Pseudomonas plecoglossicida recombinant strain for expressing green fluorescence as well as construction method and application of pseudomonas plecoglossicida recombinant strain

Abstract

The invention discloses a pseudomonas plecoglossicida recombinant strain for expressing green fluorescence as well as a construction method and application of the pseudomonas plecoglossicida recombinant strain. The construction method comprises the following steps: constructing a pBBR1-GFP plasmid, and transforming the pBBR1-GFP plasmid into pseudomonas plecoglossa NB2011, so as to obtain a recombinant strain P. plecoglossa NB2011 / pBBR1-GFP capable of stably expressing green fluorescence. The recombinant strain shows green fluorescence when being observed under a fluorescence microscope, the plasmid stability rate can reach 100% after 30 generations of passage, the strain is constructed through an electrotransformation method, positive clones are screened through chloramphenicol, the strain has high hereditary stability, and through an infection experiment, the strain has good clinical application prospects. The fluorescent recombinant strain shows good visualization ability in mouse macrophages J774A.1 and juvenile zebrafish, and the interaction between the strain and host cells and tissues can be observed in real time. The invention provides a visual and stable fluorescence labeling tool, can be used for researching the pathogenic mechanism of pseudomonas plecoglossicida, and provides a new method and technical means for researching the interaction between pathogenic bacteria and hosts.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial monitoring, and in particular, to a recombinant strain of Pseudomonas plecoglossicida expressing green fluorescence, a construction method thereof, and an application thereof. Background Art

[0002] Large yellow croaker is an important economically valuable fish species cultured in cage culture along the eastern coast of China. During its cultivation process, it is harmed by Pseudomonas plecoglossicida, resulting in visceral white spot disease, leading to high mortality and serious economic losses. In the initial stage of fish body infection, there are no obvious external symptoms. In the later stage, white nodules of different sizes appear in internal organs such as the liver, kidney, and spleen. At this time, the fish is seriously ill and cannot feed, and it is often impossible to effectively control it by the method of drug feeding. Pseudomonas plecoglossicida belongs to Gram-negative bacilli, Pseudomonas genus, Pseudomonas putida group. After the bacteria infect the fish body and enter the macrophages in the tissue, different researchers have successively observed the phenomenon of intracellular survival and proliferation, indicating that the bacteria belong to facultative intracellular pathogens. Although there are currently some studies on the pathogenic mechanism of Pseudomonas plecoglossicida, these studies have not been fully carried out and there are still large technical gaps.

[0003] Currently, the research on Pseudomonas plecoglossicida mostly focuses on its pathogenic mechanism and prevention and control methods, but there is a lack of an effective tool that can monitor and visualize the interaction between pathogens and hosts in real time. The interaction between Pseudomonas plecoglossicida and the host, especially the dynamic changes of pathogens in host cells, has not been deeply studied. This is mainly due to the lack of means and technologies to observe the pathogen infection process in real time and intuitively.

[0004] As a commonly used biological labeling method, fluorescence labeling technology can help researchers observe the process of pathogen infection of the host by labeling pathogens and host cells. Green fluorescent protein (GFP), as an ideal reporter gene, has the advantages of being able to be expressed in living cells without exogenous substrates or cofactors, and its luminescence characteristics do not require additional radiation or chemicals, avoiding radioactive contamination and other potential hazards. Therefore, GFP can be widely used in in vivo tracking experiments and become an ideal tool for studying the interaction between microorganisms and hosts and the pathogenic infection mechanism.

[0005] However, although GFP labeling technology has been used in many studies of pathogenic bacteria, there is still a lack of a recombinant strain with green fluorescence labeling in the study of Pseudomonas plecoglossicida. The existing technology cannot provide a sufficiently intuitive means to deeply understand the infection process and pathological changes of Pseudomonas plecoglossicida in the host. In addition, there are certain limitations in the expression ability, stability, and visualization observation of the recombinant strains in the existing technology in host cells and animal models. Therefore, constructing a recombinant strain of Pseudomonas plecoglossicida that can stably express green fluorescence has become an urgent need to understand the infection mechanism of pathogenic bacteria and improve the efficiency of diagnosis, prevention, and control. Summary of the Invention

[0006] The first technical problem to be solved by the present invention is to provide a recombinant strain of Pseudomonas plecoglossicida expressing green fluorescence to solve the problem that the distribution and proliferation in host cells or animals cannot be monitored in real time and dynamically in the existing technology.

[0007] To overcome the defects of the above prior art, the present invention provides a recombinant strain of Pseudomonas plecoglossicida expressing green fluorescence. The recombinant strain is a positive clone obtained by transforming Pseudomonas plecoglossicida NB2011 with plasmid pBBR1-GFP, and the recombinant strain can exhibit green fluorescence under a fluorescence microscope. The sequence of the plasmid pBBR1-GFP is shown in SEQ ID NO: 2, and the green fluorescence fragment of the plasmid pBBR1-GFP is shown in SEQ ID NO: 1.

[0008] Compared with the prior art, a recombinant Pseudomonas plecoglossicida strain expressing green fluorescence of the present invention has the following advantages: enhanced visualization ability: by introducing the green fluorescent protein (GFP) gene into Pseudomonas plecoglossicida, the growth, colonization and proliferation processes of bacteria can be observed in real time under a fluorescence microscope, overcoming the problem in traditional methods that dynamic monitoring of bacteria in host cells or animals cannot be achieved; high stability: by using the plasmid pBBR1-GFP, the constructed recombinant strain exhibits high fluorescence stability, and the plasmid remains stable during multiple generations of passage, ensuring the reliability of long-term tracking experiments. This solves the problem of unstable fluorescence labeling in the prior art and enables the sustainable application of this technology in long-term experiments; broad application prospects: this recombinant strain can not only be dynamically observed in a cell culture system, but also be used in animal models (such as zebrafish models), providing a more intuitive and reliable experimental platform for studying the interaction between bacteria and hosts, and having important application value; through the above technical advantages, the recombinant strain of the present invention also includes the following further advancements: enhanced stability of fluorescence labeling: in the prior art, fluorescence labeling methods often have the problem of unstable labeling intensity, while the pBBR1-GFP plasmid adopted in the present invention can ensure the stable expression of green fluorescent protein in Pseudomonas plecoglossicida, making it possible to perform real-time tracking of bacteria in cells and animals. Improved monitoring accuracy and reliability: by introducing green fluorescence labeling, the distribution of bacteria in the host can be monitored more accurately, avoiding the problem in traditional methods that only external symptoms can be observed; providing an effective tool: the recombinant Pseudomonas plecoglossicida strain expressing green fluorescence provided by the present invention provides an intuitive and efficient experimental tool for studying the pathogenic mechanism of bacteria, host immune response and the interaction between bacteria and hosts.

