CN1869704B - Method for quickly determining host protein interaction with pathogenic bacteria - Google Patents

Abstract

The invention is a method for fast identifying host proteins interacting with pathogenic germs, relating to a method of applying Gram positive and negative unactivated pathogenic germs to fast separate and purify host proteins interacting with them, providing a method for fast and reliably detecting and identifying host proteins interacting with pathogenic germs, comprising: using formaldehyde to unactivate pathogenic germs; using eluent to remove easy-to-drop components on outer membranes of the pathogenic germs; letting the pathogenic germs and host proteins hatch, and letting host proteins which can interact with the pathogenic germs adhere to surfaces of the pathogenic germs; collecting the host proteins interacting with the pathogenic germs by eluent. And the invention can avoid the problem that the host proteins interacting with activated germs are polluted by outer membranes of the pathogenic germs or secrete proteins. And all the obtained proteins derive from the host, having no pathogenic germ pollutants, and it is a method for in high efficiency and reliably detecting composition of proteins in host taking part in first line of defence in pathogenic germ defending system.

Description

Method for rapid identification of host proteins that interact with pathogenic bacteria

technical field

The present invention relates to a host protein, in particular to a method for quickly separating and purifying interacting host proteins, especially trace proteins, by using gram-positive and negative inactivated pathogenic bacteria.

Background technique

Multicellular organisms cannot survive without the ability to sense and eliminate microbial invasion, and identifying microbial molecules is difficult due to their molecular heterogeneity and high rate of evolution. But organisms have evolved several strategies to accomplish this task, pattern-recognition strategy is one of them, organisms recognize pathogens through the combination of pattern-recognition receptors (PRRs) and pathogenic pattern-associated molecular patterns (PAMPs), Start the messaging mechanism. The Toll-like receptor (TLR) family is the most representative PRRs. In 1996, Hoffmann's research team discovered Toll receptors as pattern recognition receptors in Drosophila. One year later, a Toll homolog (namely Toll-like receptor4, TLR4) that can stimulate acquired immunity was found from the human body. In the 1990s, it was discovered that mouse TLR4 was the receptor for recognizing lipopolysaccharide. Since then, many mammalian TLRs receptors have been discovered one after another, and they respectively recognize specific PAMPs. The discovery of TLRs made people realize that the innate immune response is not only the first line of defense against microorganisms, but also a key step in stimulating the acquired immune response. Through the interaction between pathogenic bacteria and animal host humoral proteins, and then eluting the host proteins adhered to the surface of pathogenic bacteria, it is expected to obtain some proteins involved in the recognition of invading pathogenic bacteria in the host humoral defense system, and may even find some new PRRs to further elucidate the host Messaging mechanism that initiates the immune response. Zhu et al. (Zhu Y, Thangamani S, Ho B and Ding JL. The ancient origin of the complement system. The EMBO Journal. 2005, 24: 382-394.) from the National University of Singapore used live staphylococcus (Staphylococcus aureus) to adsorb the host Limulus serum, from which CrC3, which interacts with pathogenic bacteria and has a similar function to vertebrate complement C3, was isolated. However, many bands from pathogenic bacteria appeared in the negative control group of Staphylococcus, which were difficult to distinguish from proteins from the host. , which brought a lot of inconvenience to subsequent research, especially the inability to identify low-abundance proteins.

Contents of the invention

The purpose of the present invention is to provide a method for fast and reliable detection and identification of host proteins interacting with pathogenic bacteria for the protein in the host that interacts with pathogenic bacteria.

For this reason, the technical solution adopted in the present invention is to use inactivated pathogenic bacteria to interact with host proteins in vitro, and the steps are:

1) Utilize formaldehyde to inactivate pathogenic bacteria;

2) Use the eluent to remove the components that are easy to fall off on the outer membrane of the pathogenic bacteria;

3) Incubate the pathogenic bacteria with the host protein, so that the protein in the host protein that can interact with the pathogenic bacteria adheres to the surface of the pathogenic bacteria;

4) Use the eluate to collect the interacting host proteins.

The inactivation of pathogenic bacteria in step 1) is to resuspend the pathogenic bacteria washed at least once with physiological saline with 0.5%-1% formaldehyde solution; inactivate at 70-90° C. for 30-90 minutes.

In step 2), the components that are easy to fall off on the outer membrane of pathogenic bacteria are removed by centrifuging at 4000～15000g for at least 2min to remove the formaldehyde solution; Resuspend the cells in 0.01M Tris-HCl solution (preferably 8.0), shake for 60-120min (preferably 90min), centrifuge at 4000-15000g for at least 2min, remove the supernatant, repeat at least once; finally use normal saline Wash at least once, centrifuge for at least 2min, and remove the supernatant.

