Outer membrane protein and mycobacterium detection method based on outer membrane protein

Abstract

The invention discloses an outer membrane protein and a mycobacterium detection method based on the outer membrane protein, the outer membrane protein Rv1411 is obtained by screening a mycobacterium tuberculosis secretory protein antibody library and identifying by combining an immunofluorescence method, and the amino acid sequence of the outer membrane protein Rv1411 is as shown in SEQ ID No.1; the outer membrane protein antibody can be used for specifically detecting mycobacterium bovis BCG and H37Ra, the detection ratio is as high as 99.2%, and the sensitivity of detecting the mycobacterium bovis BCG by using the outer membrane protein antibody, an indirect immunofluorescence method and a flow cytometry can respectively reach 5.5 * 10 < 3 > CFU / mL and 5.5 * 10 < 4 > CFU / mL. When the mycobacterium is detected through the outer membrane protein Rv1411, the detection process is simple to operate, short in time, high in sensitivity and low in cost, and the method can become a common, efficient and specific mode for detecting the mycobacterium bovis.

Description

Technical Field

[0001] The present invention belongs to the technical field of mycobacterium detection, and particularly relates to an outer membrane protein and a mycobacterium detection method based on the outer membrane protein. Background Art

[0002] At present, the clinical application of Mycobacterium tuberculosis antigen detection is still relatively rare, mainly due to the following reasons: the preparation of monoclonal antibodies for related antigens is relatively difficult, and traditional preparation methods are inefficient and have limited yields; there is no unified standard for the preparation method and detection process of antigen detection samples; current research on antigen detection only focuses on methodological quality and lacks reporting quality; therefore, there is still a large room for research on Mycobacterium tuberculosis antigen detection. Indirect immunofluorescence detection method is one of the commonly used methods in immunological detection and is widely used in the detection of other pathogens. However, the establishment of indirect immunofluorescence detection method for mycobacteria has been rarely reported and has great prospects; currently available detection technologies such as immunofluorescence microscopy based on fluorescent nanoparticle-labeled antibody-binding protein A (FNP-IIFM) have a detection limit of 3.6×10 5 CFU / mL.

[0003] Flow cytometry is also a promising technology that can quickly, automatically, and quantitatively collect cell data. It has become one of the best choices for rapid detection of bacteria in environmental, food, and clinical samples. However, the sensitivity of pathogen detection remains a challenge for flow cytometry. For example, existing technologies such as flow cytometry based on FDA staining for the detection of Mycobacterium tuberculosis have a detection limit of 10 7 CFU / mL. There is also a prior art method for determining the drug sensitivity of Mycobacterium tuberculosis based on fluorescein-labeled flow cytometry, in which the detection limit of Mycobacterium tuberculosis is 10 8 CFU / mL.

[0004] Detection methods for Mycobacterium tuberculosis mainly include bacteriological, immunological and molecular biological detection; among them, the bacteriological detection method has the highest credibility, but the positive detection rate is low, and it can lead to clinical misdiagnosis, so it needs to be assisted by other detection methods. Immunological detection is currently the most common detection method, but its specificity and sensitivity are not high. Molecular biological detection has the advantages of short time consumption, high sensitivity and strong specificity, but the cost of use is relatively high. In short, although there are many methods for detecting mycobacteria, there are still some shortcomings in terms of ease of operation, length of time, sensitivity and specificity. Therefore, it is necessary to develop more universal, faster, simpler, cheaper and more accurate detection technologies. Summary of the Invention

[0005] The present invention aims to provide an outer membrane protein and a method for detecting mycobacteria based on the outer membrane protein. A new outer membrane protein Rv1411 was identified by screening a library of antibodies against secretory proteins of Mycobacterium tuberculosis combined with immunofluorescence technology. The outer membrane protein antibody can be used to specifically detect Mycobacterium bovis BCG with a detection rate of up to 99.2%. Using the antibody against the outer membrane protein, the sensitivity of indirect immunofluorescence and flow cytometry for detecting Mycobacterium bovis BCG can reach 5.5×10 3 and 5.5×10 4 CFU / mL, the detection process of mycobacteria by outer membrane protein Rv1411 is simple to operate, short in time, highly sensitive and low in cost.

