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Expression vector for observing localization of bacterial protein and construction method of expression vector

A technology for expressing vectors and bacterial proteins, which is applied in the biological field, can solve the problem of fewer prokaryotic protein positioning vectors, and achieve a strong inclusive effect

Active Publication Date: 2020-01-07
武汉博欧特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the common protein spatial positioning vectors are designed for eukaryotic cells, and there are few protein positioning vectors for prokaryotic cells

Method used

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  • Expression vector for observing localization of bacterial protein and construction method of expression vector
  • Expression vector for observing localization of bacterial protein and construction method of expression vector
  • Expression vector for observing localization of bacterial protein and construction method of expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the construction of expression vector

[0036] S1. Construction of expression vector pBBRlacITac-gfp and fluorescence detection

[0037] The schematic diagram of the construction of the expression vector pBBRlacITac-gfp is shown in figure 1 As shown, use the primer pair P1 / P2 and use the pBBR1MCS-5 plasmid as a template to reversely amplify the fragments on pBBR1MCS-5 except for lacZ; use the primer pair P3 / P4 to use the pVLT33 plasmid as a template to amplify the lacI gene and tac promoter; the above two PCR products were digested with NdeI and XhoI, ligated with T4 DNA ligase, and sequenced to obtain the vector pBBRlacITac. Use the primer pair P5 / P6 to amplify the gfp gene using the pBBR1-gfp plasmid as a template; digest the gfp gene with restriction enzymes XhoI+HindIII, and then use T4 DNA ligase to connect it to the pBBRlacITac vector that has been cut with the same enzyme , Sequencing verification, confirming that the gfp gene sequence is correct,...

Embodiment 2

[0041] Embodiment 2, utilize MotA protein as target protein to detect the feasibility of the present invention

[0042] S1. Construction of expression vector pBBRlacITac-motA-gfp

[0043] The map of the expression vector pBBRlacITac-motA-gfp is as follows image 3 shown. Pseudomonas putida KT2440 is a monotendex cluster flagellate. MotA is a KT2440 flagellar matrix component protein that localizes to bacterial endpoints. The present invention uses the pBBRlacITac-gfp carrier to detect the spatial location of MotA in KT2440 to verify whether the carrier has expected functions. Using the total genomic DNA of Pseudomonas putida KT2440 as a template, the motA gene was amplified using the primer pair P7 / P8, then ligated into the pBBRlacITac-gfp vector with EcoRI and BamHI, and sequenced to verify that the motA gene was inserted into the pBBRlacITac-gfp vector Finally, the expression vector pBBRlacITac-motA-gfp was obtained. The motA coding region amplified by PCR does not have...

Embodiment 3

[0046] Embodiment 3, utilize KatG protein as target protein to detect the feasibility of the present invention

[0047] S1. Construction of expression vector pBBRlacITac-katG-gfp

[0048] The map of the expression vector pBBRlacITac-katG-gfp is as follows Figure 5 shown. KatG is a kind of catalase in Escherichia coli, which can hydrolyze hydrogen peroxide in cells and relieve oxidative stress crisis. The expression of KatG was induced by hydrogen peroxide. If no hydrogen peroxide or other oxidative stress substances were added to the medium, KatG could only be expressed in a small amount in the late growth stage of E. coli, and it was difficult to observe its spatial location. The present invention observes the spatial localization of KatG in the logarithmic growth phase of Escherichia coli under the condition of not adding hydrogen peroxide. Using Escherichia coli K-12 genomic DNA as a template, the primer pair P9 / P10 was used to amplify the katG gene, and the XbaI and H...

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Abstract

The invention discloses an expression vector for observing the localization of bacterial protein and a construction method of the expression vector. First the expression vector containing an inducibletac promoter and a green fluorescent protein gfp gene is constructed, a target protein gene is inserted into the vector, fusion expression is carried out on the target protein and green fluorescent protein GFP, then the vector is transferred into bacteria, inducible expression is carried out, and spatial orientation of the target protein is determined by observing the distribution of green fluorescence in bacterial cells under a fluorescence microscope. The real-time and intuitive detection of the spatial orientation of the target protein in living cells can be achieved, and convenience and speediness are achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an expression vector for observing bacterial protein localization and a construction method thereof. Background technique [0002] Protein is the ultimate executor of life activities. The subcellular location of protein is closely related to its function. The correct location of protein in the cell is the prerequisite for the highly orderly operation of the cell system. Studying the mechanism and rules of protein localization in cells and predicting the subcellular localization of proteins are of great significance for understanding the properties and functions of proteins, understanding the interactions between proteins, and exploring the laws and mysteries of life. It is a fast and sensitive method to study protein spatial localization by fusing the target protein with fluorescent protein and using the fluorescent properties of fluorescent protein to detect the spatial localization...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/78C12N15/65C12N15/66C12Q1/02C12R1/19C12R1/40
CPCC12N15/65C12N15/66C12N15/70C12N15/78C12N2830/002C12Q1/02G01N2333/245
Inventor 崔格特
Owner 武汉博欧特生物科技有限公司
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