Recombinant bacterium for overexpressing uranyl binding protein and application of recombinant bacterium

Abstract

The invention relates to a recombinant bacterium for overexpressing uranyl binding protein and application of the recombinant bacterium, and belongs to the technical field of microorganisms. Bacterial outer membrane protein and uranyl binding protein are expressed in host bacteria, and the uranyl binding protein is anchored on an outer membrane of recombinant bacteria through the bacterial outer membrane protein, so that the uranyl binding protein anchored on the bacterial outer membrane can adsorb uranyl ions. Meanwhile, the invention finds that the expression quantity of the uranyl binding protein can be increased by performing fusion expression on the uranyl binding protein by using LPP-OMPA, so that the adsorption effect of uranyl ions is improved. The recombinant bacterium is prepared into a freeze-dried capsule product, and the freeze-dried capsule product can penetrate through a stomach barrier and is accurately delivered to intestinal tracts so as to realize uranyl ion adsorption.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a recombinant bacterium overexpressing uranyl-binding protein and its application. Background Art

[0002] Uranium is an important natural actinide element and also an important resource for nuclear energy. Therefore, the dependence on energy will exacerbate the large-scale mining of uranium ore. Exposed waste rocks and tailings will continuously be carried into the environment by rainwater and natural wind, resulting in the release of uranium compounds into the nearby soil and groundwater and entering the ecological food chain. In addition, direct or indirect ways such as military operations and nuclear accidents will also exacerbate the entry of uranium into the human body. When the radioactive nuclides accumulated in the body exceed a certain limit, it may cause leukemia, cancer and heavy metal ion poisoning, causing serious damage to the kidneys and bones. Uranium has radioactivity and chemical toxicity and usually enters the human body in the form of hexavalent uranyl ions, entering various organs in different ways. Although two-thirds of the uranyl ions will be excreted from the body, the uranyl ions deposited in the body will increase the risk of osteosarcoma, inhibit the growth of bone cells, and cause intestinal damage.

[0003] Currently, the main method for removing uranyl in the body relies on the chelation of uranyl ions. Several chelating agents, such as Prussian blue, potassium iodide, DTPA-ZnNa3 and DTPA-CaNa3, have been approved by the US Food and Drug Administration (FDA) for removing radioactive nuclides from the body. However, before these excretion-promoting agents take effect, the radioactive toxicity and chemical toxicity of uranyl will cause serious health problems. In addition, the physical and chemical methods of entering the body to remove uranyl will also trigger safety considerations and considerations of the residence time of materials in the body. Therefore, how to select a safe method to prevent uranyl ions from causing damage has become an urgent problem to be solved. Summary of the Invention

[0004] For this reason, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a safe and efficient method for adsorbing uranyl ions in the body.

[0005] To solve the above technical problem, the present invention provides a recombinant bacterium overexpressing uranyl-binding protein and its application. The present invention uses the fusion expression of bacterial outer membrane protein and uranyl-binding protein in a host bacterium, and anchors the uranyl-binding protein on the outer membrane of the recombinant bacterium through the bacterial outer membrane protein, so that the uranyl-binding protein expressed on the bacterial outer membrane can adsorb uranyl ions. At the same time, the present invention finds that the fusion expression of uranyl-binding protein using leader peptidase lipoprotein-outer membrane protein A (LPP-OMPA) can increase the expression level of uranyl-binding protein, thereby improving the adsorption effect on uranyl ions. Preparing the recombinant bacterium of the present invention into a freeze-dried capsule product can cross the gastric barrier and accurately deliver to the intestine to realize the adsorption of uranyl ions in the body.

[0006] The first object of the present invention is to provide a recombinant bacterium overexpressing uranium-binding protein, wherein the recombinant bacterium uses a non-pathogenic bacterium as a host, and the uranium-binding protein and Braun lipoprotein-outer membrane protein A are co-expressed in the non-pathogenic bacterium.

[0007] Furthermore, Lpp-OmpA is a chimeric structure formed by fusing the lipoprotein Lpp (Lipoprotein) and outer membrane protein OmpA (Outer Membrane Protein A) of Escherichia coli. The lipoprotein structure of Lpp can anchor the fusion protein on the bacterial outer membrane, significantly improving its surface display efficiency. Thereby increasing the contact area between the recombinant bacterium and uranium, and increasing its uranium adsorption capacity.

