WO2020113917A1 - 一种用于治疗骨肉瘤的靶向多肽及其应用 - Google Patents

一种用于治疗骨肉瘤的靶向多肽及其应用 Download PDF

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WO2020113917A1
WO2020113917A1 PCT/CN2019/089086 CN2019089086W WO2020113917A1 WO 2020113917 A1 WO2020113917 A1 WO 2020113917A1 CN 2019089086 W CN2019089086 W CN 2019089086W WO 2020113917 A1 WO2020113917 A1 WO 2020113917A1
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osteosarcoma
rab22a
polypeptide
neof1
seq
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PCT/CN2019/089086
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French (fr)
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康铁邦
廖丹
钟理
沈靖南
隋建华
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中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所）
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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

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  • the present invention relates to the field of biomedical technology, in particular, to a targeting polypeptide for treating osteosarcoma and its application.
  • Osteosarcoma is a relatively rare malignant primary bone tumor, and its incidence is mainly concentrated in children and adolescents. The main cause of osteosarcoma is due to the malignant transformation of osteoblasts, which produces immature bone or bone-like tissues. Clinically, osteosarcoma is prone to lung metastasis, with high malignancy and poor prognosis.
  • osteosarcoma was mainly treated by amputation, and the five-year survival rate was only 15% to 17%. Since the 1970s, with the use of chemotherapy drugs in clinic, the survival rate of osteosarcoma patients has been greatly improved
  • the current treatment is mainly surgical resection of in situ tumors, assisted by combined chemotherapy drugs (adriamycin, cisplatin and high-dose methotrexate), the five-year survival rate of patients with osteosarcoma in situ has risen to 70 %. However, there are still 15% to 30% of patients with lung metastasis when they are first diagnosed with osteosarcoma.
  • chromosome breakage chromothripsis
  • chromosome breakage a cell is affected by a specific single mutation event, concentrated in a certain region on the chromosome thousands of times Chromosome rearrangement
  • the probability of chromosome breakage in osteosarcoma is as high as 33%, while the probability of other tumors is only 2% to 3%.
  • the most frequent driver mutation in osteosarcoma is P53, followed by RB1.
  • the mutation frequency of P53 is as high as 80%, and it mostly occurs in the structural variation segment of intron 1; although some osteosarcoma tissues have not detected the mutation of P53, a large number of them participate in the regulation of the P53 pathway
  • the molecule also has mutations, such as MDM2.
  • RB1 is the first high-frequency driving gene detected in osteosarcoma. Although its mutation frequency is not as high as P53, there are a large number of mutations related to genes related to it, such as CDK4 and cyclinD copy number amplification.
  • P53 knockout mice can spontaneously form a variety of tumors, including osteosarcoma.
  • Prx1, Osterix and CollA1 conditionally knocked out P53 the probability of spontaneous formation of osteosarcoma in mice reached nearly 100%.
  • knocking out RB1 in mice will cause embryonic lethality, and conditional knocking out RB1 mice will not spontaneously form osteosarcoma, but hybridizing RB1 conditional knockout mice with P53 knockout mice can accelerate the spontaneous formation of mice The rate of osteosarcoma.
  • Other high-frequency mutations in osteosarcoma include RECQL4, RUNX2, GRM4, ALT, ATRX, DLG2, and IGF1R, and their functions and clinical significance need to be further verified through cytology and mouse model experiments.
  • RAB22A-NeoFs is a newly identified fusion gene line in osteosarcoma, which can promote the occurrence and development of osteosarcoma; however, at present, there has not been any relevant report specifically targeting RAB22A-NeoFs fusion gene positive osteosarcoma patients.
  • the technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a RAB22A-NeoFs-iRGD fusion polypeptide for treating osteosarcoma.
  • Another object of the present invention is to provide the application of the RAB22A-NeoFs-iRGD fusion polypeptide.
  • SEQ ID NO.1 MALRELKVAL.
  • the present invention also provides another RAB22A-NeoFs-iRGD fusion polypeptide against the RAB22A-NeoFs fusion protein.
  • the amino acid sequence of the fusion polypeptide is shown in SEQ ID NO. 2:
  • SEQ ID NO. 2 MALRELKVALGGCRGDKGPDC.
  • the iRGD polypeptide sequence is looped through its terminal amino acids.
