CN114316048B - 一种可溶性b7-h5蛋白含量检测的酶联免疫检测试剂盒及其应用 - Google Patents

一种可溶性b7-h5蛋白含量检测的酶联免疫检测试剂盒及其应用 Download PDF

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CN114316048B
CN114316048B CN202111536301.2A CN202111536301A CN114316048B CN 114316048 B CN114316048 B CN 114316048B CN 202111536301 A CN202111536301 A CN 202111536301A CN 114316048 B CN114316048 B CN 114316048B
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张学光
石通国
黄子逸
张丽
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Suzhou Xuguang Kexing Antibody Biotechnology Co ltd
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Abstract

本发明公开了一种用于检测分析可溶性B7‑H5蛋白含量的酶联免疫试剂盒，其特征在于该试剂盒包括B7‑H5‑2E5和B7‑H5‑7B10两种抗人B7‑H5单克隆抗体：B7‑H5‑2E5包括重链和轻链，其中重链可变区的氨基酸序列为SEQ ID NO.1；轻链可变区的氨基酸序列为SEQ ID NO.2；B7‑H5‑7B10同样包括重链和轻链，其重链可变区的氨基酸序列为SEQ ID NO.3；B7‑H5‑7B10的轻链可变区的氨基酸序列为SEQ ID NO.4。此外，本发明还公开了该试剂盒的用途。本发明的酶联免疫试剂盒能够有效检测可溶性B7‑H5含量。利用该试剂盒检测发现，健康人血清中含有一定基础水平的可溶性B7‑H5蛋白，结直肠癌、肺癌和胃癌患者血清可溶性B7‑H5含量明显升高，与患者的临床分期和病理有相关性，肿瘤患者体液中可溶性B7‑H5含量检测具有潜在的临床价值。

Description

一种可溶性B7-H5蛋白含量检测的酶联免疫检测试剂盒及其 应用
技术领域
本发明属于生物医学技术领域，具体涉及一种对可溶性B7-H5蛋白含量进行定量分析的酶联免疫检测试剂盒及其用途。
背景技术
B7-CD28家族成员介导的共刺激信号在T细胞的活化及免疫耐受的调节中具有重要作用，随着共刺激分子相关功能研究进展，已成为免疫学研究新的热点之一。近年来，研究发现B7-CD28家族成员包括B7-H1(PD-L1)、B7-H2(PD-L2)、B7-H3、B7-H4、B7H5(VISTA)及B7-H6等在肿瘤微环境中异常表达，并参与肿瘤免疫逃逸等，对肿瘤的发生发展以及肿瘤病人的预后都起着重要的作用。
B7-H5是B7家族负性调控分子之一，研究表明，B7-H5在包括结直肠癌、非小细胞肺癌、胃癌等多种恶性肿瘤组织中高水平表达，且与患者的临床分期、预后等密切相关。另外据文献报道B7-H5在关节炎、系统性红斑狼疮、牛皮癣等自身免疫性疾病的发病和进展中发挥关键作用。但目前对于B7-H5的检测还存在检测手段缺乏的问题。
发明内容
为了克服现有技术的上述缺陷，本发明提供一种对可溶性B7-H5蛋白含量进行定量分析的酶联免疫检测试剂盒及其用途，从而弥补现有技术的不足。
本发明首先提供一种可以结合B7-H5蛋白的单克隆抗体，所述的单克隆抗体，其中包含有B7-H5-2E5活B7-H5-7B10两种抗人B7-H5单克隆抗体；