[0009] The second technical problem to be solved by the present invention is to provide a method for constructing a recombinant Pseudomonas plecoglossicida strain expressing green fluorescence, so as to solve the problems of low efficiency, difficulty in stably labeling Pseudomonas plecoglossicida, and difficulty in long-term observing the distribution and proliferation of bacteria in the host in the prior art.

[0010] To overcome the above defects of the prior art, the present invention provides a method for constructing the recombinant Pseudomonas plecoglossicida strain expressing green fluorescence, comprising the following steps: S1: Extraction of plasmid pBBR1-GFP; S2: Preparation of competent cells of Pseudomonas plecoglossicida; S3: Constructing a recombinant Pseudomonas plecoglossicida strain expressing green fluorescence by electroporation; S4: Screening of positive clones to obtain a recombinant Pseudomonas plecoglossicida strain expressing green fluorescence.

[0011] Compared with the prior art, the method for constructing a recombinant Pseudomonas plecoglossicida strain expressing green fluorescence of the present invention has the following advantages: improving transformation efficiency: the bacterial transformation methods in the prior art usually have low efficiency and it is difficult to ensure the stable expression of green fluorescence in the transformed strain. By using the electroporation method in the present invention, the transformation efficiency of Pseudomonas plecoglossicida is successfully improved, enabling the green fluorescent protein (GFP) to be highly and stably expressed in bacteria, thus solving the problems of low transformation efficiency and poor stability in the existing methods; fluorescence stability and long-term observation: traditional labeling methods often cannot ensure the long-term stable expression of the labeled gene, resulting in the weakening or disappearance of the fluorescence signal during long-term observation and unable to achieve continuous tracking research. By introducing the plasmid pBBR1-GFP, the recombinant strain of the present invention can stably express the green fluorescent protein, and even after multiple generations of passage, the expression of green fluorescence can be maintained stable, solving the problem of difficult long-term dynamic observation in the prior art; simple and efficient construction process: compared with the traditional method, the construction method of the present invention is not only easy to operate, but also has a high success rate. By the optimized electroporation method and screening process, the construction time is greatly shortened, and the experimental error caused by unstable labeling or transformation failure is reduced, making this method have wide applicability, especially suitable for large-scale experiments.

[0012] In a possible implementation manner, the step S1 includes: extracting the plasmid pBBR1-GFP by a plasmid extraction kit, and measuring the plasmid concentration after extraction to make the plasmid concentration reach 5 pg - 25 ng / μL.

[0013] Compared with the prior art, adopting the above technical solution can obtain a plasmid with high concentration and high purity, and the plasmid concentration is controlled within the range of 5 pg - 25 ng / μL, ensuring the effectiveness and transformation efficiency of the plasmid. In the subsequent electrotransformation process, the high-concentration plasmid can improve the success rate of transformation, reduce unnecessary experimental errors, ensure the stable expression of the green fluorescence gene in the target bacteria, ensure the technical effect of labeling stability, and contribute to the subsequent labeling and tracking experiments of bacteria.

[0014] In a possible implementation manner, the step S2 includes: S21: Culturing Pseudomonas plecoglossicida by shaking in an LB culture medium, measuring the OD 600 value of the culture medium, and when the OD 600 value is 0.6, ice-bathing the bacterial solution for 15 - 30 min and shaking it to ensure that the bacterial solution is fully cooled; S22: Centrifuging the bacterial solution obtained in the step S21, then recovering the cells, resuspending the cells with pure water and centrifuging and washing again to wash away the electrolyte impurities therein, and gradually resuspending and washing with glycerol, and finally resuspending the precipitate with glycerol to obtain a suspension; S23: Measure the OD value after diluting the suspension obtained in step S22, and dilute it with 10% glycerol to a concentration of 2×10 600 cells / ml, aliquot and store. 10

[0015] Compared with the prior art, adopting the above technical solution, using LB culture medium to culture Pseudomonas plecoglossicida, and performing ice bath treatment when the culture reaches an OD600 value of 0.6, which helps to cool the cells and effectively form competent cells; and further through the centrifugation and glycerol resuspension steps, removing the electrolyte impurities therein, and using glycerol as a cryoprotectant to ensure that the cells will not be damaged during cryopreservation. This method effectively improves the permeability of the cell membrane, enabling it to effectively accept the transfer of exogenous plasmids.

[0016] In a possible implementation manner, in the step S21, the conditions for shaking culture on a shaker are: 28°C, 180 rpm; in the step S22, the conditions for centrifugation are 4°C, 4000g, and the time is 10 min; in the step S23, the conditions for aliquoting and storing are: aliquoting in 100 μL / 1.5 mL EP tubes and storing at -80°C.

[0017] Compared with the prior art, adopting the above technical solution, by further optimizing the culture and centrifugation conditions, the cells are collected at the optimal growth stage, thereby obtaining high-quality competent cells. Among them, 28°C is the optimal growth temperature of Pseudomonas plecoglossicida, and the oscillation speed of 180 rpm helps to provide sufficient oxygen supply to ensure that the bacteria are in the logarithmic growth phase and the cell number reaches the most suitable level for transformation; while the low temperature of 4°C can effectively inhibit cell metabolic activities and reduce the influence of temperature on cell damage. By high-speed centrifugation (4000g), the cells can be quickly sedimented and the impurities in the culture medium can be removed; the cell suspension is aliquoted and stored at -80°C, with 100 μL / 1.5 mL EP tubes as the unit for aliquoting. Glycerol as a cryoprotectant can prevent the cells from freezing and being damaged during freezing. With the above aliquoting method, the cells can be stored at -80°C for a long time and still maintain good transformation ability after resuscitation, greatly improving the activity and transformation efficiency of the cells during the storage period.