[0008] Furthermore, the gene sequence encoding the Braun lipoprotein-outer membrane protein A is as shown in SEQ ID NO.2.

[0009] Furthermore, the gene encoding the Braun lipoprotein-outer membrane protein A and the gene encoding the uranium-binding protein are connected by a linker, and the gene sequence of the linker is as shown in SEQ ID NO.3.

[0010] Furthermore, the non-pathogenic bacterium includes Escherichia coli Nissle 1917.

[0011] Furthermore, the gene sequence of the uranium-binding protein is as shown in SEQ ID NO.1.

[0012] The second object of the present invention is to provide an application of the above recombinant bacterium in the preparation of uranium adsorption products.

[0013] The third object of the present invention is to provide a uranium adsorption product, which includes a mixture obtained by mixing the above recombinant bacterium with a lyoprotectant and freeze-drying.

[0014] Furthermore, the dosage form of the uranium adsorption product includes enteric capsule preparation.

[0015] Furthermore, when the mixture is loaded into an enteric capsule, protected by the enteric capsule, the mixture can reach the intestine through the acidic environment of the stomach and colonize in the intestine, and then by expressing uranium-binding protein and Braun lipoprotein-outer membrane protein A, the Braun lipoprotein-outer membrane protein A binds the uranium-binding protein outside the cell membrane, thereby achieving the adsorption of uranium ions in the intestine.

[0016] Due to the acidic effect of the stomach, directly instilling the mixture into the stomach will cause the inactivation of the recombinant bacterium. Therefore, it is necessary to administer an antacid in advance to neutralize the gastric acid, and then instill the mixture to play a role.

[0017] The fourth object of the present invention is to provide an application of the above-mentioned recombinant bacteria in the preparation of a therapeutic drug for uranium damage.

[0018] Furthermore, the administration method of the therapeutic drug for uranium damage is gastrointestinal administration.

[0019] Advantages of the present invention:

[0020] The present invention fuses and expresses a bacterial outer membrane protein and a uranium-binding protein in a host bacterium, and anchors the uranium-binding protein on the outer membrane of the recombinant bacterium through the bacterial outer membrane protein, so that the uranium-binding protein expressed on the bacterial outer membrane can adsorb uranium ions. At the same time, the present invention discovers that the fusion expression of the uranium-binding protein using Braun lipoprotein-bacterial outer membrane protein A (LPP-OMPA) can increase the expression level of the uranium-binding protein, thereby improving the adsorption effect on uranium ions. Preparing the recombinant bacteria of the present invention into an enteric capsule product can pass through the gastric barrier and accurately deliver to the intestine to achieve the adsorption of uranium ions. Description of the drawings

[0021] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings, where

[0022] Figure 1 is the plasmid map of LPP-OMPA-Linker-U09-pBAD His;

[0023] Figure 2 is the plasmid map of LPP-OMPA-Linker-U09-pET-17b His;

[0024] Figure 3 is the plasmid map of OMPA-Linker-U09-pET-17b;

[0025] Figure 4 is the expression of the uranium-binding protein of the recombinant bacterium EcN-Ua under arabinose induction;

[0026] Figure 5 is the expression of the uranium-binding protein of the recombinant bacteria EcN-Uc and OMPA-U09 in the constitutive expression mode;

[0027] Figure 6 is the comparison curve graph of the expression of the uranium-binding protein of EcN-Ua, EcN-Uc and OMPA-U09;

[0028] Figure 7 is the bar graph of the ability of the induced expression, constitutive expression and wild-type recombinant bacteria to bind uranium ions;

[0029] Figure 8 is the freeze-drying process of the recombinant bacteria;

[0030] Figure 9 is the detection of bacterial activity before and after freeze-drying;

[0031] Figure 10 is the colonization situation of the recombinant bacteria in the mouse intestine by gavage in liquid form;

[0032] Figure 11 is the colonization situation of the recombinant bacteria in the rat intestine after freeze-drying and filling into enteric capsules;

[0033] Figure 12 is the result graph of detecting the uranyl content in the main organs of mice after gavage with liquid recombinant bacteria;

[0034] Figure 13 is the result graph of detecting the uranyl content in the main organs of rats after feeding the rats with freeze-dried powder capsules containing recombinant bacteria. Specific embodiments

[0035] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited do not limit the present invention.

