CN108660148A - A kind of method and its application for expressing external source drug based on genetic modification probiotics - Google Patents

A kind of method and its application for expressing external source drug based on genetic modification probiotics Download PDF

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CN108660148A
CN108660148A CN201810530613.4A CN201810530613A CN108660148A CN 108660148 A CN108660148 A CN 108660148A CN 201810530613 A CN201810530613 A CN 201810530613A CN 108660148 A CN108660148 A CN 108660148A
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dapa
nissle1917
plasmid
pmalc2x
gene
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陈卫华
刘智
陈国忠
任杨柳
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Qi Yuan Technology (wuhan) Co Ltd
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Qi Yuan Technology (wuhan) Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins

Abstract

The present invention discloses a kind of method and its application for expressing external source drug based on genetic modification probiotics,Using homologous recombination and one-step method, seamless clone knocks out Escherichia coli Nissle1917 indispensable gene dapA genes,Obtain Escherichia coli auxotrophic strain Nissle1917 Δs dapA,Clone dapA genes,Build auxotrophy strain complementation plasmid pMalc2x dapA,The complementation plasmid pMalc2x dapA is electroporated in the Nissle1917 Δs dapA bacterial strains,Ampicillin resistance gene ampR on pMalc2x carriers is replaced with homologous recombination,Obtain the plasmid balance system of antibiotic-free resistance marker,External synthesis sall4 12 amino acid polypeptide sequence genes of aminoterminal,And merge carrot soft rot Erwinia pectase pelB signal peptide gene sequences in N-terminal,His protein tag gene orders are added in C-terminal,It is cloned into complementary plasmid pMalc2x dapA together,Obtain external source drug expression plasmid pMalc2x dapA sall4,The plasmid is imported into Escherichia coli auxotrophic strain Nissle1917 Δs dapA,The bacterial strain energy effective expression sall4 polypeptides of acquisition,There is remarkable result on liver cancer treatment.

Description

A kind of method and its application for expressing external source drug based on genetic modification probiotics
Technical field
The invention belongs to genetic engineering field, be related to it is a kind of based on genetic modification probiotics express external source drug method and Using.
Background technology
Liver is the main portions that the metastatic tumors such as intestinal cancer, breast cancer, cancer of pancreas occur, and the metastatic diffusion of tumour can draw 90% cancer mortality is played, and diagnosis of hepatic metastases is since small and quantity is more, to proposing very big challenge on clinical treatment.Due to swollen The microenvironment at tumor position changes, and immunization barrier is impaired, in addition the tumor center's overnutrition to addle, microorganism would generally Aggregation breeding herein.All it is directly by high concentration bacterial injections although people also used bacterium to carry out treatment of cancer in the past Into hematological system, the method lacks specificity and specific aim, and toxic side effect is big.Prebiotic type Escherichia coli Nissle1917 tools Have the advantages that safe and non-toxic, tumor-targeting is high, it can be as the internal excellent carrier for transmitting external source antitumor drug.Structure is not The plasmid balance system of the gene containing antibiotic-resistance marker imports external source drug expression vector, and structure can secreting, expressing external source base The cancer target engineering bacteria of cause, specific expression, transmission external source drug achieve the purpose that treating cancer in vivo.Periodical《It is raw Object engineering journal 2003.9.19 (5)》Huang Wei, Cao Cheng, Li Ping, Zhong Hui, Ma Qingjun (pass through Escherichia coli thyA chromosomes-plasmid Balanced lethal system structure non-resistant expression vector) describe this method in a text, but its strain gene group is except protecting It stays outside essential amino acid sequence, remaining is all knocked out.
SALL4 is a kind of oncofetal protein, in Human embryo liver expression, but is not expressed in adult human liver, in hepatocellular carcinoma Patient and prognosis deteriorate in patient, and SALL4 can again be expressed in its liver, and SALL4 can be used as potential therapy target. SALL4 Amino-terminal polypeptides can block SALL4 and nucleosome reconstruct and histone deacetylase (NuRD) composite bulk phase interaction With the SALL4 that blocking NuRD is mediated is expressed again.The carcinogenesis that SALL4 can be blocked using SALL4 12-AA peptides, can be used for Treat hepatocellular carcinoma.
