CN116590263A - 一种重组蛋白、单克隆抗体及其检测试剂盒和应用 - Google Patents
一种重组蛋白、单克隆抗体及其检测试剂盒和应用 Download PDFInfo
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Abstract
本发明公开了一种MMP11重组蛋白，所述的重组蛋白的氨基酸序列如SEQ ID NO.1所示，所述的重组蛋白密码子优化后的核苷酸序列如SEQ ID NO.2所示。本发明同时公开了一种检测MMP11蛋白的试剂盒，所述试剂盒包括有效量的重组蛋白和有效量的特异性结合所述重组蛋白的单克隆抗体以及配套的检测试剂，所述的特异性结合所述重组蛋白的单克隆抗体为单克隆抗体1B2、单克隆抗体4C8，其中单克隆抗体1B2、单克隆抗体4C8的重链可变区序列如SEQ ID NO.3和SEQ ID NO.4所示；单克隆抗体1B2、单克隆抗体4C8的轻链可变区序列如SEQ ID NO.5和SEQ ID NO.6所示。本发明制备的重组蛋白和单克隆抗体以及在此基础上制备的试剂盒，具有良好的敏感性和准确性，适合用于MMP11蛋白的检测。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种重组蛋白、单克隆抗体及其检测试剂盒和应用。
背景技术
肺癌是导致癌症死亡的主要原因之一，其病因尚很不清楚。有文献报道(CN200710111205等)通过转录组微阵列数据，分析差异表达基因，识别具有治疗潜力的肺腺癌的关键驱动基因。细胞学实验表明，基质金属蛋白酶11(matrix metallopeptidase11，MMP11)显著抑制肺腺癌细胞的增殖、迁移和侵袭。在异种移植瘤模型中也获得了一致的结果。用抗MMP11抗体处理不同的人肺腺癌细胞系，可明显延缓细胞的生长和迁移。
因此，MMP11有可能是肺癌的一个筛选靶点或治疗靶点。但，现在还没有相关的诊断试剂或诊断材料。
发明内容
为了弥补现有技术的不足，本发明的目的之一在于提供一种MMP11重组蛋白及其制备方法和应用；本发明的目的之二在于提供一种MMP11的单克隆抗体及其制备方法和应用。
因此，本发明一方面提供了一种重组蛋白，所述重组蛋白为MMP11重组蛋白，所述的重组蛋白的氨基酸序列如SEQ ID NO.1所示。
优选地，本发明所述的重组蛋白密码子优化后的核苷酸序列如SEQ ID NO.2所示。
再一方面，本发明还提供了一种用于检测MMP11蛋白的试剂盒，所述试剂盒包括有效量的所述的重组蛋白和有效量的特异性结合所述重组蛋白的单克隆抗体以及配套的检测试剂。
优选地，本发明所述的特异性结合所述重组蛋白的单克隆抗体为单克隆抗体1B2、单克隆抗体4C8，其中单克隆抗体1B2、单克隆抗体4C8的重链可变区序列如SEQ ID NO.3和SEQ ID NO.4所示，单克隆抗体1B2、单克隆抗体4C8的轻链可变区序列如SEQ ID NO.5和SEQID NO.6所示。
再一方面，本发明还提供了一种所述的重组蛋白在制备MMP11蛋白检测试剂中的应用。
再一方面，本发明还提供了一种所述的单克隆抗体1B2、单克隆抗体4C8在制备MMP11蛋白检测试剂中的应用
本发明公开的MMP11重组蛋白具有良好的免疫原性，可用于制备MMP11蛋白的检测试剂(如校准品或单克隆抗体的免疫原)。另外，本发明在制备的MMP11重组蛋白的基础上，筛选了2株特异性结合MMP11蛋白的单克隆抗体，这2株单克隆抗体具有良好的敏感性和特异性，适用于制备MMP11蛋白的检测试剂。
