Recombinant Salmonella expressing red fluorescent protein and its construction method

A red fluorescent protein and Salmonella technology, applied in the field of genetic engineering, can solve cumbersome problems, reduce false positives and save research funds

Active Publication Date: 2019-04-23
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires the participation of an intermediate host, which is too cumbersome, and green fluorescent labeling is more common. If co-localization with other target proteins is required in the experiment, the secondary antibody can no longer be fluorescently labeled with common FITC

Method used

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  • Recombinant Salmonella expressing red fluorescent protein and its construction method
  • Recombinant Salmonella expressing red fluorescent protein and its construction method
  • Recombinant Salmonella expressing red fluorescent protein and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 The construction method of Salmonella delphi (SD-pFPV-mCherry) expressing red fluorescent protein

[0061] In this example, the clinically isolated Salmonella delphi is used as the transformation recipient, and the plasmid pFPV-mCherry inserted with the red fluorescent protein gene fragment is transformed into the bacteria by electric shock, thereby constructing Salmonella capable of expressing red fluorescence. Determine the genetic stability of the plasmid of the constructed recombinant strain, and detect whether there are significant differences in colony morphology, growth curve, and virulence factors compared with the parent strain, that is, to detect whether the transfer of the plasmid affects the normal biological characteristics of Salmonella. Technical route see figure 1 .

[0062] 1. Preparation of Competent Cells

[0063] (1) Pick a single colony of Salmonella from the XLD agar medium, inoculate it in a 50ml centrifuge tube containing 20ml LB cult...

Embodiment 2

[0113] Example 2 Construction method of Salmonella infantis (SI-pFPV-mCherry) expressing red fluorescent protein

[0114] The Salmonella infantis in this example is the same as the Salmonella delphi in Example 1, all of which are clinical isolates and belong to different serotypes. In this example, the preparation of Salmonella infantis competent cells, transformation by electric shock, identification of biological characteristics, etc. were carried out using the same steps as those in Example 1. Finally, Salmonella infantis capable of expressing red fluorescent protein was successfully constructed, and the plasmid was inherited after 100 generations of self-replication. The stability is more than 95%, which is similar to the recombinant Salmonella delbeis.

[0115] Salmonella expressing red fluorescent protein can be successfully constructed by adopting the method for constructing recombinant bacteria provided by the present invention. The recombinant bacteria have the same c...

Embodiment 3

[0116] Embodiment 3 Construction of recombinant Salmonella expressing green fluorescent protein

[0117] The plasmid used in this example was replaced by the plasmid pFPV-25.1 inserted with the green fluorescent protein gene fragment, and the construction method of the recombinant bacteria used the same steps as in Example 1. Constructed recombinant Salmonella delphi (SD-pFPV-25.1) and recombinant Salmonella infantis (SI-pFPV-25.1) expressing green fluorescent protein. There was no significant difference in the colony morphology, growth performance, and virulence factors of these two strains of recombinant bacteria, but the genetic stability of the plasmid was lower than 80% after 100 generations of self-replication ( Figure 7 ), it is not suitable for long-term experimental research, and can only be used for short-term experimental projects.

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Abstract

The invention relates to recombinant salmonella capable of expressing red fluorescence protein and a building method of the recombinant salmonella. The clinically isolated salmonella is utilized as a transformation receptor, plasmids pFPV-mCherry with red fluorescence protein gene segments inserted enter a bacterium through electroporation, and the salmonella capable of expressing the red fluorescence protein is built accordingly. The built recombinant salmonella can be used for marking and tracing in-vitro and in-vivo test processes of the salmonella, and the problem that the real-time monitoring cannot be performed in the salmonella test is solved; the recombinant salmonella can express the fluorescence protein and can replace an expensive salmonella specific antibody, not only is the research grant saved, but also the false positive caused by utilization of the antibody is greatly reduced; co-localization can be performed through combination with immunofluorescent staining with related protein, and an effective means is provided for in-depth study of the clinically isolated salmonella.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant Salmonella expressing red fluorescent protein and a construction method thereof. Background technique [0002] Salmonella is the causative agent of salmonellosis. Enterobacteriaceae, Gram-negative enterobacteriaceae. Nearly a thousand Salmonella strains have been discovered, which can be divided into basic bacterial groups such as A, B, C, D, and E according to their antigenic components. Among them, those related to human diseases mainly include A. paratyphi in Group A, B. paratyphi and Bacillus typhimurium in group B, C. paratyphi and Bacillus choleraesuis in group C, typhoid bacillus and Enteritidis bacteria in group D, etc. In addition to typhoid bacillus, paratyphoid A and paratyphoid bacillus causing human diseases, most of them can only cause animal diseases such as livestock, rodents and poultry, but sometimes they can also contaminate human food and cau...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/42
CPCC07K14/00
Inventor 朱要宏王九峰胡雄于娇张伟彭玉璐
Owner CHINA AGRI UNIV
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