Chromosome-plasmid balance system is that the novel plasmid carrier system of resistant gene is replaced with nutrition selected marker gene System.Its host strain usually carries the gene mutation of important metabolic pathway on chromosome, therefore mutant strain is on minimal medium It cannot grow.After only supplementing corresponding exogenous nutrition substance or importing the complementary plasmid for carrying wildtype gene sequence, It can make its existence.Therefore in the minimal medium added without corresponding nutriment, the importing of complementary plasmid is deficiency host The requirement of bacterium growth.The system is widely used to during the developing of the attenuated vaccine of different genera at present. A kind of DNA vaccine carrier of non-resistance choosing and its construction method are described in CN200310112640.3, using chromosome-plasmid Balanced lethal system replaces antibiotics resistance gene to be used as the selected marker of DNA vaccination amplification, builds novel non-resistance sieve DNA vaccine vector is selected, compared with plasmid pVAX1, kalamycin resistance gene therein is lacked instead length is The salmonella typhimurium asd genes of 1281bp, but can not be protected in the case where not adding allogenic material and antibiotic-free selection pressure The physiological status of itself is held, the present invention also improves strain application while can maintain primary type strain own physiological state In security performance and high efficiency, applied to prepare on liver cancer treatment drug have extensive foreground.
Invention content
It is outer based on the expression of genetic modification probiotics that in response to the problems existing in the prior art, the purpose of the present invention is to provide one kind The method and its application of source drug.
To achieve the above object, the technical solution adopted by the present invention is：One kind expressing external source based on genetic modification probiotics The method of drug, includes the following steps：(1) structure Escherichia coli auxotrophic strain Nissle1917 Δs dapA：Using homologous Recombination and the seamless clone of one-step method knock out the indispensable gene dapA genes of Escherichia coli Nissle1917, obtain Escherichia coli nutrition Defect bacterial strain Nissle1917 Δs dapA；(2) structure of the auxotrophic strain complementation plasmid and conversion：Clone dapA bases Cause replaces the ampR genes on pMalc2x carriers, forms the complementary plasmid pMalc2x-dapA of auxotrophy strain；It will be described mutual It is electroporated to the Nissle1917 Δs dapA bacterial strains to mend plasmid pMalc2x-dapA, obtains the matter of antibiotic-free resistance marker Grain balance system；(3) external source drug gene is imported in the plasmid balance system, is transformed into Nissle1917 Δ dapA, built Can secreting expression of exogenous gene cancer target engineering bacteria.
The reason of present invention knockout dapA genes, is that the gene is diaminopimelic acid (Dap) biosynthesis pathway Indispensable gene exists in bacterium, is not present in mammals, and in expression in escherichia coli, and in the present invention Host strain only needs to knock out resistant gene and indispensable gene, can maintain former wild type strain own physiological state and cancer target Property；Nissle1917 is a kind of safety and widely used probiotics, and the plasmid balance system that the present invention is built is maintaining open country Can be choosing not adding exogenous nutrition ingredient and antibiotic-free while raw type strain own physiological state and tumor-targeting In the case of selecting pressure, exogenous plasmid is maintained long-term and stably, improves bacterial strain safety in the application and high efficiency While, application cost is also reduced to a certain extent.Once need terminate treatment, only need to take antibiotic can by its from It removes in vivo, moreover it is possible to reduce the toxic side effect that treatment generates.
Further, the step (1) the specific steps are：
A, the design of primers of dapA genetic fragments, the dapA genetic fragments contain the dapA genes of the quasi- knockout of 40bp The kalamycin resistance gene upstream and downstream sequence of upstream and downstream homology arm and 20bp；
B, the amplification of the PCR fragment of dapA genes is knocked out for one-step method；
C, transformed competence colibacillus Escherichia coli Nissle1917；
D, screening Escherichia coli auxotrophic strain Nissle1917 Δs dapA.
Primer sequence is as follows in a steps, the nucleotide sequence as shown in NO.5~6 sequence table SEQ ID：
DapA-Km-For:TGTTCACGGGAAGTATTGTCGCGATTGTTACTCCGATGGATGTGGGCTGGAGCTGC TTC,
DapA-Km-Re:TACAGCAAACCGGCATGCTTAAGCGCCGCTCTGACCGTCTCCTCCTTAGTTCCTAT TCC,
Further, the step (2) the specific steps are：
A, PCR generates the pMalc2x Δ ampR segments without ampR genes；
B, dapA genetic fragments are synthesized by PCR；
C, the segment obtained in the step A and step B is connected by Gibson cloning methods, obtains pMalc2x- DapA plasmids；
D, conversion defect bacterial strain Nissle1917 Δs dapA.
