WO2022227255A1 - 一种靶蛋白递送载体的制备方法 - Google Patents

一种靶蛋白递送载体的制备方法 Download PDF

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WO2022227255A1
WO2022227255A1 PCT/CN2021/102690 CN2021102690W WO2022227255A1 WO 2022227255 A1 WO2022227255 A1 WO 2022227255A1 CN 2021102690 W CN2021102690 W CN 2021102690W WO 2022227255 A1 WO2022227255 A1 WO 2022227255A1
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protein
light
cell
cells
cell membrane
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French (fr)
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刘晶
王亮
王佳一
沈丽明
康琳
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大连干细胞与精准医学创新研究院
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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    • 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
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    • C12N2740/00—Reverse transcribing RNA viruses
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    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention belongs to the technical field of cell engineering, and in particular relates to a preparation method of an efficient natural target protein delivery carrier.
  • Proteins are an important component of living organisms and play important functions in cells, including enzyme catalysis, signal transduction, and gene regulation, helping cells maintain a good balance between survival and programmed death. Intracellular delivery of functional proteins has important therapeutic implications in biological applications, including disease treatment, vaccination, and bioimaging. With the advancement of science and technology, protein drugs are an important part of the drugs launched every year, and are currently widely used to treat or relieve symptoms associated with many metabolic and tumor diseases.
  • the purpose of the present invention is to provide a preparation method for efficiently encapsulating a natural target protein delivery carrier, which is used to solve the problem of transporting protein drugs to the interior of cells through the cell membrane.
  • the host cells were transformed with light-sensitive proteins through genetic engineering. Based on the reversible binding properties of the corresponding light-sensitive proteins under specific light conditions, two corresponding light-sensitive proteins were constructed as fusion proteins with cell membrane-specific binding proteins and target proteins, respectively.
  • the host cell membrane is re-fused by physical extrusion to obtain cell membrane nanovesicles encapsulating the natural target protein derived from recombinant cells.
  • the delivery carrier can effectively deliver the natural target protein to the interior of the cell through the cell membrane.
  • the object of the present invention is to realize in the following ways:
  • a preparation method of a target protein delivery carrier comprising the following steps:
  • a recombinant cell by means of genetic engineering, the recombinant cell expresses a first fusion protein and a second fusion protein, the first fusion protein comprises a cell membrane specific binding protein and a first light-sensitive protein, and the second fusion protein comprising a target protein and a second photosensitizing protein, the first photosensitizing protein and the second photosensitizing protein can be reversibly combined under specific light irradiation conditions;
  • the cell membranes of the recombinant cells prepared in step (1) are re-fused by physical extrusion to obtain cell membrane nanovesicles.
  • the specific process of constructing the recombinant cell described in step (1) is to sequentially introduce the encoding gene of the first fusion protein and the encoding gene of the second fusion protein into the host cell by transformation or transfection .
  • the specific steps of the transfection include using a liposome or polymer transfection reagent to transfer the recombinant lentivirus carrying the encoding gene of the first fusion protein and the recombinant lentivirus carrying the second fusion protein.
  • the recombinant lentivirus encoding the gene is sequentially transfected into host cells.
  • the specific steps of the physical extrusion method in step (2) include passing the cell suspension of the reconstituted cells through an extruder of 10 ⁇ m, 5 ⁇ m and 1 ⁇ m in sequence under the irradiation of specific light.
  • a filter membrane with a pore size is repeatedly extruded for 5 to 20 times, and the obtained suspension is collected and centrifuged at 0 to 4°C and 1000 to 5000g for 5 to 30 minutes. After 30 min, the supernatant was discarded, and the obtained pellet was resuspended in buffer to obtain cell membrane nanovesicles.
  • the wavelength of the specific light is 460-650 nm.
  • the cell membrane-specific binding protein is selected from any one of CD9, CD63, CD81, CD82, apoptosis-inducing factor 6 interacting protein (ALIX) and tumor susceptibility gene 101 protein (TSG101). kind.
  • the photosensitive protein is selected from recombinant human calcium-integrin binding protein 1 (CIB), CIBN, phytochrome (PhyB), phytochrome interacting factor (PIF), photoperiod flowering time regulator (FKF1), any of clock protein (GIGANTEA), cryptochrome (CRY) and photorepair enzyme (PHR).
  • CIB human calcium-integrin binding protein 1
  • CIBN phytochrome
  • PPF phytochrome interacting factor
  • FKF1 photoperiod flowering time regulator
  • GAGANTEA any of clock protein
  • cryptochrome CRY
  • PHR photorepair enzyme
  • the first photosensitizing protein is CIB or CIBN
  • the second photosensitizing protein is CRY or PHR
  • the first photosensitizing protein and the second photosensitizing protein are sensitized by irradiating light with a wavelength of 460-490 nm. protein binding.
  • the first photosensitizing protein is PhyB
  • the second photosensitizing protein is PIF
  • the first photosensitizing protein and the second photosensitizing protein are combined by irradiating light with a wavelength of 600-650 nm.
  • the first light-sensitive protein is GIGANTEA
  • the second light-sensitive protein is FKF1
  • the first light-sensitive protein and the second light-sensitive protein are combined by irradiating light with a wavelength of 460-490 nm.
  • the host cell is selected from any one of 293T cells, B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, stem cells and tumor cells.
  • Another aspect of the present invention provides a cell membrane nanovesicle prepared by the above preparation method.
