CN114592005B - 一种糖皮质激素的检测方法 - Google Patents

一种糖皮质激素的检测方法 Download PDF

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CN114592005B
CN114592005B CN202210383164.1A CN202210383164A CN114592005B CN 114592005 B CN114592005 B CN 114592005B CN 202210383164 A CN202210383164 A CN 202210383164A CN 114592005 B CN114592005 B CN 114592005B
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黄晶
陈仕玺
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Abstract

一种糖皮质激素的检测方法，涉及检测技术领域。用人凝血因子f5基因启动子连接荧光素酶报告基因载体，构建报告质粒，将报告质粒、人糖皮质激素受体表达质粒以及内参质粒共转染HEK293T细胞，用待测样品刺激转染后的HEK293T细胞，通过检测荧光素酶的活性来检测样品中的糖皮质激素。检测方法重复性好、灵敏度高，检测结果可靠，且操作简单方便，可广泛应用于糖皮质激素检测。

Description

一种糖皮质激素的检测方法
技术领域
本发明涉及检测技术领域，尤其是涉及一种糖皮质激素的检测方法。
背景技术
糖皮质激素作为医药品，广泛用于人类和动物医疗，能够调节糖、脂肪和蛋白质的合成和代谢，主要用于抗炎反应、免疫抑制等疾病的治疗。糖皮质激素具有强极性且不易吸附于生物体的特点，使得糖皮质激素的50％～60％很快随尿液和粪便排入体外，通过各种途径进入环境。药物使用、生活污水、医疗废水、农牧渔养殖业废水是环境水体中糖皮质激素的主要来源。
激素类化合物作为潜在的污染物比其他有害的外源性物质更具有内分泌干扰特性，环境糖皮质激素可与糖皮质激素受体结合，从而模拟糖皮质激素活性，对人类生殖系统、内分泌及免疫系统产生不良影响，可导致鱼类、鸟类及哺乳动物出现不同程度的生理异常、生殖障碍、甚至种群退化，破坏微生物种群结构，影响微生物的降解能力。动物体长时间暴露高剂量糖皮质激素的，可能导致免疫反应抑制，生理状况恶化等一系列生理障碍。环境糖皮质激素对人类生殖健康的影响已经引起人们的广泛关注，建立一套快速、有效、准确的检测环境糖皮质激素的方法已经成为当务之急。
发明内容
本发明的目的在于针对现有技术存在的上述不足，提供简便、快速、可靠的一种糖皮质激素的检测方法。
本发明利用人凝血因子f5基因启动子受糖皮质激素上调的特点，建立一种通过检测糖皮质激素对上述启动子的激活情况，实现对环境糖皮质激素进行检测的方法，具体方法如下：
用人凝血因子f5基因启动子连接荧光素酶报告基因载体，构建报告质粒，将报告质粒、人糖皮质激素受体表达质粒以及内参质粒共转染HEK293T细胞(人胚肾细胞)，用待测样品刺激转染后的HEK293T细胞，通过检测荧光素酶的活性来检测样品中的糖皮质激素。
所述人凝血因子f5基因启动子核苷酸序列如SEQ ID N0：1所示。
所述荧光素酶报告基因载体为pGL3-Basic。
所述内参质粒为pRL-TK(海肾荧光素酶对照报告质粒)，作为转染实验的内参照，可以避免因转染效率的差异对结果的影响。
所述共转染HEK293T细胞所用试剂为Lipo8000转染试剂(该试剂转染效率较高、无毒性)，转染8～10h后，接种到24孔板中，在无酚红高糖DMEM培养基中继续培养12h。
所述用待测样品刺激转染后的HEK293T细胞，通过检测荧光素酶的活性来检测样品中糖皮质激素，是在样品刺激后22～26h再检测荧光素酶活性。
所述样品中的糖皮质激素为地塞米松(Dexamethasone，DHAP)，其他糖皮质激素的作用机制与地塞米松相同，本发明也可以用于检测其他种类的糖皮质激素。
pGL3-Basic萤火虫荧光素酶报告基因载体是本领域技术人员经常使用的一种荧光酶检测系统，pGL3-Basic载体不含启动子及增强子，将可能的启动子序列克隆到荧光素酶基因的上游，就可以测定其是否具有启动荧光素酶基因表达的活性。
人糖皮质激素受体表达质粒是人的受体，更适合于人启动子活性分析。HEK293T细胞为启动子的转录活性提供了较好的环境，有助于高效转录。
本发明将人凝血因子f5基因启动子序列亚克隆至pGL3-Basic载体萤火虫荧光素酶基因上游，从而构建一个报告质粒。由于人凝血因子f5基因启动子序列上含糖皮质激素反应元件(GRE)，因此能特异地对糖皮质激素刺激产生反应。
将上述构建好的报告质粒、人糖皮质激素受体表达质粒以及内参质粒(pRL-TK)共转染HEK293T细胞8～10h后，接种到24孔板中，在无酚红高糖DMEM(Gibco，含10％无激素胎牛血清)培养基中继续培养12h后，用待测样品对转染后的HEK293T细胞进行刺激，刺激后22～26h检测荧光素酶活性。根据荧光素酶活性是否明显升高可以检测待测环境样品的雄激素活性物质的存在。
