CN110835608A - Recombinant rhodotorula glutinis living cell liposome carrying exogenous polypeptide and application thereof - Google Patents

Abstract

The invention relates to a recombinant rhodotorula glutinis living cell liposome carrying exogenous polypeptide and application thereof. The invention has the beneficial effects that: the CCT is stably expressed for a long time, and the expression level of the CCT is improved by about 1.5 times; compared with wild strains, the content of lecithin and fatty acid is obviously improved, exogenous polypeptide is transferred into cells of the strains by an electroporation technology, and the exogenous polypeptide enters lipid droplets of the recombinant strains due to lipophilicity of the exogenous polypeptide, so that the exogenous polypeptide is successfully wrapped. The mouse orally takes the whole insulin-carrying strain GM4-CCT-insulin for 7 days, the content of insulin in serum and important organs (liver, lung, spleen and kidney) of the mouse is found to be increased, the blood sugar of the diabetes mouse orally takes the strain GM4-CCT-insulin, the blood sugar of the diabetes mouse is obviously reduced, and the GM4-CCT-insulin has obvious blood sugar reducing effect as exogenous insulin.

Description

Recombinant rhodotorula glutinis living cell liposome carrying exogenous polypeptide and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a rhodotorula glutinis L.carrying exogenous polypeptide living cell liposome of recombinant cytidine choline transferase and application thereof.

Background

Phospholipid (Phospholipid) is an essential component of biological membranes, wherein the hydrophilic head of the Phospholipid is positioned on the surface of the membrane, the hydrophobic tail is positioned on the inner side of the membrane, and when a plurality of Phospholipid molecules are positioned on the surface of an aqueous solution, the Phospholipid molecules usually form a Phospholipid molecule complex structure in 3 forms of micelle, liposome and Phospholipid bilayer after interacting with water molecules because water is polar. Polypeptide drugs are biological macromolecules, and have the common characteristics of being unstable in organisms and easy to be degraded by proteolytic enzymes, so that the half-life period in the organisms is short, the bioavailability of oral administration is low, and the compliance of patients is poor, thereby limiting the wide application of the polypeptide drugs. And the defects can be overcome after the liposome is coated.

The synthesis of phospholipids in eukaryotes starts with 3-phospho-glycerol, which is converted into diacylglycerol by transacylases and phospholipase enzymes, and diacylglycerol synthesizes different phospholipids by different transacylases. Therefore, in order to control the synthesis of phospholipids, the synthesis of phospholipids can be increased by increasing the activity of transacylase.

The antagonistic peptide H9 of the chemotactic factor in the early stage of inflammation, which is screened in the laboratory, can specifically block the secretion of the chemotactic factor in the early stage of inflammation, has low production cost, strong specificity, clear effect, safety and reliability, is applied for patent protection in 2011 and has the application number of 201110150794.6.

H22LP is also a broad-spectrum chemokine receptor US28 antagonistic peptide obtained by early screening in a laboratory, and H22LP can achieve the effect of inhibiting human cytomegalovirus by directly acting with virus particles.

Interferon (IFN) is a broad-spectrum antiviral agent, does not directly kill or inhibit viruses, but mainly produces antiviral proteins by the action of cell surface receptors, thereby inhibiting replication of hepatitis b virus, and is classified into three types, α - (leukocyte), β - (fibroblast), and γ - (lymphocyte), and also enhances the activities of natural killer cells (NK cells), macrophages, and T lymphocytes, thereby playing a role in immunoregulation and enhancing antiviral ability.

Type I interferons include IFN- α and IFN- β, IFN β is produced by human fibroblasts, IFN- α is produced mainly by mononuclear-macrophages, IFN- α can be synthesized by B cells and fibroblasts, IFN- β is produced mainly by fibroblasts, IFN- α/β binds to the same receptor and is widely distributed, including mononuclear-macrophages, polymorphonuclear leukocytes, B cells, T cells, platelets, epithelial cells, endothelial cells, tumor cells, and the like.

Type ii interferons: type ii interferons, i.e. gamma interferons, are produced mainly by activated T cells (including Th0, Th1 cells and almost all CD8+ T cells) and NK cells, and are one of the so-called lymphokines (lytnphkine). IFN- γ can exist in an extracellular matrix-linked form, so cell growth is controlled by a paradoxical means, which can be distributed on the surface of almost all cells except mature erythrocytes.

IFN-gamma antibacterial action: IFN-gamma can reduce the iron supply of bacteria by down-regulating transferrin receptor or directly inhibit the bacteria in cells by inducing to generate endogenous NO, and can also increase the action of phagosome of mononuclear macrophage, namely lysosome to dissolve bacteria, so as to achieve the action of killing bacteria by the above ways.

IFN-gamma antiparasitic action interferon activates macrophages (M.phi.), activated M.phi can express high level of Inducible Nitric Oxide Synthase (iNOS) to catalyze the production of NO by L-arginine, and NO has inhibitory and killing effects on inoculated pathogens.

Immunomodulatory effects of IFN- γ: the interferon involved in immune regulation is IFN-gamma, also known as immunomodulatory interferon. The immunoregulation interferon can express Fc receptor of IgG, so as to facilitate phagocytosis of antigen by macrophage, killing target cell by K, NK cell and activation of T, B lymphocyte, and enhance the immune response capability of organism. IFN-gamma can increase the expression of MHC II molecules on the surface of macrophage and enhance the antigen presenting capability. Furthermore, the Fc receptor expressed on the surface of macrophages can be enhanced, and the macrophages can be promoted to phagocytose immune complexes, antibody-coated pathogens and tumor cells. Meanwhile, the compound can stimulate neutrophils, enhance the phagocytic capacity of the neutrophils, activate NK cells, enhance the cytotoxic effect of the NK cells and the like to participate in immune regulation.

IFN-gamma is an innate and adaptive cytokine in defending tumor development, IFN-gamma is produced by T lymphocytes stimulated by specific antigens, has a structure different from that of type I interferon, is acid-resistant, is a main macrophage stimulating factor of the body, and has a multi-aspect regulating effect on the immune response of the body, can activate effector cells, improve the activity of natural killer cells, macrophages and tumor infiltrating lymphocytes, promote monocyte circulation, enhance the expression of antigens and antibodies on the surface of immune cells, stimulate the production of cytokines such as IL-2, tumor necrosis factor, interferon- α, inhibit tumor cell division, induce gene full-grown antiviral proteins and the like.

The common interferon has small molecules and short action time, and is basically and completely discharged out of the body after being injected for 12 hours generally, so that the common interferon needs to be injected for a plurality of times, thereby bringing great pain to patients. The long-acting interferon has long half-life period which is as long as 40 hours and can continuously act for 168 hours in a hepatitis B patient, so that the long-acting interferon only needs to be injected once a week, is convenient to use, improves the safety of interferon treatment, but is relatively expensive, and greatly hinders the wide application of the interferon.