本发明在制备的重组蛋白和单克隆抗体的基础上，开发了用于MMP11蛋白检测的试剂盒，该试剂盒具有较高的灵敏度和准确性。因此，该试剂盒具有较好的适用性。
附图说明
图1去除信号肽的MMP11蛋白无序区分析结果。分值越高代表该位置为无序区的概率越大，0.5作为是否为无序区的分界线。
图2MMP11重组蛋白SDS-PAGE图，其中1是纯化后的MMP11重组蛋白。
图3单克隆抗体制备生产模式图。
图4试剂盒标准曲线。
具体实施方式
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1：MMP11重组蛋白设计和制备
从NCBI(GenBank:KAI6060526.1)中挑选1个MMP11蛋白作为研究对象，首先，对MMP11蛋白的信号肽进行分析，有信号肽的概率为:99.115％,信号肽类型：SP(Sec/SPI)；切割位点：35-36,概率：76.780％；因此，去除N端35个氨基酸，对剩余序列进行蛋白无序区的分析，结果显示(图1)，aa1～aa70和aa225～aa260为无序区的概率较大，因此，去除这2个区域的氨基酸，对剩余的序列进行MMP11重组蛋白的制备，其氨基酸序列如SEQ ID NO.1所示。
将密码子优化后的MMP11重组蛋白的核苷酸序列(具体序列如SEQ ID NO.2所示)克隆到真核表达载体(如pEE12.4或pCDNA3.1等)中。以密码子优化的MMP11重组蛋白核苷酸序列为模板，分别用EcoRI和HindⅢ双酶切位点进行连接以构建含6His的重组表达质粒。将鉴定为阳性的重组质粒经测序检验正确后进行MMP11重组蛋白的表达。后续参照201611208261.8实施例1～6制备MMP11重组蛋白。
经过筛选后，共筛选到1株稳定分泌表达MMP11重组蛋白的CHO-K1细胞株，其表达产量能达到280mg/L或以上；经镍柱纯化后的MMP11重组蛋白的SDS-PAGE纯度能达到90％以上(图2所示)，SDS-PAGE分子量(图2所示)约为42kDa，比预测的分子量稍大，说明该重组蛋白有糖基化修饰。
实施例2：MMP11重组蛋白单克隆抗体的制备
使用实施例1制备的MMP11重组蛋白制备MMP11单克隆抗体。具体方法为常规的小鼠杂交瘤细胞技术(模式图如图3所示，来源于知乎)，其简述如下：
1、动物免疫：选用6～8周龄雌性Balb/c小鼠，按照制定的免疫方案进行免疫注射(一般免疫3次，在初次免疫2～3周后进行再次免疫，在再次免疫3周后进行加强免疫)，在最后一次加强免疫后第3天取脾进行融合。
2、细胞融合：采用眼球摘除放血法处死小鼠，无菌操作取出脾脏，在平皿内挤压研磨，制备脾细胞悬液。将准备好的同系骨髓瘤细胞与小鼠脾细胞按一定比例混合，并加入促融合剂聚乙二醇。在聚乙二醇作用下，各种淋巴细胞可与骨髓瘤细胞发生融合，形成杂交瘤细胞。
3、选择性培养：一般采用HAT选择性培养基。在HAT培养基中，未融合的骨髓瘤细胞因缺乏次黄嘌呤-鸟嘌呤-磷酸核糖转移酶，不能利用补救途径合成DNA而死亡。未融合的淋巴细胞虽具有次黄嘌呤-鸟嘌呤-磷酸核糖转移酶，但其本身不能在体外长期存活也逐渐死亡。只有融合的杂交瘤细胞由于从脾细胞获得了次黄嘌呤鸟嘌呤磷酸核糖转移酶，并具有骨髓瘤细胞能无限增殖的特性，因此能在HAT培养基中存活和增殖。