  • Another aspect of the present invention provides a protein pharmaceutical composition, which comprises the above-mentioned cell membrane nanovesicles, and utilizes the cell membrane nanovesicles to deliver a natural target protein to the interior of cells for action.
  • the preparation method of the engineered natural target protein delivery carrier provided by the present invention was first developed and proposed by the present inventors, which can efficiently prepare engineered cell membrane nanovesicles containing natural target proteins, and the target proteins exist in the carrier in free form , so it can be widely used in the treatment of diseases.
  • the cell membrane nanovesicles prepared by the present invention have the advantages of high loading efficiency, mass preparation, simple operation, reversible combination of target protein and carrier, and high delivery efficiency.
  • the present invention establishes a complete set of delivery systems for cell membrane transport of natural target proteins, and provides a new operation scheme for subsequent target proteins to act as therapeutic drugs in cells.
  • Fig. 1 is the flow chart of the preparation method of the engineered natural target protein delivery carrier of the present invention
  • Figure 2 is the map of the lentiviral expression vector encoding two fusion proteins according to Example 1 of the present invention
  • Figure 3 is a diagram showing the identification of recombinant cell virus transfection by flow cytometry described in Example 1 of the present invention
  • Figure 4 is a graph showing the binding of intracellular light-sensitive proteins in the recombinant cells described in Example 1 of the present invention under specific illumination conditions, wherein 1: CD9-EGFP-CIBN fusion protein, 2: mcherry-CRY2 fusion protein, 3: 488nm wavelength light After excitation, the mcherry-CRY2 fusion protein aggregated and distributed to the cell membrane, 4: The overlapping distribution position of the CD9-EGFP-CIBN fusion protein and the mcherry-CRY2 fusion protein;
  • FIG. 5 is a layout diagram of an engineered carrier prepared in a method for preparing an engineered natural target protein delivery carrier of the present invention
  • Example 6 is a transmission electron microscope image of the cell membrane nanovesicles encapsulating the target protein prepared in Example 1;
  • Example 7 is a particle size distribution diagram of nanovesicles coated with target protein cell membranes prepared by the preparation method described in Example 1;
  • Fig. 8 is a graph showing the efficiency of encapsulating target proteins prepared by cell membrane nanovesicles prepared under specific illumination conditions and without illumination by immunofluorescence protein detection;
  • FIG. 9 is a graph showing the efficiency of the cell membrane nanovesicles prepared in Example 2 for delivering target proteins into cells.
  • a fusion protein is used to co-express a tag protein, and the tag protein is used to confirm whether the host cell is successfully transformed.
  • green fluorescent protein is used as the first fusion protein tag protein
  • red fluorescent protein is used as the second fusion protein. Fusion protein-tagged proteins.
  • use the tag protein as the target protein to confirm the effect of the cell membrane nanovesicles prepared by recombinant cells to encapsulate the target protein, and confirm whether the cell membrane nanovesicles successfully encapsulate the target by confirming whether the red fluorescent protein tag is detected in the cell membrane nanovesicles. protein.
  • Example 1 Construction of an engineered delivery vector for mcherry protein
  • lentiviral vectors Two kinds of genes were constructed in lentiviral vectors, and the lentiviral expression vectors used were GV348 and CV084 (purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.), and one gene was CD9 gene, EGFP gene and CIBN gene in series, The vector is GV348; the other gene is the mcherry gene and CRY2 gene in series, and the vector is CV084;
  • Carrier enzyme digestion prepare 50 ⁇ L enzyme digestion system. Add mixed reagents, mix by pipetting gently, centrifuge briefly, and place at 37°C to react for 3h or overnight. Agarose gel electrophoresis was performed on the product of the restriction enzyme digestion of the vector, and the target band was recovered.
  • Colony PCR identification prepare an identification system, shake and mix, and centrifuge briefly. In an ultra-clean workbench, use a sterile pipette tip to pick a single colony into a 20 ⁇ L identification system, mix by pipetting, and place it in a PCR machine for reaction.
  • Fig. 2 is two recombinant lentiviral expression vector maps
  • the gene sequence of CD9-EGFP-CIBN is shown in SEQ ID NO:1, wherein the gene sequence of CD9 is shown in SEQ ID NO:2, the gene sequence of EGFP is shown in SEQ ID NO:3, and the gene sequence of CIBN is shown in Shown in SEQ ID NO:4; the gene sequence of mcherry-cry2 is shown in SEQ ID NO:5, wherein, the gene sequence of mcherry is shown in SEQ ID NO:6, and the gene sequence of cry2 is shown in SEQ ID NO:7 .
  • 293T cells were co-transfected with plasmids.
  • Virus harvesting ie, unpurified cell supernatant
  • concentration and purification method were used to obtain a high-titer lentivirus preservation solution.
  • quality standards were used to determine various indicators of lentivirus. Lentiviral particles within a certain titer range can meet the needs of most in vitro and in vivo experiments. The process is as follows.
  • the mixture is slowly added dropwise to the culture medium of 293T cells.
  • the addition process must be uniform, and the cells should not be blown up as much as possible. Mix well and culture in a 37°C, 5% CO 2 cell incubator;
  • the quality control points of lentivirus include physical state testing, sterility testing and virus titer testing.
  • the lentivirus can integrate the 5'LTR-3'LTR region of the virus into the host genome for stable expression, infect the tool cell 293T with the virus, and use an absolute quantitative method to detect the virus characteristic single-copy gene A and host characteristics in the genome of the tool cell 293T.