The primer sequence that dapA is synthesized in the step A is as follows, the nucleotide as shown in NO.7~8 sequence table SEQ ID Sequence：
pMl-DapA-For:GCAGGTCGACTCTAGATTACAGCAAACCGGCATGC
pMal-DapA-Re:CAAGGACCATAGCATATGTTCACGGGAAGTATTG
Further, the step (3) the specific steps are：Synthesize sall4 12 amino acid polypeptide sequences of aminoterminal Gene, and carrot soft rot Erwinia pectase pelB gene signal peptide gene sequences are merged in N-terminal, add His eggs in C-terminal White label gene order, is cloned into the complementary plasmid pMalc2x-dapA of balance system, obtains the plasmid of expression Sall4 polypeptides Then pMalc2x-dapA-sall4 is transformed into Nissle1917 Δ dapA, obtains corresponding engineering bacteria.
Specifically, a kind of method for expressing external source drug based on genetic modification probiotics is preparing treatment tumour medicine Application on object.
Further, the tumour medicine is the drug for treating liver cancer.
Compared with prior art, the beneficial effects of the invention are as follows：
(1) chromosome based on dapA genes-plasmid balance system is built by research object of probiotics Escherichia coli, and It further carries out in a deep going way using expression, the ability of transmission exogenous bioactive molecule in the system body and its to organism safe Correlative study achievees the purpose that utilize the systematic treating tumor disease.
(2) Nissle1917 is a kind of safety and widely used probiotics, and the system that the present invention is built is maintaining open country Can be choosing not adding exogenous nutrition ingredient and antibiotic-free while raw type strain own physiological state and tumor-targeting In the case of selecting pressure, exogenous plasmid is maintained long-term and stably, improves bacterial strain safety in the application and high efficiency While, application cost is also reduced to a certain extent.Once need terminate treatment, only need to take antibiotic can by its from It removes in vivo, moreover it is possible to reduce the toxic side effect that treatment generates.
(3) host strain in the present invention only needs to knock out resistant gene and indispensable gene, can maintain former wild type strain certainly Body physiological status and tumor-targeting.
Description of the drawings
Fig. 1 is present invention structure chromosome-plasmid BALANCE CARRIERS flow diagram；
Fig. 2 is plasmids detection result after continuous LB meat soups passage in 9 days in embodiment 2；
Fig. 3 is the vivoexpression (pET22b-SALL4in BL21) of PelB-SALL4-His6；
Fig. 4 is that SALL4Western blot are checked (different IPTG inductions, concentration nM)；
Fig. 5 is that expression of the SALL4 in Nissle1917 is (left：pMalc2x-dapA-sall4；It is right：pMalc2x- dapA)；
Fig. 6 is situation of the liver cancer cells 7721 after being inoculated with Nissle1917, and a left side is inoculation Nissle1997 and blank space Control group is managed, the right side is the processing group of inoculation engineering bacteria；
Fig. 7 is the variation that embodiment 7 is inoculated with gross tumor volume/weight after engineering bacteria；
Fig. 8 is that Western bloting check that detection of expression of the SALL4 in mouse tumor tissue is (left：Normal structure pair According to；It is right：Tumor tissues)；
Specific implementation mode
Below in conjunction with the attached drawing in the present invention, technical scheme of the present invention is clearly and completely described, it is clear that Described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the implementation in the present invention Example, all other embodiment that those of ordinary skill in the art are obtained under the conditions of not making creative work belong to The scope of protection of the invention.
