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Building method of attenuated pseudomonas aeruginosa and application of attenuated pseudomonas aeruginosa to protein transfection

A Pseudomonas aeruginosa, construction method technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria and other directions, can solve the problems of loss of cell stemness, antibiotic sterilization side effects can not be ignored, etc., to achieve the effect of efficient injection

Active Publication Date: 2018-08-03
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have shown that the use of high concentrations of antibiotics (such as ciprofloxacin) has a significant impact on the transcriptome of pluripotent stem cells (such as human embryonic stem cells), although there is no evidence that this effect will lead to the loss of their stemness , but the side effects of antibiotic sterilization cannot be ignored

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  • Building method of attenuated pseudomonas aeruginosa and application of attenuated pseudomonas aeruginosa to protein transfection
  • Building method of attenuated pseudomonas aeruginosa and application of attenuated pseudomonas aeruginosa to protein transfection
  • Building method of attenuated pseudomonas aeruginosa and application of attenuated pseudomonas aeruginosa to protein transfection

Examples

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Effect test

Embodiment 1

[0107] Example 1. Construction of genetic engineering strain Δ9

[0108] Using the genomic DNA of the Δ8 strain as a template, a homology arm fragment of about 1-kb upstream and downstream of murI was amplified by PCR. The above PCR product was digested and cloned into the plasmid pEX18Tc to construct the gene knockout plasmid pEX18Tc-murI. The knockout plasmids stored in DH5a were extracted, and the plasmids were transformed into Escherichia coli S17 competent cells by chemical transformation method. The gene knockout plasmid in S17 was transferred into the Δ8 strain by conjugative transfer method, and the murI gene in the Δ8 genome was knocked out. Single-crossover strains with homologous recombination in one homologous arm were screened on an agarose plate containing 50 μg / mL tetracycline, and then homologous homologous arms on both sides were screened on an agarose plate containing 7.5% sucrose and 10 mM D-Glu. The strain with homologous recombination finally obtained th...

Embodiment 2

[0109] Example 2. Cytotoxicity of genetically engineered strain Δ9 to mammalian HeLa cells

[0110] The wild-type strain PAK, the attenuated strain Δ8 and the auxotrophic attenuated strain Δ9 were selected as infection strains, and their cytotoxicity to mammalian HeLa cells was compared. Cytotoxicity was detected by lactate dehydrogenase (LDH) cytotoxicity detection kit. When the cells are damaged, the enzymes in the cytoplasm will be released into the culture medium, including lactate dehydrogenase LDH with relatively stable enzyme activity. Under the action of lactate dehydrogenase, NAD + It is reduced to NADH, and NADH is catalyzed to generate a strong chromogenic substance, which produces an absorption peak at a wavelength of 490nm. The quantitative analysis of cytotoxicity can be realized by quantifying the activity of LDH through colorimetry. Cultivate HeLa cells to a cell density of 70-80%; pick fresh PAK and Δ8 single colonies and inoculate them into LB medium, and in...

Embodiment 3

[0111] Embodiment 3. Growth and survival rate in vitro of genetic engineering strain Δ9

[0112] Because bacterial infection of cells ultimately involves the issue of how to remove bacteria, we used to use high concentrations of ciprofloxacin to completely remove bacteria, but through transcriptome analysis, we found that high concentrations of ciprofloxacin treatment would seriously affect the expression profile of cells. Impact. When the murI gene is mutated, the bacteria cannot synthesize the cell wall and eventually die, thus realizing the method of completely eradicating the bacteria without using antibiotics or using ordinary double antibodies, and also reducing the toxicity of high-concentration ciprofloxacin to the cells effect. Therefore, in order to confirm that the murI mutant Δ9 cannot reproduce without D-Glu supplementation, we investigated the growth of Δ9 in vitro, with and without D-Glu(+) and without D-Glu(-), each After a certain period of time, the number ...

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Abstract

The invention relates to a building method of attenuated pseudomonas aeruginosa and application of the attenuated pseudomonas aeruginosa to protein transfection. The building method is characterized in that glutamate racemase gene murI participating in cell wall peptidoglycan synthesis in a pseudomonas aeruginosa Delta8 bacterial strain is deleted, so that a D-glutamate nutritional deficiency typebacterial strain Delta9 is obtained. According to the application, the Delta9 bacterial strain has no cytotoxicity; the complete III type secretory system (T3SS) is reserved; foreign protein is effectively injected into mammalian cells through T3SS and can be applied to mammalian cell protein transfection. The Delta9 cannot grow in culture environment lacking D-glutamate, so that after the transfection, the bacterium is automatically cleared through D-glutamate nutrient limitation. The in vitro and in vivo application safety of the bacterium is interpreted by using HeLa cells and a mouse infection model. The positive significance is realized for developing the safe and efficient mammalian cell protein transfection technology.

Description

【Technical field】 [0001] The invention belongs to the field of biotechnology, and relates to a construction method of an attenuated Pseudomonas aeruginosa strain and its application in protein transfection of mammalian cells. 【Background technique】 [0002] Pseudomonas aeruginosa is a Gram-negative bacterium whose cell wall is mainly composed of peptidoglycan, which is a unique substance in prokaryotic cells. Peptidoglycan is composed of three parts: glycan backbone, tetrapeptide side chains and pentapeptide cross-linking bridges. The glycan backbone is composed of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) alternately arranged at intervals and linked by β-1,4 glycosidic bonds. The tetrapeptide side chain is composed of L-alanine, D-glutamic acid, L-lysine and D-alanine in sequence; the third L-lysine is composed of five glycines. The bridge is connected to the D-alanine at the end of the tetrapeptide side chain of the adjacent glycan backbone, thereby ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/85C12R1/385
CPCC12N9/90C12N15/85C12Y501/01003
Inventor 白芳刘颖吴卫辉靳永新程志晖刘畅许译天郑瑞萍
Owner NANKAI UNIV
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