其中所述抗人B7-H5单克隆抗体B7-H5-2E5抗体，包括重链和轻链，所述B7-H5-2E5的重链可变区(mVH)的氨基酸序列为SEQ ID NO.1：
DVKLQESGPGLVKPSQSLSLTCTVTGYSIASDYTWNWIRQFPGNKLEWMGYIDYSGATFYNPSLKSRISIIRDTSKNQFFLQLNSVTTGDTATYYCTRGFYYYGSDYWGQGTTLTVSS；
B7-H5-2E5的轻链可变区(mVL)的氨基酸序列为SEQ ID NO.2：
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHNNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYYCFQGSHVPSTFGGGTKLEIK；
所述的抗人B7-H5单克隆抗体为B7-H5-7B10抗体，包括重链和轻链，所述B7-H5-7B10的重链可变区(mVH)的氨基酸序列为SEQ ID NO.3：
EVQLKQSGPGLVAPSQSLSITCTVSGFSLTNHGVQWVRQSPGKGLEWLGVIWGDGNRNYHSALMSRLSINKDNSKSQVFLKLNRLQTDDTATYFCAKERRLGYYGEYDVMDYWGQGTSVTVSS；
B7-H5-7B10的轻链可变区(mVL)的氨基酸序列为SEQ ID NO.4：
DIVMTQSHKFMSTSVGDRVTITCKASQGVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTIGNVQSEDLADYFCQQYSSSPFTFGSGTKLEIK。
本发明所提供的单克隆抗体用于制备检测可溶性B7-H5蛋白的试剂盒。
本发明另一个方面还提供一种检测可溶性B7-H5含量的酶联免疫检测试剂盒，其中包含有B7-H5-2E5和/或B7-H5-7B10抗人B7-H5单克隆抗体；
所述的酶联免疫检测试剂盒的用途，是用于检测肿瘤、自身免疫性疾病等患者可溶性B7-H5蛋白含量。
本发明还提供一种非疾病治疗目的的检测肿瘤、自身免疫性疾病患者中可溶性B7-H5蛋白含量的方法。
本发明的酶联免疫试剂盒能够有效检测可溶性B7-H5含量，利用该试剂盒检测发现，健康人血清中含有一定基础水平的可溶性B7-H5蛋白(～350pg/ml)，结直肠癌、肺癌和胃癌患者血清可溶性B7-H5含量明显升高，与患者的临床分期和病理有相关性，肿瘤患者体液中可溶性B7-H5含量检测具有潜在的临床价值。
附图说明
图1为本发明酶联免疫试剂盒检测样品中可溶性B7-H5蛋白含量示意图。
图2：单克隆抗体检测直肠癌、肺癌和胃癌患者血清可溶性B7-H5含量图。
具体实施方式
下面结合实施例和附图对本发明进行详细的描述。
实施例1：制备抗人B7-H5单克隆抗体杂交瘤细胞的
1.1小鼠的免疫
使用B7-H5融合重组蛋白免疫BALB/c小鼠五次，前三次间隔时间为21天，在第四次免疫之后5-7天测定小鼠血液抗体效价，效价较好时则再次进行第五次加强免疫。
1.2滋养层细胞的培养
1.2.1在无菌超净台中取出7-8周BALB/c小鼠的脾脏。在200目无菌筛网中，通过研磨获得单细胞悬液。
1.2.2单细胞悬液经RPMI-1640培养基洗涤2次，用适量含15％FBS的RPMI-1640培养基重悬细胞，使用无菌巴氏玻璃吸管将细胞液均匀滴入96孔培养板中，每孔的体积约为80ul，细胞培养箱中过夜培养。
1.3细胞融合及筛选
1.3.1在无菌超净台中取出7-8周BALB/c小鼠的脾脏。在200目无菌筛网中，通过研磨获得单细胞悬液。用37℃预热的1640基础培养基依照1400rpm/5min的条件离心重悬清洗2次。
1.3.2将收集的脾脏细胞与处于生长对数期的sp2/0细胞按大约5:1的比例在离心管中混匀，并加入预热的RPMI-1640基础培养基充分混匀，之后1400rpm/5min的条件离心一次，弃上清后用手指轻弹管底来混匀两种细胞。
