WO2012126265A1 - Endospore recombinante présentant de la sérumalbumine humaine à sa surface et destinée à une administration par voie orale, et son procédé de préparation - Google Patents

Endospore recombinante présentant de la sérumalbumine humaine à sa surface et destinée à une administration par voie orale, et son procédé de préparation Download PDF

Info

Publication number
WO2012126265A1
WO2012126265A1 PCT/CN2011/084333 CN2011084333W WO2012126265A1 WO 2012126265 A1 WO2012126265 A1 WO 2012126265A1 CN 2011084333 W CN2011084333 W CN 2011084333W WO 2012126265 A1 WO2012126265 A1 WO 2012126265A1
Authority
WO
WIPO (PCT)
Prior art keywords
recombinant
human serum
serum albumin
spore
bacillus subtilis
Prior art date
Application number
PCT/CN2011/084333
Other languages
English (en)
Chinese (zh)
Inventor
陈克平
毛浪勇
宁德刚
姜姗彤
李国辉
姚勤
袁弋
Original Assignee
江苏大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏大学 filed Critical 江苏大学
Publication of WO2012126265A1 publication Critical patent/WO2012126265A1/fr

Links

Classifications

    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A61K38/385—Serum albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • 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
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to a preparation method and a product for displaying a recombinant spore of a foreign protein on the surface, and specifically relates to a recombinant spore showing a human serum albumin HSA on the surface of a Bacillus subtilis spore, belonging to the technical field of recombinant fusion protein medicine.
  • Ouman serum albumin is a single-chain aglycosylated protein consisting of 585 amino acids with a molecular mass of approximately 66.5 kD (Mingetti, et al. 1986. J. Biol. Chem. 261, 6757) The isoelectric point is between 4.7 and 4.9. HSA is the most abundant protein in human plasma, accounting for about 60% of total protein in blood. Human serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions and many therapeutic molecules in body fluids while maintaining the osmotic pressure of blood at an appropriate level.
  • HSA plays an important physiological role in the body, so it has a wide range of uses in clinical medicine practice, and is widely used to treat various shocks, burns, war wounds, Patients with blood volume reduction caused by various causes such as surgery and work accidents, as well as patients with chronic nephritis, hepatitis, cirrhosis and advanced malignant tumors, malnutrition edema, hypoalbuminemia and patients without albuminemia.
  • HSA has a high nutritional value for cells. HSA is composed of more essential amino acids and is a balanced and complete source of amino acids. About 1/3 of the proteins that nourish cells are derived from HSA synthesized by the liver.
  • HSA The daily synthesis of HSA in the liver is almost completely consumed by various tissue cells.
  • the HSA leaves the blood system, reaches the cell membrane, enters the cell, and hydrolyzes to synthesize various proteins.
  • HSA has a huge market demand.
  • HSA's global market demand is about 600t, of which China's annual demand for HSA is about 70t.
  • Clinically, HSA is mainly obtained by collecting human blood by fractional filtration.
  • human plasma has limited source, high price, and complicated source, which is easy to cause blood-borne pollution, such as hepatitis virus and HIV.
  • recombinant human serum albumin has been produced by yeast fermentation by genetic engineering, but to achieve large-scale production of drug-grade recombinant human serum albumin, the fermentation method still has many shortcomings and shortcomings, because recombinant human serum albumin The purity requirements of the product are extremely high, and the purity requirement is above 99.999999%. Any trace of miscellaneous protein may cause a serious immune reaction. This makes the separation and purification methods and steps for producing recombinant human serum albumin by fermentation method complicated, and the production cost is high.
  • Bacillus subtilis is an environmentally friendly bacterium. When nutrients are deficient, it can form spores in metabolic dormancy. Under suitable conditions, spores can germinate into vegetative cells with reproductive ability. The spores are easy to culture, are relatively easy to separate, and do not require complicated separation and purification operations. Spore is the most stress-resistant structure in the life world. It has good stability, resistance to oxidation, high temperature resistance, long-term resistance to 6 (TC high temperature, 20 minutes of survival at 120 ° C, and resistance to chemistry It is also very prominent in medicine and anti-radiation, and can survive in harsh environments for several years to several decades.
