WO2020082221A1 - 重组磷酸胆碱胞苷转移酶的粘红酵母携带外源多肽的活细胞脂质体及其应用 - Google Patents

重组磷酸胆碱胞苷转移酶的粘红酵母携带外源多肽的活细胞脂质体及其应用 Download PDF

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WO2020082221A1
WO2020082221A1 PCT/CN2018/111320 CN2018111320W WO2020082221A1 WO 2020082221 A1 WO2020082221 A1 WO 2020082221A1 CN 2018111320 W CN2018111320 W CN 2018111320W WO 2020082221 A1 WO2020082221 A1 WO 2020082221A1
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strain
insulin
recombinant
exogenous
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利时雨
孙晗笑
汪佳佳
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利时雨
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

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  • the invention relates to the technical field of genetic engineering, in particular to a living cell liposome carrying recombinant polypeptides of Rhodotorula saccharomyces with recombinant phosphocholine cytidine transferase and application thereof.
  • Phospholipid is the basic component of biological membrane. In the biological membrane, the hydrophilic head of the phospholipid is located on the surface of the membrane, and the hydrophobic tail is located on the inside of the membrane. Because water is polar, when many phospholipid molecules are on the surface of the aqueous solution, the phospholipid and After the interaction of water molecules, three types of phospholipid molecular complex structures, micelles, liposomes, and phospholipid bilayers are usually formed. Peptide drugs are all biological macromolecules. Their common feature is that they are unstable in the body and are easily degraded by proteolytic enzymes. Therefore, the half-life in the body is short, the bioavailability of oral administration is low, and the patient's compliance is poor, which limits Its wide application. After being wrapped with liposomes, these shortcomings can be overcome.
  • H22LP is also a broad-spectrum chemokine receptor US28 antagonist peptide obtained in the preliminary screening in the laboratory. H22LP can directly inhibit viral cytomegalovirus by interacting with viral particles.
  • Interferon is a broad-spectrum antiviral agent that does not directly kill or inhibit the virus, but mainly produces antiviral proteins through the action of cell surface receptors, thereby inhibiting the replication of hepatitis B virus.
  • Type ⁇ - (leukocyte) type, ⁇ - (fibroblast) type, ⁇ - (lymphocyte) type; at the same time, it can also enhance the activity of natural killer cells (NK cells), macrophages and T lymphocytes, thereby To immune regulation and enhance anti-viral capabilities.
  • Interferon is a group of active proteins (mainly glycoproteins) with multiple functions. It is a cytokine produced by monocytes and lymphocytes. They have a broad spectrum of antiviral effects on the same kind of cells, affect cell growth, and differentiate, regulate immune function and other biological activities.
  • Type I interferon includes IFN- ⁇ and IFN- ⁇ . It is called IFN ⁇ produced by human fibroblasts; IFN- ⁇ is mainly produced by monocytes-macrophages, in addition, B cells and fibroblasts can also synthesize IFN- ⁇ ; IFN- ⁇ is mainly produced by fibroblasts. Both IFN- ⁇ / ⁇ bind to the same receptor and are widely distributed, including mononuclear-macrophages, polymorphonuclear leukocytes, B cells, T cells, platelets, epithelial cells, endothelial cells and tumor cells.
  • Type II interferon is interferon ⁇ , which is mainly produced by activated T cells (including Th0, TH1 cells and almost all CD8 + T cells) and NK cells. It is one of the so-called lymphokines (LyTnPhokine) Species. IFN- ⁇ can exist in the form of extracellular matrix connection, so cell growth is controlled in a side-by-side manner, which can be distributed on almost all cell surfaces except mature red blood cells.
  • lymphokines LyTnPhokine
  • IFN- ⁇ antibacterial effect IFN- ⁇ can reduce the iron supply of bacteria by down-regulating the transferrin receptor or directly inhibit the intracellular bacteria by inducing the production of endogenous NO, and can also increase the phagosome of mononuclear macrophages—— The lysosome dissolves bacteria, and through the above methods, it can eliminate bacteria.
  • IFN- ⁇ antiparasitic effect interferon can activate macrophages (M ⁇ ), activated M ⁇ can express high levels of inducible nitric oxide synthase (iNOS) to catalyze the production of NO by L-arginine, and NO inoculates pathogens Has inhibitory and killing effects. It is reported that IFN- ⁇ can activate M ⁇ to produce NO, and promote the synthesis of NO in a dose-dependent manner. The higher the dose, the more obvious the effect. Daubener et al. (2001) found that stimulation of human brain microvascular endothelial cells (HBMEC) with IFN- ⁇ can induce its resistance to toxoplasmosis.
  • HBMEC human brain microvascular endothelial cells
  • HBMEC stimulated by IFN- ⁇ can inhibit the growth of Toxoplasma gondii and increase the appearance of TNF- ⁇ , which is related to the activity of IDO.
  • adding too much tryptophan to HBMEC culture can completely inhibit IFN- ⁇ -TNF- ⁇ -mediated toxoplasmosis, indicating that IDO can mediate its protection, and it is reported that IFN- ⁇ depends on IDO's Expression works.
  • IFN- ⁇ The interferon involved in immunomodulation is IFN- ⁇ , also known as immunomodulatory interferon.
  • Immunomodulatory interferon can express the Fc receptor of IgG, which is beneficial to the phagocytosis of antigen by macrophages, the killing of target cells by K and NK cells, and the activation of T and B lymphocytes to enhance the body's immune response ability.
  • IFN- ⁇ can increase the expression of MHC class II molecules on the surface of macrophages and enhance their antigen presentation ability.
  • it can also promote the phagocytosis of immune complexes, antibody-coated pathogens and tumor cells by enhancing the expression of Fc receptors on the surface of macrophages.
  • it can also stimulate neutrophils, enhance their phagocytic capacity, activate NK cells, and enhance their cytotoxicity to participate in immune regulation.
  • interferon is an innate and adaptive essential cytokine in the defense of tumor development.
  • IFN- ⁇ is produced by specific antigens stimulated by T lymphocytes, its structure is different from type I interferon, and it is not acid-tolerant , Is the main macrophage stimulating factor of the body, and has a variety of regulatory effects on the body's immune response.
  • IFN- ⁇ can activate effector cells, increase 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, and stimulate IL-2, tumor necrosis factor, interferon- ⁇ Such as the production of cytokines, inhibit tumor cell division, and induce gene synthesis into antiviral proteins. Siegbert (2003) and others found that IFN- ⁇ has an anti-proliferative effect on neuronal pancreatic tumor cells, and this anti-proliferative response is similar to functional and non-functional neuroendocrine tumors.
  • Rhodotorula belongs to the subfamily Hymenophora. It is round or oval, and the colony is red. Asexual, multipolar budding. Alcohol-free fermentation ability, differentiating lactose and decomposing fat. Red yeast should be grown in the environment of 25-30 °C, acidic, with a certain carbon source and nitrogen source. Red yeast is a kind of saprophytic bacteria with strong resistance to stress, which exists in nature and is widely distributed in various ecological environments.
  • Rhodotorula glutinosa The cells of Rhodotorula glutinosa are round, oval or elongated. Multilateral buds, with obvious red or yellow pigments, from the capsule to form slimy colonies.
  • This genus has good fat-producing strains, and a large amount of fat can be extracted from the bacteria. Some species have a weak oxidation effect on hydrocarbons and can synthesize a carotene.
  • the variant of this bacterium can oxidize alkanes to produce fat, and the content can reach dry biomass. Under certain conditions, alanine and glutamic acid can also be produced, and the ability to produce methionine is also strong, reaching dry biomass.
  • Rhodotorula glutinosa has a high biomass and mevalonate flux, and mevalonate is the synthesis and final synthesis of coenzyme.
  • Phytol, lycopene, penicillin and other secondary metabolites are necessary industrial precursors.
  • a strain of Rhodotorula glutinis GM4 with strong fat-producing ability was selected, and its fatty acid content could be as high as 22.54%.
  • genetic engineering transformation of the selected S. cerevisiae that is, the key enzyme of lipid metabolism, malic enzyme ME, was transfected into the strain to express it efficiently. Compared with wild bacteria, the transformed bacteria can accumulate more lipids.
  • the fatty acid is composed of palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, etc.
  • the purpose of the present invention is to provide a living cell liposome of recombinant Rhodopsyces cerevisiae carrying a recombinant phosphocholine cytidine transferase carrying foreign polypeptides and its application in view of the above-mentioned shortcomings in the prior art.
  • the present invention adopts the following technical schemes: the phosphorylated cytidine choline transferase gene is integrated into an expression vector, and the phosphorylated choline cytidine transferase (CCT) is integrated into the genome of Rhodobacter sphaeroides.
  • CCT phosphorylated choline cytidine transferase
  • the oleaginous yeast is Rhodotorula glutinosa GM4.
  • the method for constructing a cytidine phosphatidylcholine transferase gene integrated expression vector includes the following steps:
  • a preparation method for introducing liposomes into living cells of foreign polypeptides the steps are as follows:
  • the CDP-choline pathway of yeast is similar to mammals, and CCT enzymes have low activity among all enzymes involved in the CDP-choline pathway of yeast and mammals. Therefore, the CCT enzyme is the rate-limiting enzyme in the CDP-choline pathway and a key enzyme.
