CN115463081A - Preparation and application of 3D bionic implant containing yeast genetic engineering cells - Google Patents
Preparation and application of 3D bionic implant containing yeast genetic engineering cells Download PDFInfo
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Abstract
The invention relates to preparation and application of a 3D bionic implant carrying rhodotorula glutinis gene engineering cells. A linear drug-producing implant (the diameter is 3-5mm, the length is 5-10 mm) with a porous membrane structure is constructed, the outer layer is a nano-porous membrane, living cells are loaded in the nano-porous membrane, the diffusion of nutrient substances in blood vessels to tissues is simulated, and the drug release is realized at high efficiency and long time. The live cell is Rhodotorula glutinis, the sec1 gene is introduced to produce exosomes with high yield, and then vMIP expression plasmid is transferred to produce recombinant protein/peptide/nucleic acid, and liposome-drug molecules are released in exosomes under anoxic conditions. The liposome-drug molecules which are continuously secreted by the implant in a human body diffuse to tissues through a nano-pore membrane and slowly release into blood, and meanwhile, the drug is protected from enzymolysis and immunogenicity. The novel implant can be used as a platform technology, can carry live cells for transfecting and expressing different biological drug plasmids, realizes prevention and treatment of various diseases, can be implanted into different parts as required, and enhances targeting property.
Description
Technical Field
The invention belongs to the field of tissue engineering, and particularly relates to application of a novel 3D bionic implant of a genetic engineering cell.
Background
Biomacromolecule drugs encompass recombinant proteins, antibodies, vaccines, cell therapy, nucleic acid drugs, gene therapy, and the like. Recombinant protein or nucleic acid drugs play an extremely important role in the treatment of major diseases, are mostly used for treating major diseases such as tumors, AIDS, cardiovascular and cerebrovascular diseases, hepatitis and the like, and are considered to be one of the most promising fields in the research and development of 21 st century drugs. The traditional medicines such as chemical medicines, traditional Chinese medicines and the like sold in the market at present often have the defects of poor water solubility, easy rapid elimination by human bodies, poor biocompatibility, unsatisfactory distribution in vivo, low permeability to cells and the like, while biological medicines such as recombinant proteins or nucleic acids and the like, such as protein medicines, also have the problems of easy degradation, difficult cell membrane penetration, low immunogenicity, low stability and the like, so that the drug success rate of potential biological macromolecular medicines with a plurality of good targets is extremely low. How to improve the bioavailability of the biomacromolecule medicament, furthest retain the biological activity of the biomacromolecule medicament, reduce the immunogenicity of the biomacromolecule medicament and transfer the biomacromolecule medicament to a target site and target cells is also a key problem to be solved urgently for realizing the high-efficiency transfer of the biomacromolecule medicament.
In recent years, the development mode of macromolecular drugs has gradually shifted from the traditional new drug creation development mode to an innovative mode including Drug Delivery System (DDS) in the first place. Of the new drug products approved by the FDA in the united states, about half are innovative drug delivery system formulations. The traditional recombinant protein (peptide) medicament is easily degraded by enzyme in vivo, has short half-life, immunogenicity, difficult cellular uptake of recombinant nucleic acids and the like, and has greatly limited application. The cell exosome technology in recent years has been applied to drug delivery research, and particularly shows great advantages in terms of biomacromolecule drugs. Extracellular exosomes are membrane vesicles specifically secreted by conserved biological processes, having a lipid bilayer membrane structure whose major processes of production can be summarized as: (1) plasma membrane indentation of the cell to form intracellular vesicles; (2) intracellular vesicles further develop to form multivesicular bodies; (3) Multivesicular bodies fuse with the cytoplasmic membrane to release exosomes, which are received by receptor cells through ligand-receptor interactions, pinocytosis/phagocytosis or membrane fusion. According to the production process, the exosome is a vesicle of cell membrane components, can carry various components including lipid, protein, RNA, DNA and the like, can well protect the encapsulated components, and can be conveyed to specific target cells or tissues along with blood, so that the delivery of the recombinant biological medicine from the exosome in vivo is a good intercellular delivery system. The complex composition of exosomes, which can be secreted by a variety of cells, are ubiquitous in body fluids, so that exosomes exert extremely important biological effects, and the idea of using exosomes naturally produced by such cells as drug-encapsulating systems has been brought forward. Therefore, how to obtain exosomes encapsulating target proteins and nucleic acids with high efficiency is a great breakthrough. The laboratory has found that rhodotorula glutinis transformed with vesicle-like genes can produce exosomes in large quantities, and the drug molecules produced by recombinant plasmid formation in the cytoplasm can be encapsulated in the exosomes.
The exosome has unique advantages in drug transportation as a drug carrier, and is mainly reflected in that: (1) When using self-derived exosomes, the harmful immune response caused by the exosomes is very low; (2) the exosome has good stability in human blood; (3) the efficiency of transporting 'cargo' to cells is high; (4) the exosome has certain targeting property when carrying the drug; (5) The exosome diameter is between 40 and 100nm, so that the enhanced osmotic retention (EPR) effect can be well utilized to selectively infiltrate into tumor or inflammatory tissue sites. At present, research on gene therapy, tumor therapy and the like by carrying siRNA, chemical small molecule drugs and the like by exosome has been tried.