  • Spores can remain active in acidic gastric environment, can resist saliva and bile attack, can pass through the animal digestive tract, can be used as A vector for heterologous proteins or biologically active molecules in extreme environments (eg gastrointestinal tract) (Oggioni MR, et al. 2003. Vaccine. 21: 96-101). Bacillus subtilis Description
  • the sub-state After entering the digestive tract, the sub-state is rapidly revived from dormancy, and in a short period of time, it is propagated into a dominant population with high bacteria content, consumes a large amount of oxygen in the intestinal tract, and can produce hydrogen peroxide and bacteriocin, establish a micro-ecological balance, and promote beneficial.
  • the growth of anaerobic microorganisms inhibits the growth of harmful bacteria (E. coli, Salmonella), and thus prevents gastrointestinal diseases such as diarrhea and diarrhea.
  • the purpose of the invention is to utilize the unique stress resistance of spores, and use the probiotic Bacillus subtilis spore coat protein as a carrier to display human serum albumin on the spore surface by spore surface display technology, so that human serum albumin can be rapidly expressed in large quantities. At the same time, it has good resistance to stress and is suitable for rapid and large-volume preparation of pharmacologically active recombinant human serum albumin oral nutrition.
  • the product has the pharmacodynamic and nutritional functions of human serum albumin while maintaining the physiological characteristics of Bacillus subtilis spores. So far, no similar reports and invention patents have been found.
  • the present invention provides a method and a product for preparing a recombinant spore of a surface displaying human serum albumin HSA.
  • the invention uses Bacillus subtilis spore coat protein CotC as a carrier to display human serum albumin on the surface of spores of Bacillus subtilis by spore surface display technology, and can be used for direct oral administration of animals, and becomes a novel human serum albumin drug nutrition.
  • a product or animal nutrition that has similar efficacy to human HSA in terms of safety, tolerability, pharmacodynamics, and pharmacokinetics.
  • the preparation method and separation process of the surface display human serum albumin of the invention are simple and rapid, and have wider application value.
  • the invention of this product overcomes the limitation of the current human serum albumin derived from blood products, and overcomes the shortcomings of the separation and purification process of human serum albumin produced by recombinant yeast fermentation, and has a good application prospect in the pharmaceutical field.
  • Method for preparing recombinant spore of surface displaying human serum albumin HSA and product thereof using Bacillus subtilis spore coat protein gene as molecular carrier, and recombining with human serum albumin gene to construct fusion expression integrated recombinant plasmid, and Transformation of Bacillus subtilis to obtain a recombinant strain of Bacillus subtilis, inducing a recombinant strain to produce spores, and displaying human serum albumin on the surface of the spore.
  • a specific operation of the present invention is: a cDNA of human serum albumin HSA is amplified from a cDNA library of human liver tissue by RT/PCR, and the obtained gene is inserted into a cloning vector pMD18-T for storage. Then, the human serum albumin gene hsa was inserted between the ⁇ and E CO m restriction sites of the fusion-expressing plasmid pJS700 to construct the integrated recombinant plasmid pJS700-HSA, and the plasmid was used to transform Bacillus subtilis, and the depletion method was used. The host strain was induced to produce spores, and the fusion protein CotC-HSA was displayed on the surface of the spore.
  • the present invention selects a Bacillus subtilis strain which has good transforming ability and can produce spores as a transformed strain of a recombinant plasmid, such as Bacillus subtilis 168 and a series of strains thereof (Bacillus Genetic Stock Center, Department of Biochemistry, The Ohio State University) , West 12th Avenue, Columbus, Ohio, 43210 USA);
  • the spore-capsid protein gene coiC (Gene Bank SEQ ID NO: NP-389653) was selected as the molecular carrier of the surface display protein, and the human serum albumin gene fefl was selected as the recombinant gene.
