抗人CD28单链抗体基因在Sf9细胞及家蚕中的表达研究
发布时间：2018-07-20 09:55
【摘要】： 鼠源性单抗在人体使用后可产生人抗鼠抗体(Human Anti-Mouse Antibody, HAMA)反应,而且分子量大,不能有效进入病灶部位,临床使用受到限制。单链抗体(single chain Fv, ScFv)是由一条连接肽将IgG分子的重链和轻链的可变区连接而成的,减少了免疫原性,分子小,实体组织穿透力强,血浆半衰期短,因而在临床疾病的诊断和治疗中具有重要的应用价值。 将抗人CD28的鼠源单链抗体基因(mouse anti-human CD28 ScFv )亚克隆到杆状病毒转移载体pFastBacHTa中,得到重组转移载体pFastBacHTa-ScFv转化大肠杆菌DH10Bac,获得重组杆粒rBacmid-ScFv。脂质体介导转染Sf9昆虫细胞后获得重组杆状病毒rBV-ScFv。将该重组病毒感染Sf9细胞, SDS-PAGE和Weatern blot分析表明:抗人CD28 ScFv在昆虫细胞中获得高效表达,分子量约36 kDa。细胞裂解及产物可溶性分析表明:该单链抗体基因在Sf9细胞中超高量表达并以不溶性集聚体形式存在,表达产物可达细胞总蛋白的25.7%。经变性溶解集聚体、Ni-NTA亲和层析,获得纯化抗人CD28 ScFv表达产物。流式细胞分析,当CD28 SvFc结合了T-28细胞表面抗原结合表位后,单克隆抗体2F5与T-28细胞的结合率从86.9%下降至18.1%,表明抗人CD28 ScFv可有效地与CD28单克隆抗体竞争性结合CD28细胞表面抗原。 HyNPV是BmNPV和AcNPV通过基因重组后得到的一个具有BmNPV和AcNPV双重优点的新型杂交病毒,本研究还利用家蚕-HyNPV表达系统,研究了其在家蚕中的表达。 本研究在成功拼接抗人CD28单链抗体基因的基础上,应用二种Bac-to-Bac BEVS系统构建并获得了抗人CD28-ScFv的转移载体和重组病毒,并在Sf9细胞和家蚕中表达了CD28-ScFv,首次对Sf9细胞中表达的外源蛋白抗CD28 ScFv的可溶性问题进行了研究,获得了具有免疫学活性的高纯度的抗人CD28单链抗体。为抗CD28 ScFv的抗肿瘤药物的开发奠定了基础。
[Abstract]:Human Anti-Mouse Antibody (Hama) reaction can be produced after human use of murine monoclonal antibody, and its molecular weight is too large to enter the lesion effectively, so its clinical use is restricted. The single chain antibody (single chain Fv (scFv) is made of a ligated peptide that connects the heavy chain and the variable region of the light chain of the molecule. It reduces immunogenicity, is small, has strong penetration of solid tissue, and has a short plasma half-life. Therefore, it has important application value in the diagnosis and treatment of clinical diseases. The mouse scFv gene (mouse anti-human CD28 scFv) was subcloned into baculovirus transfer vector pFastBacHTa. The recombinant transfer vector pFastBacHTa-ScFv was transformed into E. coli DH10Bac.Recombinant rBacmid-ScFv was obtained. Recombinant baculovirus rBV-ScFv was obtained after transfection of Sf9 insect cells by liposome. The recombinant virus was infected with Sf9 cells. SDS-PAGE and Weatern blot analysis showed that anti-human CD28 scFv was highly expressed in insect cells with a molecular weight of about 36 kDa. Cell cleavage and soluble product analysis showed that the single chain antibody gene was expressed in high quantity in Sf9 cells and existed in the form of insoluble aggregates, and the expressed product could reach 25.775% of the total cell protein. The anti-human CD28 scFv expression product was purified by denaturing solubilizing agglomeration Ni-NTA affinity chromatography. By flow cytometry, when CD28SvFc binds to T-28 cell surface antigen binding epitopes, The binding rate of monoclonal antibody 2F5 to T-28 cells decreased from 86.9% to 18.1%, which indicated that anti-human CD28 scFv could effectively compete with CD28 monoclonal antibody to bind to CD28 cell surface antigen. HyNPV was the result of gene recombination of BmNPV and AcNPV. A novel hybrid virus with the advantages of BmNPV and AcNPV was obtained. The expression of silkworm-HyNPV in silkworm was studied by using silkworm-HyNPV expression system. On the basis of successfully splicing the single chain antibody gene against human CD28, two kinds of Bac-to-Bac BEVs system were used to construct and obtain the transfer vector and recombinant virus against human CD28-ScFv. CD28-ScFv was expressed in Sf9 cells and silkworm. The soluble expression of foreign protein against CD28 scFv in Sf9 cells was studied for the first time. A high purity anti-CD28 scFv antibody with immunological activity was obtained. It lays a foundation for the development of anti-tumor drugs for anti-CD 28 scFv.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：2133162
[Abstract]:Human Anti-Mouse Antibody (Hama) reaction can be produced after human use of murine monoclonal antibody, and its molecular weight is too large to enter the lesion effectively, so its clinical use is restricted. The single chain antibody (single chain Fv (scFv) is made of a ligated peptide that connects the heavy chain and the variable region of the light chain of the molecule. It reduces immunogenicity, is small, has strong penetration of solid tissue, and has a short plasma half-life. Therefore, it has important application value in the diagnosis and treatment of clinical diseases. The mouse scFv gene (mouse anti-human CD28 scFv) was subcloned into baculovirus transfer vector pFastBacHTa. The recombinant transfer vector pFastBacHTa-ScFv was transformed into E. coli DH10Bac.Recombinant rBacmid-ScFv was obtained. Recombinant baculovirus rBV-ScFv was obtained after transfection of Sf9 insect cells by liposome. The recombinant virus was infected with Sf9 cells. SDS-PAGE and Weatern blot analysis showed that anti-human CD28 scFv was highly expressed in insect cells with a molecular weight of about 36 kDa. Cell cleavage and soluble product analysis showed that the single chain antibody gene was expressed in high quantity in Sf9 cells and existed in the form of insoluble aggregates, and the expressed product could reach 25.775% of the total cell protein. The anti-human CD28 scFv expression product was purified by denaturing solubilizing agglomeration Ni-NTA affinity chromatography. By flow cytometry, when CD28SvFc binds to T-28 cell surface antigen binding epitopes, The binding rate of monoclonal antibody 2F5 to T-28 cells decreased from 86.9% to 18.1%, which indicated that anti-human CD28 scFv could effectively compete with CD28 monoclonal antibody to bind to CD28 cell surface antigen. HyNPV was the result of gene recombination of BmNPV and AcNPV. A novel hybrid virus with the advantages of BmNPV and AcNPV was obtained. The expression of silkworm-HyNPV in silkworm was studied by using silkworm-HyNPV expression system. On the basis of successfully splicing the single chain antibody gene against human CD28, two kinds of Bac-to-Bac BEVs system were used to construct and obtain the transfer vector and recombinant virus against human CD28-ScFv. CD28-ScFv was expressed in Sf9 cells and silkworm. The soluble expression of foreign protein against CD28 scFv in Sf9 cells was studied for the first time. A high purity anti-CD28 scFv antibody with immunological activity was obtained. It lays a foundation for the development of anti-tumor drugs for anti-CD 28 scFv.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 胡静平;抗人B7-2单链抗体基因在Tn5细胞、家蚕及哺乳动物细胞中的表达研究[D];苏州大学;2010年
，本文编号：2133162
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