WO2015106584A1 - Tat-il-24-kdel融合蛋白及其制备方法和应用 - Google Patents

Tat-il-24-kdel融合蛋白及其制备方法和应用 Download PDF

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WO2015106584A1
WO2015106584A1 PCT/CN2014/087669 CN2014087669W WO2015106584A1 WO 2015106584 A1 WO2015106584 A1 WO 2015106584A1 CN 2014087669 W CN2014087669 W CN 2014087669W WO 2015106584 A1 WO2015106584 A1 WO 2015106584A1
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tat
fusion protein
kdel
sumo
cells
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孙爱友
魏东芝
张舰
董玉国
徐瑞
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华东理工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

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  • the invention relates to bioengineering, in particular to a TAT-IL-24-KDEL fusion protein and a preparation method and application thereof.
  • IL-24 has the ability to selectively inhibit a variety of tumor growth and induce tumor cell proliferation, and this inhibition is independent of the status of tumor suppressor genes such as p53, Rb and p16, and Normal cells have no effect.
  • MDA-7/IL-24 has a broad spectrum of action and can induce tumor cells including melanoma, glioblastoma, osteosarcoma, breast cancer, cervical cancer, colon cancer, lung cancer, nasopharyngeal cancer and prostate cancer. Growth inhibition and apoptosis.
  • IL-24 not only plays an important role in activating immune cells, regulating the whole anti-cancer immune response and hematopoietic system, but also selectively induces tumor cell apoptosis and directly inhibits the proliferation and metastasis of tumor cells. Anti-tumor properties make it a new hotspot in cancer therapy research.
  • MDA-7/IL-24 phosphorylates STAT3 by activating the JAK/STAT signaling pathway by binding to the receptor complex IL-20R1/IL-20R2 and IL-22R1/IL-20R2 on the surface of tumor cell membranes. Regulation of the expression of intracellular related genes plays a role in growth inhibition and pro-apoptosis.
  • the results of Sauane et al suggest that MDA-7/IL-24 also exerts a role in inducing tumor cell apoptosis through a non-JAK/STAT-dependent pathway.
  • a large body of literature recently demonstrated that MDA-7/IL-24 can inhibit tumor cell growth and apoptosis by regulating endoplasmic reticulum stress response and mitochondrial integrity. Aiming at the endoplasmic reticulum pathway of IL-24, the use of genetic engineering technology to prepare IL-24 protein in large quantities and its use in promoting tumor cell apoptosis has not been reported.
  • the object of the present invention is to provide a TAT-IL-24-KDEL fusion protein and to provide a preparation method of the TAT-IL-24-KDEL fusion protein to obtain a high concentration and high purity TAT-IL-24-KDEL. Fusion protein, while providing a TAT-IL-24-KDEL fusion protein for tumor therapy.
  • TAT-IL-24-KDEL fusion protein which fuses TAT transmembrane peptide and endoplasmic reticulum localization signal peptide KDEL to the N-terminus and C-terminus of IL-24 protein, respectively, to form a TAT-IL-24-KDEL fusion protein.
  • the amino acid sequence of the TAT transmembrane peptide is shown in SEQ ID NO.
  • the preparation method of the above TAT-IL-24-KDEL fusion protein comprises the following steps:
  • the SUMO-TAT-IL-24-KDEL fusion protein was heterologously expressed by E. coli BL21 (DE3);
  • step A The specific method of step A is to obtain the coding gene of SUMO tag and TAT-IL-24-KDEL fusion protein by PCR amplification method, and adopt the fusion PCR method to seamlessly encode the coding genes of SUMO and TAT-IL-24-KDEL. Ligation, construction of a recombinant plasmid using the pET28a(+) expression vector containing the T7 promoter.
  • E. coli BL21 (DE3) containing the recombinant plasmid pET28a(+)/SUMO-TAT-IL-24-KDEL was cultured at 37 ° C, and when the OD 600 reached 0.6, the final concentration was added.
  • 0.1 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) was induced at 28 ° C for 10 h to heterologously express the SUMO-TAT-IL-24-KDEL fusion protein, and the fusion protein was present in the form of inclusion bodies.
  • step C The specific method of step C is to separate and purify the SUMO-TAT-IL-24-KDEL fusion protein by using His-tag affinity chromatography under denaturing conditions. After protein renaturation, the SUMO tag is excised on the fusion protein by SUMO protease. The SUMO tag was removed again by His affinity chromatography, and the TAT-IL-24-KDEL fusion protein was isolated and purified.
  • step D is to detect the apoptosis effect of the purified TAT-IL-24-KDEL fusion protein on breast cancer MCF-7 cells by flow cytometry, and to detect the intracellular localization of the fusion protein by immunofluorescence assay.
