CN116444655B - 一种鼠源性新型位点单克隆抗体阻断剂及其应用 - Google Patents
一种鼠源性新型位点单克隆抗体阻断剂及其应用 Download PDFInfo
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Abstract
本发明提供了一种鼠源性新型位点单克隆抗体阻断剂及其应用。本发明从具有高血清ROS含量的乙肝患者血液中筛选获得了乙肝病毒新型突变株的HBsAg抗原序列，以突变位点集中区域为目标抗原，将其核苷酸序列导入大肠杆菌表达载体中，制备并获得目标抗原；利用所述HBsAg抗原免疫动物，获得相应单克隆抗体，所述抗体能够高特异性结合目标抗原，并具有良好的热稳定性；利用所述单克隆抗体，制备相关检测试剂盒，该试剂盒具有高度的灵敏性和特异性。
Description
技术领域
本发明属于疾病诊断和检测领域，具体提供了一种鼠源性新型位点单克隆抗体阻断剂及其应用。
背景技术
乙型肝炎病毒 (hepatitis B，HBV) 感染是一个全球公共卫生问题，15-25% 的慢性感染者会因肝硬化、肝细胞癌或肝炎而发展为严重疾病并过早死亡。
目前，没有针对急性乙型肝炎患者的特异性抗病毒治疗药物，大约 95% 的感染免疫功能正常的成年人会自发恢复，护理的主要目标是保持舒适、缓解症状并防止患者将感染传染给他人。对于慢性 HBV 感染者，现在已经提供一些治疗药物和治疗方案，如美国食品和药物管理局已批准治疗慢性乙型肝炎干扰素-α（标准和聚乙二醇化）和口服抗病毒药物（恩替卡韦、替诺福韦、富马酸二泊西、艾拉酚替诺福韦和非首选的拉米夫定、阿德福韦二吡呋酯和替比夫定）。目前对慢性乙型肝炎的治疗可以减缓或预防肝硬化的进展，降低肝癌的发病率，提高长期生存率和生活质量，但不能治愈。因此，大多数开始乙肝治疗的患者必须终生服药，治疗的副作用和所需的定期监测增加了患者管理的难度和复杂性。因此，乙型肝炎的防治主要依赖于早期诊断和接种疫苗。
HBV是一种DNA病毒，属于嗜肝DNA病毒科，它于 1965 年由 Blumberg 博士发现的，Blumberg 博士因此项重大发现而获得了 1976 年诺贝尔医学奖。HBV 病毒，最初称为Dane 颗粒，是一种 42 纳米病毒，HBV由核衣壳核心组成，被外脂蛋白外壳（也称为包膜）包围。该病毒包含3种主要结构抗原：表面抗原（Hepatitis B surface antigen，HBsAg）、核心抗原（Hepatitis B core antigen ，HBcAg）和e抗原（Hepatitis B e antigen，HBeAg），其中HBsAg 表达量大，在受感染个体的血液中以球形和管状颗粒（约 22 nm）的形式存在，使得HBsAg成为乙肝检测试剂和疫苗开发中的重要靶点。
HBV除可导致肝炎和肝硬化之外，还是肝癌的最主要的诱因之一，据统计，全球约50-80% 的肝细胞癌 （hepatocellular carcinoma，HCC）是由 HBV 感染引起的（参见WildCP, Montesano R. A model of interaction: aflatoxins and hepatitis viruses inliver cancer aetiology and prevention. Cancer Lett. 2009;286(1):22–28），在过去的几十年中，已经证实HBV感染可以通过多种机制促进疾病进展，最终导致肝细胞的恶性转化，包括HBV基因整合、突变引起的基因组不稳定性、促癌信号通路的激活等，此外表观遗传学、外泌体、自噬、代谢调控、免疫抑制等诸多新机制的作用机理也在不断探索中（参见YuJiang, Qiuju Han,Huajun Zhao,and Jian Zhang,The Mechanisms of HBV-InducedHepatocellular Carcinoma, J Hepatocell Carcinoma. 2021; 8: 435–450.）。在众多作用机制中，氧化应激在肿瘤的发生和发展中起重要作用，HBV感染患者肝脏和血液中的活性氧（Reactive oxygen species，ROS）水平升高，这与疾病的严重程度和HBV的复制状态有关，并且这种活性氧的升高还通常与HBV的基因突变相关，但是目前临床上却缺乏对于相关基因突变的有效检测途径，不利于临床诊断和肝脏疾病的治疗。
