WO2019100777A1 - 一种抑制肿瘤血管新生的peptibody多表位疫苗及其应用 - Google Patents

一种抑制肿瘤血管新生的peptibody多表位疫苗及其应用 Download PDF

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WO2019100777A1
WO2019100777A1 PCT/CN2018/101619 CN2018101619W WO2019100777A1 WO 2019100777 A1 WO2019100777 A1 WO 2019100777A1 CN 2018101619 W CN2018101619 W CN 2018101619W WO 2019100777 A1 WO2019100777 A1 WO 2019100777A1
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bfgf
vegf
peptibody
epitope vaccine
tumor
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PCT/CN2018/101619
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French (fr)
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邓宁
邓艳瑞
张利刚
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暨南大学
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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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

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  • the invention relates to the field of vaccines, in particular to a peptibody multi-epitope vaccine for inhibiting tumor angiogenesis and application thereof.
  • neovascularization is the premise of tumor growth, and tumor microvessels provide various nutrients necessary for growth of tumor cells.
  • tumor cells promote angiogenesis of tumors by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) released by autocrine and paracrine means.
  • VEGF and bFGF have a synergistic effect in promoting angiogenesis.
  • VEGF promotes the expression of platelet-derived growth factor (PDGF-B), and bFGF promotes the expression of PDGFR- ⁇ , thereby synergistically promoting tumor angiogenesis and tumor growth.
  • PDGF-B platelet-derived growth factor
  • bFGF promotes the expression of PDGFR- ⁇
  • bevacizumab (Avastin) is a recombinant monoclonal antibody against human VEGF. It was approved by the US FDA in 2004 and inhibited by inhibiting angiogenesis. Tumors, and for the treatment of malignant colorectal cancer, non-small cell lung cancer, malignant renal cell carcinoma, breast cancer and brain cancer. Monoclonal antibodies targeting bFGF can also effectively inhibit tumor angiogenesis and have been reported.
  • the polypeptide against VEGF immunizes the body to induce the production of specific anti-VEGF antibodies, thereby inhibiting the proliferation, migration and metastasis of tumor cells.
  • the multi-epitope peptide targeting the receptor binding region of bFGF and the heparin binding region as a vaccine can produce specific anti-bFGF antibodies to reduce tumor angiogenesis and inhibit tumor growth and deterioration.
  • anti-VEGF antibody drugs have found that long-term use of bevacizumab can lead to tumor resistance, compensatoryly promote the expression of bFGF and platelet-derived growth factor (PDGF), thereby promoting tumor angiogenesis and promoting tumor recurrence. And growth, leading to resistance.
  • PDGF platelet-derived growth factor
  • the existing drugs that inhibit tumor angiogenesis are mostly directed against the inhibition of a single factor, and may cause compensatory drug resistance in long-term administration and limit its clinical effects.
  • the multi-epitope tumor vaccine designed and developed for VEGF/bFGF has dual anti-tumor effects on both VEGF and bFGF. It can induce high titers of anti-VEGF and bFGF antibodies at the same time, effectively inhibiting tumor vascular and lymphangiogenesis. Inhibit tumor growth and metastasis.
  • Overcoming the drawbacks of the previous single factor antibody or polypeptide may cause the body to stimulate the proliferation of tumor blood vessels through the compensatory proliferation of another pathway, promote the growth and metastasis of the tumor, effectively solve the problem of compensatory drug resistance, and better Inhibit the tumor. Suitable for the treatment of a variety of solid tumors.
  • the primary object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a peptibody multi-epitope vaccine that inhibits tumor angiogenesis.
  • Peptibody is a novel vaccine we designed to fuse small epitope peptides with human immunoglobulin Fc fragments. It can effectively improve the immunogenicity and stability of the vaccine, and because of the Fc fragment, the protein can be purified by Protein A, and the purification efficiency is higher.
  • the vaccine is composed of three immunophilic bFGF epitope peptides and three VEGF epitope peptides, which are ligated into a multi-epitope peptide by a flexible Linker (GGGS) and coupled to the Fc fragment of IgG1.
  • GGGS flexible Linker
  • Another object of the present invention is to provide the use of the peptibody multi-epitope vaccine for inhibiting tumor angiogenesis.