Embodiment 1：Obtain the Nissle1917 bacterial strains of GFP labels
As shown in Figure 1, by design primer, the nucleotide sequence as shown in NO.1~2 sequence table SEQ ID：Primer GFP-F：ATGACTATGATTACGGATTCTCTGGCCGTCGTATTACAACGTCGTGACTGTTTGCC CGCCAGTTGTTG, draw Object GFP-Re:CAGCTCCAGCCTACAAGGCTGGAGCTGCTTCTTAG, using SAD1702 genomes as template, by polymerase Chain reaction (PCR) obtains the GFP genetic fragments of 1359bp.Design primer simultaneously, as shown in NO.3~4 sequence table SEQ ID Nucleotide sequence：Primer Cm-F:AAGCAGCTCCAGCCTTGTAGGCTGGAGCTGCTTC, primer Cm-Re: TTATTTTTGACACCAGACCAACTGGTAATGGTAGCGACCGGCGCTCAGCTTCCTCC TTAGTTCCTATTCC, with pKD3 For template, PCR obtains chloramphenicol (Cm) genetic fragment of 1082bp, is linked together the two by overlap PCR.Polymerization The condition of enzyme chain reaction is：94 DEG C, 2 minutes, 94 DEG C, 20 seconds, 52 DEG C, 30 seconds, 72 DEG C, 1 point 15 seconds, 35 cycle, PCR production Object uses PCR Purification Kits after the detection of 1.0% agarose gel electrophoresis.GFP and Cm gene pieces after purification are taken respectively Section 500ng electroporated Nissle1917 (pKD46) competent cell, electrotransformation condition are：Voltage 2500v shocks by electricity 5 milliseconds, And be coated on the LB tablets containing 20 μ g/ml chloramphenicol and 4mg/ml X-gal, after 30 DEG C are cultivated 14~16 hours, picking is white In color colony inoculation to the LB liquid medium containing 20 μ g/ml chloramphenicol, GFP in bacterium solution is detected with microplate reader after 4~6 hours Fluorescence intensity, be used in combination PCR detect GFP genes.To the Escherichia coli Nissle1917 that mice with tumor feeding GFP is marked, lead to It crosses fluoroscopic examination and shows that Nissle1917 has tumor-targeting in Mice Body to positions of the Nissle1917 in Mice Body, grind Study carefully and shows that Nissle 1917 can be effectively enriched in mouse interior tumor tissue.
Embodiment 2：Build Escherichia coli auxotrophic strain Nissle1917 Δs dapA
Gene knockout method is knocked out using lamda-Red recombination one-step method.Operating process：Recombinant fragment is cloned, design is drawn Object, the nucleotide sequence as shown in NO.5~6 sequence table SEQ ID：Primer DapA-Km-For: TGTTCACGGGAAGTATTGTCGCGATTGTTACTCCGATGGATGTGGGCTGGAGCTGC TTC, primer DapA-Km-Re: TACAGCAAACCGGCATGCTTAAGCGCCGCTCTGACCGTCTCCTCCTTAGTTCCTAT TCC, wherein striking containing 40bp Except the homology arm upstream and downstream gene of target gene dapA and the clone resistant gene kan of 20bp, upstream and downstream sequence, using agarose Gel purification recombinates segment.The electroporated Escherichia coli Nissle1917 competent cells for having recombinant plasmid pKD46 apply It is distributed in the LB tablets containing 20 μ g/mlKan antibiotic and 20% arabinose.Single bacterium colony in picking resistant panel, utilizes inspection It surveys primer and carries out bacterium colony PCR, screen the positive recombinant of successful knockout.Picking positive colony is inoculated in LB culture mediums, 42 DEG C of trainings It supports 16 hours, scribing line picking single bacterium colony is respectively coated the LB tablets containing kanamycins and the LB without amicillin resistance is flat Plate, picking cannot be grown on ampicillin plate and the bacterium colony that can be grown on LB tablets, as eliminates recombinant plasmid Escherichia coli Nissle1917 (the dapA of pKD46::Km) engineered strain.Above-mentioned bacterial strains are prepared into competent cell, by plasmid PCP20 is transferred to Escherichia coli Nissle1917 (dapA::Km) engineered strain.Then, the bacterial strain for carrying pCP20 plasmids is turned It is connected in LB liquid medium and cultivates 12~16 hours for 42 DEG C, picking single bacterium colony of crossing is respectively coated kanamycins tablet and nothing Resistance LB tablets obtain the Escherichia coli auxotrophic strain Nissle1917 Δs of the antibiotic-free resistance of removal resistance screening DapA engineered strains.
