Phagocytosis-resistant indigo engineering bacteria as well as preparation method and application of phagocytosis-resistant indigo engineering bacteria

Abstract

The invention discloses a phagocytosis-resistant indigo engineering strain, which can sensitively respond to enteritis-related thiosulfate inflammatory factors, timely release a drug indigo to treat gastrointestinal inflammation, and release sfGFP signal molecules to assist in diagnosis and monitoring of diseases, and is very helpful for slowing down disease progression. In addition, through knockout of fhuA and trxA genes related to bacteriophage infection and knockin of an sspB-sspC-sspD-sspE gene cluster, the survival rate of the strain in the intestinal tract can be improved, and the treatment and diagnosis effects of the strain are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering bacteria, and in particular relates to a phage-resistant indigo engineering bacterium and a preparation method and application thereof. Background Art

[0002] Intestinal sulfur metabolism is associated with inflammation by poorly understood mechanisms, but interactions between the microbes and the host enable this mechanism. Sulfate-reducing bacteria (SRB) in the colon produce hydrogen sulfide (H2S) from sulfur oxides produced by the host and the diet, a process thought to be involved in colitis, while host enzymes can convert H2S to thiosulfate. Therefore, thiosulfate (S2O3 2- ) is an attractive target for studying the relationship between intestinal sulfur metabolism and inflammation. A two-component system sensor that responds to thiosulfate has been reported, including the ThsS / R two-component system and its downstream response promoter phsA. Thiosulfate can phosphorylate and activate ThsS and then activate ThsR. The activated ThsR further activates the phsA promoter to express downstream products.

[0003] Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) that causes digestive disorders and inflammation. The disease has always been a global health problem. Indigo naturalis has a high efficacy in treating ulcerative colitis and can be used for local induction therapy in patients with ulcerative colitis. It does not cause serious adverse events such as pulmonary hypertension or intussusception, and may prevent the occurrence of serious adverse events. It is a promising treatment option. The main components of indigo naturalis are indigo and indirubin, which are ligands of the endogenous aryl hydrocarbon receptor (AhR) in humans. They can upregulate IL-10 and IL-22 by activating AhR, thereby improving DSS-induced colitis in mice. Indigo, as the organic substance with the highest content in indigo naturalis, also has a certain effect on ulcerative colitis. Its precursor indole is a metabolite of the tryptophan biosynthesis pathway of Escherichia coli. Therefore, it is only necessary to introduce flavin monooxygenase (FMO) into Escherichia coli to achieve de novo synthesis of indigo.

[0004] Currently, there is no standard method for diagnosing ulcerative colitis, which mainly includes clinical evaluation, stool and blood tests, and colonoscopy. The examination steps are cumbersome, and microscopy as a first-line diagnostic procedure will also cause discomfort to patients. Although early intervention treatment strategies can solve this problem, relieve patients' pain and strive for the best treatment time, early ulcerative colitis is difficult to detect. Summary of the invention

[0005] In order to overcome the defects of the prior art and timely monitor and respond to the occurrence and development of enteritis, the present invention provides an indigo engineered bacterium that can respond to enteritis-related thiosulfate inflammatory factors, thereby releasing signal molecules and drug molecules in the intestine to monitor and treat the disease.

[0006] In one aspect, the present invention provides a phage-resistant indigo engineered bacterium, wherein the phage-resistant indigo engineered bacterium is obtained by genome modification and recombinant plasmid transformation of Escherichia coli Nissle (EcN).

[0007] The genome modification includes: knocking out the pMUT1 plasmid, knocking out the pMUT2 plasmid, knocking out the fhuA gene, knocking out the trxA gene, and inserting the sspB-sspC-sspD-sspE gene cluster between the aslA gene and the hemy gene of the genome;

[0008] The recombinant plasmid includes: thsS gene, tac promoter upstream of thsS gene, thsR gene, tac promoter upstream of thsR gene, sfGFP gene, phsA sequence upstream of sfGFP gene, fmo gene and phsA sequence upstream of fmo gene.

[0009] Preferably, the nucleotide sequence of the tac promoter is shown in SEQ ID NO:1.

[0010] Preferably, the nucleotide sequence of the thsS gene is shown in SEQ ID NO:2.

[0011] Preferably, the nucleotide sequence of the thsR gene is shown in SEQ ID NO:3.