  • the TAT-IL-24-KDEL fusion protein of the invention can effectively localize on the endoplasmic reticulum, thereby promoting apoptosis of breast cancer cell MCF-7.
  • upstream primer containing the TAT transmembrane peptide coding sequence 5'-CGCGGATCC TATGGCAGGAAGAAGCGTAGACAGAGACGTAGA GCCCAGGGCCAAGAGTTCC-3' (underlined TAT transmembrane peptide coding sequence) and downstream primer 5'- CCGCTCGAGTCA GAGCTCGTCCTT GAGCTTGTAGAATTTCTGC-3' (underlined endoplasmic reticulum
  • the localization signal peptide KDEL coding sequence is a reaction primer, and the full-length IL-24 gene is used as a template to obtain a gene encoding the TAT-IL-24-KDEL fusion protein by PCR amplification.
  • the PCR product was purified by gene purification kit, and the target gene and pET28a(+)/SUMO expression plasmid were digested with BamH I and Xho I, and T4 ligase was ligated to transform E.coli BL21(DE3). Positive clones were selected for sequencing.
  • the SUMO-TAT-IL-24-KDEL fusion protein was heterologously expressed by E. coli BL21 (DE3).
  • the correctly sequenced E. coli BL21 (DE3) containing the recombinant plasmid pET28a(+)/SUMO-TAT-IL-24-KDEL was cultured at 37 ° C. When the OD 600 reached 0.6, a final concentration of 0.1 mM was added.
  • IPTG Propyl- ⁇ -D-thiogalactopyranoside
  • the SUMO-TAT-IL-24-KDEL fusion protein was isolated and purified by His-tag affinity chromatography under denaturing conditions.
  • the SUMO tag was excised on the fusion protein with SUMO protease and the SUMO tag was removed again by His affinity chromatography to isolate and purify the TAT-IL-24-KDEL fusion protein.
  • TAT-IL-24-KDEL fusion protein In vitro detection of the pro-apoptotic effect of TAT-IL-24-KDEL fusion protein on breast cancer MCF-7 cells.
  • the activity of the purified TAT-IL-24-KDEL fusion protein was examined by flow cytometry.
  • the fusion proteins with final concentration of 0nM, 5nM, 10nM and 20nM were added to breast cancer MCF-7 cells. After 24h, the samples were treated with Annexin V-FITC/PI apoptotic double staining kit to detect the binding of fusion protein to MCF-7. Death effect.
  • Immunofluorescence technique was used to detect the effect of transmembrane peptide TAT and endoplasmic reticulum localization signal peptide KDEL on the localization of IL-24 in the fusion protein.
  • the IL-24 primary antibody and the PE-labeled secondary antibody were used to immunofluorescence the target protein, and the endoplasmic reticulum was stained with the endoplasmic reticulum staining kit, and then the two were overlapped to observe IL-24 in the cells. Positioning in the middle.
  • TAT transmembrane peptide of human immunodeficiency virus HIV-1 and the endoplasmic reticulum localization signal peptide KDEL were fused to the N-terminus and C-terminus of IL-24, respectively, to constitute a TAT-IL-24-KDEL fusion protein.
  • Construction of pET28a(+)/SUMO-TAT-IL-24-KDEL recombinant plasmid, heterologous expression, separation and purification, mass production Prepare TAT-IL-24-KDEL fusion protein.
  • the final TAT-IL-24-KDEL fusion protein was 0.6 g/L with a purity of 92%.
  • TAT transmembrane peptide of TAT-IL-24-KDEL fusion protein can transfer IL-24 protein into cells, and the endoplasmic reticulum localization signal peptide KDEL can localize IL-24 into the endoplasmic reticulum. It binds to its receptor GRP78/BiP.
  • the present invention detects the activity of the TAT-IL-24-KDEL fusion protein in vitro for the first time.
  • the results of flow cytometry and immunofluorescence experiments show that the fusion protein can efficiently localize to the endoplasmic reticulum and exert anti-tumor effect, which makes TAT-IL-24-KDEL fusion protein have great application prospects in tumor therapy.