  • the RAB22A-NeoFs-iRGD fusion polypeptide is formed by coupling the protein sequence of the fusion gene line RAB22A-NeoFs and the iRGD polypeptide sequence through two glycines. Specifically, the first 10 amino acids (SEQ ID NO. 1) of the protein expressed by the RAB22A-NeoFs fusion gene line are connected to the iRGD sequence through GG.
  • SEQ ID NO. 1 amino acids of the protein expressed by the RAB22A-NeoFs fusion gene line
  • the present invention has proved through in vitro experiments that the polypeptides shown in SEQ ID NO. 1 and SEQ ID NO. 2 can antagonize the ability of RAB22A-NeoF1 to promote the adhesion, migration and invasion of osteosarcoma cells; in vivo experiments have shown that the fusion polypeptide can antagonize The ability of RAB22A-NeoF1 to promote the metastasis of osteosarcoma cells.
  • the present invention claims the application of the polypeptide shown in SEQ ID NO. 1 or SEQ ID NO. 2 in the preparation of osteosarcoma therapeutic drugs.
  • the osteosarcoma contains RAB22A-NeoFs fusion protein.
  • the osteosarcoma contains RAB22A-NeoF1 fusion protein.
  • SEQ ID NO. 1 or SEQ ID NO. 2 polypeptide in the preparation of drugs that antagonize RAB22A-NeoF1 fusion protein to promote the adhesion, migration and invasion of osteosarcoma cells.
  • SEQ ID NO. 1 or SEQ ID NO. 2 polypeptides in the preparation of drugs that antagonize the RAB22A-NeoF1 fusion protein to promote the metastasis of osteosarcoma cells.
  • the present invention also provides a medicine for treating osteosarcoma, which contains the polypeptide shown in SEQ ID NO: 1 or SEQ ID NO. 2.
  • the dosage of the polypeptide shown in SEQ ID NO: 1 or SEQ ID NO. 2 of the present invention is 1 mg to 10 mg/kg.
  • the present invention has the following beneficial effects:
  • the present invention provides an anti-RAB22A-NeoFs fusion protein polypeptide whose amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.2; in vitro experiment results show that the polypeptide can antagonize RAB22A-NeoF1 to promote osteosarcoma cells The ability of adhesion, migration and invasion; the results of in vivo experiments show that the polypeptide can antagonize the ability of RAB22A-NeoF1 to promote the transfer of osteosarcoma cells, indicating that the polypeptide of the present invention has a large application prospect in the preparation of osteosarcoma treatment drugs.
  • Figure 1 shows the binding of RAB22A-NeoF1 to the RAP1GDS1 protein through its first 10 amino acid sequences.
  • Figure 2 shows that RBA22A-NoeF1 exerts its function of promoting osteosarcoma migration and invasion through its first 10 amino acids.
  • Fig. 3 shows that the fusion polypeptide can antagonize the ability of RAB22A-NeoF1 to promote the adhesion of osteosarcoma cells; where Pep-wt represents the fusion polypeptide and Pep-DD represents the control polypeptide.
  • Fig. 4 shows that the fusion polypeptide can antagonize the ability of RAB22A-NeoF1 to promote osteosarcoma cell migration and invasion, where Pep-wt represents the fusion polypeptide and Pep-DD represents the control polypeptide.
  • FIG. 5 shows that the fusion polypeptide can antagonize the ability of RAB22A-NeoF1 to promote osteosarcoma cell metastasis; where WT represents the fusion polypeptide and DD represents the control polypeptide.
  • test methods used in the following examples are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials.
  • RAB22A-NeoF1 exerts its function mainly through interaction with RAP1GDS1. Therefore, the RAB22A-NeoF1 and RAP1GDS1 protein expression system was constructed to identify the RAB22A-NeoF1 and RAP1GDS1 protein interaction regions.
  • reaction conditions are as follows:
  • the reaction system is as follows:
  • connection reaction is carried out at room temperature, the reaction system is as follows:
  • Example 2 Function of the first 10 amino acids of RAB22A-NeoF1 in osteosarcoma
  • TAKARA's high-fidelity enzyme MIX primSTAR was used to amplify the full-length RAB22A-NeoF1 and Del-N10 sequences with the specific primers of Example 1.