[0012] Preferably, the nucleotide sequence of the phsA sequence is shown in SEQ ID NO:4.

[0013] Preferably, the nucleotide sequence of the sfGFP gene is shown in SEQ ID NO:5.

[0014] Preferably, the nucleotide sequence of the fmo gene is as shown in SEQ ID NO:6.

[0015] Preferably, the nucleotide sequence of the sspB-sspC-sspD-sspE gene cluster is as shown in SEQ ID NO: 7.

[0016] In another aspect, the present invention provides a method for preparing a phage-resistant indigo engineered bacterium as described in any embodiment herein, the method comprising genome modification and recombinant plasmid transformation, wherein the genome modification comprises the following steps in sequence:

[0017] S1: constructing pEcCas plasmids, pTarget plasmids and donor DNA required for targeting each gene for gene editing; wherein the pTarget plasmids include pTarget-pMUT1 plasmids, pTarget-pMUT2 plasmids, pTarget-fhuA plasmids, pTarget-trxA plasmids and pTarget-ssp plasmids; the donor DNA includes donor (fhuA), donor (trxA) and donor (ssp);

[0018] S2: transformation of pEcCas plasmid;

[0019] S3: transforming the pTarget-pMUT1 plasmid, knocking out the pMUT1 plasmid, and eliminating the pTarget-pMUT1 plasmid;

[0020] S4: transforming the pTarget-pMUT2 plasmid, knocking out the pMUT2 plasmid, and eliminating the pTarget-pMUT2 plasmid;

[0021] S5: transform the pTarget-fhuA plasmid and donor(fhuA), knock out the fhuA gene in the genome, and then eliminate the pTarget-fhuA plasmid;

[0022] S6: transforming pTarget-trxA plasmid and donor(trxA), knocking out the trxA gene in the genome, and then eliminating the pTarget-trxA plasmid;

[0023] S7: transform the pTarget-ssp plasmid and donor (ssp), insert the sspB-sspC-sspD-sspE gene cluster between the aslA gene and the hemy gene of the genome, and then eliminate the pTarget-ssp and pEcCas plasmids to obtain genome-modified bacteria;

[0024] Wherein, the recombinant plasmid transformation comprises the following steps:

[0025] S1: connect the tac promoter to the upstream of the thsS gene to obtain the tac-ThsS expression frame; connect the tac promoter to the upstream of the thsR gene to obtain the tac-ThsR expression frame; connect the phsA sequence to the upstream of the sfGFP gene to obtain the phsA-sfGFP expression frame; connect phsA to the upstream of the fmo gene to obtain the phsA-fmo expression frame;

[0026] S2: inserting four expression cassettes, tac-thsS, tac-thsR, phsA-sfGFP and phsA-fmo, into the pkD184 backbone plasmid to obtain a recombinant plasmid;

[0027] S3: Transform the recombinant plasmid into genome-modified bacteria to obtain indigo engineered bacteria.

[0028] In one or more embodiments, in step S2 of genome modification, the pEcCas plasmid is transformed into a wild-type EcN strain by electroporation or chemical transformation to obtain an EcN (pEcCas) strain.

[0029] In one or more embodiments, in steps S3 to S7 of the genome modification, the specific operation of the transformation process is: the strain obtained in the previous step is induced with L-arabinose in a culture medium containing kanamycin to prepare an electroporation competent strain, and the donor DNA and the pTarget plasmid are electroporated into the competent strain, and the transformed competent strain is spread on a double-resistant plate of kanamycin and spectinomycin for overnight culture, and the strain that survives on the double-resistant plate is the successfully transformed strain.

[0030] Preferably, during the transformation process, the strain to be transformed is induced with 9-11 mM L-arabinose to prepare an electroporation competent strain. More preferably, during the transformation process, the strain to be transformed is induced with 10 mM L-arabinose to prepare an electroporation competent strain.

[0031] Preferably, during the transformation process, the concentration of kanamycin in the culture medium is 45-55 mg / L, the concentration of kanamycin in the double-resistance plate is 45-55 mg / L, and the concentration of spectinomycin is 45-55 mg / L. More preferably, during the transformation process, the concentration of kanamycin in the culture medium is 50 mg / L, the concentration of kanamycin in the double-resistance plate is 50 mg / L, and the concentration of spectinomycin is 50 mg / L.