本发明中乙肝患者血液样品中分离鉴定出导致ROS水平显著升高的新型乙肝病毒突变株，进而获得了其HBsAg氨基酸序列，并以该HBsAg为基础设计目标抗原，免疫实验动物，筛选并获得单克隆抗体，该抗体能够高特异性结合目标抗原，具有高灵敏性和特异性，为开发新型乙肝病毒检测试剂提供了新的解决方案。
发明内容
为解决上述技术问题，本发明中提供了一种鼠源单克隆抗体，包含轻链可变区和重链可变区，其轻链可变区包括氨基酸序列为SEQ ID NO:1的LCDR1，氨基酸序列为SEQ IDNO:2的LCDR2，氨基酸序列为SEQ ID NO:3的LCDR3；重链链可变区包括氨基酸序列为SEQ IDNO:4的HCDR1，氨基酸序列为SEQ ID NO:5的HCDR2，氨基酸序列为SEQ ID NO:6的HCDR3。
本发明所提供的单克隆抗体能够高度特异性结合目标抗原，所述抗原来自新型乙肝病毒突变株，所述突变株能够导致血清中ROS水平升高，使得患肝癌风险增加，为了研发相关检测试剂，实现肝癌的早诊断、早介入和早治疗提供了解决方案。
进一步的，所述轻链可变区的氨基酸序列如SEQ ID NO:7所示。
进一步的，所述重链可变区的氨基酸序列如SEQ ID NO:8所示。
提供了 一种乙肝病毒表面抗原，其氨基酸序列如SEQ ID NO:10所示。
该乙肝病毒表面抗原为发明人从乙肝患者血液样品中分离鉴定而得，其在相应位点具有氨基酸突变，所述突变与患者血清中的高水平ROS密切相关；根据序列分析，选择具有较多突变位点的区域为目标抗原，采用大肠杆菌表达载体大规模制备所述HBsAg蛋白作为目标抗原，用于筛选新型单克隆抗体。
提供了一种乙肝病毒检测试剂盒，包括本发明中所述的鼠源单克隆抗体。
进一步的，所述的试剂盒还包括标准品冻干粉、样品稀释液、显色剂、终止液和洗涤液。
进一步的，所述的试剂盒包括包被有所述单克隆抗体的96孔板中；辣根过氧化酶标记抗人IgG单克隆抗体作为酶标结合物；所述的标准品冻干粉为乙肝病毒表面抗原蛋白，其氨基酸序列如SEQ ID NO:10所示；所述的样品稀释液为0.01mM、pH值为7.4的PBS缓冲液；所述的洗涤液的溶剂为含有体积百分浓度为0.2%的吐温-20的0.01mM、pH值为7.4的PBS缓冲液；所述的终止液为2M的硫酸水溶液；所述的显色剂包括辣根过氧化物酶催化底物A液和B液；所述的辣根过氧化物酶催化底物A液是体积浓度为3%的H2O2水溶液；所述的辣根过氧化物酶催化底物B液为含0.1mg/mL的3,3',5,5'-四甲基联苯胺的0.1 mol/L、pH为6.0的磷酸缓冲液。
提供了一种所述的鼠源单克隆抗体或所述的乙肝病毒表面抗原在制备乙肝病毒检测试剂中的应用。
有益效果
本申请提供了一种鼠源性新型位点单克隆抗体阻断剂及其应用，及其在制备乙肝检测试剂中的应用，具有以下优势：
（1）筛选获得了乙肝病毒新型突变株的HBsAg抗原序列，所述突变株使得患者血清ROS水平急剧升高，导致患肝癌风险增大，以突变位点集中区域为目标抗原，将其核苷酸序列导入大肠杆菌表达载体中，制备并获得目标抗原；
（2）利用所述HBsAg抗原免疫动物，获得相应单克隆抗体，所述抗体能够高特异性结合目标抗原，并具有良好的热稳定性。
（3）利用所述单克隆抗体，制备相关检测试剂盒，该试剂盒具有高度的灵敏性和特异性。
附图说明
图1：乙肝患者血清ROS水平；
图2：HbsAg抗原核苷酸酶切鉴定图；
图3：ROC曲线；
实施方式
以下非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何形式限制本发明。凡基于本发明上述内容所实现的技术均应当属于本申请要求保护的范围之中。
以下实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂生物材料、检测试剂盒，如无特殊说明，均可从商业途径获得。
实施例1 乙肝病毒突变株鉴定
1.1导致ROS升高乙肝病毒突变株的筛选