[0018] In a possible implementation manner, the step S3 includes: S31: Prepare competent cells: Transfer freshly prepared or freeze-thawed Pseudomonas plecoglossicida competent cells to a cold sterile centrifuge tube and cool them on ice together with the sample cell of the electroporator. S32: Adding plasmid: Add 10 pg - 25 ng of the plasmid to be transformed into the sterile centrifuge tube processed in step S32, keep it ice-cold, and let it stand for 30 - 60 seconds to obtain a mixture; S33: Electroporation operation: Apply an electric pulse to the mixture in step S32 for electroporation; S34: Reviving transformed cells: After the electroporation operation in step S33 is completed, take out the mixture, add 1 mL of LB liquid medium at room temperature, and shake and culture it at 28 °C and 180 rpm to revive the bacteria; S35: Screening for positive clones: Spread different volumes of the transformed bacteria obtained after step S34 onto an LB agar plate containing chloramphenicol, and screen for recombinant bacteria with green fluorescence after cultivation; S36: Clone identification: After 24 hours, pick single colonies for positive clone identification.

[0019] Compared with the prior art, by adopting the above technical solution, the transformation efficiency is improved and a stable recombinant strain of Pseudomonas plecoglossicida with a green fluorescent label is successfully constructed through multiple optimization steps: First, by using ice-cold conditions and the freeze-thaw method to prepare competent cells (step S31), the permeability of the cell membrane can be effectively improved, enabling the cells to be in the best state to accept the transformation of exogenous plasmids. Under low-temperature conditions, the metabolic activities of the cells are inhibited, and the cell membrane is more stable, facilitating the entry of plasmids into the cells; Then, plasmid DNA is added to the cooled cells (step S32) and kept at low temperature to avoid degradation or diffusion of the plasmid due to temperature rise during the transformation process. Adding an appropriate amount of plasmid (10 pg - 25 ng) ensures sufficient DNA amount during the transformation process, thereby increasing the success rate of transformation; Through the electroporation operation (step S33), an electric pulse is applied to temporarily open the cell membrane, allowing the plasmid to enter the cell. Compared with the traditional chemical transformation method, electroporation has a higher transformation efficiency, especially better results for bacteria with a thicker cell membrane or more difficult to transform. After electroporation, the cells are revived in the medium (step S34). This process enables the transformed plasmid to be expressed under mild culture conditions and at the same time ensures that the cells return to a state suitable for proliferation. It not only helps the bacteria to revive but also enables subsequent screening; During the screening process, the transformed bacteria are spread onto an LB agar plate containing chloramphenicol (step S35). Only the bacteria that have successfully transformed the plasmid can grow in an environment containing chloramphenicol. This screening process further confirms positive clones through green fluorescent labeling; Finally, by picking single colonies for clone identification (step S36), it is ensured that each positive clone can stably express green fluorescent protein, thereby verifying the reliability and stability of the selected recombinant strain and ensuring the successful progress of the experiment.

[0020] In a possible implementation, in the step S33, the conditions of the electric pulse are as follows: the electroporator parameters are 25 uF, 25 kV, and 200 Ω, the electric field strength is 12.5 kV / cm, and the duration is 4 - 5 milliseconds; in the step S35, the conditions for culturing are: culturing at 28 °C for 12 - 16 hours.

[0021] Compared with the prior art, by adopting the above optimized technical solution, the transformation efficiency and the stability of strain screening are further improved. By precisely adjusting the electric field strength and duration during the electrotransformation process (25 uF, 25 kV, 200 Ω, and an electric field strength of 12.5 kV / cm, with a duration of 4 - 5 milliseconds), it is possible to more efficiently promote the entry of plasmids into cells, improve the transformation success rate, especially for bacteria that are difficult to transform. Through the optimized culture conditions (culturing at 28 °C for 12 - 16 hours), the recovery and proliferation of the transformed bacteria are effectively promoted, ensuring the screening effect of positive clones and making the stable expression of green fluorescence more reliable.

[0022] In a possible implementation, the step S4 includes: S41: Prepare an LB broth medium or an LB agar medium. After autoclaving and waiting for its temperature to drop to 50 - 55 °C, add chloramphenicol to make its final concentration 34 μg / mL; S42: Inoculate the transformed bacteria onto the LB liquid or agar medium containing chloramphenicol prepared in step S1, and screen out the positive clones that can tolerate chloramphenicol, or smear the cultured bacteria on a glass slide and observe under a fluorescence inverted microscope. If green fluorescence can be seen, it is a positive clone.

[0023] Compared with the prior art, by adopting the above technical solution, chloramphenicol as a screening antibiotic can effectively screen out the bacteria that have been successfully transformed and carry plasmids. By adding chloramphenicol to the medium, only the bacteria that have successfully transformed the plasmid can grow in an environment containing chloramphenicol, and other untransformed bacteria will be inhibited from growing, thereby achieving the purpose of screening positive clones. In this embodiment, by optimizing the combination of chloramphenicol screening and green fluorescence labeling screening, the screening accuracy and efficiency of positive clones are significantly improved, and it can be ensured that the screened recombinant strains can not only tolerate antibiotics but also stably express green fluorescent protein, thus providing reliable experimental strains for subsequent dynamic tracking of bacteria and host - pathogen research. This not only improves the success rate of the experiment but also ensures that the finally screened bacteria have stable labeling characteristics.

[0024] The third technical problem to be solved by the present invention is to provide an application of a Pseudomonas plecoglossicida recombinant strain expressing green fluorescence to solve the problem in the prior art that the distribution, proliferation, and infection process of bacteria in the host cannot be monitored in real - time and dynamically.

[0025] To overcome the defects of the above prior art, the present invention provides an application of the recombinant Pseudomonas plecoglossicida strain expressing green fluorescence, and the application includes the application of the recombinant strain in a cell infection model and an animal infection model, and the distribution, proliferation and infection process of bacteria in the host are monitored through the green fluorescence labeling of the recombinant strain.