  • Figure 1 is a construction process of a recombinant plasmid pET28a(+)/SUMO-TAT-IL-24-KDEL;
  • Figure 2A shows the expression of the SUMO-TAT-IL-24-KDEL fusion protein, which is mainly expressed in the form of inclusion bodies;
  • Figure 2B is a result of purification of the fusion protein by nickel column affinity chromatography under denaturing conditions
  • Figure 2C is the result of digestion of the fusion protein with SUMO protease
  • 2D is digested and purified by nickel column affinity chromatography to obtain the target protein TAT-IL-24-KDEL;
  • 2E is a result of Western blot detection of a protein of interest using an antibody of IL-24;
  • Figure 3A and Figure 3B show the pro-apoptotic effect and intracellular localization of purified TAT-IL-24-KDEL fusion protein on breast cancer MCF-7 cells;
  • Fig. 3A is the result of flow cytometry;
  • Fig. 3B is fusion Localization analysis of proteins in cells.
  • a. Obtain the gene encoding the TAT-IL-24-KDEL fusion protein.
  • the upstream primers containing the TAT transmembrane peptide coding sequence and the downstream primers containing the endoplasmic reticulum localization signal peptide KDEL coding sequence were used to amplify the TAT transmembrane peptide and endoplasmic reticulum by PCR.
  • the coding gene of the localization signal peptide KDEL was fused to the N-terminus and the C-terminus of the IL-24 gene, respectively, to obtain a TAT-IL-24-KDEL fusion protein-encoding gene.
  • Upstream primer 5'-CGCGGATCC TATGGCAGGAAGAAGCGTAGACAGAGACGTAGA GCCCAGGGCCAAGAGTTCC-3' (underlined TAT transmembrane peptide coding sequence);
  • Downstream primer 5'- CCGCTCGAGTCA GAGCTCGTCCTT GAGCTTGTAGAATTTCTGC-3' (underlined the endoplasmic reticulum localization signal peptide KDEL coding sequence).
  • the conditions of the PCR reaction were: pre-denaturation at 94 ° C for 5 min, denaturation at 94 ° C for 30 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles, and finally extension at 72 ° C for 10 min.
  • the PCR product was identified on an agarose gel and the fragment of interest was recovered using a gel recovery kit (Omega).
  • TAT-IL-24-KDEL gene fragment and the pET28a(+)/SUMO expression plasmid were digested with BamH I and Xho I.
  • T4 DNA ligase was ligated overnight at 16 ° C to obtain recombinant expression plasmid pET28a(+)/SUMO-TAT-IL-24-KDEL, and the ligation product was transformed into E. coli BL21 (DE3) competent state.
  • the positive clones were selected, and the positive clones were preliminarily confirmed by bacterial PCR and recombinant plasmid digestion, and the correct recombinant bacteria were preliminarily sampled.
  • E. coli BL21 (DE3) containing the recombinant plasmid pET28a(+)/SUMO-TAT-IL-24-KDEL was cultured in LB medium containing 50 ⁇ g/mL kanamycin at 37 ° C, when OD 600 When reached 0.6, IPTG was added to a final concentration of 0.1 mM, induced at 28 ° C for 10 h, and the SUMO-TAT-IL-24-KDEL fusion protein was heterologously expressed.
  • the cells were collected by centrifugation at 5000 x g for 15 min at 4 °C. The cells were disrupted by sonication and the supernatant and pellet were separately collected. SDS-PAGE results showed that the fusion protein was expressed almost entirely in the form of inclusion bodies.
  • the inclusion bodies were dissolved in 50 mM Tris-HCl buffer (pH 8.0) containing 8 M urea, and separated and purified by nickel column affinity chromatography under denaturing conditions.
  • the HisTrapTM HP affinity column (1 ml/min) was equilibrated with 10 CV of 50 mM Tris-HCl buffer (pH 8.0) containing 8 M urea.
  • the denatured supernatant containing the SUMO-TAT-IL-24-KDEL fusion protein was loaded at a flow rate of 1 ml/min, and the His-tagged SUMO-TAT-IL-24-KDEL fusion protein was selectively affinity-adsorbed. .
  • heterologous protein was washed with an equilibration buffer at a flow rate of 2 ml/min, and finally SUMO-TAT-IL-24-KDEL was eluted at a flow rate of 2 ml/min with 50 mM Tris-HCl buffer (pH 8.0) containing 200 mM imidazole and 8 M urea. Fusion protein.
  • the denatured purified fusion protein was dialyzed overnight in 50 mM Tris-HCl buffer (pH 8.0) containing 2 M urea, and then the renatured fusion protein was dialyzed three times in PBS to completely remove urea.
  • the fusion protein was digested with SUMO protease, and the SUMO protease and the fusion protein were mixed at a ratio of 1:1000 (w/w), and incubated at 30 ° C for 1 h or 4 ° C overnight to remove the SUMO tag.
  • the enzymatic cleavage reaction solution was concentrated and subjected to nickel column affinity chromatography.
