CN111073901A - 一种肌钙蛋白i检测试剂盒校准品的制备方法 - Google Patents
一种肌钙蛋白i检测试剂盒校准品的制备方法 Download PDFInfo
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- CN111073901A CN111073901A CN201911306223.XA CN201911306223A CN111073901A CN 111073901 A CN111073901 A CN 111073901A CN 201911306223 A CN201911306223 A CN 201911306223A CN 111073901 A CN111073901 A CN 111073901A
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Abstract
本发明公开一种肌钙蛋白I检测试剂盒校准品的制备方法，包括以下步骤：1）重组表达工程菌构建2）重组蛋白表达3）重组蛋白纯化3.1）cTnI蛋白纯化3.2）cTnC蛋白纯化4）cTnI‑cTnC二元复合抗原制备5）cTnI‑cTnC重组抗原配制化学发光检测试剂校准品配制校准品基质，并用0.22μm微孔滤膜过滤，用过滤后的校准品基质将步骤4）中制备的cTnI‑cTnC二元复合抗原作适当比例稀释，使其浓度符合试剂测试需求，作为肌钙蛋白I检测试剂盒校准品；本发明具有更好的免疫反应性和储存稳定性。
Description
技术领域
本发明属于体外诊断技术领域，具体涉及一种肌钙蛋白I检测试剂盒校准品的制备方法。
背景技术
肌钙蛋白(Tn)是骨骼肌和心肌收缩的调节蛋白，由三个结构不同的亚基通过非共价键结合组成，即肌钙蛋白I(TnI)、肌钙蛋白T(TnT)和肌钙蛋白C(TnC)。心肌肌钙蛋白I(cTnI)因其在检测特异性等方面更具优势，常被首选为急性心肌梗死(AMI)的诊断标志物。cTnI检测试剂的准确性往往受与之配套的校准质控体系性能影响，而质控体系性能又直接由关键原料cTnI抗原的稳定性及其免疫反应性所决定。cTnI抗原制备可通过多种途径实现，如从人源心脏组织中提取的天然cTnI抗原具有良好免疫反应性，但人源心脏组织来源有限难以批量制备；采用大肠杆菌表达系统制备重组cTnI抗原并制成冻干品以增加其稳定性，但冻干品形式增加了能耗成本及使用前再复溶的操作负担。研究报道cTnI在血液中主要以cTnI-cTnC二元复合物形式存在，cTnC的存在对cTnI起到保护作用，可防止cTnI被蛋白酶降解，因此cTnI-cTnC复合物更适合作为检测试剂盒的校准质控原料。文献报道采用大肠杆菌系统共表达cTnI-linker-cTnC融合蛋白用于检测试剂的质控原料，但以短肽连接形式共表达蛋白分子的空间折叠构象与天然分子形态差异大，难以保证其免疫反应性。
发明内容
本发明针对以上不足，提出了采用大肠杆菌表达系统分别制备重组cTnI与cTnC蛋白，再将两种蛋白以非共价结合形成cTnI-cTnC复合抗原的一种方法，获得更加接近人体血液中天然cTnI存在形态的二元复合抗原，以此为原料制备的cTnI检测试剂盒校准品具有更好的免疫反应性和储存稳定性。
为解决上述技术问题，本发明采用的技术方案为：一种肌钙蛋白I检测试剂盒校准品的制备方法，主要步骤包括：
1)重组表达工程菌构建
从NCBI数据库中分别查得人源cTnI(GenBank:NM_000363.5)和cTnC(GenBank:M22307.1)基因序列，分别在基因序列两端修饰合适的限制性酶切位点，并在两序列3’端添加-His纯化标签，将修饰后的基因序列送至外包服务公司进行基因合成。将合成的基因片段和选定的表达载体采用对应的限制性内切酶在合适温度下进行一定时间的酶切反应，采用T4 DNA连接酶在合适温度下反应一定时间进行连接，得到重组表达载体。最后将重组载体分别转化至合适的感受态细胞，涂Amp-LB培养基平板，在37℃条件下过夜培养，挑选单菌落进行PCR及酶切鉴定，将阳性克隆菌送至外包服务公司进行基因测序确认。
