阅读说明：本技术 一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法 (Method for engineering bacillus subtilis into multifunctional stable platform for producing nano antibody ) 是由 李嘉禾 于 2020-06-10 设计创作，主要内容包括：本发明公开了一种将枯草芽孢杆菌工程化为生产纳米抗体的多功能稳定平台的方法,通过基因工程的方法,对枯草芽孢杆菌进行改造,得到可用于生产纳米抗体的多功能稳定的平台,同时也可进一步将工程化后的枯草芽孢杆菌制为芽孢保存备用。相较于大肠杆菌法和LCE法,本发明中生产纳米抗体的方法,具有所得产品的毒素水平低、纯化工艺更简单、生产规模易于扩大、可长期保存以及在极端环境下进行生产等优点。(The invention discloses a method for engineering bacillus subtilis into a multifunctional stable platform for producing a nano antibody, which is characterized in that the bacillus subtilis is transformed by a genetic engineering method to obtain the multifunctional stable platform for producing the nano antibody, and the engineered bacillus subtilis can be further prepared into spores for storage. Compared with an escherichia coli method and an LCE method, the method for producing the nano antibody has the advantages that the toxin level of the obtained product is low, the purification process is simpler, the production scale is easy to expand, the product can be stored for a long time, the production can be carried out in an extreme environment, and the like.)

1. A method for engineering Bacillus subtilis into a multifunctional stable platform for producing a nano antibody is characterized by comprising the following steps: the method comprises the following steps:

a1: constructing a fusion gene: the fusion gene comprises a DNA sequence of a nano antibody, a FLAG peptide (DYKDDDDK) for detecting fusion protein and a6 × histidine tag (His tag) for performing metal affinity chromatography on a chromatography column containing triacetic acid (NTA); synthesizing a DNA fragment and optimizing codons encoding the DNA fragment;

a2: amplifying the DNA fragment, and purifying to obtain an amplification product;

a3: assembling the amplification product into a plasmid to obtain a recombinant plasmid;

a4: after the recombinant plasmid is transferred into escherichia coli, continuously culturing for 10-18 h at 35-40 ℃, and screening bacteria to obtain primary screened bacteria;

a5: inoculating the primary screening bacteria into a culture medium, continuously culturing for 12-18 h at 35-40 ℃, and separating to obtain expression plasmids;

a6: transferring the expression plasmid into bacillus subtilis, continuously incubating for 2-3 h, performing overnight culture at 35-40 ℃, and screening bacteria to obtain secondary screened bacteria;

a7: culturing the secondary screening bacteria in a shake flask until the cell density OD600 of the secondary screening bacteria reaches 0.6-0.8 to obtain shake flask culture bacteria;

a8: adding an inducer into the shake flask culture bacteria to induce protein expression, continuously culturing for 15-20 h at 25-30 ℃, and centrifuging to obtain bacterial supernatant containing the nano antibody; or, using the shake flask culture bacteria to prepare spores, and storing for later use.

2. The method of engineering bacillus subtilis into a multifunctional stabilization platform for the production of nanobodies according to claim 1, wherein: the nano antibody is any one of an anti-Caffeine (Caffeine) nano antibody, an anti-Methotrexate (MTX) nano antibody, an anti-mouse cytotoxic T lymphocyte-associated protein 4(CTLA-4) nano antibody and an anti-mouse programmed death ligand 1(PD-L1) nano antibody.

3. The method of engineering bacillus subtilis into a multifunctional stabilization platform for the production of nanobodies according to claim 1, wherein: the fusion gene comprises a DNA sequence of a nano antibody, a FLAG peptide (DYKDDDDK) for detecting the fusion protein and a carbohydrate binding domain (CBM), so that the fusion protein containing the carbohydrate binding domain (CBM) is obtained.

4. The method of engineering bacillus subtilis into a multifunctional stabilization platform for the production of nanobodies according to claim 1, wherein: the bacillus subtilis is a WB800N competent strain.