[0032] Preferably, during the transformation process, for every 100 μL of competent strain, 100 ng of pTarget plasmid and 400 ng of donor DNA are transformed.

[0033] In one or more embodiments, in steps S3 to S7 of the genome modification, the specific operation of eliminating the pTarget plasmid is: culturing the successfully transformed strain overnight in a liquid culture medium containing rhamnose and kanamycin, then streaking the culture medium on a kanamycin resistance plate and spotting it on a spectinomycin plate. The strain that does not grow on the spectinomycin plate but grows on the kanamycin plate is the strain from which the pTarget plasmid has been eliminated.

[0034] Preferably, during the elimination of the pTarget plasmid, the successfully transformed strain is cultured overnight in a liquid medium containing 9-11 mM rhamnose and 45-55 mg / L kanamycin. More preferably, during the elimination of the pTarget plasmid, the successfully transformed strain is cultured overnight in a liquid medium containing 10 mM rhamnose and 50 mg / L kanamycin.

[0035] In one or more embodiments, in step S7 of the genome modification, the specific operation of eliminating the pEcCas plasmid is: culturing the strain from which the pTarget plasmid has been eliminated overnight in a medium containing glucose, then streaking the culture solution on a non-resistant plate containing sucrose and glucose, and spotting a kanamycin plate, and the strain that does not grow on the kanamycin plate but grows on the non-resistant plate is the strain from which the pEcCas plasmid has been eliminated.

[0036] Preferably, in the process of eliminating the pEcCas plasmid, the strain from which the pTarget plasmid has been eliminated is cultured overnight in a medium containing 4.5 to 5.5 g / L glucose. More preferably, in the process of eliminating the pEcCas plasmid, the strain from which the pTarget plasmid has been eliminated is cultured overnight in a medium containing 5 g / L glucose.

[0037] Preferably, during the elimination of the pEcCas plasmid, the culture solution is streaked on an antibiotic-free plate containing 9-11 g / L sucrose and 4.5-5.5 g / L glucose. More preferably, during the elimination of the pEcCas plasmid, the culture solution is streaked on an antibiotic-free plate containing 10 g / L sucrose and 5 g / L glucose.

[0038] In another aspect, the present invention provides use of the phage-resistant indigo engineered bacteria as described in any embodiment herein in the preparation of a drug for treating or diagnosing gastrointestinal inflammation.

[0039] Preferably, the gastrointestinal inflammation is ulcerative colitis.

[0040] The phage-resistant indigo engineered bacteria provided by the present invention can sensitively respond to enteritis-related thiosulfate inflammatory factors, timely release the drug indigo to treat gastrointestinal inflammation, and release sfGFP signal molecules to assist in the diagnosis and monitoring of symptoms, which is very helpful in slowing down the progression of the disease. In addition, the knockout of the fhuA and trxA genes related to phage infection and the knock-in of the sspB-sspC-sspD-sspE gene cluster can increase the survival rate of the strain in the intestine, so as to improve the treatment and diagnostic effects of the strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram of the genetic circuit and therapeutic principle of phage-resistant indigo engineered bacteria.

[0042] Figure 2 This is the in vitro test result of phage-resistant indigo engineered bacteria. Figure 2 a is a response curve of the fluorescence expression level of indigo engineered bacteria under thiosulfate concentration gradient conditions; Figure 2 b is the yield and OD of indigo engineered bacteria under thiosulfate concentration gradient conditions 600 picture.

[0043] Figure 3 This is the result of in vivo testing of phage-resistant indigo engineered bacteria in mice. Figure 3 a is a schematic diagram of the in vivo testing scheme in mice; Figure 3 b is a graph showing the changes in the disease activity index of mice in each group over time. DETAILED DESCRIPTION

[0044] In the present invention, the phage-resistant indigo engineered bacteria respond to elevated thiosulfate levels in the environment, thereby promoting the principle of signaling molecules and drugs such as Figure 1 As shown, specifically: in the gastrointestinal inflammatory environment, the thiosulfate level increases, promoting the phosphorylation of thsS protein; further, the phosphorylated ThsS protein can promote the formation of dimers of ThsR protein and the phosphorylation of the dimer; further, the phosphorylated ThsR protein dimer can promote the expression of genes downstream of the phsA sequence; therefore, in the process of constructing the recombinant plasmid of the present invention, the phsA sequence is cloned upstream of the fmo gene and the sfGFP gene, respectively, so that in the environment where the thiosulfate level is increased, the expression of fmo protein and the signal molecule sfGFP is increased, and the increased fmo protein level can promote the expression activity of the shikimic acid pathway, thereby increasing the expression of the product of the pathway, indigo. Among them, the sfGFP protein can emit green fluorescence under the illumination of blue light to ultraviolet wavelengths, so it can be used as a signal molecule; indigo is a common dye, and according to the literature (DOI: 10.1007 / s00535-016-1292-z), indigo also has the function of treating gastrointestinal inflammation.