[0026] Compared with the prior art, the application of the recombinant Pseudomonas plecoglossicida strain expressing green fluorescence of the present invention has the following advantages: by introducing the green fluorescent protein (GFP) gene into Pseudomonas plecoglossicida, the present invention endows the bacteria with the characteristic of spontaneous luminescence. Through a fluorescence microscope, the growth, colonization and proliferation process of bacteria in host cells and animals can be observed in real time and dynamically. Without the need for exogenous substrates, it can emit light spontaneously in living cells, thus providing a very simple, intuitive and non-toxic labeling method for bacterial tracking research. In the prior art, it is often difficult to observe the dynamic process of bacteria in host cells or animals in real time. However, in the application process of the present invention, by using the recombinant Pseudomonas plecoglossicida strain labeled with green fluorescence, the distribution, proliferation and infection process of bacteria can be tracked in real time in both cell infection models and animal infection models, overcoming the problem of the lack of dynamic observation tools in the prior art; traditional infection monitoring methods usually rely on external symptoms or static observations and cannot directly observe the internal behavior of bacteria. However, the green fluorescence labeling of the present invention makes the bacteria clearly visible under a microscope and enables them to be observed and quantitatively analyzed at different time points and different locations, greatly improving the efficiency and accuracy of experimental monitoring; moreover, through the application of the green fluorescence-labeled strain constructed by the present invention, the stability of the fluorescence labeling is ensured, making long-term observation possible. Through this labeling system, the growth and spread of bacteria can be continuously monitored throughout the infection process, providing important support for studying host-pathogen interactions, pathogenic mechanisms and immune responses.

[0027] In a possible implementation manner, the application of the recombinant strain in a cell infection model includes: A1: Inoculate the recombinant strain into an LB medium containing chloramphenicol and culture it at 28 °C and 180 rpm / min until the logarithmic growth phase; A2: Infect macrophage J774A.1 with the cultured recombinant bacterial suspension, incubate and observe the distribution and proliferation of bacteria in the cells; A3: Observe the green fluorescence-labeled bacteria at different time points through a fluorescence microscope and analyze their colonization and proliferation processes in the cells.

[0028] The application of the recombinant strain in an animal infection model includes: B1: Inoculate the recombinant strain into an LB liquid medium containing chloramphenicol and culture it to obtain a bacterial suspension with the required concentration; B2: Expose zebrafish larvae to the recombinant strain and the control bacterial suspension respectively, incubate and monitor the infection process; B3: Observe the distribution and proliferation of fluorescent bacteria in zebrafish using a fluorescence microscope, and regularly record the changes in fluorescence intensity during the infection process.

[0029] Compared with the prior art, adopting the above technical solution, in the cell infection model, after infecting macrophage J774A.1 with the recombinant strain labeled with green fluorescence, the colonization and proliferation process of bacteria in host cells can be observed in real time using a fluorescence microscope. Through the expression of green fluorescent protein, the position and quantity of bacteria in cells can be visually displayed, enabling researchers to track the dynamic behavior of bacteria during the infection process and avoiding the problem of inability to dynamically monitor in traditional methods; in the animal infection model, exposing the recombinant strain labeled with green fluorescence to species such as zebrafish larvae can observe the distribution and proliferation of fluorescently labeled bacteria in fish in real time. By regularly recording the changes in fluorescence intensity, researchers can accurately monitor the progress of bacterial infection and host response, providing intuitive evidence for further research on immune response; the bacterial infection model provided by the application of the present invention can provide a real-time and dynamic observation tool for the study of the interaction between bacteria and host through green fluorescence labeling. Through the application in cell and animal infection models, the distribution, proliferation and infection process of bacteria in the host can be accurately tracked, which provides strong technical support for in-depth research on the pathogenic mechanism of pathogenic bacteria, host immune response and treatment strategies. Compared with the prior art, the application of the present invention has significant advantages in real-time monitoring of the dynamics of bacterial infection, improving data accuracy and experimental repeatability. Description of the Drawings

[0030] Figure 1 It is a schematic structural diagram of the fluorescence expression plasmid pBBR1-GFP in the present invention; the fluorescence plasmid map shows a length of 5836bp, and the green segment is the GFP fluorescence sequence of 720bp; Figure 2 It is a gel electrophoresis detection diagram of the fluorescence expression plasmid pBBR1-GFP in the present invention; among them, lanes 1 and 2: positive recombinant clones of pBBR1-GFP plasmid; M: DL5000 DNA Marker; Figure 3 It is Pseudomonas plecoglossicida expressing green fluorescent protein observed under the microscope in the present invention; Figure 4 It is an observation diagram of Pseudomonas plecoglossicida infecting cells under bright light field and green fluorescence light field under a fluorescence microscope in the present invention; among them, A is the observation of fluorescent bacteria infecting cells under bright light field, and B is the observation of fluorescent bacteria infecting cells under green fluorescence light field; Figure 5This is the observation diagram of Pseudomonas plecoglossicida infecting zebrafish larvae under bright light field and green fluorescence field in a fluorescence microscope in the present invention. Among them, A1, B1, and C1 are the observations of zebrafish larvae under bright light field at 1 day, 3 days, and 7 days after infection with fluorescent bacteria respectively; A2, B2, and C2 are the observations of zebrafish larvae under green fluorescence field at 1 day, 3 days, and 7 days after infection respectively. Detailed implementation manners

[0031] First of all, those skilled in the art should understand that these implementation manners are only used to explain the technical principles of the embodiments of the present invention, and are not intended to limit the protection scope of the embodiments of the present invention. Those skilled in the art can adjust them according to needs to adapt to specific application scenarios.

[0032] The present invention provides a recombinant strain of Pseudomonas plecoglossicida expressing green fluorescence. The recombinant strain is a positive clone obtained by transforming Pseudomonas plecoglossicida NB2011 with plasmid pBBR1-GFP, and this recombinant strain can exhibit green fluorescence under a fluorescence microscope. The sequence of the plasmid pBBR1-GFP is shown in SEQ ID NO:2, and the green fluorescence fragment of the plasmid pBBR1-GFP is shown in SEQ ID NO:1.

[0033] The GFP fluorescent protein gene (Green Fluorescent Protein) can autocatalyze the formation of a chromogenic structure and emit green fluorescence under blue light excitation. As a reporter gene, GFP is a luminescent protein that can be expressed in living cells without the need for exogenous substrates or cofactors. As a fluorescent labeling molecule, GFP not only has a sensitive labeling detection rate but also has no radioactive hazard. Therefore, it can realize the real-time detection of labeled strains in vivo and can be well used for the study of bacterial tracking. The present invention constructs a Pseudomonas plecoglossicida labeled with green fluorescence, which is convenient for observing the dynamic process of Pseudomonas plecoglossicida infecting the host through a fluorescence microscope in the future, laying a foundation for studying the interaction relationship between Pseudomonas plecoglossicida and the host.

