CN113088454A - Method for separating intact bacteria from infected cells - Google Patents
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The invention discloses a method for separating complete bacteria from infected cells, belonging to the technical field of bacteria and cell interaction and bacteria separation from the bacteria. The method comprises the following steps: adding Triton x-100 and/or SDS into the cell infected by the bacteria, incubating at 0-4 deg.C for 30-120min, centrifuging at 0-4 deg.C, washing, and collecting thallus; or adding Triton x-100 and/or SDS into bacteria infected cells, incubating at 0-4 deg.C for 5-15min, adding KCl, incubating at 0-4 deg.C for 20-40min, centrifuging at 0-4 deg.C, washing, and collecting thallus. The method can completely crack cells and separate the cells to obtain bacteria with complete morphological structure and unaffected vitality by 20min, and can extract high-quality RNA. The present invention greatly shortens the time to isolate intact bacteria from infected cells.
Description
Technical Field
The invention belongs to the technical field of bacteria and cell interaction and bacteria separation from the bacteria, and particularly relates to a method for quickly separating bacteria with strong activity and good integrity from infected eukaryotic cells.
Background
With the arrival of the era of completely forbidding the addition of antibiotics in feeds in China, the harm to the breeding industry is more and more serious and the public health is seriously threatened due to the problems of intestinal health and tissue and organ infection outside the intestinal tract caused by bacteria. Therefore, finding and developing new antibiotic substitutes is an effective means for preventing and treating bacterial infections, and the means is commonly recognized by more and more researchers. At present, the approaches for developing antibiotic substitutes are various, but many development ideas are based on pathogenic mechanisms of bacteria, mainly take important metabolic pathways, signal transduction systems, secretion systems or virulence factors of the bacteria as targets to screen and design corresponding inhibitors, and the inhibitors can achieve the purpose of preventing or treating bacterial infection by inhibiting the growth or pathogenic capability of the bacteria. However, few target molecules have been selected during the development of antibiotic alternatives. Although various genes related to bacterial growth and pathogenesis are discovered, antibiotic substitutes developed based thereon have far less than effective antibiotics for preventing and treating bacterial infections, and more unknown pathogens are discovered. Therefore, the identification and study of genes associated with bacterial growth and pathogenesis are a prerequisite for the development of highly effective antibiotic substitutes. Because the genome of the bacteria is large, the regulation and control network for the growth and the pathogenesis of the bacteria is complex, in order to efficiently excavate important growth or pathogenesis factors of the bacteria, researchers often separate and purify the bacteria from infected hosts and complete the research on the growth and pathogenesis of the bacteria in vitro.
In order to simulate the actual process of bacterial infection of a host in vitro, researchers often select bacteria to interact with cells in vitro, and after the bacteria infect the cells, the genes related to bacterial growth or pathogenesis are screened. As more and more bacterial genomes are being deciphered, high throughput sequencing methods (RNA-seq, ChIP-seq, Genechip) based on bacterial whole genomes have become an effective method for understanding bacterial growth or pathogenesis. In vitro experiments such as bacterial infected cells are effective methods for simulating the gene expression profile of bacteria in a host, and further compared with the bacterial gene expression profile of uninfected cells, genes which play important functions in the process of infecting the host by the bacteria can be identified, so that the method is an effective means for revealing the growth and pathogenic mechanism of the bacteria. However, the observation of the morphological structure of bacteria is seriously affected by the existence of cells, and the pure bacterial RNA is difficult to obtain, thus the application of the high-throughput screening method is seriously hindered. Therefore, the complete bacteria can be separated and purified from the infected cells, and the foundation can be laid for the subsequent bacteria morphological structure and the simple bacteria RNA extraction.
The prior art method has the defects that the number of bacteria separated from infected cells is small, the morphological structure is incomplete, RNA extracted from the separated bacteria is often polluted by cell RNA, the research on the morphological structure of the bacteria is seriously hindered, and the analysis of bacterial gene expression profiles by various high-throughput screening methods is limited due to the pollution of the cell RNA. Therefore, it is important to develop a method for separating and purifying intact bacteria from infected cells.
Disclosure of Invention
The invention aims to overcome the problems of long separation time and high bacterial death amount of the prior method for separating and purifying bacteria from infected cells, and provides a method for separating complete bacteria from infected cells. The method can separate bacteria with complete morphological structure and strong activity in a short time, and the RNA purity and quality of the extracted bacteria are high.