  • the unfused enzyme, the SUMO protease and the SUMO tag all contained a His tag, and the affinity was collected on a nickel column to collect the permeate to obtain TAT-IL. -24-KDEL fusion protein.
  • Total protein concentration was determined by the Bradford method.
  • the purified collection solution was subjected to SDS-PAGE electrophoresis, electrophoresed and stained with Coomassie Brilliant Blue R-250 for 30 min, then decolorized with a decolorizing solution, and finally the gel was scanned with a computer software Gel-Pro analyzer to analyze TAT-IL-24-KDEL. The purity, concentration and recovery rate of the fusion protein.
  • TAT-IL-24-KDEL fusion protein having a concentration of 0.6 g/L and a purity of 92% was obtained.
  • TAT-IL-24-KDEL fusion protein The pro-apoptotic effect and intracellular localization of TAT-IL-24-KDEL fusion protein on breast cancer MCF-7 cells were examined in vitro.
  • Monolayer cultured cells were digested with 0.25% trypsin, and mixed into a single cell suspension in DMEM medium containing 10% fetal bovine serum. 10 6 cells were inoculated into 60 mm culture dishes, and cultured at 37 ° C, 5% CO 2 . Cultivate in the box for 24h.
  • the fusion protein was diluted with serum-free DMEM medium to a final concentration of 5 nM, 10 nM and 20 nM, respectively, and added to the culture dish, with PBS as a negative control.
  • Q1 represents cell debris
  • Q2 represents dead cells and late apoptotic cells
  • Q3 represents living cells
  • Q4 represents early apoptotic cells.
  • transmembrane peptide TAT and endoplasmic reticulum localization signal peptide KDEL The effect of transmembrane peptide TAT and endoplasmic reticulum localization signal peptide KDEL on the localization of IL-24 in fusion protein was detected by immunofluorescence technique.
  • the specific operations are as follows:
  • monolayer cultured cells were digested with 0.25% trypsin, and mixed into a single cell suspension with DMEM medium containing 10% fetal bovine serum, and seeded into 6-well plates at 3 ⁇ 10 5 cells/mL per well.