  • Bacillus subtilis is an environmentally friendly bacterium that differentiates into dormant spores in the absence of nutrients. Spores are highly resistant to stress, are resistant to acids and alkalis, have a high ability to survive, and are relatively large in size and easy to separate and purify.
  • the cultivation method of Bacillus subtilis is simple and convenient, the growth is rapid, the nutrition requirement is simple, and it is easy to carry out industrial scale-up culture.
  • the human serum albumin is displayed on the surface of the spore of Bacillus subtilis, and the human serum albumin is rapidly expressed in a large amount, and has good stress resistance and stability, and is simple and quick to separate and purify.
  • the invention relates to a recombinant plasmid constructed by recombining the selected spore capsid protein gene coiC and the ⁇ gene coding sequence of the invention, wherein the recombinant plasmid contains a promoter of the spore capsid protein gene, a coding sequence containing no stop codon and The recombination gene of the h gene encodes a gene that expresses CotC-HSA when Bacillus subtilis differentiates to form spores.
  • the fusion gene cWC-Zira is integrated into a specific site on the B. subtilis chromosome and stably inherited, thereby avoiding the disadvantage that the plasmid is easily lost in Bacillus subtilis cells.
  • An expression vector carrying the fusion expression CotC-HSA can be transformed into a host cell by heat bombardment or electroporation.
  • Successfully transformed cells can be identified by methods well known in the art by further screening for cells carrying the fusion gene constructed by the present invention, such as collecting cells, extracting DNA after lysis, and then performing PCR identification, etc., after induction of expression. Western blot was performed using the HSA antibody.
  • the invention also relates to a recombinant strain obtained by screening a recombinant integrative plasmid expressing the CotC-HSA, which is constructed by the invention, and transformed into a Bacillus subtilis strain, and the recombinant strain induces the surface of the spore to display recombinant human serum albumin, and can be passed through Western blot.
  • the recombinant protein HSA displayed on the surface of the spore was examined.
  • the present invention relates to a primer sequence for cloning a gene hsa coding sequence fragment:
  • hsa-F ATGGTACCATGAAGTGGGTAACCTTT ( SEQ ID NO. l )
  • hsa-R GCGAATTCTTATAAGCCTAAGGCAGCT ( SEQ ID 0.2 )
  • the present invention relates to a primer sequence amplified by a fragment of the B. subtilis amylase gene amy £ (Gene Bank SEQ ID NO: NP-388186):
  • amyE-F ATTGCTCGGGCTGTATGACTGG ( SEQ ID 0.3 )
  • amyE-R GTTACACCATCACTGTTCGTTCCTT ( SEQ ID N0.4 )
  • the outstanding advantages of the present invention are as follows:
  • the present invention displays human serum albumin HSA on the surface of Bacillus subtilis, and utilizes the unique resistance of spores to make human serum albumin smoothly pass through the animal body digestion barrier.
  • Surface constructed by the present invention The manual displays the recombinant spore of Bacillus subtilis of human serum albumin HSA, which can be used for direct oral administration to animals, and becomes a novel human serum albumin nutrient or animal nutrition, compared with other injection type human serum albumin samples, preparation methods and
  • the separation process is simple and fast, and has better stability and wider application value.
  • the invention of this product overcomes the limitation of the current human serum albumin derived from blood products, and also overcomes the disadvantages of the complicated process of separation and purification of human serum albumin produced by recombinant yeast fermentation.
  • Figure 1 The human serum albumin gene coding sequence cloned by the inventors searched for alignment results in Gene Bank.
  • Fig. 2 is a construction method and a structural map of the fusion expression recombinant plasmid pJS700-HSA expressing CotC-HSA.
  • amyE 5 '-end and amyE 3 '-end respectively represent the 5' and 3' ends of the amylase gene coding sequence, and are integrated into the amylase gene of Bacillus subtilis 168 by homologous recombination.
  • Amp r ' E «/ represents the ampicillin resistance gene and the erythromycin resistance gene, respectively, and is used as a selection marker in Escherichia coli and BaciU subtilis 168 (rp-).