In step 3), the incubation of pathogenic bacteria and host protein is to mix the host protein and pathogenic bacteria, shake for 30-120min (preferably 90min), centrifuge at 4000-15000g for at least 2min, remove the supernatant; finally wash with normal saline At least once, centrifuge at 4000-15000g for at least 2min, and remove the supernatant.

In step 4), the collection of interacting host proteins with the eluent is to resuspend the bacteria with 4-5M urea Tris-HCl solution (4M urea is preferred), and centrifuge at 4000-15000g for at least 2min to obtain The bacterial cells were set aside; add 3-5 times the volume of acetone to the supernatant, place it at a temperature lower than -20°C for at least 30 minutes, centrifuge at 10000-30000g at a temperature of 3-6°C for at least 10 minutes, dry the precipitate, and use Resuspend in 0.01M Tris-HCl solution or 0.01M PBS solution with a pH of 7.4-8.5 (preferably pH 8.0), determine the protein concentration by Bradford assay, then further analyze by SDS-PAGE, and finally identify by mass spectrometry.

In step 4), the obtained cells are washed at least once with normal saline, and can be re-mixed with the host protein supernatant in step 3), and then the desired protein can be eluted according to the above steps 3) and 4). . The bacterium and host protein supernatant can be reused at least 3 times.

Compared with the existing method of capturing live bacteria, the present invention uses inactivated pathogenic bacteria to capture interacting proteins in the host, and uses inactivated pathogenic bacteria to adsorb host proteins, avoiding the interaction of host proteins due to live bacteria. The problem of contamination of the outer membrane or secreted protein of pathogenic bacteria. Therefore, the outstanding advantage of the present invention is that the obtained proteins are all derived from the host, without pathogenic bacteria pollutants, and it is an efficient and reliable method for detecting the composition of proteins in the host participating in the first line of defense in the defense system against pathogenic bacteria.

Description of drawings

Figure 1 is the SDS/PAGE analysis of amphioxus humoral proteins interacting with Escherichia coli K12. In Fig. 1, 1. Negative control of live pathogenic bacteria (inactivated staphylococcus was incubated with normal saline); 2. Host protein interacting with live pathogenic bacteria (inactivated staphylococcus was incubated with amphioxus body fluid protein).

Fig. 2 is the SDS/PAGE analysis of amphioxus body fluid proteins interacting with Staphylococcus aureus in the present invention. In Fig. 2, 1. is the host protein that interacts with pathogenic bacteria (inactivated K12 is incubated with amphioxus body fluid); 2. is the negative control of pathogenic bacteria (inactivated K12 is incubated with normal saline).

Fig. 3 is the SDS/PAGE analysis of the amphioxus humoral protein interacting with Aeropseudomonas hydrophila according to the present invention. In Fig. 3, 1. is the host protein that interacts with pathogenic bacteria (inactivated Staphylococcus aureus is incubated with amphioxus body fluid); 2. is the negative control of pathogenic bacteria (inactivated Staphylococcus aureus is only incubated with normal saline).

Fig. 4 is the SDS/PAGE analysis of Drosophila humoral proteins interacting with Escherichia coli K12 of the present invention. In Figure 4, 1. is the host protein that interacts with pathogenic bacteria (inactivated Escherichia coli K12 was incubated with Drosophila body fluid); 2. is the negative control of pathogenic bacteria (inactivated Escherichia coli K12 was only incubated with normal saline).

Fig. 5 is the SDS/PAGE analysis of the body fluid protein of mice interacting with Staphylococcus aureus of the present invention. In Figure 5, 1. is the host protein that interacts with pathogenic bacteria (inactivated Staphylococcus aureus was incubated with mouse serum); 2. is the negative control of pathogenic bacteria (inactivated Staphylococcus aureus was only incubated with normal saline).

Detailed ways

Example 1

1. Inactivation of pathogenic bacteria:

The Escherichia coli K12 (Escherichia coli K12) used in the experiment is the strain preserved in the laboratory. Pick a single colony of the strain, inoculate it in 500ml culture medium at 37°C, 200rpm, shake the bacteria for 12h, centrifuge at room temperature at 5000g for 10min, remove the supernatant; wash the bacteria three times with normal saline, centrifuge at room temperature at 5000g for 10min; Bacteria were resuspended with 1% formaldehyde solution; 80. ℃ inactivation for 90min.

