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ATP releasing agent and germ fast detection reagent containing ATP releasing agent

A detection reagent and release agent technology, applied in the field of bacterial rapid detection reagents, can solve the problems of changing cell membrane permeability, enzymatic denaturation and inactivation, and insignificant ATP release effect, and achieves simple preparation process, rapid process, and reduced ATP release. effect of steps

Active Publication Date: 2014-01-01
HANGZHOU CLONGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Dilute acid is equally destructive to enzymes and has no specificity; a certain concentration of dimethyl sulfoxide can change the permeability of cell membranes and release ATP, but it has no specific damage to ATPases in cells; commonly used surface The active agent SDS destroys the activity of luciferase and denatures and inactivates the enzyme; TritonX-100, benzalkonium bromide, etc. have no significant effect on releasing ATP

Method used

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  • ATP releasing agent and germ fast detection reagent containing ATP releasing agent
  • ATP releasing agent and germ fast detection reagent containing ATP releasing agent
  • ATP releasing agent and germ fast detection reagent containing ATP releasing agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, the construction of the recombinant expression vector containing luciferase gene

[0032] 1. Acquisition of luciferase gene

[0033]According to the Japanese firefly luciferase gene (GenBank: X66919.1), the whole gene synthesis method was adopted, and Shanghai Jierui Biological Co., Ltd. was entrusted to synthesize the whole gene, and at the same time, the protein structure was analyzed to affect the temperature of the enzyme without affecting the enzyme activity. The codons in the codon were mutated, and the 217 codon GCA encoding Ala was changed to CTT encoding Leu, the 457 codon GAA encoding Glu was changed to TCA encoding Ser, and the 465 codon AAT encoding Asn was changed to ATT encoding Ile , and add CAT and AAGCTT to its 5' and 3' ends respectively to obtain the enzyme-cleavage recognition sites CATATG and AAGCTT of endonuclease NdeI and Hind III respectively, the sequence length is 1656 bp, and the rare codons in bacteria will be replaced with bact...

Embodiment 2

[0038] Example 2. Induced expression and expanded culture of luciferase

[0039] Get the recombinant expression vector pET28a-rt-L1L that embodiment 1 obtains and transfer Escherichia coli BL21 (DE3) competent cell, cultivate on the LB culture plate that contains kanamycin, select single bacterium colony and inoculate in 5mlLB medium and screen and obtain The positive recombinant bacteria were induced to express, and the product was analyzed by SDS-PAGE, and compared with the control bacteria containing the expression vector pET28a(+), the results were as follows Figure 4 shown in the instruction manual Figure 4 Shown is the induced expression SDS-PAGE protein electrophoresis pattern of recombinant firefly luciferase, in which there are many foreign proteins.

[0040] The bacterial supernatant and total protein of the control bacteria without the target gene ( Figure 4 Compared with lanes 3 and 4 in ), the recombinant protein band with a molecular weight of about 60 kDa c...

Embodiment 3

[0042] Embodiment three, the separation and purification of luciferase

[0043] Since the recombinant expression vector pET28a-rt-L1L constructed in the above-mentioned Example 1 has a fusion tag (His·Tag) gene sequence, the expressed target protein also has a His·Tag tag. Therefore, the target protein can be purified by affinity chromatography using agarose complexed with Ni, and the affinity chromatography column is purchased from GE Healthcare.

[0044] After the expression was induced with IPTG according to the method of the above-mentioned Example 2, the bacterial cell pellet was harvested by centrifugation at 10000 rpm / min for 5 min; L cells) were resuspended, ultrasonically lysed at 250 W 90 times, each time for 5 s, with an interval of 10 s, and centrifuged at 12,000 rpm / min for 20 min at 4°C to collect the supernatant and prepare for loading.

[0045] The specific purification steps are as follows:

[0046] 1) Equilibrate the Ni column with Buffer A until the OD280 ...

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Abstract

The invention provides an ATP releasing agent and a method for preparing the ATP releasing agent which is 5-(4-ortho-pelargonic alkyl-phenyl group)-1-hexane sodium sulfonate. The invention further provides a germ fast detection reagent which comprises a germ collecting swab, luciferase, a fluorescein substrate and reaction buffer which are immersed with the ATP releasing agent. The invention relates to ATP releasing and measuring, wherein the processes of ATP releasing and measuring are finished at one step, the number of the steps of ATP releasing is reduced, the measuring method is simpler, the process is faster, and the result can be obtained within minutes. The ATP releasing agent has the advantages of being simple in preparing process, low in cost, obvious in effect and the like, and can be suitable for detecting microorganisms better.

Description

technical field [0001] The invention relates to an ATP releasing agent, more specifically to a rapid detection reagent for bacteria comprising the ATP releasing agent and luciferase. Background technique [0002] Luciferase (luciferase) is a highly efficient biocatalyst that can convert chemical energy into light energy. Firefly luciferase (abbreviated as firefly luciferase, firefly luciferase, FL) uses luciferin (Luciferin), adenosine triphosphate (ATP) and o 2 As a substrate, in the magnesium ion (Mg 2+ ) exists, convert chemical energy into light energy, and emit photons, the reaction formula is as follows: [0003] Luciferin+O 2 +ATP+Mg 2+ +Luciferase??Oxidized luciferin+CO2+AMP [0004] +Pyrophosphate+hu [0005] ATP is not only the necessary substrate for luciferase to catalyze luminescence, but also the energy source for all biological life activities. The ATP bioluminescence method is considered to be the most rapid microbial detection method at present. In t...

Claims

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Application Information

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IPC IPC(8): C07C309/24C07C303/32C12Q1/26C12N9/02
Inventor 郑曙剑殷秀飞应旭霞
Owner HANGZHOU CLONGENE BIOTECH
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