  • cotC-hsa is a gene fragment that expresses a recombinant protein of CotC-HSA in a spore of Bacillus subtilis 168, which contains a promoter sequence of the cotC gene, a CotC coding sequence which does not contain a cwC stop codon, and the entire coding sequence of HSA.
  • oriC is an E. coli replicon fragment.
  • Figure 3 recombinant strain Amylase activity analysis of 168 (rp-)/pJS700-HSA.
  • A wild type strain Bacillus subtilis 168 ( rp — ) and recombinant strain ScdZ subtilis 168 ( rp /pJS700-HSA was cultured on starch plates;
  • B iodine staining of wild type strains and recombinant strains on starch plates.
  • Figure 4 is a schematic representation of the integration of the Em r -cotC-hsa gene fragment in the Bacillus subtilis 168 chromosome.
  • FIG. 5 PCR detection of the Em r -cotC-hsa fragment in recombinant strains.
  • M 250bp DNA Ladder Marker; using Bacillus subtilis 168 ( ⁇ ') (Wild-type, WT) and Bcicillus subtilis 168 (ir ⁇ )")/pJS700-HSA (Transgenic, TG) chromosomes as templates, with amyE -F/amyE-R, hsa-F/hsa-R hsa-F/ amyE-R and amyE-F/hsa-R are four sets of primer pairs (see Figure 4 for the position of the primer pair in the chromosome). Identification of recombinant genes.
  • FIG. 6 Surface display of transgenic recombinant strains Western blot analysis of recombinant spores of HSA.
  • M is a prestained protein MW marker
  • 1 is the wild type strain Bacillus subtilis 168 ( ⁇ _) after 48 hours of culture as a control
  • 2 and 3 are the recombinant strain Bacillus subtilis 168 (irp") / pJS700-HSA culture for 8h and 48h respectively. Extract.
  • Figure 7 Effect of oral recombinant spores on serum albumin levels in mice.
  • the control group was given normal saline to the mice; the wild type group was given wild type spores at a dose of 2 mg/(gd); the low dose group and the high dose group were given recombinant spores at a dose of 1 mg respectively. /(g'd) and 2 mg/(gd).
  • Figure 8 Effect of oral spores on body weight in mice.
  • the four curves represent the changes in body weight of the control group, the wild-type spore-treated group, and the recombinant spore small-dose and high-dose treatment groups for 30 days. Detailed description of the specification
  • Example 1 Cloning, sequencing and identification of human serum albumin fe « gene
  • Human liver tissue samples were provided by Dr. Liu Jibing from Nantong University School of Medicine. Human liver tissue was taken and placed in a mortar precooled with liquid nitrogen, and liquid nitrogen was added for grinding.
  • the signal R A (mRNA) was separated by an R A extraction kit, and the cDNA sequence of the human serum albumin gene was obtained by RT/PCR.
  • the PCR amplification primer for the human serum albumin gene fragment is - hsa-F: ATGGTACCATGAAGTGGGTAACCTTT
  • hsa-R GCGAATTCTTATAAGCCTAAGGCAGCT
  • the PCR reaction system is strictly carried out according to the LA 3 ⁇ 4g instruction manual.
  • the 20 ⁇ L reaction system contains 73 ⁇ 4 R « LA Taq 0.2 L, 10 X LA PCR Buffer II (Mg 2+ Plus) 2 ⁇ L, dNTP Mixture (2.5 mM each) 3.2 ⁇ L, template DNA lOOng, upstream and downstream primers (20 ⁇ ⁇ ) 0.4 y L each, and finally add sterile ultrapure water to 20 ⁇ L.
  • the PCR conditions were: denaturation at 94 °C for 5 min, 94 °C for lmin, 56 °C for 40 s, 72 °C for 2 min, 30 cycles.
  • the PCR product was 1845 bp in size and included the coding sequence for the human serum albumin gene.
  • the upstream primer hsa-F was added with a «1 restriction site, and the downstream primer hsa-R was added ( ⁇ 1 site.
  • the amplified fragment was TaKaRa pMD18-T Simple Vector ( TaKaRa, treasure bio Engineering (Dalian) Co., Ltd.) method, cloned into pMD18-T vector, transformed into E.
  • coli DH5a screened the cloned strain, extracted the plasmid, and digested with 1 ⁇ 1 and ⁇ 3 ⁇ 4 ⁇ 1 and £/?1, agar Positive clones were identified by glycophore electrophoresis, and the recombinant plasmid was sequenced, and sequencing of the cloned fragment was performed by Biomics Biotechnology Co., Ltd.
  • the recombinant plasmid of the specification was named pMD18-T-HSA, and the recombinant strain was named DH5o/pMD18-T-HSA.
  • the positive recombinant strain was stored in LB medium containing 15% glycerol and frozen at -80 °C.
  • Example 2 Preparation and application of HSA-producing Bacillus subtilis recombinant spores using CotC as a carrier surface
  • Plasmid pJS700 Li Qian. Study on recombinant spores of Bacillus subtilis expressing WSSV envelope proteins Vp l 9 and Vp28 on the surface of molecular carrier with CotX [D].
