CN116287341A - Application of genetically engineered bacteria in preparation of preparation for treating multiple myeloma or relapsed refractory multiple myeloma - Google Patents
Application of genetically engineered bacteria in preparation of preparation for treating multiple myeloma or relapsed refractory multiple myeloma Download PDFInfo
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Abstract
The invention discloses application of genetically engineered bacteria in preparation of a preparation for treating multiple myeloma or relapsed refractory multiple myeloma. The genetically engineered bacterium is citrobacter freundii (Citrobacter freundii) with deaminase related gene mutation; the deaminase related mutated genes are imine/enamine deaminase genes andadenosine deaminase gene. The invention realizes the product NH by introducing imine/enamine deaminase mutant gene or adenosine deaminase mutant gene expression vector, carrying out homologous recombination in citrobacter freundii and replacing wild imine/enamine deaminase gene or wild adenosine deaminase gene with imine/enamine deaminase mutant gene or adenosine deaminase mutant gene 4 + Is reduced and NH is reduced 4 + And (3) a genetically engineered bacterium of the product. The invention provides a new treatment strategy for clinically treating the multiple myeloma which is difficult to treat, and enriches the existing new auxiliary treatment scheme.
Description
Technical Field
The invention relates to the field of genetic engineering and biological medicine, in particular to construction of a citrobacter freundii deaminase related gene mutant strain and application of the deaminase related gene in preparation of a preparation for diagnosing multiple myeloma, and application of the citrobacter freundii deaminase related gene mutant strain in preparation of the preparation for treating multiple myeloma or multiple myeloma with relapse refractory.
Background
Multiple Myeloma (MM) is a terminally differentiated malignant clonal disease of plasma cells, characterized by bone marrow clonal plasma cell infiltration and the appearance of monoclonal M proteins in peripheral blood and/or urine. According to 2018 global cancer report, the incidence rate of the cancer is 0.41% of all human tumors, and the incidence factors including aging and environmental factors are better for the aged, so that the trend of the aged is increasing year by year.
The engineering bacteria are novel microorganisms processed by adopting modern biological engineering technology, and have the characteristics of multifunction, high efficiency, strong adaptability and the like. Current studies indicate that imbalance in intestinal microbial homeostasis promotes the development of a variety of diseases, for example, specific flora enriched in the intestinal tract of tumor patients promotes tumor cell proliferation and induces drug resistance by encoding specific metabolites or small molecules. The homologous recombination technology is utilized to replace the gene coded by the key microorganism to obtain the microorganism without the tumor promotion effect, thereby restoring the flora balance of tumor patients, providing a direction for novel tumor targeted therapy and having wide application prospect.
The invention discovers that the citrobacter freundii (Citrobacter freund)ii, CFr) the RNA level of the deaminase related gene is increased in the intestinal tract of a patient with relapse-resistant MM, and the same result is obtained by in vivo animal experiment verification; then we construct genetic engineering bacteria Citrobacter freundii deaminase related gene mutant bacteria, and experiments in vitro and in vivo prove that NH produced by the deletion of the Citrobacter freundii deaminase related gene 4 + Reduction; finally, the established genetic engineering bacteria are planted in the intestinal tract of the MM mice, the mice are treated by using the first-line clinical medicament Bortezomib (BTZ), and compared with the combination treatment of the multi-myeloma drug-resistant related microorganism, namely the citrobacter freundii and the BTZ, the combination of the genetic engineering bacteria and the BTZ obviously reduces the tumor load of the mice and relieves the BTZ drug resistance induced by the multi-myeloma drug-resistant related microorganism, namely the citrobacter freundii. Therefore, the gene related to the citrobacter freundii deaminase can be used as a marker for predicting and diagnosing the relapse and treatment difficulty of patients with multiple myeloma, and the genetically engineered bacterium citrobacter freundii deaminase related gene mutant bacterium can be used as a new treatment mode of patients with multiple myeloma and relapse and treatment difficulty of multiple myeloma, thereby having important guiding significance for clinical new adjuvant treatment of multiple myeloma.
Disclosure of Invention
The invention provides the application of the specific intestinal microbial marker aiming at the relapse refractory MM, lays a foundation for clinically predicting and diagnosing the relapse refractory MM, and in addition, the invention provides a new treatment mode for patients with relapse refractory multiple myeloma from the aspect of intestinal microbes, proves the effectiveness of genetically engineered bacteria on the treatment of the relapse refractory multiple myeloma and provides a new scheme for the new adjuvant treatment of multiple myeloma.
An object of a first aspect of the present invention is to provide a use of an agent for detecting an expression level of a deaminase-related gene in Citrobacter freundii (Citrobacter freundii) in the intestine, the deaminase-related gene comprising: an imine/enamine deaminase gene with a sequence shown as SEQ ID NO.1 and/or an adenosine deaminase gene with a sequence shown as SEQ ID NO. 2.