[0045] In the present invention, the corresponding numbers of the partial sequences are as follows:

[0046] tac promoter, SEQ ID NO: 1;

[0047] thsS gene, SEQ ID NO: 2;

[0048] thsR gene, SEQ ID NO: 3;

[0049] phsA sequence, SEQ ID NO: 4;

[0050] sfGFP gene, SEQ ID NO: 5;

[0051] fmo gene, SEQ ID NO: 6;

[0052] sspB-sspC-sspD-sspE gene cluster, SEQ ID NO:7.

[0053] The Chinese meanings of some English terms in this invention are as follows:

[0054] Thiosulfate: Thiosulfate

[0055] Indigo: Indigo

[0056] EPR: Phage Resistance System

[0057] DSS: Dextran sulfate

[0058] Shikimic acid pathway: Shikimic acid pathway

[0059] Unless otherwise specified, the strains, plasmids and other reagents in the embodiments of the present invention can be obtained commercially.

[0060] Example 1: Construction of recombinant plasmid

[0061] This example uses the pkD184 backbone plasmid (reference DOI: 10.15252 / msb.20167416) to construct a recombinant plasmid, specifically including:

[0062] S1: The tac promoter was connected to the upstream of the thsS gene by PCR using the primer pair ThsS-F / ThsS-R to obtain the tac-ThsS expression cassette to control the expression of ThsS;

[0063] The tac promoter was linked to the upstream of the thsR gene by PCR using the primer pair ThsR-F / ThsR-R to obtain the tac-ThsR expression cassette to control the expression of ThsR;

[0064] The phsA and sfGFP gene sequences were amplified by PCR using primer pairs phsA(sfGFP)-F / phsA(sfGFP)-R and sfGFP(phsA)-F / sfGFP(phsA)-R, respectively. The PCR products were purified and then connected to the upstream of the sfGFP gene by fusion PCR using primer pairs phsA(sfGFP)-F / sfGFP(phsA)-R to obtain the phsA-sfGFP expression cassette to regulate the expression of sfGFP.

[0065] The phsA and fmo sequences were amplified by PCR using primer pairs phsA(fmo)-F / phsA(fmo)-R and fmo(phsA)-F / fmo(phsA)-R, respectively. The PCR products were purified and then connected to the upstream of the fmo gene by fusion PCR using primer pairs phsA(fmo)-F / fmo(phsA)-R to obtain the phsA-fmo expression cassette, so as to dynamically regulate the expression of Fmo and thus regulate the synthesis of indigo.

[0066] The construction of the above four expression cassettes can be carried out in any DNA vector;

[0067] S2: Four expression frames, tac-ThsS, tac-ThsR, phsA-sfGFP and phsA-fmo, were inserted into the plasmid by seamless cloning technology using primer pairs PRR(ThsS)-F / PRR(ThsS)-R&ThsS(PRR)-F / ThsS(PRR)-R, PRR(ThsR)-F / PRR(ThsR)-R&ThsR(PRR)-F / ThsR(PRR)-R, PRR(sfGFP)-F / PRR(sfGFP)-R&sfGFP(PRR)-F / sfGFP(PRR)-R, and PRR(fmo)-F / PRR(fmo)-R&fmo(PRR)-F / fmo(PRR)-R, respectively.

[0068] After the above two steps, a recombinant plasmid is obtained. The primer sequences in the above steps are shown in Table 1 below.