  • Rhodotorulaceae is a strain that produces high oil and fat, and its oil content is increased by transforming it.
  • the high fat-producing red yeast is used as a carrier to transfer the exogenous polypeptide H22LP, H9 or ⁇ -interferon into the cell by electroporation.
  • the exogenous polypeptide enters into the lipid droplets of the recombinant strain due to its own lipophilicity, which achieves the successful wrapping of the exogenous polypeptide.
  • the invention has the following beneficial effects: the foreign gene CCT expression vector pPGK1Z-rD-CCT is successfully constructed, and after double enzyme digestion identification, the target fragment on the recombinant plasmid is inserted in the correct direction; the recombinant plasmid is introduced into Rhodotorula marcescens and the transformed strain PCR identification, the recombinant plasmid was successfully introduced into Rhodotorula glutamicum and inserted into the genome of Rhodotorula maritimus, which achieved stable and long-term expression of CCT; real-time fluorescence quantitative PCR showed that the expression level of CCT increased by about 1.5 times; the content of lecithin and Compared with wild-type strains, the content of fatty acids in the transformed strains is also significantly increased compared with wild-type strains.
  • the exogenous polypeptide is transferred into the cell by electroporation technology, and the exogenous polypeptide enters into the lipid droplets of the recombinant strain due to its own lipophilicity, thereby achieving the successful wrapping of the exogenous polypeptide.
  • Figure 1 is a graph showing the expression level of Rhodopseudomonas phosphatidylcholine cytidine transferase (in which lane M is the control group; lane 1 is the phosphocholine cytidine transferase)
  • Fig. 2 is a construction diagram of Rhodotorula marcescens integrated expression vector plasmid PGK1Z-rD-phosphocholine cytidine transferase
  • Figure 3 is a PCR amplification diagram of the Saccharomyces cerevisiae promoter gene (where lane M: is the control group; lane 1 is the Saccharomyces cerevisiae promoter gene)
  • Fig. 4 is an amplification diagram of the polymerization chain reaction of the CCT gene (where lane M: is the control group; lanes 1 and 2: phosphocholine cytidine transferase gene)
  • Figure 5 is a double digestion identification diagram of recombinant plasmid pPGK1Z-rD-phosphocholine cytidine transferase (in which lane M: control group; lanes 1 and 2 are recombinant plasmid pPGK1Z-rD-phosphocholine cytidine transferase)
  • Fig. 6 is a graph showing the expression level of Rhodopseudomonas phosphatidylcholine cytidine transferase gene (where the molecular mass unit of the control group is ku; empty plasmid: pPGK1Z-rD; recombinant plasmid: pPGK1Z-rD-phosphocholine cytidine transferase)
  • Figure 7 is a graph of the concentration of polypeptides electrotransformed into the cell (wild type strain, empty plasmid, recombinant strain; * P ⁇ 0.05 compared to wild type strain)
  • Figure 8 is the concentration of insulin in serum (* p ⁇ 0.05vs Rhodotorula saccharomyces cerevisiae-phosphocholine cytidine transferase gene)
  • Figure 9 is the tissue distribution map of insulin
  • the inclined plane medium is malt agar medium; the medium is prepared according to the formulas of seed medium, fermentation basic medium and solid medium.
  • Rhodotorula glutinis GM4 was screened and stored in this laboratory;
  • Saccharomyces cervisiae 2.1445 was purchased from China General Microbial Culture Collection Management Center (CGMMCC)
  • Escherichia coli DH5 ⁇ is kept in this laboratory.
  • the expression vector pPICZ-rD (pGAPZ ⁇ A) was constructed and stored in our laboratory.
  • YPD medium YPD medium, YPD-zeocin medium, Luria-Bertani (LB) medium, LB-ampicillin medium, low-salt LB-zeocin medium, Potato dextrose agar (PDA) medium.
  • LB Luria-Bertani
  • PDA Potato dextrose agar
  • Main reagents STET buffer, TE buffer, 10% SDS (sodium dodecyl sulfate), plasmid extract, 10xDNA buffer buffer, the prepared buffer is stored at -20 °C Tricine, SDS-PAGE electrophoresis Reagents Taq enzyme (Shanghai Shenyou Bioengineering Company), dNTP (Beijing Noble Science and Technology Company), EB staining solution (Shanghai Tongwei Industrial Company), bromophenol blue (Shanghai Tongwei Industrial Company), T4DNA ligase (Shanghai Enzyme Link Biotechnology Co., Ltd.), NcoI Takara (Dalian Company), etc.
  • SDS-PAGE electrophoresis Reagents Taq enzyme Shanghai Shenyou Bioengineering Company
  • dNTP Beijing Noble Science and Technology Company
  • EB staining solution Shanghai Tongwei Industrial Company
  • bromophenol blue Shanghai Tongwei
  • Saccharomyces cerevisiae is inoculated in 50mL / 250mL YPD liquid medium, and cultured at 30 °C, 180rpm until OD660 reaches 3 centrifuge (12000r / min, 5min, 4 °C), discard the supernatant, and collect bacterial cells;
  • the PCR reaction system is:
  • the PCR reaction system is:
  • the pPGK1Z-rD plasmid was constructed and stored in this laboratory, and the plasmid was extracted according to the following steps:
  • Overlap PCR primers used for PGK1 and CCT are as follows:
  • PGK1-BamHIprimer1 and CCT-XhoIprimer2 are the upstream and downstream primers to perform Overlap PCR to obtain the PGK1-CCT fragment.
  • the enzyme digestion reaction was carried out in a water bath at 37 ° C for 3 hours.
  • the PGK1-CTT fragment and the vector pPGK1Z-rD were recovered and purified with a recovery kit, and then the double-digested vector pPGK1Z-rD and PGK1-CCT fragments were connected with T4 DNA ligase to construct a recombinant plasmid pPGK1Z-rD- CCT.
  • connection reaction system is as follows:
  • the reaction was carried out in a 16 ° C water bath overnight, and the molar ratio of the carrier to the foreign DNA fragment was controlled at 1: 3-10.
  • the digestion system is as follows:
  • the linearized recombinant plasmid was transformed into Rhodotorula glutamicum GM4.
  • the reaction system is as follows:
  • the plasmids in the correct positive clones were extracted, and the recombinant plasmids were identified by double enzyme digestion with Nco I and Bam HII, respectively, and the reaction temperature was 37 ° C.
  • the digestion reaction system is as follows:
  • Transformants of Rhodotorula glutamicum were inoculated in 40mL YPD liquid medium, and cultured with shaking at 250 rpm at 30 ° C for 24h-36h. Take 1mL of bacterial solution and dilute to 10-2, 10-3, 10-4 with sterile water, respectively, take 200 ⁇ L of different dilutions and apply them to ordinary YPD plate and YPD-Zeocin resistant plate respectively, and calculate the number of colonies , Recorded as total bacterial count and colony with integrated plasmid. Transfer to YPD liquid medium at 1% inoculation volume, shake culture at 30 ° C, 250 rpm, take 24h as 10 generations, and cultivate 50 generations in total. Plate counts are taken every 10th generation.
  • the verified recombinant plasmid was transformed into Rhodotorula marcescens GM4. Inoculate a single colony containing the expression plasmid into the culture medium, and after IPTG (1mmol / L) induces expression, continue to culture at 30 °C for 3h and 18h, take 1.5mL of the bacterial solution, centrifuge at 12000r / min for 2min, collect the bacteria, and collect the bacteria And resuspended in 100 ⁇ L 1 ⁇ SDS loading buffer, boiled in a boiling water bath for 10min to fully lyse the cells, centrifuged at 12000r / min for 10min at room temperature to precipitate cell debris and DNA, take an appropriate amount of solution to load, SDS-PAGE analysis.
  • a target band amplified around 800 bp ( Figure 3) and a target gene around 1300 bp ( Figure 4) were obtained by PCR, and the size was consistent with the size of PGK1 and CCT genes reported on GenBank.
  • the CCT gene was amplified using a high-fidelity taq enzyme and connected to a T-vector for sequencing verification.
  • the gene is 1275 bp in length and encodes 424 amino acids. After Blast comparison, the sequence homology between this product and GenBank reached 100%.
  • the CCT gene was ligated into the plasmid pGK1Z-rD to obtain the plasmid pGK1Z-rD-CCT, which was preliminarily verified by double enzyme digestion to contain the target gene fragment ( Figure 5).
  • the sequencing was verified to be correct, and the recombinant expression vector pGK1Z-rD-CCT was successfully constructed.
  • the plasmid was transformed into Rhodotorula marcescens GM4 to obtain GM4 / pGK1Z-rD-CCT.
  • Table 1-1 The stability of pPGK 1Z-rD-CCT in transformed strain
  • the high transcription level of CCT can provide enough enzymes for lecithin synthesis, so as to maintain the synthesis of lecithin at a high level.
  • the Bligh-Dyer method has been partially improved.