5. The method of engineering bacillus subtilis into a multifunctional stabilization platform for the production of nanobodies according to claim 1, wherein: the Escherichia coli is DH5a strain.

6. The method of engineering bacillus subtilis into a multifunctional stabilization platform for the production of nanobodies according to claim 4, wherein: the preparation method of the WB800N competent strain comprises the following steps: inoculating bacillus subtilis WB800N into a culture medium one day before use, wherein the culture medium comprises 1-1.1% of potassium hydrogen phosphate, 0.4-0.6% of monopotassium phosphate, 1-3% of glucose, 0.05-0.1% of sodium citrate dihydrate, 0.05-0.15% of ferric ammonium citrate, 0.2-0.3% of potassium aspartate, 0.03-0.06% of yeast extract and 8-12 mmol/L magnesium sulfate.

7. The method of engineering bacillus subtilis into a multifunctional stabilization platform for the production of nanobodies according to claim 1, wherein: the culture medium used in the shake flask is an LB culture medium, the culture temperature is 35-40 ℃, the rotation speed is 200-300 rpm, and the LB culture medium comprises 1% of trypsinogen, 1% of yeast extract and 0.5% of NaCl by mass fraction; and/or, the inducer is isopropyl thiogalactoside (IPTG).

8. The method of engineering bacillus subtilis into a multifunctional stabilization platform for the production of nanobodies according to claim 1, wherein: the spore preparation specifically comprises the following steps:

b1: coating the shake flask culture bacteria on an agar plate, incubating for 2 days at 35-40 ℃ to obtain unpurified spores, and scraping the unpurified spores from the agar plate;

b2: suspending the unpurified spores in cold water, centrifuging, and discarding the supernatant;

b3: repeating the operation in B2 for 2 times to obtain primarily purified spores, and then re-suspending the primarily purified spores in cold water at 4 ℃ overnight;

b5: the operations in B2-B3 were repeated 2 more times to finally obtain spores, which were resuspended in cold water and stored at 4 ℃.

9. The method of engineering bacillus subtilis into a multifunctional stabilization platform for the production of nanobodies according to claim 3, wherein: the carbohydrate-binding domain (CBM) -containing fusion proteins can be immobilized on carbohydrates for long-term storage and capture of small molecules.

10. The method of engineering bacillus subtilis into a multifunctional stabilization platform for nanobody production according to claim 9, wherein: the carbohydrate is Regenerated Amorphous Cellulose (RAC); and/or, the preparation of the Regenerated Amorphous Cellulose (RAC) comprises the following steps:

c1: adding microcrystalline cellulose powder into double distilled water to prepare slurry;

c2: adding ice phosphoric acid into the slurry, incubating for 2 hours on ice, then adding an alkaline agent, and adjusting the pH to 6.8-7.2;

c3: dialyzing to obtain the Regenerated Amorphous Cellulose (RAC), and freeze-drying for later use.

Technical Field

The invention relates to the field of biological medicine, in particular to a method for producing a nano antibody by utilizing bacillus subtilis.

Background

Nanobodies were a specific antibody found in llama in 1993 by belgium scientists, present in camel peripheral blood, named for the length of the molecule on the nanometer scale, consisting of only one domain of a minimal antigen binding fragment, containing the natural heavy chain variable region.

Compared with a monoclonal antibody, the nano antibody of the single-chain variable antibody fragment derived from the camelid is relatively easy and simple to prepare. In addition, because the nano antibody is easy to perform gene operation, monovalent, bivalent, bispecific and multivalent antibodies can be formed to form fusion protein for targeted therapy, and simultaneously, because the nano antibody has the advantages of small molecular weight, high solubility, good affinity, relative stability, strong penetrating power and the like, the nano antibody has translation potential proved in whole body imaging and development of new drugs for treating various diseases (including but not limited to inflammation, cancer, bone diseases, hematology and infectious diseases), and has wide prospects and applications in the fields of clinical treatment and diagnosis, medicine research and development, food science and the like.