[0069] Table 1: Primer sequences for recombinant plasmid construction

[0070]

[0071]

[0072] Example 2: Genome modification of bacterial strains

[0073] In this example, E. coli Nissle (EcN) was used as the strain for genome modification. The genome modification of the strain used CRISPR-Cas9 technology, and the genome was edited using pEcCas plasmid, pTarget plasmid for specific targets, and donor DNA, where pEcCas plasmid and pTarget plasmid were constructed according to reference (doi:10.1093 / abbs / gmab036). The specific process of genome modification is as follows:

[0074] S1: construct pTarget-pMUT1 plasmid targeting pMUT1 plasmid, pTarget-pMUT2 plasmid targeting pMUT2 plasmid, pTarget-fhuA plasmid and donor(fhuA) targeting fhuA gene, pTarget-trxA plasmid and donor(trxA) targeting trxA gene, and pTarget-ssp plasmid and donor(ssp) targeting ssp gene;

[0075] Among them, the pTarget-pMUT1 plasmid was constructed by PCR and seamless cloning technology using primer pair pT-pM1-F / pT-pM1-R; the pTarget-pMUT2 plasmid was constructed by PCR and seamless cloning technology using primer pair pT-pM2-F / pT-pM2-R; the pTarget-fhuA plasmid was constructed by PCR and seamless cloning technology using primer pair pT-fhuA-F / pT-fhuA-R; donor(fhuA) was constructed by PCR and seamless cloning technology using primer pair Up(fhuA)-F / Up(fhuA)-R and Down(fhuA)-R. huA)-F / Down(fhuA)-R were obtained by PCR and overlapping PCR techniques; donor(trxA) was obtained by PCR and overlapping PCR techniques using the primer pairs Up(trxA)-F / Up(trxA)-R and Down(trxA)-F / Down(trxA)-R; donor(ssp) was obtained by PCR and overlapping PCR techniques using the primer pairs Up(ssp)-F / Up(ssp)-R, sspBCDE-F / sspBCDE-R and Down(ssp)-F / Down(ssp)-R;

[0076] S2: transforming the pEcCas plasmid into the wild-type EcN strain to obtain the EcN (pEcCas) strain;

[0077] S3: transforming the pTarget-pMUT1 plasmid into the strain EcN(pEcCas), knocking out the cryptic plasmid pMUT1 in the strain, and then eliminating pTarget-pMUT1 to obtain the EcNΔpMUT1(pEcCas) strain;

[0078] S4: transforming the pTarget-pMUT2 plasmid into the strain EcNΔpMUT1 (pEcCas), knocking out the cryptic plasmid pMUT2 in the strain, and then eliminating pTarget-pMUT2 to obtain the EcN empty vector strain;

[0079] S5: pTarget-fhuA and donor(fhuA) were transformed into the EcN empty vector strain to knock out the fhuA gene on the genome, and then pTarget-fhuA was eliminated to obtain the EcNΔfhuA(pEcCas) strain;

[0080] S6: transforming pTarget-trxA and donor(trxA) into the EcNΔfhuA(pEcCas) strain, knocking out the trxA gene on the genome, and then eliminating pTarget-trxA to obtain the EcNΔfhuAΔtrxA(pEcCas) strain;

[0081] S7: pTarget-ssp and donor (ssp) were transformed into the EcNΔfhuAΔtrxA (pEcCas) strain, and the sspB-sspC-sspD-sspE gene cluster (sspBCDE) was inserted between the aslA and hemy genes in the genome. Then, the pTarget-ssp and pEcCas plasmids were eliminated respectively to obtain genome-modified bacteria.

[0082] Wherein, in the step S2, the pEcCas plasmid is transformed into the wild-type EcN strain by electroporation or chemical transformation to obtain the EcN (pEcCas) strain;

[0083] In the steps S3 to S7, the specific operation of the transformation process is as follows: the strain obtained in the previous step is induced with 10 mM L-arabinose in a medium containing 50 mg / L kanamycin to prepare an electroporation competent strain, and 100 ng of pTarget plasmid and 400 ng of donor DNA are electroporated into each 100 μL of the competent strain, and the transformed competent strain is spread on a double-resistance plate containing kanamycin (50 mg / L) and spectinomycin (50 mg / L) for overnight culture, and the strain that survives on the double-resistance plate is the successfully transformed strain;

[0084] In the steps S3 to S7, the specific operation of the pTarget plasmid elimination process is: culturing the successfully transformed strain in a liquid culture medium containing 10 mM rhamnose and 50 mg / L kanamycin overnight, then streaking the culture solution on a 50 mg / L kanamycin resistance plate and spotting on a 50 mg / L spectinomycin plate, and the strain that does not grow on the spectinomycin plate but grows on the kanamycin plate is the strain that has eliminated the pTarget plasmid;

[0085] In step S7, the specific operation of the pEcCas plasmid elimination process is as follows: the strain from which the pTarget plasmid has been eliminated is cultured overnight in a medium containing 5 g / L glucose, and the culture solution is streaked on a non-antibiotic plate containing 10 g / L sucrose and 5 g / L glucose, and then spotted on a 50 mg / L kanamycin plate. The strain that does not grow on the kanamycin plate but grows on the non-antibiotic plate is the strain from which the pEcCas plasmid has been eliminated.