  • a peptibody multi-epitope vaccine for inhibiting tumor angiogenesis the amino acid sequence of which is as follows:
  • peptide 4 is derived from epitope peptides 75 to 86 of bFGF protein
  • peptide 5 is derived from epitope peptides 34 to 48 of bFGF protein
  • peptide 6 is derived from epitope peptides 97 to 113 of bFGF protein
  • peptide 1 peptide 2 and peptide 3, peptide 4, peptide 5 and peptide 6 are sequentially linked by amino acid sequence GGGS; peptide 3 and peptide 4 are linked by amino acid sequence GGGGSCGGGGS; Fc fragment is derived from human lymphocyte IgG1; Antibody sequence part (provided by Shenggong Bioengineering Co., Ltd.).
  • the nucleotide sequence encoding the peptibody multi-epitope vaccine that inhibits tumor angiogenesis is as follows:
  • peptibody multi-epitope vaccine for inhibiting tumor angiogenesis in the preparation of an antitumor drug.
  • the multi-epitope vaccine provided by the present invention is a fusion multi-epitope vaccine containing human IgG1 Fc segment, which comprises three human VEGF epitopes, three bFGF epitopes and an intact Fc segment of human IgG1;
  • the anti-VEGF and anti-bFGF antibodies can simultaneously inhibit the high expression of VEGF and bFGF cell growth factors in tumors.
  • the multi-epitope vaccine can induce BABL/C mice to obtain specific immune response against VEGF/bFGF, and can effectively inhibit the growth of C57BL/6LL-2 tumor-bearing mice, and the tumor growth inhibition rate reaches 60. ⁇ 70%. Therefore, it is expected to be an auxiliary drug for cancer treatment and prevention.
  • FIG. 1 is a schematic diagram of a peptibpdy and a vector of the present invention; wherein, Figure A is an expression vector diagram; and Figure B is a construction diagram of peptibpdy.
  • Figure 2 is a PCR identification map of pET-28a-VEGF/bFGF-Fc vector; wherein, Lane A in Figure a is DNA Marker, and Lane 1 is a PCR fragment of VEGF/bFGF of pET28a-VEGF/bFGF-Fc vector; Lane M in the middle is DNA Marker, and Lane 1 is a PCR fragment of Fc of the pET28a-VEGF/bFGF-Fc vector.
  • Figure 3 is a restriction map of pET-28a-VEGF/bFGF-Fc vector; wherein lane M is DNA Marker, lane 1 is a cleavage fragment of Fc of pET28a-VEGF/bFGF-Fc vector, and lane 2 is pET28a- A cleavage fragment of VEGF/bFGF of VEGF/bFGF-Fc.
  • Figure 4 is a diagram showing the expression purification and identification of a multi-epitope vaccine; wherein Lane M is the protein Marker, Lane 1 is a purified sample of the Peptibody complex peptide, and Lane 2 is the Western Blot identification result of the purified sample.
  • Figure 5 is a graph showing the results of serum titer and antitumor effect of mice immunized with multi-epitope vaccine; wherein, Figure a is a graph showing the titer of anti-VEGF and bFGF antibodies in the serum of mice after Peptibody immunization by ELISA, and Figure b is the tumor. Growth curve, Figure c is a graph of the weight of the tumor, and Figure d is a photographic image of the isolated tumor.
  • Figure 6 is a diagram showing the immunohistochemical results of neovascularization and lymphatic vessels in tumor-bearing mice; Fig. a is a graph showing the results of CD31 and LYVE1 staining of tumor tissues, and Fig. b is a graph showing the results of quantitative analysis of microvessel density of tumor tissues. Figure c is a graph showing the results of quantitative analysis of lymphatic vessel density.
  • the VFGF immunogenic epitope and bFGF immunogenic epitope were screened, and the obtained epitope peptide and phage display technology were selected from the peptide library.
  • the linker is ligated to the Fc fragment of the IgG1 antibody, and the nucleotide sequence thereof is shown in SEQ ID NO.
  • XhoI and NheI restriction sites were added before and after the sequence shown in SEQ ID NO. 2 to obtain plasmid A.
  • SEQ ID NO. 2 The sequence of SEQ ID NO. 2 was cloned into pET-28a by XhoI and NheI sites, and the resulting expression vector is shown in Figure 1A, and the expressed multi-epitope peptide is shown in Figure 1B.