The purpose of the invention is realized by the following technical scheme:
a method of isolating intact bacteria from infected cells comprising the steps of: adding Triton x-100 and/or SDS into the cell infected by bacteria, incubating at 0-4 deg.C for 30-120min, centrifuging at 0-4 deg.C, washing, and collecting thallus.
Preferably, in the method, 0.025% Triton x-100 or 0.1% SDS is added.
Preferably, in the method, 0.0025-0.0175% of Triton x-100 and 0.03-0.09% of SDS are added.
Preferably, in the method, 0.0175% of Triton x-100 and 0.03% of SDS are added.
Preferably, the method comprises the following steps: adding Triton x-100 and/or SDS into the cell infected by bacteria, incubating at 0-4 deg.C for 5-15min, adding KCl, incubating at 0-4 deg.C for 20-40min, centrifuging at 0-4 deg.C, washing, and collecting thallus.
Preferably, the method comprises the following steps: adding 0.025% Triton x-100 into bacteria infected cells, incubating at 0-4 deg.C for 5-15min, adding 0.2% KCl, incubating at 0-4 deg.C for 30-40min, centrifuging at 0-4 deg.C, washing, and collecting thallus.
Preferably, the method comprises the following steps: adding 0.0175% Triton x-100 and 0.03% SDS into bacteria infected cells, incubating at 0-4 deg.C for 5-15min, adding 0.2% KCl, incubating at 0-4 deg.C for 20-40min, centrifuging at 0-4 deg.C, washing, and collecting thallus.
The invention has the following advantages and beneficial effects: the method can completely crack cells and separate the bacteria with complete morphological structure and unaffected vitality, can extract high-quality RNA, and can be directly used for later experimental research. Under the optimal conditions of the invention, namely adding 0.0175 percent of Triton x-100, 0.03 percent of SDS and 0.2 percent of KCl, the cells can be completely lysed in 20min without any adverse effect on bacteria, and the time for separating complete bacteria from infected cells is greatly shortened.
Drawings
FIG. 1 is a structural diagram of a form of bacteria observed by a transmission electron microscope. A: bacteria PCN033 that does not interact with cells; b: purified bacteria PCN033 were isolated from infected cells.
FIG. 2 is a graph of the growth of bacterium PCN 033. PCN 033: normal bacteria; isolated PCN 033: isolating the purified bacteria from the infected cells.
FIG. 3 is an electrophoretogram of RNA extracted from bacterium PCN 033. M: 2000bp DNA marker; 1: extracting RNA of bacteria PCN033 which does not interact with cells; 2: extraction of RNA from the bacterium PCN033 isolated and purified from the infected cells.
Detailed Description
The following examples are intended to further illustrate the present invention and should not be construed as limiting the present invention, and any other changes, modifications, substitutions, combinations and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and shall be included within the scope of the present invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
Firstly, preparing bacteria: frozen porcine-derived enteropathogenic Escherichia coli PCN033 strain was taken out from-20 ℃ and placed at room temperature, after it was dissolved, 5. mu.L of the bacterial liquid was taken on LA solid plate and streaked by partition using an inoculating silk. Then placing the culture medium in a constant temperature incubator for culture, picking up a single colony to culture in an LB culture medium after the single colony grows out, and after overnight culture, transferring the single colony to a fresh LB culture medium according to a ratio of 1:100(V/V) for shake culture in a shaking table. It is cultured to middle logarithmic phase (OD)6000.8), i.e. 5 × 10 per ml8In the case of about the number of viable bacteria (CFU), the cells were collected by centrifugation, and the collected bacteria were resuspended at 0.5X 10 in RPMI 1640 medium8CFU/mL, and placing in a refrigerator at 4 ℃ for standby.
Secondly, infection of cells by bacteria: taking out HEp-2 cells frozen at-80 deg.C, recovering, transferring to T25 cell culture bottle containing RPMI 1640 culture medium at a ratio of 1:5(V/V), transferring to T75 cell culture bottle for amplification culture after the cells are full, separating HEp-2 cells from the cell culture bottle by pancreatin treatment after the cells are full again, blowing off, and mixing at 2.5 × 106cells/well were spread evenly onto 6-well cell culture plates, after the cells were attached, the cell culture broth was discarded and washed three times with PBS, followed by 5mL of PCN033 strain prepared in advance (MOI ═ 100) per well.