  • the system is as follows:
  • reaction conditions are as follows:
  • the reaction system is as follows:
  • the connection reaction is carried out at room temperature.
  • the reaction system is as follows:
  • the culture supernatant is sterilized and filtered with a 0.45 ⁇ m non-nitrocellulose filter (cellulose acetate and other filters) to prevent the nitrocellulose from binding to the virus cell membrane protein and destroying the virus, removing cell debris and contaminated packaging cells , Temporary storage at 4 °C, long-term storage at -80 °C for future use.
  • a 0.45 ⁇ m non-nitrocellulose filter cellulose acetate and other filters
  • each medium contains the same concentration of puromycin. Adjust the concentration of puromycin according to cell death;
  • 2Hydration basement membrane add 50 ⁇ L of serum-free culture medium containing 10g/L BSA to each well and incubate at 37°C for 30min.
  • 3Cultured cells 24 hours of routine culture.
  • Cell counting take several visual fields to count the number of cells. Generally, 3 to 10 visual fields are randomly selected.
  • FIG. 2 The experimental results are shown in FIG. 2. From FIG. 2, it can be seen that the fusion protein RAB22A-NeoF1 exerts its function of promoting osteosarcoma cell migration and invasion through the first 10 amino acids.
  • the fusion polypeptide was synthesized by Sinopeptide Biochemical Co., Ltd.
  • the fusion polypeptide sequence: MALRELKVALGGCRGDKGPDC was prepared by connecting the first 10 amino acids MALRELKVAL of the protein expressed in the RAB22A-NeoFs fusion gene line through GG to iRGD sequence (CRGDKGPDC).
  • the iRGD polypeptide sequence loops through its terminal amino acids.
  • Coating of substrate add 50 ⁇ L (2 ⁇ g)/well of LN and FN to a 96-well culture plate, place in a refrigerator at 4° C. overnight, and use BSA as the control substrate;
  • test results are shown in Figure 3.
  • the results indicate that the fusion polypeptide can antagonize the ability of RAB22A-NeoF1 to promote osteosarcoma cell adhesion.
  • test results are shown in Figure 4.
  • the results show that the fusion polypeptide can antagonize the ability of RAB22A-NeoF1 to promote osteosarcoma cell migration and invasion.
  • Osteosarcoma cells (143B-Vec and 143B-RAB22A-NeoF1) were digested and collected by centrifugation. After washing twice with PBS, the cells were resuspended with PBS. The final cell density was 8 ⁇ 10 5 /20 ⁇ L, which was far from the proximal femur of the tibia 20 ⁇ L of cell suspension was injected into the end.
  • the amino acid sequence of the fusion polypeptide is MALRELKVALGGCRGDKGPDC; the amino acid sequence of the control polypeptide is MALDELDVALGGCRGDKGPDC (mutation of amino acid R and amino acid K in the fusion polypeptide sequence into amino acid D);
  • mice are divided into the following ten groups:
  • Group A negative control group 143B-Vec
  • Group B Positive control group 143B-RAB22A-NeoF1;
  • Group C tail vein injection of fusion polypeptide (1mg/kg);
  • Group D control polypeptide (1mg/kg) injected into the tail vein;
  • Group C tail vein injection of fusion polypeptide (5mg/kg);
  • Group D control polypeptide (5mg/kg) injected into the tail vein;
  • Group C tail vein injection of fusion polypeptide (10mg/kg);
  • Group D control polypeptide (10 mg/kg) injected into the tail vein;
  • Group C tail vein injection of fusion polypeptide (20mg/kg);
  • Group D tail vein injection of control polypeptide (20mg/kg);
  • the frequency of injection was once every other day for 3 weeks, and the fluorescence intensity of lung metastasis in tumor-bearing mice was observed after 3 weeks.
  • Fig. 5 The experimental results are shown in Fig. 5, from which it can be seen that the fusion polypeptide can antagonize the ability of RAB22A-NeoF1 to promote the metastasis of osteosarcoma cells, indicating that the fusion polypeptide of the present invention can be further used to prepare osteosarcoma therapeutic drugs.