[0006] The present invention is achieved through the following technical solutions: An outer membrane protein Rv1411 is obtained by screening an antibody library of secretory proteins of Mycobacterium tuberculosis in combination with immunofluorescence identification. The amino acid sequence of the outer membrane protein Rv1411 is shown in SEQ ID No. 1.

[0007] A method for detecting mycobacteria by fluorescence based on outer membrane proteins comprises the following steps: S1: Culture and pretreatment of target mycobacteria; S2: Incubate with primary antibodies against outer membrane proteins; S3: perform secondary antibody incubation for outer membrane proteins; S4: Observe the bacterial solution.

[0008] Furthermore, the specific operation methods of culturing and pre-treating the target mycobacteria include: S11: Bacterial culture: culture the target mycobacteria to the logarithmic phase; S12: Collect the bacteria by centrifuging in an EP tube at 8000 rpm for 5 min to collect 1 mL of bacteria and discard the supernatant; S13: Resuspend the cells by adding 1 mL of PBST and centrifuging again to remove the supernatant. S14: Block, incubate with BSA solution at 35℃-38℃, centrifuge again to collect the bacteria and wash with PBST.

[0009] Furthermore, the specific operation method of incubating the primary antibody of the outer membrane protein is: adding the primary antibody of the outer membrane protein to the blocked bacteria to resuspend them, incubating them at 4°C for 12h-18h; and removing unbound primary antibody by multiple washing with PBST.

[0010] Furthermore, the specific operation method of incubating the outer membrane protein with the secondary antibody is as follows: adding the fluorescent secondary antibody to the bacteria and resuspending the bacteria, and incubating at 35°C-38°C for 1 hour; after washing with PBST, resuspending the bacteria in 500µL PBST.

[0011] Furthermore, the specific operation method of observing the bacterial solution is as follows: adjusting the bacterial solution after the immune reaction to a consistent OD value of 1.0, taking a portion of the bacterial solution and adding it to the ELISA plate, and measuring the relative fluorescence intensity using the fluorescence mode of the ELISA instrument; taking an appropriate amount of bacterial solution to smear the slide, and observing the fluorescence localization of the outer membrane protein on the bacterial surface under a fluorescence microscope.

[0012] A flow cytometry method for detecting mycobacteria based on outer membrane proteins comprises the following steps: D1: Obtain samples containing target bacteria with outer membrane proteins and filter them; D2: Module division and detection by flow cytometry; D3: After every 10 samples are tested, the rinsing solution and focusing solution are loaded; D4: After all samples are tested, the flow cytometer is loaded and cleaned.

[0013] Furthermore, the specific operation method of module division and detection by flow cytometry is: the sample is set to the total cell population, single cell population and fluorescence event histogram respectively. Figure 3 modules and pre-draw doors as needed.

[0014] Furthermore, the specific operation method of loading the rinsing fluid and focusing fluid after each detection of 10 samples is: loading 3 mL of rinsing fluid at a flow rate of 1 mL / min, loading twice; loading 3 mL of focusing fluid at a flow rate of 1 mL / min, loading once.

[0015] Furthermore, the specific operation method of loading and cleaning the flow cytometer is: loading 3 mL of rinsing solution at a flow rate of 1 mL / min, loading the sample twice; loading 3 mL of focusing solution at a flow rate of 1 mL / min, loading the sample twice.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: In the present invention, a new outer membrane protein Rv1411 was identified by screening a library of antibodies against secretory proteins of Mycobacterium tuberculosis combined with immunofluorescence technology. The outer membrane protein antibody can be used to specifically detect Mycobacterium bovis BCG and Mycobacterium tuberculosis, with a detection rate of up to 99.2%. Using the antibody against the outer membrane protein, the sensitivity of indirect immunofluorescence and flow cytometry for detecting Mycobacterium bovis BCG can reach 5.5×10 3 and 5.5×10 4CFU / mL. When detecting mycobacteria through the outer membrane protein Rv1411, the detection process is simple to operate, short in time, highly sensitive and low in cost, and can become a universal and efficient method for the specific detection of bovine Mycobacterium. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings in the embodiments will be briefly introduced below. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is the fluorescence microscopy analysis of the localization of protein Rv1411 in the present invention.