  • Plasmid A and vector pET28a were digested with XhoI and NheI, respectively, and the fragment of interest was recovered and ligated to construct expression plasmid pET28a-VEGF/bFGF-Fc.
  • the enzyme digestion system is shown in Table 1:
  • connection system is shown in Table 2:
  • the ligation product was introduced into DH5 ⁇ competent cells, plated on a screening medium plate containing kanamycin, and single colonies were picked.
  • the PCR amplification system was pre-denatured at 95 ° C for 4 min, denatured at 95 ° C for 45 s, annealed at 58 ° C for 30 s, extended at 72 ° C for 1 min, and after 25 cycles, extended at 72 ° C for 10 min.
  • the primers that amplify the VEGF/bFGF fragment are as follows:
  • Upstream primer F1 5'-CCATGGGCCAGAAACGTAAACGTAAGAAATCCC-3';
  • the downstream primer R1 5'-GGATCCGGAGGTGTATTTACGGGAAC C-3'.
  • the primers for amplifying the Fc fragment are as follows:
  • Upstream primer F2 5'-GGATCC TCTTGTGACAAAACTCACAC-3';
  • Downstream primer R2 5'-CTCGAGTTTTACCCGGAGACAGGGAG-3'.
  • the enzyme digestion system is shown in Table 4:
  • the correct plasmid pET28a-VEGF/bFGF-Fc was transferred into BL21 E. coli expression strain, and the expression was induced.
  • the LB medium was inoculated at a volume ratio of 1:100, cultured at 37 ° C, 220 rpm for 5 h, and 0.1 mM / L was added. IPTG induction for 5 h.
  • the cells were collected by centrifugation at 4000 rpm, resuspended in 20 mM PB, 500 mM NaCl, pH 7.4 buffer, ultrasonic (80 Hz, open for 3 s, closed for 2 s, 20 min), and then centrifuged (8000 ⁇ g, 4 ° C, 30 min) to obtain the cells. The supernatant was disrupted for protein purification.
  • the column was equilibrated with 10 column volumes of equilibration buffer B (20 mM PB, pH 7.4), the nucleic acid protein detector was zeroed, and the flow rate was adjusted to 2 mg/ mL; the above-mentioned collected breakthrough peaks were added to the ion exchange column, and the column was again equilibrated with 5 column volumes of equilibration buffer B, respectively, with different concentrations of NaCl (500 mM, 700 mM, 1 M) elution buffer (20 mM PB, 1 M). Gradient elution was performed with NaCl, pH 7.4).
  • equilibration buffer B 20 mM PB, pH 7.4
  • the fractions eluted with 1 M NaCl were added to 2 M NaCl, and then purified by Phenyl-HP hydrophobic column using 10 column volumes of equilibration buffer C (20 mM PB, 3 M NaCl, pH 7.4) equilibrate the column, zero the nucleic acid protein detector, and adjust the flow rate to 2 mg/mL; add the collected sample to the hydrophobic column, and equilibrate the column again with 5 column volumes of equilibration buffer C, respectively, with different concentrations Gradient elution of NaCl (2M, 1M, 600 mM, 0 mM) elution buffer (20 mM PB, pH 7.4); fractions eluted with 2 M NaCl were collected and dialyzed against a 10 kDa cut-off dialysis bag for purification.
  • equilibration buffer C 20 mM PB, 3 M NaCl, pH 7.4
  • the final product was identified by SDS-PAGE (12% separation gel, 5% concentrated gel) and Western Blot (the primary antibody was a humanized monoclonal antibody against bFGF). After purification, a Peptibody complex peptide with a purity greater than 90% was obtained. 4 is shown.
  • ELISA and bFGF (50 ng / well, bFGF purchased from PROSPEC company; VEGF purchased from PEPROTECH) were coated with ELISA plate, coated at 4 ° C overnight, dried in PBS-T solution (containing 0.05% Tween) Wash with -20 of 0.015 M PBS) 3 times, 300 ⁇ l/well, 3 min/time. Then, the cells were blocked with 200 ⁇ l of 5% (w/v) skim milk powder, and sealed at 37 ° C for 1 hour, and then dried in blocking solution, 300 ⁇ l / well was added to the PBS-T solution for 3 times, 3 min / time.
  • mice were immunized with Peptibody peptide (C57BL/6 female SPF grade, 6-8 weeks old, purchased from Animal Center of Southern Medical University, license number: 44002100008628), 120 ⁇ g/immunized mice, immunological method is the same as above .