The Citrobacter freundii belongs to Citrobacter (Citrobacter of Enterobacteriaceae).
The deaminase related genes are imine/enamine deaminase genes and adenosine deaminase genes, as shown in figure 1: the 2068934-2069329 th nucleotide sequence is imine/Enamine deaminase gene (end), and the 4974011-4974986 th nucleotide sequence is adenosine deaminase gene (Adenosine deaminase, add).
The reagent for detecting the deaminase-related gene expression level in the enterobacter freundii specifically comprises a PCR reagent, in particular a reagent for detecting the RNA expression level of the imine/enamine deaminase gene and/or the adenosine deaminase gene in the stool sample to be detected by qPCR.
Further, the expression of the gene RNA level related to the deaminase of the Citrobacter freundii is significantly higher in fecal samples or anal swab samples of patients with recurrent refractory multiple myeloma than in primary patients.
The primer sequences of the detection imine/enamine deaminase genes are shown as SEQ ID NO.15 and 16, and the primer sequences of the detection adenosine deaminase genes are shown as SEQ ID NO.7 and 8.
The second aspect of the present invention is to provide a diagnostic reagent for multiple myeloma or relapsed refractory multiple myeloma, which is a reagent for detecting the expression level of deaminase-related genes in Citrobacter freundii (Citrobacter freundii) in the intestinal tract, particularly a PCR reagent, and further a reagent for detecting the RNA expression level of imine/enamine deaminase genes and/or adenosine deaminase genes in a stool sample to be detected by qPCR.
It is an object of a third aspect of the present invention to provide a NH reduction 4 + A genetically engineered bacterium of a product, the genetically engineered bacterium being a citrobacter freundii (Citrobacter freundii) that lacks and/or mutates and/or reduces expression of a deaminase-related gene; the deaminase-related gene comprises: an imine/enamine deaminase gene with a sequence shown as SEQ ID NO.1 and/or an adenosine deaminase gene with a sequence shown as SEQ ID NO. 2.
Further, the deaminase-associated gene mutation includes any mutation capable of causing the gene to be lost or weakened.
The invention carries out homologous recombination in Citrobacter freundii by introducing imine/enamine deaminase mutant gene or an expression vector of adenosine deaminase mutant gene, and replaces wild imine/enamine deaminase gene or wild adenosine deaminase gene with imine/enamine deaminase mutant gene or adenosine deaminase mutant gene.
The sequence of the constructed imine/enamine deaminase mutant gene is shown as SEQ ID NO. 23; the sequence of the constructed adenosine deaminase mutant gene is shown as SEQ ID NO. 24.
It is an object of a fourth aspect of the present invention to provide the NH reduction 4 + The application of the genetically engineered bacteria of the product in preparing the preparation for treating multiple myeloma or relapsed refractory multiple myeloma.
It is an object of a fifth aspect of the present invention to provide a therapeutic agent for treating multiple myeloma or relapsed refractory multiple myeloma, comprising said NH-reduction agent 4 + And (3) a genetically engineered bacterium of the product.
It is an object of a sixth aspect of the present invention to provide the NH reduction 4 + The application of the genetically engineered bacterium of the product in preparing a drug resistance preparation for reducing the drug resistance of bortezomib in treating multiple myeloma or patients with multiple myeloma with relapse difficulty.
The seventh aspect of the invention aims to provide a preparation for reducing the drug resistance of bortezomib in patients with multiple myeloma or multiple myeloma with complex refractory treatment, comprising the steps of reducing NH 4 + And (3) a genetically engineered bacterium of the product.
The standard of patients with multiple myeloma which are difficult to treat is diagnosed according to the guidelines for diagnosis and treatment of multiple myeloma in China (revised in 2020).
The mode of effectively inhibiting the drug resistance of the compound refractory multiple myeloma to BTZ by using the genetically engineered bacterium is that the genetically engineered bacterium is continuously and regularly irrigated to the multiple myeloma mouse model body to colonize intestinal tracts after the mice are fed with water for 2 weeks by using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L and vancomycin 0.1 g/L); reducing recurrence difficultyTreatment of NH in intestinal tract and serum of multiple myeloma mice 4 + Is contained in the composition.
Experimental results show that the drug resistance of the multiple myeloma which is difficult to treat to BTZ can be effectively inhibited by the mode of gastric lavage genetic engineering bacteria, and the tumor load of mice is reduced.
The experimental result shows that the genetically engineered bacterium reduces NH 4 + Thereby inhibiting proliferation and drug resistance of multiple myeloma cells and further alleviating drug resistance of MM to BTZ induced by drug resistance-related microorganisms (Drug resistance associated-microbes, DRAM) Citrobacter freundii. The research result of the invention has good application prospect for the new adjuvant therapy of multiple myeloma.