[0019] Figure 2 Flow cytometry combined with immunofluorescence technology was used to analyze the distribution of Rv1411 on the surface of BCG strains.

[0020] Figure 3 To analyze the sensitivity of indirect immunofluorescence assay for detecting mycobacteria based on the outer membrane protein Rv1411 antibody.

[0021] Figure 4 This is a specific analysis of mycobacteria detected by indirect immunofluorescence using antibodies against the outer membrane protein Rv1411.

[0022] Figure 5 Specificity analysis of mycobacteria detected by flow cytometry based on antibodies against the outer membrane protein Rv1411.

[0023] Figure 6 To analyze the sensitivity of flow cytometry for detection of Mycobacterium spp. BCG based on antibodies against the outer membrane protein Rv1411. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0025] Example 1: An outer membrane protein, Rv1411, was obtained by screening an antibody library of secretory proteins of Mycobacterium tuberculosis and combining it with immunofluorescence. The antibody against Rv1411 can be used to specifically detect Mycobacterium bovis BCG and Mycobacterium tuberculosis, with a detection rate of up to 99.2%. Using the antibody against Rv1411, the sensitivity of indirect immunofluorescence and flow cytometry for detecting Mycobacterium bovis BCG can reach 5.5×10 3 and 5.5×10 4 CFU / mL; the amino acid sequence of the outer membrane protein Rv1411 is shown in SEQ ID No.1.

[0026] Purification process of outer membrane protein Rv1411: 1. Amplify the target gene Rv1411 and enzymatically ligate it into the expression vector pET-28a, then transform it into the expression Escherichia coli BL21 to obtain the correct recombinant strain; 2. Activate the recombinant strain to 100 mL LB. When the OD600 of the bacterial solution reaches 0.6-0.8, add 1.2 mM IPTG (150 μl) and induce at 37°C for 6 h. 3. Bacteria collection: centrifuge at 8000 rpm for 3 min at room temperature; 4. Resuspend in 30 mL binding buffer and ultrasonically disrupt once (2 seconds of disruption and 4 seconds of rest, 99 cycles); 5. Centrifuge at 9000 rpm for 30 min at 4°C and discard the supernatant. 6. Resuspend the pellet in 10 mL of binding buffer (containing 8 M urea and 1 mM DTT), centrifuge, and retain the supernatant; 7. Add 2 mL of treated HIS beads to the supernatant and incubate for 15 minutes; 8. Centrifuge at 9000 rpm for 5 min at 4°C and remove the supernatant. 9. Elute the precipitate with 10 mL of 50 mM imidazole (containing 8 M urea and 1 mM DTT) in two fractions, 5 mL each time; 10. Centrifuge at 9000 rpm for 5 min at 4°C and remove the supernatant. 11. Elute the precipitate with 10 mL of 100 mM imidazole (containing 8 M urea and 1 mM DTT) in two steps, 5 m each time. 12. Centrifuge at 9000 rpm for 5 min at 4°C and discard the supernatant. 13. Elute the precipitate with 250 mM imidazole (containing 8 M urea and 1 mM DTT) in multiple steps until the Coomassie Brilliant Blue colorimetric solution turns blue. Elute 5 mL each time and retain the supernatant. 14. Prepare SDS-PAGE samples using the supernatant, perform gel tapping, and extract the outer membrane protein Rv1411 with PBS buffer; 15. The concentration and purity of outer membrane protein Rv1411 were analyzed by SDS-PAGE, and then mice were immunized to obtain antibodies.