与现有技术相比，本发明具有如下有益效果：
本发明用人凝血因子f5基因启动子连接荧光素酶报告基因，构建报告质粒。随后将报告质粒、人糖皮质激素受体表达质粒以及内参质粒共转染HEK293T细胞，再用待测样品刺激转染后的HEK293T细胞，通过检测荧光素酶的活性来检测样品中的糖皮质激素。本发明的检测方法重复性好、灵敏度高，检测结果可靠，且操作简单方便，可广泛应用于糖皮质激素检测，适用于环境安全、食品安全或临床医学等多领域，特别是针对环境糖皮质激素对人类生殖健康的影响问题，建立一套快速、有效、准确的检测环境糖皮质激素的方法。
附图说明
图1为实施例1中人凝血因子f5基因启动子对糖皮质激素地塞米松DHAP的剂量反应曲线。
具体实施方式
本发明利用人凝血因子f5基因启动子受糖皮质激素上调的特点，建立一种通过检测糖皮质激素对上述启动子的激活情况，实现对环境糖皮质激素进行检测的方法。以下实施例将结合附图对本发明作进一步的说明，但具体实施例并不对本发明做任何限定。
实施例1
人凝血因子f5基因启动子，其核苷酸序列如SEQ ID NO：1所示。
SEQNo.1
GCACACCCTACACTGCAAGAGAAAATCAGAGAAATGGAAAAATTCTTCATCACCTTAATATTTTACAGGGAAGAACAAATGTGTTTTACATGTTCTTTAAATCCCATGTTTAATAAATAGGTCATTGAAAGTTATATAGTATTTATCTGGAACTGATGTTAAAAAAAATAGAGAGCCAAAAATTATAGTAGTCATCTTGTCTCTGTAAAATGTAATTAAATAAATATGTCATGTACACTGTATATACATAAGCAAACATGCAAATAAAAAAGCAGCCTACTCAAAGGTTACAATAATGTTGCAACAATTCTGGTAGTAACCTTGGGATTTAGGGACACAGTGCTTTCAAAACGTTTCTCTGATACACTGCCAGTAGCATCTCTGACTGCTTATTTAAATGTCAATCAGTAGGCTAGGTGTTCTAGGACTTTGGTCTCAACCCTGTTTCCTGAGTCAGAATCTACATTTTGTAAAAGTTCCCCTGGGAATTCTGATGGTCTGCCAACTTTGGGAGACACTGATAATATTGTAGATAAAGAAGTGTAAGCAACTGTTCAGTTAGGGAGACAATAGATACCTGCATGGAATAACAAAAGGTAATTAACATGATTATATAATTTGAATAGTGTTGTAATAGTTCATAAAATGGAAAAGCCAATTTGAGCCCAGAATCTGGGAGATAGGCAGAACTCTGGACTGTAAAGAATGGCCCAGCAAAGAGGAAGAAAATGGGCATTCTAAGTTGGTGCAATAATATCAGCACAGGAGAAAAGACAGAATATTGGGGGGAGTGGGGAGGGAGGAAGAGGCTGTTGATGAGCCTGATATCATTAACAGAGACCTCATGTGAGGTGCCAAATTACAATTTATTGAATGTACTGAATGCATAAAGCATAAGACAGAAACCTGGAGCCATGCAGATACAGGGAACCAGGACAAGTCATTATGATGCCTCAGACAATATAATTCGCAGATTCTCTATCAATAGCAAAGTAAGATTTAAGGTGCTGTGAAACAGTCAGATCCTTCAAGAAAAACAAGAATACAAGGGGTCCTGGAGACAGGAAGATATGATCATAATAATAAATTTGTGATTCCTTCTTTCTCTCTTTTTCTCTTTCTTTCTTTTTCTTTCTTTCTTTTTTTTTTTTTGATGGAGTTTTGCTCTCGTTGCCCAGGCTGGAGTAAAATGGCATGATATCAGCTCACTGCAAACTCCACCTTCCAGGTTCAAGCAATTCTCTTGCCTCAATCTCTCGAGTAGGTGGGATTACAGGTGTCCACCACAACGCCCGGCTAATTTTTTATATTTTTAGTAGAGAAGGGTTTCACCATGTTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGGTGATTCACCCGCCTTGGCCTCCCAAAGTACTGGGATTACAGGCGTGAGCCACTGCGCCTAGCCTCTGTGTGTTAAGTGCTGGAACTATAGTTTACAGTAGCATTTTATCCTCCCAACATACCCTATCAGGAAAAATTATTATTGTCATCCTTATTTCACAGATGTCAAAACTGAACAGAGAGGTACAGTAACTTGCCCAAGACAACACAGCTAGTAAGTGAAACTGAAATCTGGACAACTGATTTTTTTCAGCCAAGCTCTTCAAGCCCGCTGCTATGCTGCAGCTTAGCTGGCGACAGGCCCTTA