2)重组蛋白表达
将测序正确的cTnI和cTnC重组载体质粒分别转化E.coli BL21(DE3)感受态细胞，涂Amp-LB培养基平板，在37℃条件下过夜培养后挑取单菌落，接种至含Amp-LB液体培养基中在一定温度下进行摇床培养，当菌液生长密度达到OD600＝0.8时，加入一定终浓度的IPTG，再继续培养一定时间进行诱导表达，离心收集菌体，超声破碎后取裂解物，待进行分离纯化。
3)重组蛋白纯化
3.1)cTnI蛋白纯化：取适量cTnI菌体重悬于缓冲液I中，超声破碎后进行离心，因cTnI以不可溶的包涵体形态表达，故收集离心沉淀，并重悬于一定浓度的尿素中，再离心收集上清，在
pure蛋白纯化系统上进行层析柱纯化，最后将含目的蛋白的洗脱液透析至缓冲液II中，收样待用。
3.2)cTnC蛋白纯化：取适量cTnC菌体重悬于缓冲液I中，超声破碎后进行离心，因cTnC以可溶形态表达，故收集离心上清，在
pure蛋白纯化系统上进行层析柱纯化，最后将含目的蛋白的洗脱液透析至缓冲液II中，收样待用。
4)cTnI-cTnC二元复合抗原制备
分别取适量重组表达纯化的cTnI蛋白与cTnC蛋白按一定摩尔比例混合，并添加适量浓度的CaCl2，在合适温度下搅拌反应一定时间，再将混合样液透析至缓冲液III中，经多次透析后收样待用。
4.1)抗原反应性验证
采用酶联免疫(ELISA)双抗夹心法进行cTnI-cTnC抗原反应性验证，在96孔板中包被cTnI单抗，夹心做系列梯度稀释的cTnI-cTnC复合抗原，再加HRP标记的cTnC单抗，TMB显色后加H2SO4终止反应，酶标仪测定450nm光吸收值。
4.2)抗原储存稳定性验证
将cTnI-cTnC复合抗原和cTnI单体抗原分别稀释3.125，6.25，12.5，25，50μg/L五个梯度浓度，分别置于4℃和37℃条件下，采用cTnI化学发光试剂盒检测放置14天后的抗原浓度变化情况。
5)cTnI-cTnC重组抗原配制肌钙蛋白I检测试剂校准品
配制校准品基质，并用0.22μm微孔滤膜过滤。用过滤后的校准品基质将步骤4中制备的cTnI-cTnC二元复合抗原作适当比例稀释，使其浓度符合试剂测试需求，作为肌钙蛋白抗体检测试剂盒校准品，例如可稀释成0.5ng/ml及25ng/ml，分别对应校准品1和校准品2，也可稀释成其他浓度的校准品。
通过试验验证上述方法制备的肌钙蛋白I检测试剂校准品具有特异性强，稳定性好等优点。
上述步骤1)所选用的限制性内切酶位点为BamH I，EcoR I，EcoR V，NdeI，Not I，Nhe I，Xho I，Sal I中的两种或四种；
上述步骤1)所选用的表达载体为pET-22b，pET-28a，pET-30a，pET-32a，pET-44a中的一种或两种；
上述步骤1)中限制性内切酶酶切温度为16℃～37℃，酶切时间为0.5h～4h；
上述步骤1)中DNA连接酶反应温度为4℃～37℃，反应时间为2h～24h；
上述步骤1)中所用的感受态细胞为Top10，DH5a，JM109，Stbl 3中的一种或两种；
上述步骤2)中摇菌温度为4℃～37℃；
上述步骤2)中加入的诱导剂IPTG终浓度为0.05mM～0.2mM间；
上述步骤2)中进行诱导表达的时间为2h～24h；
上述步骤3.1)与3.2)中选用的缓冲液I为磷酸盐、Tris-Hcl、硼酸盐、碳酸盐、HEPES、MOPSO缓冲液中的一种，缓冲液的使用浓度为10mM～1000mM，且pH范围在6.0～9.0之间；
上述步骤3.1)中所用的尿素浓度为4M～8M间；
上述步骤3.1)与3.2)中选用的缓冲液II为Tris-Hcl、HEPES、MOPSO缓冲液中的一种，缓冲液的使用浓度为10mM～1000mM，且pH范围在6.0～9.0之间；
上述步骤3.1)与3.2)中选用的柱层析纯化方法为阴离子交换层析、阳离子交换层析、Ni2+离子螯合亲和层析和疏水相互作用层析中的一种或几种并用；
上述步骤4)中所用的cTnI与cTnC蛋白混合的摩尔比例是1:10～10:1；
上述步骤4)中添加CaCl2的终浓度为0.1mM～100mM；