  • amyE 5 '-end and amyE 3 '-end represent the 5' and 3' ends of the amylase gene amyE (Gene Bank SEQ ID NO: NP-388186), respectively, and are integrated in Bacillus by homologous recombination.
  • the ampicillin resistance gene and the erythromycin resistance gene were respectively indicated as selection markers in Escherichia coli and Bad iw 168 ( ⁇ -).
  • cotC is the Bacillus subtilis spore capsid protein CotC gene, which contains the promoter sequence of the cotC gene (Gene Bank SEQ ID NO: NP-389653) and the CotC coding sequence which does not contain the cotC stop codon.
  • oriC is an E. coli replicon fragment.
  • the hsa fragment and pJS700 were recovered by agarose gel recovery kit with the plasmids pMD 18-T-HSA and pJS700, respectively, and the two fragments were digested with T 4 DNA ligase, and the ligated product was transformed into Escherichia coli.
  • DH5 (X competent cells, transformants were plated in LB plates containing 50 ⁇ g/mL ampicillin overnight at 37 ° C, and the next day selected positive monoclonal strains were cultured in LB liquid containing 50 ⁇ g/mL ampicillin The medium was cultured at 37 °C for 12 to 16 hours.
  • the plasmid was extracted and identified by PCR and ⁇ «1 and ⁇ 0 ⁇ 1.
  • the positive clones were identified by agarose gel electrophoresis.
  • the recombinant integrated plasmid was named pJS700- HSA
  • the positive recombinant strain was named DH5a/pJS700-HSA.
  • the recombinant integrated plasmid PJS700-HSA contained the recombinant gene cotC-hm, and the construction process and its map are shown in Fig. 2.
  • the integrated recombinant plasmid PJS700-HSA was transformed into ⁇ «7 ⁇ « subtilis 168 (trp ) (Bacillus Genetic Stock Center, Department of Biochemistry, The Ohio State University, West 12th Avenue, Columbus, Ohio, 43210, USA) with 0.4 ⁇
  • the LB plate of g/mL erythromycin purchased from SIGMA was used to screen the recombinants. Single colonies were selected from the plates and inoculated into LB plates containing 1% starch. The cells were cultured overnight at 37 ° C, and the next day was treated with iodine-potassium iodide solution. Starch plate staining was used to identify recombinant strains.
  • Preparation method of 1% starch plate 0.1 g of soluble starch was added to lOOmL LB solid medium, and the plate was inverted after autoclaving.
  • Preparation of iodine-potassium iodide solution 2 g of potassium iodide, lg of iodine, 300 mL of distilled water, first dissolve potassium iodide in a small amount of deionized water, add iodine after all dissolved, dilute to 300 mL by shaking, and store in a brown glass bottle protected from light. It can be diluted 2 ⁇ 10 times when used. Appropriate amount of iodine-potassium iodide solution was dropped on the starch plate.
  • amyE-F/amyE-R amyE-F/amyE-R
  • hsa-F/hsa-R amyE-F/hsa-R
  • amyE-F/hsa-R is a primer pair
  • the chromosome of Bacillus subtilis is used as a template to identify whether the recombinant strain contains the « -CO C-fe « gene fragment by PCR amplification.
  • a schematic diagram of the integration of the EwZ-coiC-/ ⁇ fragment in the Bacillus subtilis 168 Op-) chromosome is shown in Figure 4.
  • the PCR reaction system was subjected to the TaKaRa ⁇ instruction manual.
  • the 20 ⁇ L reaction system contained LA Taq 0.2 ⁇ L, 10 X LA PCR Buffer II (Mg 2+ Plus) 2 L, dNTP Mixture (2.5 mM each) 3.2 L, template DNA 100ng, upstream and downstream primers (20 ⁇ M) each 0.4 ⁇ L, and finally added sterile ultrapure water to 20 L.
  • the PCR reaction conditions were set separately according to the characteristics of each primer pair.
  • telomeres Approximately 5.0 kb, 1.8 kb, 4.3 kb, and 2.3 kb amplified fragments were detected in the recombinant strains, whereas only about 1.1 kp of the amylase gene was detected in the wild-type strain, and the results are shown in Fig. 5. This indicates that the recombinant strain contains the Emf-co C-foa recombinant gene on the chromosome, and the 7- CO C-/ira recombinant fragment was successfully inserted into the amylase gene.
  • the identified recombinant strain was named Bacillus subtilis 168 rp )/pJS700-HSA.
  • the method for extracting the chromosome of Bacillus subtilis is as follows: The single colony is cultured in a medium supplemented with LB and the corresponding antibiotic, and cultured in a shaker at 37 ° C to an OD 600 of 1.0 2.0. Take 1.5 mL of the bacterial solution in Eppendor f, centrifuge at 8000 rpm for 10 min, discard the supernatant, wash the pellet with 10 ml of ⁇ 1 ⁇ 118.0 (1011 ⁇ 13 ⁇ 48-1 ⁇ 1, ImMEDTA), and centrifuge to remove the supernatant.