  • single-copy gene B Calculate the average number of infected virus particles in each cell, multiply by the number of cells per well, and divide by the amount of infection to obtain the titer of the virus sample.
  • N the number of cells in the corresponding well of the 24-well plate at the time of infection
  • V volume of infected lentivirus (mL) in the corresponding well.
  • Standard preparation construct a plasmid standard A containing the conserved sequence a of the lentiviral genome and a plasmid standard B containing a single copy gene b in the 293T genome of the tool cell; the concentration is 1 ⁇ 10 10 copy/ ⁇ L, after packaging -80 °C for long-term storage.
  • Primer design and preparation qPCR primers were designed for plasmid standard A and B, respectively, and configured into a 10 ⁇ M primer working solution.
  • 293T cells were cultured in a 24-well plate at a density of 5 ⁇ 10 4 cells/well.
  • MOI multiplicity of infection
  • a cell suspension with a density of 1 ⁇ 10 5 cells/mL was prepared using a complete medium, and 4 ⁇ 10 4 cells/mL were inoculated into each well of a six-well plate.
  • the virus transfection is identified by flow cytometry, and the results are shown in Figure 3.
  • the results show that the two fusion proteins, CD9-EGFP-CIBN and mcherry-CRY2, were successfully expressed in cells by flow cytometry identification. .
  • the recombinant 293T cells were cultured in a confocal small dish, and after 24 hours of adherence, they were placed under a confocal microscope to take pictures. Under specific light (488nm wavelength), the 488nm excitation light was used to excite and take pictures to observe the red target protein and green Aggregation at the edge of the cell membrane, and dynamic observation. In the absence of specific light (460nm wavelength), the dissociation of the red target protein from the green cell membrane was recorded and analyzed.
  • Figure 4 shows the intracellular light-sensitive protein binding of recombinant 293T cells under specific light.
  • the results show that under specific light, CD9-EGFP-CIBN fusion protein and mcherry-CRY2 fusion protein are combined together.
  • the main reason is that the light-sensitive proteins CIBN and CRY2 photosensitive protein has the property of reversible binding under specific light conditions.
  • the transfected stable recombinant 293T cells were expanded and cultured (using DMEM medium containing 10% FBS, in 5% CO 2 , at 37°C), scraped off the cells with a cell scraper, and centrifuged to collect 1 ⁇ 10 7 recombinant cells with 1 mL
  • the cell suspension was successively passed through an acetate membrane (Whatman, 110615, 110613, 800319) with 10 ⁇ m, 5 ⁇ m and 1 ⁇ m pore size with an extruder (Sigma, 610000-1EA). Repeat 8 to 12 times.
  • FIG. 5 is a layout diagram of preparing cell membrane nanovesicles in the preparation method of the engineered natural target protein delivery carrier of the present invention.
  • the morphology of the prepared cell membrane nanovesicles was detected by transmission electron microscope. 10 ⁇ L of the above cell membrane nanovesicle suspension was ultrasonicated for 20 min, fixed with an equal volume of 4% paraformaldehyde for 30 min at room temperature, settled on a copper mesh, and stained with phosphotungstic acid for 5 min. After that, the excess liquid was removed and dried, and the morphology of the cell membrane nanovesicles was collected and observed by TEM.
  • Figure 6 shows that the cell membrane nanovesicles are all lipid bilayer structures observed by TEM, which are round or cup-shaped.
  • the diameter distribution and quantity of the prepared cell membrane nanovesicles were detected by nanopaticle tracking analysis (NTA). 1 mL of cell membrane nanovesicles were taken and pushed into NTA using a syringe. particle size and quantity.
  • Figure 7 shows that the average particle size of the cell membrane nanovesicles is 182.4 nm. It is calculated that 1.2 ⁇ 10 7 cells can produce 2.61 ⁇ 10 11 cell membrane nanovesicles, and a single cell can produce 21750 cell membrane nanovesicles, indicating that the method for preparing cell membrane nanovesicles of the present invention has high yield and is suitable for industrial production.
  • the invention uses immunofluorescence protein detection to detect the protein encapsulation efficiency of the prepared cell membrane nanovesicles.
  • the ratio of mcherry expression to egfp expression was quantitatively analyzed by fluorescence quantitative analysis of cell membrane nanovesicles under specific light conditions and under no light conditions, and the efficiency of cell membrane nanovesicles encapsulating mcherry under specific light conditions was judged. The results are shown in Figure 8.
  • Embodiment 2 Utilize the cell membrane nanovesicle prepared by the present invention to deliver mcherry protein into cells
  • Example 1 In order to investigate the efficiency of the cell membrane nanovesicles prepared in Example 1 in delivering target proteins to target cells, 293T cells were treated with 5 ⁇ 10 9 delivery vectors wrapped with mcherry for 24 hours, and the fluorescence intensity in 293T cells was measured, and the results are shown in the figure 9 shown.
  • Figure 9 shows that the cell membrane nanovesicles prepared by the present invention can efficiently deliver the target protein into cells.