[0036] The gene sequence of the uranyl-binding protein (U09) used in the present invention was provided by Suzhou Genewiz Biotechnology Co., Ltd., and the gene sequence of the uranyl-binding protein is shown in SEQ ID NO.1. The present invention uses pBAD and pET-17b as vectors. It should be noted that in the present invention, pBAD and pET-17b are taken as examples to construct plasmids, which does not mean that the present invention can only use pBAD and pET-17b, and other vectors capable of carrying the uranyl-binding protein are within the protection scope of the present invention.

[0037] The present invention selects a probiotic strain in Escherichia coli: E. coli Nissle 1917 (EcN) can be used as a natural vector to develop an intestinal drug delivery system. E. coli Nissle 1917 has the characteristics of genetic stability, easy engineering and strong adaptability. It can make foreign genes be highly expressed through genetic engineering technology and has the function of intestinal colonization. Therefore, the present invention selects E. coli Nissle 1917 as the competent bacteria for the construction of recombinant bacteria. However, the vectors carrying the uranyl-binding protein in the present invention are not limited to Escherichia coli strains, and can also be other non-pathogenic bacteria such as Bifidobacterium, Lactobacillus, Saccharomyces cerevisiae, etc.

[0038] Example 1: Construction of recombinant bacteria

[0039] (1) Construction of recombinant bacteria containing the LPP-OMPA-Linker-pBAD His A plasmid

[0040] To enable the fusion expression of uranyl-binding protein and membrane protein, the gene sequence of membrane protein LPP-OMPA (as shown in SEQ ID NO.2) and the gene sequence of the linker (as shown in SEQ ID NO.3) were inserted into the pBAD vector to form the LPP-OMPA-Linker-pBAD His A vector. The gene sequence of the uranyl-binding protein was inserted into the LPP-OMPA-Linker-pBAD His A vector using DNA ligase to construct a plasmid. The plasmid was introduced into an ampicillin-resistant host bacterium such as TOP10 or DH5α for plasmid cloning. The host bacterium carrying the plasmid was cultured overnight under resistant conditions, and the plasmid was extracted using a plasmid midiprep kit. The purity of the plasmid was measured as A 260 / A 280 The ratio was between 1.7 and 1.9 and the concentration was greater than or equal to 150 μg / mL, and finally the LPP-OMPA-Linker-U09-pBAD His plasmid (as Figure 1 shown) was obtained.

[0041] 10 μL of the LPP-OMPA-Linker-U09-pBAD His A plasmid was added to 200 μL of competent bacteria E. coli Nissle 1917, incubated on ice for 30 min, heat-shocked at 42 °C for 90 s, incubated on ice for another 5 min, 700 μL of antibiotic-free medium was added, and shaken at 37 °C for 1 h. 100 μL of the bacterial solution was taken and spread on a culture medium plate with ampicillin resistance, incubated overnight, and the recombinant Escherichia coli EcN-Ua was obtained through antibiotic screening.

[0042] (2) Construction of recombinant bacteria containing the LPP-OMPA-Linker-pET-17b His A plasmid

[0043] In addition to the inducible expression plasmid, for the convenience of later actual production, in this embodiment, the LPP-OMPA gene sequence and the gene sequence of the linker were inserted into the pET-17b vector to form the LPP-OMPA-Linker-pET 17b vector. The gene sequence of the uranyl-binding protein was inserted into the LPP-OMPA-Linker-pET-17b His A vector using DNA ligase to construct a plasmid. The plasmid was introduced into an ampicillin-resistant host bacterium such as TOP10 or DH5α for plasmid cloning. The host bacterium carrying the plasmid was cultured overnight under resistant conditions, and the plasmid was extracted using a plasmid midiprep kit. The purity of the plasmid was measured as A 260 / A 280 The ratio was between 1.7 and 1.9 and the concentration was greater than or equal to 150 μg / mL, and finally the LPP-OMPA-Linker-U09-pET-17b His plasmid (as Figure 2 shown) was obtained.