The main forces for the production of various biological agents including nanobodies are living cell systems such as bacteria, yeast, plant and mammalian cells, however, intracellular expression of E.coli to produce nanobodies requires lengthy steps to release intracellular proteins for purification and endotoxin removal, greatly prolonging downstream processing times. In recent years, Lyophilized Cell Extracts (LCEs) prepared from living cells have made drug production decentralised and room temperature storage possible as an alternative to the above-described methods, but cell extracts cannot replicate and are therefore difficult to scale up; some LCE systems use escherichia coli and therefore must be subjected to rigorous procedures to remove endotoxin contamination; the mixture of proteins and small molecules in the cell lysate requires extensive downstream product purification; there was lot-to-lot variability in cell-free protein expression systems.

The endotoxin level of the bacillus subtilis is extremely low, the downstream purification steps can be obviously simplified, but the bacillus subtilis can secrete a large amount of protease to the outside of cells while expressing the foreign protein, so that the foreign protein cannot exist stably. Many eukaryotic proteins are produced in poor quality in the Sec-dependent secretion pathway in bacillus subtilis, and although corresponding strains have been developed to alleviate this problem, some secreted eukaryotic proteins still have quality problems due to misfolding of the protein of interest.

Disclosure of Invention

The invention uses bacillus subtilis as a microorganism factory to produce the nano antibody, belongs to the alternative scheme of producing the nano antibody by using an escherichia coli method and an LCE method, solves various problems in the prior art, such as the reduction of the quality and the yield of the product protein caused by the error folding of the target protein, and the like, and has the following advantages: the bacillus subtilis has low toxin level, the downstream purification process is simpler, and the production scale is easy to expand; the bacillus subtilis containing the nano antibody fusion gene is converted into spores, can be stably stored for a long time, can be produced in an extreme environment, and the like.

The invention specifically adopts the technical scheme that:

the steps of engineering bacillus subtilis into a multifunctional stable platform for producing the nano antibody are as follows:

a1: constructing a fusion gene: the fusion gene comprises a DNA sequence of a nano antibody, a FLAG peptide (DYKDDDDK) for detecting fusion protein and a6 × histidine tag (His tag) for performing metal affinity chromatography on a chromatography column containing triacetic acid (NTA); synthesizing a DNA fragment and optimizing codons encoding the DNA fragment;

a2: amplifying the DNA fragment, and purifying to obtain an amplification product;

a3: assembling the amplification product into a plasmid to obtain a recombinant plasmid;

a4: after the recombinant plasmid is transferred into escherichia coli, continuously culturing for 10-18 h at 35-40 ℃, and screening bacteria to obtain primary screened bacteria;

a5: inoculating the primary screening bacteria into a culture medium, continuously culturing for 12-18 h at 35-40 ℃, and separating to obtain expression plasmids;

a6: transferring the expression plasmid into bacillus subtilis, continuously incubating for 2-3 h, performing overnight culture at 35-40 ℃, and screening bacteria to obtain secondary screened bacteria;

a7: culturing the secondary screening bacteria in a shake flask until the cell density OD600 of the secondary screening bacteria reaches 0.6-0.8 to obtain shake flask culture bacteria;

a8: adding an inducer into the shake flask culture bacteria to induce protein expression, continuously culturing for 15-20 h at 25-30 ℃, and centrifuging to obtain bacterial supernatant containing the nano antibody; or, using the shake flask culture bacteria to prepare spores, and storing for later use.

In the invention, the nano antibody can be specifically any one of an anti-Caffeine (Caffeine) nano antibody, an anti-Methotrexate (MTX) nano antibody, an anti-mouse cytotoxic T lymphocyte-associated protein 4(CTLA-4) nano antibody and an anti-mouse programmed death ligand 1(PD-L1) nano antibody.

