CN113416683B - Escherichia coli Nissle1917 genetically engineered bacterium and preparation method and application thereof - Google Patents
Escherichia coli Nissle1917 genetically engineered bacterium and preparation method and application thereof Download PDFInfo
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- CN113416683B CN113416683B CN202110607198.XA CN202110607198A CN113416683B CN 113416683 B CN113416683 B CN 113416683B CN 202110607198 A CN202110607198 A CN 202110607198A CN 113416683 B CN113416683 B CN 113416683B
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- escherichia coli
- nissle1917
- genetically engineered
- engineered bacterium
- coli nissle1917
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an escherichia coli Nissle1917 genetically engineered bacterium, a preparation method and application thereof, and the escherichia coli Nissle1917 genetically engineered bacterium with a function of improving sports injury is constructed by inserting a glucagon-like peptide gene sequence secreted by human intestinal L cells into a pseudonuclear chromosome genome of the escherichia coli Nissle 1917. The engineering probiotics can efficiently express GLP-1 in vitro, avoid the defects of unstable plasmid overexpression system, easy drug resistance and the like, have good acid resistance, bile salt resistance and oxidation resistance, and can also obviously improve the motor injury of parkinsonism mice and reduce the neuroinflammation in the brain. The escherichia coli Nissle1917 genetically engineered bacterium can be used for preparing foods or medicines capable of improving sports injury, and has important practical significance and economic value for application in the aspect of treatment of parkinsonism.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and mainly relates to an escherichia coli Nissle1917 genetic engineering bacterium, a preparation method and application thereof.
Background
Neurological diseases and injuries are one of the leading causes of disability worldwide, with Parkinson's Disease (PD) growing the fastest in prevalence, disability rate and mortality, placing a heavy burden on the patient's home and society. Unfortunately, no reliable therapeutic drug has been found that can stop or reverse PD progression. Therefore, it is important to find a safe drug capable of delaying, preventing and even reversing neurodegeneration, and protecting neurons from loss, thereby treating the diseases.
The probiotics are used as active microorganisms beneficial to hosts, and can regulate intestinal flora balance, thereby enhancing intestinal epithelial integrity, protecting intestinal barrier, regulating mucosal immune system, inhibiting pathogenic bacteria growth and the like.
In the aspect of protein medicines, glucagon-like peptide-1 (GLP-1) produced by intestinal L cells can relieve the resistance of brain to insulin, enhance the sensitivity of the brain to insulin signals, and relieve neurodegenerative diseases such as PD, alzheimer Disease (AD) and the like. Although GLP-1 is derived from human body, the curative effect is remarkable and the side effect is small. However, the amino acid sequence of GLP-1 has a site which can be recognized by dipeptidyl peptidase IV (DPP-IV), is easy to be degraded by DPP-IV in vivo, has a half-life of only a few minutes, and greatly limits the physiological efficacy; furthermore, the pain caused by repeated injections of GLP-1 can be prohibitive for many patients.
Disclosure of Invention
The invention aims to solve the defects of the prior art, and provides an escherichia coli Nissle1917 genetically engineered bacterium, wherein the nucleotide sequence of the genetically engineered bacterium is shown as SEQ ID NO. 1.
A preparation method of escherichia coli Nissle1917 genetically engineered bacteria comprises the following steps: the GLP-1 gene sequence is integrated into the genome of escherichia coli Nissle1917, and the nucleotide sequence of the genetically engineered bacterium is shown as SEQ ID NO. 1.
Preferably, the GLP-1 gene is synthesized by chemical means.
The escherichia coli Nissle1917 genetically engineered bacterium is verified and can be applied to the fields of fermentation strains, preparation of probiotics tablets for improving sports injury, preparation of foods with neuroprotection function, preparation of medicaments for treating parkinsonism and the like.
The beneficial effects of the invention are as follows: the escherichia coli Nissle1917 has obvious advantages in the aspects of gastric acid resistance, digestive tract bile salt resistance, cerebral inflammation reduction and the like. The engineering bacteria can obviously reverse the parkinsonism of mice induced by 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP), reduce inflammatory factors in the brain and increase anti-inflammatory factors. The escherichia coli Nissle1917 engineering bacteria provide a foundation for developing probiotic products with the function of improving exercise, and can be applied to the preparation of foods or medicines with the function of neuroprotection.