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Abstract

本发明公开了一种用于治疗骨肉瘤的靶向多肽及其应用；所述多肽的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示；体外实验结果显示，所述多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞黏附、迁移以及侵袭的能力；体内实验结果显示，所述多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞转移的能力，表明本发明的多肽在制备骨肉瘤治疗药物中具有较大的应用前景。

Description

一种用于治疗骨肉瘤的靶向多肽及其应用 技术领域
本发明涉及生物医学技术领域，具体地，涉及一种用于治疗骨肉瘤的靶向多肽及其应用。
背景技术
骨肉瘤(Osteosarcoma)是比较罕见的恶性原发性骨肿瘤，其发病人群主要集中在儿童和青少年。骨肉瘤发生的主要原因是由于成骨细胞发生恶性转化，产生不成熟的骨质或者类骨质组织，临床发现，骨肉瘤容易发生肺部转移，恶性程度高，预后差。
早期主要通过截肢手术治疗骨肉瘤，五年生存率仅为15％～17％，自二十世纪七十年代以来，随着化疗药物应用于临床，骨肉瘤患者的生存率得到了极大的提高，目前的治疗手段以手术切除原位肿瘤为主，辅助以联合化疗药物(阿霉素，顺铂以及大剂量甲氨蝶呤)，原位骨肉瘤的病人的五年生存率已上升到70％。但是仍然存在15％～30％的患者在初次诊断为骨肉瘤时就已经出现了肺部转移病灶，近三十年来，复发或者肺转移的患者的五年生存率一直没有得到提高，仅为20％左右，由此可以看出，寻找全新的可有效治疗骨肉瘤的药物是十分必要且急迫的，因此，从分子层面以及遗传学角度来解析骨肉瘤的发病机制、进展以及预后，对于寻找治疗骨肉瘤的靶向药物十分重要。
骨肉瘤中会发生高频率的结构变异、拷贝数变异以及单核苷酸变异，并且该变异频率远远高于其他肿瘤。最近的全基因组测序结果还发现了一个全新的遗传信息变异现象：染色体断裂(chromothripsis)，即细胞受到某一特定单个的突变事件影响，在染色体上的某一段区域集中发生成千上百次的染色体重排现象，其在骨肉瘤中发生染色体断裂的概率高达33％，而其他肿瘤的概率仅为2％～3％。
根据已有的研究报导，在骨肉瘤中发生频率最高的驱动突变为P53，其次为RB1。在所有测序的结果中，P53的突变频率高达80％，而且多发生于1号内含子的结构变异区段，尽管还有部分骨肉瘤组织没有检测到P53的突变，但是大量参与调控P53通路的分子同样存在突变，比如MDM2。而RB1是在骨肉瘤中第一个检测到的高频驱动基因，虽然其本身发生突变的频率没有P53高，但与其相 关基因同样存在大量的突变，比如CDK4和cyclin D的拷贝数扩增。另外，P53以及RB1的驱动基因功能都已经在小鼠模型中得到验证，试验证明P53敲除小鼠能自发形成多种肿瘤，其中就包括骨肉瘤。在表达了Prx1、Osterix和CollA1的小鼠中条件敲除P53，小鼠自发形成骨肉瘤的概率将近达到100％。虽然在小鼠中敲除RB1会出现胚胎致死的现象，条件敲除RB1小鼠也不会自发形成骨肉瘤，但是将RB1条件敲除小鼠与P53敲除小鼠杂交能加速小鼠自发形成骨肉瘤的速率。骨肉瘤中其他的高频突变还包括RECQL4、RUNX2、GRM4、ALT、ATRX、DLG2和IGF1R等，还需进一步的通过细胞学以及小鼠模型实验验证其功能以及临床意义。
骨肉瘤中除了基因组遗传信息的变异，大量调控肿瘤生长的关键分子的mRNA以及蛋白水平也存在异常高表达，包括IGF、VEGF、HER2、PDGFR、MET等等，为骨肉瘤分子治疗提供了大量潜在靶点。RAB22A-NeoFs是骨肉瘤中新鉴定的融合基因系，能够促进骨肉瘤的发生发展；但是目前，还未见有特异性针对RAB22A-NeoFs融合基因阳性骨肉瘤患者的多肽的相关报道。
发明内容
本发明要解决的技术问题是克服现有技术的上述不足，提供一种用于治疗骨肉瘤的RAB22A-NeoFs-iRGD融合多肽。
本发明的另一目的是提供所述RAB22A-NeoFs-iRGD融合多肽的应用。
本发明的上述目的是通过以下技术方案给予实现的：
一种抗RAB22A-NeoFs融合蛋白的多肽，所述多肽的氨基酸序列如SEQ ID NO.1所示：
SEQ ID NO.1：MALRELKVAL。
