CN110819694A - 一种rmpA的酶活反应测定方法 - Google Patents
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Abstract
本发明公开了一种rmpA的酶活反应测定方法,包括如下步骤:步骤一:制备D‑ribulose 5 phosphate和甲醛备用;步骤二:通过PCR扩链rmpA的RNA,扩链后通过无细胞蛋白表达体系进行蛋白表达;步骤三:将蛋白表达后的rmpA作为催化蛋白进行D‑ribulose 5 phosphate与甲醛的加成反应,通过检测甲醛的方法测定初始甲醛含量以及反应过程甲醛含量,计算其酶活,利用rmpA对甲醛和D‑ribulose 5 phosphate的合成催化作用,通过甲醛的含量变化来体现rmpA酶的活性,甲醛的含量检测简便;用反应的进行可以进行动态测定,确定其酶活过程的动态结果,可以更精确,更灵活的完成测定。
Description
技术领域
本发明涉及rmpA酶的检测技术领域,特别涉及一种rmpA的酶活反应测 定方法。
背景技术
超级细菌(superbug)不是特指某一种细菌,而是泛指那些对多种抗生 素具有耐药性的细菌,它的准确称呼应该是“多重耐药性细菌”。这类细菌 能对抗生素有强大的抵抗作用,能逃避被杀灭的危险,而rmpA是控制其中一 种超级细菌蛋白的基因。肺炎克雷伯菌(Kpn)是临床分离及医院感染的重要 致病菌之一,随着β-内酰胺类及氨基糖苷类等广谱抗菌素的广泛使用,细菌 易产生超广谱β-内酰胺酶(ESBLs)和头孢菌素酶(AmpC酶)以及氨基糖苷 类修饰酶(AMEs),对常用药物包括第三代头孢菌素和氨基糖苷类呈现出严重 的多重耐药性。肺炎克雷伯菌引起的医院感染率近期逐年增高,且多耐药性 菌株的不断增加常导致临床抗菌药物治疗的失败和病程迁延。
肺炎克雷伯菌耐药机制主要包括产生β-内酰胺酶、生物被膜的形成、外 膜孔蛋白的缺失、抗菌药物主动外排等,抗菌药物耐药基因水平播散是多药 耐药菌株临床加剧的重要原因。rmpA可以放大肺炎克雷伯菌不同血清型的菌 落粘液质,并作为质粒介导的囊外多糖合成调节因子。
目前关于rmpA的酶活测定方法研究较少,且酶活对于整个反应过程及结 果都有很重要的作用,但无法直接检测rmpA酶活,更无法深入进行rmpA的 研究。
发明内容
本发明的目的是提供一种检测rmpA的酶活反应测定方法,其具有检测灵 活、准确的优点。
本发明的上述技术目的是通过以下技术方案得以实现的:
一种rmpA的酶活反应测定方法,其特征在于,包括如下步骤:
步骤一:制备D-ribulose 5phosphate和甲醛备用;
步骤二:通过PCR扩链rmpA的RNA,扩链后通过无细胞蛋白表达体系进 行蛋白表达;
步骤三:将蛋白表达后的rmpA作为催化蛋白进行D-ribulose 5 phosphate与甲醛的加成反应,通过检测甲醛的方法测定初始甲醛含量以及 反应过程甲醛含量,计算其酶活。
D-ribulose 5phosphate和甲醛在rmpA催化的作用反应生成 D-arabino-hex-3-ulose 6-phosphate,反应式如下:
进一步设置:检测甲醛的方法为利用乙酰丙酮法,甲醛与乙酰丙酮及氨作用 生成黄色的3,5-二乙酰基-1,4二氢卢剔啶,用10mm比色杯在412nm波长 下测定吸光度。
进一步设置:检测甲醛的方法为利用酚试剂法,甲醛与酚试剂反应生成 丫嗪,其在酸性溶液中被铁离子氧化成蓝色,用10mm比色杯在635nm波长下 测定吸光度。
进一步设置:检测甲醛的方法为利用副品红法,亚硫酸根离子在甲醛存 在的条件下,与副品红生成紫色络合物,在570nm波长下测定吸光度。
进一步设置:步骤3中催化反应的反应温度控制为37℃。
进一步设置:步骤1中D-ribulose 5phosphate的纯度不低于98%。
综上所述,本发明具有以下有益效果:
1、利用rmpA对甲醛和D-ribulose 5phosphate的合成催化作用,通过 甲醛的含量变化来体现rmpA酶的活性,甲醛的含量检测简便;用反应的进行 可以进行动态测定,确定其酶活过程的动态结果,可以更精确,更灵活的完 成测定;
2、通过PCR技术扩链后,采用无细胞蛋白表达体系,蛋白表达纯度高且 为可溶蛋白,方便且高效的进行催化反应;
3、D-ribulose 5phosphate具有较高的纯度,避免原料中杂质对反应 的影响。
具体实施方式
下述D-ribulose 5 phosphate采购自金锦乐化学有限公司,浓度≥98%; 下述无细胞蛋白表达体系为杭州谨澳生物科技有限公司的特定的无细胞蛋白 表达体系进行蛋白表达。
实施例1:一种rmpA的酶活反应测定方法,包括如下步骤:
步骤一:制备D-ribulose 5phosphate和甲醛备用;
步骤二:通过PCR扩链rmpA的RNA,扩链后通过无细胞蛋白表达体系进 行蛋白表达;
步骤三:将蛋白表达后的rmpA作为催化蛋白进行D-ribulose 5 phosphate与甲醛的加成反应,通过酚试剂法测定初始甲醛含量以及反应过 程甲醛含量,计算其酶活。
反应体系中甲醛初始添加量为5g,rmpA蛋白10mg,D-ribulose 5 phosphate 5g,蒸馏水100ml,反应温度控制在37℃。
检测时,先将甲醛和D-ribulose 5phosphate添加进蒸馏水水中,将溶 液充分搅拌,取5ml作为标本一,然后添加rmpA蛋白10mg,搅拌均匀,添 加rmpA蛋白后30min再取5ml溶液作为标本二,并立刻将标本二进行冷冻灭 活,添加rmpA蛋白后60min再取5ml溶液作为标本三,并立刻将标本三进行 冷冻灭活,分别检测标本一、标本二和标本三的吸光度。并计算标本一和标 本二的吸光度之比A1,计算标本二和标本三的吸光度之比A2。
计算所得A1为0.78,A2为0.82。
实施例2:
一种rmpA的酶活反应测定方法,包括如下步骤:
步骤一:制备D-ribulose 5phosphate和甲醛备用;
步骤二:通过PCR扩链rmpA的RNA,扩链后通过无细胞蛋白表达体系进 行蛋白表达;
步骤三:将蛋白表达后的rmpA作为催化蛋白进行D-ribulose 5 phosphate与甲醛的加成反应,通过酚试剂法测定初始甲醛含量以及反应过 程甲醛含量,计算其酶活。
反应体系中甲醛初始添加量为5g,rmpA蛋白10mg,D-ribulose 5 phosphate 5g,溶液100ml。
检测时,先将甲醛和D-ribulose 5phosphate添加进蒸馏水水中,将溶 液充分搅拌,取5ml作为标本一,然后添加rmpA蛋白10mg,搅拌均匀,添 加rmpA蛋白后30min再取5ml溶液作为标本二,并立刻将标本二进行冷冻灭 活,添加rmpA蛋白后60min再取5ml溶液作为标本三,并立刻将标本三进行 冷冻灭活,分别检测标本一、标本二和标本三的吸光度。
计算所得A1为0.75,A2为0.81。
实施例3:一种rmpA的酶活反应测定方法,包括如下步骤:
步骤一:制备D-ribulose 5phosphate和甲醛备用;
步骤二:通过PCR扩链rmpA的RNA,扩链后通过无细胞蛋白表达体系进 行蛋白表达;
步骤三:将蛋白表达后的rmpA作为催化蛋白进行D-ribulose 5 phosphate与甲醛的加成反应,利用副品红法检测甲醛含量,亚硫酸根离子在 甲醛存在的条件下,与副品红生成紫色络合物,在570nm波长下测定吸光度。
反应体系中甲醛初始添加量为5g,rmpA蛋白10mg,D-ribulose 5 phosphate 5g,溶液100ml。