[0086] Table 2: Targets related to genome modification and their plasmids, donor DNA and primers

[0087]

[0088] The relevant primer sequences in Table 2 are shown in Table 3 below:

[0089] Table 3: Primers related to genome modification

[0090]

[0091] Example 3: Preparation of indigo engineered bacteria

[0092] The recombinant plasmid obtained in Example 1 is transformed into the genome-modified bacteria obtained in Example 2 to obtain phage-resistant indigo engineered bacteria. The specific operation of the transformation process is as follows: the strain to be transformed is induced with 10mM L-arabinose to prepare an electroporation competent strain, and 100ng of the recombinant plasmid is electroporated into the competent strain, and the transformed competent strain is coated on a double-resistance plate of kanamycin and spectinomycin (50mg / L) for overnight culture, and the surviving strain on the double-resistance plate is the successfully transformed indigo engineered bacteria.

[0093] Example 4: In vitro testing of phage-resistant indigo engineered bacteria

[0094] The indigo engineered bacteria were inoculated into LB liquid culture medium at an inoculum volume of 2% (v / v), and cultured overnight at 37° C. to obtain a fermentation liquid.

[0095] The fermentation broth was inoculated into 1 mL LB medium containing 0-1 mM sodium thiosulfate concentration gradient at an inoculum volume of 2% (v / v), and then cultured at 37°C for 8 h to obtain each culture solution; each culture solution was centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, the bacteria were washed with PBS and resuspended, and the fluorescence intensity in each resuspended solution was measured; the fluorescence intensity results of the resuspended solutions under different thiosulfate concentration gradient conditions are shown in Figure 2 As shown in a;

[0096] The fermentation broth was inoculated into 50 mL LB medium containing a 0-1 mM thiosulfate concentration gradient at an inoculum volume of 2%, and then cultured at 37°C for 24 h to obtain a culture broth; samples were taken from each culture broth and the absorbance OD at 600 nm was measured. 600nm (like Figure 2 b); then, each culture solution was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the bacteria were resuspended in DMSO; the bacteria in DMSO were ultrasonically disrupted, and the supernatant was collected after centrifugation at 12000 rpm for 10 min, and the indigo content in the supernatant was determined by HPLC (as shown in Figure 2 b).

[0097] The HPLC parameters were as follows: the chromatographic column was a reverse phase column (C18, 250 mm × 4.6 mm, Agilent Technologies, Inc., USA), the flow rate was 0.8 mL / min, the mobile phase was methanol and water (80:20, v / v), and the detection wavelength was 289 nm.

[0098] Depend on Figure 2 It can be seen that within the thiosulfate concentration range of 0 to 1 mM, as the thiosulfate concentration in the culture medium increases, the fluorescence intensity and indigo production both increase accordingly, indicating that the indigo engineered bacteria can respond to changes in the concentration of the inflammatory factor thiosulfate and regulate the expression levels of its signaling molecule sfGFP and the drug indigo accordingly.

[0099] Example 5: In vivo testing of phage-resistant indigo engineered bacteria

[0100] The phage-resistant indigo engineered bacteria, the phage-resistant indigo engineered bacteria containing mutations, and the indigo engineered bacteria without phage resistance were inoculated at an inoculum of 2% (v / v) in LB medium (containing 2 g / L tryptophan) and cultured until OD 600nm 1; take 3mL of culture medium, centrifuge at 8000rpm for 10min, discard the supernatant after centrifugation, and collect the bacteria; wash the bacteria twice with sterile PBS, and resuspend them in 150μl of sterile PBS to obtain bacterial suspensions, which are used as mouse gavage samples. A total of 8 gavage samples were prepared for each bacterium.