Drawings
FIG. 1 is a schematic diagram showing the measurement result of GLP-1 in vitro expression of engineering bacteria of Escherichia coli Nissle 1917;
FIG. 2 is a diagram showing the acid resistance measurement result of the engineering bacterium of the escherichia coli Nissle 1917;
FIG. 3 is a diagram showing the results of determining the cholate resistance of the engineering bacteria of the Escherichia coli Nissle 1917;
FIG. 4 is a schematic diagram showing the results of antioxidant assay of engineering bacteria of E.coli Nissle 1917;
FIG. 5 is a schematic diagram showing the experimental result of the E.coli Nissle1917 engineering bacteria on the pole climbing of MPTP-induced parkinsonism mice;
FIG. 6 is a schematic diagram showing the experimental results of the E.coli Nissle1917 engineering bacteria on MPTP-induced parkinsonism mice in open field;
FIG. 7 shows the effect of E.coli Nissle1917 engineering bacteria on IL-6, TNF- α, IL-1β mRNA levels in MPTP-induced parkinsonism mice brains;
FIG. 8 shows PCR conditions of example 5.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects and effects of the present invention.
Example 1: acquisition and identification of engineering bacteria of escherichia coli Nissle1917
1. Acquisition and authentication of Nissle1917
The feces of healthy human body are preserved by adopting 50% glycerol, and are subjected to gradient dilution by using sterile Phosphate Buffer (PBS), 3-5 proper gradients are selected, 0.1mL of the feces are absorbed into LB medium for streaking separation, and each dilution gradient is repeated. Placing the culture dish into a 37 ℃ incubator for aerobic culture for 48 hours, and then selecting white colonies which are smooth, convex, regular in edge and semi-transparent for microscopic examination. The suspected colonies were subjected to further isolation and purification in LB medium as described above until the colonies were completely purified. Purified colonies were serially passaged 3 times and then treated with 50% glycerol to bacterial liquid 1:1 were stored in-80 ℃.
2. Construction of engineering bacteria of escherichia coli Nissle1917
The GLP-1 target gene is synthesized by a chemical synthesis method, the nucleotide sequence of the GLP-1 target gene is shown as SEQ ID NO.2, and then the GLP-1 target gene is integrated into the genome of escherichia coli Nissle1917 to obtain genetically engineered bacteria. The construction of the engineering strain is completed by cooperation of a laboratory and Hangzhou Baisai biotechnology Co-ordination.
2.1 primer design
1917attB-upF:GAAAGCCCAATCTTCACATCAATC
1917-attB-UP-R:CCTGTGTGAAATTGTTATCCGCTAAAAAAGCAGGCTTCAAC
Pelb-hglB-F：TGAAGCCTGCTTTTTTAGCGGATAACAATTTCACACAGGA
pelB-hglB-R：CGCTCAAGTTAGTATCCCAGTCACGACGTTGTAAAACGAC
1917-attb-down-F：ACAACGTCGTGACTGGGATACTAACTTGAGCGAAACGGGA
1917attb-downR：TTACGATGGCGATAATATTTCACC
1917attb-JD-UP-F:TGCGCCGCGACCAGAAACGATAT
1917attB-JDdown-R:CGTAGTACGCATCGGTACGCCAA
2.2 repair homology arm amplification
Genome engineering repair homology arm construction
The Nissle1917 genomic DNA was used as a template and 1917attB-UP-F/1917-attB-UP R and 1917attB-downF/1917attB-down-R were used as templates for amplification, and the amplification system was as follows:
the Pelb-hglB-F/pelB-hglB-R was amplified using the previously constructed pBSC-Pelb-hglp-4 plasmid as a template, and the amplification system was as follows:
and (3) recovering the amplified fragments by using a PCR product purification kit, and performing fusion PCR amplification according to the following system, wherein the amplification conditions are as follows: 94℃5min 2cycle (94℃30sec, 50℃30sec, 72℃ 40 sec), 30cycle (94℃30sec, 50℃30sec, 72℃ 40 sec) 10℃hold on.