[0034] The present invention also provides a construction method of the recombinant strain of Pseudomonas plecoglossicida expressing green fluorescence, including the following steps: S1: Extraction of plasmid pBBR1-GFP; S2: Preparation of competent cells of Pseudomonas plecoglossicida; S3: Construction of a recombinant strain of Pseudomonas plecoglossicida expressing green fluorescence by electroporation; S4: Screening of positive clones to obtain a recombinant strain of Pseudomonas plecoglossicida expressing green fluorescence.

[0035] The technical principle and technical effect of the present invention are as follows: Technical principle: In this invention, the plasmid pBBR1-GFP is used to introduce the green fluorescent protein gene into Pseudomonas plecoglossicida, and the transformation efficiency is improved by the electrotransformation method. This plasmid enables the real-time observation of bacteria in host cells or animal models by stably expressing green fluorescent protein in bacteria. This method utilizes the property of green fluorescent protein (GFP) - it can spontaneously emit green fluorescence under blue light excitation, thus providing a simple and safe means for bacterial tracking.

[0036] Through the method of this invention, the problems of low transformation efficiency, unstable labeling, and difficulty in long-term bacterial tracking in the prior art are solved. Since the green fluorescent protein gene of the recombinant strain is stably expressed in bacteria, it can be used for real-time visualization monitoring in cells and animal models, and has good stability and reliability. The strain obtained by the construction method of this invention can provide a more intuitive and efficient experimental tool for the study of bacterial infection, host immune response, and bacterial pathogenic mechanism; in the prior art, when studying the interaction between bacteria and the host, it usually relies on traditional staining methods or directly observing the growth of bacteria. These methods often have problems such as unstable labeling, short observation period, and difficulty in long-term tracking of bacterial growth. Through the green fluorescence expression method of this invention, the dynamic monitoring of bacteria in the host can be achieved, solving the problems of inability to achieve long-term tracking and dynamic observation of bacteria in the prior art, and further promoting the research on bacterial pathogenic mechanism and its interaction with the host.

[0037] As a preferred scheme, step S1 includes: extracting the plasmid pBBR1-GFP through a plasmid extraction kit, and measuring the plasmid concentration after extraction to make the plasmid concentration reach 5 pg - 25 ng / μL.

[0038] As a preferred scheme, step S2 includes: S21: Culturing Pseudomonas plecoglossicida by shaking in an LB culture medium, measuring the OD 600 value of the culture medium. When the OD 600 value is 0.6, ice-bath the bacterial solution for 15 - 30 min, and shake it to ensure that the bacterial solution is fully cooled; S22: Centrifuge the bacterial solution obtained in step S21, then recover the cells, resuspend the cells with pure water and centrifuge and wash again to wash away the electrolyte impurities, and gradually resuspend and wash with glycerol, and finally resuspend and precipitate with glycerol to obtain a suspension; S23: Dilute the suspension obtained in step S22 and then measure the OD 600 value, dilute it with 10% glycerol to a concentration of 2×10 10 cells / ml, aliquot and store.

[0039] As a preferred solution, in the step S21, the conditions for the shaker oscillation culture are: 28 °C, 180 rpm; in the step S22, the conditions for the centrifugation are 4 °C, 4000 g, and the time is 10 min; in the step S23, the conditions for the aliquoting and preservation are: aliquoting in 100 μL / 1.5 mL EP tubes and preserving at -80 °C.

[0040] As a preferred solution, the step S3 includes: S31: Prepare competent cells: Transfer freshly prepared or freeze-thawed Pseudomonas plecoglossicida competent cells to a cold sterile centrifuge tube and cool them on ice together with the sample chamber of the electroporator. S32: Add plasmid: Add 10 pg - 25 ng of the plasmid to be transformed to the sterile centrifuge tube processed in step S32, keep it cold, and let it stand for 30 - 60 seconds to obtain a mixture. S33: Electroporation operation: Apply an electric pulse to the mixture in step S32 for electroporation. S34: Resuscitate the transformed cells: After the electroporation operation in step S33 is completed, take out the mixture, add 1 mL of LB liquid medium at room temperature, and culture it with shaking at 28 °C, 180 rpm to resuscitate the bacteria. S35: Screen for positive clones: Spread different volumes of the transformed bacteria obtained after step S34 onto an LB agar plate containing chloramphenicol, and screen for recombinant bacteria with green fluorescence after cultivation. S36: Clone identification: After 24 hours, pick single colonies for positive clone identification.

[0041] As a preferred solution, in the step S33, the conditions for the electric pulse are: the electroporator parameters are 25 uF, 25 kV, 200 Ω, the electric field strength is 12.5 kV / cm, and it lasts for 4 - 5 milliseconds; in the step S35, the conditions for the cultivation are: cultivate at 28 °C for 12 - 16 hours.

[0042] As a preferred solution, the step S4 includes: S41: Prepare LB broth medium or LB agar medium, after autoclaving, when its temperature drops to 50 - 55 °C, add chloramphenicol to make its final concentration 34 μg / mL. S42: Inoculate the transformed bacteria onto the LB liquid or agar medium containing chloramphenicol prepared in step S1 to screen for positive clones that can tolerate chloramphenicol, or smear the cultured bacteria on a glass slide and observe under a fluorescence inverted microscope. If green fluorescence can be seen, it is a positive clone.

[0043] The present invention also provides an application of the recombinant Pseudomonas plecoglossicida strain expressing green fluorescence, and the application includes the application of the recombinant strain in a cell infection model and an animal infection model, and the distribution, proliferation and infection process of bacteria in the host are monitored through the green fluorescence labeling of the recombinant strain.

[0044] As a preferred solution, the application of the recombinant strain in the cell infection model includes: A1: Inoculate the recombinant strain into an LB medium containing chloramphenicol, and culture it at 28 °C and 180 rpm / min until the logarithmic growth phase; A2: Infect macrophage J774A.1 with the cultured recombinant bacterial suspension, incubate and observe the distribution and proliferation of bacteria in the cells; A3: Observe the bacteria labeled with green fluorescence at different time points through a fluorescence microscope, and analyze its colonization and proliferation process in the cells.