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Abstract

提供了一种高效包裹天然靶蛋白递送载体的制备方法，属于细胞工程技术领域。通过基因工程对宿主细胞进行光敏蛋白质改造，将两种对应的光敏蛋白分别与细胞膜特异性结合蛋白和靶蛋白构建为融合蛋白，在特定光照下利用物理挤压方式使宿主细胞膜再融合，获取重组细胞来源的包裹天然靶蛋白的细胞膜纳米囊泡，该递送载体能够将天然靶蛋白通过细胞膜有效递送至细胞内部。细胞膜纳米囊泡具有装载效率高、可大批量制备、操作简单、递送效率高等优点，为后续靶蛋白作为治疗药物在细胞内部发挥作用提供了新方法。

Description

一种靶蛋白递送载体的制备方法 技术领域
本发明属于细胞工程技术领域，具体涉及一种高效天然靶蛋白递送载体的制备方法。
背景技术
蛋白质是生命体的重要组成成分，在细胞内发挥重要功能，包括酶催化、信号转导和基因调控等，帮助细胞在存活和程序性死亡之间保持良好的平衡。向细胞内递送功能蛋白在生物学应用中具有重要的治疗意义，包括疾病治疗、疫苗接种和生物成像。随着科技的进步，蛋白质类药物是每年上市的药物的重要组成部分，目前广泛用于治疗或缓解与许多代谢和肿瘤疾病相关的症状。
尽管有大量的蛋白药物可以用于临床疾病治疗，但是由于功能蛋白进入细胞内的过程限制，导致蛋白药物(包括细胞因子，激素和单克隆抗体等)研发仍主要集中在胞外。近年来，随着细胞内蛋白药物功能被不断发现，设计一种稳定有效的递送载体以便将这些药物输送到靶细胞内部与药物本身一样重要。因此，亟须开发一种高效将靶蛋白递送至细胞内的载体系统。
发明内容
鉴于此，本发明的目的是提供一种高效包裹天然靶蛋白递送载体的制备方法，用于解决通过细胞膜向细胞内部运送蛋白药物的问题。通过基因工程对宿主细胞进行光敏蛋白质改造，基于对应的光敏蛋白在特定光照条件下可逆结合特性，将两种对应的光敏蛋白分别与细胞膜特异性结合蛋白和靶蛋白构建为融合蛋白，在特定光照下利用物理挤压方式使宿主细胞膜再融合，获取重组细胞来源的包裹天然靶蛋白的细胞膜纳米囊泡，该递送载体能够将天然靶蛋白通过细胞膜有效递送至细胞内部。
本发明目的是通过以下方式实现：
一种靶蛋白递送载体的制备方法，包括以下步骤：
(1)通过基因工程手段构建重组细胞，所述重组细胞表达第一融合蛋白和第二融合蛋白，所述第一融合蛋白包含细胞膜特异性结合蛋白和第一光敏蛋白，所述第二融合蛋白包含靶蛋白与第二光敏蛋白，所述第一光敏蛋白和第二光敏蛋白在特定光的照射条件下能够可逆结合；
(2)在特定光的照射条件下，通过物理挤压方式使步骤(1)制备的重组细胞的 细胞膜再融合，获得细胞膜纳米囊泡。
基于以上技术方案，进一步地，步骤(1)所述构建重组细胞的具体过程为通过转化或转染方式向宿主细胞内依次导入所述第一融合蛋白的编码基因和第二融合蛋白的编码基因。
基于以上技术方案，进一步地，所述转染的具体步骤包括通过脂质体或聚合物转染试剂将携带所述第一融合蛋白的编码基因的重组慢病毒和携带所述第二融合蛋白的编码基因的重组慢病毒依次转染到宿主细胞内。
基于以上技术方案，进一步地，步骤(2)中所述物理挤压方式的具体步骤包括将所述重组细胞的细胞悬液在特定光的照射下，通过挤出器依次通过10μm、5μm、1μm孔径尺寸的滤膜，反复挤出5～20次，收集所得混悬液，于0～4℃，1000～5000g离心5～30min，取上清，于0～4℃，10000～30000g离心5～30min，弃上清，所得沉淀用缓冲液重悬得到细胞膜纳米囊泡。
基于以上技术方案，进一步地，所述特定光的波长为460～650nm。
基于以上技术方案，进一步地，所述细胞膜特异性结合蛋白选自CD9，CD63，CD81，CD82，调亡诱导因子6相互作用蛋白(ALIX)和肿瘤易感基因101蛋白(TSG101)中的任意一种。
基于以上技术方案，进一步地，所述光敏蛋白选自重组人钙-整合素结合蛋白1(CIB)、CIBN、光敏色素(PhyB)、光敏色素互作因子(PIF)、光周期开花时间调节剂(FKF1)、时钟蛋白(GIGANTEA)、隐花色素(CRY)和光修复酶(PHR)中的任意一种。
基于以上技术方案，进一步地，所述第一光敏蛋白为CIB或CIBN，所述第二光敏蛋白为CRY或PHR，通过照射波长为460～490nm的光使所述第一光敏蛋白和第二光敏蛋白结合。
基于以上技术方案，进一步地，所述第一光敏蛋白为PhyB，所述第二光敏蛋白为PIF，通过照射波长为600～650nm的光来使所述第一光敏蛋白和第二光敏蛋白结合。
基于以上技术方案，进一步地，所述第一光敏蛋白为GIGANTEA，所述第二光敏蛋白为FKF1，通过照射波长为460～490nm的光来使所述第一光敏蛋白和第二光敏蛋白结合。
基于以上技术方案，进一步地，所述宿主细胞选自293T细胞、B-淋巴细胞、T-淋巴细胞、树突细胞、巨核细胞、巨噬细胞、干细胞及肿瘤细胞中的任意一种。