  • the specific steps are as follows:
  • A is acetonitrile
  • B is 50mM aqueous ammonium acetate solution (containing 0.1% formic acid, pH ⁇ 3.65); elution procedure: 0 ⁇ 4min, 95% A, 5% B; 4 ⁇ 15min, 95% ⁇ 60% A, 5 % ⁇ 40% B; 15 ⁇ 17min, 60% A, 40% B; 17 ⁇ 17.1min, 60% ⁇ 95% A, 40% ⁇ 5% B; 17.1 ⁇ 20min, 95% A, 5% B; flow rate 0.3mL / min; injection volume: 1 ⁇ L.
  • Negative ion mode (ESI-), capillary voltage: 3.5KV, ion source temperature: 130 °C, desolvation gas temperature: 400 °C, desolvation gas flow rate: 600L / h, cone gas flow rate: 50L / h, scanning mode: SIR Mode, channel parameters are set to different phospholipid molecules, and different cone voltages are used for different phospholipid types.
  • a Bruker 450-GC instrument was equipped with a hydrogen flame ionization (FID) detector and a capillary column HP-INNOWAX (30m ⁇ 0.25mm).
  • FID hydrogen flame ionization
  • Column temperature heating program 150 °C 1min, 10min to 230 °C, and maintained at 230 °C for 2min, split ratio is 10: 1, control the retention time of fatty acid methyl ester standard, quantitative analysis of different fatty acids.
  • the experimental strain was the recombinant strain of Rhodotorula glutinosa constructed in Chapter 2 GM4-CCT.
  • Nile Red stain Dilute the purchased Nile Red mother liquor with DMSO (dinitrosulfoxide) to the target concentration.
  • the centrifuge tube, centrifuge, and incubator are the same as 2.1.4
  • Electroporation mediates the entry of foreign peptides into GM4-CCT cells
  • the resulting strains were named GM4-CCT-H22LP, GM4-CCT-H9 and GM4-CCT-IFN- ⁇ , GM4-CCT- ⁇ MSH, GM4-CCT-insulin.
  • mice 15 SPF grade BALB / c mice, weighing 18-20g / mouse, aged about 7-8 weeks, were purchased from the Experimental Animal Center of Sun Yat-sen University. Before the experiment, the mice were kept in a clean-grade animal laboratory with a room temperature of 20-25 ° C, a humidity of 60%, and daily light exposure for 12 hours. Fresh food and water were replaced once a day.
  • the blood was collected by the retrophlebular venous plexus blood collection method. After centrifugation at 2500 rpm for 15 minutes, the serum insulin concentration was measured with the ELIS kit.
  • mice were administrated with CM4-CCT-insulin and GM-CCT, and then sampled at 1.0, 2.0, 4.0, 8.0, 12.0, and 24.0h time points. The mice were immediately sacrificed and the liver, heart, spleen, lung, and lung were dissected.
  • the kidneys were rinsed 3 times with physiological saline to remove residual blood, and the tissues were blotted dry with filter paper; appropriate tissue was cut out and weighed accurately to make a homogenate; the homogenized sample was shaken at 200 rpm and 25 ° C for 20 min, Then centrifuge at 25000 rpm for 15 min; pass the supernatant through a 0.22 ⁇ m microporous filter membrane, remove impurities and use the ELISA kit to measure the insulin concentration. The method is the same as above.
  • mice take GM4-CCT-insluin and took blood through the eyeballs on days 1, 3, 5, and 7 to detect the concentration of insulin in serum with ELISA kit. Mice taking GM4-CCT served as controls. As shown in Figure 8, in the GM4-CCT-insulin group, the content of insulin in the serum increased with the number of days taken. The content of insulin increased almost linearly from 1 to 5 days, and hardly increased after 5 days.
  • the content of insulin has been maintained at a certain concentration, and the content of insulin in the GM4-CCT-insulin group is significantly higher than that of the GM4-CCT group (p ⁇ 0.05).
  • mice of clean grade Kunming were purchased from the Experimental Animal Center of Sun Yat-sen University. They were 8 weeks old, weighed 42 ⁇ 2.93g, and were kept at 20 °C -25 °C at room temperature.
  • insulin gavage group 10
  • GM4-CCT-insulin strain group 10
  • insulin injection group 10
  • Insulin gavage group was fed with insulin PBS solution (50U / kg, ig)
  • GM4-CCT-insulin strain group was fed with GM4-CCT-insulin strain
  • the daily feeding volume was 1 ⁇ 1010CFU per day, continuous intragastric administration
