WO2015188334A1 - 一种Tat蛋白及其制备方法和应用 - Google Patents

一种Tat蛋白及其制备方法和应用 Download PDF

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WO2015188334A1
WO2015188334A1 PCT/CN2014/079675 CN2014079675W WO2015188334A1 WO 2015188334 A1 WO2015188334 A1 WO 2015188334A1 CN 2014079675 W CN2014079675 W CN 2014079675W WO 2015188334 A1 WO2015188334 A1 WO 2015188334A1
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tat
seq
tat protein
hiv
protein
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张辉
耿冠男
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Sun Yat Sen University
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Priority to US15/113,013 priority patent/US9790255B2/en
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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

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  • the present invention relates to an antiviral compound, and more particularly to an improved Tat protein and a preparation method and use thereof.
  • HAART Highly Active Antiretroviral Therapy
  • latent HIV-1 Human Immunodeficiency Virus Type 1
  • the patient must take long-term medication to inhibit viral replication, and once the withdrawal is made, it will cause a rebound in viral replication.
  • How to eliminate the latent infection of HIV-1 has become a bottleneck for the complete cure of AIDS.
  • HDACi Histone Deacetylase inhibitor
  • VPA valproc acid
  • SAHA Vorinostat
  • HIV-1 Tat is a HIV-1 specific transactivator that specifically binds to HIV-1 5'
  • Tat protein is also a key factor in the latent infection of HIV-1.
  • the protein has a penetrating peptide that has been shown to function efficiently across the cell membrane; it has been designed to be used as a vaccine for HIV-1 in clinical trials and is relatively safe for humans.
  • the Tat protein has been shown to have an apoptosis-inducing function and may have an effect on immune cell function.
  • One of the objectives of the present invention is to find a new anti-HIV route
  • a Tat protein is used for the preparation of an anti-HIV drug, and the present invention uses TAT-86 to prove the effect.
  • an attenuated Tat protein for the preparation of an anti-HIV drug, the attenuated Tat protein amino acid sequence SEQ N0: 1, SEQ NO: 2, SEQ N0: 3, SEQ N0: 4.
  • an attenuated Tat protein said attenuated Tat protein amino acid sequence SEQ N0: 1 (R4M4), SEQ NO: 2 (R4M5), SEQ NO: 3 (R4M7), SEQ NO: 4 (R5M4) Shown.
  • a method for preparing the above-mentioned attenuated Tat protein is provided, which is characterized in that a site for site-directed mutagenesis is first selected, and site-directed mutagenesis of the Tat gene is carried out at these sites, and finally a large amount of transactivation function of retaining Tat is obtained. And attenuated proteins in which apoptosis and other activities are removed.
  • the object of the present invention is to provide a number of attenuated HIV-1 Tat proteins for activating HIV-1 latent infection.
  • the present invention provides an idea of using HIV-1's own transactivator Tat as a means of activating latent infection, and modifying the Tat protein by cumulative mutations makes it more plaguable in terms of safety.
  • the present invention provides four attenuated Tat proteins that specifically activate HIV-1 latent infection at the cellular level.
  • the present invention provides an improved construction of an HIV-1 in vitro latent infection model.
  • the present invention provides a novel method for activating HIV-1 latent infection by attenuating Tat protein in combination with SAHA.
  • the attenuated Tat protein provided by the present invention does not affect the function of immune cells.
  • Tat protein has a good transactivation activity and can effectively promote the transcription of HIV-1 mRNA.
  • Tat protein has good transmembrane activity and can effectively enter cells and tissues to function.
  • FIG 1 Principles of HIV1 Tat protein modification and screening.
  • FIG. 1 Schematic diagram of four attenuated Tat.
  • FIG. 3 Four attenuated Tat activates the HIV-1 promoter in Tzm-bl.
  • Figure 4 Transformation of Tat_R5M4 reduces cytotoxicity and apoptotic activity.
