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Method for separating complete bacteria from infected cells

A complete technology for infecting cells, applied in the direction of isolation of microorganisms, DNA preparation, recombinant DNA technology, etc., can solve the problems of long separation time, large amount of bacterial death, etc., and achieve the effect of shortening the time

Inactive Publication Date: 2021-07-09
WUHAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the problems of long separation time and large amount of bacterial death in the prior art methods for isolating and purifying bacteria from infected cells, and provide a method for isolating intact bacteria from infected cells

Method used

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  • Method for separating complete bacteria from infected cells
  • Method for separating complete bacteria from infected cells
  • Method for separating complete bacteria from infected cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Bacterial preparation: Take out the frozen porcine parenteral pathogenic Escherichia coli PCN033 strain from -20°C and place it at room temperature. After it dissolves, take 5 μL of the bacterial liquid on the LA solid plate, and use the inoculation silk to Its divisions are demarcated. Then place it in a constant temperature incubator for culture, wait for it to grow a single colony, pick a single colony and culture it in LB medium, after overnight culture, transfer it to fresh LB at a ratio of 1:100 (V / V) The culture is based on shaking culture in a shaker. To be cultured to mid-log phase (OD 600 =0.8), that is, each milliliter contains 5×10 8 When the number of viable cells (CFU) is about 10000000000000000000000000000000000000000000000000000 thallines were obtained, the collected bacteria were resuspended to 0.5 × 10 using RPMI 1640 medium 8 CFU / mL, store in a 4°C refrigerator for later use.

[0022] 2. Bacterial infection of cells: HEp-2 cells frozen at -80°C...

experiment approach 1

[0025] After adding bacteria to HEp-2 cells, the bacteria were thoroughly contacted with the cells by centrifugation, and then placed in 5% CO 2 constant temperature incubator for cells. After co-incubating at 37°C for 2 hours, discard the medium, add pre-cooled sterile water, and let it stand in the refrigerator at 4°C for 10 minutes, scrape off the adherent cells with a cell scraper, and transfer the cell suspension to a sterile centrifuge. tube, vortexed for 2 minutes, centrifuged at 6000r / min at 4°C for 10 minutes, discarded the supernatant, added pre-cooled PBS, resuspended the pellet, washed three times and collected the bacteria; fixed the collected bacteria with electron microscope fixative , observe the morphological structure of the bacteria through a transmission electron microscope, and measure the growth performance of the isolated and purified bacteria through the growth curve, so as to evaluate the influence of factors such as ice water, cell scraping and vortex...

experiment approach 2

[0027] After adding bacteria to HEp-2 cells, the bacteria were thoroughly contacted with the cells by centrifugation, and then placed in 5% CO 2 constant temperature incubator for cells. After co-incubating at 37°C for 2 h, Triton x-100 at different concentrations (0, 0.01%, 0.025%, 0.05% and 0.1%; V / V) were added, labeled as sample 1, sample 2, sample 3, sample 4 and For sample 5, place it on ice and incubate with shaking, observe the lysis of the cells through an inverted microscope at 0 min, 15 min, 30 min, 60 min and 120 min respectively, and at the same time count the number of viable bacteria in each sample by plate counting method; After complete lysis, transfer the cell lysate containing bacteria to a pre-cooled sterilized centrifuge tube, centrifuge at 6000r / min at 4°C for 10 min, discard the supernatant, add pre-cooled PBS, resuspend the pellet, and wash three times Finally, the bacteria were collected; the collected bacteria were fixed with an electron microscope f...

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Abstract

The invention discloses a method for separating complete bacteria from infected cells, and belongs to the technical field of bacteria and cell interaction and bacteria separation. The method comprises the following steps: adding Triton x-100 and / or SDS (Sodium Dodecyl Sulfate) into cells infected by bacteria, incubating at 0-4 DEG C for 30-120 minutes, centrifuging at 0-4 DEG C, washing and collecting thalli; or adding Triton x-100 and / or SDS into cells infected by bacteria, incubating at 0-4 DEG C for 5-15 minutes, adding KCl, incubating at 0-4 DEG C for 20-40 minutes, centrifuging at 0-4 DEG C, washing and collecting thalli. According to the method disclosed by the invention, cells can be completely split and separated within 20 minutes to obtain bacteria with complete morphological structure and unaffected activity, and high-quality RNA can be extracted. According to the invention, the time for separating complete bacteria from infected cells is greatly shortened.

Description

technical field [0001] The invention belongs to the technical field of bacteria-cell interaction and isolation of bacteria therefrom, in particular to a method for rapidly isolating bacteria with strong vitality and good integrity from infected eukaryotic cells. Background technique [0002] With the advent of the era of banning the addition of antibiotics in feed in our country, intestinal health and extraintestinal tissue and organ infections caused by bacteria are causing more and more serious harm to the aquaculture industry and a serious threat to public health. Therefore, finding and developing new alternatives to antibiotics is an effective means to prevent and treat bacterial infections, and this means has reached a consensus among more and more scientific researchers. At present, there are many ways to develop antibiotic substitutes, but many development ideas are based on the pathogenic mechanism of bacteria, and mainly target the important metabolic pathways, sign...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/02C12N15/10
CPCC12N1/02C12N15/1003
Inventor 宗冰冰张焱焱任明星刘宇付书林叶纯吴仲元邱银生
Owner WUHAN POLYTECHNIC UNIVERSITY
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