[0044] Add 10 μL of LPP-OMPA-Linker-U09-pET-17b His plasmid into 200 μL of competent bacteria E. coli Nissle 1917, incubate on ice for 30 min, heat shock at 42 °C for 90 s, incubate on ice for another 5 min, add 700 μL of antibiotic-free medium, shake at 37 °C for 1 h. Take 100 μL of the bacterial solution and spread it on the culture medium plate with ampicillin resistance, incubate overnight, and obtain recombinant Escherichia coli EcN-Uc through antibiotic screening.

[0045] (3) Construction of recombinant bacteria containing OMPA-Linker-U09-pET-17b plasmid

[0046] Select OMPA outer membrane protein (gene sequence shown in SEQ ID NO.4) to compare the advantages of this example. Insert the gene sequence of membrane protein OMPA and the gene sequence of the linker into the pET-17b vector to form the OMPA-Linker-pET-17b vector. Use DNA ligase to insert the gene sequence of the uranyl-binding protein into the OMPA-Linker-pET-17b His A vector to construct the plasmid, and introduce the plasmid into the host bacteria with ampicillin resistance such as TOP10 or DH5α for plasmid cloning. Culture the host bacteria with the plasmid overnight under resistance conditions, extract the plasmid using the plasmid midiprep kit, and measure the plasmid purity A 260 / A 280 The ratio is between 1.7 - 1.9 and the concentration is greater than or equal to 150 μg / mL, and finally obtain the OMPA-Linker-U09-pET-17b plasmid (as Figure 3 shown).

[0047] Add 10 μL of OMPA-Linker-U09-Pet-17b His plasmid into 200 μL of competent bacteria E. coli Nissle 1917, incubate on ice for 30 min, heat shock at 42 °C for 90 s, incubate on ice for another 5 min, add 700 μL of antibiotic-free medium, shake at 37 °C for 1 h. Take 100 μL of the bacterial solution and spread it on the culture medium plate with ampicillin resistance, incubate overnight, and obtain recombinant bacteria Escherichia coli OMPA-U09 through antibiotic screening.

[0048] Example 2: WB detection of the expression of uranyl-binding protein in recombinant bacteria

[0049] To detect whether EcN-Ua was successfully constructed and whether the uranyl-binding protein was expressed, EcN (wild-type E. coli Nissle 1917) and EcN-Ua+Ara (with arabinose added to EcN-U09) were designed as the experimental groups, and EcN-Ua (without arabinose added to EcN-U09) was used as the control group. The following experimental groups were designed: The single recombinant bacterium EcN-Ua in Example 1 was transferred to 5 mL of LB broth, and EcN-Ua was cultured in a bacterial shaker at 37 °C and 200 rpm for 8 hours. It was amplified at a ratio of 1:100 until 600 OD 600 = 0.4 - 0.6, then arabinose was added for induction, and it was cultured at 37 °C and 180 rpm for 4 hours. EcN-Ua after induced expression was collected by centrifugation at 5000 g for 15 minutes. Bacterial lysate was added to ultrasonically lyse the bacteria, and SDS-Page electrophoresis was used to detect the expression of the uranyl-binding protein in the bacteria of EcN-Ua+Ara (EcN-U09 induced by arabinose) and the bacteria in the control group.

[0050] The experimental results were as Figure 4 shown. Under arabinose induction, only the bacteria of EcN-Ua+Ara expressed the lanthanum uranyl-binding protein, and the molecular weight of the expressed uranyl-binding protein was approximately 28.5 kDa. Similarly, it can be considered that under arabinose induction, the recombinant bacterium expressed the uranyl-binding protein.

[0051] To detect whether the constitutive expression of the uranyl-binding protein (EcN-Uc) was successfully constructed and the expression of the uranyl-binding protein, EcN (wild-type E. coli Nissle 1917) and EcN-Uc were designed as the experimental groups, and EcN was used as the control group. The following experimental groups were designed: The single recombinant bacterium EcN-Uc in Example 1 was transferred to 5 mL of LB broth, and EcN-Uc was cultured in a bacterial shaker at 37 °C and 200 rpm for 8 hours. EcN-Uc after amplification was collected by centrifugation at 5000 g for 15 minutes. Bacterial lysate was added to ultrasonically lyse the bacteria, and SDS-Page electrophoresis was used to detect the expression of the uranyl-binding protein in the bacteria of EcN-Uc (recombinant expression of the uranyl-binding protein) and the bacteria in the control group.

