CN1227533C - 一种诊断心肌梗塞的试剂 - Google Patents
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Abstract
本发明公开了一种诊断心肌梗塞的试剂。本发明的心肌梗塞诊断试剂,是固定在支持物上与人心肌钙结合蛋白S100A1互补的单克隆或多克隆抗体。本发明诊断试剂的应用在早期性、敏感性和特异性方面均大大优于目前所使用的各种检测方法,而且诊断试剂专一,无污染,操作简便,反应时间短。
Description
技术领域
本发明涉及一种疾病的诊断试剂,特别是一种诊断心肌梗塞的试剂。
背景技术
心血管疾病是人类的主要疾病之一。随着人口老龄化程度的不断增加,急性心肌梗塞(AMI)的死亡率逐年上升。我国作为人口大国,心肌梗塞的发病率很高。对于急性心肌梗塞病人来说,时间就是生命,但是只有在确诊是心肌梗塞后,才可以开始溶解血栓的治疗,否则会造成非常严重的后果,甚至会出现生命危险。已有文献报道,有一种抑郁症的体征表现与心肌梗塞非常相似,同时心肌梗塞的病人也经常发生没有典型胸痛或心电图没有新变化的情况,对及时确诊造成了很大影响。一项欧美研究发现,有百分之六患心肌梗塞的病人因没有典型胸痛或心电图没有新变化,而被送回家中,因而有25%病人在短期内死亡,死亡率比入院接受治疗的病人多两倍。这意味着病发诊断的时期越早,时间越短,治疗就开始的越早,心肌损伤的程度就越轻,患者完全康复存活的几率就越大。如果诊断时间太长病人就会失去冠状动脉再通的机会或因心肌细胞坏死而导致死亡。如何在发病初期迅速准确地诊断,使心肌梗塞病人得到及时治疗,是挽救病人生命,降低心肌梗塞死亡率的关键。
目前,临床上急性心肌梗塞的生化检测越来越成为一种重要的检测手段,包括血清谷氨酸乙酸转氨酶/天冬氨酰转氨酶(SGOT/AST)、乳酸脱氢酶(LDH)、肌红蛋白、肌酸激酶(CK)、肌酸激酶MB同工酶(CK-MB)和肌钙蛋白(Troponin)等生化标志物均已开发作为急性心肌梗塞的生化诊断试剂。但是这些诊断试剂在灵敏性、特异性、早期性以及产品的价格等方面都或多或少存在一些问题。SGOT/AST是最早用于心肌梗塞诊断的血清酶标志物,尽管它们在心肌含量最多,但由于其大量存在于其它多种器官,如肝脏、肌肉等,临床诊断干扰比较大,因此诊断特异性较低。LDH是继AST之后一年,研究提出的另一项诊断心肌梗塞的血清酶标志物,由于LDH分子量较大,在心肌梗塞发生时血清中酶活性升高较其它酶缓慢,诊断的特异性较差。CK,特别是其同工酶的分析对于心肌梗塞的早期诊断提供了较特异、灵敏的指标。除骨骼肌外,心肌细胞含有大量的CK,当发生心肌梗塞时,释放出的CK量超过其它酶。又由于CK的分子量较小,所以发生心肌梗塞时,相对于其它酶能够较早进入血液。但是CK在体内的半衰期明显较其它酶短,48-72小时即恢复正常,因此不能用于亚急性心肌梗塞的诊断,同时,过高的生产成本也限制了CK的广泛应用。测定CK-MB的质量是目前诊断心肌梗塞的常规分析指标,但由于血液中的CK-MB升高要在心肌梗塞症状发生后4-6小时,因此不能对心肌梗塞进行更早期的诊断,也不能诊断不稳定心绞痛,并且结果还会受到骨骼肌CK-MB的影响。Troponin主要由Troponin I、Troponin T和Troponin C(分别简称为cTnI、cTnT、cTnC)三个亚基组成,当心肌细胞破损后,cTnI和cTnT能够释放到血液中。cTnI单体分子量为23500Da,临床特异性大于98.9%,明显高于CK-MB。但由于一般心肌梗塞发生7-12小时后才可在血液中检测到其明显变化,因此对于早期诊断会产生一些影响。肌红蛋白的分子量小,组织/血清浓度比大,清除率快,是目前心肌受损后最早异常变化的血清标志物,它的特殊价值只是在于排除心肌梗塞。因此,迫切需要开发一种高灵敏度、高特异性、更早期和价格合理的急性心肌梗塞生化诊断试剂。
另一方面,人心肌钙结合蛋白S100Al是一个具有93个氨基酸残基的小分子量酸性蛋白,其分子量为10415道尔顿,MEDLINE号为93041710,SWISS-PROT号为P23297。Northern blot分析表明钙结合蛋白S100A1主要存在于人类心脏中,在慢缩骨骼肌和肾组织中有少量存在,在绝大部分其它人体组织中没有。在发生心肌梗塞的患者血浆中,S100A1水平相对于正常水平呈数十倍甚至成百上千倍的增加;对于骨骼肌受到损伤或肾功能不全的患者中,S100A1在血浆中的水平不发生任何变化。此外,对照实验证明,血浆中前面所提的几个标志蛋白以及与S100A1同一家族的其他蛋白并不干扰S100A1的抗体抗原特异性反应实验。