By utilizing the characteristic that natural exosomes carry nucleic acid, lipid, protein and other substances, the exosomes can be used as an excellent carrier for carrying nucleic acid, polypeptide and protein medicines. The literatures of exosome-carried RNA drugs are reported at present, for example, exosome-carried miR-26a, long non-coding RNA (LncRNA) -H19, miR-200b and the like are used for researches on anti-tumor, diabetic trauma, intestinal fibrosis treatment and the like, and the exosome drug delivery system carrying RNA is proved to be promising to be a gene therapy means for various diseases. Protein drugs mainly comprise enzymes, polypeptides, cytokines and the like, and are important drugs for treating diseases due to special pharmacological activity, but the defects of large molecular weight, poor stability and the like limit the clinical application of the drugs. In recent years, researches show that the exosome as a drug carrier can carry various proteins, and the purposes of targeted drug delivery, enzyme activity stabilization and the like are achieved. Sterzen-bach and the like construct an exosome delivery system carrying Cre recombinase and realize brain targeted drug delivery across the blood-brain barrier, and the research realizes loading of exogenous Cre recombinase by exosome by using a molecular switch mechanism, thereby providing a new idea for constructing the exosome delivery system carrying exogenous protein. Yuan and the like carry the brain-derived neurotrophic factor by utilizing an exosome secreted by natural macrophages, realize active targeted drug delivery under the pathological changes of brain inflammation and provide a new method for treating the brain inflammation and nourishing the brain. Although initial studies to achieve targeted treatment of central nervous system diseases are common, non-brain targeted drug delivery has also been increasingly studied in recent years. For example, malhotra and the like construct an exosome delivery system carrying transferrin and lactalbumin, and realize the tumor-targeted drug delivery of systemic administration.
Exosomes can act therapeutically on recipient cells not only by endogenous inclusion, but also by loading exogenous substances. At present, two types of ways of loading medicines by utilizing exosomes are more mature and more applied, namely, the medicines and exosome source cells are co-cultured, so that the exosomes secreted by the cells naturally contain the medicines; secondly, the exosome source cell is transfected by a chemical method (such as a liposome transfection method) to carry the medicine. Exosomes are attractive vectors for delivering biomolecules. However, the mechanism of loading functional molecules into exosomes is relatively unexplained. However, the research on loading drugs by exosomes is relatively extensive at present, and the problems of duplication and difficulty of administration routes of various macromolecular drugs such as proteins, polypeptides, nucleic acids and the like are not systematically solved. Therefore, the research aims to systematically construct the genetic engineering cells for producing the medicine by the exosome, introduce the sec1 gene through homologous recombination to efficiently produce the exosome and simultaneously transfer the genetic engineering cells into the medicine protein expression plasmid (such as the vMIP expression plasmid), and the cells can release liposome-medicine protein in an exosome mode so as to realize the efficient and targeted delivery of the recombinant protein medicine.
Compared with the traditional injection and oral administration routes, the targeted administration of the medicament wrapped in an exocrine form can improve the absorption of the medicament and improve the bioavailability; the in vivo distribution of the medicament can be improved and the tissue specific concentration and the targeting property of the medicament can be improved through targeted administration; prolong the in vivo circulation time of the medicine and enhance the curative effect of the medicine. With the advent of the united states' 3D printed drug Spritam2016, 3D bioprinted drug-loaded implants have emerged in the corners, especially revolutionizing recombinant biopharmaceuticals. The further research of the project can be used as an implanted cell of a 3D biological printing drug-loaded implant, and the drug-loaded implant can release drugs into a human body through a non-injection way, control the release of the drugs in the human body for a long time and deliver the drugs to a system of a target organ.
The genetic engineering cells are suitable for most expressible proteins with definite drug targets, and the biomacromolecule drug delivery system provides a good technical platform for the in vivo efficient application of recombinant protein drugs.
Disclosure of Invention
The invention mainly aims to construct a novel 3D bionic drug-loaded implant, wherein living genetic engineering cells capable of generating a drug-containing exosome are loaded in the implant, and expressed biological drug molecules such as recombinant protein, recombinant peptide or recombinant nucleic acid and the like are continuously secreted in an exosome mode after the implant is implanted in a body (such as subcutaneous embedding). For the outer layer material of the 3D bionic drug-loaded implant, weThe nano-scale porous membrane tube is prepared by adopting poly-p-dioxanone (PPDO) material which is approved by FDA. Good biocompatibility, no antigenicity, and in vivo degradation to generate water and CO 2 (about 8 months degradation), the diameter of the lipid exosome containing the target biological medicine secreted by the loaded cells is about 200nm, the mesh is about 500nm generally, the loaded cells can not escape, and immune cells can not enter, so that the cells of the drug production engineering loaded in the cells can not have immune reaction. Hydrogel carrying cells is used as an inner layer material of the 3D bionic drug-loaded implant, and nutrient components for cell culture can be added. At present, the commonly used methacrylic acid hydrogel (GelMA) for biological 3D printing is made of a material with a cell adhesion site, is easy to biodegrade (about 6 months), has no antigenicity and good biocompatibility, and uses rhodotorula glutinis of a high-expression sec1 vesicle transport gene as a loaded cell of an implant. The Rhodotorula glutinis GM4 is subjected to homologous recombination method to knock down thymidylate synthase TS in genome thereof and enhance CCT enzyme lipid synthesis gene recombination modification, thus obtaining GM 4-delta TS-PGK1-CCT recombinant Rhodotorula glutinis strain which can secrete lipid in cytoplasm to the outside and carry polypeptide drugs, and the recombinant strain is not proliferated in vivo and only serves as a safe and controllable polypeptide drug carrier.