The invention has the advantages that:
(1) The invention realizes NH by introducing homologous mutant sequence of imine/enamine deaminase gene or homologous mutant sequence of adenosine deaminase gene into wild type citrobacter freundii 4 + The reduction of the product (53.36% and 86.69% respectively).
(2) The screening of the relapse refractory markers for diagnosing patients with multiple myeloma is verified by a large number of experiments, so that whether patients with multiple myeloma can be subjected to relapse refractory or not can be accurately predicted and diagnosed.
(3) The invention clarifies that the drug-resistance related microorganism (Drug resistance associated-microbes, DRAM) Citrobacter freundii is produced by a large amount of NH by constructing genetic engineering bacteria 4 + Induces the drug resistance of MM to BTZ.
(4) The invention provides a new treatment strategy for clinically treating the multiple myeloma which is difficult to treat, and enriches the existing new auxiliary treatment scheme.
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The nomenclature used herein and the laboratory procedures are well known and commonly employed in the art. The operations used are generally performed using standard techniques, based on the instructions and conventional specifications of the instrument consumable manufacturer and the references provided herein. It should be noted that the following figures and examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the invention, and the scope of the invention is to be considered as the scope of the invention.
Drawings
FIG. 1A gene map of Citrobacter freundii deaminase.
FIG. 2 shows the expression of the gene related to the deaminase of Citrobacter freundii.
FIG. 3 expression of the gene related to Citrobacter freundii deaminase in the cecum content of mice.
FIG. 4 shows the expression of deaminase gene end and add in feces of patients with initial diagnosis and relapse of refractory MM.
FIG. 5A flow chart (a) of the gene mutation related to Citrobacter freundii deaminase and a pKO3-km vector map (b).
FIG. 6 shows DNA gel diagram after the end and add mutations of the gene related to Citrobacter freundii deaminase.
FIG. 7 cloning identification results after mutation of the gene end and add related to Citrobacter freundii deaminase.
FIG. 8 expression of end and add after mutation of the Citrobacter freundii deaminase-related gene.
FIG. 9 NH in the cecum and serum of mice after transplantation of Citrobacter freundii deaminase-related Gene mutant bacterial lines 4 + And (5) counting content.
FIG. 10 shows the results of in vivo imaging of MM mice after transplantation of a Citrobacter freundii deaminase-related gene mutant bacterial strain.
FIG. 11 statistics of MM mice fluorescence values after transplantation of Citrobacter freundii deaminase-related gene mutant bacterial strains.
FIG. 12 statistical results of IgG2b content in MM mouse serum after transplantation of Citrobacter freundii deaminase-related gene mutant bacterial lines.
FIG. 13 Citrobacter freundii deaminase-related gene mutant bacteriaCecum and NH in serum of MM mice after line transplantation 4 + And (5) counting content.
FIG. 14 cecum of MM mice and NH in serum after transplantation of Citrobacter freundii deaminase-related Gene mutant bacterial lines 4 + And (5) content correlation analysis.
FIG. 15 statistics of end expression in the cecal content of MM mice after transplantation of Citrobacter freundii deaminase-related gene mutant bacterial lines.
FIG. 16 end expression in cecum content and NH in serum of MM mice after transplantation of Citrobacter freundii deaminase-related Gene mutant bacterial lines 4 + And (5) content correlation analysis.
Detailed Description
Example 1: collecting, preserving and RNA extracting fecal samples of primary multiple myeloma patients and recurrent refractory multiple myeloma patients and cecal contents of mice
In the examples, following the relevant ethical requirements, stool samples were taken from 10 primary patients with multiple myeloma (AD) and 10 patients with relapsed refractory multiple myeloma (Relapsed multiple myeloma, RM), all from the affiliated xiangya hospital At the university of south China. All patients with multiple myeloma were diagnosed as patients with multiple myeloma by examination of serum monoclonal immunoglobulin type and bone marrow plasma cell proportion. The diagnosis standard of patients with multiple myeloma difficult to treat is according to the diagnosis of Chinese multiple myeloma diagnosis and treatment guidelines (revised in 2020), and the treatment scheme of the patients at the initial diagnosis is based on bortezomib as the therapeutic drug. The age, sex and body mass index of healthy donors were matched to patients with multiple myeloma to exclude microbial differences caused by age, sex and body mass index.
After the fecal specimen to be detected which is freshly excreted by the MM patient or the mouse is obtained, the fecal specimen is placed into a sterile cryopreservation tube, immediately frozen in liquid nitrogen and then placed into a refrigerator at-80 ℃ for long-term storage, and the anal swab sample is directly placed into the sterile cryopreservation tube, immediately frozen in liquid nitrogen and then placed into the refrigerator at-80 ℃ for long-term storage.