  • the volume was 2 ml, and cultured in a 37 ° C, 5% CO 2 incubator for 24 h.
  • the culture medium in the 6-well plate was discarded, and the fusion protein was diluted with a serum-free DMEM medium to a final concentration of 20 nM, and added to a 6-well plate for 12 hours.

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Abstract

本发明公开了一种TAT-IL-24-KDEL融合蛋白，其是指TAT穿膜肽和内质网定位信号肽KDEL分别连接在白细胞介素-24（IL-24）的N端和C端的融合蛋白。本发明还公开了所述融合蛋白的制备方法，以及所述融合蛋白在促进乳腺癌细胞MCF-7凋亡中的应用。

Description

TAT-IL-24-KDEL融合蛋白及其制备方法和应用 技术领域
本发明涉及生物工程，尤其涉及一种TAT-IL-24-KDEL融合蛋白及其制备方法和应用。
背景技术
1995年Fisher P.B.通过差减杂交技术发现一个新的与人黑色素瘤分化相关基因，当时称为mda-7，并发现随着黑色素瘤细胞生长、发展和转移，mda-7的表达逐渐减少至最终消失，证实了其表达产物MDA-7具有抑制黑色素瘤细胞增生、促进黑色素瘤细胞终末分化的能力。2002年，Caudell等研究证实了MDA-7的细胞因子属性，重新命名为人白细胞介素24(Human interleukin-24，hIL-24)。迄今为止，已有大量实验证明IL-24具有显著地选择性抑制多种肿瘤生长、诱导肿瘤细胞增殖的能力，这种抑制作用不依赖于p53、Rb和p16等抑癌基因的状态，且对正常细胞没有影响。MDA-7/IL-24的作用谱很广，可诱导包括黑色素瘤、多形性胶质细胞瘤、骨肉瘤、乳腺癌、宫颈癌、结肠癌、肺癌、鼻咽癌及前列腺癌等肿瘤细胞的生长抑制和凋亡。IL-24作为细胞因子，不仅在激活免疫细胞、调节整个抗癌免疫反应和造血系统中起重要作用，还具有选择性诱导肿瘤细胞凋亡和直接抑制肿瘤细胞的增殖、转移作用，其显著地抗肿瘤特性使之成为肿瘤治疗研究的新热点。
早期研究表明，MDA-7/IL-24是通过与肿瘤细胞膜表面的受体复合物IL-20R1/IL-20R2和IL-22R1/IL-20R2结合，激活JAK/STAT信号途径使STAT3磷酸化而调节胞内相关基因的表达发挥生长抑制和促凋亡作用的。Sauane等的研究结果提出MDA-7/IL-24也可通过非JAK/STAT依赖途径发挥诱导肿瘤细胞凋亡的作用。最近有大量文献证明MDA-7/IL-24可以通过调控内质网应激反应和线粒体完整性抑制肿瘤细胞生长和凋亡。针对IL-24的内质网作用途径，运用基因工程技术大量制备IL-24蛋白，并将其用于促进肿瘤细胞凋亡的研究尚未见报道。
发明内容
本发明的目的，在于提供一种TAT-IL-24-KDEL融合蛋白，并提供一种TAT-IL-24-KDEL融合蛋白的制备方法，以获得高浓度高纯度的TAT-IL-24-KDEL 融合蛋白，同时提供一种TAT-IL-24-KDEL融合蛋白在肿瘤治疗上的用途。
为了达到上述目的，本发明采用了以下技术方案：
一种TAT-IL-24-KDEL融合蛋白，是将TAT穿膜肽和内质网定位信号肽KDEL分别融合到IL-24蛋白的N端和C端，构成TAT-IL-24-KDEL融合蛋白；所述TAT穿膜肽的氨基酸序列如SEQ ID NO.1所示。
上述TAT-IL-24-KDEL融合蛋白的制备方法，包括以下步骤：
A、构建pET28a(+)/SUMO-TAT-IL-24-KDEL重组质粒；
B、利用E.coli BL21(DE3)异源表达SUMO-TAT-IL-24-KDEL融合蛋白；
C、分离纯化TAT-IL-24-KDEL融合蛋白；
D、体外检测TAT-IL-24-KDEL融合蛋白对乳腺癌MCF-7细胞的促凋亡作用和融合蛋白在细胞内的定位。
步骤A的具体方法是，采用PCR扩增方法获得SUMO标签和TAT-IL-24-KDEL融合蛋白的编码基因，并采用融合PCR的方法将SUMO和TAT-IL-24-KDEL的编码基因无缝连接，利用含有T7启动子的pET28a(+)表达载体构建重组质粒。
步骤B的具体方法是，将含有pET28a(+)/SUMO-TAT-IL-24-KDEL重组质粒的E.coli BL21(DE3)在37℃下培养，当OD600达到0.6时，加入终浓度为0.1mM的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)，28℃诱导10h，异源表达SUMO-TAT-IL-24-KDEL融合蛋白，融合蛋白以包涵体形式存在。