上述步骤4)中反应温度为4℃～37℃，反应时间为1h～24h；
上述步骤4)中选用的反应搅拌方式为磁力搅拌，机械搅拌和垂直旋转混匀中的一种；
上述步骤4)中选用的缓冲液III为Tris-Hcl，HEPES，MOPSO缓冲液中的一种，使用浓度为10mM～1000mM，pH范围在6.0～9.0之间，且含有CaCl2的终浓度为0.1mM～10mM；
上述步骤5)中所述的校准品基质为磷酸盐、Tris-Hcl、MOPSO缓冲液中的一种，使用浓度为20mM～1000mM，pH范围在6.0～9.0之间。
上述步骤5)中所述的校准品基质包括20mM～150mM NaCl，0.1mM～10mM CaCl2，0.5％～10％牛血清白蛋白，0.1％～1％明胶和0.01％～0.2％防腐剂。所述的防腐剂优选为proclin300，叠氮钠，硫柳汞，庆大霉素，脱氢乙酸钠中的一种或几种。
附图说明
序列表：本发明表达的人cTnI和cTnC蛋白的氨基酸序列
图1：cTnI-pET22b(A)和cTnC-pET28a(B)重组质粒PCR和酶切鉴定图。
图2：cTnI(Lane 1&2)与cTnC(Lane 3&4)重组蛋白纯化SDS-PAGE图。
图3：cTnI单体和cTnI-cTnC抗原4℃储存条件下稳定性考察图。
图4：cTnI单体和cTnI-cTnC抗原37℃储存条件下稳定性考察图。
具体实施方式
下面用具体实施例对本发明做进一步详细说明，但本发明不仅局限于以下具体实施例。
实施例1
人肌钙蛋白cTnI和cTnC大肠表达载体构建
在cTnI和cTnC基因序列两端分别修饰NdeI，XhoI和NcoI，BamH限制性内切酶位点，基因合成后，采用NdeI，XhoI内切酶酶切cTnI基因片段和pET-22b质粒，同时采用NotI，BamH内切酶酶切cTnC基因片段和pET-28a质粒，37℃条件下酶切反应2h；在16℃条件下用T4 DNA连接酶分别进行连接16h，得到cTnI-pET22b和cTnC-pET28a重组表达载体。最后将重组载体分别转化Top10感受态细胞，涂Amp-LB培养基平板，37℃培养16h，挑选菌落进行PCR及酶切鉴定(结果见图1)，将阳性克隆菌送至外包服务公司进行基因测序确认。
实施例2
人肌钙蛋白cTnI和cTnC融合蛋白表达
将测序正确的cTnI-pET-22b和cTnC-pET-28a重组质粒分别转化E.coli BL21(DE3)感受态细胞，涂Amp-LB培养基平板，37℃培养16h后挑取单菌落，接种至含Amp的LB液体培养基中37℃摇床培养4h，当OD 600为0.8时，加入终浓度为0.4mM的IPTG，37℃继续培养4h，离心收集菌体；
实施例3
cTnI重组蛋白分离纯化
取适量cTnI菌体重悬于10mM PBS7.4缓冲液中，超声破碎后进行离心，因cTnI以不可溶的包涵体形态表达，故收集离心沉淀，并重悬于7M尿素中，再离心收集上清，在
pure蛋白纯化系统上先后进行Ni2+螯合亲和层析和SP阳离子柱纯化，最后将含目的蛋白的洗脱液透析至PBS7.4缓冲液中。
实施例4
cTnC重组蛋白分离纯化
取适量cTnC菌体重悬于10mM PBS7.4缓冲液中，超声破碎后进行离心，因cTnC以可溶形态表达，故收集离心上清，在
pure蛋白纯化系统上先后进行Ni2+螯合亲和层析和Q阴离子柱纯化，最后将含目的蛋白的洗脱液透析至PBS7.4缓冲液中。
实施例5
cTnI-cTnC二元复合蛋白制备
分别取适量重组cTnI蛋白与cTnC蛋白按摩尔比例1:1混合，并添加CaCl2至终浓度为5mM，室温条件下垂直旋转混匀2h，将混合样液透析至50mM Tris-Hcl缓冲液(pH7.5，含5mM CaCl2)中，按每次V(样液)：V(透析缓冲液)＝100比例进行三次透析后收样；
实施例6
cTnI-cTnC复合蛋白的抗原反应性验证