[0027] HIS glue bead processing process: 1. Pour 30 mL of HIS beads into 50 mL of EP, centrifuge at room temperature, 4000 g, for 3 min, and remove the supernatant; 2. Add 20 mL of sterilized dH2O, mix thoroughly by inverting, centrifuge at room temperature, 4000 g, 3 min, and remove the supernatant; 3. Add 20 mL of NiSO4, mix thoroughly by inverting, centrifuge at room temperature, 4000 g, for 3 min, and remove the supernatant; 4. Add 20 mL of binding buffer, mix by inversion, centrifuge at room temperature, 4000 g, 3 min, and remove the supernatant; 5. Repeat step 4 and wash twice; 6. Add 20 mL of binding buffer and store at 4°C.

[0028] Purification buffer formulation (1 L) Binding Buffer: 2.42g Tris-Cl + 29.22g NaCl + 0.34g imidazole, add distilled water to 1L, and adjust the pH to 7.5 with concentrated hydrochloric acid.

[0029] Example 2: A method for detecting mycobacteria by fluorescence based on outer membrane proteins comprises the following steps: S1: Culture and pretreatment of target mycobacteria; S2: Incubate with primary antibodies against outer membrane proteins; S3: perform secondary antibody incubation for outer membrane proteins; S4: Observe the bacterial solution.

[0030] When detecting mycobacteria by fluorescence detection, first perform bacterial culture: culture the target mycobacteria in 7H9 medium to the logarithmic phase; centrifuge at 8000 rpm for 5 minutes to collect 1 mL of bacteria in an EP tube and discard the supernatant; add 1 mL of PBST to resuspend the bacteria, centrifuge again at 8000 rpm for 5 minutes, and discard the supernatant; then perform the blocking operation: incubate with 500 µL of 1% BSA solution at 37°C for 1 hour, collect the bacteria after centrifugation at 8000 rpm for 5 minutes, and wash once with PBST.

[0031] Incubation with primary antibodies for outer membrane proteins: Add 100 µL of primary antibody to the EP tube (i.e., OD450 = 1 at 1 µg / mL of coating antigen), resuspend the cells, and incubate at 4°C for 12 h. Wash away unbound primary antibody and wash three times with PBST.

[0032] For secondary antibody incubation of outer membrane proteins, add 100 µL of AlexaFluor®594 or AlexaFluor®488 fluorescent secondary antibody to the EP tube, dilute it 1:200 in PBS, and resuspend the cells. Incubate at 37°C for 1 h. Wash away unbound fluorescent secondary antibody, wash three times with PBST, and resuspend the cells in 500 µL of PBST.

[0033] Observe the bacterial solution: Adjust the bacterial solution after the immune reaction to a consistent OD value of 1.0. Take 200µL of the bacterial solution and add it to the ELISA plate. Measure the relative fluorescence intensity using the fluorescence mode of the microplate reader. Take an appropriate amount to apply to the slide, cover it with a coverslip, and observe the fluorescence localization on the bacterial surface under a fluorescence microscope.

[0034] Screening of the Mycobacterium tuberculosis secretory protein antibody library revealed that the outer membrane protein Rv1411 can bind to the outer membrane of Mycobacterium bovis BCG, thereby achieving effective labeling. Furthermore, the polyclonal antibody was used to establish an indirect immunofluorescence detection method and a flow cytometry detection method for Mycobacterium bovis, with detection limits of 5.5×10 3 CFU / mL and 5.5×10 4 CFU / mL, and also has good specificity.

[0035] like Figure 1 As shown, the relative position of the fluorescent secondary antibody (red) and the bacteria after indirect immunofluorescence reaction between the outer membrane protein Rv1411 antibody and the attenuated Mycobacterium tuberculosis strain H37Ra was observed under a fluorescence microscope. The marking line is 20μm; the serum of non-immunized mice was used as a negative control (Control). Figure 1 It can be seen that the protein Rv1411 antibody bound to the fluorescent secondary antibody can be evenly distributed on the surface of the H37Ra strain, while the control serum cannot, indicating that the protein Rv1411 is the outer membrane protein of the H37Ra strain.