步骤C的具体方法是，在变性条件下，利用His标签的亲和层析分离纯化SUMO-TAT-IL-24-KDEL融合蛋白，蛋白复性以后，用SUMO蛋白酶在融合蛋白上切除SUMO标签并再次利用His亲和层析去除SUMO标签，分离纯化TAT-IL-24-KDEL融合蛋白。
步骤D的具体方法是，利用流式细胞实验检测纯化的TAT-IL-24-KDEL融合蛋白对乳腺癌MCF-7细胞的凋亡作用，利用免疫荧光实验检测融合蛋白在胞内的定位情况。
本发明的TAT-IL-24-KDEL融合蛋白能够有效定位于内质网上，从而促进乳腺癌细胞MCF-7的凋亡。
上述TAT-IL-24-KDEL融合蛋白的制备方法的具体步骤如下：
1、构建pET28a(+)/SUMO-TAT-IL-24-KDEL重组质粒
1.1、以含有TAT穿膜肽编码序列的上游引物 5’-CGCGGATCCTATGGCAGGAAGAAGCGTAGACAGAGACGTAGAGCCCAGGGCCAAGAGTTCC-3′(下划线表示TAT穿膜肽编码序列)和下游引物5’-CCGCTCGAGTCAGAGCTCGTCCTTGAGCTTGTAGAATTTCTGC-3’(下划线表示内质网定位信号肽KDEL编码序列)为反应引物，以全长的IL-24基因为模板，PCR扩增获得TAT-IL-24-KDEL融合蛋白的编码基因。
1.2、用基因纯化试剂盒对PCR产物进行纯化，将目的基因和pET28a(+)/SUMO表达质粒用BamH Ⅰ和Xho Ⅰ进行双酶切，T4连接酶连接后转化E.coli BL21(DE3)，挑选阳性克隆送样测序。
2、利用E.coli BL21(DE3)异源表达SUMO-TAT-IL-24-KDEL融合蛋白。将测序正确的含有pET28a(+)/SUMO-TAT-IL-24-KDEL重组质粒的E.coli BL21(DE3)在37℃下培养，当OD600达到0.6时，加入终浓度为0.1mM的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)，28℃诱导10h，异源表达SUMO-TAT-IL-24-KDEL融合蛋白。
3、分离纯化SUMO-TAT-IL-24-KDEL融合蛋白
3.1、由于融合蛋白以包涵体的形式表达，所以需要对其进行变复性。在变性条件下，利用His标签的亲和层析分离纯化SUMO-TAT-IL-24-KDEL融合蛋白。
3.2、利用透析复性的方法，对纯化的融合蛋白进行复性。
3.3、蛋白复性以后，用SUMO蛋白酶在融合蛋白上切除SUMO标签并再次利用His亲和层析去除SUMO标签，分离纯化TAT-IL-24-KDEL融合蛋白。
4、体外检测TAT-IL-24-KDEL融合蛋白对乳腺癌MCF-7细胞的促凋亡作用。利用流式细胞实验检测纯化的TAT-IL-24-KDEL融合蛋白的活性。分别将终浓度为0nM,5nM,10nM和20nM的融合蛋白加入乳腺癌MCF-7细胞，24h后用Annexin V-FITC/PI凋亡双染试剂盒处理样品，检测融合蛋白对MCF-7的凋亡作用。
利用免疫荧光技术检测融合蛋白中穿膜肽TAT和内质网定位信号肽KDEL对IL-24定位的影响。先用IL-24的一抗和PE标记的二抗对目的蛋白进行免疫荧光反应，再用内质网形态染色试剂盒对内质网进行染色，然后将二者重叠，观察IL-24在细胞中的定位情况。
与现有技术相比，本发明的优点在于：
(1)首次将人类免疫缺陷病毒HIV-1的TAT穿膜肽和内质网定位信号肽KDEL分别融合到IL-24的N端和C端，构成TAT-IL-24-KDEL融合蛋白。构建pET28a(+)/SUMO-TAT-IL-24-KDEL重组质粒，经过异源表达和分离纯化，大量制 备TAT-IL-24-KDEL融合蛋白。最终TAT-IL-24-KDEL融合蛋白的浓度为0.6g/L，纯度为92％。
(2)TAT-IL-24-KDEL融合蛋白的TAT穿膜肽可以将IL-24蛋白转移到细胞内，而内质网定位信号肽KDEL可以将进入细胞内的IL-24定位到内质网上，与其受体GRP78/BiP结合发挥作用。
本发明首次在体外检测了TAT-IL-24-KDEL融合蛋白的活性。流式细胞实验和免疫荧光实验结果表明，该融合蛋白能高效的定位到内质网上并发挥抗肿瘤效应，这使得TAT-IL-24-KDEL融合蛋白在肿瘤治疗上有很大的应用前景。
附图说明
图1是pET28a(+)/SUMO-TAT-IL-24-KDEL重组质粒的构建过程；
图2A是SUMO-TAT-IL-24-KDEL融合蛋白的表达情况，主要以包涵体的形式表达；
图2B是融合蛋白在变性条件下利用镍柱亲和层析纯化的结果；
图2C是融合蛋白用SUMO蛋白酶进行酶切后的结果；
图2D是酶切后用镍柱亲和层析进行纯化得到目的蛋白TAT-IL-24-KDEL；
图2E是利用IL-24的抗体对目的蛋白进行Western blot检测的结果；
