CN117285635B - 抗肝素结合蛋白的单克隆抗体组合物及应用 - Google Patents
抗肝素结合蛋白的单克隆抗体组合物及应用 Download PDFInfo
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Abstract
本发明公开了抗肝素结合蛋白的单克隆抗体组合物及应用，单克隆抗体组合物包括HBP‑Clone1和HBP‑Clone2，HBP‑Clone1的重链可变区氨基酸序列如SEQ ID NO.1所示，HBP‑Clone1的轻链可变区氨基酸序列如SEQ ID NO.2所示；HBP‑Clone2的重链可变区氨基酸序列如SEQ ID NO.3所示，HBP‑Clone2的轻链可变区氨基酸序列如SEQ ID NO.4所示。制备方法：将亲和纯化后的肝素结合蛋白与弗氏佐剂进行等体积混合乳化，采集小鼠血液分离血清，筛选免疫较好的小鼠进行杂交瘤融合实验，以HBP作为酶标板包被抗原，取上清进行酶联免疫吸附测定，筛选出优质阳性细胞株，并进行两轮的细胞亚克隆。同时也提供了上述抗体的化学发光诊断检测试剂盒，从而提高诊断试剂的特异性和灵敏度。
Description
技术领域
本发明属于抗体的制备及序列测定领域，具体涉及抗肝素结合蛋白的单克隆抗体组合物及应用。
背景技术
肝素结合蛋白（Heparin-binding protein，简称HBP），是中性粒细胞释放的一种重要的颗粒蛋白，它能够激活巨噬细胞和单核细胞，有着较强的抗菌活性、趋化及调节炎症反应的作用。HBP基因位于人体19号染色体短臂末端。HBP前体由251个氨基酸组成，在成熟的过程中会从C末端、N末端分别去除3个及26个氨基酸残基，从而形成一条分子量为24000的单链蛋白，HBP结构中含有8个半胱氨酸，使HBP与中性粒细胞弹性蛋白酶相似，具有同源性。HBP属于丝氨酸蛋白酶家族的成员，但第41位的组氨酸残基被丝氨酸残基代替、第175位的丝氨酸残基被甘氨酸残基代替，因此不具备蛋白酶活性。如今HBP作为一种国际新型感染标志物，因其能够预测由脓毒症引起的器官功能障碍而逐渐被人们所熟悉。《中国严重脓毒症/脓毒性休克治疗指南(2014)》中指出了HBP能够作为脓毒症，尤其是严重细菌感染的早期诊断标志物。因此，它作为创新型感染标志物，有着非常广阔的应用前景。
HBP是蛋白酶样丝氨酸蛋白酶家族中的成员之一，可以通过调节血管内皮细胞的通透性，影响炎症反应；调节单核巨噬细胞的功能，激活相关炎症反应；介导线粒体途径来调节细胞凋亡。而且血浆HBP水平能够反应脓毒症患者的病情严重程度，还可以作为脓毒症患者早期诊断、评估预后的标志物。传统的感染监测指标，如血培养、C反应蛋白、降钙素原及白细胞计数等，由于检测耗时、滞后、敏感度和特异度低等缺点延误了早期对感染性疾病的诊断。HBP作为新型感染指标，其诊断效能优于传统实验室指标，在与某些指标联合运用时可提高感染性疾病的诊断率。
开发性能优异的HBP免疫诊断试剂的核心原料是抗HBP单克隆抗体，一般厂商的特异性和灵敏度不高，所以开发性能优异的HBP诊断试剂非常有必要，同时也期望能够进一步推动诊断试剂的国产化。
发明内容
为解决灵敏性、特异性的问题，本发明提供了抗肝素结合蛋白的单克隆抗体组合物。该抗体性能优异、准确性高。同时提供了基于上述HBP抗体的化学发光试剂盒。
本发明提供如下技术方案：
抗肝素结合蛋白的单克隆抗体组合物，包括HBP-Clone1和HBP-Clone2，
HBP-Clone1的重链可变区氨基酸序列：
DVQLQESGPGLVKPSQSLSLTCTGTGYSITSDYAWNEIRQFPGNKLEWMGYISYIGGTNYNPSLKSIRSILRSTSKNQFFLQLNSVATEDTATYFCARSSFGYDERGYDMLYWGQGTSVTVSS
HBP-Clone1的轻链可变区氨基酸序列：
QTVLTQPPAVGGSLGQQVSITSSGSGSNIGNGAYVSWYHQHPGSGLKTIIYGTTTRSSGVPDRSSGSTNGNTATLIITALQAEDEADYYCCTSDKSIPSRMFGSGTRLTVL
HBP-Clone2的重链可变区氨基酸序列：
QVQLQQSGPPLVRPGVSVKISNKGSGYRFTDYALHLLKQSHLKSLEQIGVISTYSGNNDYNQKFKGKATMTVDQSSSTAYMEAARLTSDDSAIYYCSRMNDYGGDYFFDHWGQGTLLLVSS
HBP-Clone2的轻链可变区氨基酸序列：