氧化应激是导致乙型肝炎癌变的重要途径，为了筛选可能导致乙肝患者活性氧自由基水平升高的乙肝病毒突变株，本实施例中收集58例乙肝转化为肝癌或肝硬化患者血液样本，3 000 r/min 离心 10 min，收集血清，使用ROS的ELISA试剂盒（购自上海 BestBio贝博生物科技有限公司）检测血清中的活性氧水平，具体步骤参照试剂盒说明书进行。
结果如图1所示，本实施例中共筛选出明显高于乙肝患者血清ROS平均水平的乙肝病毒株12例，用于后续进行序列分析。
1.2 乙肝病毒突变株的序列鉴定
使用TIANamp Virus DNA/RNA Kit（购自TIANGEN公司）提取HBV DNA，具体步骤按照试剂盒操作说明书进行。
采用巢式PCR对样本DNA的 S 片段进行靶向扩增，扩增引物序列见表1。
表1 巢式PCR引物序列
| 引物名称 | 引物序列（5’-3‘） |
| 外侧正向引物OR | CCTAGGA CCCCTGCT |
| 外侧反向引物OF | AGAAAATTGGTAATAG |
| 内侧正向引物IR | TTGACAA GAATCCTCAC |
| 内侧反向引物IF | ATACCACATCATCC |
使用巢式PCR试剂盒（购自TAKATA公司）进行扩增反应，第一轮PCR反应体系表2所示，反应条件为：94℃ 3min；94℃ 30s，58℃ 30s，72℃ 60s，30个循环；72℃ 5min。
表2 第一轮PCR反应体系
| 试剂 | 体积/μL |
| DNA样品 | 2 |
| 10×Primer STAR 缓冲液 | 5 |
| dNTP Mixture | 3 |
| 引物OR | 1 |
| 引物OF | 1 |
| Taq DNA聚合酶 | 1 |
| Rnase-free H2O | 37 |
第二轮PCR反应体系表3所示，反应条件为：94℃ 3min；94℃ 30s，58℃ 30s，72℃60s，30个循环；72℃ 5min。
表3第二轮PCR反应体系
| 试剂 | 体积/μL |
| 第一轮PCR产物 | 5 |
| 10×Primer STAR 缓冲液 | 5 |
| dNTP Mixture | 3 |
| 引物IR | 1 |
| 引物IF | 1 |
| E-Taq DNA聚合酶 | 1 |
| Rnase-free H2O | 34 |
扩增产物经琼脂糖凝胶电泳后出现目的条带，则将巢式PCR 产物送上海生物工程公司进行基因测序。从 NCBI 网站下载不同基因型参考序列，利用网站 HIV sequencedatabase ( https: / /www．hiv．lanl．gov /content /sequence /VESPA /vespa．html)序列分析工具和相关生物信息学软件对基因测序结果进行分析。野生型HBsAg S 区域序列如SEQ ID NO:9所示，而鉴定出的新型HBsAg S 区域的突变体，经序列比对，其区别区域主要位于：R112W，G145S，P153缺失，L176F。
为了筛选能够识别所述突变位点的新型抗体，本发明将突变位点集中的100-190区域作为目标区域，所述目标抗原的氨基酸序列如SEQ ID NO:10所示。
实施例2 HbsAg抗原制备
人工合成编码实施例1中所获得的HBsAg抗原的核苷酸序列，并在其两端引入BamHI和XhoI限制性内切酶位点，经PCR反应扩增富集后，使用限制性内切酶BamHI和XhoI对扩增产物在37℃水浴进行双酶切，酶切体系为：目标基因16μL，BamHI和XhoI各1μL，10×buffer 2μL。然后使用核酸纯化试剂盒（购自上海生工生物工程有限公司）对连接产物进行纯化。pET28a(+)质粒也按照类似的方法用限制性内切酶BamHI和XhoI进行双酶切及纯化。然后使用T4 DNA Ligase（购自Takara公司）连接经双酶切后的质粒和基因，连接体系为：目标基因4μL，质粒4μL，T4 DNA Ligase 1μL和10×buffer 1μL，连接体系在4℃冰箱过夜连接，获得质粒载体pET28a(+)-HBsAg。将连接后的质粒载体pET28a(+)-HBsAg电转化至BL21(DE3)感受态细胞，将其涂布含氨苄霉素的LB琼脂培养基，在37℃恒温培养箱过夜培养。挑取单菌落置于含氨苄霉素的LB液体培养基中，37℃ 200rpm 震荡培养过夜，3000rpm 5min离心收集菌体，使用质粒提取试剂盒（购自上海生工生物工程有限公司）提取质粒，酶切鉴定筛选阳性克隆，结果如图2所示；阳性克隆经基因测序鉴定，核酸序列正确，命名为BL21(DE3)- HBsAg。