本发明另一方面提供一种细胞膜纳米囊泡，所述细胞膜纳米囊泡是由上述制备方法制得。
本发明另一方面提供一种蛋白质药物组合物，所述药物组合物包含上述细胞膜纳米囊泡，并利用细胞膜纳米囊泡将天然靶蛋白递送至细胞内部发挥作用。
本发明相对于现有技术具有的有益效果如下：
1.本发明提供的工程化天然靶蛋白递送载体的制备方法是本发明人首先开发并提出的，能够高效制备包含天然靶蛋白的工程化细胞膜纳米囊泡，靶蛋白在载体内以游离形式存在，因此可以广泛应用于疾病的治疗。
2.本发明制备的细胞膜纳米囊泡具有装载效率高、可大批量制备、操作简单、靶蛋白与载体可逆性结合、递送效率高等优点。
3.本发明建立了一整套细胞膜运输天然靶蛋白的递送体系，为后续靶蛋白作为治疗药物在细胞内部发挥作用提供了新的操作方案。
附图说明
为了更清楚地说明本发明实施例，下面将对实施例涉及的附图进行简单地介绍。
图1为本发明工程化天然靶蛋白递送载体的制备方法流程图；
图2为本发明实施例1所述编码两种融合蛋白的慢病毒表达载体图谱；
图3为本发明实施例1所述流式细胞术鉴定重组细胞病毒转染情况图；
图4为本发明实施例1所述重组细胞在特定光照情况下胞内光敏蛋白结合情况图，其中，1：CD9-EGFP-CIBN融合蛋白，2：mcherry-CRY2融合蛋白，3：488nm波长光激发后，mcherry-CRY2融合蛋白向细胞膜处聚集分布，4：CD9-EGFP-CIBN融合蛋白和mcherry-CRY2融合蛋白重叠分布位置；
图5为本发明工程化天然靶蛋白递送载体的制备方法中制备工程化载体布局图；
图6为实施例1制备的包裹靶蛋白细胞膜纳米囊泡透射电镜图；
图7为实施例1所述制备方法制得的包裹靶蛋白细胞膜纳米囊泡粒径分布图；
图8为免疫荧光蛋白检测在特定光照条件下和不照光情况下制备的细胞膜纳米囊泡包裹靶蛋白效率图；
图9为实施例2制备的细胞膜纳米囊泡向细胞内传递靶蛋白效率图。
具体实施方式
下面结合实施例对本发明进行详细的说明，但本发明的实施方式不限于此，显而易见地，下面描述中的实施例仅是本发明的部分实施例，对于本领域技术人员来讲，在不付出创造性劳动性的前提下，获得其他的类似的实施例均落入本发明的保护范围。
为确认宿主细胞是否改造成功，使用融合蛋白共同表达标签蛋白，所述标签蛋白是为了确认宿主细胞是否改造成功而使用，例如使用绿色荧光蛋白作为第一融合蛋白标签蛋白，红色荧光蛋白作为第二融合蛋白标签蛋白。同样，使用标签蛋白作为靶蛋白来确认重组细胞制备的细胞膜纳米囊泡包裹靶蛋白效果，通过确认细胞膜纳米囊泡中是否检测到红色荧光蛋白标签，来确认所述细胞膜纳米囊泡是否成功包裹靶蛋白。
实施例1:构建mcherry蛋白的工程化递送载体
1.1重组慢病毒表达载体的构建
将两种基因构建于慢病毒载体中，使用的慢病毒表达载体为GV348和CV084(购买于上海吉凯基因化学技术有限公司)，一种基因为依次串联的CD9基因、EGFP基因和CIBN基因，载体为GV348；另一种基因为依次串联的mcherry基因和CRY2基因，载体为CV084；
具体步骤如下：
1.利用限制性内切酶消化获得线性化载体
(1)载体酶切：配制50μL酶切体系。加入混合试剂，用移液器轻轻吹打混匀，短暂离心，置于37℃反应3h或过夜。对载体酶切产物进行琼脂糖凝胶电泳，回收目的条带。
(2)目的基因片段的获取：配制反应体系，轻轻吹打混匀，短暂离心，置于PCR仪中进行反应。
(3)PCR产物与载体进行交换：于冰水浴中配制PCR反应体系，用移液器轻轻吹打混匀，短暂离心，避免产生气泡，于37℃反应30min，随后置于冰水浴中冷却5min后立即转化。
(4)转化：将10μL交换反应产物加入到100μL感受态细胞中，轻弹管壁数下混匀，在冰上放置30min，42℃热激90s，冰水浴孵育2min，加入500μL LB培养基，置于37℃摇床振荡培养1h。取适量菌液均匀涂布在含有相应抗生素的平板上，在恒温培养箱中倒置培养12-16h。
(5)菌落PCR鉴定：配制鉴定体系，震荡混匀，短暂离心。在超净工作台中， 用无菌的枪头挑取单个菌落至20μL鉴定体系中，吹打混匀，置于PCR仪中进行反应。
(6)测序：将鉴定出的阳性克隆转化子接种于适量含相应抗生素的LB液体培养基中，37℃培养12-16h，取适量菌液进行测序。对测序结果与目的基因序列进行比对分析。比对结果说明：测序结果与目标序列完全一致。
图2为两种重组慢病毒表达载体图谱；
CD9-EGFP-CIBN的基因序列如SEQ ID NO:1所示，其中，CD9的基因序列如SEQ ID NO:2所示，EGFP的基因序列如SEQ ID NO:3所示，CIBN的基因序列如SEQ ID NO:4所示；mcherry-cry2的基因序列如SEQ ID NO:5所示，其中，mcherry的基因序列如SEQ ID NO:6所示，cry2的基因序列如SEQ ID NO:7所示。