Embodiment 3：The structure of auxotrophic strain complementation plasmid and conversion
Design primer, using Nissle1917 genomes as template, PCR amplification obtains the dapA genetic fragments of 869bp, PCR Reaction condition is：94 DEG C, 2 minutes, 94 DEG C, 20 seconds, 52 DEG C, 30 seconds, 72 DEG C, 1 point, 32 cycles, PCR product purifying obtains DapA genetic fragments, design primer, the nucleotide sequence as shown in NO.7~8 sequence table SEQ ID：pMl-DapA-For: GCAGGTCGACTCTAGATTACAGCAAACCGGCATGC, pMal-DapA-Re: CAAGGACCATAGCATATGTTCACGGGAAGTATTG, using pMalc2x plasmids as template, PCR amplification obtains no ampR's PMalc2x Δ ampR segments mix two kinds of PCR products, are connected with the method for Gibson cloning, condition of contact is：It is used in It is connected 15 minutes at 50 DEG C in Gibson cloning reagents, the electroporated Nissle 1917 containing pKD46 of connection product is felt By state cell, the LB tablets without DAP are coated on, after 37 DEG C are cultivated 14~16 hours, 5 colony inoculations of picking are in LB culture solutions In, it cultivates 5~6 hours, PCR detects bacterium solution, and PCR product is detected with 1.0% agarose gel electrophoresis, and two pipe bacterium solutions is taken to be inoculated into In LB culture solutions, Plasmid DNA is extracted with plasmid extraction kit after 12 hours, digestion verification positive colony is named as PMalC2x-dapA plasmids, as complementary plasmid.Bacterial strain Nissle1917 (Δ dapA, pMalc2x- containing complementary plasmid DapA) bacterium solution is transferred in LB broth bouillons and repeatedly passes on, and detects plasmid stability.Continuous passage 9 days in LB meat soups, often Left-right gradient is diluted and is coated on LB agar plates within 3 days, is obtained and is detached good single bacterium colony, and single bacterium colony is then inoculated with LB meat Soup extracts plasmid, and plasmid loss situation is verified by electrophoresis.To further ensure that the presence of plasmid, usually we also with PCR come Verify the presence or absence of dapA genes.As shown in Fig. 2, continuous passage in 9 days the result shows that, 100% plasmid stabilisation exists.
Embodiment 4：Expression of the sall4 aminopeptidase genes terminal polypeptide in pET systems
Known sall4 amino terminals polypeptide sequence is MSRRKQAKPQHI, by NCBI to sall4 gene sequencings, Determine that the base sequence of sall4 aminoterminals 12-AA is, as shown in sequence table SEQ ID NO.9： ATGTCGAGGCGCAAGCAGGCGAAACCCCAGCACATC, pelB signal peptide sequence are, such as sequence table SEQ ID NO.10 institutes The nucleotide sequence shown：GGCATCGCCGGCTGGGCAGCGAGGAGCAGCAGACCAGCAGCAGCGGTCGGCAGCAGGTATTT CAT by 12-AA peptide fusions to pelB gene signal peptide sequences, and adds histidine tag His6 in C-terminal.Segment is merged to return After receipts, pET22b expression vectors are built into, convert Escherichia coli Ecoli DH5 α competent cells, the Amp resistance screening positives turn Beggar, upgrading grain convert Escherichia coli Ecoli BL21 competent cells, and Kan resistance screening positive transformants, transformant is through fast Speed cracking, bacterium colony PCR and digestion identification, screen correct positive transformant.The above-mentioned positive transformant access 5ml of picking contains 100 μ In the LB culture mediums of g/ml ampicillins, 37 DEG C of overnight incubations.By 1：50 are transferred in 50ml LB ammonia benzyl culture mediums, 37 DEG C The IPTG of final concentration of 1mmol/L, 37 DEG C of induction 4h is added until light absorption value OD600 reaches 0.4-0.6 in shaken cultivation 1.5h or so. Thalline is collected, 2xSDS sample-loading buffers, boiling water bath 5min is added, centrifuging and taking supernatant carries out SDS-PAGE electrophoresis, western Blotting (His6 monoclonal antibodies) detects 12-AA polypeptides in the expression of pET systems, and the results are shown in Figure 3；SDS-PAGE and Western blotting testing results show specific protein band, and the results are shown in Figure 4.