Total microbial RNA in fecal samples was extracted using fecal microbial RNA extraction kit (HiPure Stool RNA Kit, R4185-02) from Meiy (Magen) company, as described in reference to the instructions, and stored at-80℃until use. To examine the quality of the total microbial DNA extracted, the concentration of DNA and the OD 260/280 and OD 260/230 values were determined using Nanodrop, and 1% agarose gel was prepared, 500ng RNA was spotted, running at 100V voltage in a DNA electrophoresis tank for 10min, and whether RNA was degraded was observed under a blue light.
Example 2: fluorescent quantitative PCR (qPCR) for detecting deaminase gene expression in stool or cecum contents
Early studies showed that Citrobacter freundii (Citrobacter freundii, CFr) is a microorganism involved in MM resistance and can produce a large amount of NH 4 + While NH 4 + Resistance of MM to BTZ can be further induced, see in particular patent 202111079749.6.NH (NH) 4 + Can be produced by regulating deaminase in bacteria, a Citrobacter freundii 4974986bp NZ_CP033744.1 gene sequence is found in NCBI database, and primers of 9 deaminase related genes are designed by utilizing an on-line tool Primer Blast in NCBI. In addition, in this example, a pair of internal reference primers (Maeda H, et al, quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, porphyromonas gingivalis, prevotella intermedia, tetQ gene and total bacteria.FEMS Immunology) is required in addition to the gene primers&Medical microbiology.2003, 39:81-86), the primer sequences are shown in SEQ ID NO.3-22. Subsequently, total microbial RNA in the fecal sample was extracted as in example 1, and the expression of 9 deaminase genes in CFr was detected by qPCR, and it was found that the 9 deaminase genes were all expressed in CFr (fig. 2); subsequently, the CFr induced expression of 9 deaminase genes in the cecal content of MM resistant mice was examined and found to be significantly up-regulated in the CFr group and CFr +btz expression of the imine/Enamine deaminase genes (Enamine/imine deaminase, end, primer sequences see SEQ ID nos. 15 and 16) and adenosine deaminase genes (Adenosine deaminase, add, primer sequences see SEQ ID nos. 7 and 8) (fig. 3); finally, we found that the expression of end and add in RM patients was significantly up-regulated by examining the expression of end and add in the feces of AD and RM patients (fig. 4). The results show that deaminase genes end and add are at MThe intestinal faeces of M recurrent drug-resistant patients are obviously up-regulated. The statistical method uses Unpaired t-test.
The experimental method for qPCR detection of microbial gene expression in fecal samples comprises the following steps: firstly, determining the optimal annealing temperature of each primer through gradient annealing temperature PCR; then, respectively carrying out qPCR by using specific primers of each gene by taking cDNA reverse transcribed from RNA extracted from each sample as a template, and recording the cycle number (Ct value) of the corresponding primers of each gene; finally, the relative expression of the genes in each sample was calculated, and the relative abundance of any gene i (Relative abundance, ra) can be formulated as: ra [ ] i )＝(1/2)^(Ct i －Ct c ) Wherein Ct is i Represents the cycle number, ct, of gene i c The number of cycles of the universal primer is shown.
The gradient annealing temperature PCR reaction system is as follows:
and a total of 20. Mu.L. PCR amplification was accomplished on a Biorad PCR apparatus under the following reaction conditions: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, gradient annealing temperature (50℃to 60 ℃) for 15s, extension at 72℃for 20s,32 cycles; further extension was carried out at 72℃for 3min. Preparing 1% agarose gel, spotting 10 mu L of PCR amplified products, running gel for 20min under 100V voltage in a DNA electrophoresis tank, observing the band specificity and brightness of each annealing temperature under a blue light lamp, and determining the annealing temperature of each bacterial primer.
The qPCR reaction system is as follows:
and a total of 20. Mu.L. The qPCR reaction conditions were: 1) Incubating at 50 ℃ for 2min; 2) Pre-denaturation at 95℃for 2min; 3) Denaturation at 95℃for 15s, annealing at 52℃for 15s, elongation at 72℃for 1min, and repeating 40 cycles; 4) Melting curve analysis, default procedure.