  • the mouse lung cancer cell LLC (LL/2) (Wuhan Punosei Life Technology Co., Ltd., item number CL-0140) in logarithmic growth phase was inoculated subcutaneously in mice at 1 ⁇ 10 6 /only. .
  • the weight of the excised tumor was recorded, and the inhibition rate was calculated.
  • the inhibition rate was 57.7% compared with the PBS control group (Fig. 5b, c and d).
  • Immunohistochemical analysis of the neovascular and lymphatic vessels revealed microvascular and lymphatic tumors. Tube generation was significantly suppressed as shown in Figure 6.

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Abstract

一种抑制肿瘤血管新生的peptibody多表位疫苗及其应用。该peptibody多表位疫苗的氨基酸序列如SEQ ID NO.1所示，其含人IgG1Fc段的融合多表位疫苗，其中包含三个人VEGF表位、三个bFGF表位和完整的人IgG1的Fc段；其具有同时诱导机体产生高滴度抗VEGF和抗bFGF抗体的作用，能同时抑制肿瘤高表达的VEGF和bFGF细胞生长因子。通过实验表明，该多表位疫苗可以诱导BABL/C小鼠获得针对VEGF/bFGF的特异性免疫应答，并能有效抑制C57BL/6LL-2荷瘤小鼠肿瘤的生长，肿瘤生长抑制率达到60～70％。因此，其可用于制备抗肿瘤药物。

Description

一种抑制肿瘤血管新生的peptibody多表位疫苗及其应用 技术领域
本发明涉及疫苗领域，特别涉及一种抑制肿瘤血管新生的peptibody多表位疫苗及其应用。
背景技术
在实体肿瘤的进展中，新生血管是肿瘤增长的前提，肿瘤微血管为肿瘤细胞提供生长所必须的各种营养物质。在血管的形成过程中，肿瘤细胞通过自分泌和旁分泌的方式释放的血管内皮生长因子(VEGF)和碱性成纤维生长因子(bFGF)，促进肿瘤的血管新生。而且，VEGF和bFGF在促血管新生中存在协调作用，VEGF通过促进血小板源性生长因子(PDGF-B)的表达，bFGF促进PDGFR-β的表达，进而协同促进肿瘤血管新生和肿瘤生长。靶向VEGF和bFGF抑制可有效抑制肿瘤血管新生，例如，贝伐单抗(bevacizumab，Avastin)是一种重组的抗人VEGF的单克隆抗体，2004年获得美国FDA批准，通过抑制血管新生来抑制肿瘤，并用于恶性结直肠癌、非小细胞肺癌、恶性肾细胞癌、乳腺癌和脑癌的治疗。靶向于bFGF的单克隆抗体也能够有效的抑制肿瘤的血管新生也有相应的研究报道。针对VEGF的多肽免疫机体，能诱导产生特异性的抗VEGF抗体，从而能够抑制肿瘤细胞的增殖、迁移和转移。针对bFGF的受体结合区域与肝素结合区域的多表位肽作为疫苗能产生特异性的抗bFGF抗体减少肿瘤的血管新生、抑制肿瘤的生长和恶化。
针对抗VEGF抗体药物的研究发现，较长时间使用贝伐单抗会导致肿瘤耐药，会代偿性的促进bFGF和血小板源性生长因子(PDGF)表达，进而促进肿瘤血管新生，促进肿瘤复发和生长，导致耐药。现有的抑制肿瘤血管新生的药物多是针对单一因子的抑制作用，在长期用药中可能会产生代偿性耐药而限制其临床作用。