4、杂交瘤阳性克隆的筛选与克隆化：在HAT培养基中生长的杂交瘤细胞，只有少数是分泌预定特异性单克隆抗体的细胞，因此，必须进行筛选和克隆化。通常采用有限稀释法进行杂交瘤细胞的克隆化培养。采用灵敏、快速、特异的免疫学方法，筛选出能产生所需单克隆抗体的阳性杂交瘤细胞，并进行克隆扩增，及时进行冻存。
5、单克隆抗体的大量制备：采用动物体内诱生法和体外培养法。
(1)体内诱生法：取6～8周Balb/c小鼠，首先腹腔注射0.5ml液体石腊或降植烷进行预处理。1～2周后，腹腔内接种杂交瘤细胞。杂交瘤细胞在小鼠腹腔内增殖，并产生和分泌单克隆抗体。约1～2周，可见小鼠腹部膨大。用注射器抽取腹水，经过ProteinG柱纯化后即可获得大量单克隆抗体，分装保存于-80℃冰箱待用。
(2)体外培养法：将杂交瘤细胞置于培养瓶中进行培养。在培养过程中，杂交瘤细胞产生并分泌单克隆抗体，收集培养上清液，离心去除细胞及其碎片，，经过ProteinG柱纯化后即可获得大量单克隆抗体，分装保存于-80℃冰箱待用。但这种方法产生的抗体量有限。
经过以上步骤，使用实施例1的MMP11重组蛋白共筛选了2株较好的杂交瘤细胞，并在此基础上，制备了2株单克隆抗体，分别为单克隆抗体1B2、单克隆抗体4C8。
实施例3：两株单克隆抗体的性能检测
1、采用SouthernBiothech公司的SBA Clonotyping System-HRP试剂盒，按照说明书的操作来鉴定单克隆抗体重链和轻链的亚型。经过检测，两株单克隆抗体重链亚型均为IgG1，轻链均为Kappa。
2、根据抗体亚型结果，采用基于RACE技术路线的方法克隆抗体基因序列。收集生长状态良好的杂交瘤细胞，使用总RNA提取试剂盒获得杂交瘤细胞总RNA，按照Takara公司的SMARTer RACE说明书的操作方法将mRNA逆转录为cDNA，并扩增目的抗体全长序列。经过测序和分析，单克隆抗体1B2、单克隆抗体4C8的重链可变区序列如SEQ ID NO.3和SEQ IDNO.4所示；单克隆抗体1B2、单克隆抗体4C8的轻链可变区序列如SEQ ID NO.5和SEQ IDNO.6所示。
3、使用商品化试剂盒(HRP偶联试剂盒-Lightning-(ab102890))对两株单克隆抗体进行HRP标记。使用双抗体夹心方法对两株单克隆抗体的配对进行检测，以验证两株单克隆抗体的配对效果。结果显示(如表1所示)，两株单克隆抗体配对良好，且使用单克隆抗体1B2包被、单克隆抗体4C8检测取得的效果最好。
配对检测：使用两株单克隆抗体分别包被酶标板，100ng/孔，0.1ml/孔，4℃包被过夜；洗涤3次后，使用含2％的BSA的PBST封闭，37℃孵育2h；用PBS将实施例1制备的MMP11稀释至100ng/ml，再加到包被的酶标板中，0.1ml/孔，37℃孵育1h；洗涤3次后，交叉加入1:20000倍稀释的HRP标记的单克隆抗体(如单克隆抗体1B2包被时，使用单克隆抗体4C8进行检测)，0.1ml/孔，37℃孵育30min；洗涤3次后，加入0.1ml TMB单组分显色液，37℃孵育10min；加入终止液(2M硫酸)，测定OD450nm值，以OD450nm值最高为最好。
表1两株单克隆抗体交叉配对结果
| 项目 | 单克隆抗体1B2包被 | 单克隆抗体4C8包被 |
| 单克隆抗体1B2检测 | 未检测 | 2.12 |
| 单克隆抗体4C8检测 | 2.78 | 未检测 |
实施例4：MMP11蛋白的检测
1、标准品配制：使用稀释液(含1％BSA的PBST缓冲液)将本发明制备的MMP11重组蛋白稀释为6个梯度，分别为1000ng/ml、500ng/ml、250ng/ml、125ng/ml、62.5ng/ml、0ng/ml。
