CN106443010A - 一种血清acy1抗体定量检测试剂盒 - Google Patents
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Abstract
本发明涉及一种血清ACY1抗体定量检测试剂盒，可以特异性定量检测血清标本中ACY1抗体的水平。本发明将ACY1抗原蛋白包被于96孔酶标板中，通过酶联免疫吸附法定量测定血清标本中的ACY1抗体水平，根据血清ACY1抗体水平高低来评估肝硬化患者的疾病状态。本试剂盒提供了一种新的肝硬化筛查及早期诊断方法，可以用来区分慢性乙肝和肝硬化患者。
Description
技术领域
本发明专利涉及一种血清ACY1抗体定量检测试剂盒，可用于肝硬化患者的筛查和早期诊断。
背景技术
慢性乙肝-肝硬化-肝细胞癌(HCC)是总所周知的肝癌发生三部曲。由于HCC缺乏早期症状，大部分病人出现症状时已属中晚期，失去有效治疗的机会，存活期一般少于1年，早期HCC通过切除术或肝移植，5年生存率在60-70％以上。因此，对高危人群如肝硬化的筛查和早期诊断、准确区分慢性乙肝和肝硬化，对指导病人的及时治疗至关重要。目前肝硬化的诊断主要通过肝脏活检和影像学检查，肝脏活检虽然一直被认为是金标准，但是由于取样误差以及侵入性特点等限制了其广泛应用；影像学检查如B超对临床医生的个人经验要求较高。比较而言，血清标志物因为获取方便，便于重复操作和动态观察，易于被患者接受，非常适合在人群中筛查肝硬化病例，但目前临床上仍缺乏敏感度高、特异性好，可用于区分乙肝和肝硬化的血清学标志物。
近年来的研究表明，在疾病发生发展的过程中，由于内外因素的影响，机体某些与疾病发生发展相关的蛋白有可能由于基因突变、异常表达或细胞定位异常等改变而产生抗原性，患者细胞中会出现针对这些自体细胞抗原的抗体免疫反应，通过检测外周血中的这些自身抗体可以进行疾病的诊断。近年来的研究还发现，自身抗体不仅可以出现在自身免疫性疾病如自身免疫性肝病、糖尿病、风湿性关节炎以及系统性红斑狼疮，还可以出现在非自身免疫性疾病如肿瘤等疾病中。最近的一项小样本研究发现，alpha-enolase(α-ENO1，烯醇化酶)在肝纤维化早中期能够激发机体的自身免疫反应，其自身抗体水平在肝纤维化的诊断中具有潜在的价值。
在国家自然科学基金面上项目“肝细胞癌早期诊断血清自身抗体标志物的筛选与分析”(项目编号81071973)和“人肝癌转移特异性标志物的筛选及验证”(项目编30572128)、以及留学人员择优资助北京市重点项目“肝癌早期诊断血清自身抗体标志物的筛选与临床考核”(项目编号：20110323)的资助下，我们通过大样本研究对肝硬化、慢性肝炎及健康人血清的蛋白芯片高通量检测筛选出是一种肝硬化特异自体抗原蛋白，肝硬化患者血清中自身抗体水平显著高于慢性肝炎(P＜0.001)，具有很好的区分肝硬化与慢性乙肝的能力，AUC值达0.874。
发明内容
本发明的目的在于提供一种检测试剂盒，该试剂盒能够定量检测血清中ACY1抗体水平，可用于区分乙肝和肝硬化，将有助于肝硬化的筛查及早期诊断。
本发明的检测试剂盒采用酶联免疫法测定，抗体检测反应板为包被有ACY1抗原蛋白的酶标96孔板，待测血清中的ACY1抗体能够与反应孔中的ACY1蛋白特异性结合而吸附于反应孔内，加入HRP标记的兔抗人IgG二抗孵育，充分洗涤后加入显色底物，终止反应后用酶标仪测定各反应孔的吸光度OD值，其OD值与ACY1抗体水平呈正比。过标准品的检测绘制标准曲线并对各血清标本中的ACY1抗体水平进行定量。
本发明所述检测试剂盒的制备包括如下步骤：
1.重组ACY1抗原蛋白的制备：本课题组制备的ACY1抗原蛋白为带有His标签的His-ACY1融合蛋白。采用pET21a载体构建的人ACY1重组表达载体转化到感受态大肠杆菌，提取质粒并鉴定重组表达体基因序列。将测序正确的重组表达体质粒转化到感受态大肠杆菌并诱导其表达His-ACY1融合蛋白，收集菌体并处理得到菌液上清，利用Ni-NTA亲和层析柱纯化得到His-ACY1抗原蛋白。
2.检测试剂盒标准品的选定：本课题组在前期工作基础上，已通过蛋白芯片技术定量检测筛选出一批高水平表达ACY1抗体的血清标本，可以作为本检测试剂盒的标准品进行质量控制和抗体定量。