我们设计开发的针对VEGF/bFGF的多表位肿瘤疫苗同时针对VEGF和bFGF，具有双重抗肿瘤作用，可诱导机体同时产生高滴度的抗VEGF和bFGF抗体，有效抑制肿瘤血管和淋巴管新生、抑制肿瘤的生长和转移。克服了既往针对单一因子抗体或多肽可能导致机体通过另一通路代偿性增殖而刺激肿瘤血管的新生，促进肿瘤的生长和转移的弊端，有效解决了代偿性耐药问题，能更好的抑制肿瘤。适用于多种实体肿瘤的治疗。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种抑制肿瘤血管新生的peptibody多表位疫苗。Peptibody是我们设计的一种新型疫苗，是将小分子的表位多肽与人免疫球蛋白Fc片段融合而成。能有效提高疫苗的免疫原性和稳定性，而且由于有Fc片段，可通过Protein A纯化蛋白，纯化效率更高。
该疫苗是由具有免疫原性的三个bFGF表位肽和三个VEGF表位肽，通过柔性Linker(GGGS)串联成多表位肽，并与IgG1的Fc片段偶连。构建多表位peptibody疫苗，该疫苗可刺激机体产生高滴度抗VEGF和抗bFGF抗体，有效抑制肿瘤血管新生，同时，由于多表位肽与人免疫球蛋白Fc片段连接，Fc段作为融合蛋白既可有效的提高免疫原性，还有利于可溶性表达，方便Protein A纯化等特点。
本发明的另一目的在于提供所述抑制肿瘤血管新生的peptibody多表位疫苗的应用。
本发明的目的通过下述技术方案实现：一种抑制肿瘤血管新生的peptibody多表位疫苗，其氨基酸序列如下所示：
Figure PCTCN2018101619-appb-000001
其中，肽1来自VEGF蛋白的第125～136位表位肽；肽2来自VEGF蛋白的第105～117位表位肽；肽3来源于噬菌体多肽库，通过抗VEGF抗体从肽库中筛选获得，模拟VEGF表位；肽4来自bFGF蛋白的第75～86位表位肽；肽5来自bFGF蛋白的第34～48位表位肽；肽6来自bFGF蛋白的第97～113位表位肽；肽1、肽2和肽3之间，肽4、肽5和肽6之间 分别通过氨基酸序列GGGS依次连接；肽3和肽4之间通过氨基酸序列GGGGSCGGGGS连接；Fc段来自人淋巴细胞IgG1抗体序列部分(生工生物工程有限公司提供)。
编码所述的抑制肿瘤血管新生的peptibody多表位疫苗的核苷酸序列如下所示：
Figure PCTCN2018101619-appb-000002
所述的抑制肿瘤血管新生的peptibody多表位疫苗在制备抗肿瘤药物中的应用。
本发明相对于现有技术具有如下的优点及效果：
本发明提供的多表位疫苗是一种含人IgG1Fc段的融合多表位疫苗，其中包含三个人VEGF表位、三个bFGF表位和完整的人IgG1的Fc段；其具有同时诱导机体产生抗VEGF和抗bFGF抗体的作用，能同时抑制肿瘤高表达的VEGF和bFGF细胞生长因子。
通过实验表明，该多表位疫苗可以诱导BABL/C小鼠获得针对VEGF/bFGF的特异性免 疫应答，并能有效抑制C57BL/6LL-2荷瘤小鼠肿瘤的生长，肿瘤生长抑制率达到60～70％。因此，其有望作为肿瘤治疗和预防的辅助药物。
附图说明
图1是本发明的peptibpdy及载体示意图；其中，图A为表达载体图；图B为peptibpdy的构建图。
图2是pET-28a-VEGF/bFGF-Fc载体PCR鉴定图；其中，图a中的泳道M为DNA Marker，泳道1为pET28a-VEGF/bFGF-Fc载体的VEGF/bFGF的PCR片段；图b中的泳道M为DNA Marker，泳道1为pET28a-VEGF/bFGF-Fc载体的Fc的PCR片段。
图3是pET-28a-VEGF/bFGF-Fc载体的酶切鉴定图；其中，泳道M为DNA Marker，泳道1为pET28a-VEGF/bFGF-Fc载体的Fc的酶切片段，泳道2为pET28a-VEGF/bFGF-Fc的VEGF/bFGF的酶切片段。
图4是多表位疫苗的表达纯化和鉴定图；其中，泳道M为蛋白Marker，泳道1为Peptibody复合肽的纯化样品，泳道2是纯化后样品的Western Blot鉴定结果。
图5是多表位疫苗免疫小鼠后的血清效价和抑瘤作用结果图；其中，图a是ELISA检测Peptibody免疫后小鼠血清中抗VEGF和bFGF抗体滴度图，图b是肿瘤的生长曲线图，图c是肿瘤的重量结果图，图d是分离的肿瘤的照片图。