The 3D bionic implant provided by the invention is used as a platform type technology, can carry the vMIP protein, and can carry various other biomacromolecule drugs, recombinant proteins, recombinant polypeptides and the like at the later stage, so that the effects of treating and preventing different diseases are achieved. Compared with the prior art, the 3D bionic genetic engineering cell implant not only increases the continuous action of the medicine in vivo and plays a good role in prevention, but also increases the stability of the medicine in vivo and reduces the immunogenicity by using a mode that living cells secrete exosomes to produce the medicine.
Drawings
FIG. 1 agarose gel electrophoresis analysis of total RNA from Saccharomyces cerevisiae. ( M is DL5000 DNA Marker; left panel, lane 1, saccharomyces cerevisiae total RNA; lane 1 of the right panel is the sec1 electrophoresis lane (2175 bp) )
FIG. 2 shows PCR detection and double restriction enzyme identification of recombinant plasmid pPICZ-PGK1-CCT/sec1 (left: M is DL2000 DNA Marker; lane 1 is sec1 gene PCR product; right: lane 1 is recombinant plasmid pPICZ-PGK1-CCT/sec1; lane 2 is plasmid pPICZ-PGK 1-CCT)
FIG. 3 SDS-PAGE detection of target protein in recombinant GM 4-. DELTA.TS-CCT/sec 1 (note: lane 1 is a strain transformed with pPICZ-PGK1-CCT (empty plasmid); lane 2 is a strain transformed with pPICZ-PGK1-CCT/sec 1)
FIG. 4Western Blot to detect recombinant Sec1/Munc18 protein (note: 1 is control group; 2 is recombinant strain GM 4-. DELTA.TS-CCT/Sec 1).
FIG. 5 shows PCR (left) and double-restriction identification (right) of the plasmid pMD18-T-Kex2-EK colony. (Note: left: M: DL500 DNA Marker; lane 1: a strain transfected with pMD18-T-Kex2-EK plasmid; lane 2: a strain transfected with pMD18-T vector; right: M1: DL4500 DNA Marker; M2: DL500 DNA Marker; lane 1: pMD18-T-Kex2-EK plasmid; lane 2.
FIG. 6 shows colony PCR detection (left) and double restriction enzyme identification (right) of recombinant plasmid pPICZ alpha-vMIP-II. ( Note: left: m is DL2000 DNA Marker; lane 1 shows the PCR product of the Kex2+ vMIP-II gene; and (3) right: m is DL15000 DNA Marker; lane 1 is recombinant plasmid pPICZ alpha-vMIP-II double digestion; lane 2 is pPICZ alpha plasmid double digestion )
FIG. 7 shows that expression of target protein is detected by SDS-PAGE after transferring pPICZ alpha-vMIP-II into GM 4-delta TS-CCT/sec1 cell. (Note: M is protein Marker; lane 1 is vMIP-II standard; lane 2 is pPICZ α -vMIP-II plasmid transferred group containing no signal peptide; lane 3 is pPICZ α -vMIP-II plasmid transferred group containing signal peptide; and lane 4 is pPICZ α plasmid transferred group).
Fig. 8a schematic view of a 3d biomimetic implant.
Figure 9 exosome particle size qNano analysis chart.
Detailed Description
The present invention will be described in further detail with reference to examples.
EXAMPLES study of cell systems expressing recombinant proteins or nucleic acid drugs secreted from exosomes
1 genetic engineering cell (GM 4-delta TS-CCT/sec 1) of auxotrophic rhodotorula glutinis high-yield lipid-exosome is modified in the early stage, thymidylate synthase TS in the genome of the discovered rhodotorula glutinis reduced by a homologous recombination method for GM4, and CCT enzyme lipid synthesis gene recombination modification is enhanced, so that a GM 4-delta TS-PGK1-CCT recombinant rhodotorula glutinis strain which can secrete lipid in cytoplasm to the outside and carry polypeptide drugs is obtained, and the recombinant strain is not proliferated in vivo and only serves as a safe and controllable polypeptide drug carrier. Therefore, the present study was further modified on the basis of this recombinant strain.
(1) Extraction of total RNA from Saccharomyces cerevisiae
Picking a single colony on YPD medium with saccharomyces cerevisiae normally growing, culturing in fresh YPD medium overnight at 30 ℃, diluting the overnight culture solution according to the ratio of 1 600 When the concentration reaches 0.8-1.0, the thalli are collected to extract the total RNA, and the specific steps are carried out according to the thermal phenol method in the literature.
(2) Cloning of Saccharomyces cerevisiae vesicle transport Gene sec1
According to the sequence alignment result of the saccharomyces cerevisiae vesicle transport gene sec1 reported in GenBank, corresponding cloning primers are designed. The first strand of the Saccharomyces cerevisiae cDNA was synthesized from the total RNA extracted according to TaKaRa Prime script RT-PCR Kit protocol, and then PCR was performed using the synthesized cDNA as a template with the designed primers pF _ sec1 and pR _ sec1, in the following reaction system: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 2min for 10s, extension at 72 ℃ for 10min after 35 cycles, and preservation at 4 ℃. The PCR reaction products were analyzed by 0.75% agarose gel electrophoresis. After PCR amplification of the target gene, gel electrophoresis is carried out to separate and purify the PCR product, and the target gene is recovered. Connecting a target gene to a pMD-T vector, transforming escherichia coli DH5 alpha, identifying positive clones through plasmid electrophoresis and enzyme digestion screening, selecting positive recombinants to carry out sequence determination, and carrying out sequence comparison analysis on a sequencing result and a reported sequence by Bioedit software. The PCR amplification primers are shown below:
pF-sec1：5’-ATGTCTGATTTAATTGAATTACAGA-3’；
pR-sec1：5’-TCATTTATCATGGTGAGATTTTCTT-3’。
(3) Construction of GM 4-. DELTA.TS-CCT/sec 1 Strain (cell)
The recombinant plasmid pPICZ-PGK1-CCT/sec1 was constructed by Sangon Biotech (Shanghai) Co., ltd., and the recombinant plasmid was electrically transformed into a GM 4. DELTA. TS strain, i.e., a GM 4. DELTA. TS-CCT/sec1 recombinant strain was obtained. Positive clones were determined by colony PCR and enzyme digestion screening of recombinant strain GM 4-. DELTA.TS-CCT/sec 1.