[0101] The phage-resistant indigo engineered bacteria containing mutations were prepared according to the methods of Examples 1 to 3. The difference between the phage-resistant indigo engineered bacteria containing mutations and the indigo engineered bacteria is that, in the construction of the recombinant plasmid, when the thsR gene is synthesized, its 57th amino acid is mutated to alanine; the indigo engineered bacteria without phage resistance were prepared according to the method of Example 1 and the transformation method of Example 3, that is, by transforming the recombinant plasmid into E. coli Nissle (EcN) without the cryptic plasmids pMUT1 and pMUT2.

[0102] C57BL / 6J mice (6-8 weeks old) that had been acclimated for at least one week were divided into 5 groups, with 5 mice in each group. DSS0 Group, PBS DSS5 Group, engineered bacteria EPR-DSS5 Group, D57A EPR-DSS5 Group and engineered bacteria DSS5 PBS DSS5 The date of modeling in each group was day 0, in which PBS DSS0 The PBS group was not subjected to DSS (dextran sulfate) inflammation modeling, but was gavaged with PBS for 8 days (from -3rd day to 4th day) as the normal mouse group; DSS5 The DSS inflammation model was established on day 0, and PBS was administered intragastrically for 8 days (day -3 to day 4) to serve as the inflammation mouse group; EPR-DSS5 The DSS inflammation model was established on day 0 in the group, and the suspension of the mutant indigo engineered bacteria was gavaged for 8 days (day -3 to day 4); DSS5 The DSS inflammation model was established on day 0, and the suspension of indigo engineered bacteria without phage resistance was gavaged for 8 days (day -3 to day 4) as the engineered bacteria treatment group without EPR; EPR-DSS5 The DSS inflammation model was established on day 0, and the suspension of phage-resistant indigo engineered bacteria was gavaged for 8 days (day -3 to day 4) as the engineered bacteria treatment group. The disease activity index (DAI) data of mice were collected every day during the test.

[0103] The disease activity index is a comprehensive score based on the percentage of weight loss of the patient (sick animal) (weight unchanged is 0, 1-5 is 1 point, 5-10 is 2 points, 10-15 is 3 points, and more than 15 is 4 points), stool consistency (normal is 0, loose stool is 2 points, and diarrhea is 4 points), and stool bleeding (normal is 0, occult blood is 2 points, and overt bleeding is 4 points). The total score of the three results is divided by 3 to get the DAI value. That is, DAI = (body weight index + stool shape + bleeding) / 3.

[0104] Depend on Figure 3 It can be seen that PBS DSS5 The disease activity index of mice in the group continued to increase after DSS modeling (starting from day 0), indicating that the inflammatory mouse modeling was successful; compared with PBS DSS5 Group, D57A EPR-DSS5 The disease activity index of mice in the control group was only slightly reduced, indicating that the engineered indigo bacteria containing mutations also have certain therapeutic effects, but the effect is relatively limited; EPR-DSS5 Group and engineered bacteria DSS5 The worsening trend of the disease in the mice in the group was suppressed on the fourth day, and their condition began to improve on the fifth day, but the engineered bacteria DSS5Compared with the engineered bacteria EPR-DSS5 The disease remission trend in the group was smaller, indicating that the anti-phage system increased the survival rate of the strain in the intestine, which was beneficial to improving the therapeutic effect of the strain. This proved that phage-resistant indigo engineered bacteria had a significant therapeutic effect on DSS-induced gastrointestinal inflammation.

Claims

1. A phage-resistant indigo engineered bacterium, characterized in that: The phage-resistant indigo engineered bacteria are obtained by genome modification and recombinant plasmid transformation of Escherichia coli Nissle (EcN). The genome modification includes: knocking out the pMUT1 plasmid, knocking out the pMUT2 plasmid, knocking out the fhuA gene, knocking out the trxA gene, and inserting the sspB-sspC-sspD-sspE gene cluster between the aslA gene and the hemy gene of the genome; The recombinant plasmid includes: thsS gene, tac promoter upstream of thsS gene, thsR gene, tac promoter upstream of thsR gene, sfGFP gene, phsA sequence upstream of sfGFP gene, fmo gene and phsA sequence upstream of fmo gene.

2. The phage-resistant indigo engineered bacteria according to claim 1, characterized in that: The nucleotide sequence of the phsA sequence is shown in SEQ ID NO:

4.

