抗人CD28单链抗体基因在Sf9细胞及家蚕中的表达研究
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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。
The mouse anti-human CD28 ScFv gene was subcloned into Bac-to-Bac transfer plasmid pFastBacHTa, and the recombinant plasmid pFastBacHTa-ScFv was transformed into DH10Bac competent cells. The rBacmid-ScFv virus was obtained by screening of white plaque and identification by PCR. The rBacmid-ScFv DNA was transfected into Sf9 cells using lipofectin and the virus rBV-ScFv was harvested. After Sf9 cells were infected with the rBV-ScFv, the expression of protein was analyzed by SDS-PAGE and Western blot. The highly expressed specific protein was approximately 36 kDa and reached to 25.7% of total protein, resulting in the form of insoluble aggregate. The purified protein of CD28 ScFv was obtained by Ni-NTA Resin after solubilization. Flow cytometry assays showed the specific binding ability of CD28 ScFv to T-28 cells (T cell transfected with CD28 gene), which could complete with the antibody 2F5 produced by a mouse anti-Human CD28 2F5 hybridoma.
HyNPV is a new type of baculovirus which was derived from recombining of BmNPV and AcNPV. In this study, silkworm-HyNPV expression system was used to express the CD28 ScFv gene.
Based on design of the fusing gene of CD28 single-chain antibody, two Bac-to-Bac BEVS were applied for construction of anti-Human CD28-ScFv transfer vectors and recombinant viruses. Finally, the recombinant viruses were obtained and were expressed in SF9 cells and silkworm. In the first time, we obtained highly purified products of anti-Human CD28 single-chain antibody, and discussed insoluble problem of the products. This is the first report for high level expression of heterologous protein in aggregate form in insect cells and its subsequent solubilization and purification. This work will be helpful for the development of anti-tumor drugs.
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。
The mouse anti-human CD28 ScFv gene was subcloned into Bac-to-Bac transfer plasmid pFastBacHTa, and the recombinant plasmid pFastBacHTa-ScFv was transformed into DH10Bac competent cells. The rBacmid-ScFv virus was obtained by screening of white plaque and identification by PCR. The rBacmid-ScFv DNA was transfected into Sf9 cells using lipofectin and the virus rBV-ScFv was harvested. After Sf9 cells were infected with the rBV-ScFv, the expression of protein was analyzed by SDS-PAGE and Western blot. The highly expressed specific protein was approximately 36 kDa and reached to 25.7% of total protein, resulting in the form of insoluble aggregate. The purified protein of CD28 ScFv was obtained by Ni-NTA Resin after solubilization. Flow cytometry assays showed the specific binding ability of CD28 ScFv to T-28 cells (T cell transfected with CD28 gene), which could complete with the antibody 2F5 produced by a mouse anti-Human CD28 2F5 hybridoma.
HyNPV is a new type of baculovirus which was derived from recombining of BmNPV and AcNPV. In this study, silkworm-HyNPV expression system was used to express the CD28 ScFv gene.
Based on design of the fusing gene of CD28 single-chain antibody, two Bac-to-Bac BEVS were applied for construction of anti-Human CD28-ScFv transfer vectors and recombinant viruses. Finally, the recombinant viruses were obtained and were expressed in SF9 cells and silkworm. In the first time, we obtained highly purified products of anti-Human CD28 single-chain antibody, and discussed insoluble problem of the products. This is the first report for high level expression of heterologous protein in aggregate form in insect cells and its subsequent solubilization and purification. This work will be helpful for the development of anti-tumor drugs.
引文
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[1]邱玉华,於葛华,陈永井,等.激发型CD28单抗直接激发的T细胞及其表型.上海免疫学杂志,2003,23(5):324-326.
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[2]邱玉华,张学光,季玉红等.鼠抗人CD28分子单克隆抗体的研制及生物学特性研究.细胞与分子免疫学杂志,2001,17(4):368~370
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