检测时,先将甲醛和D-ribulose 5phosphate添加进蒸馏水水中,将溶 液充分搅拌,取5ml作为标本一,然后添加rmpA蛋白10mg,搅拌均匀,添 加rmpA蛋白后30min再取5ml溶液作为标本二,并立刻将标本二进行冷冻灭 活,添加rmpA蛋白后60min再取5ml溶液作为标本三,并立刻将标本三进行 冷冻灭活,分别检测标本一、标本二和标本三的吸光度。
计算所得A1为0.78,A2为0.84。
以上所述的实施方式,并不构成对该技术方案保护范围的限定。任何在 上述实施方式的精神和原则之内所作的修改、等同替换和改进等,均应包含 在该技术方案的保护范围之内。
Claims (6)
1.一种rmpA的酶活反应测定方法,其特征在于,包括如下步骤:
步骤一:制备D-ribulose 5phosphate和甲醛备用;
步骤二:通过PCR扩链rmpA的RNA,扩链后通过无细胞蛋白表达体系进行蛋白表达;
步骤三:将蛋白表达后的rmpA作为催化蛋白进行D-ribulose 5phosphate与甲醛的加成反应,通过检测甲醛的方法测定初始甲醛含量以及反应过程甲醛含量,计算其酶活。
2.根据权利要求1所述的rmpA的酶活反应测定方法,其特征在于,检测甲醛的方法为利用乙酰丙酮法,甲醛与乙酰丙酮及氨作用生成黄色的3,5-二乙酰基-1,4二氢卢剔啶,用10mm比色杯在412nm波长下测定吸光度。
3.根据权利要求1所述的rmpA的酶活反应测定方法,其特征在于,检测甲醛的方法为利用酚试剂法,甲醛与酚试剂反应生成丫嗪,其在酸性溶液中被铁离子氧化成蓝色,用10mm比色杯在635nm波长下测定吸光度。
4.根据权利要求1所述的rmpA的酶活反应测定方法,其特征在于,检测甲醛的方法为利用副品红法,亚硫酸根离子在甲醛存在的条件下,与副品红生成紫色络合物,在570nm波长下测定吸光度。
5.根据权利要求1所述的rmpA的酶活反应测定方法,其特征在于,步骤3中催化反应的反应温度控制为37℃。
6.根据权利要求1所述的rmpA的酶活反应测定方法,其特征在于,步骤1中D-ribulose5phosphate的纯度不低于98%。
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JP2007068424A (ja) * | 2005-09-05 | 2007-03-22 | Kyoto Univ | 二機能性ホルムアルデヒド固定酵素遺伝子 |
CN104995293A (zh) * | 2012-12-17 | 2015-10-21 | 基因组股份公司 | 用于在甲醇的存在下提高还原当量的可用性,以及用于生产与此有关的己二酸、6-氨基己酸、六亚甲基二胺或己内酰胺的微生物体和方法 |
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JP2007068424A (ja) * | 2005-09-05 | 2007-03-22 | Kyoto Univ | 二機能性ホルムアルデヒド固定酵素遺伝子 |
CN104995293A (zh) * | 2012-12-17 | 2015-10-21 | 基因组股份公司 | 用于在甲醇的存在下提高还原当量的可用性,以及用于生产与此有关的己二酸、6-氨基己酸、六亚甲基二胺或己内酰胺的微生物体和方法 |
Non-Patent Citations (2)
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YASUYOSHI SAKAI等: "Organization of the genes involved in the ribulose monophosphate pathway in an obligate methylotrophic bacterium,Methylomonas aminofaciens 77a" * |
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