图3A、图3B是纯化的TAT-IL-24-KDEL融合蛋白对乳腺癌MCF-7细胞的促凋亡作用和胞内定位情况；其中，图3A是流式细胞实验结果；图3B是融合蛋白在细胞内的定位分析。
具体实施方式
实施例1
TAT-IL-24-KDEL融合蛋白的制备
1、构建pET28a(+)/SUMO-TAT-IL-24-KDEL重组质粒
a.获取TAT-IL-24-KDEL融合蛋白的编码基因。以全长的IL-24基因为模板，利用含有TAT穿膜肽编码序列的上游引物和含有内质网定位信号肽KDEL编码序列的下游引物，通过PCR扩增将TAT穿膜肽和内质网定位信号肽KDEL的编码基因分别融合到IL-24基因的N端和C端，获得TAT-IL-24-KDEL融合蛋白编码基因。
上游引物： 5’-CGCGGATCCTATGGCAGGAAGAAGCGTAGACAGAGACGTAGAGCCCAGGGCCAAGAGTTCC-3′(下划线表示TAT穿膜肽编码序列)；
下游引物：5’-CCGCTCGAGTCAGAGCTCGTCCTTGAGCTTGTAGAATTTCTGC-3’(下划线表示内质网定位信号肽KDEL编码序列)。
PCR反应的条件是：94℃预变性5min，94℃变性30s，58℃退火30s，72℃延伸1min，循环30次，最后72℃延伸10min。PCR产物经琼脂糖凝胶鉴定，用胶回收试剂盒(Omega)回收目的片段。
b.利用BamH Ⅰ和Xho Ⅰ将所得的TAT-IL-24-KDEL基因片段和pET28a(+)/SUMO表达质粒双酶切。T4 DNA连接酶16℃连接过夜，获得重组表达质粒pET28a(+)/SUMO-TAT-IL-24-KDEL，将连接产物转化E.coli BL21(DE3)感受态。挑选阳性克隆，通过菌液PCR和重组质粒上酶切初步验证阳性克隆，将初步验证正确的重组菌送样测序。
2、利用E.coli BL21(DE3)异源表达SUMO-TAT-IL-24-KDEL融合蛋白
将测序正确的含有pET28a(+)/SUMO-TAT-IL-24-KDEL重组质粒的E.coli BL21(DE3)在含有50μg/mL卡那霉素的LB培养基中37℃培养，当OD600达到0.6时，加入终浓度为0.1mM的IPTG，28℃诱导10h，异源表达SUMO-TAT-IL-24-KDEL融合蛋白。在4℃下，5000×g离心15min，收集菌体。超声破碎细胞，分别收集上清和沉淀。SDS-PAGE结果显示，融合蛋白几乎全部以包涵体的形式表达。
3、SUMO-TAT-IL-24-KDEL融合蛋白的变复性和纯化
a.SUMO-TAT-IL-24-KDEL融合蛋白的变性和纯化
将包涵体溶解在含有8M尿素的50mM Tris-Hcl缓冲液(pH8.0)中，在变性条件下利用镍柱亲和层析进行分离纯化。用10CV的含有8M尿素的50mM Tris-Hcl缓冲液(pH8.0)平衡HisTrapTM HP亲和柱(1ml/min)。将含有SUMO-TAT-IL-24-KDEL融合蛋白的变性上清液以1ml/min的流速上样，带有His标签的SUMO-TAT-IL-24-KDEL融合蛋白被选择性的亲和吸附。用平衡缓冲液以2ml/min的流速冲洗去除杂蛋白，最后用含有200mM咪唑和8M尿素的50mM Tris–HCl缓冲液(pH 8.0)以2ml/min流速洗脱SUMO-TAT-IL-24-KDEL融合蛋白。
b.SUMO-TAT-IL-24-KDEL融合蛋白的复性
将变性纯化得到的融合蛋白在含有2M尿素的50mM Tris-Hcl缓冲液(pH8.0)中透析过夜进行复性，然后将复性的融合蛋白在PBS中透析三次以彻底除尽尿素。
c.去除SUMO标签，分离纯化得到TAT-IL-24-KDEL融合蛋白
利用SUMO蛋白酶对融合蛋白进行酶切，将SUMO蛋白酶和融合蛋白以1:1000(w/w)的比例混合，30℃孵育1h或4℃过夜，去除SUMO标签。将酶切反应液浓缩后进行镍柱亲和层析，未酶切完全的融合蛋白、SUMO蛋白酶和SUMO标签均含有His标签，能够亲和在镍柱上，收集穿出液，得到TAT-IL-24-KDEL融合蛋白。
4、纯化的TAT-IL-24-KDEL蛋白的定量
总蛋白浓度采用Bradford法来测定。将纯化得到的收集液进行SDS-PAGE电泳，电泳后用考马斯亮蓝R-250染色30min，然后用脱色液脱色，最后用计算机软件Gel-Pro analyzer扫描凝胶，分析TAT-IL-24-KDEL融合蛋白的纯度，浓度及纯化工艺的回收率。
通过以上变复性和纯化操作，获得浓度为0.6g/L，纯度为92％的TAT-IL-24-KDEL融合蛋白。
实施例2
TAT-IL-24-KDEL融合蛋白的活性检测
体外检测TAT-IL-24-KDEL融合蛋白对乳腺癌MCF-7细胞的促凋亡作用和细胞内定位作用。
1、采用流式细胞实验检测TAT-IL-24-KDEL融合蛋白对乳腺癌MCF-7细胞的促凋亡作用，具体操作如下：