采用酶联免疫(ELISA)双抗夹心法进行cTnI-cTnC抗原反应性验证，在96孔板中包被2.0μg/ml的cTnI单抗，37℃孵育2h，添加2％BSA封闭1h后，加入cTnI-cTnC复合抗原，首孔50μg/L，按列对倍稀释，37℃孵育1h，PBST洗涤后，再加入1.0μg/ml HRP标记的cTnC单抗，37℃反应1h。TMB显色后加2M H2SO4终止反应，酶标仪测定450nm光吸收值。由表1中数据可见，与对照抗原比较，重组cTnI-cTnC复合蛋白与抗体反应信号较强，说明具有良好的抗原反应性；并且cTnI-cTnC复合蛋白能够同时被cTnI和cTnC抗体识别，说明cTnI-cTnC结合形成的复合物状态单一稳定。
表1自制复合蛋白的免疫反应性验证
注：cTnI单抗和cTnC单抗均是外购抗体；对照抗原为外购的cTnI/C二元复合抗原；空白对照组为以2％BSA替代夹心抗原
实施例7
cTnI-cTnC二元复合抗原的稳定性考察
采用SIMENS超敏肌钙蛋白I测定试剂盒分别测得重组cTnI单体和cTnI-cTnC二元复合物蛋白的浓度，稀释五个浓度梯度样液(50ng/ml，25ng/ml，12.5ng/ml，6.25ng/ml，3.125ng/ml)，分别于4℃和37℃条件下放置14天后再检测各样液的cTnI浓度变化情况。如图3和图4所见，不论是4℃还是37℃储存条件下，cTnI单体蛋白在放置14天后的浓度较起始浓度均具有较大幅度降低，降幅达30％～80％之间，可能原因是在储存过程中发生蛋白降解导致其测值大幅降低，且37℃条件下加速了其降解速率；而cTnI-cTnC二元复合蛋白的浓度变化较小，即使在37℃热加速条件下浓度变化幅度均在±15％间，说明cTnI-cTnC二元复合蛋白因cTnC对cTnI的空间保护作用具有良好的储存稳定性。
实施例8
cTnI-cTnC重组抗原配制化学发光检测试剂校准品
(1)配制pH 7.0，浓度为100mM的MOPSO缓冲液，向其中加入150mM NaCl，10mMCaCl2，5％牛血清白蛋白，0.5％明胶和0.2％叠氮钠。并用0.22μm微孔滤膜过滤；
(2)向过滤后的校准品基质中加入cTnI-cTnC二元复合抗原，稀释终浓度分别为0.5ng/ml和25ng/ml；
(3)将其分别混合均匀后，作为肌钙蛋白抗体检测试剂盒校准品；
上述实施例中所用pH值的缓冲液、防腐剂可替换成前述限定的任意一种或多种的组合。
实施例9
配制试剂校准品的抗原反应性验证
cTnI的标准参考物质SRM2921是公认的cTnI检测试剂的对照品，标准参考物质的标示浓度为(31.2±1.4)mg/L，将其以健康人血清分别进行稀释至0.5ng/ml和25ng/ml，计为S1和S2。同时用cTnI-cTnC二元复合抗原制备浓度为0.5ng/ml和25ng/ml的自制校准品，计为s1和s2。
采用SIMENS超敏肌钙蛋白I测定试剂盒同时测定标准参考物质及自制的校准品。由表2结果可见，该校准品与标准参考物质在同样的配制浓度条件下，实测浓度均与理论浓度相近，说明两者具有相同的免疫反应性。
表2自制校准品与标准参考物质的免疫反应性
实施例10
配制试剂校准品的稳定性考察
(1)热加速稳定性
将配置的cTnI检测试剂校准品，于37℃恒温箱中密封分别放置7天，14天后取样测定浓度，与起始时间比较，计算浓度变化率。由表3所示结果可见，自制cTnI检测试剂校准品，于37℃恒温箱中密封放置14天后，浓度变化率<5％，说明本发明的校准品热加速稳定性良好。
表3cTnI检测试剂校准品热加速稳定性
注：上述测值均由SIMENS超敏肌钙蛋白I测定试剂盒测得。
(2)开封稳定性
将配置的cTnI检测试剂校准品，试剂瓶开封后，再放置于2℃～8℃条件下，并于7天、14天、21天、28天取样测定浓度，与起始时间比较，计算浓度变化率。由表4所示结果可见，开封7天、14天、21天、28天的cTnI检测试剂校准品的测定浓度变化率均小于5％，说明本发明的cTnI检测试剂校准品开封稳定性良好。
表4cTnI检测试剂校准品开封稳定性
注：上述测值均由SIMENS超敏肌钙蛋白I测定试剂盒测得。
(3)效期稳定性
将配制的cTnI检测试剂校准品，于2℃～8℃条件下密封放置12个月，分别于3，6，9，12个月后取样测定浓度，与起始时间比较，计算浓度变化率。由表5所示结果可见，自制cTnI检测试剂校准品于2℃～8℃条件下密封放置12个月测值变化率<10％，说明本发明的自制校准品效期稳定性良好。
