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Attenuated salmonella typhimurium mediated eukaryocyte plasmid transfection method

A Salmonella typhi, cytoplasmic technology, applied in the directions of viruses/phages, biochemical equipment and methods, nucleic acid vectors, etc., can solve the problem of high cost and achieve the effects of low cost, low toxicity and uniform transfection efficiency

Inactive Publication Date: 2017-04-26
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the technical defects of the prior art, and provides an attenuated Salmonella typhimurium-mediated eukaryotic cell plasmid transfection method to solve the technical problem of high cost of the eukaryotic cell plasmid transfection method in the prior art

Method used

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  • Attenuated salmonella typhimurium mediated eukaryocyte plasmid transfection method

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Embodiment 1

[0041] 1. Construction of recombinant plasmid pTriex-TEM1-EGFP

[0042] The full length of TEM1 was amplified by PCR technology, and then the TEM1 fragment and the pTriex expression vector were double-digested with endonuclease, and the TEM1 fragment was inserted into the multiple cloning site of the pTriex expression vector using T4 ligase. The TEM1 fragment is located before the EGFP gene in the vector pTriex, and the constructed vector is named pTriex-TEM1-EGFP.

[0043] 2. Competent preparation of attenuated Salmonella typhimurium VNP20009

[0044]Streak inoculate the attenuated Salmonella typhimurium VNP20009 stored at -80°C on a non-resistant LB plate, and culture at 37°C overnight; pick a single colony in 5ml LB, shake and culture at 37°C for 12 hours; inoculate at a ratio of 1:100 on Shake culture in 100ml LB until the bacteria OD=0.4; after ice bath for 20min, centrifuge at 3000rpm at 4°C for 10min; Wash the cell pellet with 1 / 100 volume of pre-cooled 10% glycerol a...

Embodiment 2

[0050] A method for eukaryotic cell plasmid transfection mediated by attenuated Salmonella typhimurium, comprising the following steps:

[0051] 1) Prepare competent cells of attenuated Salmonella typhimurium VNP20009 strain;

[0052] 2) taking the plasmid to be transfected, introducing it into the competent cells described in step 1) by electroporation, and screening positive clones to obtain recombinant bacteria;

[0053] 3) Take HEK293-T cells and the recombinant bacteria obtained in step 2), mix them in a cell culture plate at a ratio of 1:20, culture them for 4 hours, and then replace the medium in the cell culture plates with Antibiotic cell culture medium against Salmonella typhimurium, cultivated for 40 h.

[0054] On the basis of the above technical solutions, the following conditions are met:

[0055] The amount of HEK293-T cells added in step 3) was 8000 / well.

[0056] After the mixing in step 3), the total amount of liquid in each well of the cell culture plate ...

Embodiment 3

[0069] A method for eukaryotic cell plasmid transfection mediated by attenuated Salmonella typhimurium, comprising the following steps:

[0070] 1) Prepare competent cells of attenuated Salmonella typhimurium VNP20009 strain;

[0071] 2) taking the plasmid to be transfected, introducing it into the competent cells described in step 1) by electroporation, and screening positive clones to obtain recombinant bacteria;

[0072] 3) Take HEK293-T cells and the recombinant bacteria obtained in step 2), mix them in a cell culture plate at a ratio of 1:200, culture them for 16 hours, and then replace the culture medium in the cell culture plate with Antibiotic cell culture medium against Salmonella typhimurium, cultured for 56h.

[0073] On the basis of the above technical solutions, the following conditions are met:

[0074] The amount of HEK293-T cells added in step 3) was 12000 / well.

[0075] After the mixing in step 3), the total amount of liquid in each well of the cell culture...

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Abstract

The invention provides an attenuated salmonella typhimurium mediated eukaryocyte plasmid transfection method. According to the transfection method, based on an intracellular invasion characteristic of the attenuated salmonella typhimurium, recombinant plasmids are carried to enter eukaryocytes to realize specific protein expression. Specifically, recombinant eukaryotic expression plasmids are introduced into an attenuated salmonella typhimurium VNP20009 strain in an electric shock transformation way; then recombinant bacteria and the eukaryocytes are cultured in a co-incubation manner; and finally, the attenuated salmonella typhimurium inside and outside cells is killed by a serum culture medium containing penicillin and streptomycin, and the recombinant plasmids are released to the eukaryocytes to realize the specific protein expression. Compared with conventional calcium sulfate transfection, liposome transfection and electro-transformation, the transfection method disclosed by the invention takes viable bacteria as a bearing host of specific plasmids, so that a larger number of target vectors can be obtained by only conventional culture; and in addition, the plasmid transfection operating steps are simplified, the cost is lowered, and the transfection efficiency and the cytotoxicity are comprehensively better than those in the conventional transfection method.

Description

technical field [0001] The invention relates to the technical field of gene recombination, in particular to a eukaryotic cell plasmid transfection method mediated by attenuated Salmonella typhimurium. Background technique [0002] Salmonella typhimurium is an invasive intracellular bacterium. After entering the digestive tract of the host, Salmonella typhimurium first adheres to and invades the epithelial cells of the small intestine. Patches, PPs). M cells (Microfold cells) are microfold cells sparsely distributed on the surface of PPs, tightly surrounded by intestinal epithelial cells. The high permeability of cells allows the transport not only of soluble macromolecules but also of particulate matter and intact microorganisms. Its main function is to transport the antigenic substances on the mucosal surface into the cells through the intestinal epithelial barrier, and present the antigens to lymphocytes to trigger the body's primary immune response. Salmonella typhimur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
CPCC12N15/85C12N2800/107
Inventor 陈廷涛辛洪波王鑫
Owner NANCHANG UNIV
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