2. Remove the components that are easy to fall off on the surface of pathogenic bacteria:

Centrifuge at 12000g at room temperature for 5min to remove the formaldehyde solution; then resuspend the cells with 0.01M Tris-HCl (pH8.0) solution containing 4.5M urea, shake gently for 90min, centrifuge at 12000g at room temperature for 5min, remove the supernatant, repeat 3 times ;Finally washed 3 times with 8.5% physiological saline (12000g, 5min centrifugation, room temperature to remove the supernatant).

3. Pathogen and host protein incubation:

Divide the pathogenic bacteria into two groups, one group is mixed with the amphioxus body fluid, and the other group is used as the control group and mixed with an equal volume of normal saline; both are placed at room temperature and shaken gently for 60 minutes; centrifuged at 12000g for 5 minutes at 4°C; Temporarily stored at ℃; the last two groups were washed 3 times with 8.5% normal saline.

4. Protein elution

Resuspend the bacteria in 0.01M Tris-HCl (pH 8.0) solution containing 4M urea, shake gently at room temperature for 60min, centrifuge at 12000g, 4°C, 5min respectively, and collect the bacteria at 4°C respectively; add 4 Double the volume of acetone, place at -20°C overnight, centrifuge at 12000g, 4°C for 20min, air-dry the precipitate, resuspend with 0.01M Tris-HCl (pH 8.0) solution, and then further analyze the results by SDS-PAGE as shown in Figure 1 , and finally identified by mass spectrometry, it was found that protein No. 1 was histone h4, and the protein score was significant. The test results are shown in Figure 1.

Example 2

1. Inactivation of pathogenic bacteria:

Staphylococcus aureaus was used in the experiment as a strain preserved in our laboratory. Pick a single colony of the strain, inoculate it in 500ml culture medium at 37°C, 200rpm, shake the bacteria for 12h, centrifuge at room temperature at 5000g for 10min, remove the supernatant; wash the bacteria three times with normal saline, centrifuge at room temperature at 5000g for 10min; Bacteria were resuspended with 1% formaldehyde solution; inactivated at 70°C for 90 minutes.

2. Remove the components that are easy to fall off on the surface of pathogenic bacteria:

Centrifuge at 12000g at room temperature for 5min to remove the formaldehyde solution; then resuspend the cells with 0.01M Tris-HCl (pH8.0) solution containing 4.5M urea, shake gently for 90min, centrifuge at 12000g at room temperature for 5min, remove the supernatant, repeat 3 times ;Finally washed 3 times with 8.5% physiological saline (12000g, 5min centrifugation, room temperature to remove the supernatant).

3. Pathogen and host protein incubation:

Divide the pathogenic bacteria into two groups, one group is mixed with the amphioxus body fluid, and the other group is used as the control group and mixed with an equal volume of normal saline; both are placed at room temperature and shaken gently for 60 minutes; centrifuged at 12000g for 5 minutes at 4°C; Temporarily stored at ℃; the last two groups were washed 3 times with 8.5% normal saline.

4. Protein elution

Resuspend the bacteria in 0.01M Tris-HCl (pH 8.0) solution containing 4M urea, shake gently at room temperature for 60min, centrifuge at 12000g, 4°C, 5min respectively, and collect the bacteria at 4°C respectively; add 4 Double the volume of acetone, place at -20°C overnight, centrifuge at 12000g, 4°C for 20min, air-dry the precipitate, resuspend with 0.01M Tris-HCl (pH 8.0) solution, and then further analyze the results by SDS-PAGE as shown in Figure 2 , and finally identified by mass spectrometry, it was found that protein No. 1 was pol protein, and the protein score was significant. The test results are shown in Figure 2.

Example 3

1. Inactivation of pathogenic bacteria:

The Aeromonas hydrophila used in the experiment is the strain preserved in our laboratory. Pick a single colony of the strain, inoculate it in 500ml culture medium at 37°C, 200rpm, shake the bacteria for 12h, centrifuge at room temperature at 5000g for 10min, remove the supernatant; wash the bacteria three times with normal saline, centrifuge at room temperature at 5000g for 10min; The bacteria were resuspended with 1% formaldehyde solution; inactivated at 80°C for 90min.

2. Remove the components that are easy to fall off on the surface of pathogenic bacteria:

Centrifuge at 12000g at room temperature for 5min to remove the formaldehyde solution; then resuspend the cells with 0.01M Tris-HCl (pH8.0) solution containing 4.5M urea, shake gently for 90min, centrifuge at 12000g at room temperature for 5min, remove the supernatant, repeat 3 times ;Finally washed 3 times with 8.5% physiological saline (12000g, 5min centrifugation, room temperature to remove the supernatant).