  • Tat-R5M4 effectively penetrates the membrane into cells and tissues.
  • Figure 6 Construction of an HIV-1 in vitro latent infection model.
  • Tat-R5M4 is effective in activating the HIV-1 in vitro latency model.
  • FIG 8 Tat-R5M4 is effective in activating CD4 + T cells from latent infection in peripheral blood of clinical patients.
  • Figure 9 Acute toxicity test and immunogenicity test. Concrete real
  • Example 1 Modification and Activity Detection of Tat Proteins
  • the structure and function of HIV-1 Tat have been studied in detail over the past few decades.
  • the 36th valine was replaced with alanine
  • the 66th glutamine was substituted with alanine
  • the 67th valine was replaced with alanine
  • the 68th position The serine is replaced with alanine.
  • the 39th isoleucine was substituted with alanine
  • the 66th glutamine was substituted with alanine
  • the 67th valine was replaced with alanine
  • the 68th The serine at position is substituted with alanine.
  • the 66th glutamine was replaced with alanine
  • the 67th valine was replaced with alanine
  • the 68th serine was replaced with alanine
  • the 77th serine was used. Alanine substitution.
  • the 36th valine was replaced with alanine
  • the 66th glutamine was substituted with alanine
  • the 67th valine was replaced with alanine
  • the 68th position was replaced with alanine
  • the serine was replaced with alanine
  • the serine at position 77 was substituted with alanine.
  • R4M4, R4M5, R4M7 and R5M4 were cloned into prokaryotic expression vector for expression and purification, and the obtained protein was treated with Tzm-bl cells. The activity of Firefly Luciferase was detected 48 hours later.
  • Tat-86 or Tat-R5M4 were co-incubated with Tzm-bl cells, and cell viability was measured by MTS method 48 hours later.
  • 0. lug, 0. 5ug, lug, 2ug of Tat-86 (prokaryotic expression according to conventional techniques, cited by Expression of full length Tat in E. coli and its purification) and Tat_R5M4 and Jurkat cells were co-incubated. After 48 hours, Annexin_V_FITC and PE were double-stained, and the proportion of apoptosis was detected by flow cytometry.
  • Tat_R5M4 significantly reduces cytotoxicity and the ability to induce apoptosis.
  • Tat-R5M4 affects transmembrane activity after mutation
  • the purified Tat_R5M4 protein was labeled with NHS-Rhodanmine, and the labeled proteins were treated with PBMC and Jurkat cells for 6 hours and then flow-detected. Tat enters the state of the cell. Tat_R5M4 has good membrane penetration activity.
  • Tzm_bl is fine. The cells were treated with purified Tat-R5M4 protein and immunofluorescence was detected with Tat_R5M4 antibody.
  • Tat-R5M4 was mostly distributed in the cytoplasm after entering the cell, and only a small amount of protein entered the nucleus.
  • rhodamine-labeled Tat_R5M4 protein was injected into mice through tail vein injection. After 6 hours, the spleen, thymus, brain and small intestine were sliced and detected. It was found that Tat_R5M4 was clearly distributed. In these organizations.
  • Primary CD4 + T cells activated by infection with a pseudovirus carrying the gene usually have a positive rate between 5% and 10%.
  • Three days after infection we sorted GFP-positive cells by flow sorting, continued culture with PRM1640 medium supplemented with IL-2, and added CD3 and CD28 antibodies for reactivation, and changed to no cells after one week.
  • Factor PRM1640 medium after 3-4 weeks of culture, IL-2 was withdrawn to allow the cells to gradually enter a resting state. These resting cells can be used for the detection of latent activators.
  • Example 5 Activation of Tat-R5M4 in HIV-1 in vitro latent infection model and clinical patient-derived CD4 + T cells
  • the latent infection system constructed in vitro was used to detect the ability of Tat-R5M4 to activate latent infection, while CD3/CD28 and SAM were used as positive controls, and the proportion of GFP-positive cells was observed by fluorescence microscopy three days later.