[0045] The application of the recombinant strain in the animal infection model includes: B1: Inoculate the recombinant strain into an LB liquid medium containing chloramphenicol and culture it to obtain a bacterial suspension with the required concentration; B2: Expose zebrafish larvae to the recombinant strain and the control bacterial suspension respectively, incubate and monitor the infection process; B3: Observe the distribution and proliferation of fluorescent bacteria in zebrafish using a fluorescence microscope, and regularly record the changes in fluorescence intensity during the infection process.

[0046] The following provides specific examples that combine the above technical solutions and specific operation methods and application methods to further expand the above technical solutions of the present invention: Example 1: This example provides a method for constructing a recombinant Pseudomonas plecoglossicida strain expressing green fluorescence, and the construction method of the recombinant strain is as follows: S1: Extraction of plasmid pBBR1-GFP Use a plasmid extraction kit to extract plasmid pBBR1-GFP. After extraction, verify whether the extracted plasmid is the target plasmid through agarose gel electrophoresis. Use the Plasmid Mini Kit kit provided by OMEGA company to extract the plasmid, and use a 5000bp DNA molecular weight marker for determination. The plasmid band should appear above 5000bp, as Figure 1 shown, Figure 1 is the structural schematic diagram of the fluorescence expression plasmid pBBR1-GFP in the embodiment of the present invention. The fluorescence plasmid map shows a length of 5836bp, and the green segment is the GFP fluorescence sequence of 720bp; Figure 2This is the gel electrophoresis detection diagram of the fluorescent expression plasmid pBBR1-GFP in the present invention; among them, lanes 1 and 2: positive recombinant clones of pBBR1-GFP plasmid; M: DL5000 DNA Marker; the length of this fluorescent plasmid is 5836 base pairs, which is consistent with the band shown in the gel electrophoresis detection diagram, proving that the extracted plasmid is the fluorescent expression plasmid pBBR1-GFP. The sequence of the plasmid pBBR1-GFP is shown in SEQ ID NO:2, and the green fluorescent fragment of the plasmid pBBR1-GFP is shown in SEQ ID NO:1; S2: Preparation of Pseudomonas plecoglossicida competent cells Use LB culture medium to shake and culture Pseudomonas plecoglossicida in a shaker at 28 °C and 180 rpm. When the OD600 value of the culture medium reaches 0.6, quickly ice-bath the bacterial solution for 15 - 30 minutes to ensure that the bacterial solution is fully cooled. Then, centrifuge at 4 °C and 4000g for 10 minutes to recover the cells. Resuspend the cells with ice-cold pure water, centrifuge and wash again to remove electrolyte impurities. Finally, resuspend the cells with glycerol, precipitate and store the competent cells. After measuring the OD600 value of the diluted suspension, dilute it to 2×10 10 cells / ml with 10% glycerol, aliquot it into EP tubes, store at -80 °C, and prepare for electroporation transformation.

[0047] As Figure 3 shown, Figure 3 This is Pseudomonas plecoglossicida expressing green fluorescent protein observed under a microscope in the above-mentioned embodiment of the present invention; the wild strain of Pseudomonas plecoglossicida cannot be observed under the fluorescence field of a fluorescence microscope, while Pseudomonas plecoglossicida transfected with the fluorescent plasmid pBBR1-GFP can be clearly observed under a fluorescence microscope; S3: Construction of recombinant strains expressing green fluorescence by electroporation Add freshly prepared or freeze-thawed Pseudomonas plecoglossicida competent cells to an ice-cold sterile microcentrifuge tube, place the sample cell on ice for cooling, add 10 pg - 25 ng of pBBR1-GFP plasmid to each centrifuge tube, and let it stand on ice for 30 - 60 seconds. Set the electroporator parameters to 25 μF, 25 kV, and 200 Ω, and transform the cells by electric pulse. After transformation, add 1 mL of LB medium and resuscitate the cells by shaking at room temperature. Spread the transformed cells on LB agar medium containing chloramphenicol and place it in an incubator at 28 °C for 12 - 16 hours. Check whether the bacterial cells show green fluorescence under a fluorescence microscope to screen for positive clones.

[0048] S4: Screening of positive clones Use LB liquid or solid medium containing chloramphenicol to culture bacteria. Only bacteria containing plasmids can grow on chloramphenicol medium, and these bacteria are positive clones. Through observation with a fluorescence microscope, if the bacteria show green fluorescence under a green fluorescence light field, it is confirmed that they are positive clones, and the construction is completed.

[0049] Morphological identification, growth curve determination and genetic stability analysis of the recombinant bacteria expressing fluorescence: Activate the cryopreserved recombinant bacteria expressing fluorescence constructed above and subculture them for 3 - 4 generations in LB medium containing chloramphenicol, and at the same time subculture the original Pseudomonas piscicida in ordinary medium.

[0050] Spread the subcultured bacterial cells evenly on LB agar medium containing chloramphenicol at an appropriate concentration. Similarly, spread the original Pseudomonas piscicida on ordinary LB medium, and culture at 28 °C for 12 - 14 h to compare the morphological characteristics of the bacteria.

[0051] By comparing the colony morphology of the wild strain and the recombinant bacteria on the plate, it was observed that there was no difference in morphology between the wild strain and the recombinant bacteria; thus, it can be seen that the transfer of plasmid pBBR1 - GFP did not affect the colony morphology of Pseudomonas piscicida.

[0052] Dilute the constructed recombinant strain with sterile water and spread it on a glass slide, and observe and take pictures with a fluorescence microscope at a magnification of 40×10. Take out the cryopreserved Pseudomonas piscicida from the -80 °C refrigerator and activate it by the ordinary method, and subculture it for 3 - 4 generations. Chloramphenicol should be added throughout the whole process of culturing the recombinant bacteria, the same below. Use a 500 mL Erlenmeyer flask pre - added with 300 mL of LB medium, and add the bacterial liquid cultured in the previous step according to a ratio of 1:100, and culture it at 28 °C with shaking at 180 rpm. Take out the bacterial liquid every 1 h and measure the OD 600 value with a spectrophotometer to monitor the trend of the growth curve, and stop measuring until the late logarithmic phase.

[0053] Statistically organize the data results. Through analysis, the wild strain of Pseudomonas piscicida and the recombinant bacteria are basically the same in growth ability without difference; thus, it can be seen that the transfer of plasmid pBBR1 - GFP did not affect the growth ability of Pseudomonas piscicida.