[0036] like Figure 2 As shown, the R2 region represents the proportion of bacterial particles carrying fluorescent groups, and the serum of non-immunized mice was used as a negative control (Control). Figure 2 It can be seen that 99.23% of the H37Ra strain surface can be incubated with the fluorescent secondary antibody labeled with the protein Rv1411 antibody, while the non-immune mouse serum cannot, indicating that the protein Rv1411 is located on the surface of the H37Ra strain.

[0037] Example 3: A flow cytometry method for detecting mycobacteria based on outer membrane proteins comprises the following steps: D1: Obtain samples containing target bacteria with outer membrane proteins and filter them; D2: Module division and detection by flow cytometry; D3: After every 10 samples are tested, the rinsing solution and focusing solution are loaded; D4: After all samples are tested, the flow cytometer is loaded and cleaned.

[0038] When testing mycobacteria by flow cytometry, the target strain (Escherichia coli, Mycobacterium bovis, or Mycobacterium smegmatis) is first cultured to the logarithmic phase. An indirect immunofluorescence assay is then performed on the target strain using the corresponding antibody, and the fluorescence intensity signal is recorded by flow cytometry. The sample to be tested is filtered through a 40µm cell sieve and tested on the flow cytometer within 10 minutes. A pre-test is performed using a negative sample. Three modules are set for the total cell population, single cell population, and fluorescence event histogram, respectively. The voltage is adjusted to center the event signal in the image. The appropriate fluorescence channel and fluorescence intensity are set, and then pre-gating is performed as needed. The samples to be tested are injected sequentially for testing at a flow rate of ≤100µL / min, and a set of data is recorded for each sample. After every 10 samples were tested, the flow cytometer was loaded with rinsing solution and focusing solution: 3 mL of rinsing solution was loaded twice at a flow rate of 1 mL / min; 3 mL of focusing solution was loaded once at a flow rate of 1 mL / min.

[0039] After all samples have been tested, the flow cytometer is cleaned by loading 3 mL of rinse solution at a flow rate of 1 mL / min twice; loading 3 mL of focusing solution at a flow rate of 1 mL / min twice; finally, run a Sanitize run to remove sample residues to ensure the accuracy of the next test and extend the life of the instrument. After the run is completed, the sample stage is lowered and the test is complete.

[0040] like Figure 3 As shown in the figure, the fluorescent secondary antibody could not label the BCG strain incubated with non-immune mouse serum, but could label the BCG strain incubated with protein Rv1411 antibody (concentration as low as 5.5×103 CFU / mL) under the same experimental conditions, indicating that the indirect immunofluorescence method based on outer membrane protein Rv1411 antibody has high sensitivity for detecting mycobacteria.

[0041] like Figure 4Figure 2 shows the relative fluorescence intensity values ​​detected after immunofluorescence assays with different concentrations of BCG strains. Error bars represent the error range of three biological replicates. (A) Comparison of relative fluorescence intensity values ​​after immunofluorescence assays of BCG (Mycobacterium bovis), Msm (Mycobacterium smegmatis), and DH5α (Escherichia coli). Error bars represent the error range of three biological replicates. (B) 5.5×10 3 Indirect immunofluorescence detection micrograph of BCG strain with CFU / mL. Green fluorescent secondary antibody was used, and the bar is 20 μm. Fluorescence micrograph of immunofluorescence reaction of BCG, Msm and DH5α strains, using green fluorescent secondary antibody, and the bar is 20 μm. Figure 4 It can be seen that indirect immunofluorescence based on the outer membrane protein Rv1411 antibody can identify BCG strains, but not Msm and DH5α strains, indicating that this method can specifically detect BCG strains.

[0042] like Figure 5 and Figure 6 As shown, fluorescence event analysis after flow cytometric detection of BCG, Msm, and DH5α strains was performed. The bacterial concentration was 5.5×108 CFU / mL. Fluorescence event analysis after flow cytometric detection of different BCG strains was performed. The negative control (control) had a BCG bacterial concentration of 5.5×106 CFU / mL. This indicates that this method can specifically detect some pathogenic mycobacteria (such as BCG) and has a high sensitivity, reaching a minimum of 5.5×104 CFU / mL.