Thirdly, purification and separation of bacteria
1. Experimental protocol 1
After adding bacteria to HEp-2 cells, the bacteria were thoroughly contacted with the cells by centrifugation, and then placed in the presence of 5% CO2The cell constant temperature incubator. After incubating for 2h at 37 ℃, removing the culture medium, adding precooled sterile water, after standing for 10min in a refrigerator at 4 ℃, scraping adherent cells by using a cell scraper, transferring the cell bacterial suspension into a sterile centrifuge tube, carrying out vortex oscillation for 2min, centrifuging for 10min at 4 ℃ at 6000r/min of a centrifuge, removing the supernatant, adding precooled PBS, carrying out heavy suspension precipitation, washing for three times, and collecting thalli; fixing the collected thalli by using an electron microscope fixing solution, observing the morphological structure of the bacteria by using a transmission electron microscope, and simultaneously measuring the growth performance of the separated and purified bacteria by using a growth curve so as to evaluate the influence of factors such as ice water, cell scraping and vortex oscillation on the growth activity and morphology of the bacteria; and finally, extracting the total RNA of the separated and purified bacteria by a Trizol cracking method, and evaluating the influence of the cracking solution on the RNA of the separated and purified bacteria by determining the quality and the purity of the extracted total RNA.
2. Experimental scheme 2
After adding bacteria to HEp-2 cells, the bacteria were thoroughly contacted with the cells by centrifugation, and then placed in the presence of 5% CO2The cell constant temperature incubator. After incubating for 2h at 37 ℃, adding Triton x-100 with different concentrations (0, 0.01%, 0.025%, 0.05% and 0.1%; V/V), respectively marking as sample 1, sample 2, sample 3, sample 4 and sample 5, placing on ice for shaking incubation, respectively observing the cell lysis condition through an inverted microscope at 0min, 15min, 30min, 60min and 120min, and simultaneously counting the viable bacteria number in each sample by a plate counting method; after the cells are completely lysed, transferring the cell lysate containing bacteria to a sterilized centrifugal tube precooled in advance, centrifuging for 10min at a 4 ℃ centrifuge of 6000r/min, discarding the supernatant, adding PBS precooled in advance, resuspending the precipitate, washing for three times, and collecting thalli; fixing the collected thallus with electron microscope fixing liquid, observing the morphological structure of the bacteria with a transmission electron microscope, and measuring the growth performance of the separated and purified bacteria with a growth curve to evaluate the fissionInfluence of the lysate Triton x-100 on the growth vigor and morphology of bacteria; and finally, extracting the total RNA of the separated and purified bacteria by a Trizol cracking method, and evaluating the influence of the cracking solution on the RNA of the separated and purified bacteria by determining the quality and the purity of the extracted total RNA.
3. Experimental protocol 3
After adding bacteria to HEp-2 cells, the bacteria were thoroughly contacted with the cells by centrifugation, and then placed in the presence of 5% CO2The cell constant temperature incubator. After incubating for 2h at 37 ℃, adding SDS with different concentrations (0, 0.1%, 0.25%, 0.5% and 1%; W/V), respectively marking as a sample 1, a sample 2, a sample 3, a sample 4 and a sample 5, placing the samples on ice for shaking incubation, respectively observing the cell lysis condition through an inverted microscope at 0min, 15min, 30min, 60min and 120min, and simultaneously counting the viable count in each sample by a plate counting method; after the cells are completely lysed, transferring the cell lysate containing bacteria to a sterilized centrifugal tube precooled in advance, centrifuging for 10min at a 4 ℃ centrifuge of 6000r/min, discarding the supernatant, adding PBS precooled in advance, resuspending the precipitate, washing for three times, and collecting thalli; fixing the collected thalli by using an electron microscope fixing solution, observing the morphological structure of the bacteria by using a transmission electron microscope, and simultaneously determining the growth performance of the separated and purified bacteria by using a growth curve so as to evaluate the influence of the SDS lysate on the growth activity and morphology of the bacteria; and finally, extracting the total RNA of the separated and purified bacteria by a Trizol cracking method, and evaluating the influence of the cracking solution on the RNA of the separated and purified bacteria by determining the quality and the purity of the extracted total RNA.