(4) Induction expression and verification of target protein in GM 4-delta TS-CCT/sec1 strain
The GM 4-delta TS-CCT/sec1 recombinant strain is inoculated into YPD liquid culture medium containing thymidylate resistance with the same concentration according to the inoculation amount of 1 percent for propagation, after constant temperature culture at 37 ℃ until OD600 reaches 0.6, IPTG (isopropyl-beta-D thiogalactoside) with the final concentration of 0.6mmol/L is added for induction expression at 20 ℃. 1mL of the culture was collected by centrifugation at 12000rpm, and the cells were suspended in 100. Mu.L of 1 XSDS loading buffer at pH 7.0. The thalli is placed in an ultrasonic crusher for crushing, and the supernatant is collected for the next protein purification and SDS-PAGE analysis. Protein purification was performed using Ni-NTA His-BindTM Resin, according to the Qiagen catalog. The IPTG-induced cells, supernatant and precipitated samples after disruption were electrophoretically transferred to a PVDF membrane by SDS-PAGE, and then Western Blotting was carried out using Sec1 (yD-19) antibody as a primary antibody and phosphatase (Ap) -labeled goat anti-guinea pig IgG as a secondary antibody.
(5) The GM 4-delta TS-CCT/sec1 strain is characterized in that during the process of passage, a single clone is picked from a YPD plate containing thymidylate resistance (50 mu g/mL), inoculated into a YPD liquid culture medium containing thymidylate resistance (50 mu g/mL), cultured at 37 ℃ until the density of bacteria is 5 x 109, diluted by 1 x 104 times, inoculated into a nonselective YPD culture medium, transferred into the YPD culture medium after growing for 20 generations (12 h), and cultured for 20 generations continuously, and the like. After the culture was plated on non-selective YPD plates every 20 passages and grown overnight at 37 ℃, 100 colonies were picked from each plate to YPD plates containing thymidylate-resistant (50. Mu.g/mL) and kanamycin, and the number of colonies grown was counted by culturing at 37 ℃. Single colonies were picked from the YPD plates and subjected to small-scale expression assay.
2. Design, synthesis and construction of secretion signal peptide
(1) Design of secretory signal peptide Kex2-EK
Modification of the Kex2 cleavage site sequence of the signal peptide is one of the common strategies for improving the secretion efficiency of foreign proteins. The Kex2 protease is Ca-containing protease encoded by yeast itself 2+ The dependent serine protease belongs to the subtilisin family, and the specific enzyme cutting sites for processing protein precursors and cutting signal peptides are Lys-Arg/Arg-Arg/Pro-Ar. Based on the most commonly used secretion signal of the s.cerevisiae alpha-factor prepro-leader, we therefore used the carboxy-terminal end of the s.cerevisiae alpha-factor secretion signal (LEKR) as the leader peptide for the protein of interest, which contains the Kex2 cleavage site, followed by the spacer peptide (eeaeaeaeaeaepk), and finally linked to the protein of interest.
The secretion signal peptide sequence is shown below:
Leu Glu Lys Arg Glu Glu Ala Glu Ala Glu Ala Glu Pro Lys CTC GAG AAG AGA GAA GAA GCT GAA GCT GAA GCT GAA CCA AAG GAG CTC TTC TCT CTT CTT CGA CTT CGA CTT CGA CTT GGT TTC
(2) Synthesis of Kex2-EK
The designed Kex2-EK peptide fragment was sent to Dalibao for synthesis.
(3) Construction and identification of plasmid pMD18-T-Kex2-EK
EcoRI/EcoRV was introduced at both ends of the synthesized Kex2-EK fragment and cloned into pMD18-T by homologous recombination to construct the pMD18-T-Kex2-EK plasmid.
3 construction of drug expression vector expressed by prokaryotic cell
Construction of vMIP-II expression plasmid pPICZ alpha-vMIP-II
pPICZ alpha-vMIP-II was constructed in this laboratory. The construction method comprises the following steps: according to the principle of primer design, primer design software Primer5.0 is adopted, and the design primers are as follows:
| name of the leadBalance | Sequence of |
| vMIP-ⅡFP | 5'ACCATGGGTGACACCCTGGGTGC3' |
| vMIP-ⅡRP | 5'GCGAGCGGTAACCGGCAGT 3' |
| 5'AOX1 | 5'GACTGGTTCCAATTGACAAGC 3' |
| 3'AOX1 | 5'GCAAA T GGCA T TCT GACA TCC3' |
The PCR reaction system was 10 Xbuffer 8. Mu.l, 25mmol/L MgSO 4 Mu.l, 8. Mu.l of 10mmol/L dNTP, 2. Mu.l of 20. Mu. Mol/L vMIP-IIFP, 2. Mu.l of 20. Mu. Mol/L vMIP-IIRP, 4. Mu.l of KOD-plus polymerase, and water to 80. Mu.l. The PCR reaction conditions were as follows: after denaturation at 94 ℃ for 5min, 30 cycles were performed, with the program: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 80s. The band containing the desired fragment was excised from the agarose gel and recovered according to the instructions of the gel recovery kit. Plasmid extraction, enzyme digestion, ligation reaction, caCl 2 The method transformation and recombinant identification were performed as reference, and recombinants were selected on LB medium plates containing 25. Mu.g/ml Zeocin. And (4) identifying the recombinants by using a bacteria liquid PCR method, and sending the clones identified as correct to the organism company for sequencing.