活化BL21(DE3)- HBsAg菌种，然后按5%接种量接种于含抗生素的LB培养基，37℃200rpm 震荡培养4h，加入终浓度0.5mM IPTG，降低培养温度至25℃，诱导培养16h；后8000rpm离心5min收集菌泥，用1/10体积的无菌PBS在4℃下重悬菌泥，超声波破碎后10000rpm离心15min，取上清用0.45μm滤膜过滤，使用镍离子亲和层析柱分离获得目标蛋白抗原，经测定所示蛋白纯度达95%以上，符合后续实验要求。经过测序，所得到的蛋白其氨基酸序列如SEQ ID NO:10所示。
实施例3 单克隆抗体的筛选和获得
以实施例2中获得的纯化HBsAg蛋白为免疫原配制免疫原液，浓度为50mg/mL，皮下多点注射6-8周龄的雌性Balb/C小鼠，剂量200μL/只，每周一次共进行三至四次免疫。小鼠尾静脉采血用间接ELISA方法测定血清抗体效价，选择效价高的小鼠在细胞融合前3天，腹腔注射200μL的HBsAg蛋白免疫原液加强免疫。无菌采集免疫小鼠脾细胞，与SP2/0细胞按一定比例进行混合，再用PEG4000按常规方法进行细胞融合。用HBsAg蛋白包被96孔板，使用间接ELISA筛选阳性细胞克隆，共获得10株稳定分泌单克隆抗体的杂交瘤细胞株，分别命名为1A3、1D2、1F4、2A5、2A6、3B2、3D1、3D5、4B3、5B8。
取6-8周龄健康雌性Balb/c小鼠，腹腔注射灭菌液体石蜡0.5mL/只；7天后，每只小鼠腹腔接种1×106个/mL上述杂交瘤细胞，杂交瘤细胞在接种前使用无血清培养基洗涤三次，去掉血清蛋白避免干扰。注射7-10天后，小鼠腹部明显胀大，收集腹水；3-5天后，腹部再次胀大后继续收集，一般可收集2-3次。腹水收集于离心管，4000r/min离心l0分钟，取中间无色透明层液体，应用Protein A亲和层析法纯化单克隆抗体。
利用蛋白相互作用分子动力学检测仪 ForteBio Octet Red 96e 检测所述抗体与HBsAg蛋白的亲和力，配置浓度为5 μg/mL 的HBsAg 蛋白液，取200μL蛋白液加入至生物感应器中，以 PBS 作为对照组，30℃反应 5 min 后，检测抗体结合情况，通过软件 OctetData Analysis Software 计算解离常数 KD、结合速率常数 (Kon ) 和解离速率常数(Koff )。
使用热稳定性检测仪LightCycler480 II测量所述抗体的Tm值，实验参数为：激发波长为465nm，发射波长为580nm，温度梯度设定为25-95℃。Acquisition Mode设为Continuous，变化率设为0.01℃/s，Acquisitions(per℃)设为50。结果如表4所示，选择与目标抗原的亲和力高且热稳定性好的的4B3抗体，用于后续研究和实验。经序列鉴定和分析，该抗体轻链可变区包括氨基酸序列为SEQ ID NO:1的LCDR1，氨基酸序列为SEQ ID NO:2的LCDR2，氨基酸序列为SEQ ID NO:3的LCDR3；重链链可变区包括氨基酸序列为SEQ ID NO:4的HCDR1，氨基酸序列为SEQ ID NO:5的HCDR2，氨基酸序列为SEQ ID NO:6的HCDR3；所述轻链可变区的氨基酸序列如SEQ ID NO:7所示；所述重链可变区的氨基酸序列如SEQ ID NO:8所示。
表4 抗体性能检测
| 抗体编号 | 亲和力KD值 | 热稳定性Tm/℃ |
| 1A3 | 5.36E-08 | 57.6 |
| 1D2 | 2.05E-10 | 65.8 |
| 1F4 | 6.77E-09 | 75.4 |
| 2A5 | 1.22E-08 | 63.9 |
| 2A6 | 6.92E-09 | 66.5 |
| 3B2 | 4.35E-09 | 58.9 |
| 3D1 | 7.60E-09 | 70.2 |