同时，本发明还提供另外一种抗RAB22A-NeoFs融合蛋白的RAB22A-NeoFs-iRGD融合多肽，所述融合多肽的氨基酸序列如SEQ ID NO.2所示：
SEQ ID NO.2：MALRELKVALGGCRGDKGPDC。
优选地，所述iRGD多肽序列通过其末端氨基酸成环。
所述RAB22A-NeoFs-iRGD融合多肽通过两个甘氨酸将靶向融合基因系RAB22A-NeoFs的蛋白序列与iRGD多肽序列偶联而成。具体是将RAB22A-NeoFs融合基因系所表达蛋白的前10个氨基酸(SEQ ID NO.1)通过 GG连接于iRGD序列。通过将肿瘤靶向多肽iRGD与靶向融合基因系RAB22A-NeoFs的蛋白序列相连，可以更好的发挥SEQ ID NO.1所述多肽的靶向治疗作用。
本发明通过体外实验已证明，SEQ ID NO.1和SEQ ID NO.2所示的多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞黏附、迁移以及侵袭的能力；通过体内实验证明，该融合多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞转移的能力。
因此，本发明请求保护SEQ ID NO.1或SEQ ID NO.2所示多肽在制备骨肉瘤治疗药物中的应用。
优选地，所述骨肉瘤含有RAB22A-NeoFs融合蛋白。
更优选地，所述骨肉瘤含有RAB22A-NeoF1融合蛋白。
同时，下列应用也在本发明保护范围内：
SEQ ID NO.1或SEQ ID NO.2所示多肽在制备拮抗RAB22A-NeoF1融合蛋白促进骨肉瘤细胞黏附、迁移、侵袭的药物中的应用。
SEQ ID NO.1或SEQ ID NO.2多肽在制备拮抗RAB22A-NeoF1融合蛋白促进骨肉瘤细胞肺转移的药物中的应用。
另外，本发明还提供一种骨肉瘤治疗药物，所述药物含有SEQ ID NO:1或SEQ ID NO.2所示多肽。
优选地，本发明SEQ ID NO:1或SEQ ID NO.2所示多肽的剂量为1mg～10mg/kg。
与现有技术相比，本发明具有以下有益效果：
本发明提供了一种抗RAB22A-NeoFs融合蛋白的多肽，其氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示；体外实验结果显示，所述多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞黏附、迁移以及侵袭的能力；体内实验结果显示，所述多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞转移的能力，表明本发明的多肽在制备骨肉瘤治疗药物中具有较大的应用前景。
附图说明
图1为RAB22A-NeoF1通过其前10个氨基酸序列与RAP1GDS1蛋白的结合。
图2为RBA22A-NoeF1通过其前10个氨基酸发挥其促进骨肉瘤迁移和侵袭的功能。
图3为融合多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞黏附的能力；其中，Pep-wt表示融合多肽，Pep-DD表示对照多肽。
图4为融合多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞迁移以及侵袭的能力，其中，Pep-wt表示融合多肽，Pep-DD表示对照多肽。
图5为融合多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞转移的能力；其中，WT表示融合多肽，DD表示对照多肽。
具体实施方式
下面结合说明书附图及具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1 RAB22A-NeoF1与RAP1GDS1蛋白相互作用区域的鉴定
本发明人团队经过前期的大量研究发现，RAB22A-NeoF1发挥其功能主要是通过与RAP1GDS1的相互作用。因此通过构建RAB22A-NeoF1与RAP1GDS蛋白表达体系来鉴定RAB22A-NeoF1与RAP1GDS1蛋白相互作用区域。
具体包括如下步骤：
一、蛋白表达载体的构建
1、RAB22A-NeoF1与RAP1GDS1全长以及截断体表达载体构建
(1)引物序列：
①RAP1GDS1-F:
Figure PCTCN2019089086-appb-000001
RAP1GDS1-R:
Figure PCTCN2019089086-appb-000002
②RAB22A-NeoF1-Full-length-F:
Figure PCTCN2019089086-appb-000003
RAB22A-NeoF1-Full-length-R:
Figure PCTCN2019089086-appb-000004
③RAB22A-NeoF1-de l 1-10-F:
Figure PCTCN2019089086-appb-000005
RAB22A-NeoF1-del 1-10-R:
Figure PCTCN2019089086-appb-000006
④RAB22A-NeoF1-del 1-20-F:
Figure PCTCN2019089086-appb-000007
RAB22A-NeoF1-del 1-20-R:
Figure PCTCN2019089086-appb-000008
⑤RAB22A-NeoF1-del 1-30-F:
Figure PCTCN2019089086-appb-000009
RAB22A-NeoF1-del 1-30R:
Figure PCTCN2019089086-appb-000010
⑥RAB22A-NeoF1-del 1-40-F:
Figure PCTCN2019089086-appb-000011
RAB22A-NeoF1-del 1-40-R:
Figure PCTCN2019089086-appb-000012
⑦RAB22A-NeoF1-1-38-F:
Figure PCTCN2019089086-appb-000013
RAB22A-NeoF1-1-38-R:
Figure PCTCN2019089086-appb-000014
(2)序列扩增
使用TAKARA的高保真酶MIX primSTAR和上述引物扩增序列，体系如下：
Figure PCTCN2019089086-appb-000015
将其混匀后置于PCR仪器中，反应条件如下：
95℃预变性5min后，下列条件进行38个循环：
98℃                                15s
55℃～60℃                          30s
72℃                                5kb/min
38个循环过后，再进行一步72℃，10min延长反应，此后回收扩增产物；
(3)酶切(包括过表达载体PBABE和扩增产物)
在200μL PCR管中进行酶切反应。反应体系如下：
Figure PCTCN2019089086-appb-000016
37℃酶切1h，回收酶切产物。
(4)连接
在室温进行连接反应，反应体系如下：
Figure PCTCN2019089086-appb-000017
室温连接过夜。
(5)转化
将10μL连接产物加入到感受态细胞(Stbl3)中，轻轻混匀，冰浴20min，42℃热激60s，随后冰浴2min，加入500μL无抗生素的LB培养基，摇床37℃，200rpm，30min。使用无菌玻璃珠在需转化质粒对应的抗性固体LB培养板上涂板，放入37℃细菌培养箱中，培养过夜，第二天挑取单克隆菌落，送SANGER测序验证克隆是否成功。
2、质粒瞬时转染
利用Lipofectamine2000进行质粒瞬时转染，以细胞培养6孔板为例：
1)细胞接种密度为70％，转染前换成无血清完全培养基。
2)将转染试剂Lipofectamine 2000与目的质粒按照体积质粒比1:1加入500uL Opti-MEM优化培养基中，充分混匀，静置10min。
3)将孵育好的转染液加入细胞中，6h后换成10％FBS完全培养基，继续培养48～72h后进行目的实验。
3、蛋白免疫共沉淀(Co-Immunoprecipitation)
在293t中共转染SFB-tagged-RAB22A-NeoF1-Full-length或者各个截断体(1-38/△1-10/△1-20/△1-30/△1-40)与HA-tagged-RAP1GDS1。48小时后收集蛋白，在蛋白裂解液中加入S-beads或者V5-agarose，4℃摇床孵育6小时，10000rpm X 1min，弃上清，加入RIPA清洗，再次离心弃上清，重复以上步骤5次，加入loading buffer变性，然后进行Western blotting，使用HA抗体和Flag抗体检测蛋白之间的相互作用。
二、检测结果