发明内容
本发明的目的是提供一种能够较早期、高灵敏、高特异性、价格较低的心肌梗塞诊断试剂。
一种心肌梗塞诊断试剂,是固定在支持物上与人心肌钙结合蛋白S100A1互补的单克隆或多克隆抗体。
本发明试剂诊断心肌梗塞是基于ELISA法基础之上的。
所述支持物采用一般固相试剂的支持物均可。用常规方法即可将与人心肌钙结合蛋白S100A1互补的单克隆或多克隆抗体固定在支持物上。
制备与人心肌钙结合蛋白S100A1互补的多克隆抗体的方法,包括以下步骤:
1)人心肌钙结合蛋白S100A1基因在大肠杆菌中表达,获得人心肌钙结合蛋白S100A1;
2)用人心肌钙结合蛋白S100A1免疫动物,采集被免疫动物的血液,纯化血清中的IgG。
制备与人心肌钙结合蛋白S100A1互补的单克隆抗体的方法,包括以下步骤:
1)人心肌钙结合蛋白S100A1基因在大肠杆菌中表达,获得人心肌钙结合蛋白S100A1;
2)用人心肌钙结合蛋白S100A1免疫小鼠;
3)将被免疫小鼠脾脏细胞与骨髓瘤细胞融合,得到杂交瘤细胞;
4)筛选获得分泌抗人心肌钙结合蛋白S100A1的单克隆分泌细胞株。
本发明巧妙地利用人心肌钙结合蛋白S100A1在心肌梗塞患者血浆中的特异表现,将经标记后的人心肌钙结合蛋白S100A1单克隆或多克隆抗体固定在支持物上作为检测试剂,采用ELISA方法或金标法,根据检测试剂与样品中抗原反应后颜色的变化测定血液中抗原一心肌钙结合蛋白S100A1的含量,作出诊断结论。心肌梗塞患者血液中心肌钙结合蛋白S100A1初始浓度升高的平均时间为2.8±2.3h,达浓度峰值最高的平均时间为11.3±6.5h,恢复正常所需的时间为29.4±18.0h,平均最高浓度为358±1188ng/l,因此本发明诊断试剂的应用在早期性、敏感性和特异性方面均大大优于目前所使用的各种检测方法,而且诊断试剂专一,无污染,操作简便,反应时间短。选取17位心肌梗塞患者(病发后2-15小时不等)的血样,同时用10位正常人的血样作为对照,测定血液中S100A1的含量。测定结果:17位患者中有16位为强阳性,1位为弱阳性,而10位对照正常人的血样均为阴性。说明本发明选用S100A1作为急性心肌梗塞的诊断指标具有很高的灵敏度和准确性,病发后诊断时间也大大提前。
本发明利用基因工程的方法使人心肌钙结合蛋白S100A1在大肠杆菌中得到高效表达,并且得到大量的单克隆或多克隆抗体,成本较低,能够为广大的普通消费者所接受,为本发明试剂的商业化生产提供了保障。另外,由于人心肌钙结合蛋白S100A1的特异型及敏感性非常高,用多克隆抗体制成的试剂完全能够满足检测的需要,也是降低成本的重要因素。
下面结合具体实施例对本发明做进一步说明。
具体实施方式
实施例1、人心肌钙结合蛋白S100A1基因在大肠杆菌中的表达和纯化
1.表达质粒的构建和克隆
根据基因库中的人心肌钙结合蛋白S100A1的基因序列(X58079),设计两个引物,引物在5’端和3’端分别带上两个不同的限制性内切酶位点。
引物1为:5’-CATATGGGCTCTGAGCTGGAGA-3’
引物2为:5’-GGATCCTCAACTGTTCTCCCA-3’
提取人心肌的总mRNA,经反转录得到总cDNA后,用引物1和引物2进行PCR扩增。
PCR扩增体系如下:
样品 | 体积(μl) |
H2010×Reaction Buffer4×dNTP5’端引物P13’端引物P2模板Ex Taq聚合酶总体积 | 3754111150 |
反应程序如下:94℃5min;94℃30s,60℃30s,72℃30s,30个循环;2℃5min;4℃保存。
扩增产物回收后经双酶切(37℃),切胶回收。
挑取含pET25b载体质粒的单菌落,接种于5ml含100ug/ml氨苄青霉素(Amp)的LB培养基中,37℃培养过夜。取1.5ml菌液15,000rpm离心5分钟,弃上清。加入100ul溶液I(50mM葡萄糖,10mMEDTA,25mMTris,pH8.0),振荡混匀,加入200ul溶液II(0.2M NaOH,1%SDS)混匀后加入100ul溶液III(60ml5M KAC,11.5ml冰乙酸,28.5ml水),混匀5分钟后,15000rpm离心10分钟,取上清。加入等体积酚/氯仿抽提,15000rpm离心10分钟,取上清。加入等体积的异丙醇,混合后-20℃放置30分钟,15000rpm离心10分钟,弃上清,沉淀用70%乙醇清洗2次,风干,溶于无菌水。将pET25b载体质粒进行双酶切(37℃)。切胶回收得到pET25b载体大片段。