| 3D5 | 8.51E-08 | 65,8 |
| 4B3 | 1.63E-10 | 70.1 |
| 5B8 | 2.40E-07 | 63,4 |
实施例4检测试剂盒的构建和乙肝病毒突变株的检测
4.1 构建检测试剂盒
为有效检测乙肝病毒突变株，本发明提供了一种检测试剂盒，所述试剂盒包括实施例3中所提供的抗乙肝病毒表面抗原的单克隆抗体，还包括标准品冻干粉、样品稀释液、显色剂、终止液和洗涤液。所述的试剂盒包被有单克隆抗体的96孔板；辣根过氧化酶标记抗人IgG单克隆抗体作为酶标结合物；其中所述的标准品冻粉为乙肝病毒表面抗原蛋白，其制备方法参见实施例2，其氨基酸序列如SEQ ID NO:10所示；所述的样品稀释液为0.01mM、pH值为7.4的PBS缓冲液；所述的洗涤液的溶剂为含有体积百分浓度为0.2%的吐温-20的0.01mM、pH值为7.4的PBS缓冲液；所述的终止液为2M的硫酸水溶液；所述的显色剂包括辣根过氧化物酶催化底物A液和B液；所述的辣根过氧化物酶催化底物A液是体积浓度为3%的H2O2水溶液；所述的辣根过氧化物酶催化底物B液为含0.1mg/mL的3,3',5,5'-四甲基联苯胺的0.1 mol/L、pH为6.0的磷酸缓冲液。
4.2乙肝病毒突变株的检测
收集另外43例乙肝患者血清，经核酸检测确定其中8例为高ROS乙肝病毒突变株，使用ELISA法检测血清样品，具体步骤包括：按1:200比例稀释血清样本，将血清稀释液加入到4.1中所制备试剂盒的96孔板中，4℃过夜孵育；弃去孵育液，加入洗涤液，置于摇床上100rpm洗涤10min，共洗涤3次；洗涤结束后，加入按1:200比例稀释的二抗溶液，室温放置1h；弃去孵育液，加入洗涤液，置于摇床上100rpm洗涤10min，共洗涤3次；洗涤结束后，拍干洗涤液，室温干燥10min；每孔加入100 μL显色液，37℃反应15 min,；加入终止液终止反应，50 μL/孔，于 450 nm下检测OD值。
实验结果使用GraphPad Prism 8软件进行分析，绘制ROC曲线，如图3所示，检测乙肝突变病毒株的特异性为87.50%，灵敏性为74.29%，AUC为0.8482能够满足检测要求。
Claims (7)
1.一种鼠源单克隆抗体，包含轻链可变区和重链可变区，其轻链可变区包括氨基酸序列为SEQ ID NO:1的LCDR1，氨基酸序列为SEQ ID NO:2的LCDR2，氨基酸序列为SEQ ID NO:3的LCDR3；重链可变区包括氨基酸序列为SEQ ID NO:4的HCDR1，氨基酸序列为SEQ ID NO:5的HCDR2，氨基酸序列为SEQ ID NO:6的HCDR3。
2.如权利要求1所述的鼠源单克隆抗体，其特征在于，所述轻链可变区的氨基酸序列如SEQ ID NO:7所示。
3.如权利要求1所述的鼠源单克隆抗体，其特征在于，所述重链可变区的氨基酸序列如SEQ ID NO:8所示。
4.一种乙肝病毒检测试剂盒，包括权利要求1-3中任一项所述的鼠源单克隆抗体。
5.如权利要求4所述的乙肝病毒检测试剂盒，其特征在于：所述的试剂盒还包括标准品冻干粉、样品稀释液、显色剂、终止液和洗涤液。
6.如权利要求5所述的乙肝病毒检测试剂盒，其特征在于：所述的试剂盒包括包被有所述单克隆抗体的96孔板；辣根过氧化酶标记抗人IgG单克隆抗体作为酶标结合物；所述的标准品冻干粉为乙肝病毒表面抗原蛋白，其氨基酸序列如SEQ ID NO:10所示；所述的样品稀释液为0.01mM、pH值为7.4的PBS缓冲液；所述的洗涤液为含有体积百分浓度为0.2%的吐温-20的0.01mM、pH值为7.4的PBS缓冲液；所述的终止液为2M的硫酸水溶液；所述的显色剂包括辣根过氧化物酶催化底物A液和B液；所述的辣根过氧化物酶催化底物A液是体积浓度为3%的H2O2水溶液；所述的辣根过氧化物酶催化底物B液为含0.1mg/mL的3,3',5,5'-四甲基联苯胺的0.1 mol/L、pH为6.0的磷酸缓冲液。
7.权利要求1-3中任一项所述的鼠源单克隆抗体在制备乙肝病毒检测试剂中的应用。
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