  • CD3/CD28 antibody and SAHA were used as positive controls, and the isolated clinical samples of CD4 + T cells were treated with Tat_R5M4 or Tat_R5M4 in combination with SAHA. After 18 hours, the supernatant was taken and RNA was extracted. HIV-1 RNA content in the Qing Dynasty.
  • Tat-R5M4 can effectively activate the HIV-1 in vitro latent infection model and the latent state of CD4 + T cells derived from clinical patient samples, and the combination with SAM can enhance its transactivation effect.
  • Experiment 6 Acute toxicity test and immunogenicity test In order to provide some theoretical support for subsequent animal experiments, the toxicity of Tat_R5M4 protein was further tested. First, acute toxicity experiments with Babl/c mice showed that mice still survived well when the tail vein dose reached 40 mg/ml. Mouse alanine aminotransferase or alanine
  • ALT aspartate aminotransferase or aspartate
  • BREA urea nitrogen
  • CR CR
  • Tat-86 and Tat_R5M4 subcutaneously, and measured the concentration of Tat antibody at 7 days, 14 days, and 21 days, respectively. It was found that the amount of antibody produced by Tat-R5M4 was significantly lower than that of Tat_86. .
  • This experiment demonstrates that the modified Tat-R5M4 is relatively safe to mice and has significantly reduced immunogenicity, and is more suitable for in vivo experiments than wild-type Tat-86.

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Abstract

一种Tat蛋白，所述的Tat蛋白的氨基酸序列如SEQ NO:1、SEQ NO:2、SEQ NO:3、SEQ NO:4所示。本发明的Tat蛋白能够作为HIV-1潜伏感染激活的潜在药物进行研究和开发。

Description

一种 Tat蛋白及其制备; 和应用
技术领域
本发明涉及一种抗病毒化合物， 更具体地，涉及一种改良的 Tat蛋白及其制 备方法和应用。
背景技术
高效抗逆转录病毒治疗 (Highly Active Antiretroviral Therapy, HAART) 治疗可以有效地将病人体内的病毒数量控制到检测不到的程度，但是潜伏感染的 HIV-1 (人类免疫缺陷病毒一型） 前病毒整合进宿主的基因组中形成储存库以后 很难去除。病人必须长期用药以抑制病毒复制， 一旦撤药就会造成病毒复制的回 弹。 怎样清除 HIV-1的潜伏感染已经成为彻底治愈艾滋病的瓶颈问题。