实验结果结果如图1所示，从图1可以看出，融合蛋白RAB22A-NeoF1通过其前10个氨基酸与RAP1GDS1相互作用。
实施例2 RAB22A-NeoF1前10个氨基酸在骨肉瘤中的功能
一、方法
1、稳定过表达载体的构建
(1)RAB22A-NeoF1全长以及Del-N10(去除RAB22A-NeoF1前10个氨基酸)的序列扩增
使用TAKARA的高保真酶MIX primSTAR，以实施例1的特异性引物扩增RAB22A-NeoF1全长以及Del-N10序列，体系如下：
Figure PCTCN2019089086-appb-000018
将其混匀后置于PCR仪器中，反应条件如下：
95℃预变性5min后，下列条件进行38个循环：
98℃                               15s
55-60℃                            30s
72℃                           5kb/min
38个循环过后，再进行一步72℃，10min延长反应，此后回收扩增产物；
(2)酶切(包括过表达载体PBABE和扩增产物)
在200μL PCR管中进行酶切反应。反应体系如下：
Figure PCTCN2019089086-appb-000019
Figure PCTCN2019089086-appb-000020
37℃酶切1h，回收酶切产物。
(3)连接
在室温进行连接反应，反应体系如下:
Figure PCTCN2019089086-appb-000021
室温连接过夜
(4)转化
将10μL连接产物加入到感受态细胞(Stbl3)中，轻轻混匀，冰浴20min，42℃热激60s，随后冰浴2min，加入500μL无抗生素的LB培养基，摇床37℃，200rpm，30min。使用无菌玻璃珠在需转化质粒对应的抗性固体LB培养板上涂板，放入37℃细菌培养箱中，培养过夜，第二天挑取单克隆菌落，送SANGER测序验证克隆是否成功。
2、包装病毒
(1)转染前一天在直径10cm平皿中接种293T细胞，用10％FBS的DMEM培养基培养；
(2)包装细胞50％融合时共转染质粒pBABE(vector,fusion,Del-N10)与病毒的包装质粒PIK各6μg以及30μL标准的脂质体转染试剂LipofectamineTM2000；
(3)6h后换液，加入10mL的新鲜培养基；
(4)48～72h后培养上清用0.45μm的非硝酸纤维滤器(醋酸纤维素等滤器)消毒过滤，以防止硝酸纤维与病毒细胞膜蛋白结合而破坏病毒，清除细胞碎屑及污染的包装细胞，4℃暂时保存，-80℃长期保存备用。
3、逆转录病毒感染目标细胞
(1)感染前一天(18～24h)，目标细胞铺板(六孔板)；
(2)待细胞生长至50％密度，2mL的含病毒上清液加入8μg/mL 1000×的Polybrene；
(3)6h后更换正常培养基10％FBS的DMEM，通常感染2次；
(4)48h后开始用浓度0.5～1.0μg/mL(范围500～1000μg/mL，每次递减100μg/mL)嘌呤霉素筛选；
(5)每1～2天更换培养基，培养过程中各培养基均含有相同的嘌呤霉素浓度，根据细胞死亡情况调整嘌呤霉素浓度；
(6)约1周后细胞构建成功。
4、细胞迁移以及侵袭实验
(1)Transwell小室制备：包被基质胶的Transwell的小室制备
①包被基底膜：将50mg/L Matrigel 1:8稀释液包被Transwell小室底部膜的上室面，4℃风干(或冰箱4℃过夜)。
②水化基底膜：每孔加入50μL含10g/L BSA的无血清培养液，37℃培养30min。
(2)制备细胞悬液
取成功构建得到的过表达细胞系，消化细胞，终止消化后离心弃去培养液，用PBS洗1～2遍，用无血清或低血清的培养基重悬，调整细胞密度至1×10 5～10×10 5。
(3)接种细胞
①吸出培养板中残余液体，取细胞悬液100～200μL加入Transwell上室；
②24孔板下室一般加入500μL含FBS或趋化因子的培养基；
③培养细胞：常规培养24h。
5、结果统计：通过计数穿过膜的细胞数来比较侵袭和迁移的能力。
(1)用棉签擦去基质胶和上室内的细胞。
(2)0.1％结晶紫染色，方法如下：1)4％多聚甲醛固定15min，冲洗；2)浸泡，0.05％结晶紫染色15～30min；3)PBS冲洗2～3次。
(3)细胞计数：取若干个视野计数细胞个数一般随机选取3～10个视野。
二、检测结果