QTVLTQPPASSGSLGQRVSITCSGSGSRIGGGAYVSWYQQHPGSGLKTIIYGTTTRSSGVPDRFSGGTSGNTATLLATALQAEDEADYYCAASSKSIPSRMFASGTRLTVL
抗肝素结合蛋白的单克隆抗体组合物的制备方法：
肝素结合蛋白的表达生产：
在NCBI官网上查找人HBP氨基酸序列，在蛋白氨基酸的C端加上组氨酸标签，利用密码子优化软件在哺乳动物细胞表达系统中优化密码子，基因合成后利用EcoRI/BamHI限制性内切酶酶切位点，将基因克隆至pTT5表达载体中，构建pTT5-HBP表达质粒，并测序验证基因序列；
将pTT5-HBP表达质粒与转染试剂PEI混匀后，转染HEK293细胞，同时在OPM-293CD03 Medium细胞培养基中加入增强剂和辅料进行培养，当细胞活率<70%后，收集细胞培养物，离心并收集细胞上清。
肝素结合蛋白的亲和纯化：细胞上清用盐和咪唑充分重悬过滤后，用预装重力柱进行亲和纯化，通过梯度咪唑浓度进行洗杂和洗脱，SDS-PAGE跑胶检测蛋白纯度，将蛋白透析至磷酸盐缓冲液中，0.22μm滤器无菌过滤后，BCA测定蛋白浓度。
将亲和纯化后肝素结合蛋白与弗氏佐剂进行等体积混合乳化，免疫计量为100μg/只小鼠，3周后进行加强免疫，免疫计量为50μg/只小鼠，后期间隔2周每次，采集小鼠血液分离血清，酶联免疫吸附测定抗体效价，筛选免疫较好的小鼠进行杂交瘤融合实验，经过HAT筛选培养基筛选培养后，以HBP蛋白作为酶标板包被抗原，取上清进行酶联免疫吸附测定，筛选出优质阳性细胞株，并进行两轮的细胞亚克隆，克隆出单克隆细胞株，用无血清培养和发酵细胞株，收集培养上清。
抗肝素结合蛋白的单克隆抗体组合物在制备脓毒症早期免疫诊断试剂中的应用。
单克隆抗体组合物用于 HBP化学发光试剂盒。试剂盒使用HBP-Clone1和HBP-Clone2分别作为包被和检测抗体。
与现有技术相比，本发明的有益效果是：
本发明提供了抗肝素结合蛋白，用于制备诊断用单克隆抗体，本发明的抗 肝素结合蛋白的单克隆抗体组合物包括HBP-Clone1和HBP-Clone2，能够识别免疫原，此抗体组合具有特异性和高亲和力的特点，同时也提供了基于上述抗体的化学发光检测试剂盒，从而提高诊断试剂的特异性和灵敏度，可以对脓毒症等细菌感染性疾病的患者进行早期诊断病情评估，具有重要意义，精准的检测结果对后续临床治疗指导用药提供重要的医学价值。
附图说明
图1为本发明检测试剂盒与同类产品相关性对比图。
实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 肝素结合蛋白表达载体的构建
在NCBI官网上查找人HBP氨基酸序列，在蛋白氨基酸的C端加上组氨酸标签，利用密码子优化软件在哺乳动物细胞表达系统中优化密码子，基因合成后利用EcoRI/BamHI限制性内切酶酶切位点，将基因克隆至pTT5表达载体中，构建pTT5-HBP表达质粒，并测序验证基因序列。
实施例2 肝素结合蛋白的表达生产
转染前一天，细胞以0.8×106/ml接种于细胞培养瓶，以OPM-293 CD03 Medium培养液培养，置于37℃，5%CO2培养箱中，至细胞融合度达到80%-90%，活力大于95%时进行转染，pTT5-HBP表达载体与PEI混匀后，室温孵育15-30min，缓慢加入到HEK293细胞中，加入增强剂和辅料，于 37℃，5%CO2培养箱中继续培养。当细胞活率<70%后，收集细胞培养物，转速8000 g，离心10 min，收集上清液，再通过转速12000 g，4℃离心30 min，收集细胞上清。
实施例3 肝素结合蛋白的亲和纯化
细胞上清中加入NaCl和咪唑，并将工作浓度分别调整至300 mM和5 mM后再用0.22μm滤膜过滤，Ni TED-6FF beads预装重力柱进行亲和纯化，20mM PB，300mM NaCl，10%甘油，50mM咪唑，pH8.0溶液进行洗杂，20mM PB，300mM NaCl，10%甘油，250mM咪唑，pH8.0溶液进行洗脱，SDS-PAGE跑胶检测蛋白纯度，将蛋白透析至20mM PB，300mM NaCl，10%甘油，pH8.0缓冲液中，0.22μm滤器无菌过滤后，BCA测定蛋白浓度。
实施例4 单克隆抗体的制备
将肝素结合蛋白与弗氏佐剂进行等体积混合乳化，免疫计量为100μg/只小鼠，皮下多点注射，3周后进行加强免疫，免疫计量为50μg/只小鼠，后期间隔2周每次，采集小鼠血液分离血清，酶联免疫吸附法测定抗体效价，筛选免疫较好的小鼠进行杂交瘤融合实验，经过10天左右的HAT筛选培养基筛选培养后，以肝素结合蛋白作为酶标板包被抗原，取上清进行酶联免疫吸附法测定，筛选出优质阳性细胞株，并进行两轮的细胞亚克隆，克隆出单克隆细胞株，用无血清培养和发酵细胞株，收集培养上清，进行抗体纯化，将纯化后的抗体进行诊断性能分析。