Embodiment 5：PelB-sall4 genetic fragments are in balance pUC pUC expression
By PCR pET22b-pelB-sall4 plasmids, pelB-sall4 genetic fragments are obtained, are cloned into complementary expression matter In grain pMalc2x-dapA, pMalc2x-dapA-sall4 plasmids are obtained, are transformed into Nissle1917 Δ dapA bacterial strains, obtain work Journey bacterium Nissle 1917 (Δ dapA, pMalc2x-dapA-sall4).It is inoculated in the engineering bacteria to LB liquid medium and shakes for 37 DEG C Overnight incubation is swung, the LB liquid medium of 10mL, 37 DEG C of shaken cultivations to OD600 to 0.5, IPTG are forwarded to 2% ratio Induced expression 5h.Collect thalline, the expression of western blotting detection 12-AA polypeptides, as shown in figure 5, left：pMalc2x- dapA-sall4；It is right：pMalc2x-dapA；There is destination protein specific band.
Embodiment 6：The tumor cell in vitro of engineering bacteria is tested
Liver cancer cells 7721 are bought, a 25cm is passed2Bottle is used as Monolayer growth of cells in culture medium (DMEM), is aided with 15% fetal calf serum (FBS) and 2mM glutamine (complete DMEM), condition of culture are 37 DEG C, contain 5% CO2, it is denoted as original Generation --- P0, if cell state is bad can to change liquid daily, 1 after covering with:3 passages, are denoted as first generation P1, then according to the life of cell Long speed cover with after by 1:6 passages, are denoted as P2, and so on, the algebraically of cell is recorded in detail.It to be observed before cell passage thin Born of the same parents can be passed on when cell coverage reaches 80%-90%.By tumor cell inoculation in 24 hole tissue culturing plates (BDFalcon) (10 in5A cells/well), inoculation engineering bacteria Nissle 1917 (Δ dapA, pMalc2x-dapA-sall4) is trained Nutrient solution, 0.6 μ l/well, while with same amount of Escherichia coli Nissle1917 is inoculated with and space management compares, at 37 DEG C and 5%CO2Lower growth 20 hours, detects the growing state of tumour cell, as shown in fig. 6, left is blank control group, the right side is processing Group, the tumour cell quantity for being inoculated with engineering bacteria are obviously rounded, and are reduced, hence it is evident that cause cell death.
Embodiment 7：Engineering bacteria inoculated tumour mouse
The mouse for choosing 5 week old obtains the mouse with liver cancer cells tumour by being subcutaneously injected.Mouse grows to 6 week old After, when tumour reaches a volume in 200-400mm3Between, mouse is randomly divided into experimental group.Take engineering bacteria Nissle1917(ΔdapA,pMalc2x-dapA-sall4)1:100, which are added to LB culture mediums, is incubated overnight, bacterium solution centrifugation after with 1×108、1×107、1×106CFU/mL is resuspended in sterile PBS, and every intragastric administration on mice dosage is 100ul, while the agent such as gavage The Nissle1917 and PBS of amount are compareed, and the growing state of mouse tumor after gavage is handled 15 days are observed and recorded, such as Fig. 7 institutes Show；And tumor resection tissue, expression of the Western blot detection 12-AA polypeptides in Mice Body, as shown in figure 8, result is aobvious It is shown with specific protein band.
Comparative example：
Escherichia coli thymine synthetase thyA genes are knocked out, build plasmid balance system in the same fashion, and carry out Plasmid stability is tested, and discovery is passed on 9 days in LB meat soups, also without discovery plasmid loss, but in zoopery, tumour SALL4 is not detected in tissue, analyzes reason, it may be possible to which animal reservoir's environment provides thymidine, causes Δ thyA prominent Becoming bacterial strain can equally grow in the case of no plasmid.Thymidine is further added in LB culture mediums, finds plasmid Loss Rate often on behalf of 10% or so, further demonstrates the stability pUC pUC that dapA must be gene constructed and is more suitable as place Main experiment.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace And modification, the scope of the present invention is defined by the appended.
