因片段通过全基因合成制备得到,并连入pUC57载体。
(2)编码基因片段的扩增和纯化:以上述亚克隆载体为模板,通过特异性引物P1和P2扩增所述探针的编码基因片段,并在其两端添加NdeI和SalI酶切位点,扩增条件为:第一步,98℃,5min;第二步,98℃,20s;第三步,60℃,20s;第四步,72℃,60s;第二步至第四步循环25次;第五步,72℃,10min;第六步,4℃,30s,所得扩增产物上样至1%(w/v)琼脂糖凝胶中,恒压100V电泳30min,然后将凝胶置于0.5%(w/v)溴化乙锭溶液中染色10min,在紫外灯下切割含有编码基因片段的胶块,按照琼脂糖凝胶回收试剂盒(Magen公司生产)说明书推荐步骤回收该片段;
(3)酶切、连接及转化:第(2)步所得编码基因片段和pET-26b(+)载体分别通过限制性核酸内切酶NdeI和SalI进行双酶切,其条件为37℃反应2h,将酶切后的基因片段和质粒载体按照摩尔比5:1混合后,通过T4DNA连接酶进行连接,连接条件为16℃反应4h,所得连接产物转化至大肠杆菌DH5α感受态细胞,并挑取3个转化子进行测序,测序结果正确的表达载体保存于-20℃冰箱中待用;
实施例2:生物发光探针的表达与纯化
将重组载体转化至大肠杆菌BL21(DE3)感受态细胞,挑取阳性转化子到含有30μg/mL卡那霉素的50mL LB液体培养基(1%蛋白胨,0.5%酵母提取物,1%NaCl,以上均为质量体积比)中,于30℃,150r/min震荡培养至菌液OD600为0.5时,加入终浓度为0.5mM的IPTG,并继续于30℃,150r/min震荡培养12h,所得菌液于10,000g离心10min收集菌体沉淀,并用6mL细菌裂解缓冲液(20mM Tris-Cl,500mM NaCl,pH=7.4)重悬菌体沉淀,通过超声波对上述菌体进行破碎,超声条件为:80W,超声4s,停4s,共计重复50次,所得裂解液于30000g离心30min,上清液中含有的生物发光探针通过镍亲和层析试剂盒(康为世纪公司)进行纯化,操作步骤参照试剂盒说明进行,纯化所得探针的结构如图1所示。
该探针无需化学修饰即可产生明显的生物发光信号,其最大发射光波长为452.5nm,如图2所示。
实施例3:一种新型生物发光探针在人流感病毒血凝素抗原免疫印迹分析中的应用
(1)待检测蛋白质的分离:将40μL具有人类流感病毒血凝素抗原(HA)的待检测蛋白质与10μL 5×上样缓冲液(购自康为世纪公司)混匀后,于100℃加热10min,取10μL上述溶液上样至变性聚丙烯酰胺凝胶中,恒流25mA电泳1h。
(2)待检测蛋白质的转印:取出第(1)步得到的凝胶,用超纯水漂洗5min,与此同时,将PVDF膜和滤纸于甲醇中浸泡2min,将经过漂洗的凝胶和甲醇处理过的滤纸和PVDF膜于转移缓冲液中(3.03g Tris碱,14.42g甘氨酸,200mL甲醇,用超纯水定容至1L)平衡5min,按照从负极到正极的顺序将滤纸、凝胶、PVDF膜、滤纸平铺在转印仪上,接通电源后,于恒流200mA的条件下转印20min,完成转印后,将PVDF膜在10mL含有5%(w/v)脱脂奶粉的PBS缓冲液(购自Amersco公司)中封闭1h,倒去封闭液,向10mL含有5%(w/v)脱脂奶粉的PBS缓冲液中加入5μL浓度为1mg/mL的抗HA的一级抗体(购自康为世纪公司),上述一抗杂交液与经过封闭的PVDF膜于室温下孵育1h,倒去一抗杂交液,用PBS缓冲液漂洗PVDF膜3次,每次5min,向10mL含有5%(w/v)脱脂奶粉的PBS缓冲液中加入5μL浓度为0.5mg/mL的生物发光探针,上述探针杂交液与经过一抗杂交的PVDF膜于室温下孵育1h,倒去探针杂交液,用PBS缓冲液漂洗PVDF膜3次,每次5min,最后向PVDF膜上添加100μL萤光素酶底物缓冲液(购自Promega公司),并通过成像仪拍摄结果,上述检测过程的原理如图3所示,实验结果表明:基于所述生物发光探针的免疫印迹方法可对HA抗原进行高灵敏度检测,检测结果如图4所示,图4中1至8号泳道分别对应:7.2pmol、5.8pmol、4.8pmol、4.1pmol、3.6pmol、3.2pmol、2.88pmol和2.6pmol待测蛋白质。