  • insulin injection Groups were injected subcutaneously with insulin PBS solution (50U / kg, sc), and 20 ⁇ L of blood was collected from the tail vein of the mice at 0, 1, 2, 4, 6, 9, 12, and h. After the blood coagulated, the temperature was low, and the temperature was 12,000 r / min. Centrifuge for 4 min. Accurately measure 20 ⁇ L of supernatant and measure its blood glucose value by gop-pod method.
  • mice After 72 hours of modeling, none of the mice died, and diet, urine output, and body weight were not significantly different from those before the experiment. All mice used for modeling have different degrees of reduced activity, increased water intake, and increased urine output.
  • the insulin PBS solution was administered to the diabetic mice, and the blood sugar decrease was not obvious, which may be related to the degradation and digestion of insulin by protease and trypsin in the gastrointestinal tract.
  • the blood glucose concentration of mice can only be kept at a low level for 2 hours. This is due to the short half-life when insulin is directly injected into the body.
  • the GM4-CCT-insulin strain after taking the GM4-CCT-insulin strain by intragastric administration, the blood glucose of the mice decreased significantly within 1-4 hours, and remained basically at a low level within the next 6 hours. After that, the strain enters the body and is not completely destroyed, and can effectively lower blood sugar. Therefore, GM4-CCT-insulin can be used as an exogenous insulin for the treatment of diabetes and has a significant effect of lowering blood sugar.

Abstract

涉及一种重组粘红酵母携带外源多肽的活细胞脂质体及其应用，同源重组法构建外源磷酸胞苷胆碱转移酶基因CCT整合表达载体，并以CCT基因重组的工程菌为载体，成功将外源多肽引入重组的工程菌的体内。其有益的效果：实现了CCT的稳定、长久表达，且CCT的表达水平提高了1.5倍左右；与野生型菌株相比，卵磷脂及脂肪酸含量显著提高，电穿孔技术将外源多肽转入菌株胞内，外源多肽因其自身亲脂性而进入重组菌株的脂滴内，实现了外源多肽的成功包裹。小鼠口服整个携带胰岛素的菌株GM4-CCT-insulin 7天，发现小鼠血清和重要器官(肝、肺、脾、肾)中insulin的含量增加，糖尿病小鼠口服菌株GM4-CCT-insulin，其血糖显著降低，GM4-CCT-insulin作为外源性胰岛素具有显著的降血糖的作用。

Description

[根据细则26改正21.12.2018]　重组磷酸胆碱胞苷转移酶的粘红酵母携带外源多肽的活细胞脂质体及其应用 技术领域
[根据细则26改正21.12.2018]　
本发明涉及基因工程技术领域，具体是涉及重组磷酸胆碱胞苷转移酶的粘红酵母携带外源多肽的活细胞脂质体及其应用。
背景技术
磷脂(Phospholipid)是生物膜的基本成分，在生物膜中磷脂的亲水头位于膜表面，而疏水尾位于膜内侧，由于水是极性的，当众多的磷脂分子位于水溶液表面时，磷脂与水分子相互作用后通常会形成微团、脂质体、磷脂双层3种形式的磷脂分子复合物结构。多肽类药物都是生物大分子,其共同特点是在生物体内不稳定,易于被蛋白水解酶降解,因而在生物体内的半衰期较短,口服给药生物利用率低，且患者依从性差，因而限制了其广泛应用。而用脂质体包裹后,可克服这些缺点。
真核生物体内其磷脂的合成其起始物为3-磷酸-甘油，3-磷酸-甘油在转酰基酶及磷脂酸酶作用下转变成二酰甘油，而二酰甘油在不同的转酰基酶的作用下合成不同的磷脂。因此，为控制磷脂的合成，可以通过提高转酰基酶的活性而使得磷脂的合成增多。
本实验室前期筛选到的一种炎症前期趋化因子拮抗肽H9，,此拮抗肽可特异阻断炎症前期趋化因子的分泌,生产成本低,特异性强、功效明确,安全可靠，已于2011年申请专利保护，申请号为201110150794.6。
H22LP也是实验室前期筛选得到的广谱趋化因子受体US28拮抗肽，H22LP可以通过直接与病毒颗粒作用来达到抑制人巨细胞病毒的作用。
干扰素(IFN)是一种广谱抗病毒剂，并不直接杀伤或抑制病毒， 而主要是通过细胞表面受体作用使细胞产生抗病毒蛋白，从而抑制乙肝病毒的复制，其类型分为三类，α-(白细胞)型、β-(成纤维细胞)型，γ-(淋巴细胞)型；同时还可增强自然杀伤细胞(NK细胞)、巨噬细胞和T淋巴细胞的活力，从而起到免疫调节作用，并增强抗病毒能力。干扰素是一组具有多种功能的活性蛋白质(主要是糖蛋白)，是一种由单核细胞和淋巴细胞产生的细胞因子。它们在同种细胞上具有广谱的抗病毒、影响细胞生长，以及分化、调节免疫功能等多种生物活性。
Ⅰ型干扰素包括IFN-α与IFN-β等。由人成纤细胞产生的称IFNβ；IFN-α主要由单核-巨噬细胞产生，此外B细胞和成纤维细胞也能合成IFN-α；IFN-β主要由成纤维细胞产生。IFN-α/β二者结合相同受体，分布广泛，包括单核-巨噬细胞、多形核白细胞、B细胞、T细胞、血小板、上皮细胞、内皮细胞与肿瘤细胞等。
Ⅱ型干扰素：Ⅱ型干扰素即γ干扰素，主要由活化的T细胞(包括Th0、TH1细胞和几乎所有的CD8+T细胞)和NK细胞产生，是所谓的淋巴因子(LyTnPhokine)的一种。IFN-γ可以以细胞外基质相连的形式存在，故通过旁邻方式控制细胞生长，其可以分布在除成熟红细胞以外的几乎所有细胞表面。
IFN-γ抗菌作用：IFN-γ能通过下调转铁蛋白受体减少细菌供铁量或通过诱导产生内源性NO直接抑制细胞内细菌，还能增加单核巨噬细胞的吞噬小体——溶酶体溶解细菌作用，通过以上途径共同达到消灭细菌的作用。
IFN-γ抗寄生虫作用：干扰素可激活巨噬细胞(Mφ)，活化的Mφ可表达高水平的诱导型-氧化氮合酶(iNOS)催化L-精氨酸产生NO，NO对接种病原体有抑制和杀伤作用。并据报道，IFN-γ能激活Mφ产生NO，同时促进NO合成作用受剂量依赖性，剂量越高作用越明 显。Daubener等(2001)发现用IFN-γ刺激人脑微血管内皮细胞(HBMEC)能诱导其抗弓形虫病。IFN-γ刺激后的HBMEC能抑制弓形虫生长，提高TNF-α的出现，这与IDO的活性有关。另外，在HBMEC的培养中加入过量的色氨酸能完全抑制IFN-γ-TNF-α介导的抗弓形虫病，表明IDO能介导其保护性，并且据报道，IFN-γ依赖IDO的表达而起作用。
IFN-γ的免疫调节作用：参与免疫调节的为干扰素为IFN-γ，又称为免疫调节作用干扰素。免疫调节干扰素可对IgG的Fc受体表达，从而有利于巨噬细胞对抗原的吞噬，K、NK细胞对靶细胞的杀伤以及T、B淋巴细胞的激活，增强机体免疫应答能力。IFN-γ可使巨噬细胞表面MHCⅡ类分子的表达增加，增强其抗原递呈能力。此外还可以通过增强巨噬细胞表面表达Fc受体，促进巨噬细胞吞噬免疫复合物、抗体包被的病原体和肿瘤细胞。同时还可以刺激中性粒细胞，增强其吞噬能力，活化NK细胞，增强其细胞毒作用等作用来参与免疫调节。
IFN-γ的抗肿瘤作用：干扰素是一个在防御肿瘤发展的先天的和适应的必需细胞因子，IFN-γ由特异性抗原刺激T淋巴细胞产生，其结构与I型干扰素不同，不耐酸，为机体主要的巨噬细胞刺激因子，对机体免疫反应有多方面的调节作用。能激活效应细胞，提高自然杀伤细胞、巨噬细胞和肿瘤浸润淋巴细胞的活性，促进单核细胞循环，增强免疫细胞表面抗原和抗体的表达，刺激IL-2、肿瘤坏死因子、干扰素-α等细胞因子的产生，抑制肿瘤细胞分裂，诱导基因全成抗病毒蛋白等。Siegbert(2003)等发现，IFN-α对神经型胰腺肿瘤细胞有抗增殖的作用，且此种抗增殖反应与机能性和非机能性神经内分泌瘤是相似的。
