CN102796807B - 一种筛选PknB抑制剂的反应体系及其筛选方法 - Google Patents
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Abstract
本发明提供一种用于筛选PknB抑制剂的反应体系,以及利用该体系筛选PknB抑制剂的方法。本发明的筛选方法包括配置反应体系、设置反应组、制备荧光素酶反应液和筛选。本发明PknB抑制剂的筛选方法简单可靠,效率高,成本低,是一种理想的抗生素筛选方法。
Description
技术领域
本发明涉及生物医药领域,具体涉及一种PknB抑制剂的反应体系及其筛选方法。
背景技术
结核分枝杆菌(Mycobacteria tuberculosis,Mtb)是人类肺结核(TB)的病原体。肺结核是一种严重威胁人类健康的传染病,在全球范围内每年造成多达三百万人死亡。AIDS的蔓延和多重耐药菌株的出现使肺结核的防控形势异常严峻,传统药物已不能满足需要。因此,必须加紧新型抗结核药物的研发。药物筛选模型的建立及潜在药物的筛选处于药物研发链条的最上游,为了开发出更加有效安全的新型抗结核药物,首先必须建立高效的药物筛选模型。
蛋白激酶PknB是其中的一种丝氨酸/苏氨酸蛋白激酶,可以磷酸化包括其自身在内的多种底物蛋白。已有研究结果显示,PknB参与多种重要生理进程的调控,为结核分枝杆菌生存所必需,具体包括:1.调控生长分裂,细胞形态以及细胞壁合成;2.调控热应激等多种应激反应;3.调控谷氨酸代谢。有关PknB的生理功能仍在进一步的研究中,但其作为潜在药物靶点的地位已得到广泛认同。但采用哪些反应体系、以及如何更快、更有效地筛选PknB抑制剂,是亟待解决的技术问题。
发明内容
因此,本发明要解决的技术问题是开发一种用于筛选PknB抑制剂的反应体系,并建立一种基于蛋白激酶PknB的药物筛选方法。
为了解决上述技术问题,本发明提供一种用于筛选PknB抑制剂的反应体系,以及一种筛选PknB抑制剂的方法。
本发明的技术方案如下。
本发明用于筛选PknB抑制剂的反应体系含有如下浓度的反应物:
优选地,本发明的反应体系含有如下浓度的反应物:
本发明还提供一种筛选PknB抑制剂的方法,包括如下步骤:
步骤(1)配置如下反应体系:
步骤(2)设置如下各反应组:
i)样品实验组:取步骤(1)所述的反应体系,加入1μL浓度为1mg/mL~10mg/mL的样品溶液;
ii)阴性实验组:取步骤(1)所述的反应体系,加入1μL DMSO;
iii)阳性对照组:取步骤(1)所述的反应体系,加入终浓度为60μg/ml的星形孢菌素;
步骤(3)制备荧光素酶反应液
采用Pomega公司的KinaseLuminescent Kinase Assay试剂盒进行活性测定,试剂盒中包括Kinase缓冲液和Kinase底物;将Kinase缓冲液在37℃下溶解,与Kinase底物充分混合,形成绿色澄清的溶液,即荧光素酶反应液;
步骤(4)筛选
将步骤(1)的各反应组在酶标仪中分别反应180min后,加入荧光素酶反应液,测定各反应组荧光值,计算反应抑制率,对反应具有抑制作用的即为阳性样品。
优选地,所述反应体系为:
优选地,其中步骤(2)中样品为化合物时,向50μL反应体系中加入1μL的1mg/mL的样品溶液作为样品实验组。
优选地,其中步骤(4)为将步骤(2)中各反应组分别混匀后加入同一块96孔板中,置于酶标仪内,开启温控为37℃进行反应,反应180min后取出,每孔均加入50μL荧光素酶反应液,置于酶标仪中测定每孔荧光值,每孔测定持续时间1s,测定次数1次;按以下公式计算各孔的抑制率:
其中:ΔFN是阴性对照孔荧光值,ΔFP是阳性对照孔荧光值,ΔFS是样品实验孔荧光值。
优选地,抑制率大于30%的样品为阳性样品。
本发明的有益效果如下。
本发明选择的蛋白激酶PknB作为药物筛选的靶点具有其独特的优点。PknB与其它蛋白激酶在结构以及催化特性方面的差异将有利于避免交叉耐药的问题。另外,PknB在不同细菌中的序列比较保守,而且与真核生物的同种功能的蛋白之间不具有相关性,有利于筛选到对结核分枝杆菌有效的抗生素,而且所筛选到的化合物对人体的毒性也会很小。因此用PknB筛选抗结核的药物的靶点,建立崭新、低毒、高效的筛选模型。
因此,本发明筛选PknB抑制剂的方法所选择的靶点特异性高,并通过分子生物学技术和细胞生物学技术建立体外高通量筛选模型。本发明的PknB酶活测定体系与传统的方法相比,简单可靠,方便易行。本发明PknB抑制剂的筛选方法简单可靠,效率高,成本低,是一种理想的抗生素筛选方法。
具体实施方案
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1样品筛选
本发明中以结核分枝杆菌PknB酶为例,说明抗菌药物的筛选方法。
一、重组PknB蛋白的制备
1、pET28ab/pknB质粒的构建
以结核分枝杆菌H37Rv基因组DNA为模板,通过PCR方法扩增pknB基因N端序列。引物序列为:上游引物5’TAACATATGACCACCCCTTCCCA3’;下游引物5’ATAAGCTTCTAACCGTTGTGCACGCGGA 3’。斜体部分分别是Nde I和Hind III的酶切位点。
PCR反应体系包括:
PCR反应的条件为:
将回收纯化的PCR产物和质粒载体pET28a经过Nde I和HindIII双酶切后,用T4DNA连接酶于16℃连接16h。
连接反应体系如下:
PCR产物与pET20b载体的摩尔比应该约为3∶1。
连接产物转化大肠杆菌DH5α,涂布于含有40μg/ml硫酸卡那霉素的LB平板上,37℃培养16h筛选重组子,提取质粒,双酶切鉴定插入片段后,然后进行测序。
2、目的蛋白的诱导表达以及分离纯化
将测序无误的重组质粒pET28a/pknB转化大肠杆菌Bl21(λDE3),得到Bl21/pknB(该生物材料大肠埃希氏菌(Escherichia coli)Bl21/pknB,已于2011年5月10日保藏于中国普通微生物菌种保藏管理中心(北京市朝阳区北辰西路1号院3号),保藏编号为CGMCCNo.4840)。挑取转化子在含有40μg/mL的硫酸卡那霉素的LB液体培养基(LB液体培养基(g/L:NaCl 10,胰蛋白胨10,酵母提取物5,pH 7.0)中。37℃200rpm振荡培养至培养液变浑浊后,按1∶50的比例将上述培养物转接于含40μg/mL的硫酸卡那霉素的LB液体培养基中,37℃200rpm振荡培养至OD600=0.8左右,加入终浓度1.0mMIPTG,20℃诱导表达过夜。
离心收集菌体,高压裂解菌体后,将裂解液在4℃,15000g离心30min,上清用0.45μm滤膜过滤。通过explorer系统,使用1ml HisTrapTM HP镍离子亲和层析柱纯化重组PknB蛋白。
二.星形孢菌素的活性测定
星形孢菌素是已知的、非特异性的蛋白激酶抑制剂。因此将其选为该模型的参考阻抑物以评价模型的有效性。按照上述样品活性检测的方法对星形孢菌素进行活性测定。将星形孢菌素(Sigma)1mg溶于100μL DMSO中,浓度为10mg/mL。稀释并加入到50μL反应体系,使其终浓度分别为1.25、2.5、5.0、10、20、40、60、80和100μg/ml。测定结果如表1所示。
表1星形孢菌素的酶活抑制量效关系
浓度(μg/mL) | 1.25 | 2.5 | 5.0 | 10 | 20 | 40 | 60 | 80 | 100 |
抑制率(%) | 22.12 | 24.40 | 29.29 | 38.50 | 55.53 | 76.99 | 84.86 | 85.72 | 88.58 |
三、样品制备
化合物样品:1mg纯品化合物溶到1ml DMSO中,取1μl作用于50μl反应体系,使其终浓度为20μg/ml。
四、样品活性检测
配制如下反应体系(每孔反应体系总体积为50μl):
按以下三个分组分别向反应体系中添加组分:
阴性对照组:加入1μl DMSO。
阳性对照组:加入终浓度为60μg/ml的星形孢菌素。
样品实验组:加入1μl待测样品。
活性测定采用Pomega公司的KinaseLuminescent KinaseAssay试剂盒,试剂盒中包括Kinase缓冲液和Kinase底物。将Kinase缓冲液在37℃下溶解,与Kinase底物充分混合,形成绿色澄清的溶液,即荧光素酶反应液备用。
将上述各组反应体系分别混匀后加入同一块96孔板中,置于Berthold公司的TriStarLB941酶标仪内,开启温控为37℃进行反应。反应180min后取出每孔均加入50μL荧光素酶反应液,置于酶标仪中测定每孔荧光值。每孔测定持续时间1s,测定次数1次。
结果分析:
依下列公式计算各孔的抑制率;
其中:
ΔFN是阴性对照孔荧光值,
ΔFP是阳性对照孔荧光值,
ΔFS是样品实验孔荧光值。
筛选结果为:从化合物库及天然产物库中共筛选5000个样品(以抑制率大于30%为筛选阳性标准),阳性样品率为0.10%,得到活性化合物5个,编号分别为YH-1、YH-2、YH-3、YH-4和YH-5,它们的抑制率分别为61.78%、61.22%、43.27%、31.15%、30.39%。
实施例2
按照实施例1同法进行试验,不同之处仅在于“样品活性检测”项下,配制如下反应体系(每孔反应体系总体积为50μl):
采用该反应体系进行试验,与实施例1的筛选结果一致。
实施例3
按照实施例1同法进行试验,不同之处仅在于“样品活性检测”项下,配制如下反应体系(每孔反应体系总体积为50μl):
采用该反应体系进行试验,与实施例1的筛选结果一致。
Claims (7)
3.一种筛选PknB抑制剂的方法,包括如下步骤:
(1)配置如下反应体系:
(2)设置如下各反应组:
i)样品实验组:取步骤(1)所述的反应体系,加入1μL浓度为1mg/mL~10mg/mL的样品溶液;
ii)阴性实验组:取步骤(1)所述的反应体系,加入1μL DMSO;
iii)阳性对照组:取步骤(1)所述的反应体系,加入终浓度为60μg/ml的星形孢菌素;
(3)制备荧光素酶反应液
(4)筛选
将步骤(2)的各反应组在酶标仪中分别反应180min后,加入步骤(3)配制的所述荧光素酶反应液,测定各反应组荧光值,计算反应抑制率,对反应具有抑制作用的即为阳性样品。
5.根据权利要求3所述的方法,其特征在于,其中步骤(2)中样品为化合物时,向50μL反应体系中加入1μL的1mg/mL的样品溶液作为样品实验组。
7.根据权利要求6所述的方法,其特征在于,抑制率大于30%的样品为阳性样品。
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