TCAAGGCAGAACTGTGCCTTGATAGAGGCCAGGAAGGCGATTCAGAGCAGTAGGCCTTGTCCATGTGTTTGTTCCCTGCTTCTTCACCACTTGAAAATAGTGTTTCAGTGCCCAGTAACAGGGCACAATACTCTTCTCCTCAGATTAAAAGGGGAGCCATCTGCTTAAGGTGGTTGGGTGCCACTGCACTGCACAGAAGGTTCTCCAAGTGTGAGCTTGGACCCAAGCAGGCCTCAGGTCAGTGGTAACGGACGACTGCTTCCGTGGTGTTGGCAGGTGGAAATGGAGGGATACAGCTTTGTCCGGCTCATGAATCCCTGGGCATTGTCCCGCCTGCTAGAGCCTCTGATCTCTGCCCCTTCTTCACCTGCAGTAAAACAGTCACTAGTTTGGTTGCTCTCCCTAATACCTCTGGTCACTGGGAGCTGTGATCTCACCAAACCCCTGCCAGGAAAAGCCCCAGAAAAAGCGGAGGGAGTGAGAGCCAGAGGCTGCTGCTGCCGTTTGCAAGAACTGCAGGGGAGGAGGACGCTGCCACCCACAGCCTCTAGAGCTCATTGCAGCTGGGACAGCCCGGAGTGTGGTTAGCAGCTCGGCAAGCGCTGCCCAGGTCCTGGGGTGGTGGCAGCCAGCGGGAGCAGGAAAGGAAGC
本实施例采用Promega公司的pGL3-Basic萤火虫荧光素酶报告基因载体。
本发明将上述人凝血因子f5基因启动子序列亚克隆至pGL3-Basic载体萤火虫荧光素酶基因上游，常规方法构建一个报告质粒，命名为pGL3-hf5。
本实施例的人凝血因子f5基因启动子序列上含糖皮质激素反应元件(GRE，糖皮质激素反应元件核苷酸序列如SEQ ID NO:2所示：TGCACAGAAGGTTCT)及其它天然辅助元件，因此能特异地对糖皮质激素刺激产生反应。
采用碧云天公司的Lipo8000把报告质粒(3000ng)、人糖皮质激素受体表达质粒(1500ng)、以及Promega公司的内参质粒(pRL-TK，150ng)共转染到HEK293T细胞中，8～10h后，将HEK293T细胞转移至24孔板(每孔105细胞)，在无酚红高糖DMEM(Gibco，含10％无激素胎牛血清)培养基中继续培养12h后，用待测样品(本实施例地塞米松)对转染细胞进行刺激，刺激后培养24h裂解细胞，采用双萤光素酶报告基因检测系统(Promega公司产品)测定荧光素酶活性。通过荧光素酶活性是否升高就可以检测待测环境样品的糖皮质激素活性物质的存在。
根据检测的荧光素酶活性图，如图1示。
从图1可以看出，地塞米松对人凝血因子f5基因启动子活性具有剂量依赖上调作用。
本发明利用人凝血因子f5基因启动子受糖皮质激素上调的特殊性，通过检测糖皮质激素对这种启动子的激活情况而检测环境糖皮质激素的存在，采用的技术方案基于检测糖皮质激素的生物学活性，结果可靠、重复性好、灵敏度高，且操作简单方便。
序列表
<110> 厦门大学
<120> 一种糖皮质激素的检测方法
<160> 2
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 人(Homo sapiens)
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gcacacccta cactgcaaga gaaaatcaga gaaatggaaa aattcttcat caccttaata 60
ttttacaggg aagaacaaat gtgttttaca tgttctttaa atcccatgtt taataaatag 120
gtcattgaaa gttatatagt atttatctgg aactgatgtt aaaaaaaata gagagccaaa 180
aattatagta gtcatcttgt ctctgtaaaa tgtaattaaa taaatatgtc atgtacactg 240
tatatacata agcaaacatg caaataaaaa agcagcctac tcaaaggtta caataatgtt 300
gcaacaattc tggtagtaac cttgggattt agggacacag tgctttcaaa acgtttctct 360
gatacactgc cagtagcatc tctgactgct tatttaaatg tcaatcagta ggctaggtgt 420
tctaggactt tggtctcaac cctgtttcct gagtcagaat ctacattttg taaaagttcc 480
cctgggaatt ctgatggtct gccaactttg ggagacactg ataatattgt agataaagaa 540