[0054] Spread the recombinant strain on an LB agar plate added with chloramphenicol and culture it at 28 °C for 16 h. Subsequently, pick a single colony and inoculate it into LB medium containing chloramphenicol, and culture it overnight for 12 h to make the bacteria reach the middle and late logarithmic growth phase. Then, take an appropriate amount of the bacterial solution and perform three-zone subculture on LB solid plates with and without chloramphenicol. After culturing at 28 °C for 12 h, pick single colonies from the well-cultured plates respectively and repeat the above steps; Repeat the above operation 8 times (According to the colony counting method, Pseudomonas plecoglossicida can self-replicate 12 - 13 generations every 12 h of culture. It can be considered that after 24 h of subculture, bacteria that can self-replicate 25 generations can be obtained; In the experiment of plasmid genetic stability, the stability of the plasmid is detected when the bacteria self-replicate 100 generations or longer); The results of the stability experiment show that when the fluorescent recombinant bacteria are passed to 30 generations, the plasmid stability rate can reach 100%. This plasmid has high stability. Thus, it can be seen that the recombinant strain of Pseudomonas plecoglossicida can stably inherit and express green fluorescence.

[0055] Example 2: This example provides an application of a recombinant strain of Pseudomonas plecoglossicida expressing green fluorescence, specifically including its application in a cell infection model: Visualization of the fluorescent expression strain in murine macrophage J774A.1: Pick a single colony of the recombinant bacteria and inoculate it into LB medium containing chloramphenicol. Culture overnight at 28 °C, 180 rpm / min; Seed the macrophages after 3 generations of subculture into a 24-well plate at a density of 106 cells / well, and culture in a 37 °C, 5% CO2 incubator until the cells are nearly confluent in a monolayer; Infect the cells with the test bacterial suspension. According to the counting results of macrophages in the 96-well plate, add a DMEM medium containing a quantitatively resuspended bacterial suspension to the plate at a multiplicity of infection (MOI) of 10∶1, and continue to culture in a 30 °C, 5% CO2 incubator for 1 h. After washing the cells in the 96-well plate 3 times with PBS, add DMEM medium containing 200 µg / mL gentamicin and incubate at 30 °C for 2 h, 4 h, 8 h, 16 h, 24 h, and observe the situation of the fluorescent expression strain infecting macrophages under an inverted fluorescence microscope;

[0056] As Figure 4As shown, in bright light field, bacteria can be clearly seen on the surface of macrophages and near the cells, but the presence of bacteria inside the cells cannot be accurately judged; when adjusted to fluorescence light field, not only can the bacteria on the surface and near the macrophages be observed, but also the aggregated fluorescent bacteria inside the cells can be observed; at 2 h of incubation, a small amount of fluorescent bacteria adhered to the cell surface, and with the extension of incubation time, the bacteria began to aggregate inside the cells; as the cells lysed and died, the number of fluorescent bacteria gradually increased and reached the peak at 24 h; through the observation of the green fluorescence of the recombinant bacteria, the distribution and localization of the bacteria inside the cells can be accurately detected, providing an important means for the visualization of bacteria inside the cells.

[0057] Example 3: This example provides an application of a recombinant strain of Pseudomonas plecoglossicida expressing green fluorescence, specifically including its application in an animal infection model: Visualization of the recombinant bacteria expressing fluorescence in zebrafish: Purchase zebrafish embryos and incubate them in an illumination incubator at 28.5 °C under the condition of 12 h light + 12 h darkness. Pick out the hatched healthy zebrafish larvae and place them in a 24-well plate, with three in a group, and set up an experimental group and a control group;

[0058] Coat the recombinant strain on an LB agar plate containing chloramphenicol and culture it at 28 °C for 16 h. Pick out the positive clones and inoculate them into LB liquid containing chloramphenicol for overnight culture; inoculate the liquid medium into 500 mL of LB liquid containing chloramphenicol at a ratio of 1:100 for culture to obtain a bacterial suspension of 1×107 cells / mL; and prepare the wild-type bacterial liquid in the same way; Soak the control group and the experimental group in the wild-type and recombinant bacterial suspensions respectively for 20 min, take out the experimental fish, rinse them three times with zebrafish culture medium and then place them in clean 24-well plates for culture for 2 weeks. Randomly take 3 from the experimental group and the control group every 24 h, anesthetize them and observe and take pictures under a fluorescence microscope.

[0059] Figure 5Microscopic observation images of zebrafish larvae infected with Pseudomonas plecoglossicida expressing fluorescence. Through observation, it can be found that zebrafish larvae are not highly sensitive to Pseudomonas plecoglossicida, which can meet the requirement of no high mortality rate within the experimental period. Under the bright light field of a fluorescence microscope, various parts and internal conditions of the fish body can be clearly observed, but the localization of Pseudomonas plecoglossicida in fish organs or tissues cannot be judged. Under the fluorescence light field, it can be clearly observed that the bacteria aggregate in the abdominal cavity of zebrafish larvae. During the observation period, the fluorescence intensity in the body of zebrafish larvae at 3 days after hatching is significantly stronger than that at 1 day after hatching. It can be speculated that with the increase of the infection time, the fluorescent bacteria proliferate in the abdominal cavity and the number continues to increase. However, due to the pathogenicity of the bacteria and the continuous development and growth of zebrafish larvae, they no longer remain translucent. After 10 days, that is, when the fish body basically completely loses its translucency, green fluorescence can no longer be observed at the corresponding site.

[0060] Through the above embodiments, it is further proved that the present invention provides a recombinant strain of Pseudomonas plecoglossicida expressing green fluorescence, and its construction method and application. By stably expressing green fluorescent protein (GFP), this recombinant strain can not only effectively observe the bacterial infection process in real time and dynamically in cell and animal models, but also provide an accurate and reliable experimental platform for the study of the interaction between bacteria and hosts. Through this technology, researchers can track the distribution, proliferation and infection process of bacteria in the host, making up for the defect in the prior art that the dynamic changes of bacteria cannot be observed in real time. The construction method of the present invention uses electroporation and chloramphenicol screening, which significantly improves the transformation efficiency and strain stability, ensures the stability of fluorescence labeling, and enables long-term bacterial tracking research. The recombinant strain labeled with green fluorescence not only shows good visualization ability in cell culture, but also has excellent application effects in animal infection models.