Rhodotorula belongs to the subdivision Deuteromycotina. The colonies are red in shape of circles or ovals. Asexual, multi-polar sprouting. Has no alcoholic fermentation ability, and can be used for assimilating lactose and decomposing fat. The Rhodotorula should grow at 25-30 deg.C, and be acidic and have certain carbon and nitrogen source. Rhodotorula is a saprophytic bacteria with strong stress resistance, exists in nature, and is widely distributed in various ecological environments.

The cells of Rhodotorula glutinis are round, oval or elongated. Multilateral budding, with distinct red or yellow pigments, forms slime-like colonies from the capsule. The strain has good fat production, and a large amount of fat can be extracted from the strain. Some species have a weak oxidizing effect on hydrocarbons and can synthesize a carotene. The variant of the bacterium can oxidize alkane to produce fat with content of dry biomass. Can also produce alanine and glutamic acid under certain conditions, has strong capacity of producing methionine, and can reach dry biomass. The strain has the other advantages that the strain can grow on a plurality of cheap culture mediums, such as corn flour, syrup and the like, can also grow on industrial waste materials such as molasses, bean pulp, monosodium glutamate waste water and the like, can reach high biomass, and is easy to culture at high density. The biomass of the strain can reach OD600=80 and can be cultured in ultrahigh density according to reports.

The fermentation related reports demonstrate that Rhodotorula glutinis has a high biomass and a flux of mevalonate, whereas mevalonate is synthesized and eventually a coenzyme. Essential precursors of secondary metabolites such as fusel alcohol, lycopene, artemisinin and the like are potential industrial strains.

Rhodotorula glutinis GM4 with strong lipid production ability is screened in the early stage of the laboratory, and the fatty acid content can reach 22.54%. And carrying out genetic engineering transformation on the screened rhodotorula glutinis, namely transfecting a key enzyme malic enzyme ME for lipid metabolism into a strain body to efficiently express the malic enzyme ME, wherein the transformed strain can accumulate more lipid compared with wild strain, and the fatty acid of the transformed strain is detected by gas chromatography and consists of palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid and the like.

In order to improve the synthesis amount of the phospholipid in the Rhodotorula glutinis GM4 strain in vivo, the key enzyme for synthesizing the phospholipid, especially the rate-limiting enzyme for synthesizing the lecithin, is prepared by the following steps of: the Cytidine Choline Transferase (CCT) is transferred into a strain body to be over-expressed, so that the synthesis of phospholipid in the strain body is further improved, and the possibility of the cytidine choline transferase serving as a medicine living cell liposome is researched.

Disclosure of Invention

The invention aims to provide a rhodotorula glutinis of recombinant cytidine phosphate choline transferase living cell liposome carrying exogenous polypeptide and application thereof aiming at the defects in the prior art.

In order to solve the technical problems, the invention adopts the following technical scheme: the cytidine choline phosphate transferase gene is integrated with an expression vector, and cytidine choline phosphate transferase (CCT) is integrated into the genome of rhodotorula glutinis.

Preferably, the oleaginous yeast is Rhodotorula glutinis GM 4.

The construction method of the cytidine choline phosphate transferase gene integration expression vector comprises the following steps:

(1) extracting an expression vector plasmid;

(2) connecting a strong promoter PGK1 gene with a CCT gene fragment;

(3) the PGK1-CCT fragment and the expression vector plasmid are subjected to double enzyme digestion reaction and then are connected to construct a recombinant plasmid pPGK 1Z-rD-CCT.

A preparation method of exogenous polypeptide living cell liposome is provided, which comprises the following steps:

(1) preparing competent cells of an engineering strain GM4-CCT strain.

(2) Dissolving modified exogenous polypeptides H22LP, H9, IFN-gamma, α MSH and insulin respectively, and mixing with competent cells.

(3) Electric shock treatment, exogenous polypeptide H22LP, H9, IFN-gamma, α MSH and insulin are transferred into the allelochemicals.

The CDP-choline pathway of yeast is similar to that of mammals, whereas CCT enzymes are less active in all enzymes involved in the CDP-choline pathway of yeast and mammals. Thus, CCT enzymes are rate-limiting enzymes in the CDP-choline pathway and are also key enzymes.

The Rhodotorula glutinis is a strain with high oil content, the oil content of the Rhodotorula glutinis is increased by modifying the Rhodotorula glutinis, the high oil content is used as a carrier, exogenous polypeptides H22LP, H9 or gamma-interferon are transferred into cells by electroporation, and the exogenous polypeptides enter lipid droplets of a recombinant strain due to lipophilicity of the exogenous polypeptides, so that the exogenous polypeptides are successfully wrapped.

The invention has the following beneficial effects: successfully constructs an exogenous gene CCT expression vector pPGK1Z-rD-CCT, and the insertion direction of a target fragment on the recombinant plasmid is correct through double enzyme digestion identification; introducing the recombinant plasmid into the rhodotorula glutinis, and successfully introducing the recombinant plasmid into the rhodotorula glutinis and inserting the recombinant plasmid into a genome of the rhodotorula glutinis through PCR identification of a transformed strain, so that stable and long-term expression of CCT is realized; real-time fluorescent quantitative PCR shows that the expression level of CCT is improved by about 1.5 times; the content of lecithin is obviously improved compared with that of a wild strain, and the content of fatty acid in the transformed strain is also obviously improved compared with that of the wild strain through gas chromatography analysis. Exogenous polypeptide is transferred into cells by an electroporation technology, and enters lipid droplets of the recombinant strain due to lipophilicity of the exogenous polypeptide, so that the exogenous polypeptide is successfully wrapped.

Drawings

FIG. 1 is a diagram showing the expression level of the CCT gene of Rhodotorula glutinis (wherein lane M is marker.; lane 1 is CCT enzyme).

FIG. 2 is a construction diagram of Rhodotorula glutinis integration expression vector pPGK 1Z-rD-CCT.

FIG. 3 is a PCR amplification of the PGK1 gene (wherein lane M: marker; lane 1 is the PGK1 gene).

FIG. 4 is a PCR amplification plot of the CCT gene (wherein lane M: marker; lanes 1 and 2: CCT gene).

FIG. 5 shows the double restriction pattern of recombinant plasmid pPGK1Z-rD-CCT (wherein, Lane M: marker; Lane 1 and 2 are pPGK 1Z-rD-CCT).

FIG. 6 is a diagram showing the expression level of the CCT gene of Rhodotorula glutinis (Molecular-mass markers (ku); Vec: pPGK1Z-rD; Rec: pPGK 1Z-rD-CCT).

FIG. 7 is a graph showing the concentration of polypeptides electrotransformed into cells (WT: wild-type strain, vector: empty plasmid, GM 4-CCT: recombinant strain; P <0.05 compared with WT).

