Recombinant plasmid pMDMcherry, construction method of recombinant plasmid and method for marking Edwardsiella ictaluri with red fluorescent protein gene

A red fluorescent protein, recombinant plasmid technology, applied in microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., to achieve the effects of reducing costs, high stability, and simple and intuitive methods

Inactive Publication Date: 2017-05-10
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, there are few domestic research reports on the pathogenic mechanism of Edwardsiella spp., and the research on the pathogenic mechanism of bacteria inevitably involves the relationshi

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  • Recombinant plasmid pMDMcherry, construction method of recombinant plasmid and method for marking Edwardsiella ictaluri with red fluorescent protein gene
  • Recombinant plasmid pMDMcherry, construction method of recombinant plasmid and method for marking Edwardsiella ictaluri with red fluorescent protein gene
  • Recombinant plasmid pMDMcherry, construction method of recombinant plasmid and method for marking Edwardsiella ictaluri with red fluorescent protein gene

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Embodiment 1

[0029] The construction method of embodiment 1 recombinant plasmid PMDMcherry

[0030] The construction method of the recombinant plasmid PMDMcherry of the present embodiment comprises the following steps:

[0031] (1) Preparation of promoter fragments:

[0032] Using the plasmid PRUAL as a template, ppS (SEQ ID No: 1) and ppA (SEQ ID No: 2) as upstream and downstream primers, the PCR reaction was carried out according to conventional methods, and the PCR products were subjected to 1% agarose electrophoresis with Gel Extraction Kit (day Root Biochemical Technology (Beijing) Co., Ltd.) for gel recovery to obtain the promoter fragment, the sequence number is SEQ ID No:3. The PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2:

[0033] ppS: 5'CGCCTAGGATACGCACACCGTGGAAAC3' (SEQ ID No: 1)

[0034] ppA: 5'CCTTGCTCACCATTTTTTCTTCCTCCA3' (SEQ ID No: 2)

[0035] Table 1 PCR amplification system

[0036]

[0037]

[0038] Table 2 PCR ...

Embodiment 2

[0050] Embodiment 2 The preparation method of red fluorescent protein gene marker Edwardsiella

[0051] The preparation method of the red fluorescent protein gene marker Edwardsiella of the present embodiment comprises the following steps:

[0052] (1) Using conventional operating methods, isolate the highly virulent strain of Edwardsiella spp. from the diseased zebrafish;

[0053] (2) Prepare the competent state of Edwardsiella spp.

[0054] Methods as below:

[0055] (a) Inoculate Edwardsiella sibriculi into BHI liquid medium, cultivate overnight at 28° C. on a shaker at 280 rpm;

[0056] (b) transfer to 500ml BHI liquid culture medium according to the amount of 1:500, 280rpm, 2.5-3.5h, cultivate until OD600 is 0.6;

[0057] (c) Immediately quench in an ice-water bath: You can choose to place a large container with ice-water mixture on a shaker and quickly rotate and shake to cool down the temperature of the culture as soon as possible;

[0058] (d) Centrifuge in a 250ml...

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Abstract

The invention discloses a recombinant plasmid pMDMcherry, a construction method of the recombinant plasmid and a method for marking Edwardsiella ictaluri with a red fluorescent protein gene. A base sequence of the recombinant plasmid pMDMcherry is shown as SEQ ID NO:7. The Edwardsiella ictaluri, in which a pMDMcherry recombinant vector is introduced, shows red fluorescence when observed under a fluorescence microscope. A stability test shows that a stability rate of the plasmid can reach 100% when the recombinant bacterium is transferred to the 25th generation. With the introduction of the red fluorescent protein gene, a simple and intuitive relation is provided for researching a relation between the Edwardsiella ictaluri and a host.

Description

technical field [0001] The invention belongs to the technical field of biological monitoring, and in particular relates to a recombinant plasmid pMDMcherry and a construction method thereof, and a method for marking Edwardsiella spp. with red fluorescent protein gene. Background technique [0002] Zebrafish is an emerging model animal developed in the past ten years. As a vertebrate, it has many advantages that mice cannot achieve: zebrafish is small in size, low in feeding and maintenance costs; short in reproductive cycle and large in egg production; more importantly, zebrafish embryos are fertilized in vitro , the developmental process can be directly and continuously observed. Zebrafish are increasingly favored by pathogenic microbiologists as infection models for infectious diseases. [0003] Edwardsiella is a bacterium belonging to the genus Edwardsiella in the Enterobacteriaceae family. In 1979, Hawke first discovered the infection of channel catfish by Edwardsiell...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/65C12N1/21C12R1/01
CPCC12N15/74C07K14/43595C12N15/65
Inventor 刘春王芳李凯彬王庆曾伟伟石存斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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