[0052] To highlight the advantages of the LPP-OMPA membrane protein and the successful construction of OMPA-U09, EcN and EcN-Uc were used as the control groups, and OMPA-U09 was used as the experimental group. The following experimental groups were designed: The single recombinant bacterium OMPA-U09 in Example 1 was transferred to 5 mL of LB broth, and OMPA-U09 was cultured in a bacterial shaker at 37 °C and 200 rpm for 8 hours. OMPA-U09 after amplification was collected by centrifugation at 5000 g for 15 minutes. Bacterial lysate was added to ultrasonically lyse the bacteria, and SDS-Page electrophoresis was used to detect the expression of the uranyl-binding protein in the bacteria of OMPA-U09 and the bacteria in the control group.

[0053] The experimental results are as shown in Figure 4 、 5 、and Figure 6. The inducible expression EcN-Ua, constitutive expression EcN-Uc, and OMPA-U09 have all been successfully constructed and can all express uranium-binding proteins. However, with the same protein loading amount, the expression level of OMPA-U09 is significantly lower than that of the constitutive expression of EcN-Uc.

[0054] Example 3: Detecting the ability of bacteria expressing uranium-binding proteins to bind uranium ions

[0055] (1) Binding ability of recombinant Escherichia coli EcN-Ua to uranium ions

[0056] Transfer the single recombinant bacterium EcN-Ua colony in Example 1 to 5 mL of LB broth respectively. EcN-U09 is cultured in a bacterial shaker at 37 °C and 200 rpm for 8 hours, and amplified 1:100 until OD 600 = 0.4 - 0.6, then add arabinose for induction, and culture at 37 °C and 180 rpm for 4 hours. The bacteria are centrifuged at 5000 g for 15 minutes. The collected bacteria are concentrated to OD 600 = 1, add 50, 100, 1000, 2000 uranium ions, shake for 4 hours, then centrifuge to collect the bacteria and supernatant for nitrification treatment. The content of uranium ions in the bacteria and supernatant is detected by inductively coupled plasma optical emission spectrometer (ICP-OES). The percentage of bacteria binding uranium ions is calculated by dividing the uranium ions in the bacteria by the sum of the uranium ions in the bacteria and the uranium ions in the supernatant.

[0057] (2) Binding ability of recombinant Escherichia coli EcN-Uc to uranium ions

[0058] Verify the bacteria EcN-Uc with constitutive expression of uranium-binding protein constructed in Example 1. Pick a single constitutive Escherichia coli EcN-Uc colony, transfer the single EcN-Uc colony to 5 mL of LB broth, and culture in a bacterial shaker at 37 °C and 200 rpm for 8 hours. The collected bacteria are concentrated to OD 600 = 1, add 50, 100, 1000, 2000 uranium ions, shake for 4 hours, then centrifuge to collect the bacteria and supernatant for nitrification treatment. The content of uranium ions in the bacteria and supernatant is detected by ICP-OES. The percentage of bacteria binding uranium ions is calculated by dividing the uranium ions in the bacteria by the sum of the uranium ions in the bacteria and the uranium ions in the supernatant.

[0059] (3) Binding ability of recombinant Escherichia coli OMPA-U09 to uranium ions

[0060] The single recombinant bacterium OMPA-U09 colonies constructed in Example 1 were separately transferred to 5 mL of LB broth and cultured in a bacterial shaker at 37 °C and 200 rpm for 8 hours. The collected bacteria were concentrated to OD 600 = 1, and uranyl ions at 50, 100, 1000, and 2000 ppm were added. After shaking for 4 hours, the bacteria and supernatant were collected by centrifugation and then nitrified. The content of uranyl ions in the bacteria and supernatant was detected by ICP-OES. The percentage of uranyl ions bound by the bacteria was calculated by dividing the uranyl ions in the bacteria by the sum of the uranyl ions in the bacteria and the uranyl ions in the supernatant.