TABLE 1 primers involved in example 2
Example 3: construction of genetically engineered bacterium CFr deaminase gene end/add mutant bacterial system
The construction flow is shown in FIG. 5a. Fusion PCR: PCR amplification is carried out on the primer pair of SEQ ID NO.25/SEQ ID NO.26 (169 bp of the length of the amplified product) and SEQ ID NO.27/SEQ ID NO.28 (208 bp of the length of the amplified product), and then the primer pair of SEQ ID NO.25/SEQ ID NO.26 and SEQ ID NO.27/SEQ ID NO.28 is recovered; mixing the purified SEQ ID NO.25/SEQ ID NO.26 and SEQ ID NO.27/SEQ ID NO.28 products at a ratio of 1:1, diluting to a total content of 30ng/ul, performing fusion PCR as a template, performing PCR amplification on a primer pair of SEQ ID NO.25/SEQ ID NO.28 (the length of the amplified product is 287 bp), and recovering a primer pair product glue of SEQ ID NO.25/SEQ ID NO. 28; the product of SEQ ID No.25/SEQ ID No.28 was ligated to pEASY-Blunt Zero Cloning Vector (Transgene pEASY-Blunt Zero Cloning Kit), and the ligation product was transformed into DH 5. Alpha; bacterial liquid PCR identification of positive clones: extracting plasmid after positive cloning amplification, carrying out enzyme digestion on the plasmid NotI by 5 mug, and recovering 287bp fragments by gel; the plasmid NotI of pKO3-km was digested at 5. Mu.g, the 6939bp fragment was recovered in the gel, the product fragment of SEQ ID NO.25/SEQ ID NO.28 was ligated (Transgene T4 DNA Ligase) at 25℃overnight with pKO3-km (FIG. 5 b), DH 5. Alpha. Was transformed with the ligation product, plated (LB-Kana) and incubated overnight at 30℃to select clones for bacterial liquid PCR, and after positive clones were identified, 220rpm was used at 30℃to extract plasmids. Transformation CFr:100 mu L of competent CFr is added with 400ng of plasmid, gently flicked and evenly stirred, added into a precooled 0.2cm electric shock cup at-80 ℃, placed on ice for 15min, electric shocked under the parameters of 25 mu F,200 omega and 2.5Kv, immediately transferred into 10mL of LB after electric transformation, plated with all transformants (LB-Kana) at 30 ℃ for 3h at 200rpm, cultured overnight at 30 ℃, clone identification is selected, positive clones are recombined in two rounds, and finally the CFr bacterial strain with deaminase gene end deletion is obtained. The add mutation primer sequence is shown in SEQ ID NO.29-32, and the construction method is the same as that described above (FIG. 6). 7-8 clones were selected from each of the recombinant mutants: end 1-7 and add 1-8, and detecting NH in the culture medium after amplification culture for 18h 4 + Content (FIG. 7), end 5 (CFr-end) mut ) And add 1 (CFr-add mut ) Carrying out subsequent experiments; extraction of CFr-end mut And CFr-add mut Bacterial RNA was tested for end and add expression, respectively, and it was found that deaminase gene mutation was followed by down-regulation of end and add expression, respectively (FIG. 8). The result shows that the genetically engineered bacterium CFr-end mut And CFr-add mut The bacterial system was constructed successfully.
TABLE 2 primers involved in example 3
Example 4: genetically engineered bacterium CFr-end mut And CFr-add mut Bacterial lines against cecum and NH in serum in the mouse intestinal tract 4 + Influence of (2)
For detecting the genetically engineered bacterium CFr-end mut And CFr-add mut Bacterial lines are colonized in the mouse intestinal tract followed by NH in cecum and serum 4 + Is performed using normal C57BL6 mice. 15 mice were randomly divided into 3 groups of 5 mice each, CFr-WT and CFr-end respectively mut Group sum CFr-add mut A group. CFr-WT group: mice were fed with water for 2 weeks using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L, vancomycin 0.1 g/L), followed by replacement with normal drinking water and lavage CFr-WT (2X 10) 8 200 μl), frequency of 3 times per 1 week, and 2 weeks of gavage; CFr-end mut Group: mice were fed with water for 2 weeks using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L, vancomycin 0.1 g/L), followed by replacement with normal drinking water and lavage CFr-end mut (2×10 8 200 μl), frequency of 3 times per 1 week, and 2 weeks of gavage; CFr-add mut Group: mice were fed with water for 2 weeks using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L, vancomycin 0.1 g/L), followed by replacement with normal drinking water and lavage CFr-add mut (2×10 8 200. Mu.L), frequency was 3 times per 1 week, and stomach was irrigated for 2 weeks. After termination of the experiment cheek blood was collected and cecal content was collected.
The animal experiment result shows that compared with the CFr-WT group, CFr-end mut Group sum CFr-add mut Group cecum and NH in serum 4 + Significantly reduced, and CFr-end mut The group reduction was more pronounced (fig. 9). The statistical method uses Unpaired t-test. This result indicates CFr-end mut And CFr-add mut Colonisation in the gut reduces NH in cecum and serum 4 + Is contained in the composition.
