Gene encoding of firefly luciferase, its preparation method and application

A technology that encodes genes and amino acids, applied in the fields of molecular enzymology and biology, can solve problems such as poor thermal stability and poor resistance to protease degradation

Inactive Publication Date: 2011-09-21
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although firefly luciferase has important application value, due to the poor thermal stability of the enzyme itself and the poor resistance to protease degradation, there are many improvements aimed at improving the stability or enzyme activity of firefly luciferase. In order to better play and realize the function and role of firefly luciferase

Method used

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  • Gene encoding of firefly luciferase, its preparation method and application
  • Gene encoding of firefly luciferase, its preparation method and application
  • Gene encoding of firefly luciferase, its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Embodiment 1 A method for preparing a gene encoding luciferase mutant strain protein with high thermostability and high enzymatic activity characteristics, the steps are:

[0063] 1. Preparation of PCR template

[0064] Activate the pGL3-Basic Vector plasmid Escherichia coli strain with firefly luciferase gene purchased from Promega in the laboratory in solid LB, pick a single clone in 5ml LB liquid medium and cultivate it on a shaker at 37°C, then use OMEGA The company's plasmid extraction kit extracts the plasmid and uses it as a template for PCR.

[0065] 2. Overlap PCR amplification to obtain a firefly luciferase fragment containing 4 point mutations

[0066] (1) Two sets of mutagenic primers are designed (see Table 1 for mutagenic primers), and the first set of mutagenic primers A1, B1, C1, and D1 are used to amplify according to the following conditions: the wild-type luciferase gene is used as a template, and the primers A1, B1 amplified fragment A1B1, accordin...

Embodiment 2

[0085] Example 2: Detection of mutant protein FIREFLY LUCIFERASE-E354K, I423L, D436G, L530R activity and other enzymatic properties.

[0086] Luminous intensity analysis:

[0087] The BCA protein concentration assay kit produced by Pierce was used to determine the concentration, and BSA was used as the standard curve. Prepare 0.1mg / ml protein solution after protein quantification, and perform luminescence measurement with E1500 luciferase detection kit from Promega Company. The measurement system is 10u10.1mg / ml protein solution, 30ul pH7.4, 50mMTris-Hcl, 10mM MgCl 2 buffer and 10ul of substrate mix. Using Promega's 20 / 20Luminometer for measurement, such as Figure 4 As shown, under the same conditions, the modified protease FIREFLYLUCIFERASE-E354K, I423L, D436G, and L530R increased by about 20 times compared with the wild-type FIREFLYLUCIFERASE when the substrate was diluted 100 times and 1000 times, which reflected the enzyme activity. improvement.

[0088] Thermal Sta...

Embodiment 3

[0090] Embodiment 3: Firefly luciferase detects in the total number of bacteria (comprising Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, yeast, Salmonella, Listeria, cholera and other common environment and food contamination bacteria) That is, the application in ATP detection) (the application of an isolated protein in bacterial detection).

[0091] Detection principle: ATP exists in all organisms from microorganisms to higher animal and plant cells, and is the direct source of cellular energy. When other reactants in the firefly luciferase reaction are excessive, the amount of ATP is linearly related to the luminescence value. By measuring The change of ATP content after adding lysate to a sample can determine the number of cells or microorganisms in a sample.

[0092] The BCA protein concentration assay kit produced by Pierce was used to determine the concentration, and BSA was used as the standard curve. After measuring the protein c...

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Abstract

The invention relates to a gene encoding of firefly luciferase, its preparation method and application, which comprises the following steps: 1. introducing mutation sites of mutant enzymes; 2. identifying recombinant plasmids; 3. sequencing to obtain polymerase chain reaction (PCR) fragment with 1060 mutation sites; 4. obtaining escherichia by using restriction endonucleasecoli purifying after recovering and purifying through gel; 5. identifying recombinant plasmids to obtain a separating protein gene; 6. culturing escherichia coli, 7. converting recombinant plasmids into BL21 competent cell to obtain bacterial strain capable of expressing mutant enzymes, 8. culturing the obtained BL21 expression bacterial strain; 9. centrifuging bacteria liquid of protein, centrifuging to obtain supernatant through ultrasonic fragmentation; 10. determining to obtain firefly luciferase with high heating stability and high enzyme activity. The invention relates to application of firefly luciferase gene on foodstuff, medicine, thalline detection. The luminous intensity of the mutant enzyme is about 15 to 20 times than that of wild type, and the mutant enzyme has good heat stability than that of the wild type.

Description

technical field [0001] The invention relates to molecular enzymology and biotechnology, more specifically to a firefly luciferase encoding gene with high thermal stability and high enzymatic activity, and to a preparation method of the mutant strain encoding gene. The high thermal stability and high enzymatic activity firefly luciferase mutant strain protein encoded by the mutant gene can be widely used in the fields of food, medicine, biology and the like. technical background [0002] Luciferase is one of the most important tool enzymes in the field of biology today. North American firefly luciferase is one of the most widely used luciferases. Since it does not require an excitation light source, the luminous energy mainly comes from chemical reactions and the luminescence can last for a certain period of time. Time is favored by scientific research, medicine and food industry. [0003] In the field of scientific research, the more prominent application of firefly lucifer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N9/02C12Q1/66C12Q1/06C12R1/19
Inventor 危宏平赵金凤王殿冰张治平张先恩
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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