实施例4:一种新型生物发光探针在小鼠β-微管蛋白免疫印迹分析中的应用
将40μL小鼠肝脏组织裂解液与10μL 5×上样缓冲液混匀后,于100℃加热10min,其余电泳、转印、杂交和结果拍摄条件参见实施例3,唯一不同之处在于使用抗β-微管蛋白抗体作为一级抗体,以上实验结果表明:基于所述生物发光探针的免疫印迹方法可对小鼠β-微管蛋白进行特异性检测,不受组织中其他蛋白质的干扰,检测结果如图5所示,图5中1至5号泳道分别对应:小鼠肝脏组织裂解液稀释5倍、10倍、15倍、20倍、25倍的样品。
以上描述是对本发明的解释,不是对本发明的限定,本发明所限定的范围参见权利要求,在不违背本发明的精神的情况下,本发明可以做任何形式的修改。
序列表
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Claims (4)
1.一种用于免疫印迹分析的生物发光探针,其特征在于:所述探针的氨基酸序列如SEQID NO.1所示,包括:可结合一级抗体的串联Z结构域;具有生物发光功能的萤光素酶结构域;上述两个结构域的连接区。
2.一种编码权利要求1所述氨基酸序列的基因片段,其特征在于:所述基因片段的核苷酸序列如SEQ ID NO.2所示,包括:串联Z结构域的编码序列;萤光素酶结构域的编码序列;上述两个结构域连接区的编码序列。
3.权利要求1所述的用于免疫印迹分析的生物发光探针的制备方法,其特征在于包括以下步骤:
(1)编码基因片段的设计与制备:对Z结构域和萤光素酶结构域的编码序列和连接区编码序列进行拼接,然后根据大肠杆菌密码子偏好性,对拼接序列进行序列优化,从而使其在大肠杆菌中具有更高的表达效率,优化后的基因片段通过全基因合成制备得到,并连入pUC57载体;
(2)编码基因片段的扩增和纯化:以上述pUC57载体为模板,通过特异性引物P1和P2扩增所述探针的编码基因片段,并在其两端添加NdeI和SalI酶切位点,所得扩增产物通过琼脂糖凝胶电泳回收,即制得编码基因片段;所述特异性引物P1和P2的核苷酸序列分别如SEQID NO.3和SEQ ID NO.4所示;
(3)酶切、连接及转化:第(2)步所得编码基因片段和pET-26b(+)载体分别通过限制性核酸内切酶NdeI和SalI进行双酶切,将酶切后的基因片段和质粒载体混合后,通过T4DNA连接酶进行连接,所得连接产物转化至大肠杆菌DH5α感受态细胞,并挑取转化子进行测序,测序结果正确的表达载体保存、待用;
(4)生物发光探针的表达与纯化:挑取含有所述表达载体的大肠杆菌到含有卡那霉素的LB液体培养基中,震荡培养,加入IPTG并继续震荡培养,所得菌液离心收集菌体沉淀,并用细菌裂解缓冲液重悬菌体沉淀,通过超声波对上述菌体进行破碎,所得裂解液离心,最后通过镍亲和层析对上清液中含有的生物发光探针进行纯化,即制得所述用于免疫印迹分析的生物发光探针。
4.利用权利要求1所述生物发光探针进行蛋白质免疫印迹分析的方法,其特征在于包括以下步骤:
(1)待检测蛋白质的分离和转印:通过SDS聚丙烯酰胺凝胶电泳分离待检测蛋白质,并通过半干法将凝胶中分离的蛋白质转印到聚偏氟乙烯(PVDF)膜上;
(2)免疫印迹分析:用脱脂奶粉封闭上述转印有待检测蛋白质的PVDF膜,然后使用一级抗体对膜上的待检测蛋白质进行杂交,再通过所述生物发光探针对一级抗体进行杂交,最后在膜表面添加萤光素酶底物缓冲液,并通过成像仪拍摄结果。
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