2、检测步骤和判定方法：使用本发明制备的单克隆抗体1B2作为包被抗体，包被于酶标板上，每孔100ng/0.1ml，2～8℃包被过夜；洗涤后，使用含1％BSA的PBST缓冲液封闭，每孔0.2ml，37℃封闭2h；洗涤后，加入样品(根据样品确定稀释度，使其落入标准曲线之间)和各个梯度的标准品，每孔0.1ml，37℃孵育30min；洗涤后，加入20000倍稀释的HRP标记的单克隆抗体4C8(实施例3制备)，每孔0.1ml，37℃孵育30min；洗涤后，加入TMB显色液，每孔0.1ml，37℃孵育10min；加入终止液(2M硫酸)，每孔0.05ml，立即检测OD450nm值。以标准品浓度为横坐标，以对应的标准品OD450nm值为纵坐标，拟合标准曲线(如图4所示)，并生成标准曲线函数和R2，其中R2应≥0.9。将稀释后待测样品OD450nm值代入标准曲线函数中，计算得到稀释后待测样品浓度，以此浓度*待测样品稀释倍数，即为待测样品的浓度。具体试剂盒配制如表2所示：
表2试剂盒组分与数量
| 试剂 | 数量 |
| 单克隆抗体1B2包被板 | 1块 |
| 标准品S1～S6 | 1ml/管，共7管 |
| 样品稀释液(含1％BSA的PBST缓冲液) | 30ml/瓶 |
| 25×浓缩洗涤液(25×PBST缓冲液) | 15ml/瓶 |
| 20000倍稀释HRP标记的单克隆抗体4C8 | 12ml/瓶 |
| TMB底物溶液 | 12ml/瓶 |
| 终止液 | 6ml/瓶 |
3、试剂盒灵敏度检测：对零标准溶液进行20次重复测试，取零定标溶液测定的平均值加上3倍的标准偏差，即为本试剂盒的检测限。经过检测和计算，本试剂盒的检测限为6ng/ml。
4、样品检测：对市场采购的3批重组人MMP11蛋白(购自abcam，货号为ab92861)进行检测，结果显示，本试剂盒检测的结果与标识浓度基本一致，偏差均在5％左右。具体见表3所示。同时使用本试剂盒对临床血清样品进行检测，检测结果显示，检测结果与临床具有较好的关联性。
表3试剂盒检测结果
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (6)
1.一种重组蛋白，所述重组蛋白为MMP11重组蛋白，其特征在于，所述的重组蛋白的氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的重组蛋白，其特征在于，所述的重组蛋白密码子优化后的核苷酸序列如SEQ ID NO.2所示。
3.一种检测MMP11蛋白的试剂盒，其特征在于，所述试剂盒包括有效量的权利要求1所述的重组蛋白和有效量的特异性结合权利要求1所述重组蛋白的单克隆抗体以及配套的检测试剂。
4.根据权利要求3所述的试剂盒，其特征在于，所述的特异性结合权利要求1所述重组蛋白的单克隆抗体为单克隆抗体1B2、单克隆抗体4C8，其中单克隆抗体1B2、单克隆抗体4C8的重链可变区序列如SEQ ID NO.3和SEQ ID NO.4所示，单克隆抗体1B2、单克隆抗体4C8的轻链可变区序列如SEQ ID NO.5和SEQ ID NO.6所示。
5.一种如权利要求1所述的重组蛋白在制备MMP11蛋白检测试剂中的应用。
6.一种如权利要求4所述的单克隆抗体1B2、单克隆抗体4C8在制备MMP11蛋白检测试剂中的应用。
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