普通干扰素分子小、作用时间短，一般情况下，普通干扰素注射12小时后基本完全排出体外，因而需要多次注射，因此给患者带来了极大的痛苦。长效干扰素半衰期长，长效干扰素的半衰期长达40小时，可以在乙肝患者体内持续作用168个小时，因而，长效干扰素一周只需要注射一次，使用比较方便，而且提高了干扰素治疗的安全性，但长效干扰素价格相对较贵，这大大阻碍了干扰素的广泛应用。
红酵母属属于半知菌亚门。呈圆形或卵圆形,菌落为红色。无性,多极出芽。无酒精发酵能力,不同化乳糖,分解脂肪。红酵母宜生长在25-30℃,偏酸性,有一定碳源和氮源的环境。红酵母是一类抗逆性较强的腐生菌,存在于自然界,广泛分布于各种生态环境中。
粘红酵母的细胞圆形、卵形或长形。多边芽殖,有明显的红色或黄色色素,由荚膜而形成粘质状菌落。本属中有较好产脂肪的菌种,可由菌体提取大量脂肪。有的种对烃类有弱氧化作用,并能合成一胡萝卜素。该菌的变种能氧化烷烃生产脂肪,含量可达干生物量的。在一定条件下还能产生一丙氨酸和谷氨酸,产蛋氨酸的能力也狠强,可达干生物量的。该菌的另一个优势是可以在很多便宜的培养基上生长,如玉米粉,糖浆等另外还可以在糖蜜,豆粕,味精废水等工业废料上生长,并且可以达到很高的生物量,容易进行高密度培养。现已有报道证明该菌的生物量可达到OD600＝80以上可以进行超高密度培养。
发酵方面的相关报道证明粘红酵母有很高的生物量和甲经戊酸通量,而甲轻戊酸是合成并最终合成辅酶。、幽醇类、番茄红素、青篙素等次级代谢产物的必要前体物,是很有潜力的工业菌种。
本实验室前期筛选到一株产脂能力较强的粘红酵母(Rhodotorula glutinis)GM4，其脂肪酸含量可以高达22.54％。并对筛选到的粘红酵母进行基因工程改造，即将脂质代谢的关键酶苹果酸酶ME转染到菌株体内使其高效表达，与野生菌相比，转化菌可以积累更多的脂质， 通过气相色谱检测，其脂肪酸由棕榈酸、硬脂酸、棕榈油酸、油酸、亚油酸等组成。
此实验中为提高粘红酵母GM4菌株体内磷脂的合成量，通过基因工程改造技术，即将磷脂合成的关键酶，尤其是卵磷脂合成的限速酶：磷酸胞苷胆碱转移酶(CCT)将其转入到菌株体内使其过表达，进一步提高菌株体内磷脂的合成，研究其作为药物活细胞脂质体的可能性。
发明内容
[根据细则26改正21.12.2018]　
本发明目的是针对上述现有技术存在的不足，提供一种重组磷酸胆碱胞苷转移酶的粘红酵母携带外源多肽的活细胞脂质体及其应用。
[根据细则26改正21.12.2018]　
为解决上述的技术问题，本发明采用如下技术方案：磷酸胞苷胆碱转移酶基因整合表达载体，将磷酸胆碱胞苷转移酶(CCT)整合到粘红酵母菌的基因组。
优选的，所述产油脂酵母菌是粘红酵母GM4。
磷酸胞苷胆碱转移酶基因整合表达载体的构建方法,包括如下步骤：
(1)抽提表达载体质粒；
(2)强启动子PGK1基因和CCT基因片段连接；
(3)PGK1-CCT片段与表达载体质粒进行双酶切反应后连接构建重组质粒pPGK1Z-rD-CCT。
一种引入外源多肽活细胞脂质体的制备方法，步骤如下：
(1)制备工程菌株GM4-CCT菌株的感受态细胞。
(2)将经过修饰的外源多肽H22LP、H9、IFN-γ、αMSH及胰岛素分别溶解后与感受态细胞混合。
(3)电击化，将外源多肽H22LP、H9、IFN-γ、αMSH及胰岛素转入感受态菌株体内。
酵母的CDP-胆碱途径与哺乳动物类似，而CCT酶在参与酵母和哺乳动物的CDP-胆碱途径的所有酶中活性低。因此，CCT酶是CDP-胆碱途径中的限速酶，也是关键酶。
粘红酵母是一株高产油脂的菌株，通过对其改造使其油脂含量增多，以高产油脂的粘红酵母为载体通过电穿孔将外源多肽H22LP、H9或γ-干扰素转入胞内，外源多肽因其自身亲脂性而进入重组菌株的脂滴内，实现了外源多肽的成功包裹。
本发明具有如下有益效果：成功构建了外源基因CCT表达载体pPGK1Z-rD-CCT，经双酶切鉴定，重组质粒上的目的片段插入方向正确；将重组质粒导入粘红酵母中，经转化菌株PCR鉴定，重组质粒成功导入粘红酵母中并插入到粘红酵母的基因组中，实现了CCT的稳定、长久表达；实时荧光定量PCR表明CCT的表达水平提高了1.5倍左右；卵磷脂的含量与野生型菌株相比有显著提高，经气相色谱法分析，转化菌株中的脂肪酸含量较野生菌株也有明显的提高。通过电穿孔技术将外源多肽转入胞内，外源多肽因其自身亲脂性而进入重组菌株的脂滴内，实现了外源多肽的成功包裹。
附图说明
[根据细则26改正21.12.2018]　　
图1是粘红酵母磷酸胆碱胞苷转移酶表达水平图(其中泳道M为对照组；泳道1为磷酸胆碱胞苷转移酶)
[根据细则26改正21.12.2018]　
图2是粘红酵母整合表达载体质粒PGK1Z-rD-磷酸胆碱胞苷转移酶的构建图
[根据细则26改正21.12.2018]　
图3是酿酒酵母启动子基因的PCR扩增图(其中泳道M:为对照组；泳道1为酿酒酵母启动子基因)
[根据细则26改正21.12.2018]　
图4是CCT基因的聚合链式反应扩增图(其中泳道M:为对照组；泳道1和2：磷酸胆碱胞苷转移酶基因)
[根据细则26改正21.12.2018]　
图5是重组质粒pPGK1Z-rD-磷酸胆碱胞苷转移酶双酶切鉴定图(其中泳道M:对照组；泳道1和2为重组质粒pPGK1Z-rD-磷酸胆碱胞苷转移酶)
[根据细则26改正21.12.2018]　
图6是粘红酵母磷酸胆碱胞苷转移酶基因表达水平图(其中对照组分子质量单位为ku；空质粒:pPGK1Z-rD；重组质粒:pPGK1Z-rD-磷酸胆碱胞苷转移酶)
[根据细则26改正21.12.2018]　
图7是电转化进入胞内的多肽的浓度图(野生型菌株，空质粒，重组菌株；*与野生型菌株相比P<0.05)
[根据细则26改正21.12.2018]　
图8是血清中胰岛素的浓度(*p<0.05vs 粘红酵母-磷酸胆碱胞苷转移酶基因)
[根据细则26改正21.12.2018]　
图9是胰岛素的组织分布图
图10糖尿病小鼠血糖变化曲线
具体实施例
为了更好地阐述本发明内容，下面用若干较佳的具体实施例进行说明。但这些具体实施例只是为了说明本发明的内容，而不是对本发明内容进行限制。
实施例一 粘红酵母GM4体内CCT表达水平的检测
1.1 菌种和培养基
1.1.1 菌种：粘红酵母(Rhodotorula glutinis)GM4
1.1.2 培养基
斜面培养基为麦芽琼脂培养基；根据种子培养基、发酵基础培养基、固体培养基的配方配制培养基。
1.2 发酵方法
1.2.1 种子液制备
配制50ml种子培养基于250ml锥形瓶中,115℃,0.169MPa条件下高压蒸汽灭菌30min。在超净工作台中,用灭过菌的接种环从固体培养基上挑取一环单菌落接种到种子培养基中,于30℃,250rmp/min条件下培养,作为种子液。
1.2.2 发酵培养
同样方法制备50ml发酵培养基,高压蒸汽灭菌后,于超净工作台中接种,接种量为5％。同样的培养条件发酵培养。
1.3 CCT酶的检测
将粘红酵母GM4的单菌落接种于培养基中，IPTG(1mmol/L)诱 导表达后，30℃继续培养18h后取菌液1.5mL，12000r/min离心2min，收集菌体，收集菌体并重悬于100μL 1×SDS加样缓冲液，沸水浴中煮沸10min以充分裂解细胞，12000r/min室温离心10min使细胞碎片及DNA等沉淀，取适量溶液上样，SDS-PAGE分析。
1.4 实验结果
在IPTG诱导表达后，收集菌株，裂解、离心，取上清进行SDS-PAGE。电泳结果(图2)显示在45k附近有一条表达带，和CCT酶分子质量理论值相符合。
实施例二 高产磷脂工程菌株GM4-CCT的构建
2.1 实验材料与仪器
2.1.1 菌种
粘红酵母(Rhodotorula glutinis)GM4为本实验室筛选并保存；
酿酒酵母(Saccharomyces cervisiae)2.1445购自中国普通微生物菌种保藏管理中心(CGMMCC)
大肠杆菌(Escherichia coli)DH5α为本实验室保存。
2.1.2 质粒
表达载体pPICZ-rD(pGAPZαA)，为本实验室构建并保存。
2.1.3 培养基及主要试剂
培养基：YPD培养基、YPD-zeocin培养基、Luria-Bertani(LB)培养基、LB-ampicillin培养基、低盐LB-zeocin培养基、Potato dextrose agar(PDA)培养基。
主要试剂：STET缓冲液、TE缓冲液、10％SDS(十二烷基硫酸钠)、质粒提取液、10xDNA buffer链接缓冲液，配制好的的缓冲液保存于-20℃Tricine、SDS-PAGE电泳试剂Taq酶(上海申友生物工程公司)、dNTP(北京诺博莱德科技公司)，EB染色液(上海通蔚实业公司)、溴酚蓝(上海通蔚实业公司)、T4DNA连接酶(上海酶联生物科技有限公司)、NcoI Takara(大连公司)等。
2.1.4 主要仪器
高压灭菌锅、低速离心机、光学显微镜，超净工作台、高速离心机、Hettich凝胶成像系统等。
2.2 实验方法
2.2.1 目的基因的分离与扩增
2.2.1.1 酿酒酵母PGK1基因的分离与扩增
1、分离：
(1)酿酒酵母接种于50mL/250mL YPD液体培养基中，30℃，180rpm培养至OD660达到3时离心(12000r/min，5min，4℃)，弃上清，收集菌体；
(2)无菌水冲洗菌体两次，离心后用冷的无菌水重悬菌体，菌悬液转移至含有液氮和1g矾土的研钵中研磨破碎菌体细胞；
(3)将破碎的菌体细胞转移至5mL DNA提取缓冲液中(50mM TRIS,10mM MgCl2,50mM NaCl，1％(wt/vol)SDS，pH 7.4)的离心管中；