FIG. 8 is the concentration of insulin in serum (. p <0.05 vs GM 4-CCT).

FIG. 9 is a tissue distribution map of insulin.

FIG. 10 is a graph showing the blood glucose profile of diabetic mice.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

In order to better illustrate the present disclosure, several preferred embodiments are described below. However, these specific examples are only for illustrating the present invention and are not intended to limit the present invention.

Example detection of expression level of CCT in Rhodotorula glutinis GM4 in vivo

1.1 strains and media

1.1.1 strains: rhodotorula glutinis (A)Rhodotorula glutinis）GM4

1.1.2 culture Medium

The slant culture medium is malt agar culture medium; the culture medium is prepared according to the formula of the seed culture medium, the fermentation basal culture medium and the solid culture medium.

1.2 fermentation Process

1.2.1 seed liquid preparation

50ml of seed culture medium is prepared and sterilized in a 250ml conical flask under the conditions of 115 ℃ and 0.169MPa by high-pressure steam for 30 min. In a clean bench, a ring of single colony is selected from a solid culture medium by a sterilized inoculating ring and inoculated into a seed culture medium, and the single colony is cultured at 30 ℃ and 250rmp/min to be used as seed liquid.

1.2.2 fermentation culture

50ml of fermentation medium was prepared in the same manner, sterilized with high pressure steam, and inoculated in a clean bench with an inoculum size of 5%. The same culture conditions are used for fermentation culture.

1.3 detection of CCT enzymes

Inoculating a single colony of Rhodotorula glutinis GM4 in a culture medium, after IPTG (1 mmol/L) induction expression, continuing to culture at 30 ℃ for 18 h, taking 1.5 mL of bacterial liquid, centrifuging at 12000r/min for 2min, collecting thalli, suspending in 100 muL of 1 xSDS sample adding buffer solution, boiling in a boiling water bath for 10min to fully crack cells, centrifuging at 12000r/min for 10min at room temperature to precipitate cell debris, DNA and the like, taking a proper amount of solution for sampling, and analyzing by SDS-PAGE.

1.4 results of the experiment

After IPTG induction expression, the strains were collected, lysed, centrifuged, and the supernatant was subjected to SDS-PAGE. The electrophoresis result (figure 2) shows that an expression band is formed near 45 k, and the molecular mass of the CCT enzyme is consistent with the theoretical value.

EXAMPLE two construction of high-yield phospholipid engineering Strain GM4-CCT

2.1 Experimental materials and instruments

2.1.1 Strain

Rhodotorula glutinis (Rhodotorula glutinis) GM4 was screened and stored for this laboratory;

saccharomyces cerevisiae 2.1445 was purchased from China general microbiological culture Collection center (CGMCC)

Escherichia coli DH5 α was stored in the laboratory.

2.1.2 plasmids

Expression vector pPICZ-rD (pGAPZ α A), was constructed and stored for this laboratory.

2.1.3 Medium and Main reagents

Culture medium: YPD medium, YPD-zeocin medium, Luria-Bertani (LB) medium, LB-ampicilin medium, low-salt LB-zeocin medium, Potato Dextrose Agar (PDA) medium.

The main reagents are as follows: STET buffer, TE buffer, 10% SDS (sodium dodecyl sulfate), plasmid extract, 10xDNA buffer, prepared buffer was stored in Tricine at-20 ℃, SDS-PAGE electrophoresis reagent Taq enzyme (Shanghai Shenyou bioengineering Co., Ltd.), dNTP (Beijing Nobel Biolaide technology Co., Ltd.), EB staining solution (Shanghai Tong Wei Kogyo Co., Ltd.), bromophenol blue (Shanghai Tong Yu Kogyo Co., Ltd.), T4DNA ligase (Shanghai enzyme-Linked Biotech Co., Ltd.), NcoITakara (Dalian Co., Ltd.), etc.

2.1.4 Main Instrument

An autoclave, a low-speed centrifuge, an optical microscope, an ultra-clean workbench, a high-speed centrifuge, a Hettich gel imaging system and the like.

2.2 Experimental methods

2.2.1 isolation and amplification of the Gene of interest

2.2.1.1 isolation and amplification of the Saccharomyces cerevisiae PGK1 Gene

1. Separation:

(1) inoculating Saccharomyces cerevisiae into 50 mL/250 mL YPD liquid culture medium, culturing at 30 deg.C and 180 rpm until OD660 reaches 3, centrifuging (12000 r/min, 5min, 4 deg.C), removing supernatant, and collecting thallus;

(2) washing the thalli twice with sterile water, centrifuging, then re-suspending the thalli with cold sterile water, transferring the bacterial suspension into a mortar containing liquid nitrogen and 1g of alumina, and grinding and crushing thalli cells;

(3) transferring the disrupted somatic cells to a centrifuge tube in 5 mL of DNA extraction buffer (50 mM TRIS, 10 mM MgCl2,50 mM NaCl, 1% (wt/vol) SDS, pH 7.4);

(4) after repeated phenol/chloroform extraction and ethanol precipitation steps, the Saccharomyces cerevisiae DNA was isolated.

2. And (3) PCR amplification:

primers were designed based on the PGK1 gene sequence.

Forward primer 5'-CGCGGATCCTATTTAGATTCCTGACTTCAACTC-3' (Bam HI);

reverse primer 5'-TATCCGCTCGAGTGTTTTATATTTGTTGAAAAAGTAG-3' (Xho I)

The PCR reaction system is as follows:

and (3) PCR reaction conditions:

after pre-denaturation, 30 cycles were performed according to the parameters indicated in the table above. 5. mu.L of PCR amplification product was analyzed by 2% agarose gel electrophoresis.

2.2.2 isolation and amplification of Saccharomyces cerevisiae CCT Gene

2.2.2.1 isolation:

method for separating same 1.2.1.1 Saccharomyces cerevisiae PGK1 gene

2.2.2.2 PCR amplification:

genbank databases were searched and PCR primers were designed using Prmier 5.0 software based on CCT gene sequence information.

CCT-F: 5'-ATGGCAAACCCAACAACAGG-3' (Xho I)，

CCT-Ｒ: 5'-GTTCGCTGA TTGTTTCTTCTTCTG-3' (NcoI)

The PCR reaction system is as follows:

and (3) PCR reaction conditions:

after pre-denaturation, 30 cycles were performed according to the parameters indicated in the table above. mu.L of the PCR amplification product was taken and analyzed by 2% agarose gel electrophoresis.