The Rhodotorula glutinis GM 4-delta TS-CCT/sec1 competent cells were prepared by reference to the yeast lithium acetate transformation method. 80 μ l of competent cells were mixed with 10 μ l (5-10 μ g) of Xha I linearized recombinant expression plasmid pPICZ α -vMIP-II, respectively, and then electrotransformed in a Bio-Rad Gene Pluser (voltage: 1500V; capacitance: 25 μ F; time parameter: 5.4 ms) and spread on YPD resistant selection plates containing 50 μ g/mL thymidylate and 100 μ g/mL Zeocin to screen for recombinant yeasts.
4. Cell construction capable of efficiently secreting drug exosomes
(1) Target drug carrier transfection GM 4-delta TS-CCT/sec1 cell
The Rhodotorula glutinis GM 4-delta TS-CCT/sec1 competent cell is prepared by referring to a yeast lithium acetate conversion method. 80 μ L of competent cells were mixed with 10 μ L (5-10 μ g) of pPIC-vMIP-II (Zeocin) for expression of vMIP-II protein, pET-28a-PI (kanamycin) for expression of insulin, and pYES2-miR377 (ampicillin Amp) for expression of miR377, respectively, and the mixture was subjected to electrotransformation (voltage: 1500V; capacitance: 25 μ F; time parameter: 5.4 ms) using a Bio-Rad Gene Pluser electrotransfer, and spread on YPD resistance selection plates containing thymidylate and the corresponding resistance, respectively, to select recombinant yeasts. Inoculating to YPD liquid culture medium containing thymidylate (50 μ g/mL) and corresponding resistance according to 1% of inoculum size, culturing at constant temperature of 37 deg.C until OD 600 After reaching 0.6, IPTG was added to a final concentration of 0.6mmol/L to induce expression at 20 ℃. 1mL of the culture was collected by centrifugation at 12000rpm, and the cells were suspended in 100. Mu.L of 1 XSDS loading buffer at pH 7.0. The thalli is placed in an ultrasonic crusher for crushing, and the supernatant is collected for SDS-PAGE analysis.
Single clones were picked from the YPD plates containing thymidylate resistance (50. Mu.g/mL) and the corresponding resistance, inoculated into YPD liquid media containing thymidylate resistance (50. Mu.g/mL) and the corresponding resistance, respectively, and cultured at 37 ℃ to a bacterial density of 5X 10 9 Dilution 1X 10 4 After doubling, the cells were inoculated into a non-selective YPD medium, grown for 20 passages (12 hours), transferred to a YPD medium, and cultured for another 20 passages, and so on. After every 20 passages, the culture was plated on non-selective YPD plates and grown overnight at 37 ℃ and 100 colonies were picked from each plate to YPD plates containing thymidylate resistance (50. Mu.g/mL) and the corresponding resistance, and the number of colonies grown was counted by culturing at 37 ℃.
(2) Extracellularly detecting content of drug-loaded exosome and expression level of target protein
A. Extraction and separation of GM 4-delta TS-CCT/sec1 extracellular secretion after plasmid transformation
Transformation of plasmids from electrotransforms by modification (Chutkan et al, 2013) as described previouslyExosomes were purified in GM4- Δ TS-CCT/sec1 strain. Briefly, when the GM 4-. DELTA.TS-CCT/sec 1 strain after transformation had grown to logarithmic phase (OD 600. Apprxeq.0.5-06), the cell-free supernatant was collected by centrifugation at 10000 Xg for 10min at 4 ℃. The supernatant was further filtered through a 0.45 μm filter and the exosomes were precipitated by ultracentrifugation at 400000 × g for 1.5 hours in a Beckman NVTTM65 rotor at 4 ℃. After removal of the supernatant, the exosomes were resuspended in 300 μ L sterile PBS. Preparing sucrose solutions with mass fractions of 8%, 30%, 45% and 60%, sequentially adding sucrose solutions with different concentrations into a centrifuge tube, and slowly adding the resuspension solution on the sucrose solution with mass fraction of 8% [12-15] And centrifuging at 36000rpm for 1.5h, combining the upper two layers of strips, adding water for dilution, centrifuging again to remove sucrose, and resuspending the precipitate with PBS.
And extracting and separating the transfected iPS-Rab27A cell exosome
Collecting culture supernatant of 3 rd generation human iPS-Rab27A cells transfected with different drug expression vectors, and extracting exosome by adopting high-speed ultracentrifugation method [15] The concrete steps are briefly described as follows: (1) collecting 200mL of culture supernatant of the 3 rd generation transfected human iPS-Rab27A cells through starvation pretreatment; (2) centrifuging at 300 Xg low speed for 10min at 4 deg.C; (3) centrifuging at 2000 Xg for 10min at 4 deg.C; (4) subpackaging the collected supernatant again, centrifuging according to the centrifugation parameters (10000 Xg, 30min, 4 ℃), and collecting the supernatant; (5) ultracentrifugation is carried out for 70min under the conditions of 4 ℃ and 100000 Xg, supernatant is discarded, and exosome can be obtained and is resuspended in PBS and stored at minus 80 ℃ for standby.