[0061] The experimental results are as Figure 7 shown. EcN-Ua containing LPP-OMPA and EcN-Uc containing LPP-OMPA had better uranium adsorption effects than OMPA-U09, far exceeding the uranium adsorption effect of wild-type Escherichia coli.

[0062] Example 4: Preparation of freeze-dried powder of recombinant bacteria

[0063] Engineered bacteria can be used as a drug delivery system, but it is more cumbersome for liquid bacteria to enter the body. Processing liquid bacteria into solids will help commercialize recombinant bacteria.

[0064] Step (1): Separate single wild-type Escherichia coli colonies and single EcN-Uc colonies in Example 1 were transferred to 50 mL of LB broth medium respectively. The bacteria were cultured in a bacterial shaker at 37 °C and 200 rpm for 8 hours and amplified 1:100 until the OD of the bacteria 600 = 0.8 - 1, and the bacterial liquid was collected from the medium. The LB broth medium was centrifuged, and the precipitate was collected by centrifugation at 5000 g for 10 minutes. Subsequently, deionized water was used to repeat the above steps twice. After centrifugation, the precipitate was resuspended with deionized water, freeze-drying protectant (10% lactose) was added and mixed evenly. The bottle mouth was sealed with a sealing film, leaving 5 - 8 exhaust holes. Then the sealed bacterial liquid was placed in a -80 °C refrigerator for quick freezing. After the bacterial liquid became solid, it was placed in a freeze-drying machine for 48 hours to obtain freeze-dried powder, as Figure 8 shown.

[0065] Step (2): To test whether the freeze-drying process affects the activity of recombinant bacteria, in this example, 1 mL of EcN-Uc with an OD value of 0.8 was freeze-dried, and then freeze-dried powder was obtained. The freeze-dried powder obtained after freeze-drying and the bacterial liquid with an OD value of 0.8 of the liquid were separately added to 50 mL of sterilized medium, and then the OD values of the two media were detected. The results are as Figure 9 shown. There was no significant difference between the two. It shows that freeze-drying has little effect on recombinant bacteria.

[0066] Example 5: Detection of the ability of recombinant bacteria to colonize in vivo

[0067] Step (1): Take the recombinant bacteria (EcN-Ua) that inducibly expresses uranyl-binding protein and the recombinant bacteria (EcN-Uc) that constitutively expresses uranium protein in the steps of Examples 2 and 3 for intestinal colonization experiments. First, take 3 female balb / c mice weighing 20 g and instill the gastric acid neutralizing reagent (1.3% (w / v) and 365 mg, 3.6% of Mg(OH)₂ dissolved in 10 mL of deionized water) into each mouse through a gavage needle at a dose of 0.2 mL per mouse. After 30 minutes, take 0.2 mL of EcN-Ua with an OD of 0.8 and instill it into the mice. Repeat the above steps for the next two days. After the bacteria injection, take 0.1 g of mouse feces at 1, 3, 5, 7, 14, 21, and 30 days respectively, inject it into 1 mL of PBS for homogenization, take 0.1 mL of the diluted homogenate and add it to a solid medium with ampicillin resistance for culturing for 24 hours, and then count the number of bacteria through bacterial counting.

[0068] Step 2: Similar to the above steps, take 3 female SD rats weighing 200 g, load the freeze-dried recombinant bacterial powder into enteric-coated capsules, and use a gavage device to inject the enteric-coated capsules filled with the freeze-dried powder into the esophagus of the rats. Repeat the above steps for the next two days. After the enteric-coated capsules are instilled, take 0.1 g of rat feces at 1, 3, 5, 7, 14, 21, and 30 days respectively, inject it into 1 mL of PBS for homogenization, take 0.1 mL of the diluted homogenate and add it to a solid medium with ampicillin resistance for culturing for 24 hours, and then count the number of bacteria through bacterial counting.

[0069] Subsequent experimental results show that (for details, see Figure 10 and 11 ), both the liquid recombinant bacteria and the freeze-dried recombinant bacteria can colonize in animals for a long time.