实施例5 单克隆抗体性能分析
基于双抗体夹心法和吖啶酯发光法原理制备试剂盒。
1、羧基磁珠包被抗体
将购买的羧基磁珠充分混匀，取出相应量的磁珠置于离心管，使用磁力架去除上清液，清洗后加入包被活化液，混匀后加入包被活化液溶解的EDC，充分混匀后室温颠倒活化0.5-0.8h；
超声分散磁珠，使用包被活化液清洗三次，加入本发明的包被抗体，充分混匀后室温颠倒反应1.5-2h；
超声分散磁珠，加入封闭剂，充分混匀后室温颠倒封闭1-1.5h；
超声分散磁珠，使用磁力架去除上清液，清洗三次，加入磁珠保存液将磁珠稀释至浓度为0.25mg/mL待用。
上述磁珠保存液配方为50mM Tris、0.2% Tween-20、0.85% NaCl、2% BSA、0.2%Proclin-300。
2、吖啶酯标记抗体
将NHS酯化的吖啶酯（NSP-DMAE-NHS）与本发明的包被抗体按照分子摩尔比10：1的比例在1×PBS中颠倒混匀标记反应1.5-2h；
反应结束后将标记液转移至14kD透析袋中，1×PBS中旋转透析，换液6次，每次1.5-2h；
透析过夜后取出标记液，使用分光光度计测试标记液中蛋白含量，计算并添加保护液，充分混匀后可置于18-21℃保存，上机测试时使用标记保存液将蛋白浓度稀释至0.5μg/mL待用。
上述标记保存液的配方为0.1M MES、0.2% Tween-20、0.85% NaCl、2% BSA、0.2%Proclin-300。
3、配制校准品和质控品
使用抗原稀释液将HBP抗原分别稀释至0、2、10、20、100、200、1000 ng/mL，分装至校准品管中。
使用抗原稀释液将HBP抗原稀分别释液至2、20 ng/mL，分装至质控品管中。
上述抗原稀释液配方为1×PBS、1%牛血清白蛋白。
检测试剂盒与同类产品对比：
实施例中的HBP化学发光检测试剂盒与对照公司的产品在120例临床标本上进行了相关性对比，数据如图1所示。相关系数R2为0.9908。说明本发明的试剂盒能准确检测HBP含量，为临床诊断提供依据，能够充分满足临床体外诊断检测需求。
检测试剂盒灵敏度和精密度考察：
按照临床和实验室标准协会灵敏度验证分析指南文件CLSI:EP17-A2对本发明中的试剂盒的空白限和检测限进行了研究，结果表明空白限和检测限分别为1 ng/mL和2 ng/mL。
按照临床和实验室标准协会精密度验证分析指南文件CLSI:EP5-A3对本发明的试剂盒进行精密度研究，使用3个批号的试剂和校准品，对2个水平的质控品进行了检测，每天上下午各检测两次，连续考察5天，结果表明各批次室内总精密度小于5%。
检测试剂盒抗干扰能力分析：
在HBP浓度约为2 ng/mL和20 ng/mL浓度水平的两个血清样本中分别添加不同浓度的干扰物，以未添加组为对照，添加组和对照组两者相对偏差小于5%为干扰可接受标准，研究不同干扰物的最大浓度水平（不超过临床可见最高浓度）。
表1 检测试剂盒抗干扰能力分析数据
结果表明表1中的浓度时对检测结果干扰不明显。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (5)
1.抗肝素结合蛋白的单克隆抗体组合物，其特征在于：包括HBP-Clone1和HBP-Clone2，
所述HBP-Clone1的重链可变区氨基酸序列如SEQ ID NO.1所示，HBP-Clone1的轻链可变区氨基酸序列如SEQ ID NO.2所示；
所述HBP-Clone2的重链可变区氨基酸序列如SEQ ID NO.3所示，HBP-Clone2的轻链可变区氨基酸序列如SEQ ID NO.4所示。
2.权利要求1所述的抗肝素结合蛋白的单克隆抗体组合物在制备脓毒症早期免疫诊断试剂中的应用。
3.根据权利要求2所述的抗肝素结合蛋白的单克隆抗体组合物在制备脓毒症早期免疫诊断试剂中的应用，其特征在于：单克隆抗体组合物用于HBP化学发光试剂盒。
4. HBP化学发光试剂盒，其特征在于：含有权利要求1中的HBP-Clone1和HBP-Clone2。
5.根据权利要求4所述的HBP化学发光试剂盒，其特征在于：试剂盒使用HBP-Clone1和HBP-Clone2分别作为包被抗体和检测抗体。
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