Preferably, the fusion gene comprises a DNA sequence of a nanobody, a FLAG peptide (DYKDDDDK) for detecting the fusion protein and a carbohydrate-binding domain (CBM), so as to obtain the fusion protein containing the carbohydrate-binding domain (CBM).

Preferably, the bacillus subtilis is a WB800N competent strain.

Preferably, the E.coli is DH5a strain.

The preparation method of the WB800N sensitive strain comprises the following steps: inoculating bacillus subtilis WB800N into a culture medium one day before use, wherein the culture medium comprises 1-1.1% of potassium hydrogen phosphate, 0.4-0.6% of monopotassium phosphate, 1-3% of glucose, 0.05-0.1% of sodium citrate dihydrate, 0.05-0.15% of ferric ammonium citrate, 0.2-0.3% of potassium aspartate, 0.03-0.06% of yeast extract and 8-12 mmol/L magnesium sulfate.

Preferably, in the steps a7 and A8, the culture medium used in the shake flask is an LB culture medium, the culture temperature is 35 to 40 ℃, the rotation speed is 200 to 300rpm, and the LB culture medium comprises 1% by mass of trypsinogen, 1% by mass of yeast extract and 0.5% by mass of NaCl.

Preferably, the inducer used in step A8 is isopropyl thiogalactoside (IPTG).

Preferably, in step A8, the preparation of spores specifically comprises the following steps:

b1: coating the shake flask culture bacteria on an agar plate, incubating for 2 days at 35-40 ℃ to obtain unpurified spores, and scraping the unpurified spores from the agar plate;

b2: suspending the unpurified spores in cold water, centrifuging, and discarding the supernatant;

b3: repeating the operation in B2 for 2 times to obtain primarily purified spores, and then re-suspending the primarily purified spores in cold water at 4 ℃ overnight;

b5: the operations in B2-B3 were repeated 2 more times to finally obtain spores, which were resuspended in cold water and stored at 4 ℃.

Preferably, the fusion protein comprising a carbohydrate binding domain (CBM) can be immobilized on a carbohydrate for long term storage and capture of small molecules.

Preferably, the carbohydrate is Regenerated Amorphous Cellulose (RAC).

Preferably, the preparation of the Regenerated Amorphous Cellulose (RAC) specifically comprises the following steps:

c1: adding microcrystalline cellulose powder into double distilled water to prepare slurry;

c2: adding ice phosphoric acid into the slurry, incubating for 2 hours on ice, then adding an alkaline agent, and adjusting the pH to 6.8-7.2;

c3: dialyzing to obtain the Regenerated Amorphous Cellulose (RAC), and freeze-drying for later use.

The invention has the beneficial effects that:

firstly, the modified bacillus subtilis strain WB800N is adopted, 8 extracellular proteases are deleted, and the problem that the yield and the quality of secreted proteins are obviously reduced due to the secretion of protease in culture supernatant of bacillus subtilis can be solved.

Second, the constructed fusion gene: the fusion gene consists of a DNA sequence of a nano antibody, a FLAG peptide (DYKDDDDK) for detecting fusion protein and a6 × histidine tag (His tag) for performing metal affinity chromatography on a chromatography column containing triacetic acid (NTA). The His-tag may also be replaced with a carbohydrate-binding domain (CBM), such as a thermostable cellulose-binding domain (CBD) from the cipA gene of clostridium thermoanaerobicum, to achieve immobilization or affinity purification of a cellulose-based fusion protein, wherein:

and (3) fixing the CBD-nanobody fusion protein: the fusion protein can be fixed on cellulose and used for producing cellulose paper-based biosensing or biological storage materials;

affinity purification: the cellulose fixed with the fusion protein is used as a stationary phase in affinity chromatography and can be used for capturing corresponding small molecules. If the secreted CBD-caffeine nanobody fusion protein is fixed on cellulose and loaded into a syringe, a simple chromatographic system is obtained, and the simple chromatographic system can be used for capturing caffeine. The method can be used for purifying other substances by changing the types of the nano antibodies.