  • the formulation of the DSM medium is: 0.8% nutrient broth (Difo), 0.1% CL 0.025% MgS0 4 - 7H 2 0, l.OmM Ca(N0 3 ) 2 ⁇ 4 ⁇ 2 0, 10 ⁇ MnCl 2 , l. ( ⁇ MFeS0 4 .
  • the wild type strain fiadZfe ⁇ Mfc 168 ) and the recombinant strain were inoculated separately in DSM medium, the recombinant strains were treated with 8h and 48h cultures respectively, and the wild type strains were treated for 48 hours.
  • Method for treatment of spore protein The cells were collected by centrifugation at 12000 rpm for 10 min, resuspended in the same volume of GTEBuffer (50 mM glucose, 20 mM pH 7.5 Tris-HCl, 10 mM EDTA, 2 mg/mL lysozyme), and treated at 37 ° C for 30 minutes for destruction.
  • GTEBuffer 50 mM glucose, 20 mM pH 7.5 Tris-HCl, 10 mM EDTA, 2 mg/mL lysozyme
  • mice Male SPF-grade ICR mice (5 weeks old, 18-22 g) were acclimated for two weeks in the experimental environment prior to the experiment. One cage per 10 mice, culture conditions are: Maintain room temperature 20 ⁇ 2 ° C, free of diet and drinking water. After adaptation, we first performed an acute toxicological analysis of the recombinant spores. Twenty ICR mice (half male and female) were taken and the mice were intragastrically administered with recombinant spores at a dose of 5 m g /fe ⁇ .
  • mice were then observed for two weeks, and no adverse reactions occurred in the experimental mice. Then, 40 mice were randomly divided into 4 groups: a blank control group (administered saline to mice), and a wild-type treatment group (wild-type spores were administered to mice at a dose of 2 mg/(gd). ), two recombinant spore treatment groups (a low-dose group at a dose of 1 mg/Cg_d), a large dose group at a dose of 2 mg/(gd)) o Collected spores were vacuum-dried and weighed and resuspended in physiology In salt water, it is used separately.
  • mice were intragastrically administered with spores resuspended in physiological saline, and the mice were observed and weighed daily during the intragastric administration. After 30 days, the eyeballs of the mice were dug, blood was taken, serum was separated, and biochemical parameters were detected. The biochemical detection of mouse serum was performed by the affiliated Hospital of Jiangsu University (Jiangbin Hospital). The experimental data is expressed as the mean standard deviation. Statistical analysis was performed using a t-test of independent samples. PO.05 indicates that the experimental results are statistically significant. The experimental results showed that albumin in the serum of mice with large doses of recombinant spores was significantly higher than other groups, P ⁇ 0.01, see Figure 7. This indicates that oral recombinant spores can increase the albumin content in the serum of animals. There was no significant difference in the body weight of the mice, as shown in Figure 8, which indicates that oral spores did not cause discomfort in the gastrointestinal tract of mice.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Virology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé de préparation d'une endospore de Bacillus subtilis présentant à sa surface une protéine de fusion de la sérumalbumine humaine, ainsi qu'un produit destiné à une administration par voie orale à base de celle-ci. Le gène cotC de la protéine de la capside de l'endospore de Bacillus subtilis, qui sert de vecteur moléculaire, est recombiné avec le gène hsa de la sérumalbumine humaine, en vue de l'édification d'un plasmide recombinant intégré qui s'exprime sous la forme d'un produit de fusion. Le plasmide recombinant exprimant la HSA (sérumalbumine humaine) sous la forme d'un produit de fusion est transformé dans Bacillus subtilis, un gène exogène étant intégré à un gène amyE de l'amylase au niveau d'un site spécifique du chromosome par recombinaison homologue. La souche recombinante ne présente pas d'activité amylase du fait de l'inactivation insertionnelle du gène de l'amylase. Une souche recombinante de Bacillus subtilis, exprimant CotC-HAS sous la forme d'un produit de fusion, fait l'objet d'un criblage et l'on induit la production par la souche recombinante d'une endospore présentant de la sérumalbumine humaine à sa surface. L'endospore produite par la souche recombinante peut être directement administrée par voie orale à des animaux.
PCT/CN2011/084333 2011-03-21 2011-12-21 Endospore recombinante présentant de la sérumalbumine humaine à sa surface et destinée à une administration par voie orale, et son procédé de préparation WO2012126265A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201110067373.7 2011-03-21
CN 201110067373 CN102181469A (zh) 2011-03-21 2011-03-21 枯草芽孢杆菌表面展示人血清白蛋白重组芽孢及其制备方法