[0043] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the scope of protection of the present invention.

Claims

1. An outer membrane protein, characterized in that The outer membrane protein Rv1411 was identified by screening the antibody library of secretory proteins of Mycobacterium tuberculosis in combination with immunofluorescence. The amino acid sequence of the outer membrane protein Rv1411 is shown in SEQ ID No.

1.

2. A method for detecting mycobacteria by fluorescence based on outer membrane proteins, characterized in that: The following steps are involved: S1: Culture and pretreatment of target mycobacteria; S2: Incubate with primary antibodies against outer membrane proteins; S3: perform secondary antibody incubation for outer membrane proteins; S4: Observe the bacterial solution.

3. The method for detecting mycobacteria based on outer membrane protein fluorescence according to claim 2, wherein: The specific operation method of culturing and pre-treating the target mycobacterium includes: S11: Bacterial culture: culture the target mycobacteria to the logarithmic phase; S12: Collect the bacteria by centrifuging in an EP tube at 8000 rpm for 5 min to collect 1 mL of bacteria and discard the supernatant; S13: Resuspend the cells by adding 1 mL of PBST and centrifuging again to remove the supernatant. S14: Block, incubate with BSA solution at 35℃-38℃, centrifuge again to collect the bacteria and wash with PBST.

4. The method for detecting mycobacteria based on outer membrane protein fluorescence according to claim 2, wherein: The specific operation method of incubating the primary antibody of the outer membrane protein is as follows: adding the primary antibody of the outer membrane protein to the blocked bacteria to resuspend them, incubating them at 4°C for 12h-18h; and removing the unbound primary antibody by washing with PBST multiple times.

5. The method for detecting mycobacteria based on outer membrane protein fluorescence according to claim 2, wherein: The specific operation method of the secondary antibody incubation for outer membrane protein is as follows: adding fluorescent secondary antibody to the bacteria and resuspending the bacteria, and incubating at 35°C-38°C for 1 hour; after washing with PBST, resuspending the bacteria in 500µL PBST.

6. The method for detecting mycobacteria based on outer membrane protein fluorescence according to claim 2, wherein: The specific operation method of observing the bacterial solution is as follows: adjusting the bacterial solution after the immune reaction to a consistent OD value of 1.0, taking a portion of the bacterial solution and adding it to an ELISA plate, and measuring the relative fluorescence intensity using the fluorescence mode of the ELISA instrument; taking an appropriate amount of the bacterial solution to apply to a slide, and observing the fluorescence localization of the outer membrane protein on the bacterial surface under a fluorescence microscope.

7. A flow cytometry method for detecting mycobacteria based on outer membrane proteins, characterized in that: The following steps are involved: D1: Obtain samples containing target bacteria with outer membrane proteins and filter them; D2: Module division and detection by flow cytometry; D3: After every 10 samples are tested, the rinsing solution and focusing solution are loaded; D4: After all samples are tested, the flow cytometer is loaded and cleaned.

8. The method for detecting mycobacteria by flow cytometry based on outer membrane proteins according to claim 7, wherein: The specific operation method of module division and detection by flow cytometry is: setting the sample into three modules of total cell population, single cell population and fluorescence event histogram, and performing pre-gate operation as needed.

9. The method for detecting mycobacteria by flow cytometry based on outer membrane proteins according to claim 7, wherein: The specific operation method of loading the rinsing solution and focusing solution after each detection of 10 samples is as follows: loading 3 mL of rinsing solution at a flow rate of 1 mL / min, loading twice; loading 3 mL of focusing solution at a flow rate of 1 mL / min, loading once.

10. The method for detecting mycobacteria by flow cytometry based on outer membrane proteins according to claim 7, wherein: The specific operation method of the flow cytometer sample loading and cleaning is: loading 3 mL of rinsing solution at a flow rate of 1 mL / min, loading the sample twice; loading 3 mL of focusing solution at a flow rate of 1 mL / min, loading the sample twice.

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