The vector plasmid used in the invention contains a strong promoter of a groE gene operon derived from bacillus subtilis, and on the basis, an escherichia coli-derived lactose operon is introduced to be converted into an IPTG inducible promoter. Downstream of the promoter region are the Ribosome Binding Site (RBS) and the amyQ gene secretion signal sequence. Considering that extracellular protein transport of amyQ secretion signal sequences is dependent on the bacterial Sec pathway, the transporters must perform a protein-spreading reaction, which relies on the mechanism of protein transport molecules immobilized to bacterial membranes. During this process, problems often arise in that the secretion of most mammalian recombinant proteins does not reach detectable levels due to degradation or misfolding. Whereas in the present invention, the fusion gene comprises a recombinant protein having high thermostability and/or relatively small molecular weight, i.e., a nanobody of interest, in this combination, the above-mentioned problems do not occur in the Sec-dependent secretion pathway of bacillus subtilis.

In general, the multifunctional stable platform based on engineered bacillus subtilis obtained by the method of the invention for producing nanobodies has the following advantages:

1. the yield is high: 15-20 mg of nano antibody can be produced in each liter of culture supernatant in the shake flask culture mode, and the yield of the nano antibody can be further improved through feeding, batch culture and the like, so that the productivity is enlarged.

2. Low cost, long shelf life: the secreted nano antibody containing the carbohydrate binding domain (CBM) has good heat resistance, can be fixed on substrates such as carbohydrates, such as cellulose and the like, has low cost, can be stored for a long time, and can detect small molecules under extreme conditions (such as high temperature, ultraviolet irradiation and acid-base environment). It is known that the recombinant fusion protein is prepared mainly by using Escherichia coli as an expression platform, and the process needs to go through a step of decomposing bacteria by using lysozyme, while the Sec-dependent secretion pathway in Bacillus subtilis is extracellular production of the recombinant protein. In addition, the small size and high thermal stability of CBM's help to achieve rapid renaturation of Bacillus subtilis and resistance to protease degradation.

3. Can be used in the field of cancer immunotherapy: for example, anti-PD-Ll and anti-CTLA-4 can be used for Immune Checkpoint Blockade (ICB), at present, most anti-PD-Ll and anti-CTLA-4 nano antibodies are mostly produced in escherichia coli cells, but a long-time bacterial lysis and endotoxin removal process can greatly increase the production cost, the process is dependent on certain integrated production equipment and is high in cost, and the nano antibodies are produced by the engineered bacillus subtilis, so that the process can be simplified, the remarkable cost advantage is shown, and the nano antibodies can be stably stored for a long time after being made into spores;

4. can be used for the actual development of sensitive diagnosis. The nano antibody can realize targeted recognition on a low percentage (< 1%) of cells, and can also be directly used for detecting proteins on the cell surface by using the bacterial supernatant containing the nano antibody.

5. Can be used in the field of inflammation treatment: for example, the expression of inflammatory cytokines such as TNF-alpha is blocked through the generation of nano antibodies;

6. can be used in the field of gastrointestinal disease treatment: the tolerance capability of the spores to the acidic environment also shows the application prospect of producing the nano antibody by using the bacillus subtilis as a carrier so as to improve gastrointestinal diseases.

7. Has potential for use in combination with other technologies: such as covalent conjugation of the nanobody with a fluorescent dye or other peptide sequences through Sortase-mediated protein ligation.

Drawings

The invention will be further explained with reference to the drawings.