白介素 2 Cnterleukin 2 IL-2)和 anti_CD3等细胞因子曾被用于激活 HIV-1 的潜伏感染， 这些细胞因子使细胞在整体水平上发生了激活， 对机体造成了极大 的毒副作用。 多种组蛋白乙酰化酶抑制剂 （Histone Deacetylase inhibitor, HDACi ) 也是目前研究比较多的潜伏激活剂， 一方面由于 HDACi对基因的激活也 是广谱效应， 容易引起其他基因的异常表达， 另一方面在体外效果比较好的 HDACi , 如 valproc acid (VPA)禾口 Vorinostat (即 suberanilohydroxamic acid, SAHA)等， 在临床上的表现都不佳， 不能够投入实际应用。 因此， 寻找新的特异 性强、 高效而安全的潜伏激活剂是当务之急。
HIV-1 Tat是 HIV-1特异性的反式激活因子， 该蛋白特异地结合到 HIV-1 5'
LTR的 TAR上， 将 HIV-1 mRNA的转录抬高数百倍。 Tat 蛋白在 HIV-1的潜伏感 染中也是关键的因子。 该蛋白带有穿膜肽， 被证明具有高效穿过细胞膜的功能； 曾被设计作为 HIV-1的疫苗进入临床实验使用，对人体相对安全。但 Tat蛋白被 证明具有诱导凋亡的功能， 可能对免疫细胞功能造成影响。
发明内容
本发明的目的之一就是寻找一种新的抗 HIV途径，
首先提供一种 Tat蛋白在制备抗 HIV药物中的应用，本发明采用的是 TAT-86 来证明效果。
更进一步提供一种减毒 Tat蛋白在制备抗 HIV药物中的应用， 所述的减毒 Tat蛋白氨基酸序列 SEQ N0: 1、 SEQ N0: 2、 SEQ N0: 3、 SEQ N0: 4所示。 更进一步提供一种减毒 Tat蛋白，所述的减毒 Tat蛋白氨基酸序列 SEQ N0: 1 (R4M4)、 SEQ NO: 2 (R4M5)、 SEQ NO: 3 (R4M7)、 SEQ NO: 4 (R5M4) 所示。
进一步提供一种上述的减毒 Tat蛋白的制备方法，其特征在于， 首先选择定 点突变的位点， 对这些位点在 Tat的基因上进行定点突变， 最终得到大量保留 Tat 的反式激活功能， 并去除其中凋亡和其他活性的减毒蛋白。
本发明具有以下优点：
1. 本发明的目的在于提供若干减毒的 HIV-1 Tat蛋白用于激活 HIV-1潜伏 感染。
2. 本发明提供了一种利用 HIV-1 自身的反式激活蛋白 Tat作为激活潜伏感 染工具的思路， 并且通过累计突变对 Tat蛋白进行改造， 使其在安全性上更加可 罪。
3. 本发明提供四种减毒的 Tat蛋白， 能够在细胞水平上特异性激活 HIV-1 潜伏感染。
4. 本发明提供了一种改良的 HIV-1体外潜伏感染模型的构建方式。
5. 本发明提供一种减毒 Tat蛋白跟 SAHA联用激活 HIV-1潜伏感染的新方 法。
6. 本发明提供的减毒 Tat蛋白对免疫细胞的功能不造成影响。
7. 本发明发现 Tat蛋白的具备较好的反式激活活性， 能够有效抬高 HIV-1 mRNA的转录。
8. Tat蛋白具备良好的穿膜活性， 能够有效进入细胞和各个组织发挥功能 附图说明
图 1 : HIV1 Tat蛋白改造及筛选原理。
图 2: 四种减毒 Tat的模式图。
图 3: 四种减毒 Tat能够在 Tzm-bl中激活 HIV-1的启动子。
图 4: Tat_R5M4的改造降低了细胞毒性和凋亡活性。
图 5: Tat-R5M4能有效地穿膜进入细胞以及各个组织。
图 6: HIV-1体外潜伏感染模型的构建。
图 7: Tat-R5M4能有效激活 HIV-1体外潜伏模型。
图 8: Tat-R5M4能有效激活来自临床病人外周血的潜伏感染状态的 CD4+T细胞。 图 9: 急性毒性实验和免疫原性检测。 具体实 式