And detecting the form of the exosome by a transmission electron microscope
Sucking 10 μ L of the exosome suspension by using a pipette gun, slowly dripping the exosome suspension on a 200-mesh sample-carrying copper net, and standing for 2min at room temperature; drying the liquid on one side of the sample-carrying copper net by using a thin filter strip, then dropwise adding 20 mu L of 0.1% phosphotungstic acid on the same sample-carrying copper net, and carrying out negative dyeing for 5min at room temperature; and (3) sucking the dye-laden liquid from the side surface of the sample-carrying copper mesh by using a long and thin filter paper strip, drying the dye-laden liquid under an incandescent lamp, putting the sample-carrying copper mesh under a transmission electron microscope, observing the form of suspended particulate matters at the voltage of 80kV, and determining the size of the exosomes.
qNano measuring exosome particle size distribution
And (4) installing the nano-pore plate, and debugging the instrument until no air bubbles exist in the nano-pore plate. Taking 1 mu L of standard particles, adding 1mL of special Buffer to dilute 1000 times, and filtering by using a 0.22 mu m filter for later use; and (3) adding PBS 45 mu L of exosome of 5 mu L each to dilute by 10 times, filtering by a 0.22 mu m filter, loading the exosome on a loading machine for testing, wherein the loading amount is 30 mu L each time, the number of the test particles is about 400, clicking a stop button, and automatically processing data by an instrument to generate a particle size distribution diagram.
BCA method for determining exosome and protein concentration
Preparing a BCA working solution according to the ratio of solution A to solution B = 50; diluting the protein standard substance to 0.5g/L, adding the standard substance into corresponding wells according to the amount of 0, 1, 2, 4, 8, 12, 16 and 20 μ L, and adding the standard substance diluent to 20 μ L; adding 2 mu L of sample to be detected into each hole, and adding the standard substance diluent to 20 mu L; adding 200 mu L of BCA working solution into each well, and incubating for 30min at 37 ℃; a562 of each well was measured by a microplate reader, a standard curve was drawn, and the amount of protein was calculated.
Laser confocal detection of cell and exosome distribution
After the iPS-Rab27A cells grow to about 80%, carrying out cell membrane red fluorescence labeling on the exosome by using PKH-26, then co-culturing the exosome and the iPS-Rab27A cells for 24h, adding DAPI (Dapi) into the cell to carry out cell nucleus blue fluorescence labeling on the iPS-Rab27A cells, and washing the cell nucleus blue fluorescence labeling to carry out fluorescence confocal microscope detection.
Extraction of exosome and cell total RNA and cDNA synthesis
And removing the culture medium from the exosomes or transfected cells obtained by extraction and separation, adding 1mL of TRIzol reagent to lyse the cells, and extracting exosome or total RNA of the cells according to the reagent instruction. Mu.g of total RNA was used for reverse transcription reaction according to the iScript cDNA synthesis protocol.
And detecting the expression level of miR377 by real-time fluorescent quantitative PCR
This was done by the Bio-Rad CFX96 Real-Time PCR detection system. 30 μ l reaction: 2 × 1. Mu.l each of 10. Mu. Mol/L gene-specific primers, 1. Mu.l each of 2 × 1 SYBR Green Master Mix, 2. Mu.l of cDNA template, and ddH 2 And O is supplemented to 30 mu l. PCR cycling conditions: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 10s, annealing at 60 DEG CExtension 60s, 40 cycles in total. The expression level of the target drug protein/nucleic acid in a cell sample takes beta-actin as an internal reference gene, the expression level in exosomes takes U6 as the internal reference gene for homogenization, and the relative quantitative detection of the target drug protein/nucleic acid uses 2 -△△Ct And (4) calculating. The detection primer is synthesized by Oncorhynchus corporation.
Western blot detection of the expression level of surface protein and target protein of exosome
20 mu L of each exosome is taken to carry out SDS-PAGE protein separation, the protein is electrically transferred to a PVDF membrane, 5 percent skimmed milk powder blocking liquid is used for blocking nonspecific antigen binding sites, then Anti-CD63 antibody (ab 216130), anti-TSG101 antibody (ab 70974), vMIP-II monoclonal antibody, anti-Insulin antibody and corresponding HRP-labeled II antibody are added, ECL is used for developing color, darkroom X film development is carried out, and strip gray scale analysis is carried out after photographing.
5. Construction of 3D bionic implant capable of carrying genetic engineering cells
(1) Selection and preparation of outer layer material of nano porous membrane tube
For the outer layer material of the 3D bionic drug-loaded implant, the nano-scale porous membrane tube is prepared by adopting poly (p-dioxanone) (PPDO) material which is approved by FDA. Good biocompatibility, no antigenicity, and in vivo degradation to generate water and CO 2 (about 8 months degradation), the diameter of the lipid exosome containing the target biological medicine secreted by the loaded cells is about 200nm, the mesh is about 500nm generally, the loaded cells can not escape, and immune cells can not enter, so that the cells of the drug production engineering loaded in the cells can not have immune reaction.
The PPDO preparation of the nanoscale porous structure is intended to be carried out in three ways:
A. electrostatic spinning: and (3) performing electrostatic spinning on the surface of the inner hydrogel material which continuously rolls to wrap the nanofiber membrane for the inner material.
B. Coaxial 3D printing: the inner layer is made of hydrogel material, the outer layer is made of a mixture of dissolved PPDO and a pore-foaming agent, and the outer layer porous structure is formed after printing.
C. Hydrogel perfusion: preparing hollow outer layer PPDO porous drums by a particle sintering method or a phase separation method and the like, and then pouring inner layer hydrogel.
