Application of whole-cell biosensors in the preparation of reagents for in vivo localization and detection of gastrointestinal bleeding

Abstract

The present invention discloses the application of whole-cell biosensors in the preparation of reagents for in vivo localization and detection of gastrointestinal bleeding, and belongs to the field of synthetic biology and clinical diagnosis. The present invention uses a mouse model animal experiment to gavage the recombinant engineered bacteria YES601 into a gastrointestinal bleeding mouse model, and detects by using a fluorescence and chemiluminescence imaging system that the recombinant engineered bacteria YES601 can achieve in vivo localization detection of gastrointestinal bleeding. The present invention breaks through the limitations of existing gastrointestinal bleeding detection methods. In future clinical practice, the non-invasive and non-invasive characteristics of the present invention provide a potential strategy for the accurate diagnosis of gastrointestinal bleeding.

Description

Technical Field

[0001] The present invention relates to the fields of synthetic biology and clinical diagnosis, and in particular to the application of whole-cell biosensors in the preparation of reagents for in vivo localization and detection of gastrointestinal bleeding. Background Art

[0002] Gastrointestinal bleeding is a common clinical problem for clinicians in both inpatient and outpatient settings, and is a symptom and clinical manifestation of various gastrointestinal diseases. It refers to gastrointestinal bleeding from the esophagus to the anus, and has a high incidence rate. 5% of gastrointestinal bleeding cases are classified as occult gastrointestinal bleeding (OGIB). As a type of gastrointestinal bleeding disease, the bleeding site of OGIB is often unclear in clinical practice. Moreover, the vast majority of OGIB lesions are located in the small intestine. Existing detection methods (gastroenteroscopy) are highly invasive to the human body, and it is difficult to effectively detect microbleeding lesions, which often leads to missed diagnosis or misdiagnosis.

[0003] In recent years, with the development of synthetic biology, genetically engineered bacterial biosensors have received extensive attention and application in the detection of various diseases such as cancer, inflammatory bowel disease (IBD), infection, metabolic diseases, etc. This emerging technology combines gene editing and synthetic biology techniques to enable microbial cells to sense, process and respond to various environmental conditions by relying on recognition elements and genetic sensing circuits. Although engineered bacterial biosensors show potential for detecting intestinal diseases, most studies rely on the isolation and detection of engineered bacterial samples from feces, which makes in vivo analysis impossible. Although existing cutting-edge methods can achieve real-time monitoring of gastrointestinal bleeding, they still cannot accurately locate the lesion site, which brings difficulties to the etiology diagnosis and prevention and treatment of the disease. Therefore, it is very important to develop engineered bacterial sensors and explore their potential applications in the localization of intestinal diseases. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a whole-cell biosensor in the preparation of a reagent for locating and detecting gastrointestinal bleeding in vivo, so as to solve the problems existing in the above-mentioned prior art, and to realize the visualization of the recombinant engineered bacteria YES601 in the intestine through a fluorescence or chemiluminescence imaging system, thereby realizing sensitive detection and precise positioning of hidden gastrointestinal bleeding.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of a whole-cell biosensor in preparing a reagent for locating and detecting gastrointestinal bleeding in vivo and in vitro. The whole-cell biosensor is a bacterium that is highly sensitive to heme and can emit a bioluminescent signal under the induction of heme.

[0007] Preferably, the bacterium is the recombinant engineered bacterium YES601.

[0008] Preferably, the bacteria achieve in vivo localization detection of gastrointestinal bleeding by means of a fluorescence or chemiluminescence imaging system.

[0009] Preferably, the bacteria enter the gastrointestinal bleeding receptors in a non-invasive manner to achieve in vivo localization of the receptors and detection of gastrointestinal bleeding.

[0010] Preferably, the induction concentration of the bacteria to heme in vivo is ≤1 μM, and the minimum bacterial count is 10 8 CFU.

[0011] Preferably, the gastrointestinal bleeding includes occult gastrointestinal bleeding.