4. Experimental protocol 4
After adding bacteria to HEp-2 cells, the bacteria were thoroughly contacted with the cells by centrifugation, and then placed in the presence of 5% CO2The cell constant temperature incubator. After incubation for 2h at 37 ℃ 0.025% Triton x-100, 0.0225% Triton x-100 and 0.01% SDS, 0.02% Triton x-100 and 0.02% SDS, 0.0175% Triton x-100 and 0.03% SDS, 0.015% Triton x-100 and 0.04% SDS, 0.0125% Triton x-100 and 0.05% SDS, 0.01% Triton x-10, 0.7% Triton x-100 and 0.04% SDS, were added to sample 1, sample 3, and the like0 and 0.06% SDS, 0.0075% Triton x-100 and 0.07% SDS in sample 8, 0.005% Triton x-100 and 0.08% SDS in sample 9, 0.0025% Triton x-100 and 0.09% SDS in sample 10 and 0.1% SDS in sample 11, placing them on ice and shaking and incubating them, observing cell lysis by inverted microscope at 0min, 15min, 30min, 60min and 120min of experiment, and simultaneously counting the number of viable bacteria in each sample by plate counting method; after the cells are completely lysed, transferring the cell lysate containing bacteria to a sterilized centrifugal tube precooled in advance, centrifuging for 10min at a 4 ℃ centrifuge of 6000r/min, discarding the supernatant, adding PBS precooled in advance, resuspending the precipitate, washing for three times, and collecting thalli; fixing the collected thallus by using an electron microscope fixing solution, observing the morphological structure of the bacteria by using a transmission electron microscope, and simultaneously measuring the growth performance of the separated and purified bacteria by using a growth curve so as to evaluate the influence of lysates with different compositions on the growth activity and morphology of the bacteria; and finally, extracting the total RNA of the separated and purified bacteria by a Trizol cracking method, and evaluating the influence of the cracking solution on the RNA of the separated and purified bacteria by determining the quality and the purity of the extracted total RNA.
5. Experimental protocol 5
After adding bacteria to HEp-2 cells, the bacteria were thoroughly contacted with the cells by centrifugation, and then placed in the presence of 5% CO2The cell constant temperature incubator. After co-incubation for 2h at 37 ℃, adding physiological saline with the same dosage into the sample 1, incubating on ice for 10min, and adding 0.2% KCl; sample 2 was incubated with 0.025% Triton x-100 on ice for 10min and 0.2% KCl was added; adding 0.1% SDS into the sample 3, incubating on ice for 10min, and adding 0.2% KCl; adding 0.0175% Triton x-100 and 0.03% SDS into the sample 4, incubating on ice for 10min, and adding 0.2% KCl; observing the cell lysis condition by an inverted microscope at 0min, 10min, 20min and 30min of the experiment, and counting the viable bacteria number in each sample by a plate counting method; after the cells are completely lysed, transferring the cell lysate containing bacteria to a sterilized centrifugal tube precooled in advance, centrifuging for 10min at a 4 ℃ centrifuge of 6000r/min, discarding the supernatant, adding PBS precooled in advance, resuspending the precipitate, washing for three times, and collecting thalli; using electron microscope to fixFixing the collected thalli by using a stationary liquid, observing the morphological structure of the bacteria by using a transmission electron microscope, and simultaneously measuring the growth performance of the separated and purified bacteria by using a growth curve so as to evaluate the influence of lysates with different compositions on the growth activity and morphology of the bacteria; and finally, extracting the total RNA of the separated and purified bacteria by a Trizol cracking method, and evaluating the influence of the cracking solution on the RNA of the separated and purified bacteria by determining the quality and the purity of the extracted total RNA.
Fourthly, experimental results:
as shown in table 1: experimental protocol 1 bacteria separated from infected cells by ice water, cell scraping and vortex shaking using conventional methods were significantly reduced to only initial amounts (2.5X 10)8CFU) 84%.