3.制备酶标反应板：用棋盘法确定酶标板的最佳包被浓度，各反应孔内加入最佳浓度的ACY1抗原蛋白100ul，37℃孵育2h后，4℃过夜后甩掉蛋白溶液，用洗涤液PBS-T(0.01mol/LPBS，pH 7.2-7.4，0.05％Tween 20)洗板1次。然后各反应孔内加入10％的NBS封闭液200ul以封闭反应孔内未结合的空白位点，37℃孵育2h后甩掉封闭液，甩干后于室温晾干过夜，于4℃保存备用。
本发明提供的检测试剂盒的优势之处在于：提供了一种新的肝硬化筛查及早期诊断方法。
附图说明：
图1：pET21a-ACY1表达质粒的酶切验证。1为pET21a-ACY1，2为pET21a-ACY1 NdeI和XhoI双酶切产物，M为DNA Marker。
图2：人ACY1原核表达载体测序鉴定。
图3：BL21(DE3)菌株在0.5mmol/L IPTG诱导下融合蛋白的表达情况，其中2为表达菌体裂解上清，3为表达菌体裂解沉淀，4为未诱导对照。1为ProteinRuler II Marker。
图4：大肠杆菌诱导表达产物的Western-Blot结果。为BL21(DE3)菌株的融合蛋白表达 情况。
图5：融合蛋白纯化结果。
图6：融合蛋白纯化产物的Western-Blot结果。验证是否为相关融合蛋白纯化产物。
具体实施方式
实施例1ACY1重组蛋白的制备
一、材料
DNA marker(1k-10k)彩虹预染蛋白marker(14-120KD)均购自北京天根生化科技有限公司；化学发光蛋白Marker ProteinRuler II Marker(20-90KD)购自北京全式金生物技术有限公司；大肠杆菌BL21(DE3)为本实验室保存；异丙基硫代卢-D-半乳糖苷(IPTG)为Amresco产品；HRP标记的山羊抗人IgG购自美国Earthox公司；其他试剂均为国产分析纯。
二、人ACY1原核表达载体的构建及鉴定
本课题组重组表达的蛋白为ACY1蛋白，并在其C端加上了组氨酸His标签，共包含408个氨基酸，分子量为45.885KD。
1.人ACY1原核表达载体的构建
本课题组通过对1224bp ACY1基因序列进行密码子优化并人工合成，其中5’端添加NdeI酶切位点序列，3’端添加XhoI酶切位点序列。将合成的ACY1基因序列克隆入pET21a载体(表达蛋白C端将带有His标签)，酶切及测序鉴定成功后将重组质粒转化BL21感受态细胞。取5ul转化到感受态大肠杆菌BL21(DE3)内，涂布于LB(含Amp抗生素)平板，37℃倒置培养过夜。
2.人ACY1原核表达载体的鉴定
对人ACY1原核表达载体进行酶切及测序鉴定，结果见附图1及附图2。其基因序列与GeneBank上公布的基因序列一致。
三、His-ACY1融合蛋白大肠杆菌菌株诱导表达
1.BL21(DE3)菌株融合蛋白的诱导表达
测序正确地重组表达质粒转化到感受态大肠杆菌BL21(DE3)细胞内，挑取单个菌落放入3ml液体LB培养基(含Amp)内，37℃振荡培养过夜。次日，按7％的比例将上述菌液加入到100ml液体LB培养基中，37℃振荡培养3.5h，测得OD600值为0.5，加入IPTG至终浓度为0.5mmol/L，在16℃振荡培养15h。对照组不加IPTG诱导，同样在16℃振荡培养15h。收集菌液到1.5ml离心管中，8000rpm/min离心3min后收集表达菌体经超声裂解后，4℃12000rpm离心10分钟取上清进行SDS-PAGE电泳验证蛋白表达情况。加入上样缓冲液煮沸10min，进行12％SDS-PAGE电泳，考马斯亮蓝染色观察融合蛋白的表达情况，结果见附图3
2.表达产物的Western-Blot鉴定
将重组菌菌体蛋白经SDS-PAGE电泳，然后进行蛋白质电转移，将蛋白质转移至硝酸纤维素滤膜上(300mA，1h)；膜用含5％脱脂奶粉的Tris缓冲盐-吐温缓冲液(TBST)室温缓摇封闭1h；然后用TBST缓冲液洗涤3次后，用含ACY1抗体的TBST溶液4℃孵育反应过夜；用TBST缓冲液洗膜3次后加入1∶8000的HRP标记的山羊抗人IgG，室温下缓摇孵育1h，TBST缓冲液洗膜3次，在吸水纸上沥干缓冲液后将膜浸入化学发光液中，30s后于显像仪下呈像，结果见附图4。