实验结果如图2所示，从图2可知，融合蛋白RAB22A-NeoF1通过其前10个氨基酸发挥其促进骨肉瘤细胞迁移以及侵袭的功能。
实施例3 融合多肽的治疗价值
1、融合多肽的合成
所述融合多肽为中肽生化有限公司合成的，所述融合多肽序列：MALRELKVALGGCRGDKGPDC，通过将RAB22A-NeoFs融合基因系所表达蛋白的前10个氨基酸MALRELKVAL通过GG连接于iRGD序列(CRGDKGPDC)制备得到。所述iRGD多肽序列通过其末端氨基酸成环。
2、细胞黏附实验
(1)基质的包被：将LN、FN各50μL(2μg)/孔加至96孔培养板，置4℃冰箱过夜备用，BSA为对照基底；
(2)用前PBS洗两次重新水化，10％BSA 50μL/孔以封闭结合位点，孵育60min，PBS漂洗三次×5min备用；
(3)取生长良好的细胞制备成单细胞悬液浓度为8×10 4个/mL，按200μL/孔加入包被不同基质(Fn或Ln)的96孔板，37℃，5％CO 2条件下孵育60min；
(4)弃去培养基，加入PBS轻柔洗去未粘附细胞，共3次；
(5)加入4％的多聚甲醛进行固定10分钟；
(6)弃去4％的多聚甲醛，加入结晶紫进行染色30分钟；
(7)弃去结晶紫，PBS洗3次，显微镜下拍照计数；
检测结果如图3所示，该结果表明融合多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞黏附的能力。
3、细胞迁移以及侵袭实验
本实验的操作及条件与实施例2步骤4基本相同，不同之处在于基质胶不需包被。
检测结果如图4所示，结果表明融合多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞迁移以及侵袭的能力。
4、动物实验
构建RAB22A-NeoF1阳性骨肉瘤小鼠模型。将骨肉瘤细胞(143B-Vec与143B-RAB22A-NeoF1)消化并离心收集，用PBS洗两次后，用PBS重悬细胞，最终细胞密度为8×10 5/20μL，于胫骨近端股骨远端注射20μL细胞悬液。融合多肽的氨基酸序列为MALRELKVALGGCRGDKGPDC；对照多肽的氨基酸序列为MALDELDVALGGCRGDKGPDC(将融合多肽序列上的第4位氨基酸R和第 7位氨基酸K突变成了氨基酸D)；
将小鼠分为以下十组：
A组：阴性对照组143B-Vec；
B组：阳性对照组143B-RAB22A-NeoF1；
C组：尾静脉注射融合多肽(1mg/kg)；
D组：尾静脉注射对照多肽(1mg/kg)；
C组：尾静脉注射融合多肽(5mg/kg)；
D组：尾静脉注射对照多肽(5mg/kg)；
C组：尾静脉注射融合多肽(10mg/kg)；
D组：尾静脉注射对照多肽(10mg/kg)；
C组：尾静脉注射融合多肽(20mg/kg)；
D组：尾静脉注射对照多肽(20mg/kg)；
注射频率为隔天1次，持续3周，3周后观察荷瘤小鼠肺部转移荧光强度。
实验结果如图5所示，由此可以看出，融合多肽能够拮抗RAB22A-NeoF1促进骨肉瘤细胞转移的能力，表明本发明的融合多肽可进一步用于制备骨肉瘤治疗药物。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (9)

  1. 一种抗RAB22A-NeoFs融合蛋白的多肽，其特征在于，所述多肽的氨基酸序列如SEQ ID NO.1所示。
  2. 一种抗RAB22A-NeoFs融合蛋白的RAB22A-NeoFs-iRGD融合多肽，其特征在于，所述融合多肽的氨基酸序列如SEQ ID NO.2所示。
  3. 根据权利要求2所述的融合多肽，其特征在于，所述iRGD多肽序列通过其末端氨基酸成环。
  4. 权利要求1或2所述多肽在制备骨肉瘤治疗药物中的应用。
  5. 根据权利要求4所述应用，其特征在于，所述骨肉瘤含有RAB22A-NeoFs融合蛋白。
  6. 根据权利要求4所述应用，其特征在于，所述骨肉瘤含有RAB22A-NeoF1融合蛋白。
  7. 权利要求1或2所述多肽在制备拮抗RAB22A-NeoF1融合蛋白促进骨肉瘤细胞黏附、迁移、侵袭的药物中的应用。
  8. 权利要求1或2所述多肽在制备拮抗RAB22A-NeoF1融合蛋白促进骨肉瘤细胞肺转移的药物中的应用。
  9. 一种骨肉瘤治疗药物，其特征在于，所述药物含有SEQ ID NO.1或SEQ ID NO.2所示多肽。
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