下面结合附图和具体实施例进一步详细说明本发明。除非特别说明， 本发明 采用的试剂、设备和方法为本技术领域常规市购的试剂、设备和常规使用的方法。 实施例一： 对 Tat蛋白的改造和活性检测 在过去的几十年中， HIV-1 Tat的结构和功能已经有很详细的研究。 在本发 明中， 我们通过积累突变改造出去除凋亡活性， 同时保留大部分反式激活活性的 Tat蛋白。 由于 Tat 蛋白前三个结构域 （氨基酸 1-59 ) 的研究已经非常详尽， 所以我们首先选择研究较少的氨基酸 60-72进行点突变，将突变的基因连接到真 核表达载体上转染 Tzm-bl细胞后检测荧光素酶活性，其中只有 M36， M39, M51， M66， M67， M68， M69， M77 保留了 80%的反式激活活性。 接着我们把这些突变 进行组合叠加，进行了一共六轮突变，最后得到 4个候选蛋白： R4M4, R4M5, R4M7, R5M4, 其中 R5M4积累了最多 5个点突变。 其中 R4M4在定点突变中， 将第 36位的缬氨酸用丙氨酸取代， 第 66位的谷 氨酰胺用丙氨酸取代， 第 67位的缬氨酸用丙氨酸取代， 第 68位的丝氨酸用丙氨 酸取代。 其中 R4M5在定点突变中， 将第 39位的异亮氨酸用丙氨酸取代， 第 66位的 谷氨酰胺用丙氨酸取代， 第 67位的缬氨酸用丙氨酸取代， 第 68位的丝氨酸用丙 氨酸取代。 其中 R4M7在定点突变中， 第 66位的谷氨酰胺用丙氨酸取代， 第 67位的缬 氨酸用丙氨酸取代， 第 68位的丝氨酸用丙氨酸取代， 第 77位的丝氨酸用丙氨酸 取代。 其中 R5M4在定点突变中， 将第 36位的缬氨酸用丙氨酸取代， 第 66位的谷 氨酰胺用丙氨酸取代， 第 67位的缬氨酸用丙氨酸取代， 第 68位的丝氨酸用丙氨 酸取代， 第 77位的丝氨酸用丙氨酸取代。
将 R4M4， R4M5, R4M7, R5M4克隆到原核表达载体中进行表达及纯化， 获得 的蛋白对 Tzm-bl细胞进行处理， 48小时后检测 Firefly Luciferase的活性。
该实验证明改造的 R4M4， R4M5, R4M7, R5M4在 Tzm-bl中具有良好的反式激 活活性。
实施例二： 细胞毒性和凋亡活性检测
分别将 ΙΟηΜ, 50ηΜ， ΙΟΟηΜ, 500nM， luM, 2uM, 3uM, 4uM 的 Tat-86或 Tat-R5M4与 Tzm-bl细胞共同孵育， 48小时后用 MTS法检测细胞活力。 分别用 0. lug, 0. 5ug, lug, 2ug的 Tat-86 (根据常规的技术进行原核表达， 引用文献： Expression of full length Tat in E. coli and its purification) 禾口 Tat_R5M4 和 Jurkat细胞共孵育， 48小时后用 Annexin_V_FITC和 PE双染色， 通过流式细 胞术检测凋亡比例。
本实验证明 Tat_R5M4明显降低了细胞毒性和诱导凋亡的能力。
实施例三： 检测 Tat-R5M4的穿膜活性
为了验证 Tat-R5M4在突变后是否影响穿膜活性， 纯化的 Tat_R5M4蛋白用 NHS-罗丹明 （NHS-Rhodanmine) 进行了标记， 标记过的蛋白分别处理 PBMC禾口 Jurkat细胞 6小时后用流式检测 Tat进入细胞的状态。 Tat_R5M4具有良好的穿 膜活性。 为了检测 Tat-R5M4进入细胞以后在胞质跟胞质的分布情况， Tzm_bl细 胞用纯化的 Tat-R5M4蛋白处理后用 Tat_R5M4的抗体进行免疫荧光检测，我们发 现 Tat-R5M4进入细胞后大部分分布在胞质， 仅有少量蛋白进入胞核。 为了检测 Tat-R5M4蛋白在小鼠体内的分布情况， 罗丹明标记的 Tat_R5M4蛋白通过尾静脉 注射进入小鼠体内， 6小时后脾脏、 胸腺、 大脑和小肠等组织制作切片后检测， 发现 Tat_R5M4明显分布在这些组织中。
本实验证明了改造后的蛋白完整保留了其穿膜特性，能够高效进入细胞和组 织发挥功能。
实施例四： HIV-1体外潜伏感染模型的构建