表5cTnI检测试剂校准品效期稳定性
注：上述测值均由SIMENS超敏肌钙蛋白I测定试剂盒测得。
以上仅是本发明的特征实施范例，对本发明保护范围不构成任何限制。凡采用同等交换或者等效替换而形成的技术方案，均落在本发明权利保护范围之内。
序列表
<110> 美康生物科技股份有限公司
<120> 一种肌钙蛋白I检测试剂盒校准品的制备方法
<130> 2019
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 216
<212> PRT
<213> cTnI蛋白氨基酸序列(人)
<400> 1
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Ala Pro Ile Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala Thr Glu
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Pro His Ala Lys Lys Lys Ser Lys Ile Ser Ala Ser Arg Lys Leu Gln
35 40 45
Leu Lys Thr Leu Leu Leu Gln Ile Ala Lys Gln Glu Leu Glu Arg Glu
50 55 60
Ala Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala Leu Ser Thr Arg Cys
65 70 75 80
Gln Pro Leu Glu Leu Ala Gly Leu Gly Phe Ala Glu Leu Gln Asp Leu
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Cys Arg Gln Leu His Ala Arg Val Asp Lys Val Asp Glu Glu Arg Tyr
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Asp Ile Glu Ala Lys Val Thr Lys Asn Ile Thr Glu Ile Ala Asp Leu
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Thr Gln Lys Ile Phe Asp Leu Arg Gly Lys Phe Lys Arg Pro Thr Leu
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Arg Arg Val Arg Ile Ser Ala Asp Ala Met Met Gln Ala Leu Leu Gly
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Ala Arg Ala Lys Glu Ser Leu Asp Leu Arg Ala His Leu Lys Gln Val
165 170 175
Lys Lys Glu Asp Thr Glu Lys Glu Asn Arg Glu Val Gly Asp Trp Arg
180 185 190
Lys Asn Ile Asp Ala Leu Ser Gly Met Glu Gly Arg Lys Lys Lys Phe
195 200 205
Glu Ser His His His His His His
210 215
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<212> PRT
<213> cTnC蛋白氨基酸序列(人)
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Met Thr Asp Gln Gln Ala Glu Ala Arg Ser Tyr Leu Ser Glu Glu Met