[0012] The present invention also provides a reagent for locating and detecting gastrointestinal bleeding in vivo, comprising a recombinant engineered bacterium YES601, wherein the bacterial concentration of the recombinant engineered bacterium YES601 is ≥10 8 CFU / 200μL.

[0013] The present invention discloses the following technical effects:

[0014] The present invention provides a non-invasive method for detecting intestinal bleeding that is different from traditional methods, and in particular can detect and locate bleeding points of gastrointestinal occult blood, providing a potential strategy for the accurate diagnosis of clinical gastrointestinal bleeding in the future.

[0015] The whole-cell biosensor YES601 strain provided by the present invention is highly sensitive to heme and can emit a strong bioluminescent signal under heme induction. The uniqueness of the strain itself provides a promising method for the timely and accurate diagnosis of gastrointestinal bleeding. The present invention uses the YES601 strain to locate and detect gastrointestinal bleeding. Compared with traditional methods, it no longer relies on naked eye observation and can enhance the visibility of bleeding lesions by improving the luminescent signal. In addition, the inherent mobility of the YES601 strain allows it to spread freely in the intestine, thereby facilitating its rapid positioning to bleeding lesions, including those that cannot be reached by conventional gastroscopy methods. In essence, the recombinant engineered bacteria YES601 exhibits sensitivity and unique visualization capabilities in detecting gastrointestinal bleeding, thereby providing a new diagnostic method for solving the huge clinical challenges brought by OGIB. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The time distribution diagram of the whole-cell biosensor YES601 strain in vivo; A: Schematic diagram of the construction principle of the YES601 strain; B: Bioluminescence response curves of different concentrations of heme; C: Determination results of luminescence values ​​in LB medium (control), simulated gastric fluid (SGF), and intestinal fluid (SIF); D: Luminescence response curves of different concentrations of blood; E: Animal experiment operation flow chart; F: In vivo bioluminescence signal imaging diagram; G: Gastrointestinal bioluminescence imaging diagram;

[0018] Figure 2 The whole-cell biosensor YES601 strain was gavaged into mice to sense heme and respond to luminescence detection results;

[0019] Figure 3 The validation results of the whole-cell biosensor YES601 strain being able to locate the site of intestinal bleeding in mice; A: Modeling flow chart; B: In vivo bioluminescent signal imaging; C: Gastrointestinal bioluminescent signal imaging;

[0020] Figure 4 The whole-cell biosensor YES601 strain can detect intestinal bleeding in colitis mice. A: Schematic diagram of the experimental operation process; B: Disease activity index; C: In vivo bioluminescent signal imaging; D: Gastrointestinal bioluminescent signal imaging; E: HE staining microscope image;

[0021] Figure 5 Biosafety evaluation of the whole-cell biosensor YES601 strain; A: Body weight changes; B: Changes in TNF-α cytokine content; C: Changes in IL-6 cytokine content; D: Changes in liver function-related enzyme content; E: Changes in the number of YES601 bacteria in the intestine over time; F: Electron microscope image of HE staining. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] (1) Reagents and materials used in the following examples:

[0028] Nude mice (SPF grade, 5-6 weeks old) and C57BL / 6 mice (SPF grade, 5-6 weeks old) were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd. (Hangzhou, Zhejiang, China).

[0029] Other chemicals including streptomycin were purchased from Sigma-Aldrich.

[0030] Strain YES601 is a recombinant engineered bacterium constructed by the applicant in the early stage, which has been disclosed in the invention patent "CN114921478B A method for constructing a whole-cell biosensor and its application". Strain YES601 was cultured in lysogeny broth (LB) medium, and appropriate antibiotics were added: streptomycin (50 μg / mL).