2.2.3 gel electrophoresis and PCR product recovery

1. Gel electrophoresis:

firstly, preparing 2% agarose gel, pouring the agarose gel into a placed gel preparation tank when the agarose gel liquid is cooled to about 70 ℃, cooling until the agarose gel is formed, taking out the gel and the gel tank, placing the gel and the gel tank into an electrophoresis tank, and adding 1xTAE electrophoresis buffer solution until the gel is immersed; mixing a loading buffer solution (bromophenol blue) to a DNA sample, and adding the sample into a small groove of a gel plate by using a pipette; after sample adding, electrifying immediately for electrophoresis, wherein the voltage is 120V, and when bromophenol blue is close to the top end of the gel, stopping electrophoresis; taking out the gel, staining with EB/1xTAE solution containing 0.5 μ g/ml for 30min, and washing with distilled water for 15 min; and (4) observing under an ultraviolet lamp, and taking a picture by using a gel imaging system for storage after a strip is observed.

2. And (3) recovering a PCR product:

developing the target DNA band by using ultraviolet light, cutting the corresponding agar band by using a slicing knife, and placing the cut agar band into a centrifugal tube.

2.2.4 construction of expression exogenous CCT integration vector pPGK1Z-rD-CCT

2.2.4.1 construction route map of integration vector pPGK1Z-rD-CCT

The related method of the reference document utilizes the homologous recombination principle to design a CCT gene integration expression vector, and the construction technical route is shown in figure 2.

2.2.4.2 extraction of expression vector pPGK1Z-rD-CCT

The pPGK1Z-rD plasmid was constructed and stored in the laboratory and was extracted according to the following procedure:

(1) escherichia coli DH5 α containing pPGK1Z-rD plasmid was inoculated into 5 mL LB-amp medium and cultured overnight at 37 ℃ with shaking at 250 rpm;

(2) sucking 1.5 mL of overnight bacterium liquid into a centrifuge tube, centrifuging for 1 minute at 12000 rpm at 4 ℃, and discarding the supernatant;

(3) the liquid culture medium in the centrifuge tube is sucked dry by filter paper, the thalli sediment is suspended in 200 mu L STET buffer solution and is fully and uniformly mixed by a vortex mixer;

(4) adding 4 mL of newly prepared lysozyme solution, uniformly mixing, and standing for 5min at room temperature;

(5) putting the centrifuge tube on a float frame, placing the centrifuge tube in a boiling water bath, accurately timing for 45s, taking out and immediately centrifuging at 12000 rpm for 5 minutes;

(6) picking and discarding precipitates in a centrifuge tube by using a sterile toothpick, adding 8 mL of 5% CTAB into the supernatant of the centrifuge tube, uniformly mixing by using a mixer, centrifuging at 13000 rpm for 5 minutes, discarding the supernatant, and sucking the liquid in the centrifuge tube by using filter paper;

(7) adding 300 mL of 1.2M NaCl, fully dissolving the precipitate, adding 750 mL of precooled ethanol, fully and uniformly mixing, centrifuging at 13000 rpm for 15 minutes, and removing the supernatant;

(8) slowly leaching the inner wall of a centrifuge tube by using 1mL of 70% cold ethanol, centrifuging for 15 min at 13000 rpm, removing supernatant, sucking liquid on the tube wall by using filter paper, and naturally drying the nucleic acid precipitate for 5-10 min at room temperature;

(9) the precipitate was dissolved in 50mL TE buffer, mixed well in a mixer and stored at-20 ℃ for further use.

2.2.4.3 overlapping PCR joining PGK1 and CCT

In order to realize the high-efficiency expression of an exogenous gene CCT under the drive of a strong promoter PGK1, an Overlap PCR is used for connecting a strong promoter PGK1 gene and a CCT gene fragment so as to ensure that no other sequence exists between the two gene fragments, and thus the abnormal expression of a target gene caused by the introduction of other genes is avoided.

The Overlap PCR primers for PGK1 and CCT were as follows:

PGK1-Bam HI primer1：

5′-CGCGGATCCTATTTAGATTCCTGACTTCAACTC-3′ (Bam HI)

PGK1-Xho I primer 2：

5-TATCCGCTCGAGTGTTTTATATTTGTTGAAAAAGTAGATGTCGCCTATTATT-3′ (XhoI)

CCT- Xho I primer1：

5′-TCTGCTTTCTTCGCTCCGCTCGAGATGTCAGGGCAAACTCGAG-3′ (Xho I)

CCT-Xho I primer2：

5′-CATGCCATGGATCATCTAAAACATCTTTTGAGAG-3′ (Nco I)

PGK1 and CCT fragments are recovered after 5 cycles by respectively amplifying PGK1 and CCT with PGK1-Bam HI primer1, PGK1-Xho I primer2, CCT-Xho I primer1 and CCT-Xho I primer2, and OVerlap PCR is carried out by taking the recovered PGK1 and CCT fragments as common templates and the recovered PGK1-Bam primer1 and CCT-Xho I primer2 as upper and lower primers to obtain PGK1-CCT fragments.

The Overlap PCR reaction system is as follows:

and (3) PCR reaction conditions:

after pre-denaturation, 30 cycles were performed according to the parameters indicated in the table above. mu.L of the PCR amplification product was taken and analyzed by 3% agarose gel electrophoresis.

2.2.4.4 fragment and vector double enzyme digestion reaction, connection

1. The Overlap PCR product and pPGK1Z-rD were subjected to Nco I and Bam HI double digestion reaction systems respectively for plasmid pPGK1Z-rD Nco I/Bam HI double digestion reaction systems:

fragment PGK1-CCT Nco I/Bam HI double enzyme digestion reaction system:

the enzyme reaction was carried out in a water bath at 37 ℃ for 3 hours.

2.2.4.5 ligation reaction

After the enzyme digestion reaction, the PGK1-CTT fragment and the vector pPGK1Z-rD are respectively recovered and purified by a recovery kit, and then T4DNA ligase is used for connecting the double enzyme digested vector pPGK1Z-rD and the PGK1-CCT fragment to construct a recombinant plasmid pPGK 1Z-rD-CCT.

The ligation reaction system is as follows:

reacting in water bath at 16 deg.c overnight, and controlling the molar ratio of carrier to exogenous DNA segment in 1: 3-10.

2.2.5 recombinant plasmid pPGK1Z-rD-CCT electrotransformation of Rhodotorula glutinis competent cells

2.2.5.1 preparation of Rhodotorula glutinis competence

1. Selecting a yeast single colony, inoculating the yeast single colony in 5 mL YPD liquid culture medium, and carrying out shaking culture at 30 ℃ and 250 rpm for overnight;

2. transferring the cells to 100mL YPD liquid culture medium with the inoculation amount of 1%, and carrying out shaking culture at 30 ℃ and 250 rpm until the OD600 of the cells is approximately equal to 1.4;

3. centrifuging the bacterial solution at 4 ℃ for 5min at 3000 g to precipitate cells, discarding supernatant, and resuspending with 100mL sterile water;

4. repeating the third step;

5. centrifuging at 4 deg.C for 2min at 3000 g to precipitate cells, discarding supernatant, and resuspending cells with 20 mL of ice-precooled 1M sorbitol;

6. centrifuging at 4 deg.C for 2min at 3000 g to precipitate cells, discarding supernatant, and resuspending cells with 200 μ L of 1M sorbitol for transformation;

2.2.6 plasmid linearization

About 10 mu g of recombinant plasmid pPGK1Z/rD/CCT is subjected to single enzyme digestion by Sac I, the dosage of enzyme and the temperature during enzyme digestion are referred to manufacturer specifications, the time for complete enzyme digestion needs to be specially noted, and the enzyme digestion can not be carried out partially or the plasmid can not be digested, which has very important effect on the efficiency of electric conversion.