gtgtaagcaa ctgttcagtt agggagacaa tagatacctg catggaataa caaaaggtaa 600
ttaacatgat tatataattt gaatagtgtt gtaatagttc ataaaatgga aaagccaatt 660
tgagcccaga atctgggaga taggcagaac tctggactgt aaagaatggc ccagcaaaga 720
ggaagaaaat gggcattcta agttggtgca ataatatcag cacaggagaa aagacagaat 780
attgggggga gtggggaggg aggaagaggc tgttgatgag cctgatatca ttaacagaga 840
cctcatgtga ggtgccaaat tacaatttat tgaatgtact gaatgcataa agcataagac 900
agaaacctgg agccatgcag atacagggaa ccaggacaag tcattatgat gcctcagaca 960
atataattcg cagattctct atcaatagca aagtaagatt taaggtgctg tgaaacagtc 1020
agatccttca agaaaaacaa gaatacaagg ggtcctggag acaggaagat atgatcataa 1080
taataaattt gtgattcctt ctttctctct ttttctcttt ctttcttttt ctttctttct 1140
tttttttttt ttgatggagt tttgctctcg ttgcccaggc tggagtaaaa tggcatgata 1200
tcagctcact gcaaactcca ccttccaggt tcaagcaatt ctcttgcctc aatctctcga 1260
gtaggtggga ttacaggtgt ccaccacaac gcccggctaa ttttttatat ttttagtaga 1320
gaagggtttc accatgttgg ccaggctggt cttgaactcc tgacctcagg tgattcaccc 1380
gccttggcct cccaaagtac tgggattaca ggcgtgagcc actgcgccta gcctctgtgt 1440
gttaagtgct ggaactatag tttacagtag cattttatcc tcccaacata ccctatcagg 1500
aaaaattatt attgtcatcc ttatttcaca gatgtcaaaa ctgaacagag aggtacagta 1560
acttgcccaa gacaacacag ctagtaagtg aaactgaaat ctggacaact gatttttttc 1620
agccaagctc ttcaagcccg ctgctatgct gcagcttagc tggcgacagg cccttatcaa 1680
ggcagaactg tgccttgata gaggccagga aggcgattca gagcagtagg ccttgtccat 1740
gtgtttgttc cctgcttctt caccacttga aaatagtgtt tcagtgccca gtaacagggc 1800
acaatactct tctcctcaga ttaaaagggg agccatctgc ttaaggtggt tgggtgccac 1860
tgcactgcac agaaggttct ccaagtgtga gcttggaccc aagcaggcct caggtcagtg 1920
gtaacggacg actgcttccg tggtgttggc aggtggaaat ggagggatac agctttgtcc 1980
ggctcatgaa tccctgggca ttgtcccgcc tgctagagcc tctgatctct gccccttctt 2040
cacctgcagt aaaacagtca ctagtttggt tgctctccct aatacctctg gtcactggga 2100
gctgtgatct caccaaaccc ctgccaggaa aagccccaga aaaagcggag ggagtgagag 2160
ccagaggctg ctgctgccgt ttgcaagaac tgcaggggag gaggacgctg ccacccacag 2220
cctctagagc tcattgcagc tgggacagcc cggagtgtgg ttagcagctc ggcaagcgct 2280
gcccaggtcc tggggtggtg gcagccagcg ggagcaggaa aggaagc 2327
<210> 2
<211> 15
<212> DNA
<213> 人(Homo sapiens)
<400> 2
tgcacagaag gttct 15