[0070] Example 6: Evaluate the effect of recombinant bacteria on preventing the absorption of uranyl by the body

[0071] Recombinant bacteria have the effect of adsorbing uranyl both in vivo and in vitro. To verify the protective effect of the present invention on the body, a mouse uranyl injury model was established. Twenty female balb / c mice were randomly divided into 4 groups: Untreated group (non-treated group), Uranyl group (add 40 ppm of uranyl to the drinking water on day 0), E.coli + Uranyl group (inject E.coli bacteria into the mice on days -3, -2, and -1 according to the method in step (1) of Example 6, and then add 40 ppm of uranyl to the drinking water on day 0), EcN-Ua group (instill EcN-Ua bacteria into the mice on days -3, -2, and -1 according to the method in step (1) of Example 6, and then add 40 ppm of uranyl to the drinking water on day 0). All the mice were sacrificed on day 30, and the kidney, bone, and fecal tissues of the mice were taken for digestion to detect the content of uranyl deposition in the organs.

[0072] In addition to verifying the liquid recombinant bacteria, the present invention also verified the effect of freeze-dried powder of recombinant bacteria on preventing uranyl adsorption. A rat uranyl injury model was established. Fifteen female SD rats were randomly divided into three groups: the Untreated group (non-treated group), the Uranyl group (40 ppm uranyl was added to the drinking water on day 0), and the EcN-Uc group (EcN-Uc bacteria were infused into the mice in the manner of step (1) of Example 6 on days -3, -2, and -1, and then 40 ppm uranyl was added to the drinking water on day 0). All rats were sacrificed on day 30, and the kidney, bone, and fecal tissues of the rats were taken for digestion to detect the content of uranyl deposition in the organs.

[0073] The experimental results are as Figure 12 and Figure 13 shown. The experimental results indicate that, compared with the untreated group, the Uranyl group and the E. coli+Uranyl group had the most deposition in the kidneys and bones, while the two recombinant bacteria groups, EcN-Ua and EcN-Uc, had the least deposition. However, the fecal results were exactly the opposite. The EcN-Ua group and the EcN-Uc group had a high content of uranyl, while the Uranyl group and the E. coli+Uranyl group had a very low content of uranyl in the feces. Therefore, it can be considered that the recombinant bacteria of the present invention will carry away the uranyl with the body metabolism after adsorbing uranyl, but the non-recombinant bacteria group can only allow the body to absorb uranyl, indicating that the recombinant bacteria of the present invention can effectively prevent uranyl absorption and achieve the effect of protecting the body.

[0074] Obviously, the above examples are only for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A recombinant bacterium overexpressing a uranyl-binding protein, characterized in that: The recombinant bacteria uses non-pathogenic bacteria as a host and expresses a fusion protein of uranyl binding protein and Braun lipoprotein-bacterial outer membrane protein A in the non-pathogenic bacteria.

2. The recombinant bacterium according to claim 1, characterized in that The gene sequence encoding the Braun lipoprotein-bacterial outer membrane protein A is shown in SEQ ID NO.

2.

3. The recombinant bacterium according to claim 1, characterized in that The gene encoding the lauren lipoprotein-bacterial outer membrane protein A and the gene encoding the uranyl binding protein are connected via a linker, and the gene sequence of the linker is shown in SEQ ID NO.

3.

4. The recombinant bacterium according to claim 1, characterized in that The non-pathogenic bacteria include Escherichia coli Nissle1917.

5. The recombinant bacterium according to claim 1, characterized in that The gene sequence encoding the uranyl binding protein is shown in SEQ ID NO.

1.

6. Use of the recombinant bacteria according to any one of claims 1 to 5 in the preparation of a uranyl adsorption product.

7. A uranyl adsorption product, characterized in that: The uranyl adsorption product comprises a mixture obtained by mixing the recombinant bacteria according to any one of claims 1 to 5 with a freeze-drying protectant and freeze-drying.

8. The uranyl adsorption product according to claim 7, characterized in that The dosage form of the uranyl adsorption product includes an enteric-coated capsule preparation.

9. Use of the recombinant bacteria according to any one of claims 1 to 5 in preparing a drug for treating uranyl injury.

10. The use according to claim 9, characterized in that The administration mode of the uranyl injury treatment drug is gastrointestinal administration.

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