Example 5: genetically engineered bacterium CFr-end mut Effect of bacterial lines colonization in the mouse intestinal tract on multiple myeloma BTZ resistance
The 5TGM1 mouse MM model was used. 35 mice were randomly divided into 7 groups of 5, PBS group, BTZ group, CFr-WT group, CFr-WT+BTZ group, CFr-end mut Group, CFr-end mut +BTZ group and CFr-end mut +BTZ+NH 4 And Cl group. PBS group: mice were fed with water for 2 weeks using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L, vancomycin 0.1 g/L), then replaced with normal drinking water and lavage PBS (200. Mu.L/L), frequency 3 times for 1 week, and 1X 10 by tail vein injection after 1 week of lavage 6 5TGM1 cells with luciferase reporter (5 TGM 1-Luc) followed by lavage with PBS (200. Mu.L/dose) at a frequency of 3 times 1 week; BTZ group: mice were fed with water for 2 weeks using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L, vancomycin 0.1 g/L), then replaced with normal drinking water and lavage PBS (200. Mu.L/L), frequency 3 times for 1 week, and 1X 10 by tail vein injection after 1 week of lavage 6 5TGM1 cells with luciferase reporter gene (5 TGM 1-Luc), lavage PBS (200. Mu.L/dose), frequency 3 times per week 1, and intraperitoneal administration of BTZ (0.75 mg/kg) 3 times per week 1; CFr-WT group: mice were fed with water for 2 weeks using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L, vancomycin 0.1 g/L), followed by replacement with normal drinking water and lavage CFr-WT (2X 10) 8 200. Mu.L) at a frequency of 3 times per 1 week, 1X 10 by tail vein injection after 1 week of intragastric administration 6 5TGM1 cells with luciferase reporter (5 TGM 1-Luc) followed by lavage CFr-WT (2X 10) 8 200 μl), frequency was 3 times per week 1; CFr-WT+BTZ group: mice were fed with water for 2 weeks using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L, vancomycin 0.1 g/L), followed byChanging into normal drinking water and irrigating stomach CFr-WT (2×10) 8 200. Mu.L) at a frequency of 3 times per 1 week, 1X 10 by tail vein injection after 1 week of intragastric administration 6 5TGM1 cells with luciferase reporter (5 TGM 1-Luc), gastric lavage mice CFr-WT (2X 10) 8 200 μL) 1 week later, BTZ (0.75 mg/kg) was administered intraperitoneally, at a frequency of 3 times per week 1, with lavage CFr-WT alternating with intraperitoneal administration of BTZ; CFr-end mut Group: mice were fed with water for 2 weeks using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L, vancomycin 0.1 g/L), followed by replacement with normal drinking water and lavage CFr-WT (2X 10) 8 200. Mu.L) at a frequency of 3 times per 1 week, 1X 10 by tail vein injection after 1 week of intragastric administration 6 5TGM1 cells with luciferase reporter (5 TGM 1-Luc) followed by lavage CFr-end mut (2×10 8 200 μl), frequency was 3 times per week 1; CFr-end mut +btz group: mice were fed with water for 2 weeks using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L, vancomycin 0.1 g/L), followed by replacement with normal drinking water and lavage CFr-end mut (2×10 8 200. Mu.L) at a frequency of 3 times per 1 week, 1X 10 by tail vein injection after 1 week of intragastric administration 6 5TGM1 cells (5 TGM 1-Luc) with luciferase reporter gene, gastric lavage mice CFr-end mut (2×10 8 200 μL) for 1 week, BTZ (0.75 mg/kg) was administered intraperitoneally 3 times at a frequency of 1 week, and gastric lavage CFr-end mut Alternating with intraperitoneal administration of BTZ; CFr-end mut +BTZ+NH 4 Cl group: mice were fed with water for 2 weeks using tetranectin antibiotics (ampicillin 0.2g/L, neomycin 0.2g/L, metronidazole 0.2g/L, vancomycin 0.1 g/L), followed by replacement with normal drinking water and lavage CFr-end mut (2×10 8 200. Mu.L) at a frequency of 3 times per 1 week, 1X 10 by tail vein injection after 1 week of intragastric administration 6 5TGM1 cells (5 TGM 1-Luc) with luciferase reporter gene, gastric lavage mice CFr-end mut (2×10 8 200 μl), frequency of 3 times per 1 week, and NH of intragastric mice 4 Cl, frequency was daily, after 1 week, BTZ (0.75 mg/kg) was administered intraperitoneally, frequency was 3 times per week, and stomach was irrigated CFr-end mut And NH 4 Cl and celiac BTZ administration are alternated. Collecting mouse faecal specimens before water feeding antibiotics and before cell injection, and after cell injectionLiving imaging and cheek blood sampling were performed for 6 weeks.