1.1、用0.25％胰蛋白酶消化单层培养细胞，用含有10％胎牛血清的DMEM培养基配成单细胞悬浮液，将106个细胞接种到60mm培养皿内，37℃，5％CO2培养箱中培养24h。
1.2、将融合蛋白用无血清的DMEM培养基稀释成终浓度为5nM,10nM和20nM的溶液，分别加入培养皿中，以PBS为阴性对照。
1.3、24h后，先用不含EDTA的0.125％的胰酶消化，加入3mL完全培养基配成单细胞悬浮液后离心，1000r，5min。再用PBS洗两次并离心，1000r，5min。 加入100μL Binding buffer和FITC标记的Annexin V(20μg/mL)10μL，室温避光孵育30min。再加入PI(50μg/mL)5μL，避光反应5min后，加入400μL PBS，立即用FACScan进行流式细胞术定量检测，同时设置Annexin V-FITC及PI单染各一管作为对照。
1.4、统计分析实验结果。Q1表示细胞碎片，Q2表示死细胞和晚期凋亡细胞，Q3表示活细胞，Q4表示早期凋亡细胞。
2、利用免疫荧光技术检测融合蛋白中穿膜肽TAT和内质网定位信号肽KDEL对IL-24定位的影响，具体操作如下：
2.1、用0.25％胰蛋白酶消化单层培养细胞，用含有10％胎牛血清的DMEM培养基配成单细胞悬浮液，以每孔3×105个/mL细胞接种于6孔板，每孔体积2ml，37℃，5％CO2培养箱中培养24h。
2.2、倒掉6孔板中的培养液，将融合蛋白用无血清的DMEM培养基稀释成终浓度为20nM的溶液，加入6孔板中，培养12h。
2.3、倒掉培养液，用PBS洗涤两次，每次5min。然后用1mL4％多聚甲醛室温固定20-30min。用PBS洗涤两次，每次5min。再用0.2％的Triton X-100透化10min后，PBS洗涤两次，每次5min。
2.4、用10％的脱脂奶粉37℃封闭30min后，加入抗IL-24的一抗，37摄氏度孵育1.5h。PBS洗涤3次，每次5min。然后加入PE标记的二抗，37℃孵育1h。PBS洗涤3次，每次5min。
2.5、加入1mL清理液和10μL GENMED染色工作液，对内质网进行染色，混匀后，室温下孵育10-30s，吸弃染色液并加入500μL清理液，即刻在10min内置于荧光显微镜下观察。

Claims (7)

  1. 一种TAT-IL-24-KDEL融合蛋白，其特征在于：将TAT穿膜肽和内质网定位信号肽KDEL分别融合到IL-24蛋白的N端和C端，构成TAT-IL-24-KDEL融合蛋白；所述TAT穿膜肽的氨基酸序列如SEQ ID NO.1所示。
  2. 权利要求1所述TAT-IL-24-KDEL融合蛋白的制备方法，其特征在于，包括以下步骤：
    A、构建pET28a(+)/SUMO-TAT-IL-24-KDEL重组质粒；
    B、利用E.coli BL21(DE3)异源表达SUMO-TAT-IL-24-KDEL融合蛋白；
    C、分离纯化TAT-IL-24-KDEL融合蛋白；
    D、体外检测TAT-IL-24-KDEL融合蛋白对乳腺癌MCF-7细胞的促凋亡作用和融合蛋白在细胞内的定位。
  3. 根据权利要求2所述的方法，其特征在于：步骤A的具体方法是，采用PCR扩增方法获得SUMO标签和TAT-IL-24-KDEL融合蛋白的编码基因，并采用融合PCR的方法将SUMO和TAT-IL-24-KDEL的编码基因无缝连接，利用含有T7启动子的pET28a(+)表达载体构建重组质粒。
  4. 根据权利要求2所述的方法，其特征在于：步骤B的具体方法是，将含有pET28a(+)/SUMO-TAT-IL-24-KDEL重组质粒的E.coli BL21(DE3)在37℃下培养，当OD600达到0.6时，加入终浓度为0.1mM的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)，28℃诱导10h，异源表达SUMO-TAT-IL-24-KDEL融合蛋白，融合蛋白以包涵体形式存在。
  5. 根据权利要求2所述的方法，其特征在于：步骤C的具体方法是，在变性条件下，利用His标签的亲和层析分离纯化SUMO-TAT-IL-24-KDEL融合蛋白，蛋白复性以后，用SUMO蛋白酶在融合蛋白上切除SUMO标签并再次利用His亲和层析去除SUMO标签，分离纯化TAT-IL-24-KDEL融合蛋白。
  6. 根据权利要求2所述的方法，其特征在于：步骤D的具体方法是，利用流式细胞实验检测纯化的TAT-IL-24-KDEL融合蛋白对乳腺癌MCF-7细胞的凋亡作用，利用免疫荧光实验检测融合蛋白在胞内的定位情况。
  7. TAT-IL-24-KDEL融合蛋白在促进乳腺癌细胞MCF-7的凋亡中的应用。
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