用带有 基因的假病毒感染激活的原代 CD4+T细胞， 通常阳性率在 5%-10%之间。 感染 3天后， 我们通过流式分选把 GFP阳性的细胞分选出来， 用添 加了 IL-2的 PRM1640培养基继续培养，同时添加 CD3和 CD28抗体进行再次激活， 一周后换成不添加任何细胞因子 PRM1640培养基， 培养 3-4周后撤除 IL-2使细 胞逐步进入静息状态。 这些静息状态的细胞即可用于潜伏激活剂的检测。
实施例五： Tat-R5M4在 HIV-1体外潜伏感染模型和临床病人来源的 CD4+T细胞 中的激活作用
用体外构建的潜伏感染系统对 Tat-R5M4激活潜伏感染的能力进行检测， 同 时用 CD3/CD28和 SAM处理作为阳性对照，三天后通过荧光显微镜观察 GFP阳性 细胞的比例。
用 CD3/CD28抗体和 SAHA作为阳性对照，同时用 Tat_R5M4或者 Tat_R5M4和 SAHA联用处理分离的临床样本 CD4+T细胞， 18小时后取上清， 提取 RNA检测上 清中的 HIV-1 RNA含量。 以上实验证明 Tat-R5M4能有效地激活 HIV-1体外潜伏感染模型和临床病人 样本来源的潜伏状态的 CD4+T细胞， 同时与 SAM联用能增强其反式激活效应。 实验六： 急性毒性实验和免疫原性检测 为了给后续的动物实验提供了一定的理论支持， Tat_R5M4蛋白的毒性被进 行进一步的检测。 首先， 用 Babl/c小鼠进行的急性毒性实验表明， 当尾静脉注 射剂量达到 40mg/ml时小鼠依然生存良好。 小鼠的丙氨酸氨基转移酶或谷丙
(ALT ) , 天冬氨酸氨基转移酶或谷草 （AST)、 尿素氮 （BREA) 和 CR (肌酐） 等 指标经检测都处于正常水平， 病理切片也显示， 除了野生型 Tat-86蛋白引起了 肝部局部炎症细胞的浸润， 小鼠的心脏、 肝脏、 脾脏、 肺部和肾都未显示明显损 伤，表明 Tat-R5M4对小鼠实验而言是安全的。同时，我们发现尾静脉注射 Tat-86 一周后， 小鼠的脾脏有明显的肿大， 说明 Tat-86会引起明显的免疫反应， 但 Tat-R5M4不会引起类似的现象。 继而我们分别用 Tat-86和 Tat_R5M4皮下注射 免疫小鼠， 在 7天， 14天， 21天时分别断尾取血检测 Tat抗体的浓度， 发现 Tat-R5M4剌激产生的抗体量明显要低于 Tat_86。 本实验证明改造后的 Tat-R5M4对小鼠相对安全， 且免疫原性显著降低， 比 野生型 Tat-86更适于进行体内实验。 SEQUENCE LISTING
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Claims

权 利 要 求 书
1. 一种 Tat蛋白在制备抗 HIV药物中的应用。
2. 一种减毒 Tat蛋白在制备抗 HIV药物中的应用，其特征在于，所述的减毒 Tat 蛋白氨基酸序列 SEQ N0:1、 SEQ N0:2、 SEQ N0:3、 SEQ N0:4所示。
3. 一种减毒 Tat蛋白， 其特征在于， 所述的减毒 Tat蛋白氨基酸序列 SEQ NO: 1、 SEQ NO: 2、 SEQ NO: 3、 SEQ NO: 4所示。
4. 一种根据权利要求 3所述的减毒 Tat蛋白的制备方法， 其特征在于， 首先选 择定点突变的位点， 对这些位点在 Tat的基因上进行定点突变， 最终得到大量保 留 Tat 的反式激活功能， 并去除其中凋亡和其他活性的减毒蛋白。
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CN103073625A (zh) * 2013-01-21 2013-05-01 中国人民解放军第二军医大学 一种缺失疏水区的人类免疫缺陷病毒I型Tat蛋白突变体序列及其应用

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CN103073625A (zh) * 2013-01-21 2013-05-01 中国人民解放军第二军医大学 一种缺失疏水区的人类免疫缺陷病毒I型Tat蛋白突变体序列及其应用

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