Animal experiment results show that compared with PBS group, the fluorescence value of BTZ group is obviously reduced, compared with BTZ group, the fluorescence value of CFr-WT+BTZ group is obviously increased, compared with CFr-WT+BTZ group, CFr-end mut The fluorescence value of +BTZ group is obviously reduced, and CFr-end mut CFr-end compared to +BTZ group mut +BTZ+NH 4 Cl group fluorescence value increased significantly (fig. 10, fig. 11); the same results were obtained for the content of IgG2b secreted by MM cells in serum (fig. 12); then, we examined the NH in cecum and serum 4 + Is found to be CFr-end compared to CFr-WT transplantation mut Post-implantation NH 4 + Is reduced in content in CFr-end mut Post-implantation replenishment of NH 4 Cl, NH thereof 4 + In turn (FIG. 13), and NH in cecum and serum of each group 4 + Is positively correlated (fig. 14); finally, we examined the expression of end in cecal content and found CFr-end compared to CFr-WT transplantation mut Reduced expression of end after transplantation (FIG. 15), CFr-end mut +BTZ+NH 4 After Cl removal (NH is removed) 4 Cl interference), we analyzed end expression and NH in cecum and serum 4 + Is found to be positively correlated (fig. 16). Statistical methods used Unpaired t-test, correlation analysis Correlation analysis. This result indicates CFr-end mut Colonisation in the gut reduces NH in cecum and serum 4 + Thereby alleviating CFr-WT induced MM resistance to BTZ.
The imine/enamine deaminase gene sequence is as follows: SEQ ID NO.1
ATGAGAAAAGTTATTGCAACCGAATGTGCGCCAGGGGCTATCGGGCCTTACGTACAGGGTGTGGATCTGGGCAGCATGGTGTTGACGTCAGGTCAAATCCCGGTGTGTCCACAGACCGGTGAAGTGGCTGAAAACGTATCCGATCAAGCGCGTCAAAGCCTGGAAAACGTGAAAGCGATTGTCGAGTCTGCAGGTTTGAAAGTGAGCGATATCGTCAAGACCACCGTTTTCGTTTCCGACCTGAACGACTTCGCCACCATCAATCAGGTGTACCAGCAGTTCTTTGATGAGCATAAGGCAATCTACCCTACGCGCAGCTGCGTGCAGGTCGCCCGCTTACCAAAGGATGTGAAGCTGGAGATTGAAGCCATCGCCGTACGTGGCGACACGCTGTAA
The gene sequence of the adenosine deaminase is shown as SEQ ID NO.2
CGACGTCCATCGCCACCTTGATGGTAACATCCGTGCCCAAACGATTCTGGATCTGGGTCGTCAGTTCAATTTAACGCTCCCGGCACAAACGTTGGAAACGCTGATCCCTCATGTGCAAGTGACATCCACTGAGCCAGATTTAGTGAGCTTTTTATCCAAGCTCGACTGGGGCGTGAAGGTACTGGCCTCGCTGGATGCCTGTCGCCGCGTGGCATTTGAAAATATTGAGGATGCAGCACGTAACGGTCTGCACTATGTAGAATTACGTTTTTCACCAGGCTATATGGCGATGGCGCACCAACTCCCGGTGGCAGGTGTGGTTGAAGCCGTCATCGCAGGCGTGCGCGAGGGCTGCAAGACCTTTGGCGTCGAAGCACGCTTAATTGGCATTATGAGCCGTACTTTTGGCGAAGCCGCCTGTCTGCAGGAACTGGATGCGCTGTTAGCCCACCGAGATCATATTACGGCACTGGATCTAGCCGGTGACGAACTGGGATTCCCTGGCAGTCTGTTCCTGTCCCATTTCAACCAGGCACGCGACGCCGGCTGGCATATTACCGTGCATGCGGGCGAAGCCGCGGGACCAGAAAGCATCTGGCAGGCCATTAAAGAGCTGGGCGCTGAGCGTATTGGTCACGGCGTTAAAGCCGTTGAAGATCGTGCGTTGATGGATTACCTCGCCGAGCATCGTATCGGCATCGAATCCTGCCTGACATCAAACATTCAGACCAGCACCGTTGCCTCACTAGCGAACCATCCGCTGAAAACGTTCCTTGAGCATGGCGTTATTGCCAGTTTGAATACGGACGACCCGGCTGTTCAGAGTGTGGATATTATTCACGAGTACACCATTGCCGCACCAGCTGCAGGCCTAACGCGCGAACAGATCCGCCAGGCGCAAATCAACGGTCTGGAAATGGCATTCCTGAGTAACGAAGAAAAACGAGCCTTGCGCGAGAAAGTCGGCGCAGCGTAA
The imine/enamine deaminase gene homologous mutation sequence is as follows: SEQ ID NO.23
ATGAGAAAAGTTATTGCAACCGAATGTGCGCCAGGGGCTATCGGGCCTTACGTACAGGGTGTGGATCTGGGCAGCATGGTGTTGACGTCAGGTCAAATCCCGGTGTGTCCACAGACCGGTGAAGTTCCGACCTGAACGACTTCGCCACCATCAATCAGGTGTACCAGCAGTTCTTTGATGAGCATAAGGCAATCTACCCTACGCGCAGCTGCGTGCAGGTCGCCCGCTTACCAAAGGATGTGAAGCTGGAGATTGAAGCCATCGCCGTACGTGGCGACACGCTGTAA
The homologous mutation sequence of the adenosine deaminase gene is as follows: SEQ ID NO.24