2.质粒抽提
将测序正确的菌液转接于10mL含相应抗生素的LB液体培养基中，37℃培养过夜，用天根无内毒素质粒小提中量试剂盒进行质粒抽提，抽提合格的质粒进入下游流程。详细操作步骤如下：
(1)收集过夜培养的菌液于标记好的5mL离心管，12000rpm，离心2min收菌；
(2)弃上清，加入250μL细胞重悬液，充分振荡，使菌块悬浮均匀；
(3)加入250μL细胞裂解液，再加入10μL蛋白酶K，上下颠倒5-6次，轻轻混匀；静置1-2min，致使菌体裂解澄清；
(4)加入350μL中和液，上下颠倒混匀，使蛋白完全析出，冰浴静置5min；
(5)10000rpm离心10min，弃蛋白，收集上清于另一干净无菌的1.5mL EP管；
(6)12000rpm离心5min，同时准备标记好的回收柱，将上清转秱至回收柱中，12000rpm离心1min，弃下层废液；
(7)加入600μL预先配置好的漂洗液，12000rpm离心1min，弃下层废液，重复一次，12000rpm空离2min，进一步除去残留的漂洗液；
(8)在超净台中将回收柱转移至新的1.5mL EP管中，静置10-20min，自然晾干；
(9)往回收柱中加入95μL Nuclease-Free Water，静置2min，12000rpm离心2min，收集样品做好编号，电泳、测定浓度，进行质检。
3.质粒转染与慢病毒收获
采用质粒共转染293T细胞。在转染完成后的48-72h进行病毒收获(即未纯化的 细胞上清液)，根据不同的实验需求，确定采用相应的浓缩纯化方式得到高滴度的慢病毒保存液，最后根据严格的质量标准测定慢病毒的各项指标。在一定滴度范围内的慢病毒颗粒可以满足大部分体内外实验需求，流程如下。
(1)转染前24h，用胰蛋白酶消化对数生长期的293T细胞，以含10％血清的培养基调整细胞密度约5 x 10 6细胞/15mL，重新接种于10cm细胞培养皿，37℃、5％CO 2培养箱内培养。24h待细胞密度达70％～80％时即可用于转染；
(2)转染前2h更换为无血清培养基；
(3)向一支灭菌离心管中加入所制备的各DNA溶液(GV348载体质粒20μg、pHelper1.0载体质粒15μg、pHelper 2.0载体质粒10μg)，与相应体积的Fugene6转染试剂(Boehringer Mannheim)混合均匀，调整总体积为1mL，在室温下温育15min；
(4)混合液缓慢滴加至293T细胞的培养液中，加入过程一定要均匀，尽可能地不要将细胞吹起，混匀，于37℃、5％CO 2细胞培养箱中培养；
(5)培养6h后弃去含有转染混合物的培养基，加入10mL的PBS液清洗一次，轻柔晃动培养皿以洗涤残余的转染混合物后倒弃；
(6)缓慢加入含10％血清的细胞培养基20mL，于37℃、含5％CO 2培养箱内继续培养48-72h。
4.慢病毒浓缩与纯化
(1)根据细胞状态，收集转染后48h(转染即0h计起)的293T细胞上清液；
(2)于4℃，4000g离心10min，除去细胞碎片；
(3)以0.45μm滤器过滤上清液于40mL超速离心管中；
(4)分别配平样品，将带有病毒上清液的超速离心管逐一放入至Beckman超速离心机内,设置离心参数为25000rpm，离心时间为2h，离心温度控制在4℃；
(5)离心结束后，弃去上清，尽量去除残留在管壁上的液体，加入病毒保存液(可用PBS或细胞培养基替代)，轻轻反复吹打重悬；
(6)经充分溶解后，高速离心10000rpm，离心5min后，取上清按要求分装；
(7)准备样品待检测。
5.慢病毒质量检测
慢病毒的质量控制要点包括物理状态检测、无菌检测及病毒滴度检测。
(1)物理指标检测
1)颜色判定：通过肉眼判定，慢病毒保存液呈粉红色澄清液体状；
2)粘稠度判定：用20-200μL规格移液器缓慢吸取50μL慢病毒保存液体，无明显粘稠感或吸液滞后现象；
(2)无菌检测：将病毒加入293T细胞验证，正常培养24h后镜检，无任何细菌及真菌污染情况，同时参照空细胞组，细胞间隙无明显颗粒存在，培养基澄清透明。
(3)滴度检测及分析：绝对定量qPCR法
慢病毒可以将病毒的5’LTR—3’LTR区整合入宿主基因组中稳定表达，通过病毒感染工具细胞293T，用绝对定量方法检测出工具细胞293T基因组中的病毒特征单拷贝基因A和宿主特征单拷贝基因B。计算出每个细胞中平均感染病毒颗粒数，再乘以每孔的细胞个数，除以感染量，即可得出病毒样品的滴度。
计算公式：
qPCR滴度(TU/mL)＝N*C/V
N＝感染时24孔板中对应孔的细胞数量；
C(每个细胞中含有的慢病毒个数)＝(A拷贝数/B拷贝数)*2；
V＝对应孔中感染的慢病毒体积(mL)。
实验步骤：
1)标准品制备：构建含有慢病毒基因组保守序列a的质粒标准品A和含有工具细胞293T基因组内单拷贝基因b的质粒标准品B；浓度1×10 10copy/μL，分装后-80℃长久保存。