1.3.3向两种混合细胞中滴加预热的PEG溶液1ml，期间不停轻轻震荡离心管，加速两种细胞融合。
1.3.4融合完毕后，滴入14ml RPMI-1640基础培养基来终止融合过程。离心，去上清。
1.3.5加入RPMI-1640全培养基重悬融合后的细胞，随后使用巴氏玻璃将含有滋养层细胞的96孔培养板中，每孔大约80μl/孔，培养箱中培养，每隔3-4天用HT培养基进行换液，之后每孔取少量上清液进行ELISA检测。
1.3.6挑选抗体检测值较高的孔进行亚克隆，每次亚克隆后都进行ELISA检测，通常经过3-4次亚克隆后才能获得稳定分泌抗体表型的细胞，并对最后一次亚克隆后的细胞进行冻存。
实施例2：抗人B7-H5单克隆抗体轻重链可变区序列的测定
测定抗人B7-H5单克隆抗体重链和轻链可变区的方法包括以下步骤：
2.1杂交瘤细胞cDNA的获取
从目标的杂交瘤细胞中获取RNA，通过逆转录技术将获得的RNA反转录为cDNA。利用特定设计的上下游引物PCR克隆该杂交瘤细胞的重链可变区(mVH)和轻链可变区(mVL)。
2.2分别将重链可变区(mVH)和轻链可变区(mVL)与pJET cloning vector克隆载体连接。随后，将连接的产物转化至DH5a感受态细菌后，将转化后的菌液均匀涂布于LB固体培养基上。
2.3挑选LB固体培养基上边缘清晰、生长良好的菌落进行测序鉴定。
2.4根据测序结果保留候选的轻重链可变区序列，再次通过PCR克隆出可与表达载体连接的轻链和重链的可变区序列，并将可变区序列与表达载体连接，随后将连接产物转化至DH5a。将转化菌液均匀涂布LB固体培养基，培养过夜。
2.5挑选生长良好的细菌测序，并对比两次测序结果从而得到具有正确的序列的转化细菌，通过扩大培养后进行质粒抽提。
2.6将连有当克隆抗体重链和轻链可变区基因的表达载体一起共同转染真核表达细胞293。
2.7 293细胞用无血清培养基SFM4Transfx-293 without L-Glutamine悬浮培养，在转染时培养基替换为无血清培养基
Figure BDA0003411372620000041
FreeStyleTM 293 Expression Medium。
2.7利用ELISA试剂盒检测含有目的抗体的上清液，结果显示良好。
通过上述方法来获得抗人B7-H5-2E5单克隆抗体重链可变区：
DVKLQESGPGLVKPSQSLSLTCTVTGYSIASDYTWNWIRQFPGNKLEWMGYIDYSGATFYNPSLKSRISIIRDTSKNQFFLQLNSVTTGDTATYYCTRGFYYYGSDYWGQGTTLTVSS；
B7-H5-2E5单克隆抗体轻链可变区:
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHNNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYYCFQGSHVPSTFGGGTKLEIK；
B7-H5-7B10单克隆抗体重链可变区
EVQLKQSGPGLVAPSQSLSITCTVSGFSLTNHGVQWVRQSPGKGLEWLGVIWGDGNRNYHSALMSRLSINKDNSKSQVFLKLNRLQTDDTATYFCAKERRLGYYGEYDVMDYWGQGTSVTVSS；
B7-H5-7B10单克隆抗体轻链可变区
DIVMTQSHKFMSTSVGDRVTITCKASQGVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTIGNVQSEDLADYFCQQYSSSPFTFGSGTKLEIK