[0031] (2) The whole cell biosensor YES601 in the following examples is applied using the following method:

[0032] Nude mice and C57BL / 6 mice were housed under a 12-h light / 12-h dark cycle, with an ambient temperature of 22±2°C. The acclimatization period was one week, and they had free access to drinking water and food. The gavage volume was 200 μL per nude mouse. The acquisition of luminescent signal imaging in the living state requires anesthesia of the nude mice, which is achieved by a single intraperitoneal injection of 75 mg / mL sodium pentobarbital. The fluorescent and chemiluminescent imaging system (ChemiScope 6100) can be used to obtain luminescent signal imaging of the gastrointestinal tract obtained in vivo and dissected, with an exposure time of 1 min for in vivo imaging and 2 min for gastrointestinal imaging. All experimental animals were euthanized by cervical dislocation.

[0033] According to the inoculation standard of 1% of the volume of the culture medium, the YES601 strain was inoculated into 5mL of LB culture medium with streptomycin resistance (100mg / mL), and placed in a constant temperature shaker (37°C, 220rpm) for 12h. After activation, it was inoculated into 30mL of LB culture medium with streptomycin resistance according to the inoculation amount of 0.5%, and placed in a constant temperature shaker for 3h to OD of 0.6-0.8 to obtain the YES601 bacterial solution. Hemoglobin was added to the LB culture medium after the secondary activation of YES601 to a final concentration of 20μM, and it was placed in a constant temperature shaker for induction for 2h to obtain the YES601 bacterial solution after hemoglobin in vitro induction.

[0034] Example 1 Time distribution of whole cell biosensor in the intestine

[0035] The whole-cell biosensor strain YES601 is an engineered strain that has been developed for the detection of gastrointestinal bleeding (see Figure 1 YES601 strain can sense different concentrations of heme and show strong bioluminescent signals (see Figure 1 (middle B).

[0036] In vitro simulated gastric and intestinal fluid environment: The engineered strain was activated and cultured in a 30 mL shake flask to the logarithmic growth phase, and the bacteria were collected in a 50 mL sterile centrifuge tube, centrifuged at 7000 rpm for 10 min, the supernatant was removed, and 2 mL of LB medium with streptomycin antibiotics was taken with a pipette, and the bacterial solution was evenly resuspended, 100 μL was taken for gradient dilution and then coated with streptomycin resistance plate for counting, and the initial bacterial amount without gastric juice incubation was obtained, and then 1 mL of artificial gastric juice prepared in advance was added and allowed to stand for 5 min, and centrifuged again at 7000 rpm for 10 min after 5 min, the supernatant was removed, 2 mL of LB was added for resuspending, 200 μL was taken and added to a 96-well plate with a white edge and a transparent bottom, and the luminescence intensity was measured by an enzyme marker, and 100 μL was taken for gradient dilution and then coated with resistance plate for counting, and the remaining bacterial amount after gastric juice incubation was obtained. The survival rate and luminescence intensity detection steps in intestinal fluid were the same as above, but the incubation time in intestinal fluid was 30 min.

[0037] By simulating gastric and intestinal fluid environments in vitro, such as Figure 1 As shown in C, the results show that the bioluminescence performance of the YES601 strain was not significantly affected.

[0038] In addition, in a microaerobic environment, the pre-cultured YES601 (OD 600 ≈0.5) can also detect blood rapidly and respond to even minor bleeding levels (see Figure 1 These results demonstrate the potential of strain YES601 for detection in the gastrointestinal environment.

[0039] The bacterial solution after in vitro induction with hemoglobin was collected into a 50 mL sterile tube, centrifuged in a centrifuge (4°C, 7000 rpm, 10 min) for 10 min, and then washed and resuspended with sterile PBS to obtain a gavage sample. The negative control group was set to be the YES601 bacterial solution, and the positive group was set to be the YES601 bacterial solution after in vitro induction with hemoglobin. Through the fluorescence and chemiluminescence imaging system, the present invention collected the luminescence signal imaging of the living body and the gastrointestinal tract of nude mice at 15 min, 30 min, 60 min, 90 min, and 3 h after gavage. The specific process is shown in Figure 1 Middle E. The results showed that compared with the uninduced group, all experimental groups showed strong luminescence signals, and over time, the location of the luminescence signal shifted from the small intestine to the colon (see Figure 1 F and G). Quantitative data showed that the integrated density of the luminescent signal continued to increase until 60 min and then gradually decreased. In addition, the luminescent signal was significantly weakened 3 h after oral administration. These results indicate that YES601 can be effectively used for in vivo bioluminescent imaging and is excreted from mice within 3 h.