The enzyme digestion system is as follows:

2.2.7 electrotransformation

The linearized recombinant plasmid was electrically transformed into Rhodotorula glutinis GM 4.

1. Mixing 80. mu.L of prepared competent cells with 20. mu.L (about 10. mu.g) of linearized plasmid DNA to be transformed, and adding the mixture into a 0.2 cm pre-cooled electric transformation cup;

2. carrying out ice bath on the electric transformation cup filled with the mixed solution for 5 min;

3. the parameters of the electrotransformation instrument are adjusted, wherein the voltage is 1.5 kV, the capacitance is 25uF, the resistance is 200 omega, and the electric shock duration is about 5 ms;

4. immediately adding 1mL of precooled 1M sorbitol solution into the transformation cup after the electric shock is finished, slightly sucking and uniformly mixing by using a gun, then transferring the mixture into a sterilized centrifugal tube, standing at 30 ℃ for l h, then adding 1mL of fresh YPD culture medium, and shaking at 30 ℃ and 200rpm for 1 hour;

5. centrifuging at 3000 rpm for 4min at room temperature, and resuspending the cells with 200. mu.L ddH 2O;

6. the resulting suspension was spread on YPD plates (containing 50. mu.L of Zeocin), and cultured in an incubator at 30 ℃ for 2-3 days until single colonies were grown.

1.2.8 PCR detection of recombinant Rhodotorula glutinis

1. Selecting a single colony with better growth on the YPD resistance plate, transferring the single colony into a YPD liquid culture medium, and carrying out shaking culture at 30 ℃ and 250 rpm until the OD600 is more than 2;

2. centrifuging 1mL of bacterial solution at 12000 rpm for 2min, removing the supernatant, adding 100 μ L of TE buffer solution to resuspend the thallus, boiling in water bath for 5-10 min, immediately freezing in a refrigerator at-20 deg.C for 15 min, standing at room temperature to dissolve the bacterial suspension, centrifuging at 12000 rpm for 5min, and taking 5 μ L of the supernatant as a template for PCR detection.

The reaction system is as follows:

and (3) PCR reaction conditions:

after pre-denaturation, 35 cycles were performed according to the parameters indicated in the table above. mu.L of the PCR amplification product was taken and analyzed by 2% agarose gel electrophoresis.

2.2.9 restriction enzyme verification

After the PCR preliminary verification of the bacterial liquid, plasmids in the correct positive clones are extracted, and the recombinant plasmids are subjected to double enzyme digestion identification by using Nco I and Bam HII respectively, wherein the reaction temperature is 37 ℃.

The enzyme digestion reaction system is as follows:

2.2.10 transformant stability test

The Rhodotorula glutinis transformants are inoculated into 40 mL YPD liquid medium and cultured for 24 h-36h at 30 ℃ under 250 rpm shaking. 1mL of bacterial liquid is respectively diluted to 10-2,10-3 and 10-4 by sterile water, 200 mu L of diluent with different dilution degrees is respectively coated on a common YPD plate and a YPD-Zeocin resistance plate, and the colony number is calculated and respectively recorded as the total bacterial number and the colony number with the integrated plasmid. The cells were inoculated to YPD liquid medium at 1% inoculum size, cultured at 30 ℃ under shaking at 250 rpm for 50 generations of 24 hours at 10 generations. Plate counts were performed every 10 generations.

Plasmid stability was calculated as follows:

stability (%) = number of colonies with integrated plasmid ÷ total bacteria number × 100%

2.2.11 transformant CCT expression level determination

The recombinant plasmid which is verified to be correct is transferred into Rhodotorula glutinis GM 4. Inoculating a single colony containing an expression plasmid into a culture medium, after IPTG (1 mmol/L) induction expression, continuing to culture for 3 h and 18 h at 30 ℃, taking 1.5 mL of bacterial liquid, centrifuging at 12000r/min for 2min, collecting thalli, suspending in 100 mu L of 1 xSDS sample adding buffer solution, boiling in a boiling water bath for 10min to fully crack cells, centrifuging at 12000r/min at room temperature for 10min to precipitate cell debris, DNA and the like, taking a proper amount of solution for sampling, and analyzing by SDS-PAGE.

2.3 results of the experiment

2.3.1 PCR amplification to detect fragments of interest

Taking the genome of S.cerevisiae as a template, obtaining a target band amplified by about 800 bp (figure 3) and a target gene (figure 4) of about 1300 bp by PCR, wherein the size of the target band is consistent with the sizes of PGK1 and CCT genes reported on GenBank.

1.3.2 recombinant plasmid pPGK1Z-rD-CCT double enzyme digestion identification

CCT gene is amplified by using high fidelity taq enzyme and is connected into a T-carrier for sequencing verification, the total length of the gene is 1275 bp, and 424 amino acids are coded. The homology of the product with the sequence on GenBank reaches 100 percent through Blast comparison. Then, the CCT gene is connected into the plasmid pGK1Z-rD to obtain a plasmid pGK1Z-rD-CCT, the target gene fragment is preliminarily verified to be contained by double enzyme digestion (figure 5), the sequencing verification is correct, and the recombinant expression vector pGK1Z-rD-CCT is successfully constructed. The plasmid is transformed into Rhodotorula glutinis GM4 to obtain GM4/pGK 1Z-rD-CCT.

2.3.2 recombinant plasmid stability testing

In order to detect the genetic stability of the recombinant plasmid pPGK1Z-rD-CCT, the transformed strain is subjected to shake flask culture for 60 generations under non-selective pressure, and the strain liquid is smeared on a Zeocin resistance plate and cultured, and the colony number is calculated. The results are shown in Table 1-1, which shows that the stability of the transformed Rhodotorula glutinis strain can still reach 99.23% after continuous culture for 60 generations, indicating that the plasmid in Rhodotorula glutinis has good genetic stability under non-selection pressure.