图6是荷瘤小鼠肿瘤组织的新生血管和淋巴管的免疫组化结果图；其中，图a为肿瘤组织的CD31和LYVE1染色结果图，图b是肿瘤组织的微血管密度定量分析结果图，图c是淋巴管密度的定量分析结果图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
一、序列的设计
据计算机模拟、生物信息学方法等对VFGF免疫源性表位和bFGF免疫源性表位进行筛 选，得到的表位肽与噬菌体展示技术从肽库中筛选的模拟肽通过柔性Linker将不同的表位链接，并与IgG1抗体的Fc片段相连，其核苷酸序列如SEQ ID NO.2所示。在合成(生工生物工程(上海)股份有限公司合成)时，在如SEQ ID NO.2所示的序列的前后分别添加上XhoI和NheI酶切位点，得到质粒A。
将如SEQ ID NO.2的序列通过XhoI和NheI位点克隆入pET-28a中，得到的表达载体如图1A所示，表达的多表位肽如图1B所示。
二、实验验证
(1)pET-28a-VEGF/bFGF-Fc载体的构建
将质粒A与载体pET28a分别通过XhoI和NheI双酶切，胶回收目的片段，进行连接，构建表达质粒pET28a-VEGF/bFGF-Fc
酶切体系如表1所示：
表1
Figure PCTCN2018101619-appb-000003
37℃酶切4h。琼脂糖凝胶电泳后，胶回收质粒A和pET28a双酶切的目的条带，进行连接。
连接体系如表2所示：
表2
Figure PCTCN2018101619-appb-000004
Figure PCTCN2018101619-appb-000005
16℃连接过夜。将连接产物导入DH5α感受态细胞，涂布在含卡那霉素的筛选培养基平板，挑取单菌落。
(2)表达质粒pET28a-VEGF/bFGF-Fc的鉴定
将提取质粒进行PCR和酶切验证，结果如图2和图3，表明已经VEGF/bFGF-Fc已经连接入pET28a载体中。其中，PCR扩增体系如表3所示：
表3
Figure PCTCN2018101619-appb-000006
PCR扩增体系为：95℃预变性4min，95℃变性45s，58℃退火30s，72℃延伸1min，25个循环后，72℃延伸10min。
扩增VEGF/bFGF段的引物如下：
上游引物F1：5’-CCATGGGCCAGAAACGTAAACGTAAGAAATCCC-3’；
下游引物R1：5’-GGATCCGGAGGTGTATTTACGGGAAC C-3’。
扩增Fc段的引物如下：
上游引物F2：5’-GGATCC TCTTGTGACAAAACTCACAC-3’；
下游引物R2：5’-CTCGAGTTTTACCCGGAGACAGGGAG-3’。
酶切体系如表4所示：
表4(在分别进行Fc段和VEGF/bFGF多表位段时，除了所用的酶不同，其他试剂相同，相同的试剂为共性试剂)
Figure PCTCN2018101619-appb-000007
37℃，酶切4h。
实施例2
一、Peptibody蛋白的表达与纯化
将鉴定正确的质粒pET28a-VEGF/bFGF-Fc转入BL21大肠杆菌表达菌株，鉴定正确后诱导表达，以体积比1:100接种于LB培养基，37℃、220rpm培养5h，加入0.1mM/L IPTG诱导5h。4000rpm离心收集菌体，用20mM PB、500mM NaCl，pH7.4的缓冲液重悬，超声(80Hz，开3s、关2s，20min)破碎后离心(8000×g，4℃，30min)获取菌体破碎上清，进行蛋白纯化。
纯化的具体步骤如下：用10倍柱体积的平衡缓冲液A(20mM PB、500mM NaCl，pH7.4) 平衡Protein A(广州佰路生物科技有限公司)亲和层析柱，对核酸蛋白检测仪进行调零，并调整其流速为2mg/mL；将破碎后的菌体上清加入纯化柱中，收集穿出峰，以5倍柱体积的平衡缓冲液再次平衡柱子；调整流速为1mg/mL，用洗脱缓冲液(Tris·HCL，pH2.7)进行洗脱；收集洗脱组分。