3. Pathogen and host protein incubation:

Divide the pathogenic bacteria into two groups, one group is mixed with the amphioxus body fluid, and the other group is used as the control group and mixed with an equal volume of normal saline; both are placed at room temperature and shaken gently for 60 minutes; centrifuged at 12000g for 5 minutes at 4°C; Temporarily stored at ℃; the last two groups were washed 3 times with 8.5% normal saline.

4. Protein elution

Resuspend the bacteria in 0.01M Tris-HCl (pH 8.0) solution containing 4M urea, shake gently at room temperature for 60min, centrifuge at 12000g, 4°C, 5min respectively, and collect the bacteria at 4°C respectively; add 4 Double the volume of acetone, place at -20°C overnight, centrifuge at 12000g, 4°C for 20min, air-dry the precipitate, resuspend with 0.01M Tris-HCl (pH 8.0) solution, and then further analyze the results by SDS-PAGE as shown in Figure 3 , and finally identified by mass spectrometry, it was found that protein No. 1 was Actin, muscle, and the protein score was significant. The test results are shown in Figure 3.

Example 4

1. The Escherichia coli K12 (Escherichia coli K12) used in the experiment is the strain preserved in this laboratory. Pick a single colony of the strain, inoculate it in 500ml culture medium at 37°C, 200rpm, shake the bacteria for 12h, centrifuge at room temperature at 5000g for 10min, remove the supernatant; wash the bacteria three times with normal saline, centrifuge at room temperature at 5000g for 10min; The bacteria were resuspended with 1% formaldehyde solution; inactivated at 80°C for 90min.

2. Remove the components that are easy to fall off on the surface of pathogenic bacteria:

Centrifuge at 12000g at room temperature for 5min to remove the formaldehyde solution; then resuspend the cells with 0.01M Tris-HCl (pH8.0) solution containing 4.5M urea, shake gently for 90min, centrifuge at 12000g at room temperature for 5min, remove the supernatant, repeat 3 times ;Finally washed 3 times with 8.5% physiological saline (12000g, 5min centrifugation, room temperature to remove the supernatant).

3. Pathogen and host protein incubation:

Divide the pathogenic bacteria into two groups, one group is mixed with Drosophila body fluid, and the other group is used as a control group and mixed with an equal volume of normal saline; both are placed at room temperature and gently shaken for 60 minutes; centrifuged at 12000g 4°C for 5 minutes; amphioxus serum 4 Temporarily stored at ℃; the last two groups were washed 3 times with 8.5% normal saline.

4. Protein elution

Resuspend the bacteria in 0.01M Tris-HCl (pH 8.0) solution containing 4M urea, shake gently at room temperature for 60min, centrifuge at 12000g, 4°C, 5min respectively, and collect the bacteria at 4°C respectively; add 4 Double the volume of acetone, place at -20°C overnight, centrifuge at 12000g, 4°C for 20min, air-dry the precipitate, resuspend with 0.01M Tris-HCl (pH 8.0) solution, and then further analyze the results by SDS-PAGE, see Figure 4 , and finally identified by mass spectrometry, it was found that protein No. 1 was glycogen phosphorylase; protein No. 2 was ATP synthase beta subunit; and the protein scores were all significant. The test results are shown in Figure 4.

Example 5

1. Inactivation of pathogenic bacteria:

Staphylococcus aureaus was used in the experiment as a strain preserved in our laboratory. Pick a single colony of the strain, inoculate it in 500ml culture medium at 37°C, 200rpm, shake the bacteria for 12h, centrifuge at room temperature at 5000g for 10min, remove the supernatant; wash the bacteria three times with normal saline, centrifuge at room temperature at 5000g for 10min; Bacteria were resuspended with 1% formaldehyde solution; inactivated at 70°C for 90 minutes.

2. Remove the components that are easy to fall off on the surface of pathogenic bacteria:

Centrifuge at 12000g at room temperature for 5min to remove the formaldehyde solution; then resuspend the cells with 0.01M Tris-HCl (pH8.0) solution containing 4.5M urea, shake gently for 90min, centrifuge at 12000g at room temperature for 5min, remove the supernatant, repeat 3 times ;Finally washed 3 times with 8.5% physiological saline (12000g, 5min centrifugation, room temperature to remove the supernatant).

