阅读说明：本技术 一种rmpA的酶活反应测定方法 (Enzyme activity reaction determination method of rmpA ) 是由 王尧 卢彩婷 林鹏 徐涵瑜 叶锦浩 刘欣 王再花 于 2019-10-11 设计创作，主要内容包括：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。(The invention discloses an rmpA enzyme activity reaction determination method, which comprises the following steps: the method comprises the following steps: preparing D-ribose 5phosphate and formaldehyde for later use; step two: carrying out PCR chain extension on the RNA of rmpA, and carrying out protein expression through a cell-free protein expression system after chain extension; step three: performing addition reaction of D-ribose 5phosphate and formaldehyde by using the rmpA after protein expression as catalytic protein, determining initial formaldehyde content and formaldehyde content in the reaction process by a formaldehyde detection method, calculating enzyme activity, reflecting the activity of the rmpA by using the synthetic catalysis of the rmpA on the formaldehyde and the D-ribose 5phosphate through the content change of the formaldehyde, and simply and conveniently detecting the content of the formaldehyde; the dynamic determination can be carried out by using the reaction, and the dynamic result of the enzyme activity process can be determined, so that the determination can be completed more accurately and more flexibly.)

1. An rmpA enzyme activity reaction determination method is characterized by comprising the following steps:

the method comprises the following steps: preparing D-ribose 5phosphate and formaldehyde for later use;

step two: carrying out PCR chain extension on the RNA of rmpA, and carrying out protein expression through a cell-free protein expression system after chain extension;

step three: and (3) performing addition reaction of D-ribose 5phosphate and formaldehyde by using rmpA after protein expression as catalytic protein, determining initial formaldehyde content and formaldehyde content in the reaction process by a formaldehyde detection method, and calculating enzyme activity of the rmpA.

2. The method for determining enzymatic activity of rmpA according to claim 1, wherein the formaldehyde is detected by reacting formaldehyde with acetylacetone and ammonia to produce 3, 5-diacetyl-1, 4-dihydrolutidine in yellow color, and measuring the absorbance at 412nm using a 10mm cuvette.

3. The enzymatic activity assay method of rmpA according to claim 1, characterized in that the method for detecting formaldehyde is a method using a phenol reagent, formaldehyde reacts with a phenol reagent to form azine, which is oxidized to blue by iron ions in an acidic solution, and absorbance is measured at 635nm using a 10mm cuvette.

4. The method for measuring enzymatic activity of rmpA according to claim 1, wherein the formaldehyde is detected by the parafuchsin method, wherein a purple complex is formed between sulfite ions and parafuchsin in the presence of formaldehyde, and the absorbance is measured at a wavelength of 570 nm.

5. The method for measuring enzymatic activity of rmpA according to claim 1, wherein the reaction temperature of the catalytic reaction in step 3 is controlled to 37 ℃.

6. The method for determining enzymatic activity reaction of rmpA according to claim 1, wherein the purity of D-ribose 5phosphate in step 1 is not less than 98%.

Technical Field

The invention relates to the technical field of detection of rmpA enzyme, in particular to a method for determining enzyme activity reaction of rmpA.

Background

The super bacteria (superbug) is not specific to a certain bacteria, but generally refers to bacteria with drug resistance to multiple antibiotics, and the precise name of the super bacteria is 'multi-drug resistant bacteria', the bacteria have strong resistance to antibiotics and can escape the danger of killing, and rmpA is a gene for controlling one of super bacteria proteins, Klebsiella pneumoniae (Kpn) is one of important pathogenic bacteria for clinical separation and hospital infection, along with the wide use of broad-spectrum antibiotics such as β -lactams and aminoglycosides, the bacteria are easy to generate ultra-broad spectrum β -lactamase (ESBLs), cephalosporins (AmpC) and Aminoglycoside Modified Enzymes (AMEs), and present serious multi-drug resistance to common drugs including third-generation cephalosporins and aminoglycosides, the hospital infection rate caused by the Klebsiella pneumoniae is increased year by year recently, and the continuous increase of multi-drug resistance strains often leads to the failure and the prolongation of clinical antibacterial drug therapy.

The drug resistance mechanism of Klebsiella pneumoniae mainly comprises β -lactamase generation, biofilm formation, outer membrane porin deletion, active antibacterial drug discharge and the like, and the horizontal spreading of the drug resistance gene of the antibacterial drug is an important reason for clinical exacerbation of multidrug resistant strains.

At present, the enzyme activity determination method of the rmpA is less researched, the enzyme activity plays an important role in the whole reaction process and the result, but the enzyme activity of the rmpA cannot be directly detected, and the rmpA cannot be deeply researched.

Disclosure of Invention

The invention aims to provide an enzyme activity reaction determination method for detecting rmpA, which has the advantages of flexibility and accuracy in detection.

The technical purpose of the invention is realized by the following technical scheme:

an rmpA enzyme activity reaction determination method is characterized by comprising the following steps:

the method comprises the following steps: preparing D-ribose 5phosphate and formaldehyde for later use;

step two: carrying out PCR chain extension on the RNA of rmpA, and carrying out protein expression through a cell-free protein expression system after chain extension;

step three: and (3) performing addition reaction of D-ribose 5phosphate and formaldehyde by using rmpA after protein expression as catalytic protein, determining initial formaldehyde content and formaldehyde content in the reaction process by a formaldehyde detection method, and calculating enzyme activity of the rmpA.

D-ribose 5phosphate reacts with formaldehyde under the catalysis of rmpA to generate D-arabino-hex-3-ulose 6-phosphate, and the reaction formula is as follows:

further setting: the formaldehyde is detected by reacting formaldehyde with acetylacetone and ammonia by acetylacetone method to obtain yellow 3, 5-diacetyl-1, 4-dihydrolutidine, and measuring absorbance with 10mm cuvette at 412nm wavelength.

Further setting: the method for detecting formaldehyde comprises the steps of utilizing a phenol reagent method, enabling formaldehyde to react with a phenol reagent to generate azine, oxidizing the azine into blue by iron ions in an acid solution, and measuring absorbance at 635nm by using a 10mm cuvette.

Further setting: the formaldehyde was detected by the method of parafuchsin, in which sulfite ions formed a violet complex with parafuchsin in the presence of formaldehyde, and the absorbance was measured at a wavelength of 570 nm.

Further setting: the reaction temperature of the catalytic reaction in step 3 was controlled to 37 ℃.

Further setting: the purity of the D-ribose 5phosphate in the step 1 is not lower than 98%.

In conclusion, the invention has the following beneficial effects:

1. the activity of the rmpA enzyme is reflected by the change of the content of formaldehyde by utilizing the synthetic catalysis of the rmpA on the formaldehyde and the D-ribose 5phosphate, and the content detection of the formaldehyde is simple and convenient; the dynamic determination can be carried out by using the reaction, and the dynamic result of the enzyme activity process can be determined, so that the determination can be completed more accurately and more flexibly;

2. after chain extension is carried out by a PCR technology, a cell-free protein expression system is adopted, the protein expression purity is high, the protein is soluble protein, and the catalytic reaction is conveniently and efficiently carried out;

3. the D-ribose 5phosphate has higher purity, and avoids the influence of impurities in the raw materials on the reaction.

Detailed Description

The following D-ribose 5phosphate was purchased from Jinjinle chemical Co., Ltd at a concentration of 98% or more; the following cell-free protein expression system was protein expression using a specific cell-free protein expression system of Hangzhou Hai Australia Biotech Co., Ltd.