1 5 10 15
Ile Ala Glu Phe Lys Ala Ala Phe Asp Met Phe Asp Ala Asp Gly Gly
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Gly Asp Ile Ser Val Lys Glu Leu Gly Thr Val Met Arg Met Leu Gly
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Gln Thr Pro Thr Lys Glu Glu Leu Asp Ala Ile Ile Glu Glu Val Asp
50 55 60
Glu Asp Gly Ser Gly Thr Ile Asp Phe Glu Glu Phe Leu Val Met Met
65 70 75 80
Val Arg Gln Met Lys Glu Asp Ala Lys Gly Lys Ser Glu Glu Glu Leu
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Pro Gly Glu Leu Ala Glu Ile Phe Arg Ala Ser Gly Glu His Val Thr
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Gly Arg Ile Asp Phe Asp Glu Phe Leu Lys Met Met Glu Gly Val Gln
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His His His His His His
165
Claims (7)
1.一种肌钙蛋白I检测试剂盒校准品的制备方法，其特征在于，包括以下步骤：
1)重组表达工程菌构建
从NCBI数据库中分别查得人源cTnI(GenBank:NM_000363.5)和cTnC(GenBank:M22307.1)基因序列，分别在基因序列两端修饰合适的限制性酶切位点，并在两序列3’端添加-His纯化标签，将修饰后的基因序列进行基因合成；将合成的基因片段和选定的表达载体采用对应的限制性内切酶在合适温度下进行一定时间的酶切反应，再用T4 DNA连接酶在合适温度下反应一定时间进行连接，得到重组表达载体；最后将重组载体分别转化至合适的感受态细胞，涂Amp-LB培养基平板，在37℃下过夜培养，挑选单菌落进行PCR及酶切鉴定，将阳性克隆菌进行基因测序确认；
2)重组蛋白表达
将测序正确的cTnI和cTnC重组载体质粒分别转化E.coliBL21(DE3)感受态细胞，涂Amp-LB培养基平板，在37℃下过夜培养后挑取单菌落，接种至含Amp-LB液体培养基中在一定温度下进行摇床培养，当菌液生长密度达到OD600＝0.8时，加入一定终浓度的IPTG，再继续培养一定时间进行诱导表达，离心收集菌体，超声破碎后取裂解物，待进行分离纯化；
3)重组蛋白纯化
3.1)cTnI蛋白纯化：取适量cTnI菌体重悬于缓冲液I中，超声破碎后进行离心，收集离心沉淀，并重悬于一定浓度的尿素中，再离心收集上清，在
pure蛋白纯化系统上进行层析柱纯化，最后将含目的蛋白的洗脱液透析至缓冲液II中，收样待用；
3.2)cTnC蛋白纯化：取适量cTnC菌体重悬于缓冲液I中，超声破碎后进行离心，因cTnC以可溶形态表达，故收集离心上清，在
pure蛋白纯化系统上进行层析柱纯化，最后将含目的蛋白的洗脱液透析至缓冲液II中，收样待用；
4)cTnI-cTnC二元复合抗原制备
分别取适量重组表达纯化的cTnI蛋白与cTnC蛋白按一定摩尔比例混合，并添加适量浓度的CaCl2，在合适温度下搅拌反应一定时间，再将混合样液透析至缓冲液III中，经多次透析后收样待用；
5)cTnI-cTnC重组抗原配制化学发光检测试剂校准品
