Astaxanthin synthetic gene recombinant plasmid as well as preparation method and application of astaxanthin synthetic gene recombinant plasmid

A technology for synthesizing genes and astaxanthin, applied in biochemical equipment and methods, recombinant DNA technology, and using vectors to introduce foreign genetic material, etc., can solve the problems of cumbersome operation steps and low efficiency, and achieve simple methods, low cost, Effects built quickly

Inactive Publication Date: 2014-05-21
HEBEI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, researchers still have a lot of confusion about the detection of the function of the main enzyme system in the synthetic and metabolic pathway of the target receptor astaxanthin during the research and development process, such as through in vitro biochemical reactions or the use of gene mutants for functional complementation. Its operation steps are more loaded down with trivial details, and efficiency is not high

Method used

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  • Astaxanthin synthetic gene recombinant plasmid as well as preparation method and application of astaxanthin synthetic gene recombinant plasmid
  • Astaxanthin synthetic gene recombinant plasmid as well as preparation method and application of astaxanthin synthetic gene recombinant plasmid
  • Astaxanthin synthetic gene recombinant plasmid as well as preparation method and application of astaxanthin synthetic gene recombinant plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Construction of recombinant expression plasmid pET-Ast

[0058] (1) Gene amplification:

[0059] The total RNA of Phaffia rhodozyma CGMCC2.1557 (purchased from the China Agricultural Microorganism Culture Collection Management Center) was extracted using an ultra-pure RNA extraction kit (purchased from Kangwei Century Biotechnology Co., Ltd.), and the HiFi-MMLV cDNA first-strand synthesis kit was used to extract (purchased from Kangwei Century Biotechnology Co., Ltd.) was reverse-transcribed to obtain the first strand of cDNA, and RT-PCR was used to amplify the coding regions of crtS and crtR genes, ExTaq DNA polymerase (purchased from Takara), and corresponding primers (synthesized by Beijing Sanbo Polygala) ) See Table 1.

[0060] Genomic DNA of Pantoea agglomerans ACCC10495 (purchased from China Agricultural Microorganism Culture Collection and Management Center) was extracted by phenol-form extraction method, and used as a template for PCR amplification....

Embodiment 2

[0070] Example 2: Enzyme digestion verification of recombinant expression plasmid pET-Ast

[0071] The recombinant plasmid pET-Ast constructed in Example 1 was digested with NdeI and HpaI to verify the connection of crtE ( image 3 , lane 1); after HpaI and MfeI double digestion, the connection of crtB was verified ( image 3 , lane 2); after MfeI and NheI double digestion, the connection of crtI was verified ( image 3 , lane 3); after NheⅠ and AhaⅢ double digestion, the connection of crtY was verified ( image 3 , lane 4); the connection of crtS was verified by AhaⅢ and ApaLI double enzyme digestion ( image 3 , lane 5); after NotⅠ and ApaLI double digestion, the connection of crtR was verified ( image 3 , lane 6).

Embodiment 3

[0072] Embodiment 3: the fermentation detection of recombinant bacterial strain

[0073] The recombinant plasmid pET-Ast was introduced into Escherichia coli BL21 (Escherichia coli strain BL21 was purchased from Takara Company) for expression, and the main components of the fermentation product were determined by high performance liquid chromatography (HPLC) to detect whether the constructed recombinant expression plasmid accumulated astaxanthin .

[0074] The specific conditions for high performance liquid chromatography in this example are: chromatographic column: Hypersil ODS25um, 250×4.6mm; mobile phase: methanol: acetone: water; UV detection wavelength: 480nm; flow rate: 1.0mL / min; : 20 μL.

[0075] (1) Precisely weigh 10mg of astaxanthin, first dissolve it with a small amount of dichloromethane and then dilute it with acetonitrile to make a solution with a concentration of 0.1mg / mL, and use it as a standard solution for later use;

[0076] Introduce the recombinant pla...

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Abstract

The invention discloses an astaxanthin synthetic gene recombinant expression plasmid pET-Ast with a base sequence as shown in SEQ ID NO: 1. The invention also discloses a preparation method of the recombinant expression plasmid pET-Ast, an application of the recombinant plasmid pET-Ast to the detection of astaxanthin synthetase activity of a target receptor, and an application method. After the astaxanthin synthetic gene recombinant expression plasmid pET-Ast provided by the invention is introduced to an Escherichia coli BL21 strain, the accumulation of astaxanthin in a cell body of Escherichia coli is successfully realized, so that an astaxanthin production strain can be accurately and rapidly established. The preparation method of the astaxanthin synthetic gene recombinant expression plasmid pET-Ast is simple and convenient, easy to operate and low in cost. By using the method disclosed by the invention, a gene sequence to be detected in the target receptor can be replaced with a related gene sequence in the recombinant expression plasmid pET-Ast through digestion and connection, and then, the function of a gene to be detected in the target receptor can be simply and effectively detected and verified through detecting whether the astaxanthin is accumulated in a host cell body.

Description

technical field [0001] The invention relates to a recombinant plasmid and its preparation method and application, in particular to an astaxanthin synthetic gene recombinant plasmid and its preparation method and application. Background technique [0002] Astaxanthin is a ketocarotenoid that is not a source of vitamin A. Pharmacological and physiological studies in recent years have found that astaxanthin has strong biological antioxidant properties. In addition, it also has the functions of promoting antibody production, enhancing immunity and resisting ultraviolet radiation. Therefore, it is widely used in medicine, food, aquaculture, cosmetics, etc. It has broad application prospects. At present, the production methods of astaxanthin mainly include chemical synthesis, shrimp shell extraction and biotechnology. Due to the complex chemical synthesis process and the low content of astaxanthin in shrimp shell waste, the production costs of chemical synthesis and shrimp shell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12Q1/68C12Q1/32C12Q1/25C12Q1/26
Inventor 张利平汤晖孔敏于淼
Owner HEBEI UNIVERSITY
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