本发明从表达抗人B7-H5单克隆抗体的杂交瘤细胞中提取到抗体的重链和轻链的可变区，根据测序结果保留候选的轻重链可变区序列，并通过PCR扩增出与表达载体匹配的轻重链可变区序列，连接PCR产物与双酶切预处理的表达载体。将连有目的单抗轻链和重链可变区的表达载体共同转染真核表达细胞株293，收取培养后的上清含有目的抗体，说明得到的重链和轻链可变区序列是正确的。
实施例3：检测可溶性B7-H5蛋白含量的ELISA试剂盒的检测方法
3.1试剂盒的组成
本发明的ELISA试剂盒包括包被于酶标板上的实施案例1中制备的B7-H5包被抗体(B7-H5-2E5)、经生物素标记的B7-H5检测抗体(B7-H5-7B10)、可溶性B7-H5蛋白标准品(R&DSystems)、辣根过氧化物酶(HRP)、样本稀释液、洗涤液(PBST)、显色液(TMB)及终止液组成。
3.2样本的采集及处理
3.2.1收集一批医院结直肠癌、肺癌和胃癌患者的血清，分装后-80℃冰箱保存，并避免反复冻融。
3.3.2血清样本检测前，提前将样本常温平衡半小时，并震荡混匀，取适量病人血清用样本用稀释液稀释进行50倍稀释。
3.3可溶性B7-H5蛋白含量的测定方法
3.3.1使用包被液(Na2CO3和NaHCO3)稀释包被抗体B7-H5-2E5(1μg/ml),随后将包被抗体加入96孔酶标板(100μl/孔)，4℃孵育过夜。
3.3.2第二天，用洗涤液(PBST)洗板3次，随后每孔加入3％牛血清封闭液100μl，37℃封闭1h。
3.3.3去除封闭液，使用移液器加入100μl稀释好的待测血清样品以及梯度稀释好的可溶性B7-H5蛋白标准品(从2ng/ml开始对比稀释，共设置7个浓度，最后一空加样品稀释液为空白对照，每个梯度做3个重复)，室温条件下75rpm震荡孵育1h。
3.3.4PBST洗液洗板三次，加入生物素标记的检测抗体B7-H5-7B10(100μl/孔)，室温条件下75rpm震荡孵育1h。
3.3.PBST洗液洗板洗板三次，加入辣根过氧化物酶(HRP，100μl/孔)，室温条件下75rpm震荡孵育1h。
3.3.5PBST洗液洗板六次，加入显色液TMB(100μl/孔)，室温震荡孵育10min，之后加入终止液(100μl/孔)终止颜色反应，通过酶标仪测定每孔的OD450值。
3.3.6以B7-H5标准品浓度乘以样品稀释倍数(×50)的值为纵坐标，以相应测得的OD450值为横坐标制作标准曲线(如图1)，并得到计算公式，根据待测样品的OD450值计算出样品中可溶性B7-H5的含量。
本发明试剂盒按照方法学鉴定，可达到以下指标：
标准曲线线性：R2＝0.9990；最低检测限≤7.285pg/ml。
3.3.7本发明的酶联免疫试剂盒能够有效检测可溶性B7-H5含量。利用该试剂盒检测发现，健康人血清中含有一定基础水平的可溶性B7-H5蛋白(～350pg/ml)，结直肠癌、肺癌和胃癌患者血清可溶性B7-H5含量明显升高(图2)，与患者的临床分期和病理有相关性，肿瘤患者体液中可溶性B7-H5含量检测具有潜在的临床价值。
由于目前国内外市场上没有检测可溶性B7-H5含量的酶联免疫试剂盒，由此可见本发明的试剂盒，对后期的数据采集等工作的具有重大突破意义。
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Claims (3)

1.一种抗人B7-H5蛋白的单克隆抗体，其特征在于，所述的单克隆抗体包括重链和轻链，其中的重链可变区的氨基酸序列为SEQ ID NO.3，轻链可变区的氨基酸序列为SEQ IDNO.4。
2.权利要求1所述的单克隆抗体在制备检测可溶性B7-H5蛋白的试剂盒中的应用。
3.一种检测可溶性B7-H5含量的酶联免疫检测试剂盒，其特征在于，所述的检测试剂盒中包含有权利要求1所述的单克隆抗体。
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