配制校准品基质，并用0.22μm微孔滤膜过滤，用过滤后的校准品基质将步骤4)中制备的cTnI-cTnC二元复合抗原作适当比例稀释，使其浓度符合试剂测试需求，作为肌钙蛋白I检测试剂盒校准品。
2.根据权利要求1所述的肌钙蛋白I检测试剂盒校准品的制备方法，其特征在于，步骤1)中所用的限制性内切酶位点为BamH I，EcoR I，EcoR V，Nde I，Not I，Nhe I，Xho I，SalI中的两种或四种；所选用的表达载体为pET-22b，pET-28a，pET-30a，pET-32a，pET-44a中的一种或两种；所用的限制性内切酶酶切温度为16℃～37℃，酶切时间为0.5h～4h；所用的DNA连接酶反应温度为4℃～37℃，反应时间为2h～24h；所用的感受态细胞为Top10，DH5a，JM109，Stbl 3中的一种或两种。
3.根据权利要求1所述的肌钙蛋白I检测试剂盒校准品的制备方法，其特征在于，步骤2)中所用的摇菌温度为4℃～37℃；所用的诱导剂IPTG终浓度为0.05mM～0.2mM；所用的诱导表达时间为2h～24h。
4.根据权利要求1所述的肌钙蛋白I检测试剂盒校准品的制备方法，其特征在于，步骤3.1)与3.2)中选用的缓冲液I为磷酸盐、Tris-Hcl、硼酸盐、碳酸盐、HEPES、MOPSO缓冲液中的一种，缓冲液的使用浓度为10mM～1000mM，且pH范围在6.0～9.0之间；3.1)中所用的尿素浓度为4M～8M间；3.1)与3.2)中选用的缓冲液II为Tris-Hcl、HEPES、MOPSO缓冲液中的一种，缓冲液的使用浓度为10mM～1000mM，且pH范围在6.0～9.0之间；3.1)与3.2)中选用的柱层析纯化方法为阴离子交换层析、阳离子交换层析、Ni2+离子螯合亲和层析和疏水相互作用层析中的一种或几种并用。
5.根据权利要求1所述的肌钙蛋白I检测试剂盒校准品的制备方法，其特征在于，步骤4)中所用的cTnI与cTnC蛋白混合的摩尔比例是1:10～10:1；所用的CaCl2终浓度为0.1mM～100mM；所用的反应温度为4℃～37℃，反应时间为1h～24h；选用的反应搅拌方式为磁力搅拌，机械搅拌和垂直旋转混匀中的一种；选用的缓冲液III为Tris-Hcl，HEPES，MOPSO缓冲液中的一种，使用浓度为10mM～1000mM，pH范围在6.0～9.0之间，且含有CaCl2的终浓度为0.1mM～10mM。
6.根据权利要求1所述的肌钙蛋白I检测试剂盒校准品的制备方法，其特征在于，步骤5)中所用的的校准品基质为磷酸盐、Tris-Hcl、MOPSO缓冲液中的一种，使用浓度为20mM～1000mM，pH范围在6.0～9.0之间；所述的校准品基质包括20mM～150mM NaCl，0.1mM～10mMCaCl2，0.5％～10％牛血清白蛋白，0.1％～1％明胶和0.01％～0.2％防腐剂。
7.根据权利要求6所述的肌钙蛋白I检测试剂盒校准品的制备方法，其特征在于，所述的防腐剂为proclin300，叠氮钠，硫柳汞，庆大霉素，脱氢乙酸钠中的一种或几种。
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| WO2021120856A1 (zh) * | 2019-12-18 | 2021-06-24 | 美康生物科技股份有限公司 | 一种肌钙蛋白i检测试剂盒校准品的制备方法 |
| CN114034869A (zh) * | 2020-10-16 | 2022-02-11 | 广东菲鹏生物有限公司 | 一种心肌肌钙蛋白i检测方法和试剂盒 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998029726A2 (en) * | 1996-12-27 | 1998-07-09 | Coulter International Corp. | Method of detecting and determining the concentration of total troponin i in a biological sample |