2.3Crisp integration
Preparation of the MG1655 delta lacZ Strain the electrotransformation competent transformation knockout plasmid and identification of repair homology arm coated plates
Integration authentication 1917attb-JD-UP-F: TGCGCCGCGACCAGAAACGAT
1917attB-JDdown-R:CGTAGTACGCATCGGTACGC
Wherein, the successful insertion is 2579bp, and the unsuccessful insertion is 1739bp.
3. GLP-1 in vitro expression determination result of escherichia coli Nissle1917 engineering bacteria
GLP-1 expression in the fermentation supernatant of the engineering bacteria is detected in vitro by referring to the instructions of the human GLP-1ELISA detection kit of Abcam company.
As shown in FIG. 1, the engineering bacterium of the escherichia coli Nissle1917 can efficiently express GLP-1 protein (88-104 pg/mL).
Example 2: acid resistance experiment of E.coli Nissle1917 engineering bacteria
E.coli Nissle1917 and engineering bacteria were inoculated at a volume of 1/100 and subcultured in LB liquid medium for 18h. Centrifuging at 8000rpm for 5min, reserving the precipitate, discarding the supernatant, washing with sterile PBS buffer solution for 2 times, respectively sub-packaging into 5 1.5mL centrifuge tubes, centrifuging at 8000rpm for 5min, and obtaining 5 parts of wild E.coli Niss le1917 and engineering bacteria respectively. And then respectively adding pre-prepared PBS buffer solutions with pH values of 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0, placing the mixture in a 37 ℃ incubator for static culture for 2 hours, taking the ratio (10 times) of 0h and 2h bacterial suspension to dilute after the culture is finished, measuring the viable count by a plate counting method, and calculating the survival rate of wild escherichia coli Nissle1917 and engineering bacterial strains.
The results are shown in FIG. 2, in which wild type E.coli was cultured in PBS at pH=4 for 2 hours in an acid-proof experimentThe number of Nissle1917 and engineering bacteria can reach 10 8 The CFU/mL is higher than that, and the acid resistance is stronger. ( Coliinissle 1917: wild type E.coli 1917-attb-pelB-GLP-1: engineering bacteria group )
EXAMPLE 3 bile salt tolerance experiment of E.coli Nissle1917 engineering bacteria
E.coli Nissle1917 and engineering bacteria were inoculated at a volume of 1/100 and subcultured in LB liquid medium for 18h. Centrifuging at 8000rpm for 5min, reserving the precipitate, discarding the supernatant, washing with sterile PBS buffer solution for 2 times, respectively sub-packaging into 5 1.5mL centrifuge tubes, centrifuging at 8000rpm for 5min, and obtaining 5 parts of wild Escherichia coli Nissle1917 and engineering bacteria respectively. And then respectively adding PBS buffer solutions with the pre-prepared bile salt concentration of 0%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, placing in a 37 ℃ incubator for static culture for 2 hours, taking the ratio (10 times) of 0h and 2h bacterial suspension to dilute after the culture is finished, measuring the viable count by a plate counting method, and calculating the survival rate of the wild escherichia coli Nissle1917 and the engineering bacterial strain.
As shown in FIG. 3, in the cholate-resistant experiment, both engineering bacteria and wild E.coli Nissle1917 showed good cholate resistance, and both bacteria could reach 10 when cultured for 2h at 0.3% cholate concentration 9 CFU/mL or more. ( Coliinissle 1917: wild type E.coli 1917-attb-pelB-GLP-1: engineering bacteria group )
Example 4: antioxidant experiment of engineering bacteria of escherichia coli Nissle1917
(1) Respectively culturing wild E.coli Nissle1917 and engineering bacteria with LB liquid culture medium, centrifuging at 10000rpm for 10min to precipitate thallus, sucking supernatant, and storing in refrigerator.
(2) Measurement of DPPH radical scavenging ability:
1mL of supernatant was mixed with 1mL of the prepared methanol solution of DPPH free radical, and after shaking uniformly, the mixture was reacted at room temperature for 30min under the dark condition, and the OD value was measured at 517nm wavelength (deionized water was used as a blank control).
