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Rumen green fluorescent protein (GFP) genetically engineered bacterium and building method and application thereof

A technology of green fluorescent protein and genetically engineered bacteria, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of no bacterial growth and growth, unable to fully reflect the dynamic state of the rumen, and achieve objective and reliable results. , weak phototoxicity, and the effect of the study is close to the real state

Inactive Publication Date: 2014-05-14
YANGZHOU UNIV
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Problems solved by technology

The fluorescein (Fluorescein) dye that our laboratory explored in the early stage labeled rumen bacteria, but the research was also static dead bacteria after staining (Wang Mengzhi et al., 2010), without the reproduction and growth of the labeled bacteria, it could not fully reflect the real dynamics of the rumen state

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  • Rumen green fluorescent protein (GFP) genetically engineered bacterium and building method and application thereof
  • Rumen green fluorescent protein (GFP) genetically engineered bacterium and building method and application thereof
  • Rumen green fluorescent protein (GFP) genetically engineered bacterium and building method and application thereof

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Embodiment Construction

[0023] The present invention includes the following steps: 1) construction of prokaryotic expression vector pET15b for green fluorescent protein GFP gene; 2) construction of rumen butyricobacterium fibrolyticus green fluorescent protein GFP genetic engineering bacteria; 3) green fluorescence of rumen butyricivibriolyticus Test the expression performance of green fluorescent protein in protein GFP genetically engineered bacteria; 4) Place rumen vibrio fibrolyticus green fluorescent protein GFP genetically engineered bacteria in Hengate rolling tube solid medium and store them in a refrigerator at 4°C.

[0024] Each step is described in detail below:

[0025] The construction method of the enhanced green fluorescent protein GFP gene prokaryotic expression vector pET15b in the step 1) of the present invention is as follows: the enhanced green fluorescent protein EGFP gene is used to amplify the EGFP coding sequence by PCR. The amplified sequence was directly routinely subcloned a...

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Abstract

The invention relates to a rumen green fluorescent protein (GFP) genetically engineered bacterium and a building method and application thereof. The rumen green fluorescent protein (GFP) genetically engineered bacterium is rumen butyrivibrio fibrisolven containing a prokaryotic expression vector pET15b-EGFP, wherein the prokaryotic expression vector pET15b-EGFP is obtained by subcloning and connecting the enhanced green fluorescent protein (GFP) gene into a BamH1 site of the prokaryotic expression vector pET15b. The rumen green fluorescent protein (GFP) genetically engineered bacterium disclosed by the invention can be applied to rumen micro-ecological microcirculation digestion and metabolism detection; genetic manipulation is carried out by using the GFP gene, and the target protein is marked, so that the time and space change of target protein can be monitored through a biological camera in real time.

Description

technical field [0001] The invention relates to a method for constructing a rumen butyrivibrio fibrolyticus green fluorescent protein GFP genetically engineered bacterium used for the research and detection of rumen microecology of ruminants. Background technique [0002] It is well known that one of the important digestive processes in the digestion of ruminants is the digestion of rumen microorganisms (Zhu Weiyun, 2004). However, due to the difficulty of rumen microecological research, it is still limited to the digestibility of nutrients in the rumen, the cultivation of rumen dominant flora, or the use of drugs to control rumen microorganisms (Koenig et al., 2000; Han Chunyan et al., 2002 ; Hristov et al., 2004). For the interaction between microorganisms in the rumen microecology, such as the symbiosis of bacteria, protozoa and fungi; the phagocytosis of bacteria and fungi by protozoa is still unknown, the main reason is that there is no better research method for this ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12Q1/02C12R1/63
Inventor 王梦芝经语佳高健皮宇王洪荣喻礼怀
Owner YANGZHOU UNIV
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