3. Pathogen and host protein incubation:

Divide the pathogenic bacteria into two groups, one group is mixed with the mouse serum, and the other group is used as the control group and mixed with an equal volume of normal saline; both are placed at room temperature and gently shaken for 60 minutes; centrifuged at 12000g for 5 minutes at 4°C; the serum of the mice is 4 Temporarily stored at ℃; the last two groups were washed 3 times with 8.5% normal saline.

4. Protein elution

Resuspend the bacteria in 0.01M Tris-HCl (pH 8.0) solution containing 4M urea, shake gently for 60min, and centrifuge at 12000g, 4°C, 5min respectively, and the bacteria are collected at 4°C respectively; the supernatants are added 4 times volume of acetone, placed at -20°C overnight; 12000g, 4°C, centrifuged for 20min, the precipitate was air-dried, resuspended with 0.01M Tris-HCl (pH 8.0) solution, and then analyzed by SDS-PAGE. The results are shown in Figure 5, and finally carried out Mass spectrometry identified protein No. 1 as immunoglobulin heavy chain variable region, and the protein score was significant. The test results are shown in Figure 5.

Claims (9)

1. The method for quickly identifying the host protein interacting with pathogenic bacteria, characterized in that its steps are: 1) Utilize formaldehyde to inactivate pathogenic bacteria; 2) Use the eluent to remove the components that are easy to fall off on the outer membrane of the pathogenic bacteria; 3) Incubate the pathogenic bacteria with the host protein, so that the protein in the host protein that can interact with the pathogenic bacteria adheres to the surface of the pathogenic bacteria; 4) The interacting host proteins were collected with the eluate, and the protein concentration was determined by Bradford detection method, then further analyzed by SDS-PAGE, and finally identified by mass spectrometry. 2. The method for quickly identifying host proteins interacting with pathogenic bacteria as claimed in claim 1, characterized in that in step 1), the inactivated pathogenic bacteria is that the pathogenic bacteria washed at least once with normal saline are washed with 0.5 Resuspend in %-1% formaldehyde solution; inactivate at 70-90°C for 30-90 minutes. 3. The method for rapid identification of host proteins interacting with pathogenic bacteria as claimed in claim 1, characterized in that in step 2), the components that are easy to fall off on the outer membrane of pathogenic bacteria are removed by centrifugation at 4000～15000g for at least 2min, Remove the formaldehyde solution; then resuspend the bacteria with 0.01M Tris-HCl solution containing 4-5M urea and pH 7.4-8.5, shake for 60-120min, centrifuge at 4000-15000g for at least 2min, remove the supernatant, repeat at least once ;Finally wash at least once with normal saline, centrifuge for at least 2min, and remove the supernatant. 4. the rapid identification as claimed in claim 3 and the method for the host protein of pathogenic bacterium interaction, it is characterized in that after removing formaldehyde solution described in step 2), use the 0.01M Tris-HCl that contains 4.5M pH to be 8.0 Resuspend the bacteria in the solution and shake for 90 minutes. 5. The method for quickly identifying host proteins interacting with pathogenic bacteria as claimed in claim 1, characterized in that the incubation of pathogenic bacteria and host proteins described in step 3) is to mix the host protein and pathogenic bacteria and shake for 30-120min , centrifuge at 4000-15000g for at least 2min, remove the supernatant; finally wash with normal saline at least once, centrifuge at 4000-15000g for at least 2min, remove the supernatant. 6. The method for quickly identifying host proteins interacting with pathogenic bacteria as claimed in claim 5, characterized in that the shaking described in step 3) is 90min. 7. the method for rapid identification as claimed in claim 1 and the host protein that pathogenic bacterium interacts is characterized in that the host protein that collects interaction with eluent described in step 4) is to use the Tris of 4～5M urea - Resuspend the bacteria in HCl solution, centrifuge at 4000-15000g for at least 2min, and the obtained bacteria are ready for use; add 3-5 times the volume of acetone to the supernatant, and place it at a temperature lower than -20°C overnight. Centrifuge at 10000-30000g for at least 10min at ℃, dry the precipitate, and resuspend with 0.01M Tris-HCl solution or 0.01M PBS solution with pH 7.4-8.5. 8. the method for rapid identification as claimed in claim 7 and the host protein of pathogenic bacteria interaction, it is characterized in that in step 4) the concentration of the Tris-HCl solution described in is 4M urea; The pH of 0.01M Tris-HCl solution is 8.0. 9. The method for quickly identifying host proteins interacting with pathogenic bacteria as claimed in claim 1, characterized in that in step 4), the obtained thalline is washed at least once with normal saline, and then combined with the protein in step 3) The host protein supernatant was mixed again, and then the desired protein was eluted according to steps 3) and 4).

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