FIG. 1 is a schematic diagram of engineered Bacillus subtilis spore production as a nano-antibody stabilization platform;

FIG. 2 is a process route for the production of nanobodies;

FIG. 3 shows the result of detection by denaturing polyacrylamide gel electrophoresis (SDS-PAGE);

FIG. 4 shows the results of an immunoblotting (western blot) test for the presence or absence of a target protein;

FIG. 5 shows the result of a secretion kinetics detection experiment (SDS-PAGE) of CBD-anti-caffeine nanobody;

FIG. 6 shows the results of a test for the secretion kinetics of CBD-anti-caffeine nanobody (western blot);

FIG. 7 shows the stability test of CBD-anti-caffeine nanobody;

FIG. 8 is a specific target recognition and verification experiment of CBD-anti-caffeine nanobody;

FIG. 9 shows the flow cytometry detection result of the binding of the anti-PD-L1 nano antibody to DC-2.4 cells;

FIG. 10 shows the results of the detection of the target recognition of a low percentage of cells by anti-CTLA-4 nanobody;

FIG. 11 shows the results of heat resistance tests of spores obtained by the present invention;

FIG. 12 shows the results of detection of spores obtained by the present invention after acid resistance test.

Detailed Description

The present invention will be further described with reference to the following embodiments.

Materials and methods

Chemical products:

unless otherwise indicated, all chemicals and cell culture media were purchased from the united states flying world science (cambridge, massachusetts, usa) and were of the highest purity or analytical grade commercially available.

The Gibson Assembly Master Mix kit and cloning enzymes were purchased from New England Biolabs Inc. (NEB) (Epstein, Mass., USA). DNA oligonucleotides were designed and supplied by Sigma Aldrich (St. Louis, Missouri, USA). Plasmid extraction, agarose gel DNA purification, and PCR clean-up kits were purchased from CoWinBiosciences (cambridge, ma, usa). DC protein assay kits (#500-0116) were purchased from Bio-Rad biomedical products, Inc. (Helkris, Calif., USA).

The nanobody sequences against mouse PD-L1 and against mouse CTLA-4 were derived from the RCSB protein structure database (PDB) and gene fragments were synthesized by Twist Bioscience corporation (san francisco, ca, usa). Coli expression vectors encoding anti-MTX and anti-caffeine nanobodies were gifted by James horns doctor of the university of beilinois.

Plasmids and strains:

the plasmid and the Bacillus subtilis WB800N strain were generously given to Boshi, the national university academy of sciences (Vietnam university), where the plasmid had been digested with BamHI and XbaI. Bacillus subtilis WB800N was cultured in Luria Bertani (LB, 1% trypsinogen, 1% yeast extract and 0.5% NaCl) medium. Chloramphenicol and neomycin were used as selection antibiotics at concentrations of 5. mu.g/ml and 10. mu.g/ml, respectively. Plasmid construction and propagation were carried out using E.coli strain DH5 a.

Primers used in the construction of the fusion gene:

note: capital letters represent sequences complementary to the gene of interest, and lowercase letters are sequences overlapping the plasmid backbone.

Preparation of bacillus subtilis WB800N competent cells:

step 1: inoculating bacillus subtilis WB800N into a culture medium one day before use, wherein the culture medium comprises 1-1.1% of potassium hydrogen phosphate, 0.4-0.6% of monopotassium phosphate, 1-3% of glucose, 0.05-0.1% of sodium citrate dihydrate, 0.05-0.15% of ferric ammonium citrate, 0.2-0.3% of potassium aspartate, 0.03-0.06% of yeast extract and 8-12 mmol/L of magnesium sulfate;

step 2: shaking at 37 deg.C until the cell density OD600 of Bacillus subtilis WB800N in the medium was 1.0.

Preparation of Regenerated Amorphous Cellulose (RAC):

step 1: adding 1g of microcrystalline cellulose powder with the average particle size of 50pm into 3ml of double distilled water to prepare slurry;

step 2: slowly adding 12.5ml of 85% ice phosphoric acid, and incubating for 2h on ice;

and step 3: 12.5g of solid sodium hydroxide are added and the pH is adjusted to 7;

and 4, step 4: transferring the sample to a dialysis tube with molecular weight cutoff of 3500, adding 4L of double distilled water, dialyzing for 48h, and replacing the double distilled water every 6 h;

and 5: the RAC samples obtained by final dialysis were lyophilized for further use.