TABLE 1 lysis of cells and bacteria by ice water, cell scraping and vortex shaking
| Number of cells (Cell) | Number of bacteria (CFU) |
| 0 | 2.1×108 |
As shown in table 2: experimental protocol 2 different concentrations of Triton x-100 lysate showed different lysis effects on cells at different time points and Triton x-100 lysate also showed different lysis effects on bacteria with increasing time and concentration, wherein 0.025% Triton x-100 lysate was added and incubated on ice for 120min (sample 3 in Table 2), without reducing the number of bacteria (2.5X 10) on the premise of complete cell lysis8CFU)。
TABLE 2 lysis of cells and bacteria at different time points with different concentrations of Triton x-100
As shown in table 3: experimental protocol 3 different concentrations of SDS showed different lysis of cells at different time points and SDS showed different lysis of bacteria with increasing time and concentration, wherein 0.1% SDS was added and incubated on ice for 120min (sample 2 in Table 3) without reducing the number of bacteria (2.5X 10) with complete lysis of cells8CFU)。
TABLE 3 lysis of cells and bacteria at different time points with different concentrations of SDS
As shown in table 4: experimental protocol 4 Triton x-100 lysate and SDS were simultaneously added to the bacteria-infected cells at different ratios, and the lysis effect exhibited by the cells at different time points was different depending on the concentrations of the Triton x-100 lysate and SDS added, in which 0.0175% Triton x-100 and 0.03% SDS were added and incubated on ice for 30min (sample 4 in Table 4), without reducing the number of bacteria (2.5X 10) on the premise of completely lysing the cells (2.5X 10)8CFU)。
TABLE 4 lysis of cells and bacteria at different time points with different concentrations of Triton x-100 and SDS mixtures
As shown in table 5: experimental protocol 5 addition of 0.2% KCl under optimum conditions for the protocol promotes lysis of bacteria by Triton x-100 and SDS and further shortens the time for lysis of eukaryotic cells by Triton x-100 and SDS, wherein 0.0175 was added% Triton x-100, 0.03% SDS and 0.2% KCl and incubated on ice for 20min (sample 4 of Table 5) without reducing the number of bacteria (2.5X 10) with complete lysis of the cells8CFU)。
TABLE 5 Effect of 0.2% KCl addition on Triton x-100 and SDS lysis of cells and bacteria
The observation result of the transmission electron microscope shows that the morphological structure of the bacterial cell separated from the infected HEp-2 cell (sample 4 in Table 5) is clear and complete (figure 1), and the growth characteristic result shows that the growth characteristic of the separated and purified bacteria (sample 4 in Table 5) is not affected (figure 2); the final RNA extraction showed that the total RNA extracted from the isolated and purified bacteria from the infected cells (sample 4 in Table 5) was high in concentration (Table 6) and purity (FIG. 3), and could be used directly in the subsequent experimental studies.
TABLE 6 RNA of isolated and purified bacterium PCN033
Claims (7)
1. A method of isolating intact bacteria from infected cells, comprising: the method comprises the following steps: adding Triton x-100 and/or SDS into the cell infected by bacteria, incubating at 0-4 deg.C for 30-120min, centrifuging at 0-4 deg.C, washing, and collecting thallus.
2. The method of claim 1, wherein: 0.025% Triton x-100 or 0.1% SDS was added.
3. The method of claim 1, wherein: 0.0025-0.0175% Triton x-100 and 0.03-0.09% SDS were added.
4. The method of claim 1, wherein: 0.0175% Triton x-100 and 0.03% SDS were added.
5. The method of claim 1, wherein: the method comprises the following steps: adding Triton x-100 and/or SDS into the cell infected by bacteria, incubating at 0-4 deg.C for 5-15min, adding KCl, incubating at 0-4 deg.C for 20-40min, centrifuging at 0-4 deg.C, washing, and collecting thallus.
6. The method of claim 5, wherein: the method comprises the following steps: adding 0.025% Triton x-100 into bacteria infected cells, incubating at 0-4 deg.C for 5-15min, adding 0.2% KCl, incubating at 0-4 deg.C for 30-40min, centrifuging at 0-4 deg.C, washing, and collecting thallus.
7. The method of claim 5, wherein: the method comprises the following steps: adding 0.0175% Triton x-100 and 0.03% SDS into bacteria infected cells, incubating at 0-4 deg.C for 5-15min, adding 0.2% KCl, incubating at 0-4 deg.C for 20-40min, centrifuging at 0-4 deg.C, washing, and collecting thallus.
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