Construction method and applications of engineering strain for delivering mammalian cell protein

A technology of mammals and engineering strains, applied in the biological field, can solve problems such as inapplicability to large-scale production, cumbersome operation, and carcinogenesis induced by exogenous nucleic acid fragments, and achieve an effect suitable for large-scale production

Inactive Publication Date: 2017-08-29
NANKAI UNIV
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  • Abstract
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Problems solved by technology

[0002] At present, the most widely used intracellular protein introduction technology is based on DNA / RNA transfection method, and its two major defects are: ①Exogenous nucleic acid fragments may be randomly integrated into the genome to induce cancer or other unforeseen consequences
② Once the expression of foreign protein is started, it cannot be effectively terminated
At present, there have been many reports on protein import methods, such as microinjection, transfection reagent (TransfectionReagent) and protein transfer domain (ProteinTranslocationDomain) mediated protein import method, but the protein delivery efficiency of these methods is very low, and the transfection The staining reagent is very toxic to the target cells, and the latter is limited to the introduction of small molecular weight proteins
In addition, the above methods all involve the separation and purification of the target protein, which is cumbersome and costly, and is not suitable for large-scale production

Method used

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  • Construction method and applications of engineering strain for delivering mammalian cell protein
  • Construction method and applications of engineering strain for delivering mammalian cell protein
  • Construction method and applications of engineering strain for delivering mammalian cell protein

Examples

Experimental program
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Effect test

Embodiment 1

[0058] Embodiment 1. Construction of genetic engineering strain Δ5

[0059] Using the genomic DNA of the PAK strain as a template, about 1-kb homology arm fragments upstream and downstream of exoS, exoT, exoY, ndk and popN were amplified by PCR. The above PCR products were digested and cloned into plasmid pEX18Tc to construct gene knockout plasmids pEX18Tc-exoS, pEX18Tc-exoT, pEX18Tc-exoY, pEX18Tc-ndk and pEX18Tc-popN. 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 PAK strain by conjugative transfer method, and the exoS, exoT, exoY, ndk and popN genes in the PAK genome were knocked out one by one. The principle of gene knockout is as figure 1 As shown, single-crossover strains with homologous recombination in one homologous arm were screened on an agarose plate containing 50 μg / mL tetracycline, and homo...

Embodiment 2

[0060] Example 2. Cytotoxicity of Genetic Engineering Strain Δ5 to Various Mammalian Cells

[0061] The wild-type strain PAK and the genetically engineered strain Δ5 were selected as infection strains, and their cytotoxicity against mammalian cells HeLa, A549, mouse embryonic stem cells (mESC) J1 and human embryonic stem cells (hESC) H9 were 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. Culture HeLa, A549, mESCJ1 and hESCH9 cells to 70-80% cell densi...

Embodiment 3

[0062] Example 3. Inject mApple protein into HeLa cells using genetically engineered strain Δ5

[0063] The mApple gene was amplified by PCR and cloned into the expression vector pExoS 54 In this method, the target protein (mApple) to be injected and the T3SS secretion signal peptide ExoS 54 Fusion, constructed into the expression vector pExoS 54 -mApple( image 3 ). The expression vector was electrotransformed into Pseudomonas aeruginosa Δ5 strain to construct mApple delivery strain Δ5 / pExoS 54 -mApple. After the strain contacts the target cells, its T3SS is activated, and the bacterial expression vector pExoS 54 -mApple expresses ExoS efficiently 54 -mApple fusion protein, which can be injected into target cells through T3SS. ExoS 54 There is a Flag tag behind the sequence, so the expression of fusion protein can be detected by WesternBlot method. Such as Figure 4 showed that the exogenous protein mApple can secrete the signal ExoS at the T3SS 54 Injected into HeLa...

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Abstract

The invention relates to a construction method and applications of engineering strain for delivering mammalian cell protein. The construction method comprises the following steps: deleting four virulence factors including exoS, exoT, exoY and ndk genes from pseudomonas aeruginosa PAK strain genome, so as to have no cytotoxicity to mammalian cells; further deleting popN gene, for inhibiting bacterial III type secretory system (T3SS), from chromosome, so as to remarkably increase the injection quantity of T3SS-mediated protein, thus constructing the engineering strain delta5. The constructed engineering strain delta5 can be used for delivering proteins of various mammalian cell linings, performing gene editing, cell reprogramming, protein function research and other work. The types of delivered cells are wide, including human and mice skin cells, muscle cells, intestinal cells, liver cells, multipotent stem cells and the like.

Description

【Technical field】 [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing a Pseudomonas aeruginosa genetic engineering strain used for protein delivery in mammalian cells and an application thereof. 【Background technique】 [0002] At present, the most widely used intracellular protein introduction technology is based on DNA / RNA transfection, and its two major defects are: ①Exogenous nucleic acid fragments may be randomly integrated into the genome to induce cancer or other unforeseen consequences. ② Once the expression of foreign protein is started, it cannot be terminated effectively. If the molecule of interest can be delivered directly into the target cell in the form of "protein", the above disadvantages can be overcome. First, protein introduction avoids genome instability caused by exogenous DNA / RNA. Secondly, since the exogenous protein enters the cell, it will be degraded by the intracellular protease, so that its action...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/85C12R1/385
CPCC07K14/21C12N15/85C12N2800/107
Inventor 白芳金守光靳永新刘颖李振鹏吴卫辉程志辉
Owner NANKAI UNIV
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