将两个回收片段按T.Maniatis等的分子克隆操作指南进行连接转化大肠杆菌BL21(DE3)。筛选阳性克隆。测序鉴定,表达的产物不含有多余的氨基酸,为阅读框架正确的阳性克隆。
2.S100A1蛋白的诱导表达
挑取一个单菌落,接种到含有100ug/mlAmp的50mlLB培养基中。37℃,200rpm培养过夜。按1%接种量转瓶,37℃,200rpm培养。4小时后(OD595=0.6-0.8),加入IPTG至终浓度为1mM。37℃继续诱导培养8-10小时。将菌液于15℃下5,000rpm,离心15min。收集菌体。
3.重组S100A1蛋白的纯化
重组S100A1蛋白的纯化主要分为二步。第一步是阴离子交换柱;第二步用疏水柱。
将菌体用Buffer A(25mM Tris,pH8.0)重悬,超声裂解菌体后,15000rpm离心30分钟,收集上清。将得到的上清样品上样到用Buffer A平衡好的阴离子交换柱中,用Buffer A清洗2个柱体积,然后用Buffer A和Buffer B(25mM Tris,1M NaCl,pH8.0)作线性梯度洗脱,共5个柱体积。电泳检测目的蛋白的位置后收集样品。
将收集的样品上到用Buffer C(25mM Tris,pH8.0)平衡好的疏水柱中,再用BufferC清洗5个柱体积后,用Buffer C和Buffer D(25mM Tris,pH8.0,50mM EDTA)作线性梯度洗脱,洗脱峰便是纯化的人心肌钙结合蛋白S100A1,16%SDS-PAGE检测重组蛋白的纯度大于95%。
实施例2、人心肌钙结合蛋白S100A1多克隆抗体的制备
取雄性大白兔(5-7周龄),用纯化的人心肌钙结合蛋白S100A1(0.1mg/只)加等体积的完全佐剂对雄性大白兔进行腿部初次免疫;三周后用纯化的人心肌钙结合蛋白S100A1(0.1mg/只)加等体积的非完全佐剂对大白兔进行腿部二次免疫;每两周后用第二次免疫的量对大白兔进行免疫,共免疫四次。采血前对已免疫的大白兔用0.1mg/只的量进行加强免疫,免疫结束后对大白兔进行采血,纯化其血清中的IgG,即为人心肌钙结合蛋白S100A1的多克隆抗体。
实施例3、人心肌钙结合蛋白S100A1单克隆抗体的制备
1、动物免疫
取BalB/C雌性小鼠(5-7周龄),用纯化的人心肌钙结合蛋白S100A1(0.05mg/只)加等体积的完全佐剂进行腹腔初次免疫,三周后用等量抗原加等体积的非完全佐剂对小鼠进行二次免疫,两周后用等量抗原加等体积的非完全佐剂对小鼠进行腹腔第三次免疫。细胞融合前三天,用抗原(0.05mg/只)对已免疫的小鼠进行直接脾脏注射,不加佐剂。
2、细胞融合
无菌条件下,取免疫的小鼠脾脏,收集脾细胞计数。在完全培养液中培养骨髓瘤细胞,取对数生长期的骨髓瘤细胞与脾细胞按1∶5比例混合,1000rpm离心十分钟,去上清,用非完全培养液洗细胞一次,同上法离心去上清,打散细胞聚集块,加50%PEG4000进行细胞融合。融合细胞加HAT选择培养液于培养板(105个细胞/孔)培养。一周后用1/2体积新鲜HAT选择培养液替换原有1/2体积细胞培养液,继续培养。待杂交瘤细胞数大于105个后,检测细胞培养液中是否含有抗体。方法如下:包被抗原为纯化的人心肌钙结合蛋白S100A1(0.01mg/ml),96孔酶标板每孔加0.05ml,4℃过夜,洗涤后加入待测培养液,37℃孵育1小时,洗涤后加入HRP-羊抗鼠IgG,37℃保温30分钟,洗涤后加底物H2O2及四甲基联苯胺进行显色,450nm测定光吸收,大于0.5以上即为阳性。
3、克隆化
用有限稀释法克隆阳性细胞,重复至阳性率100%,常规方法筛选获得分泌抗人心肌钙结合蛋白S100A1的单克隆分泌细胞株。
Claims (1)
1、一种心肌梗塞诊断试剂,是固定在支持物上与人心肌钙结合蛋白S100A1互补的单克隆或多克隆抗体。
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