TABLE 1-1 stability testing of recombinant plasmids

Table 1-1 The stability of pPGK1Z-rD-CCT in transformed strain

2.3.3 expression analysis of transformed Rhodotorula glutinis Strain CCT

Selecting positive bacterial colonies, inoculating the positive bacterial colonies into 3 mL of liquid culture medium, culturing at 37 ℃ overnight, taking 1mL of bacterial liquid, transferring the bacterial liquid into a culture medium containing antibiotics, culturing at 37 ℃ for about 3 h, adding IPTG to enable the final concentration to reach 1 mmol/L, continuing culturing at 30 ℃ for 3 h and 18 h, taking 1.5 mL of bacterial liquid, centrifuging at 12000r/min for 2min, collecting thalli, and performing SDS-PAGE. The electrophoresis result (figure 5) shows that an expression band is arranged near 45 k, the expression band is consistent with the molecular mass theoretical value of CCT enzyme, the protein expression is obviously enhanced at 3 h, and the expression quantity is larger at 18 h and is about 2 times of that of the wild strain. A high transcription level of CCT may provide sufficient enzymes for lecithin and synthesis to maintain the amount of lecithin synthesized at a high level.

EXAMPLE III phospholipid composition analysis of the transformant GM4-CCT

3.1 extraction of Total Phospholipids from Strain GM4-CCT

The Bligh-Dyer method is partially improved, and the specific steps are as follows:

(1) accurately weighing 0.1 g of dry thallus in a test tube with a plug, adding 1.2 ml of deionized water, 1.5 ml of chloroform and 3 ml of methanol, carrying out vortex oscillation, uniformly mixing, and carrying out ultrasound for 30 min;

(2) adding 1.5 ml chloroform, vortex shaking, mixing well, and performing ultrasonic treatment for 30 min;

(3) adding 1.5 ml of water, shaking in a vortex, uniformly mixing, and performing ultrasonic treatment for 30 min;

(4) adding 0.6 m L saturated sodium chloride solution, washing the precipitate, standing overnight to ensure sufficient extraction;

(5) the overnight placed samples were centrifuged (4500 rpm, 10 min). After centrifugation, the solution was divided into three layers, the upper layer was methanol and water, the middle thin white layer was the cell residue, the lower layer was chloroform, and phospholipids were present in the chloroform layer. Transferring the lower layer solution into a glass bottle;

(6) adding 1.5 ml chloroform into the rest solution and residue, vortex vibrating, mixing, ultrasonic treating for 30min, and centrifuging (4500 rpm, 10 min);

(7) mixing the lower clear solution with the previous solution, blowing nitrogen, and dissolving the obtained solid in

1ml chloroform/methanol solution (2: 1, v/v), stored at-20 ℃ and tested.

3.2 phospholipid quantification method

(1) Chromatographic test conditions

Waters BEH HILIC chromatography column (100 mm. times.1 mm. times.1.7 μm); mobile phase:

a is acetonitrile, B is 50 mM ammonium acetate in water (containing 0.1% formic acid, pH 3.65); elution procedure: 0-4 min, 95% of A and 5% of B; 4-15 min, 95-60% of A and 5-40% of B; 15-17 min, 60% of A and 40% of B; 17-17.1 min, 60-95% of A and 40-5% of B; 17.1-20 min, 95% of A and 5% of B; the flow rate was 0.3 m L/min; sample introduction amount: 1 μ L.

(2) Mass spectrum experimental conditions:

negative ion mode (ESI-), capillary voltage: 3.5KV, ion source temperature: 130 ℃, desolventizing gas temperature: 400 ℃, desolventizing air flow rate: 600L/h, taper hole air flow rate: 50L/h, scanning mode: SIR mode, channel parameters are set for different phospholipid molecules, and different cone hole voltages are used for different phospholipid species.

3.3 analysis of fatty acid component of the transformant GM4-CCT

1. Extraction of strain GM4-CCT total fatty acid

(1) Collecting strain cultured in seed YPD liquid culture medium to stationary phase, centrifuging, washing with distilled water for three times, centrifuging at 4 deg.C for 5min to 5000g, collecting thallus, oven drying to constant weight, weighing, grinding into powder in mortar, wrapping with filter paper, and oven drying again to constant weight.

(2) And (3) putting the dried powder with constant weight into an extraction cylinder, injecting anhydrous ether to immerse the sample, refluxing in a constant-temperature water bath at 70 ℃ for about 8h, and removing the solvent to obtain grease for later use after extraction is finished when no oil exists in the ether.

(3) 1ml of 2% (wt/vol) sulfuric acid-methanol was added to the grease, and esterification was carried out at 60 ℃ for 2 hours.

(4) After the esterification, the mixture was naturally cooled to room temperature, 1ml of hexane was added thereto, and the mixture was vortexed for 10min to extract fatty acid methyl ester.

(5) 800. mu.L of hexane from (4) was taken and added to a glass bottle for GC analysis.

2. GC analysis

A Bruker 450-GC instrument was used with a hydrogen Flame Ionization (FID) detector and a capillary chromatography column HP-INNOWAX (30 m.times.0.25 mm). Column temperature program: and (3) raising the temperature to 230 ℃ for 1min at 150 ℃, keeping the temperature at 230 ℃ for 2min, controlling the retention time of the fatty acid methyl ester standard substance according to the split ratio of 10:1, and carrying out quantitative analysis on different fatty acids.

3.4 results of the experiment

3.3.1 accumulation Studies of lecithin

In order to determine whether the Rhodotorula glutinis transformed strain expressing CCT gene can produce higher level lecithin, the phospholipid component of the Rhodotorula glutinis transformed strain is determined and analyzed in the experimental process, and the result is shown in Table 1-2, and the lecithin content of the transformed strain is found to be remarkably increased from 42.8% to 65.7%, so that the transformed strain can accumulate more lecithin compared with the wild strain.

TABLE 1-2 phospholipid composition of transformed and wild strains

Table 2-2 Relative phosphatidylcholine content of the wild-type strainand the transformed strain

Phospholipid content	Wild strain	Transformed strain
			PC	42.8±0.3%	65.7±0.2%
PI	20.7±0.6%	13.3±0.9%
			PA	16.4±3%	8.7±3.1%
PE	9.4±0.5%	15.4±0.3%
			PG	5.5±0.2%	3.3±0.5%

3.3.2 composition of fatty acids in transformed and wild-type strains

Fatty acid content (w/w,%)	Wild strain	Transformed strain
			Palmitic acid	22.21±1.52	20.86±1.52
Stearic acid	7.75±0.54	7.12±0.38
			Palmitoleic acid	5.34±0.27	5.79±0.26
Oleic acid	43.12±1.58	48.51±1.92
			Linoleic acid	15.86±0.75	19.32±0.51
Gamma-linolenic acid	3.18±0.15	5.25±0.94

Through fatty acid analysis, compared with a wild strain, the content of each fatty acid of the transformed strain is higher than that of the wild strain, which shows that the synthesis and accumulation of unsaturated fatty acid in the rhodotorula glutinis strain can be obviously improved through CCT enzyme transfer.