(4)经过反复的苯酚/氯仿抽提以及乙醇沉淀步骤，酿酒酵母DNA被分离出来。
2、PCR扩增：
根据PGK1基因序列，设计引物。
正向引物5′-CGCGGATCCTATTTAGATTCCTGACTTCAACTC-3′(Bam HI)；
反向引物5′-TATCCGCTCGAGTGTTTTATATTTGTTGAAAAAGTAG-3′(Xho I)
PCR反应体系为：
组分 用量/μl
DNA模板 0.4
10×buffer 5
正向引物 0.7
反向引物 0.7
dNTP 5
Tag酶 0.4
ddH2O 37.8
总计 50
PCR反应条件：
94℃ 预变性 5min
94℃ 变性 0.5min
54℃ 退火 0.5min
72℃ 延伸 1min
72℃ 再延伸 5min
在预变性之后，按照上表所示的参数循环30次。取5μL PCR扩增产物，经2％琼脂糖凝胶电泳分析。
2.2.2 酿酒酵母CCT基因的分离与扩增
2.2.2.1 分离：
同1.2.1.1酿酒酵母PGK1基因的分离方法
2.2.2.2 PCR扩增：
搜索Genbank数据库，根据CCT基因序列信息，用Prmier 5.0软件设计PCR引物。
CCT-F:5'-ATGGCAAACCCAACAACAGG-3'(Xho I)，
CCT-R:5'-GTTCGCTGA TTGTTTCTTCTTCTG-3'(NcoI)
PCR反应体系为：
组分 用量/μl
DNA模板 0.4
10×buffer 5
正向引物 0.7
反向引物 0.7
dNTP 5
Tag酶 0.4
ddH2O 37.8
总计 50
PCR反应条件：
94℃ 预变性 5min
94℃ 变性 1min
60℃ 退火 50s
72℃ 延伸 1.5min
72℃ 再延伸 10min
在预变性之后，按照上表所示的参数循环30次。取5μL PCR扩增产物待经2％琼脂糖凝胶电泳分析。
2.2.3 凝胶电泳与PCR产物回收
1、凝胶电泳：
首先制备2％琼脂糖凝胶，待琼脂糖凝胶液冷却至70℃左右，倒入已放好的制胶槽内，冷却直至成凝胶，将凝胶及胶槽取出，放置于电泳槽内，加入1xTAE电泳缓冲液直至浸没凝胶；把上样缓冲液(溴酚蓝)混合到DNA样品，用移液枪将样品加入到凝胶板的小槽内；加样后，立即通电进行电泳，电压120V，当溴酚蓝将至凝胶顶端时，停止电泳；取出凝胶，用含有0.5μg/ml的EB/1xTAE溶液染色30min，再用蒸馏水清洗15min；在紫外灯下观察，观察到条带后， 采用凝胶成像系统拍照保存。
2、PCR产物回收：
使用紫外光显影出目的DNA条带，用切片刀切割相应的琼脂带，置入离心管中。
2.2.4 表达外源CCT整合载体pPGK1Z-rD-CCT的构建
2.2.4.1 整合载体pPGK1Z-rD-CCT的构建路线图
参考文献的相关方法，利用同源重组原理设计CCT基因整合表达载体，构建技术路线见图2。
2.2.4.2 表达载体pPGK1Z-rD-CCT的抽提
pPGK1Z-rD质粒为本实验室构建并保存的，按照以下步骤抽提质粒：
(1)将含有pPGK1Z-rD质粒的大肠杆菌DH5α接种到装有5mL LB-amp培养基中，于37℃下250rpm振荡培养过夜；
(2)吸取1.5mL过夜菌液于离心管中，4℃下12000rpm离心1分钟，弃上清；
(3)用滤纸吸干离心管中的液体培养基，将菌体沉淀悬浮于200μL STET缓冲液中，用涡旋混合器充分混匀；
(4)加入4mL新配制的溶菌酶溶液，混匀后于室温下静置5min；
(5)用漂子架住离心管，置于沸水浴中，精确记时45s，取出后立刻12000rpm离心5分钟；
(6)用无菌牙签挑取离心管中的沉淀物弃去，离心管的上清液中加入8mL 5％CTAB，用混合器混匀后，13000rpm离心5分钟，弃上清液，用滤纸吸干离心管中的液体；
(7)加入1.2M NaCl 300mL，充分溶解沉淀物，再加入750mL的预冷乙醇，充分混匀后，13000rpm离心15分钟，弃上清液；
(8)取1mL 70％冷乙醇，缓慢淋洗离心管内壁，13000rpm离心15min，弃上清液，用滤纸吸干管壁上的液体，置室温中使核酸沉淀自然干燥5-10min；
(9)沉淀物溶于50mL TE缓冲液中，混合器混匀，于-20℃保存备用。
2.2.4.3 重叠PCR连接PGK1和CCT
为了实现外源基因CCT在强启动子PGK1的带动下高效表达，我们用Overlap PCR将强启动子PGK1基因和CCT基因片段连接，以保证两个基因片段之间无其他序列，从而避免因其他基因的引入造成目的基因表达异常。
用于PGK1和CCT的Overlap PCR引物如下：
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)
分别以PGK1-Bam HI primer1、PGK1-Xho I primer 2和CCT-Xho I primer1、CCT-Xho I primer2分别扩增PGK1和CCT，5个循环后回收PGK1和CCT片段，以回收的PGK1和CCT片段为共同模板，PGK1-Bam HI primer1和CCT-Xho I primer2为上、下游引物进行Overlap PCR，得到PGK1-CCT片段。
Overlap PCR反应体系为：
组分 用量/μl
回收的DNA片段模板 80
10×buffer 7.5
dNTP 7.5
反向引物 0.7
dNTP 5
PGK1-Bam HI primer1 0.4
CCT-Xho I primer2 37.8
Tag酶 1.5
ddH2O 112.5
总计 150
PCR反应条件：
94℃ 预变性 5min
94℃ 变性 1min
60℃ 退火 50s
72℃ 延伸 1.5min
72℃ 再延伸 10min
在预变性之后，按照上表所示的参数循环30次。取5μL PCR扩增产物待经3％琼脂糖凝胶电泳分析。
2.2.4.4 片段与载体的双酶切反应、连接
1、将Overlap PCR产物与pPGK1Z-rD分别进行进行Nco I和Bam  HI双酶切质粒pPGK1Z-rD Nco I/Bam HI双酶切反应体系：
组分 用量/μl
质粒pPGK1Z-rD 15
Nco I内切酶 2
Bam HI内切酶 2
10×buffer 5
1％BSA 5
ddH2O 26
总计 50
片段PGK1-CCT Nco I/Bam HI双酶切反应体系：
组分 用量/μl
片段PGK1-CTT 10
Nco I内切酶 2
Bam HI内切酶 2
10×buffer 5
1％BSA 5
ddH2O 26
总计 50
酶切反应在37℃水浴反应3小时。
2.2.4.5 连接反应
酶切反应后,PGK1-CTT片段和载体pPGK1Z-rD分别用回收试剂盒回收纯化,然后用T4DNA连接酶连接双酶切后的载体pPGK1Z-rD和PGK1-CCT片段,构建重组质粒pPGK1Z-rD-CCT。
连接反应反应体系如下:
组分 用量/μl
片段PGK1-CCT酶切纯化产物 6
载体pPGK1Z-rD酶切纯化产物 2
10×T4 DNA Ligase Buffer 1
T4 DNA连接酶 1
总计 10
16℃水浴过夜反应,载体与外源DNA片段的摩尔比要控制在1:3-10。
2.2.5 重组质粒pPGK1Z-rD-CCT电转化粘红酵母感受态细胞
2.2.5.1 粘红酵母感受态的制备
1、挑取酵母单菌落接种在5mL YPD液体培养基，于30℃，250rpm振荡培养过夜；
2、以1％接种量转接至100mL YPD液体培养基，于30℃，250rpm振荡培养至细胞OD600≈1.4；
3、菌液于4℃，3000g离心5min沉淀细胞，弃上清，用100mL无菌水重悬；
4、重复步骤三；
5、4℃，3000g离心2min沉淀细胞，弃上清，用20mL冰预冷的1M山梨醇重悬细胞；
6、4℃，3000g离心2min沉淀细胞，弃上清，用200μL 1M山梨醇重悬细胞，用于转化；
2.2.6 质粒线性化
将约10μg重组质粒pPGK1Z/rD/CCT用Sac I进行单酶切,酶的用量、酶切时的温度,参照厂家说明书,完全酶切所用的时间要特别注意,既不能部分酶切,亦不能将质粒消化掉,这对电转的效率有着十分重要的作用。
酶切体系如下所示:
组分 用量/μL
Sac I 1
10×buffer 5
质粒pPGK1Z-rD-CCT 10
ddH2O 7
总计 20
2.2.7 电转化
将线性化好的重组质粒电转化至粘红酵母GM4。
1、将80μL己制备好的感受态细胞与20μL(约10μg)已线性化好的待转化质粒DNA混合,加入到0.2cm已预冷的电转化杯中；
2、将装有混合液的电转化杯冰浴5min；
3、调整好电转仪的参数:电压1.5kV，电容25uF，电阻200Ω，电击持续时间约为5ms左右；
4、电击结束后，立即向转化杯中加入1mL预冷的1M山梨醇溶液,用枪轻微吸打混匀,然后将其转至灭过菌的离心管中,30℃静置l h，然后加入1mL新鲜的YPD培养基，30℃，200rpm摇1小时；
5、室温下3000rpm，离心4min，用200μL ddH2O重悬菌体；
6、将消液涂布于YPD平板(含50μL Zeocin)，置于30℃培养箱中培养2-3d,直到长出单菌落为止。
1.2.8 重组粘红酵母的PCR检测
1、挑取YPD抗性平板上长势较好的单菌落转接到YPD液体培养基中，30℃，250rpm振荡培养至OD600大于2；
2、取1mL菌液12000rpm离心2min，弃上清，加入100μL TE缓冲液重悬菌体，水浴煮沸5-10min，立即置于-20℃冰箱冻存15 min，室温下静置使菌悬液溶解，12000rpm离心5min，取上清液5μL为模板进行PCR检测。
反应体系如下：
组分 用量/μL