Publications (1)

Publication Number Publication Date
WO2012126265A1 true WO2012126265A1 (fr) 2012-09-27

Family

ID=44567757

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2011/084333 WO2012126265A1 (fr) 2011-03-21 2011-12-21 Endospore recombinante présentant de la sérumalbumine humaine à sa surface et destinée à une administration par voie orale, et son procédé de préparation

Country Status (2)

Country Link
CN (1) CN102181469A (fr)
WO (1) WO2012126265A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103520706A (zh) * 2013-10-09 2014-01-22 天津林达生物科技有限公司 口服用重组人血清白蛋白融合蛋白制剂配方、产品及用途

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102181469A (zh) * 2011-03-21 2011-09-14 江苏大学 枯草芽孢杆菌表面展示人血清白蛋白重组芽孢及其制备方法
CN103045631B (zh) * 2012-12-26 2014-12-24 江苏大学 口服降血糖重组人胰岛素原的制备方法
CN105349566B (zh) * 2015-11-18 2019-04-30 江苏大学 一种补充人体必需氨基酸的口服重组营养多肽的制备方法
CN108410786B (zh) * 2018-02-09 2021-10-15 武汉瑞法医疗器械有限公司 高效表达纤溶酶的枯草芽孢杆菌工程菌以及制备方法
CN109220911B (zh) * 2018-09-04 2021-05-14 南开大学 葡萄糖和乙醇在协同调控斑马鱼心血管发育中的应用
CN109609425B (zh) * 2018-12-29 2022-04-05 广西壮族自治区农业科学院农产品加工研究所 一种利用枯草芽孢杆菌整合位点的酶的活性恢复筛选整合重组子的方法
CN113817657A (zh) * 2021-07-23 2021-12-21 中国农业科学院饲料研究所 芽孢和营养体阶段均具群体淬灭活性的枯草芽孢杆菌及其构建方法与应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0143836A1 (fr) * 1983-05-19 1985-06-12 The Public Health Research Institute Of The City Of New York, Inc. Vecteurs d'expression et de secretion et procede de formation de ces vecteurs
WO2002046388A1 (fr) * 2000-12-08 2002-06-13 Genofocus Co., Ltd. Methode d'expression de proteines a la surface d'une spore
CN101475954A (zh) * 2008-12-05 2009-07-08 江苏大学 表面展示具有催化活性的脂肪酶的重组芽孢的制备方法
CN102174557A (zh) * 2011-03-16 2011-09-07 江苏大学 一种表面展示家蚕乙醇脱氢酶重组芽孢及其制备方法
CN102181469A (zh) * 2011-03-21 2011-09-14 江苏大学 枯草芽孢杆菌表面展示人血清白蛋白重组芽孢及其制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0143836A1 (fr) * 1983-05-19 1985-06-12 The Public Health Research Institute Of The City Of New York, Inc. Vecteurs d'expression et de secretion et procede de formation de ces vecteurs
WO2002046388A1 (fr) * 2000-12-08 2002-06-13 Genofocus Co., Ltd. Methode d'expression de proteines a la surface d'une spore
CN101475954A (zh) * 2008-12-05 2009-07-08 江苏大学 表面展示具有催化活性的脂肪酶的重组芽孢的制备方法
CN102174557A (zh) * 2011-03-16 2011-09-07 江苏大学 一种表面展示家蚕乙醇脱氢酶重组芽孢及其制备方法
CN102181469A (zh) * 2011-03-21 2011-09-14 江苏大学 枯草芽孢杆菌表面展示人血清白蛋白重组芽孢及其制备方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KIM, JUNEHYUNG ET AL.: "Display of proteins on Bacillus subtilis endospores", CELL.MOL.LIFE SCI., vol. 66, no. 19, 25 June 2009 (2009-06-25), pages 3127 - 3136 *
SAUNDERS, C.W. ET AL.: "Secretion of Human Serum Albumin from Bacillus subtilis", JOURNAL OF BACTERIOLOGY, vol. 169, no. 7, 30 July 1987 (1987-07-30), pages 2917 - 2925 *
XU, XIAOMAN ET AL.: "Recent progress of the research on spore surface display", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 26, no. 10, 25 October 2010 (2010-10-25), pages 1404 - 1409 *
ZHOU, ZHENWEN ET AL.: "Immunogenicity of recombinant Bacillus subtilis spores expressing Clonorchis sinensis tegumental protein", PARASITOLOGY RESEARCH, vol. 102, no. 2, 9 October 2007 (2007-10-09), pages 293 - 297 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103520706A (zh) * 2013-10-09 2014-01-22 天津林达生物科技有限公司 口服用重组人血清白蛋白融合蛋白制剂配方、产品及用途
CN103520706B (zh) * 2013-10-09 2019-02-26 天津林达生物科技有限公司 口服用重组人血清白蛋白融合蛋白制剂配方、产品及用途