The chemical properties of the material are studied by nuclear magnetic resonance, infrared spectroscopy and X-ray diffraction, and the physical characteristics of the material are studied by mechanical testing, in vivo degradation testing and swelling testing.
(2) Material selection and preparation of hydrogel inner matrix
Hydrogel carrying cells is used as an inner layer material of the 3D bionic drug-loaded implant, and nutrient components for cell culture can be added. At present, methacrylic acid hydrogel (GelMA) is commonly used for biological 3D printing, and the material has a cell adhesion site, is easily biodegraded (about 6 months), has no antigenicity and has good biocompatibility.
And (3) carrying out hydrogel printing of agarose cell-GelMA on a low-temperature 3D printing platform, and researching the requirements of different reaction systems on gelling conditions. Cell growth and expression of medicinal molecules are detected by live cell staining, western bolt and the like, and the cell/glue ratio for enabling the cells to survive well is researched, so that the survival time of the loaded cells in the implant is long and the secretion effect is good.
6. In vivo pharmacodynamic study of exosome type genetically engineered cells
The constructed 3D biomimetic genetically engineered cell implants were embedded subcutaneously in the neck with SD rats and the animals were sacrificed at different times.
(1) Drug release concentration in animal serum at different times
(1) Exosome detection
Exosomes are extracted through ultracentrifugation, and indexes of particle size (electron microscope) and lipid staining (rhodotorula glutinis exosomes) are detected to determine changes of the exosomes before and after implantation.
(2) Detection of blood concentration of drug biomolecules
The blood of a rat is extracted at different time to detect the concentration of the drug in the serum, and the therapeutic half-life, AUC, kd, metabolic kinetic characteristics and the like of the 3D bionic gene engineering cell implant in vivo are calculated through the drug release curve in the animal body.
(2) Histocompatibility and degradation characteristics of 3D bionic gene engineering cell implant
The embedded 3D bionic gene engineering cell implant is subjected to an anatomy, histology and immunohistochemistry staining method, and the histocompatibility in vivo is known. The degradation characteristic is mainly characterized in that the internal structure of the sample is observed noninvasively through Micro-CT scanning with high resolution and no damage to the sample, so that the volume and morphological characteristics of an observation region of the implant are known.
Results of the experiment
Construction and identification of GM4-delta TS-CCT/sec1 Strain
(1) Cloning of Saccharomyces cerevisiae vesicle transport genes
Firstly, total RNA of the saccharomyces cerevisiae is extracted, the detection result is shown in figure 1 (left), the RNA extraction effect is good, 28s RNA and 18s RNA bands can be obviously seen, the ratio of the obtained RNA in A260/A280 is determined to be 1.79, and the extracted total RNA is relatively complete and has good quality. The total RNA was reverse transcribed into cDNA, and PCR amplification was carried out using primers pF-sec1 and pR-sec1, as a result, as shown in FIG. 1 (right), the size of the PCR product was similar to the expected product size (2175 bp) in comparison with the Marker in the lane 1 band; cutting a 2175bp band, recovering, connecting a recovered product with a cloning vector, transforming the product into escherichia coli, and selecting positive cloning for sequencing; in the absence of mutations determined by PCR, the complete sequence of the Saccharomyces cerevisiae vesicle trafficking gene sec1 was obtained.
(2) Identification of recombinant plasmid pPICZ-PGK1-CCT/sec1
The recombinant plasmid pPICZ-PGK1-CCT/sec1 was constructed by the Biotechnology engineering (Shanghai) Inc. (Sangon Biotech (Shanghai) Co., ltd.). The pPICZ-PGK1-CCT/sec1 expression vector constructed above was electrically transformed into Rhodotorula glutinis GM4- Δ TS competent cells, and colony PCR was performed to obtain a gene of about 2175bp, which was equal in size to the sec1 gene reported in GenBank (FIG. 2). At the same time, the recombinant plasmid was identified by double restriction enzyme digestion, as shown in FIG. 3.
(3) Sec1 expression analysis of recombinant strain GM 4-delta TS-CCT/sec1
The successfully constructed positive recombinant expression strain GM 4-delta TS-CCT/sec1 is subjected to induction expression, expression products are analyzed by SDS-PAGE, electrophoresis detection results are shown in figure 4, compared with a control, a target band appears, the size of the target band is consistent with that of a predicted protein and is about 65kDa respectively, and the result shows that the success is achieved when the saccharomyces cerevisiae sec1 vesicle transport gene is independently expressed in Rhodotorula glutinis GM 4-delta TS competent cells. Bands appeared at 45kDa, indicating that CCT enzyme was also expressed.
The purified proinsulin protein sample was transferred to a PVDF membrane by SDS-PAGE electrophoresis, and then Western Blot analysis was carried out using Sec1 (yD-19) antibody as a primary antibody and phosphatase (Ap) -labeled goat anti-guinea pig IgG as a secondary antibody, respectively. As shown in FIG. 4, there was a clear band in the Western blotting of PVDF membrane, indicating that the recombinant Sec1/Munc18 protein could hybridize with the antibody, and thus the Sec1/Munc18 protein obtained by purification was immunologically active.
(4) Stability of plasmid during passage of GM 4-Delta TS-CCT/sec1 Strain
The determination results of the bacterial liquid of passage 20, 40, 60 and 80 show that: calculated according to 20 passages per 12h, after the expression bacteria are passaged for 80 passages, the stability of the two recombinant plasmids can still respectively reach 97.7 percent and 95.1 percent, and the plasmids have good stability in the passage process and meet the requirements of genetic engineering bacteria.