Claims (6)

1.一种糖皮质激素的检测方法，其特征在于其具体步骤为：用人凝血因子f5基因启动子连接荧光素酶报告基因载体，构建报告质粒，随后将报告质粒、人糖皮质激素受体表达质粒以及内参质粒共转染HEK293T细胞，再用待测样品刺激转染后的HEK293T细胞，通过检测荧光素酶的活性来检测样品中的糖皮质激素；所述人凝血因子f5基因启动子的核苷酸序列如序列表 SEQ ID N0：1所示。
2.根据权利要求1所述的一种糖皮质激素的检测方法，其特征在于所述荧光素酶报告基因载体为pGL3-Basic。
3.根据权利要求1所述的一种糖皮质激素的检测方法，其特征在于所述内参质粒为pRL-TK。
4.根据权利要求1所述的一种糖皮质激素的检测方法，其特征在于所述共转染HEK293T细胞所用试剂为Lipo8000，且转染8～10h后，接种到24孔板中，在无酚红高糖DMEM培养基中继续培养12h。
5.根据权利要求1所述的一种糖皮质激素的检测方法，其特征在于所述用待测样品刺激转染后的HEK293T细胞，通过检测荧光素酶的活性来检测样品中的糖皮质激素，是在样品刺激后22～26h再检测荧光素酶活性。
6.根据权利要求1所述的一种糖皮质激素的检测方法，其特征在于所述糖皮质激素为地塞米松。
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WO2001040494A1 (en) * 1999-12-01 2001-06-07 Centre For Translational Research In Cancer Drug inducible system and use thereof
KR20030046896A (ko) * 2001-12-07 2003-06-18 학교법인 포항공과대학교 당질코르티코이드에 의해 발현이 조절되는 재조합 리포터유전자를 가지는 형질전환 세포주 및 이를 이용한당질코르티코이드 유사물질 및 저해물질의 생물학적검색방법
JP2006265173A (ja) * 2005-03-24 2006-10-05 Sumitomo Chemical Co Ltd グルココルチコイド受容体リガンド結合ドメインとリガンドとの複合体を含む結晶
CN102753572A (zh) * 2009-10-06 2012-10-24 百时美施贵宝公司 应用糖皮质激素在哺乳动物细胞培养物中生产糖蛋白的方法
CN109022467A (zh) * 2018-09-20 2018-12-18 中山大学 新型多功能双荧光素酶报告基因质粒

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001040494A1 (en) * 1999-12-01 2001-06-07 Centre For Translational Research In Cancer Drug inducible system and use thereof
KR20030046896A (ko) * 2001-12-07 2003-06-18 학교법인 포항공과대학교 당질코르티코이드에 의해 발현이 조절되는 재조합 리포터유전자를 가지는 형질전환 세포주 및 이를 이용한당질코르티코이드 유사물질 및 저해물질의 생물학적검색방법
JP2006265173A (ja) * 2005-03-24 2006-10-05 Sumitomo Chemical Co Ltd グルココルチコイド受容体リガンド結合ドメインとリガンドとの複合体を含む結晶
CN102753572A (zh) * 2009-10-06 2012-10-24 百时美施贵宝公司 应用糖皮质激素在哺乳动物细胞培养物中生产糖蛋白的方法
CN109022467A (zh) * 2018-09-20 2018-12-18 中山大学 新型多功能双荧光素酶报告基因质粒

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