模板(上清液) 5
10×buffer 5
dNTP 5
CCT正向引物 1
CCT反向引物 1
Tag酶 0.5
ddH2O 32.5
总计 50
PCR反应条件:
94℃ 预变性 5min
94℃ 变性 0.5min
54℃ 退火 0.5min
72℃ 延伸 1.5min
72℃ 再延伸 5min
在预变性之后，按照上表所示的参数循环35次。取5μL PCR扩增产物待经2％琼脂糖凝胶电泳分析。
2.2.9 质粒的酶切验证
经菌液PCR初步验证后，提取正确阳性克隆中的质粒，用Nco I和Bam HII分别对重组质粒进行双酶切鉴定，反应温度为37℃。
酶切反应体系如下：
组分 用量/μl
质粒 2
10×buffer 5
Nco I 1
Bam HII 1
ddH2O 41
总计 50
2.2.10 转化子稳定性检测
将粘红酵母转化子接种于40mL YPD液体培养基中，30℃，250rpm振荡培养24h-36h。取1mL菌液用无菌水分别稀释到10-2,10-3,10-4，各取200μL不同稀释度的稀释液分别涂布于普通YPD平板和YPD-Zeocin抗性平板，计算菌落数，分别记为总菌数和带有整合质粒的菌落数。以1％接种量转接至YPD液体培养基，30℃，250rpm振荡培养，以24h为10世代，共培养50个世代。每隔10世代进行一次平板计数。
以下式计算质粒稳定性：
稳定性(％)＝带有整合质粒的菌落数÷总菌数×100％
2.2.11 转化子CCT表达水平测定
将验证正确的重组质粒转入粘红酵母GM4。将含有表达质粒的单菌落接种于培养基中，IPTG(1mmol/L)诱导表达后，30℃继续培养3h及18h后取菌液1.5mL，12000r/min离心2min，收集菌体，收集菌体并重悬于100μL 1×SDS加样缓冲液，沸水浴中煮沸10min以充分裂解细胞，12000r/min室温离心10min使细胞碎片及DNA等沉淀，取适量溶液上样，SDS-PAGE分析。
2.3 实验结果
2.3.1 PCR扩增检测目的片段
以S.cerevisiae基因组为模板，经PCR得到一个800bp(图3)左右扩增的目的条带以及1300bp左右的目的基因(图4)，大小和GenBank上报道的PGK1和CCT基因大小相符。
1.3.2 重组质粒pPGK1Z-rD-CCT双酶切鉴定
使用高保真taq酶将CCT基因扩增出来，连入T-载体进行测序验证，该基因全长1275bp，编码424个氨基酸。经过Blast比较，该产物与GenBank上的序列同源性达到100％。再将CCT基因连入质粒pGK1Z-rD，得到质粒pGK1Z-rD-CCT，经双酶切初步验证含有目的基因片段(图5)，测序验证无误，重组表达载体pGK1Z-rD-CCT构建成功。将质粒转化粘红酵母GM4，得到GM4/pGK1Z-rD-CCT。
2.3.2 重组质粒稳定性检测
为了检测重组质粒pPGK1Z-rD-CCT的遗传稳定性，我们对转化菌株在非选择压力下摇瓶培养60世代，沾取菌液于Zeocine抗性平板上涂布、培养，计算菌落数。结果如表1-1所示，显示转化的粘红酵母菌株在连续培养60世代后，稳定性仍然可以达到99.23％，表明在非选择压力下，粘红酵母中的质粒具有良好的遗传稳定性。
表1-1重组质粒的稳定性检测
Table 1-1 The stability of pPGK 1Z-rD-CCT in transformed strain
Figure PCTCN2018111320-appb-000001
2.3.3 转化粘红酵母菌株CCT的表达分析
挑取阳性菌落，接种于3mL液体培养基中，于37℃培养过夜 后，取1mL菌液转接到含抗生素的培养基中，37℃培养约3h，加入IPTG使其终浓度达到1mmol/L，30℃继续培养3h及18h后取菌液1.5mL，12000r/min离心2min，收集菌体，进行SDS-PAGE。电泳结果(图5)显示在45k附近有一条表达带，和CCT酶分子质量理论值相符合，3h时，该蛋白表达明显增强，到18h时表达量更大，约是野生型菌株的2倍。CCT的高转录水平可以为卵磷脂的和合成提供所需的足够的酶，从而使卵磷脂的合成量维持在较高的水平。
实施例三 转化菌株GM4-CCT的磷脂组成分析
3.1 菌株GM4-CCT总磷脂的提取
对Bligh-Dyer法进行了部分改进，具体步骤如下：
(1)准确称取0.1g干菌体于具塞试管中，添加1.2ml去离子水，1.5ml氯仿，3ml甲醇，漩涡震荡，混合均匀，超声30min；
(2)加入1.5ml氯仿，漩涡震荡，混合均匀，超声30min；
(3)加入1.5ml水，漩涡震荡，混合均匀，超声30min；
(4)加入0.6m L饱和氯化钠溶液，洗涤沉淀，放置过夜，保证充分提取；
(5)将放置过夜的样品离心(4500rpm，10min)。离心后溶液分为三层，上层为甲醇和水层，中间薄薄的白色层为菌体残渣，下层为氯仿层，磷脂存在氯仿层中。将下层溶液转移至玻璃瓶内；
(6)加入1.5ml氯仿于剩余的溶液和残渣中，漩涡震荡，混合均匀，超声30min后离心(4500rpm，10min)；
(7)将下层澄清溶液与之前的溶液混合，氮吹，所得固体溶于1ml氯仿/甲醇溶液(2:1，v/v)，-20℃保存，待测。
3.2、磷脂定量方法
(1)色谱实验条件
Waters BEH HILIC色谱柱(100mm×1mm×1.7μm)；流动相：
A是乙腈，B是50mM醋酸铵水溶液(包含0.1％的甲酸，pH≈3.65)；洗脱程序：0～4min，95％A，5％B；4～15min，95％～60％A，5％～40％B；15～17min，60％A，40％B；17～17.1min，60％～95％A，40％～5％B；17.1～20min，95％A，5％B；流速0.3mL/min；进样量：1μL。
(2)质谱实验条件：
负离子模式(ESI-)，毛细管电压：3.5KV，离子源温度：130℃，脱溶剂气温度：400℃，脱溶剂气流速：600L/h，锥孔气流速：50L/h，扫描模式：SIR模式，通道参数设置为不同的磷脂分子，对不同的磷脂种类采用不同的锥孔电压。
3.3 转化菌株GM4-CCT脂肪酸组分的分析
1、菌株GM4-CCT总脂肪酸的提取
(1)收集子啊YPD液体培养基培养至稳定期的菌株，离心后用蒸馏水清洗三遍后，5000g于4℃下离心5min，收集菌体后烘干至恒重，称重后置于研钵中研磨成粉末，用滤纸包好粉末，再次烘干至恒重。
(2)将烘干至恒重的粉末放于抽提筒中，注入无水乙醚浸没样品，70℃下恒温水浴中回流8h左右，当乙醚中无油为抽提结束，除去溶剂后的油脂备用。
(3)向油脂中加入1ml 2％(wt/vol)硫酸-甲醇，60℃酯化2h。
(4)酯化结束后，自然冷却至室温，加入1ml己烷，涡旋10min萃取脂肪酸甲酯。
(5)取(4)中己烷800μL己烷加到玻璃瓶中进行GC分析。
2、GC分析
用Bruker 450-GC仪器配氢火焰离子化(FID)检测器和毛细管 色谱柱HP-INNOWAX(30m×0.25mm)。柱温升温程序：150℃ 1min，10min升至230℃,并在230℃保持2min，分流比为10:1，对照脂肪酸甲酯标准品的保留时间，对不同脂肪酸进行定量分析。
3.4 实验结果
3.3.1 卵磷脂的积累研究
为了测定表达CCT基因的粘红酵母转化菌株是否会产生较高水平的卵磷脂，实验过程中对其磷脂成分进行了测定及分析，结果如表1-2所示，发现转化菌株的卵磷脂含量显著增高，由原来的42.8％提高到65.7％，由此可以看出，与野生菌株相比，转化菌株可以积累更多的卵磷脂。
表1-2转化菌株和野生菌株的磷脂组成
Table 2-2 Relative phosphatidylcholine content of the wild-type strain and the transformed strain
磷脂含量 野生菌株 转化菌株
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 转化菌株和野生型菌株脂肪酸的组成
脂肪酸含量(w/w,％) 野生菌株 转化菌株
棕榈酸 22.21±1.52 20.86±1.52
硬脂酸 7.75±0.54 7.12±0.38
棕榈油酸 5.34±0.27 5.79±0.26
油酸 43.12±1.58 48.51±1.92
亚油酸 15.86±0.75 19.32±0.51
γ-亚麻酸 3.18±0.15 5.25±0.94
经过脂肪酸分析，与野生型菌株相比，转化菌株的各脂肪酸含量均高于野生型菌株，说明CCT酶的转入能显著提高粘红酵母菌株体内不饱和脂肪酸的合成与积累。
实施例四 外源多肽进入转化菌株GM4-CCT菌株胞内的定量分析
4.1 实验材料
4.1.1 试验菌株
实验菌株为第二章中构建的粘红酵母重组菌株GM4-CCT。
4.1.2 培养基
同2.1.3
4.1.3 主要试剂
0.5mg/mL的尼罗红染色剂：将购买的尼罗红母液用DMSO(二硝基亚砜)稀释至目标浓度。
FITC修饰的H22LP、H9、IFN-γ、αMSH及胰岛素。
4.1.4 主要设备
离心管、离心机、培养箱同2.1.4
4.2 实验方法
4.2.1 电穿孔方法介导外源多肽进入GM4-CCT胞内
1、制备工程菌株GM4-CCT菌株感受态细胞，方法同1.2.5。
2、将10μg修饰了的H22LP、H9、IFN-γ、αMSH及胰岛素分别溶解后与感受态细胞混合后共100μL，加入到0.2cm已预冷的 电转化杯中；
3、电击过程同1.2.7；
4、电击结束后，立即向转化杯中加入900μL预冷的1M山梨醇溶液，用枪轻微吸打混匀，然后将其转至灭过菌的离心管中，30℃静置lh，离心后加入1mL新鲜的YPD培养基重悬菌体，30℃，200rpm摇1小时；