DPPH radical clearance: [1-A ] 517 (sample)/A 517 (blank)]×100％
(3) Determination of the ability to scavenge hydroxyl radicals:
1mL of the supernatant was put into a glass test tube (sample tube), and 1mL of ddH was put into a blank tube 2 O, 1mL of 3mmol/L salicylic acid, 1mL of 1mmol/L FeSO, respectively, are added 4 1mL of 3mmol/L H 2 O 2 After being uniformly mixed, the mixture is reacted for 15min in a water bath kettle at 37 ℃, and the absorbance is measured at the wavelength of 510nm by a spectrophotometer, and the hydroxyl radical clearance (%) = [1-A ] 510 (sample)/A 510 (blank)]×100％。
(4) Determination of the ability to scavenge superoxide radicals:
preparing Tris-HCl buffer (0.05 mol/L, 1mmol/L Na) 2 EDTA), 0.5mL of the supernatant was added to a glass test tube No.1, 2mL of Tris-HCl solution and 1mL of pyrogallol solution were added, no.2 tube was added with 0.5mL of deionized water, 2mL of Tris-HCl solution and 1mL of pyrogallol solution, no. 3 tube was added with 1.5mL of deionized water, 2mL of Tris-HCl solution, no. 4 tube was added with 1mL of deionized water, 0.5mL of bacterial supernatant and 2mL of Tris-HCl solution, and the superoxide radical scavenger (%) = [1- (A) 11 -A 10 )/(A 01 -A 00 )]X 100%, wherein a00: no sample and pyrogallol (3); a01: no sample contains pyrogallol (2); a10: the sample contained no pyrogallol (4); a11: comprises a sample and pyrogallol (1).
(5) Determination of ferrous ion chelating ability:
0.5mL of supernatant was added into a glass tube, 0.1mL of 2mol/L FeSO4,0.1mL of 1% vitamin C and 1mL of 0.2mol/L NaOH solution were added, the mixture was uniformly mixed, the reaction was continued for 20min at 37℃and 10% trichloroacetic acid was further added, and the precipitate protein was centrifuged at 600 rpm for 10min at 4℃of a refrigerated centrifuge. 0.4mL of the supernatant was taken and 4mL of phenanthroline was added. The blank was added with 0.5mL ddH 2 0, the others are the same. Measuring absorbance at 536nm after reacting for 10min at room temperature, fe 2+ Chelating ability= (a Blank space -A Sample of )/A Blank space ×100％。
(6) Determination of the reduction Activity:
1mL of the bacterial supernatant was taken, 1mL of 0.2mol/L PBS buffer and 1mL of 1% potassium ferricyanide were added, and after mixing, the mixture was reacted at 50℃for 20miAfter n 1mL of 10% trichloroacetic acid was added and the reaction was centrifuged at 6000rpm for 10min. 1mL of the mixture was taken and 4mL of ddH was added 2 0 and 0.4mL of 0.1% FeCl 3 The solution was reacted at room temperature for 10min. Blank control tube plus 1mL ddH 2 0, the other added reagents are the same as the treatment. Absorbance was measured at a wavelength of 700nm after standing.
As shown in FIG. 4, the oxidation resistance of the two strains is evaluated, and the oxidation resistance test results show that the clearance rate of the E.coli 1917-attb-pelB-GLP-1 and the clearance rate of the E.coli 1917-pelB-GLP-1 and the clearance rate of the E.coli Nissle1917 are high, and the clearance rates are not significantly different (95.4% vs 95.2% and 81.3% vs 78.6%). Superoxygen radical scavenging Rate and Fe of E.coli 1917-attb-pelB-GLP-1 and Nissle1917 2+ The chelation rate is low.
Example 5: e.coli 1917-attb-pelB-GLP-1 Effect on motor function in Parkinson mice
1. Preparation of the experimental strains: inoculating E.coli 1917-attb-pelB-GLP-1 into LB medium, culturing at 37deg.C overnight, inoculating LB medium at 1%, culturing at 37deg.C again to OD 600 Centrifugation at 3000rpm for 4min at 0.6 to collect cells, washing with sterile physiological saline for 2 times, re-suspending with gelatin physiological saline containing 0.1% to adjust the number of cells to 1×10 7 cfu/mL。
2. Experimental animals and group treatment: the 15 week old C57/BL6 male mice (purchased from Hunan Stokes Seda Co., ltd.) were kept for one week with adaptability, and the experiment was started after weighing the markers.