[0040] Example 2 Whole-cell biosensor can sense heme and respond with luminescence

[0041] In order to determine the threshold level of bacterial number and heme concentration required to observe bioluminescence phenomenon in vivo, the present invention collected the corresponding in vivo bioluminescence signal images. In order to determine the minimum number of bacteria used in vivo, the present invention detected the luminescence signals of different bacterial amounts of YES601 induced by 20μM heme. The results are as follows: Figure 2 As shown, the results show that the luminescence intensity is positively correlated with the number of bacteria and induction time. 10 The CFU group showed obvious luminescence signals at 30 minutes, while the 10 7 The CFU group did not show any detectable bioluminescent signal at any time point. 10The bioluminescence rate of the CFU group was the fastest and most obvious. Therefore, the optimal amount of bacteria for heme detection in YES601 strain was 10 10 CFU, minimum bacterial count is 10 8 CFU. Then, the minimum heme concentration that causes the YES601 strain to luminesce in vivo was determined. The amount of occult blood bleeding in the human gastrointestinal tract is 5mL / day. If a human weighing 60kg is converted to a mouse weighing 20g, the amount of occult blood bleeding in the mouse gastrointestinal tract is 1.67μL / day, which is equivalent to a heme concentration of 8.35μM. Except for the control group, the YES601 strain can respond to luminescence in vivo to heme as low as 1μM. This shows that YES601 can detect and generate bioluminescent signals with high sensitivity even in the case of microbleeding.

[0042] Example 3 Whole-cell biosensor can locate the site of intestinal bleeding in mice

[0043] In order to further study whether the YES601 strain can achieve accurate positioning and imaging of bleeding sites, the present invention combines bleeding detection with engineered bacteria. 10 CFU of YES601 bacterial solution 1 hour later, the intestines of nude mice were surgically cut to produce bleeding points (see Figure 3 (A). Figure 3 As shown in B, the in vivo signal image shows that the luminescent signal increases over time. At 60 minutes, both the small intestine and cecal bleeding experimental groups showed luminescent signals, indicating that the YES601 strain has the ability to contact and respond to blood. 60 minutes after the operation, the nude mice were dissected to obtain gastrointestinal luminescent images. The results are shown in Figure 3 As shown in C, there is only one luminous site in the gastrointestinal imaging of the single incision test group. Multiple luminous spots appeared in the multi-incision experimental group. More importantly, the present invention confirms that the luminous site is consistent with the surgical incision site and shows a strong luminescent signal. The results of the present invention show that the YES601 strain can detect the fixed point of gastrointestinal bleeding with high sensitivity and accuracy.

[0044] Example 4 Whole-cell biosensor can detect intestinal bleeding in colitis mice

[0045] To investigate whether the YES601 strain can detect and localize intestinal bleeding caused by colitis in mice, colitis mice were given 10 10 Nude mice were allowed to drink water containing 3% dextran sulfate sodium (DSS) freely for 5 consecutive days, and then water without DSS was replaced (see Figure 4 The disease activity index of mice increased significantly after drinking DSS for 3 days, and gradually recovered to a healthy state after the removal of DSS (see Figure 4 (B) Figure 4As shown in C and D, the in vivo and gastrointestinal images show that mice began to bleed on the third day after 3% DSS induction, and the luminescence signal increased with the increase in the degree of colitis. After switching to water, the luminescence signal weakened as the disease activity index of the mice decreased, and no luminescence signal was detected on the 23rd day.