3. The phage-resistant indigo engineered bacteria according to claim 1, characterized in that: The nucleotide sequence of the sfGFP gene is shown in SEQ ID NO:

5.

4. The phage-resistant indigo engineered bacteria according to claim 1, characterized in that: The nucleotide sequence of the fmo gene is shown in SEQ ID NO:

6.

5. A method for preparing the phage-resistant indigo engineered bacteria as claimed in claim 1, the method comprising genome modification and recombinant plasmid transformation, wherein: The genome modification comprises the following steps in sequence: S1: constructing pEcCas plasmids, pTarget plasmids and donor DNA required for targeting each gene for gene editing; wherein the pTarget plasmids include pTarget-pMUT1 plasmids, pTarget-pMUT2 plasmids, pTarget-fhuA plasmids, pTarget-trxA plasmids and pTarget-ssp plasmids; the donor DNA includes donor(fhuA), donor(trxA) and donor(ssp); S2: transformation of pEcCas plasmid; S3: transforming the pTarget-pMUT1 plasmid, knocking out the pMUT1 plasmid, and eliminating the pTarget-pMUT1 plasmid; S4: transforming the pTarget-pMUT2 plasmid, knocking out the pMUT2 plasmid, and eliminating the pTarget-pMUT2 plasmid; S5: transform the pTarget-fhuA plasmid and donor(fhuA), knock out the fhuA gene in the genome, and then eliminate the pTarget-fhuA plasmid; S6: transforming pTarget-trxA plasmid and donor(trxA), knocking out the trxA gene in the genome, and then eliminating the pTarget-trxA plasmid; S7: transform the pTarget-ssp plasmid and donor (ssp), insert the sspB-sspC-sspD-sspE gene cluster between the aslA gene and the hemy gene of the genome, and then eliminate the pTarget-ssp and pEcCas plasmids to obtain genome-modified bacteria; Wherein, the recombinant plasmid transformation comprises the following steps: S1: connect the tac promoter to the upstream of the thsS gene to obtain the tac-ThsS expression frame; connect the tac promoter to the upstream of the thsR gene to obtain the tac-ThsR expression frame; connect the phsA sequence to the upstream of the sfGFP gene to obtain the phsA-sfGFP expression frame; connect phsA to the upstream of the fmo gene to obtain the phsA-fmo expression frame; S2: inserting four expression cassettes, tac-thsS, tac-thsR, phsA-sfGFP and phsA-fmo, into the pkD184 backbone plasmid to obtain a recombinant plasmid; S3: Transform the recombinant plasmid into genome-modified bacteria to obtain phage-resistant indigo engineered bacteria.

6. The method according to claim 5, characterized in that In step S2 of genome modification, the pEcCas plasmid is transformed into the wild-type EcN strain by electroporation or chemical transformation to obtain the EcN (pEcCas) strain.

7. The method according to claim 5, characterized in that In steps S3 to S7 of the genome transformation, the specific operation of the transformation process is: the strain obtained in the previous step is induced with L-arabinose in a culture medium containing kanamycin to prepare an electroporation competent strain, and the donor DNA and the pTarget plasmid are electroporated into the competent strain, and the transformed competent strain is spread on a double-resistant plate of kanamycin and spectinomycin for overnight culture, and the strain that survives on the double-resistant plate is the successfully transformed strain.

8. The method according to claim 5, characterized in that In steps S3 to S7 of the genome transformation, the specific operation of eliminating the pTarget plasmid is: culturing the successfully transformed strain overnight in a liquid culture medium containing rhamnose and kanamycin, then streaking the culture solution on a kanamycin resistance plate and spotting it on a spectinomycin plate. The strain that does not grow on the spectinomycin plate but grows on the kanamycin plate is the strain that has eliminated the pTarget plasmid.

9. The method according to claim 5, characterized in that In step S7 of the genome modification, the specific operation of the pEcCas plasmid elimination process is as follows: the strain from which the pTarget plasmid has been eliminated is cultured overnight in a medium containing glucose, and the culture solution is then streaked on a non-resistant plate containing sucrose and glucose, and then on a kanamycin plate. The strain that does not grow on the kanamycin plate but grows on the non-resistant plate is the strain from which the pEcCas plasmid has been eliminated.

10. Use of the phage-resistant indigo engineered bacteria according to any one of claims 1 to 4 in the preparation of a drug for treating or diagnosing gastrointestinal inflammation.

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