SEQUENCE LISTING
<110>Strange member science and technology（Wuhan）Co., Ltd
<120>A kind of method and its application for expressing external source drug based on genetic modification probiotics
<130>
<160> 10
<170> PatentIn version 3.3
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<213> pMal-DapA-Re
<400> 8
caaggaccat agcatatgtt cacgggaagt attg 34
<210> 9
<211> 36
<212> DNA
<213>Artificial sequence
<400> 9
atgtcgaggc gcaagcaggc gaaaccccag cacatc 36
<210> 10
<211> 65
<212> DNA
<213>Artificial sequence
<400> 10
ggcatcgccg gctgggcagc gaggagcagc agaccagcag cagcggtcgg cagcaggtat 60
ttcat 65

Claims (6)

1. a kind of method for expressing external source drug based on genetic modification probiotics, which is characterized in that include the following steps：
(1) structure Escherichia coli auxotrophic strain Nissle1917 Δs dapA：Utilize homologous recombination and the seamless clone of one-step method The indispensable gene dapA genes of Escherichia coli Nissle1917 are knocked out, Escherichia coli auxotrophic strain Nissle1917 Δs are obtained dapA；
(2) structure of the auxotrophic strain complementation plasmid and conversion：DapA genes are cloned, the ampR bases of pMalc2x are replaced Cause forms the complementary plasmid pMalc2x-dapA of auxotrophy strain；It arrives the complementary plasmid pMalc2x-dapA is electroporated The Nissle1917 Δs dapA bacterial strains obtain the plasmid balance system of antibiotic-free resistance marker；
(3) external source drug gene is imported in the plasmid balance system, is transformed into Nissle1917 Δ dapA, structure can secrete table Up to the cancer target engineering bacteria of foreign gene.
2. a kind of method for expressing external source drug based on genetic modification probiotics as described in claim 1, which is characterized in that institute State step (1) the specific steps are：
A, the design of primers of dapA genetic fragments, the dapA genetic fragments contain the upper and lower of the dapA genes of the quasi- knockout of 40bp Swim the kalamycin resistance gene upstream and downstream sequence of homology arm and 20bp；
B, the amplification of the PCR fragment of dapA genes is knocked out for one-step method；
C, transformed competence colibacillus Escherichia coli Nissle1917；
D, screening Escherichia coli auxotrophic strain Nissle1917 Δs dapA.
3. a kind of method for expressing external source drug based on genetic modification probiotics as described in claim 1, which is characterized in that institute State step (2) the specific steps are：
A, PCR generates the pMalc2x Δ ampR segments without ampR genes；
B, dapA genetic fragments are synthesized by PCR；
C, the segment obtained in the step A and step B is connected by Gibson cloning methods, obtains pMalc2x-dapA Plasmid；
D, conversion defect bacterial strain Nissle1917 Δs dapA.
4. a kind of method for expressing external source drug based on genetic modification probiotics as described in claim 1, which is characterized in that institute State step (3) the specific steps are：The gene of sall4 12 amino acid polypeptide sequences of aminoterminal is synthesized, and Hu trailing plants is merged in N-terminal Foretell soft rot Erwinia pectase pelB gene signal peptide gene sequences, adds His protein tag gene orders in C-terminal, be cloned into The complementary plasmid pMalc2x-dapA of balance system obtains the plasmid pMalc2x-dapA-sall4 of expression sall4 polypeptides, then Nissle1917 Δ dapA are transformed into, corresponding engineering bacteria is obtained.
5. a kind of method for expressing external source drug based on genetic modification probiotics of claim 1-4 any one of them is controlled in preparation Treat the application on tumour medicine.
6. application according to claim 5, which is characterized in that the tumour medicine is the drug for treating liver cancer.
CN201810530613.4A 2018-05-29 2018-05-29 A kind of method and its application for expressing external source drug based on genetic modification probiotics Pending CN108660148A (en)

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CN111304140A (en) * 2020-03-05 2020-06-19 清华大学 Recombinant intestinal bacterium for producing (R) -3-hydroxybutyric acid and construction method thereof
CN114456992A (en) * 2020-11-05 2022-05-10 中国科学院深圳先进技术研究院 Self-lysis salmonella strain, preparation method thereof and application thereof in tumor treatment
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CN114522184A (en) * 2020-11-05 2022-05-24 中国科学院深圳先进技术研究院 Cell lysate of engineering bacteria and application thereof in tumor treatment
CN114456992B (en) * 2020-11-05 2024-04-19 中国科学院深圳先进技术研究院 Salmonella strain capable of being automatically cracked, preparation method thereof and application thereof in tumor treatment
CN112481181A (en) * 2020-12-04 2021-03-12 中国科学院分子植物科学卓越创新中心 Engineering probiotics producing STING agonist with tumor inhibition effect
CN115029365A (en) * 2022-06-09 2022-09-09 江南大学 Construction and application of antibiotic-free efficient stable expression system of escherichia coli probiotics EcN
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