2)引物设计和制备：分别设计针对质粒标准品A和B设计qPCR引物，并配置成10μM的引物工作液。
3)样品制备：
a)感染前24h，在24孔板中培养293T细胞，密度为5×10 4cell/孔。
b)感染时，收集2-3孔空细胞，分别计数每孔内感染时细胞总数N，每孔感染病毒体积V mL，每个病毒感染3个复孔。
c)感染后24h，每孔加入1000μL完全培养基，小心操作，不要吹起细胞。
d)感染后72h，吸去上清，荧光拍照；同时分别收集孔内细胞。
e)用天根《细胞、血液基因组提取试剂盒》提取收集细胞的基因组。
4)标准品稀释：
10倍梯度稀释法，以10 9、10 8、10 7、10 6、10 5、10 4、10 3、10 2、10 1梯度稀释质量标准品A和B，以及待测样品。
5)配制PCR反应体系。
6)PCR反应：
设定程序为两步法Real-Time定量。预变性95℃，15s，之后每一步变性95℃，5s，退火延伸60℃，30s，共进行40个循环。每次在延伸阶段读取吸光值。PCR结束后，制作溶解曲线，在95℃变性1min，然后冷却至60℃，1min，使DNA双链充分结合。从60℃开始，每步增加0.5℃，保持30s，同时读取吸光值。
7)滴度结果
根据计算公式：qPCR滴度(TU/mL)＝N*C/V，计算样品的平均滴度。
1.2重组细胞的构建
使用含10％FBS的DMEM培养基培养293T细胞，然后以感染复数(MOI)＝5的病毒滴度依次添加上述方法制得的两种慢病毒感染293T细胞，从而构建得到CD9-EGFP-CIBN和mcherry-CRY2过表达的重组细胞。由于病毒载体具有抗性，经过含有puromycin和neomycin的抗性培养基筛选高表达的重组293T细胞。
具体步骤如下：
(1)使用完全培养基基制备密度为1×10 5个/mL细胞悬液，以4×10 4个/mL接种至六孔板的每个孔中。
(2)待细胞贴壁后，对细胞进行换液，根据细胞MOI＝5加入相应数量CD9-EGFP-CIBN病毒。37℃培养12-16h，更换完全培养基继续培养。
(3)感染后约72小时，观察感染效率。感染效率80％为最佳的感染效率。如感染效率尚未达到预期，使用2μg/mL的嘌呤霉素进行筛选。
(4)选择生长良好、感染效率高的细胞进行下一步实验。
(5)使用相同方法对上述稳转株使用相同方法进行mcherry-CRY2病毒转染，同时使用新霉素进行筛选。
本发明通过流式细胞术进行鉴定病毒转染情况，结果如图3所示，结果表明，经流式细胞术鉴定，CD9-EGFP-CIBN和mcherry-CRY2两种融合蛋白均在细胞中成功表达。
1.3重组细胞内部mcherry蛋白在特定光照下在细胞膜周围富集的确认
将重组293T细胞在共聚焦小皿中培养，贴壁24h后，放置于共聚焦显微镜下进行拍照，在特定光照(488nm波长)情况下，利用488nm激发光进行激发并拍摄，观察红色靶蛋白和绿色细胞膜边缘的聚集情况，并进行动态观察。在无特定光照(460nm 波长)情况下，红色靶蛋白与绿色细胞膜的解离情况，并记录分析。
图4为重组293T细胞在特定光照下胞内光敏蛋白结合情况图，结果表明，在特定光照下，CD9-EGFP-CIBN融合蛋白和mcherry-CRY2融合蛋白结合在一起，主要原因是光敏蛋白CIBN和CRY2光敏蛋白在特定光照条件下存在可逆性结合的特性。
1.4富含mcherry蛋白的工程化细胞膜纳米囊泡的制备
将转染稳定的重组293T细胞进行扩大培养(使用含有10％FBS的DMEM培养基，在5％CO 2，37℃培养)，用细胞刮刀将细胞刮下，离心收集1x10 7个重组细胞用1mL PBS重悬后，在460nm波长的光照射下，用挤出器(Sigma，610000-1EA)将细胞悬液依次通过10μm、5μm、1μm孔径尺寸的醋酸膜(Whatman，110615，110613，800319)，反复挤出8～12次。收集挤膜完成的混悬液，于4℃，3000g离心15min去除细胞碎片，取上清，于4℃，20000g离心15min，弃上清，沉淀用200-500μL PBS重悬得到包裹mcherry蛋白的细胞膜纳米囊泡；全程置于冰上或者低温环境中，防止蛋白变性。
图5为本发明工程化天然靶蛋白递送载体的制备方法中制备细胞膜纳米囊泡的布局图。
利用透射电镜对制备的细胞膜纳米囊泡进行形态学检测，取上述细胞膜纳米囊泡悬液10μL超声20min，用等体积4％多聚甲醛室温固定30min，沉降在铜网上，用磷钨酸染色5min后，除去多余的液体并干燥，通过TEM采集并观察细胞膜纳米囊泡的形貌。
图6为TEM观察到细胞膜纳米囊泡均为脂质双分子层结构，呈圆形或杯口形。
利用纳米颗粒跟踪分析(nanopaticle tracking analysis，NTA)对制备的细胞膜纳米囊泡的直径分布和数量进行检测，取1mL细胞膜纳米囊泡，使用针筒注射器推入NTA，同时检测的细胞膜纳米囊泡的粒径和数量。