5、最后得到的菌株分别命名为GM4-CCT-H22LP、GM4-CCT-H9和GM4-CCT-IFN-γ、GM4-CCT-αMSH、GM4-CCT-胰岛素。
4.2.2 荧光分光光度计定量分析
(1)分别配制FITC-H22LP、FITC-H9及FITC-IFN-γ、FITC-αMSH及FITC-胰岛素的浓度梯度标准液；并取1mL标准液到1毫升比色皿，待测定荧光强度Fs；
(2)分别移取1毫升FITC-H22LP、FITC-H9及FITC-IFN-γ、FITC-αMSH及FITC-胰岛素菌液到1毫升比色皿，待测荧光强度Fx；
(3)配制空白溶液于1毫升比色皿，待测荧光强度F0；
(4)做(Fs-F0)与待测样品标准品浓度C之间的标准曲线；
(5)放进荧光分光光度计测读：激发波长514nm，散发波长492nm，确定活体细胞H22LP、H9、IFN-γ、αMSH及胰岛素的荧光强度进行定量；
(6)根据(Fx-F0)从标准曲线上求得样品的含量。
4.3 实验结果
4.3.1 荧光分光光度法测定胞内多肽FITC-H22LP、FITC-H9及FITC--IFN-γ的浓度
电转化后，我们离心出去溶液中的多肽，收集菌液，运用荧光分光光度法测定胞内多肽FITC-H22LP、FITC-H9及FITC--IFN-γ的含量， 实验结果如图7所示，经检测三种多肽的的含量差别不大，均别为4mg/mL左右，而最初电转化时多肽的浓度为10mg/mL，说明电转化后胞内多肽的浓度可到达最初浓度的2/5，而且三种多肽在胞内的浓度没有显著性差异，αMSH和胰岛素实验结果与上述结果相一致。实施例五小鼠体内研究
5.1 实验材料与仪器
5.1.1 实验菌株
前一章得到的菌株GM4-CCT-insulin
5.1.2 主要试剂
insulin ELISA试剂盒
5.1.3 主要仪器
多功能酶标仪Molecular Device，美国；高速匀浆机XHF-D高速分散器宁波新芝生物科技股份有限公司。
5.1.4 实验小鼠
15只SPF级BALB/c小鼠，体重为18-20g/只，年龄在7-8周左右，购买于中山大学实验动物中心。在实验前，小鼠饲养在清洁级动物实验室，室温为20-25℃，湿度为60％，每天接受光照12h，每天更换新鲜食品和水一次。
5.2 实验方法
5.2.1 GM4-CCT-insulin的小鼠体内研究
5.2.1.1 insulin在血清中的浓度
1、小鼠分组
(1)GM4-CCT-insulin组，取6只正常小鼠，每天除饲喂正常的饲料，还饲喂灭活的GM4-D6D-CCT-insulin菌株，每只每天饲喂量为1×1010CFU；
(2)GM4-CCT组，取6只正常小鼠，每天除饲喂正常的饲料， 还饲喂灭活的GM4-CCT菌株，每只每天饲喂量为1×1010CFU，做为对照；
2、小鼠眼球取血
在饲喂后的第1,3,5,7天，采用眼球后静脉丛取血法取血，在2500rpm下离心15min后用ELIS试剂盒测血清中insulin浓度。
5.2.1.2 insulin组织分布
分别给小鼠灌服CM4-CCT-胰岛素和GM-CCT后再1.0、2.0、4.0、8.0、12.0、24.0h各时间点取样后，立即处死小鼠并剖取肝、心、脾、肺和肾，用生理盐水漂洗3次，以除去残留血液，用滤纸将组织吸干；剪取适量组织，精确称重后制成匀浆；制成匀浆的样品在200rpm，25℃下震荡20min，然后在25000rpm下离心15min；将上清过0.22μm微孔滤膜，去除杂质后用ELISA试剂盒测insulin浓度，方法同上。
5.3 实验结果
5.3.1 insulin在血清以及组织中的检测
为了检测GM4-CCT-insulin运载insilin的效果，我们使小鼠服用GM4-CCT-insluin后，在第1、3、5、7天通过眼球取血，用ELISA试剂盒检测血清中insulin的浓度，服用GM4-CCT的小鼠作对照。如图8所示，在GM4-CCT-insulin组，insulin在血清中的含量随着服用天数的增加而增加，在1至5天，insulin的含量几乎呈直线增加，在5天后，几乎不增加并且趋向于平稳，而在GM4-CCT组，insulin的含量在一定浓度一直保持平衡状态，且GM4-CCT-insulin组insulin在血清中的含量显著高于GM4-CCT组(p<0.05)。
由小鼠组织分布图(图9)可见，GM4-CCT-insulin运载insulin在小鼠脏器的分布依次为肝、肾、心、肺、脾，在2h时达到最高， 尤其是在肝脏其浓度达到5.36。这可能是与脂质主要在肝脏代谢的原因，我们猜测insulin很可能是随着脂质形成乳糜微粒而被携带，并一起进入淋巴系统，最后进入血液，运送到身体各个组织，尤其是脂质代谢的主要器官肝脏。
实施例六 糖尿病小鼠体内实验
6.1 材料与方法
6.1.1 实验动物：
清洁级昆明种雄性小鼠40只，购自中山大学实验动物中心，8w龄，体重42±2.93g，室温保持在20℃-25℃。
6.1.2 实验菌种
菌株GM4-CCT-insulin
6.1.3 主要实验试剂及仪器
四氧嘧啶、胰岛素标准品、PBS缓冲溶液
血糖仪、紫外分光光度仪、台式离心机、显微镜
6.3 实验方法
6.3.1 实验动物造模：
糖尿病小鼠模型的建立，取健康雄性昆明种小鼠数只，禁食，正常给水，24h后称重，按40mg/kg剂量在小鼠尾静脉注射质量分数为3％的四氧嘧啶溶液，正常喂养3d后继续禁食12h，于其尾静脉取血200μL。待血液凝固后，于低温4℃，转数12000r/min离心4min后，量取上清液(血清)20μL，采用葡萄糖氧化酶法(gop-pod)测其血糖含量，血糖值高于16.67mmol/l的为试验用小鼠。
6.3.1 实验动物分组及给药
取上述糖尿病小鼠30只，称重，随机分成3组，胰岛素灌胃组(n＝10)、GM4-CCT-insulin菌株组(n＝10)、胰岛素注射组(n＝10)。禁食12h，可自由饮水，灌胃给药。胰岛素灌胃组分别灌给予胰岛素 PBS溶液(50U/kg,ig)、GM4-CCT-insulin菌株组饲喂GM4-CCT-insulin菌株，每只每天饲喂量为1×1010CFU连续灌胃，胰岛素注射组皮下注射胰岛素PBS溶液(50U/kg，sc)，在0，1,2,4,6,9,12,h于小鼠尾部静脉取血20μL，待血液凝固后，低温，12000r/min转数下离心4min。精密量取上清液20μL，采用gop-pod法测定其血糖值。
6.4 实验结果
6.4.1 造模后小鼠情况
造模72h后，小鼠均无死亡，饮食、尿量、体重等均与实验前无明显差别。各用于造模的所有小鼠均有不同程度的活动减少，饮水量、尿量增多等表现。
6.4.2 血糖的检测
由图10可看出，对糖尿病小鼠灌胃胰岛素PBS溶液，血糖降低并不明显，可能与胃肠道中的蛋白酶、胰蛋白酶等消化酶对胰岛素降解消化有关。而皮下注射胰岛素PBS溶液，小鼠血糖浓度只能保持在较低水平2h，这是由于胰岛素直接注射进入体内时，半衰期较短。但在灌胃服用GM4-CCT-insulin菌株之后，小鼠血糖在1-4h内降低幅度明显，而后6h内基本保持在较低水平，由此可看出在灌胃服用GM4-CCT-insulin菌株之后，菌株进入体内并没有被完全破坏，而且能够有效地降低血糖，因此，GM4-CCT-insulin作为外源性胰岛素可以用来糖尿病的治疗，具有显著的降血糖的作用。

Claims (8)

  1. 一种重组粘红酵母携带外源多肽的活细胞脂质体及其应用，其特征在于，以经磷酸胞苷胆碱转移酶基因工程菌为载体，引入外源多肽被包裹进经磷酸胞苷胆碱转移酶基因工程的菌株体内。
  2. 根据权利要求1所述的一种重组粘红酵母携带外源多肽的活细胞脂质体及其应用，其特征在于,所述的工程菌是粘红酵母菌GM4。
  3. 根据权利要求1所述的一种重组粘红酵母携带外源多肽的活细胞脂质体及其应用，其特征在于,所述的磷酸胞苷胆碱转移酶(CCT)由酿酒酵母CCT基因分离扩增得到。
  4. 根据权利要求3所述的一重组粘红酵母携带外源多肽的活细胞脂质体及其应用，其特征在于，包括如下步骤：
    (1)抽提表达载体质粒；
    (2)强启动子PGK1基因和CCT基因片段连接；
    (3)PGK1-CCT片段与表达载体质粒进行双酶切反应后连接构建重组质粒pPGK1Z-rD-CCT。
  5. 根据权利要求1所述引入外源多肽的制备方法，其特征在于，包括如下步骤：
    (1)制备工程菌株GM4-CCT菌株的感受态细胞，
    (2)将经FITC修饰的外源多肽H22LP、H9、IFN-γ、αMSH及胰岛素分别溶解后与感受态细胞混合。
    (3)通过电击将外源多肽H22LP、H9、IFN-γ、αMSH及胰岛素转入感受态菌株体内。
  6. 根据权利要求1所述的一种重组粘红酵母携带外源多肽的活细胞脂质体及其应用，其特征在于，携带外源多肽的GM4-CCT菌株经口服途径在体内可达到有效的生物利用度和肠道吸收的缓控作用。
  7. 根据权利要求1所述的一种重组粘红酵母携带外源多肽的活细胞脂质体及其应用，其特征在于，用于外源亲脂类分子、多肽类、核酸类或化合物的药品的口服引入载体。
  8. 根据权利要求1所述的一种重组粘红酵母携带外源多肽的活细胞脂质体及其应用，其特征在于，用于外源亲脂类分子、多肽类、核酸类或化合物的药品或/和食品的应用。
PCT/CN2018/111320 2018-10-23 2018-10-23 重组磷酸胆碱胞苷转移酶的粘红酵母携带外源多肽的活细胞脂质体及其应用 WO2020082221A1 (zh)

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