| CN103951734A (zh) * | 2014-04-21 | 2014-07-30 | 江苏省农业科学院 | 边界病病毒重组E2蛋白及其IgG抗体检测试剂盒 |
| CN107216381A (zh) * | 2017-04-24 | 2017-09-29 | 郑忠亮 | cTnI‑cTnC‑cTnT三聚体蛋白及其制备方法和cTnI检测试剂盒 |
| CN109912713A (zh) * | 2019-01-08 | 2019-06-21 | 美康生物科技股份有限公司 | 用于免疫诊断试剂配制的肌钙蛋白i抗体的制备方法及获得的菌株 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935354A (zh) * | 2010-07-08 | 2011-01-05 | 温州医学院 | 人心肌肌钙蛋白t/人心肌肌钙蛋白i融合蛋白 |
| WO2016127318A1 (zh) * | 2015-02-10 | 2016-08-18 | 深圳市新产业生物医学工程股份有限公司 | 心肌肌钙蛋白i超敏检测试剂盒及其超敏检测方法 |
| CN108375559B (zh) * | 2018-02-08 | 2021-01-15 | 南京岚煜生物科技有限公司 | 基于微流控芯片的心肌肌钙蛋白试剂盒及制备和检测方法 |
| CN111073901B (zh) * | 2019-12-18 | 2023-04-28 | 美康生物科技股份有限公司 | 一种肌钙蛋白i检测试剂盒校准品的制备方法 |
-
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-
2020
- 2020-10-27 WO PCT/CN2020/123919 patent/WO2021120856A1/zh active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998029726A2 (en) * | 1996-12-27 | 1998-07-09 | Coulter International Corp. | Method of detecting and determining the concentration of total troponin i in a biological sample |
| CN103951734A (zh) * | 2014-04-21 | 2014-07-30 | 江苏省农业科学院 | 边界病病毒重组E2蛋白及其IgG抗体检测试剂盒 |
| CN107216381A (zh) * | 2017-04-24 | 2017-09-29 | 郑忠亮 | cTnI‑cTnC‑cTnT三聚体蛋白及其制备方法和cTnI检测试剂盒 |
| CN109912713A (zh) * | 2019-01-08 | 2019-06-21 | 美康生物科技股份有限公司 | 用于免疫诊断试剂配制的肌钙蛋白i抗体的制备方法及获得的菌株 |
Non-Patent Citations (1)
| Title |
|---|
| 武强等: "重组cTnI-cTnC 复合抗原的制备及其性能验证" * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021120856A1 (zh) * | 2019-12-18 | 2021-06-24 | 美康生物科技股份有限公司 | 一种肌钙蛋白i检测试剂盒校准品的制备方法 |
| CN114034869A (zh) * | 2020-10-16 | 2022-02-11 | 广东菲鹏生物有限公司 | 一种心肌肌钙蛋白i检测方法和试剂盒 |
| CN112903836A (zh) * | 2021-01-13 | 2021-06-04 | 上海凯宝药业股份有限公司 | 体外培育熊胆粉中异丙基-β-D-硫代吡喃半乳糖苷的测定方法 |
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