图7结果显示，细胞膜纳米囊泡的平均粒径为182.4nm。经计算，1.2×10 7个细胞制备出2.61×10 11个细胞膜纳米囊泡，单个细胞能够制备21750个细胞膜纳米囊泡，表明本发明制备细胞膜纳米囊泡的方法产率高，适宜工业化生产。
本发明利用免疫荧光蛋白检测对制备的细胞膜纳米囊泡进行包裹蛋白效率的检测。通过分别对在特定光照条件下与不照光条件下的细胞膜纳米囊泡进行荧光定量分析mcherry表达量与egfp表达量的比值，判断在特定光照条件下细胞膜纳米囊泡包裹mcherry的效率。结果如图8所示。
图8结果表明：在蓝光照射条件下制备的细胞膜纳米囊泡包裹mcherry的效率明 显高于未照光条件的对照组。
实施例2:利用本发明制备的细胞膜纳米囊泡向细胞内递送mcherry蛋白
为了考察实施例1制备的细胞膜纳米囊泡向靶细胞递送靶蛋白的效率，使用5x10 9个包裹mcherry的递送载体对293T细胞处理24h，对293T细胞中的荧光强度进行了测定，其结果如图9所示。
图9结果显示，本发明制备的细胞膜纳米囊泡能够将靶蛋白高效地递送到细胞内。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Figure PCTCN2021102690-appb-000001
Figure PCTCN2021102690-appb-000002
Figure PCTCN2021102690-appb-000003
Figure PCTCN2021102690-appb-000004
Figure PCTCN2021102690-appb-000005
Figure PCTCN2021102690-appb-000006

Claims (10)

  1. 一种靶蛋白递送载体的制备方法，其特征在于，包括以下步骤：
    (1)通过基因工程手段构建重组细胞，所述重组细胞表达第一融合蛋白和第二融合蛋白，所述第一融合蛋白包含细胞膜特异性结合蛋白和第一光敏蛋白，所述第二融合蛋白包含靶蛋白与第二光敏蛋白，所述第一光敏蛋白和第二光敏蛋白在特定光的照射条件下能够可逆结合；
    (2)在特定光的照射条件下，通过物理挤压方式使步骤(1)制备的重组细胞的细胞膜再融合，获得细胞膜纳米囊泡。
  2. 根据权利要求1所述的制备方法，其特征在于，步骤(1)中所述构建重组细胞的具体过程为通过转化或转染方式向宿主细胞内依次导入所述第一融合蛋白的编码基因和第二融合蛋白的编码基因。
  3. 根据权利要求2所述的制备方法，其特征在于，所述转染的具体步骤包括通过脂质体或聚合物转染试剂将携带所述第一融合蛋白的编码基因的重组慢病毒和携带所述第二融合蛋白的编码基因的重组慢病毒依次转染到宿主细胞内。
  4. 根据权利要求1所述的制备方法，其特征在于，步骤(2)中所述物理挤压方式的具体步骤包括将所述重组细胞的细胞悬液在特定光的照射下，通过挤出器依次通过10μm、5μm、1μm孔径的滤膜，反复挤出5～20次，收集所得混悬液，于0～4℃，1000～5000g离心5～30min，取上清，于0～4℃，10000～30000g离心5～30min，弃上清，所得沉淀用缓冲液重悬得到细胞膜纳米囊泡。
  5. 根据权利要求1所述的制备方法，其特征在于，所述特定光的波长为460～650nm。
  6. 根据权利要求1-5任一项所述的制备方法，其特征在于，所述细胞膜特异性结合蛋白选自CD9、CD63、CD81、CD82、ALIX和TSG101中的任意一种；所述光敏蛋白选自CIB、CIBN、PhyB、PIF、FKF1、GIGANTEA、CRY和PHR中的任意一种；所述宿主细胞选自293T细胞、B-淋巴细胞、T-淋巴细胞、树突细胞、巨核细胞、巨噬细胞、干细胞和肿瘤细胞中的任意一种。
  7. 根据权利要求6所述的制备方法，其特征在于，所述第一光敏蛋白为CIB或CIBN，所述第二光敏蛋白为CRY或PHR，通过照射波长为460～490nm的光使所述第一光敏蛋白和第二光敏蛋白结合。
  8. 根据权利要求6所述的制备方法，其特征在于，所述第一光敏蛋白为PhyB，所述第二光敏蛋白为PIF，通过照射波长为600～650nm的光来使所述第一光敏蛋白和 第二光敏蛋白结合；
    或者，所述第一光敏蛋白为GIGANTEA，所述第二光敏蛋白为FKF1，通过照射波长为460～490nm的光来使所述第一光敏蛋白和第二光敏蛋白结合。
  9. 一种细胞膜纳米囊泡，其特征在于，由权利要求1-8任一项所述的制备方法制得。
  10. 一种药物组合物，其特征在于，所述药物组合物包含权利要求9所述的细胞膜纳米囊泡。
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