EXAMPLE quantitative analysis of the entry of four exogenous polypeptides into the cells of the transformant GM4-CCT Strain

4.1 Experimental materials

4.1.1 test strains

The experimental strain is Rhodotorula glutinis recombinant strain GM4-CCT constructed in chapter II.

4.1.2 culture Medium

Same as 2.1.3

4.1.3 Primary reagents

0.5 mg/mL nile red stain: the purchased nile red mother liquor was diluted with DMSO (dinitrosulfoxide) to the target concentration.

FITC-modified H22LP, H9, IFN-. gamma. α MSH, and insulin.

4.1.4 Main Equipment

Centrifuge tube, centrifuge, and incubator 2.1.4

4.2 Experimental methods

4.2.1 electroporation method mediates the exogenous polypeptide to enter GM4-CCT cells

1. The method for preparing competent cells of the engineering strain GM4-CCT is the same as 1.2.5.

2. Dissolving 10 μ g of modified H22LP, H9, IFN-gamma, α MSH and insulin respectively, mixing with competent cells to obtain 100 μ L, and adding into a 0.2 cm pre-cooled electric conversion cup;

3. the electric shock process is the same as 1.2.7;

4. immediately adding 900 mu L of precooled 1M sorbitol solution into the transformation cup after the electric shock is finished, slightly sucking and uniformly mixing by using a gun, then transferring the transformation cup into a sterilized centrifuge tube, standing at 30 ℃ for L h, centrifuging, adding 1mL of fresh YPD culture medium to suspend thallus, and shaking at 30 ℃ and 200rpm for 1 hour;

5. the finally obtained strains are respectively named as GM4-CCT-H22LP, GM4-CCT-H9, GM 4-CCT-IFN-gamma, GM4-CCT- α MSH and GM 4-CCT-insulin.

4.2.2 quantitative analysis by Fluorospectrophotometer

(1) Respectively preparing FITC-H22LP, FITC-H9, FITC-IFN-gamma, FITC- α MSH and FITC-insulin concentration gradient standard solutions, putting 1mL of the standard solution into a 1mL cuvette, and determining the fluorescence intensity Fs;

(2) transferring 1ml of FITC-H22LP, FITC-H9, FITC-IFN-gamma, FITC- α MSH and FITC-insulin bacterial liquid to a 1ml cuvette respectively, and measuring the fluorescence intensity Fx;

(3) preparing a blank solution in a 1ml cuvette, and measuring the fluorescence intensity F0;

(4) making a standard curve between (Fs-F0) and the concentration C of the standard sample to be detected;

(5) reading with a fluorescence spectrophotometer with excitation wavelength of 514 nm and emission wavelength of 492 nm, and determining fluorescence intensity of living cells H22LP, H9, IFN-gamma, α MSH and insulin for quantification;

(6) the content of the sample was determined from the standard curve according to (Fx-F0).

4.3 results of the experiment

4.3.1 Fluorospectrophotometry for determining the concentration of intracellular polypeptides FITC-H22LP, FITC-H9 and FITC-IFN-gamma

After electric conversion, centrifuging polypeptides in a solution, collecting a bacterial solution, and determining the contents of intracellular polypeptides FITC-H22LP, FITC-H9 and FITC-IFN-gamma by using a fluorescence spectrophotometry, wherein the experimental result is shown in figure 7, the content of three detected polypeptides is not very different and is about 4mg/mL, the concentration of the polypeptide during initial electric conversion is 10 mg/mL, which indicates that the concentration of the intracellular polypeptides after electric conversion can reach 2/5 of the initial concentration, the intracellular concentrations of the three polypeptides have no significant difference, and the experimental result of α MSH and insulin is consistent with the result.

Example five in vivo studies in mice

5.1 Experimental materials and instruments

5.1.1 test strains

The strain GM4-CCT-insulin obtained in the previous chapter

5.1.2 Primary reagents

insulin ELISA kit

5.1.3 Main Instrument

Multifunctional microplate reader Molecular Device, usa; high speed refiner XHF-D high speed disperser Ningbo Xinzhi Biotech GmbH.

5.1.4 Experimental mice

15 SPF-grade BALB/c mice, weighing 18-20 g/mouse, aged around 7-8 weeks, were purchased from the center of laboratory animals of Zhongshan university. Before the experiment, the mice are raised in a clean-grade animal laboratory, the room temperature is 20-25 ℃, the humidity is 60%, the mice receive illumination for 12 hours every day, and fresh food and water are replaced once a day.

5.2 Experimental methods

5.2.1 in vivo study of GM4-CCT-insulin in mice

5.2.1.1 concentration of insulin in serum

1. Grouping of mice

(1) In the GM4-CCT-insulin group, 6 normal mice are taken and fed with inactivated GM4-D6D-CCT-insulin strain in addition to normal feed every day, and the feeding amount of each mouse is 1 multiplied by 1010CFU per day;

(2) in the GM4-CCT group, 6 normal mice are taken and fed with inactivated GM4-CCT strain in addition to normal feed every day, and the feeding amount of each mouse per day is 1 × 1010CFU, and the control is used;

2. blood sampling from mouse eyeball

On days 1,3,5 and 7 after feeding, blood was taken by retrobulbar venous plexus bleeding method, centrifuged at 2500 rpm for 15 min, and then the concentration of insulin in serum was measured by ELIS kit.

5.2.1.2 tissue distribution of insulin

After the mice are respectively drenched with CM 4-CCT-insulin and GM-CCT and then sampled at each time point of 1.0, 2.0, 4.0, 8.0, 12.0 and 24.0 h, the mice are immediately killed and the liver, the heart, the spleen, the lung and the kidney are dissected and taken out, and are rinsed for 3 times by physiological saline to remove residual blood, and tissues are sucked dry by filter paper; shearing a proper amount of tissue, accurately weighing and preparing homogenate; the homogenized sample was shaken at 25 ℃ for 20 min at 200rpm and then centrifuged at 25000 rpm for 15 min; the supernatant was filtered through a 0.22 μm microporous membrane, and after impurities were removed, the concentration of insulin was measured by using an ELISA kit in the same manner as described above.

5.3 results of the experiment

5.3.1 detection of insulin in serum and tissue

To examine the effect of GM4-CCT-insulin in carrying insulin, mice were bled by the eyeball on days 1,3,5, and 7 after being administered GM4-CCT-insulin, and the concentration of insulin in serum was examined using an ELISA kit, and mice administered GM4-CCT were used as controls. As shown in FIG. 8, in the GM4-CCT-insulin group, the content of insulin in serum increases with the number of days of administration, the content of insulin almost linearly increases in 1 to 5 days, hardly increases and tends to level after 5 days, while in the GM4-CCT group, the content of insulin maintains an equilibrium state at a certain concentration until, and the content of insulin in the GM4-CCT-insulin group is significantly higher than that in the GM4-CCT group (p < 0.05).