TABLE 1 plasmid stability of recombinant plasmid strains at passage
2. Synthesis and characterization of secretory signal peptides
The artificially synthesized Kex2-EK peptide fragment was cloned into pMD18-T vector to construct plasmid pMD18-T-Kex2-EK, and the recombinant plasmid was identified as described above, and colony PCR revealed that about 40bp fragment existed in lane 1, while no target band existed in lane 2 of the empty plasmid-transfected strain in the control group (FIG. 5, left), and further, the double digestion indicated that Kex2-EK peptide fragment was successfully cloned into pMD18-T vector (FIG. 5, right).
3. Construction and identification of drug expression vector expressed by prokaryotic cell
Construction and identification of pPICZ alpha-vMIP-II plasmid for expressing vMIP-II protein
The recombinant plasmid pPICZ α -vMIP-II was constructed by Sangon Biotech (Shanghai) Co., ltd. The pPICZ alpha-vMIP-II expression vector constructed above is electrically transformed into Escherichia coli DH5 alpha competent cells, and a gene of about 330bp is obtained through colony PCR, and the size of the gene is equal to that of Kex2 (41 bp) + vMIP-II (285 bp) (figure 6 left). Meanwhile, the recombinant plasmid is subjected to double enzyme digestion identification, as shown in the right side of figure 6.
Expression of 4-purpose protein medicine in GM 4-delta TS-CCT/sec1 cell
pPICZ alpha-vMIP-II transferred to GM 4-delta TS-CCT/sec1 cell
The plasmid pPICZ alpha-vMIP-II for expressing the vMIP-II protein is electrically converted and transferred into GM 4-delta TS-CCT/sec1 cells, the transferred cells are expanded and cultured, and the fermentation supernatant is analyzed by SDS-PAGE, so that the transferred plasmids containing signal peptide and without signal peptide have specific protein bands with relative molecular mass of about 8.5kDa, the specific protein bands are consistent with the size of the vMIP-II standard protein, and the bacterial liquid transferred into the pPICZ alpha vector does not see the bands, as shown in figure 7. The purity of the purified target protein reaches 98.1 percent through analysis of Bandscan software.
5. Construction of 3D bionic implant capable of carrying genetic engineering cells
The 3D bionic gene engineering cell implant constructed by the research, namely a linear drug-producing implant (the diameter is 3-5mm, the length is 5-10 mm) with a porous membrane structure, the outer layer is a membrane wall with nano pores, and living cells capable of releasing drugs for a long time are loaded in the implant. The loaded drug-producing cells select rhodotorula glutinis, and are introduced into the sec1 gene through homologous recombination to efficiently produce exosomes, and meanwhile, the exosomes-drug molecules can be released in an exosome mode under the anoxic condition by transferring expression plasmids to produce recombinant proteins or recombinant peptides or recombinant nucleic acids. As shown in figure 8, the novel drug-producing implant can continuously secrete the biomolecule drug encapsulated by the liposome after being implanted into a human body, and the biomolecule drug can be diffused to the surrounding tissues through the outer layer of the nanopore membrane to be slowly released into the blood, so that the in-vivo long-acting drug release and the cell targeting delivery of the recombinant biological drug can be realized. The linear 3D bionic gene engineering cell implant has the characteristic of long-term drug-production slow-release blood-injection, has the function of isolating immune cells, and allows biomolecules to escape enzymolysis and immunogenicity through an exosome.
6. Detection of exosomes
Exosome particle size was measured using the qNano method. And (4) installing the nano-pore plates in a distributed manner, and debugging the instrument until no air bubbles exist in the nano-pore plates. Taking 1 mu L of standard particles, adding 1mL of special Buffer to dilute 1000 times, and filtering by using a 0.22 mu m filter for later use; and (3) adding PBS 45 mu L of exosome into 5 mu L of exosome respectively to dilute by 10 times, filtering by a 0.22 mu m filter, loading the exosome into a sample loader for testing, wherein the loading amount is 30 mu L each time, the number of test particles is about 400, clicking a stop button, and automatically processing data by an instrument to generate a particle size distribution diagram 9.
Claims (8)
1. Preparation and application of a 3D bionic implant carrying rhodotorula glutinis gene engineering cells.
2. The 3D biomimetic implant with Rhodotorula glutinis genetically engineered cells as in claim 1, wherein the nano-scale porous membrane tube is fabricated by biological 3D printing technology using, but not limited to, cyclohexanone dioxide (PPDO) as raw material.
3. The 3D biomimetic implant with cells engineered from Rhodotorula glutinis as claimed in claim 1, wherein the carried cells include but are not limited to Rhodotorula glutinis.
4. The genetically engineered cell of Rhodotorula glutinis harbored by the 3D biomimetic implant according to claim 3, wherein sec1 vesicle trafficking gene in Rhodotorula glutinis expressed by homologous recombination to stably produce a large amount of exosomes.
5. The Rhodotorula glutinis genetically engineered cell loaded with the 3D biomimetic implant according to claim 3, wherein the vMIP plasmid is transfected to stably express the recombinant protein vMIP.
6. The genetically engineered Rhodotorula glutinis cell loaded on the 3D biomimetic implant according to claim 3, wherein the cell can continuously produce a large amount of exosomes in vivo and release the drug in the form of exosomes.
7. The design scheme, the use method through subcutaneous implantation and the treatment and/or prevention effect on HIV/AIDS high risk group and/or patient of 3D bionic implant carrying Rhodotorula glutinis gene engineering cells in claim 1.
8. The 3D biomimetic implant with cells engineered from Rhodotorula glutinis according to claim 1, wherein any kind of cells capable of expressing the recombinant protein vMIP are used.
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