CGACGTCCATCGCCACCTTGATGGTAACATCCGTGCCCAAACGATTCTGGATCTGGGTCGTCAGTTCAATTTAACGCTCCCGGCACAAACGTTGGAAACGCTGATCCCTCATGTGCAAGTGACATCCACTGAGCCAGATTTAGTGAGCTTTTTATCCAAGCTCGACTGGGGCGTGAAGGTACTGGCCTCGCTGGATGCCTGTCGCCGCGTGGCATTTGAAAATATTGAGGATGCAGCACGTACCAGCACCGTTGCCTCACTAGCGAACCATCCGCTGAAAACGTTCCTTGAGCATGGCGTTATTGCCAGTTTGAATACGGACGACCCGGCTGTTCAGAGTGTGGATATTATTCACGAGTACACCATTGCCGCACCAGCTGCAGGCCTAACGCGCGAACAGATCCGCCAGGCGCAAATCAACGGTCTGGAAATGGCATTCCTGAGTAACGAAGAAAAACGAGCCTTGCGCGAGAAAGTCGGCGCAGCGTAA。
Claims (10)
1. Use of a reagent for detecting the expression level of a deaminase-related gene in enterobacter freundii (Citrobacter freundii) for the preparation of a diagnostic formulation for multiple myeloma or relapsed refractory multiple myeloma, characterized in that the deaminase-related gene comprises: an imine/enamine deaminase gene with a sequence shown as SEQ ID NO.1 and/or an adenosine deaminase gene with a sequence shown as SEQ ID NO. 2.
2. The use according to claim 1, wherein the reagent for detecting the level of deaminase-related gene expression in Citrobacter enterica comprises specifically PCR reagents, in particular reagents for detecting the level of imine/enamine deaminase gene and/or adenosine deaminase gene RNA expression in a fecal sample to be tested by qPCR.
3. The use according to claim 2, wherein the primer sequences for detecting imine/enamine deaminase gene are shown in SEQ ID NO.15 and 16, and the primer sequences for detecting adenosine deaminase gene are shown in SEQ ID NO.7 and 8.
4. A diagnostic reagent for multiple myeloma or relapsed refractory multiple myeloma is characterized in that the reagent is used for detecting the expression level of deaminase related genes in Citrobacter freundii (Citrobacter freundii) in intestinal tracts, particularly a PCR reagent, and further a reagent for detecting the RNA expression level of imine/enamine deaminase genes and/or adenosine deaminase genes in a fecal sample to be detected by qPCR.
5. NH reduction 4 + A genetically engineered bacterium of a product, characterized in that the genetically engineered bacterium is a citrobacter freundii (Citrobacter freundii) that lacks and/or mutates and/or reduces expression of a deaminase-related gene; the deaminase-related gene comprises: imine/enamine deaminase gene with sequence shown as SEQ ID NO.1 and/or sequence shown as SEQ ID NO.2Adenosine deaminase gene of (a).
6. The NH-reducing device of claim 5 4 + Genetically engineered bacterium which is a product, characterized in that said deaminase-related gene mutation comprises any mutation which is capable of causing a loss or attenuation of the function of the gene.
7. The NH reduction of claim 5 or 6 4 + The application of the genetically engineered bacteria of the product in preparing the preparation for treating multiple myeloma or relapsed refractory multiple myeloma.
8. A therapeutic agent for treating multiple myeloma or relapsed refractory multiple myeloma, comprising the NH-reducing agent according to claim 5 or 6 4 + And (3) a genetically engineered bacterium of the product.
9. The NH reduction of claim 5 or 6 4 + The application of the genetically engineered bacterium of the product in preparing a drug resistance preparation for reducing the drug resistance of bortezomib in treating multiple myeloma or patients with multiple myeloma with relapse difficulty.
10. A preparation for reducing resistance of bortezomib in patients with multiple myeloma or multiple myeloma with a relapse refractory, comprising the NH-reducing agent according to claim 5 or 6 4 + And (3) a genetically engineered bacterium of the product.
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