[0046] The present invention performed hematoxylin and eosin (HE) staining and microscopic observation on each group of tissue sections, and performed pathological analysis. The results are as follows Figure 4 As shown in Figure E, arrows of different colors represent different pathological changes. Under the HE staining microscope, no obvious abnormalities were observed in the groups at 0, 1, 2, and 23 days. In the 3-day group, a large number of intestinal villi were separated from the lamina propria at the top of the mucosal epithelium (black arrow), and vascular congestion was seen near the bottom of the lamina propria (green arrow). In the 5-day group, in addition to occasional vascular congestion at the bottom of the lamina propria (green arrow), small focal aggregations of lymphocytes (blue arrow) were also seen, indicating that the colitis model was successfully established. On the 14th day of the recovery period, it was observed that the number of intestinal villi decreased, the villi were loosely arranged, the gaps were larger (yellow arrow), and a small number of villi were fused (red arrow). The pathological changes in tissue sections were consistent with the results of luminescent signal imaging, proving the ability of the YES601 strain to locate the site of intestinal bleeding in colitis mice.

[0047] Example 5 Biosafety Evaluation of Whole Cell Biosensor

[0048] After confirming that the YES601 strain could accurately localize the site of intestinal bleeding caused by mechanical injury and colitis in mice, the biosafety of YES601 was evaluated, which is critical for clinical application.

[0049] Figure 5 Figure A shows the weight changes of mice after oral administration of PBS and YES601 strains. The results show that there was no significant change in weight between the YES601 group and the PBS group. Subsequently, on the 1st, 3rd and 5th day after oral administration of YES601 bacteria, the levels of IL-6 and TNF-α in mouse serum, as well as liver function-related enzyme markers, were tested using the corresponding biological detection kits. Figure 5 As shown in Figure BC, statistical analysis showed that the YES601 strain did not induce an inflammatory response in mice, and there was no significant difference in the levels of the two pro-inflammatory factors compared with the PBS control group. Figure 5 As shown in D, none of the three enzyme markers showed abnormalities. The YES601 strain had no statistically significant effect on the levels of ALT, AST, and γ-GT, indicating that the YES601 strain had no effect on the liver function of mice.

[0050] Evaluating the colonization ability of the YES601 strain in the mouse intestine is a key step in evaluating its biosafety. Figure 5Middle E shows that the number of YES601 strains in feces is negatively correlated with time. Twelve hours after gavage, the number of YES601 strains in the gastrointestinal tract is about 1×10 4 CFU. However, after 48 hours, the presence of the YES601 strain was almost undetectable. These results suggest that the YES601 strain has a relatively weak ability to colonize the intestine and can only persist in the body for 48 hours. The relatively weak intestinal colonization ability of the YES601 strain may help reduce the risk of adverse intestinal reactions.

[0051] In order to evaluate whether the YES601 strain induces pathological changes in the intestinal tissue of mice, the present invention conducted microscopic observation and pathological analysis on the small intestine and colon tissue sections of two groups of mice. Figure 5 The micrographs shown in F show that there were no abnormal or obvious inflammatory responses in the intestinal and colonic tissues of mice in the PBS group and the YES601 group. Therefore, the YES601 strain is a highly sensitive and accurate method for detecting intestinal bleeding, has good biosafety, and does not cause pathological changes in the intestinal tissues of mice.

[0052] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a whole-cell biosensor in the preparation of a reagent for in vivo localization and detection of gastrointestinal bleeding, characterized in that: The whole-cell biosensor is a bacterium that is highly sensitive to heme and can emit a bioluminescent signal under the induction of heme; The bacteria can achieve in vivo localization detection of gastrointestinal bleeding through a fluorescence or chemiluminescence imaging system; The bacteria enter the gastrointestinal bleeding receptors in a non-invasive manner, thereby achieving the in vivo localization of the receptors and detecting gastrointestinal bleeding; The induction concentration of the bacteria to heme in vivo is ≤1 μM, and the minimum bacterial count is 10 8 CFU; The bacterium is the recombinant engineering bacterium YES601; The gastrointestinal bleeding includes occult gastrointestinal bleeding.

2. A reagent for locating and detecting gastrointestinal bleeding in vivo, characterized in that: The recombinant engineering bacterium YES601 has a bacterial concentration of ≥10 8 CFU / 200μL.

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