四、HIS-ACY1融合蛋白的纯化
1.Ni-NTA亲和层析柱纯化His-ACY1融合蛋白
挑取含重组质粒的BL21(DE3)单菌落置3ml液体LB培养基(含Amp)中37℃震荡培养过夜。次日，按7％的比例将上述菌液加入到1000ml液体LB培养基(含Amp)中，37℃振荡培养至OD600值为0.5，加入IPTG使终浓度为1.0mmol/L，16℃振荡培养6h，按上述同样方法收集菌体，用缓冲液PBS(PH 7.0)悬浮菌体，进行超声破碎60min，12000rpm/min离心去沉淀。将上清液分为3组，分别加入到经PBS(PH 7.0)预平衡的Ni-NTA亲和层析柱，用5倍柱体积的PBS(PH 7.0)充分洗涤后，以250mmol/L的咪唑洗脱，收集洗脱液，进行12％SDS-PAGE电泳，并对产物进行Western-Blot验证。结果见附图5及附图6。
2.His融合蛋白产品的制备
将上述咪唑洗脱液再经过透析处理，最终制备的蛋白浓度为0.5mg/ml。
实施例2酶标板的制备
一、材料
96孔酶标板购自美国Thermo公司；重组ACY1抗原蛋白为本实验室制备；NBS购自购自美国Thermo公司；TMB显色液及终止液均购自北京索莱宝科技有限公司；HRP标记的兔抗人IgG购自美国Sigma公司；其他试剂均为国产分析纯。
二、检测试剂盒中标准品的制备
本课题组在前期研究过程中，已通过蛋白芯片技术定量检测了一批肝癌患者血清ACY1抗体水平，将其中筛选出的含有高水平ACY1抗体的血清(蛋白芯片检测信号值大于6000)的20例血清标本混合，作为本检测试剂盒的标准品。
三、酶标板包被浓度的确定
1.酶标板的包被
采用棋盘法确定酶标板的最佳包被浓度。用CB(0.05mol/L，pH 9.16)稀释ACY1蛋白至不同浓度(0.625-2.5ug/ml)进行包被，每个浓度重复两列，各反应孔加入上述蛋白100ul置于37℃恒温箱孵育2h，后于4℃冰箱过夜；然后甩掉液体，各反应孔加入400ul洗涤液PBS-T(0.01mol/L PBS，pH 7.2-7.4，0.05％Tween 20)洗涤1次，在实验台上适度晃动酶标板后甩掉 洗涤液，在吸水纸上拍干酶标板。
2.酶标板的封闭
向上述酶标板的各反应孔中加入10％的NBS封闭液200ul，37℃孵育2小时后甩掉封闭液，甩干后与室温过夜晾干。
3.酶标板检测
各反应孔中加入样本稀释液(10％NBS-PBS)100ul，各反应孔内加入样品10ul，将酶标板置于37℃恒温箱孵育1h。甩掉液体后洗板6次。然后各反应孔加1∶8000的HRP-兔抗人IgG100ul，于37℃恒温箱孵育30min后洗板6次。配置TMB显色液(等体积A液+B液)，各反应孔内加入显色液100ul，避光条件下37℃恒温箱反应10min，然后各反应孔加入终止液50ul以终止反应。在酶标仪450nm、630nm条件下测定各反应孔的OD值。
Claims (3)
1.一种血清ACY1抗体定量检测试剂盒，其特征在于由包被有一定浓度的重组ACY1抗原蛋白的酶标板、标准品、酶标二抗、显色底物及浓缩洗涤液组成。待测血清或标准品中的特异性ACY1抗体结合固定在酶标板微孔表面的ACY1抗原，酶标记抗体识别ACY1抗体并与之结合，形成ACY1抗原-ACY1抗体-酶的复合物，酶与底物反应显色后测出吸光度，通过标准品的检测绘制标准曲线并计算待测血清中ACY1抗体的水平。
2.如权利要求1所述的试剂盒，其特征在于，所述酶标板包被的ACY1抗原蛋白为带有His标签的His-ACY1融合蛋白，采用化学合成的方法合成基因，与可溶性表达载体pET21a构建表达人ACY1重组表达质粒，转化到感受态大肠杆菌BL21(DE3)并诱导其表达可溶性His-ACY1融合蛋白，收集菌体后处理得到菌体上清，利用Ni-NTA亲和层析柱纯化得到重组ACY1抗原蛋白。
3.如权利要求1所述的试剂盒，其特征在于，所述标准品为经蛋白芯片技术定量检测所筛选出的含高水平ACY1抗体的血清标本混合液。
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