[0061] Through the technical means provided by the present invention, the pathogenic mechanism of Pseudomonas plecoglossicida, the host immune response and the interaction between pathogenic bacteria and the host can be deeply understood, providing new ideas and methods for the research in related fields, and having important scientific research significance and broad application prospects.

[0062] In the description of the present invention, the descriptions referring to terms such as "one embodiment", "some embodiments", "in this embodiment", "specific examples", or "some examples", etc., mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0063] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A recombinant strain of Pseudomonas aeruginosa expressing green fluorescence, characterized in that: The recombinant strain is a positive clone obtained by transforming the Pseudomonas ayutans NB2011 with the plasmid pBBR1-GFP, and the recombinant strain can present green fluorescence under a fluorescence microscope. The sequence of the plasmid pBBR1-GFP is shown in SEQ ID NO: 2, and the green fluorescence fragment of the plasmid pBBR1-GFP is shown in SEQ ID NO:

1.

2. A method for constructing a recombinant strain of Pseudomonas aeruginosa expressing green fluorescence as claimed in claim 1, characterized in that: The following steps are involved: S1: Extraction of plasmid pBBR1-GFP; S2: Preparation of Pseudomonas ayuminogensis competent cells; S3: Construction of a recombinant strain of Pseudomonas aeruginosa expressing green fluorescence by electroporation; S4: Screening of positive clones to obtain recombinant strains of Pseudomonas aeruginosa expressing green fluorescence.

3. The construction method according to claim 2, characterized in that: The step S1 comprises: extracting the plasmid pBBR1-GFP by using a plasmid extraction kit, and measuring the plasmid concentration after extraction to make the plasmid concentration reach 5pg-25ng / μL.

4. The construction method according to claim 2, characterized in that: The step S2 comprises: S21: Using LB culture medium to culture Pseudomonas aeruginosa by shaking on a shaker, and measuring the OD of the culture medium 600 Value, waiting for OD 600 When the value is 0.6, place the bacterial solution in an ice bath for 15-30 minutes and shake it to ensure that the bacterial solution is fully cooled; S22: centrifuging the bacterial solution obtained in step S21, then recovering the cells, resuspending the cells with pure water and centrifuging again to wash the cells to remove electrolyte impurities, and gradually resuspending and washing the cells with glycerol, and finally resuspending the cells with glycerol to obtain a suspension; S23: Dilute the suspension obtained in step S22 and measure OD 600 value, and diluted it with 10% glycerol to a concentration of 2 × 10 10 cells / ml, and store after aliquoting.

5. The construction method according to claim 4, characterized in that: In the step S21, the shaking culture conditions are: 28°C, 180rpm; in the step S22, the centrifugation conditions are 4°C, 4000g, and the time is 10min; in the step S23, the packaging and storage conditions are: packaging in 100 μL / 1.5 mL EP tubes and storing at -80°C.

6. The construction method according to claim 2, characterized in that: The step S3 comprises: S31: Preparation of competent cells: transfer freshly prepared or frozen-thawed Pseudomonas auriculatus competent cells into an ice-cold sterile centrifuge tube and cool on ice together with the electroporation instrument sample cell; S32: Adding plasmid: Add 10 pg-25 ng of the plasmid to be transformed into the sterile centrifuge tube treated in step S32, keep it ice-cold, and let it stand for 30-60 seconds to obtain a mixed solution; S33: Electroconversion operation: applying electric pulses to the mixed solution of step S32 for electroconversion; S34: Resuscitating transformed cells: After the electroporation operation in step S33 is completed, the mixed solution is taken out, 1 mL of LB liquid culture medium is added at room temperature, and shaking culture is carried out at 28° C. and 180 rpm to resuscitate the bacteria; S35: Screening positive clones: taking different volumes of transformed bacteria obtained after the treatment in step S34 and applying them to LB agar plates containing chloramphenicol, and screening out recombinant bacteria containing green fluorescence after cultivation; S36: Clone identification: After 24 hours, single colonies were picked for positive clone identification.

7. The construction method according to claim 6, characterized in that: In step S33, the conditions of the electric pulse are: the parameters of the electroporator are 25uF, 25kV, 200Ω, the electric field strength is 12.5 kV / cm, and the duration is 4-5 milliseconds; in step S35, the conditions of the culture are: culture at 28°C for 12-16 hours.

8. The construction method according to claim 2, characterized in that: The step S4 comprises: S41: Prepare LB broth medium or LB agar medium, sterilize it by high pressure, and when the temperature drops to 50-55°C, add chloramphenicol to make the final concentration 34 μg / mL; S42: Inoculate the transformed bacteria into the LB liquid or agar medium containing chloramphenicol obtained in step S1 to screen out positive clones that can tolerate chloramphenicol, or spread the cultured bacteria on a glass slide and observe under a fluorescent inverted microscope. If green fluorescence can be seen, it is a positive clone.

9. An application of the recombinant strain of Pseudomonas aeruginosa expressing green fluorescence according to claim 1, characterized in that: The application includes the application of the recombinant strain in a cell infection model and an animal infection model, and the distribution, proliferation and infection process of bacteria in a host are monitored through the green fluorescent labeling of the recombinant strain.

10. The use of the recombinant strain of Pseudomonas aeruginosa expressing green fluorescence according to claim 9, characterized in that: The application of the recombinant strain in the cell infection model includes: A1: inoculating the recombinant strain into LB medium containing chloramphenicol, and culturing at 28° C. and 180 rpm / min until the logarithmic growth phase; A2: Infect macrophages J774A.1 with the cultured recombinant bacterial suspension, incubate and observe the distribution and proliferation of bacteria in the cells; A3: Observe the green fluorescent-labeled bacteria at different time points using a fluorescence microscope to analyze their colonization and proliferation process within the cells; The application of the recombinant strain in an animal infection model includes: B1: inoculating the recombinant strain into LB liquid culture medium containing chloramphenicol to obtain a bacterial suspension of a desired concentration; B2: Expose zebrafish larvae to the recombinant strain and control bacterial suspensions, incubate and monitor the infection process; B3: Use a fluorescence microscope to observe the distribution and proliferation of fluorescent bacteria in zebrafish, and regularly record changes in fluorescence intensity during the infection process.

Images