a. Control group (12 weeks of feeding and treatment with 0.1% gelatin in physiological saline, n=12);
b. parkinson model group (n=12): MPTP is dissolved in sterile physiological saline to make the concentration of MPTP be 20mg/kg, and the mice are injected intraperitoneally for 7 days continuously;
c. parkinson model + Nissle1917 treatment group (n=12): after parkinsonism modeling, nissle1917 was dissolved in physiological saline containing 0.1% gelatin as drinking water treatment (1×10) 7 cfu/day);
d. parkinson model+e.coll 1917-attb-pelB-GLP-1 treatment group (n=12): e.coli 1917-attb-pelB-GLP-1 in physiological saline containing 0.1% gelatin as drinking waterTreatment (1X 10) 7 cfu/day);
e. parkinson model + GLP-1 treatment group (n=12): GLP-1 was dissolved in sterile physiological saline to a final concentration of 24nmol/kg and was intraperitoneally injected into mice.
After evaluating the open field experiment and the pole climbing experiment, mice were sacrificed and brain tissues of the mice were collected.
3. Experimental animal pole climbing experiment: the motor delay of PD mice was evaluated by the metal bar test. The mouse was placed head down on top of a gauze wrapped metal rod. The time the mouse climbs from the top of the pole to the bottom of the pole was recorded manually. Each mouse was repeated three times with one pause between each experiment.
4. Experimental animals open field experiment: each mouse was placed in a (90 cm x 30 cm) dark box activity room for 10 minutes and the total distance of movement of the mouse and time to central area was measured using activity monitor video tracking software. To minimize odor interference, the darkroom was cleaned with 75% ethanol after each run.
5. Determining brain tissue inflammatory factors of experimental animals:
(1) Extraction of brain tissue RNA
Grinding brain tissue into powder in liquid nitrogen, adding 1mL of Rizol solution into 50-100mg of tissue, grinding, taking the total volume of the sample not exceeding 10% of the volume of the used Rizol, repeatedly blowing with a gun or shaking vigorously to lyse cells, and standing at room temperature of 15-30 ℃ for 5 minutes; adding chloroform in a proportion of 0.2mL for each 1mL of Trizol solution, covering the centrifuge tube tightly, shaking the centrifuge tube vigorously by hand for 15 seconds, and standing for 2-3 minutes at room temperature; centrifuging at 12000rpm and 4 ℃ for 15min; taking the upper water phase into a new centrifuge tube, adding isopropanol into the centrifuge tube according to the proportion that 0.5mL of isopropanol is added into each 1mL of Trizol solution, and standing for 10 minutes at room temperature; centrifuging at 12000rpm and 4 ℃ for 15min; discarding the supernatant, adding 1mL of 75% ethanol into each 1mL of Trizol solution for washing, mixing by vortex, centrifuging at 12000rpm and 4 ℃ for 15min, and repeating twice; removing the supernatant, and allowing the precipitated RNA to naturally dry or vacuum dry at room temperature for 5-10min, wherein the excessive drying is not required, otherwise, the RNA loses solubility, so that A260/280 is less than 1.6; adding a proper amount of RNase-freecoat to dissolve RNA precipitate, repeatedly sucking with a gun head, and mixing; water bath at 65 ℃ for 15min, and preserving at-80 ℃ for standby; the concentration and purity of RNA were determined by UV absorption.
(2)Real Time PCR：
Real-time quantitative fluorescence measurement of RNA was performed according to TAKARA reverse transcription and real-time quantitative fluorescence kit instructions. The PCR conditions are shown in FIG. 8, and the primers are shown in Table 1.
TABLE 1 primer sequences
The results in FIGS. 5-7 show that the parkinsonism model mice were slow to move, stay on the rod longer, move a shorter total distance, and have significantly increased levels of inflammatory factors TNF-alpha and IL-1 beta in the brain compared to the control group. The engineering bacteria of the escherichia coli Nissle1917 reverse the sports injury caused by the MPTP and reduce the expression level of the inflammatory factors increased in brain tissues. ( C: normal control group, M: model group, ME, E.coli 1917-attb-pelB-GLP-1 treatment group, MG, GLP-1 treatment group, MN, nissle1917 treatment group )
While the present invention has been described in considerable detail and with particularity with respect to several described embodiments, it is not intended to be limited to any such detail or embodiments or any particular embodiment, but is to be construed as providing broad interpretation of such claims by reference to the appended claims in view of the prior art so as to effectively encompass the intended scope of the invention. Furthermore, the foregoing description of the invention has been presented in its embodiments contemplated by the inventors for the purpose of providing a useful description, and for the purposes of providing a non-essential modification of the invention that may not be presently contemplated, may represent an equivalent modification of the invention.