Also Published As

Publication number Publication date
CN102181469A (zh) 2011-09-14

Similar Documents

Publication Publication Date Title
WO2012126265A1 (fr) Endospore recombinante présentant de la sérumalbumine humaine à sa surface et destinée à une administration par voie orale, et son procédé de préparation
CN110157654B (zh) 一种纳豆芽孢杆菌重组菌及其构建方法与应用
CN110055204B (zh) 一种敲除spoⅡQ和pcf基因提高地衣芽孢杆菌发酵产酶的方法及应用
WO2021063355A1 (fr) Procédé de construction de bactéries recombinantes de bacillus subtilis dégradant l'éthanol de qualité alimentaire
WO2022121968A1 (fr) Probiotiques modifiés capables de dégrader la phénylalanine
WO2012122765A1 (fr) Spore recombinant ayant une alcool déshydrogénase de bombyx mori présente sur sa surface et son procédé de préparation
CN106967659A (zh) 一种表达谷氨酸脱羧酶的无抗生素抗性重组枯草芽孢杆菌的构建及发酵方法
WO2022148362A1 (fr) Probiotique d'ingénierie ayant une phénylalanine ammoniac-lyase à affichage de surface
US11970723B2 (en) Strain producing D-allulose 3-epimerase and application thereof
CN103864914A (zh) 高纯度白细胞介素-24包涵体的制备方法
WO2022100543A1 (fr) Polynucléotide pour augmenter le rendement de c-di-amp lorsqu'il est utilisé dans l'expression bactérienne de daca
CN106085934B (zh) 食品级纳豆激酶表达菌
CN109486834B (zh) 高产乳酸链球菌素的重组乳酸乳球菌及构建方法
CN117756898A (zh) 重组表达阿克曼氏菌Amuc_1631蛋白及其应用
CN110172433B (zh) 一种产猪表皮生长因子的重组枯草芽孢杆菌工程菌及应用
CN113234699A (zh) α-1,2-岩藻糖基转移酶及其应用
WO2023045682A1 (fr) Procédé d'augmentation de la quantité d'expression soluble d'un polypeptide
CN115948316B (zh) 一种提升乳酸菌耐酸性的方法
WO2021062886A1 (fr) Procédés de préparation et de transformation pour cellule compétente de bacillus subtilis
CN113699092B (zh) 一种重组枯草芽孢杆菌及其构建方法和应用
CN110669710A (zh) 重组乳酸乳球菌和罗非鱼无乳链球菌病疫苗
WO2020143708A1 (fr) Bactérie d'ingénierie de bacillus licheniformis (b. licheniformis) capable de limiter l'autolyse des plantes et procédé de construction, et application pour la bactérie d'ingénierie de b. licheniformis
CN110257314B (zh) 一种产抗菌肽Cecropin B的重组枯草芽孢杆菌、构建方法及其应用
Chou et al. Optimization of the host/vector system and culture conditions for production of penicillin acylase in Escherichia coli
WO2023102816A1 (fr) Bactérie génétiquement modifiée et procédé de préparation de l-ornithine à partir d'une bactérie génétiquement modifiée

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11861696

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11861696

Country of ref document: EP

Kind code of ref document: A1