As can be seen from the tissue distribution map of the mouse (FIG. 9), the distribution of GM4-CCT-insulin delivery insulin in the mouse organs is liver, kidney, heart, lung and spleen in sequence, and the highest concentration is reached at 2h, especially at 5.36 in the liver. This may be the reason for the major metabolism of lipids in the liver, and we hypothesize that insulin is likely to be carried along with the lipids forming chylomicrons and into the lymphatic system, and finally into the blood, for transport to various tissues of the body, especially the liver, the major organ of lipid metabolism.

Example six in vivo experiments in diabetic mice

6.1 materials and methods

6.1.1 Experimental animals:

40 male mice of Kunming species of clean grade purchased from the center of laboratory animals of Zhongshan university, aged 8w, weighed 42 + -2.93 g, and kept at room temperature between 20 ℃ and 25 ℃.

6.1.2 Experimental species

Strain GM4-CCT-insulin

6.1.3 Main Experimental reagents and instruments

Alloxan, insulin standard, PBS buffer solution

Glucometer, ultraviolet spectrophotometer, desk centrifuge, microscope

6.3 Experimental methods

6.3.1 modeling of experimental animals:

establishing a diabetes mouse model, taking a plurality of healthy male Kunming mice, fasting, supplying water normally, weighing after 24 h, injecting a alloxan solution with the mass fraction of 3% into the tail vein of the mice according to the dose of 40mg/kg, continuously fasting for 12h after feeding the mice normally for 3d, and taking 200 mu L of blood from the tail vein. After blood coagulation, centrifuging at the low temperature of 4 ℃ and the rotation number of 12000r/min for 4min, measuring 20 mu L of supernatant (serum), and measuring the blood sugar content of the blood by adopting a glucose oxidase method (gop-pod), wherein the mice for the test have the blood sugar value higher than 16.67 mmol/L.

6.3.1 grouping and administration of Experimental animals

30 diabetic mice were weighed, and randomly divided into 3 groups, an insulin gavage group (n = 10), a GM4-CCT-insulin strain group (n = 10), and an insulin injection group (n = 10). Fasting for 12h, free drinking water and gastric lavage for administration. The insulin gastric perfusion group is respectively perfused with insulin PBS solution (50U/kg, ig) and GM4-CCT-insulin strain group to feed GM4-CCT-insulin strain, the feeding amount of each GM is 1 multiplied by 1010CFU per day for continuous gastric perfusion, the insulin injection group is injected with insulin PBS solution (50U/kg, sc) subcutaneously, 20 mu L of blood is taken from the tail vein of the mouse at 0, 1,2,4,6,9,12 h, after the blood is coagulated, the blood is centrifuged at low temperature and the rotation number of 12000r/min for 4 min. The supernatant was measured precisely to 20. mu.L, and the blood glucose level was measured by the gop-pod method.

6.4 results of the experiment

6.4.1 post-model mouse Condition

After 72h of molding, the mice do not die, and the diet, urine volume, weight and the like are not obviously different from those before the experiment. All mice used for molding have the performances of reduced activity, increased water intake and urine output and the like in different degrees.

6.4.2 detection of blood glucose

As can be seen from FIG. 10, the decrease of blood sugar is not obvious when the insulin PBS solution is perfused into diabetic mice, and may be related to the degradation and digestion of insulin by digestive enzymes such as protease and trypsin in the gastrointestinal tract. And the blood sugar concentration of the mice can only be kept at a lower level for 2 hours by subcutaneous injection of insulin PBS (phosphate buffered saline) solution, because the half-life of insulin is short when the insulin is directly injected into the body. However, after the GM4-CCT-insulin strain is taken by intragastric administration, the blood sugar of the mouse is obviously reduced within 1-4 h, and then is basically kept at a lower level within 6h, so that the situation that the bacterial strain is not completely destroyed after entering the body and can effectively reduce the blood sugar after the GM4-CCT-insulin strain is taken by intragastric administration can be seen, and therefore, the GM4-CCT-insulin as an exogenous insulin can be used for treating diabetes and has a remarkable blood sugar reducing effect.

Claims (8)

1. A recombinant Rhodotorula glutinis living cell liposome carrying exogenous polypeptide and its application are characterized by that it uses cytidine choline phosphate transferase gene engineering bacteria as carrier, and introduces exogenous polypeptide to be wrapped in the bacterial strain body which is undergone cytidine choline phosphate transferase gene engineering.

2. The recombinant Rhodotorula glutinis liposome of living cell carrying exogenous polypeptide and its use according to claim 1, wherein said engineering bacterium is Rhodotorula glutinis GM 4.

3. The living cell liposome carrying exogenous polypeptide of recombinant Rhodotorula glutinis and use thereof as claimed in claim 1, wherein said Cytidine Choline Transferase (CCT) is obtained by separation and amplification of Saccharomyces cerevisiae CCT gene.

4. The living cell liposome of claim 3, wherein the recombinant Rhodotorula glutinis harboring exogenous polypeptide and the use thereof, comprises the following steps:

(1) extracting an expression vector plasmid;

(2) connecting a strong promoter PGK1 gene with a CCT gene fragment;

(3) the PGK1-CCT fragment and the expression vector plasmid are subjected to double enzyme digestion reaction and then are connected to construct a recombinant plasmid pPGK 1Z-rD-CCT.

5. The method for preparing an exogenous polypeptide according to claim 1, comprising the steps of:

(1) preparing competent cells of an engineering strain GM4-CCT strain;

(2) respectively dissolving FITC-modified exogenous polypeptides H22LP, H9, IFN-gamma, α MSH and insulin, and mixing with competent cells;

(3) exogenous polypeptides H22LP, H9, IFN-gamma, α MSH and insulin are transferred into the allelochemicals by electric shock.

6. The living cell liposome carrying exogenous polypeptide of recombinant Rhodotorula glutinis and use thereof as claimed in claim 1, wherein GM4-CCT strain carrying exogenous polypeptide can achieve effective bioavailability and slow control effect of intestinal absorption in vivo by oral route.

7. The recombinant rhodotorula glutinis viable cell liposome carrying exogenous polypeptide and use thereof according to claim 1, wherein the recombinant rhodotorula glutinis viable cell liposome is used as an oral introduction carrier for drugs of exogenous lipophilic molecules, polypeptides, nucleic acids or compounds.

8. The recombinant rhodotorula glutinis viable cell liposome carrying exogenous polypeptide and use thereof according to claim 1, wherein the recombinant rhodotorula glutinis viable cell liposome is used for medicine or/and food of exogenous lipophilic molecules, polypeptides, nucleic acids or compounds.

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