SEQUENCE LISTING
<110> university of Nanchang
<120> an escherichia coli Nissle1917 genetically engineered bacterium, and preparation method and application thereof
<130> 2021
<160> 2
<170> PatentIn version 3.3
<210> 1889
<211> 25
<212> DNA
<213> Escherichia coli Nissle1917
<400> 1
gaaagcccaa tcttcacatc aatcggtttt tcacccgtac cgtacagagt aattccaccc 60
ggagcggcag ggacatacac cgttccctga tactcaccag gcatcacggc aatatactgg 120
cgcttgttgg tacgcttgat aattgccgca tctaccgccg cctgaatcgt ggtatgcgtt 180
acaccttgag tacccgccgg gccgacaaca aagtcaggtt gcgcaggcag ggtaatcggg 240
gaaggattcc acgctgccgc acctggtgtc agggatgcaa aatagtgttg agcatcgaaa 300
ttctgcgctt cttttgccga cagaatcggg cgcgaagagg taccaggcgc ggtttgatca 360
gaaggacgtt gatcgggcgg tgttgagcta caggcggtca gcgtcacgcc aaaagccaat 420
gccagcgcca gacgggaaac tgaaaatgtg ttcacaggtt gctccgggct atgaaataga 480
aaaatgaatc cgttgaagcc tgcttttttc aggaaacagc tatgaccatg attacgccaa 540
gcttgatctc tccttcacag attcccaatc tcttgttaaa taacgaaaaa gcatcaatta 600
aaacggcggc atgtctttct atattccagc aatgttttat aggggacata ttgatgaaga 660
tgggtatcac cttagtaaaa aaaaagaatt gctataagct gctctttttt gttcgtgata 720
tactgataat aaattgaatt ttcacacttc tggaaaaagg agatatacca tggaagagct 780
cggtaccatg aaatacctgc tgccgaccgc tgctgctggt ctgctgctcc tcgctgccca 840
gccggcgatg gccatggata tcggaattaa ttcggatccg atgcatgatg aatttgaacg 900
tcatgctgaa ggtactttta cgagtgatgt tagttcatat ttagaaggtc aagctgcaaa 960
ggaatttatt gcatggttgg ttaagggtcg gggttaactc gagggctgtt ttggcggatg 1020
agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca 1080
gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt 1140
gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca 1200
ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt 1260
tgtcggtgaa cgctctcctg agtaggacaa atgaattcac tggccgtcgt tttacatact 1320
aacttgagcg aaacgggaag gtaaaaagac aaaaagttgt ttttaatacc tttaagtgat 1380
accagatggc attgcgccat ctggcagagt gattaactaa acatcgcagt aatcgaggca 1440
ctcgccagag agtgaaaatg aacgttaaac ccgaccatcg cgccgctggc accttcatcg 1500
acatcaatac gttctacatc cagcgcgtga acggtaaaaa tgtagcgatg ggtttcgcct 1560
ttcggcggcg ctgcgccatc gtacccggtt ttaccaaagt cggtacgcgt ctgcaaaacg 1620
ccgtctggca tagctaccag accagagcca aacccttgcg gtaatacgcg ggtatcagcg 1680
ggtaaattaa caactaccca gtgccaccag ccggagccgg ttggcgcatc cgggtcatag 1740
caggtgacaa caaaactttt cgttcccaca ggaacatcat cccacgccag atgcggtgaa 1800
atattatcgc catcgtaacc catgccgtta aagacatgac gatgcggcag cttatcgcca 1860
tcgcgcagat cgttactgat gagtttcat 1889
Claims (1)
1. The application of the escherichia coli Nissle1917 genetically engineered bacterium in preparing medicaments for treating parkinsonism is characterized in that the preparation method of the escherichia coli Nissle1917 genetically engineered bacterium comprises the following steps: the GLP-1 gene sequence was integrated into the genome of E.coli Nissle 1917.
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