洗脱组分再进行SP-HP离子交换柱纯化，用10倍柱体积的平衡缓冲液B(20mM PB、pH 7.4)平衡柱子，对核酸蛋白检测仪进行调零，并调整其流速为2mg/mL；将上述收集的穿出峰加入离子交换柱，用5倍柱体积的平衡缓冲液B再次平衡柱子，分别用不同浓度NaCl(500mM，700mM，1M)的洗脱缓冲液(20mM PB、1M NaCl，pH 7.4)进行梯度洗脱；收集1M NaCl洗脱下来的组分加入2M NaCl后，再进行Phenyl-HP疏水柱纯化，用10倍柱体积的平衡缓冲液C(20mM PB、3M NaCl，pH 7.4)平衡柱子，对核酸蛋白检测仪进行调零，并调整其流速为2mg/mL；将上述收集样品加入疏水柱，用5倍柱体积的平衡缓冲液C再次平衡柱子，分别用不同浓度NaCl(2M，1M，600mM，0mM)的洗脱缓冲液(20mM PB，pH 7.4)进行梯度洗脱；收集2M NaCl洗脱下来的组分，用10kDa截留量的透析袋进行透析后作为纯化后终产物。对产物进行SDS-PAGE(12％分离胶，5％浓缩胶)和Western Blot鉴定(一抗为抗bFGF的人源化单抗)，纯化后获得了纯度大于90％的Peptibody复合肽，如图4所示。
二、Peptibody蛋白的免疫原性和抑瘤作用分析
(1)将纯化获得Peptibod复合肽免疫BABL/C小鼠(BABL/C雌鼠SPF级别，6-8周龄，购自南方医科大学动物中心，许可证号：44002100008628)，初次免疫将120μg蛋白与弗氏完全佐剂按体积比1:1混合乳化，皮下多点对小鼠进行免疫；二、三次免疫120μg蛋白与弗氏不完全佐剂按体积比1:1混合乳化，皮下多点免疫，每次免疫间隔两周。三免后十天，对小鼠尾静脉取血，分离血清，测定血清中抗VEGF、bFGF抗体的滴度，是通过间接ELISA法测定血清中抗VEGF和抗bFGF抗体的滴度，具体步骤如下：
分别用VEGF和bFGF(50ng/孔，bFGF购自PROSPEC公司；VEGF购自PEPROTECH公司)包被ELISA板，4℃包被过夜，甩干包被液，加入PBS-T溶液(含0.05％吐温-20的0.015M PBS)洗涤3次，300μl/孔，3min/次。再以200μl 5％(w/v)脱脂奶粉进行封闭，37℃封闭1h后，甩干封闭液，300μl/孔加入PBS-T溶液洗涤3次，3min/次。加入系列稀释的免疫血清，37℃孵育1h，再以300μl/孔加入PBS-T洗涤3次，3min/次；加入100μl HRP标记的山羊抗鼠的IgG抗体(1:4000稀释)，37℃孵育40min，以300μl/孔加入PBS-T洗涤5次，3min/ 次；加入TMB室温避光显色10min，加入2mol/L硫酸终止显色；测定OD450nm的值。结果表明血清中抗VEGF抗体的效价为1:8000，抗bFGF抗体的效价为1:32000，结果如图5a所示。
(2)Peptibody复合肽免疫C57BL/6小鼠后抑瘤作用分析
用Peptibody复合肽免疫C57BL/6小鼠(C57BL/6雌鼠SPF级别，6-8周龄，购自南方医科大学动物中心，许可证号：44002100008628)，120μg/只免疫小鼠，免疫方法同上。三免后十天，将处于对数生长期的小鼠肺癌细胞LLC(LL/2)(武汉普诺赛生命科技有限公司，货号CL-0140)以1×10 6/只接种于小鼠皮下。待肿瘤长至可触摸大小后，每隔一天测量肿瘤的大小，肿瘤体积计算公式为V＝π/6(a×b 2)。记录剥离肿瘤的重量，计算抑制率，与PBS对照组相比其抑制率为57.7％(图5b、c和d)，对新生血管和淋巴管进行免疫组化分析，结果表明肿瘤的微血管和淋巴管生成受到了显著的抑制，如图6所示。
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 (3)

  1. 一种抑制肿瘤血管新生的peptibody多表位疫苗，其特征在于：其氨基酸序列如SEQ ID NO.1所示。
  2. 编码权利要求1所述的抑制肿瘤血管新生的peptibody多表位疫苗的核苷酸序列如SEQ ID NO.2所示。
  3. 权利要求1所述的抑制肿瘤血管新生的peptibody多表位疫苗在制备抗肿瘤药物中的应用。
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