携带全长抗CD20抗体的嵌合型腺病毒Ad5F11b-Anti-CD20载体构建与表达研究  

Construct and Expression of Chimeric Adenovirus Ad5F11b-Anti-CD20 Carrying Full Length CD20 Antibody

作　　者：杨秋梅[1] 吴红平[2] 周秀梅[1] 李林芳[2] 王春红[2] 姜梨华[2] 钱其军[1,2] 

机构地区：[1]浙江理工大学生命科学学院,杭州310018 [2]第二军医大学东方肝胆外科医院,上海200438

出　　处：《浙江理工大学学报（自然科学版）》2009年第5期743-746,共4页Journal of Zhejiang Sci-Tech University(Natural Sciences)

基　　金：国家高技术研究发展计划"863"重点项目资助(2007AA021108);浙江省重中之重学科开放基金资助(SWYX0809)

摘　　要：构建Ad5F11b-Anti-CD20嵌合型腺病毒载体并探索该腺病毒在体外表达全长抗体的能力。合成部核糖体进入位点(IRES)序列,将其与美罗华抗体重轻链连接,克隆到pDC338质粒载体上,并与以11b型腺病毒(Ad11b)Fiber替代5型腺病毒(Ad5)Fiber的嵌合型腺病毒骨架质粒pAd5F11b进行重组,获得Ad5F11b-Anti-CD20嵌合型腺病毒。将鉴定正确的病毒空斑感染293细胞,ELISA检测其表达水平以及间接免疫荧光检测该病毒表达抗体的特异性。结果表明,嵌合型腺病毒Ad5F11b-Anti-CD20在293细胞中的表达量高达3.78μg/ml±0.30μg/ml,间接免疫荧光(IFA)显示该病毒表达的抗体只与CD20+的Raji细胞有特异性结合,而与CD20-的Molt-4细胞无结合能力。成功构建了高效表达抗CD20抗体的嵌合型腺病毒Ad5F11b-Anti-CD20。To construct vector carrying Rituximab light and heavy chain bridged by internal ribonsome entry site（IRES） and investigate full length antibody expression ability of chimeric adenovirus vector in which AdS fiber is replaced by Adllb fiber. Method： Synthesized IRES sequence is used to bridge light and heavy chain, which is subsequently cloned to pDC338 vector. This construct is then homologously recombined with pAd5F11B adenovirus backbone plasmid and the recombined adenovirus is confirmed by PCR. Antibody expression and specificity are detected by ELISA and indirect fluorescence assay（IFA） respectively after virus infection of 293 cells. Results： ELISA assay demonstrates the expression of the antibody is 3. 78μg/ml±0. 3μg/ml and IFA indicate that antibody expressed by this adenovirus show specific binding capacity to CD20 positive Raji cells and no binding with CD20 negative Molt-4 cells. Conclusion： chimeric adenovirus Ad5F11b-Anti-CD20 carrying full length CD20 antibody is successfully constructed and in vitro assays suggest the expression of anti-CD20 antibody with high efficiency and specificity.

关 键 词：全长抗体 嵌合型腺病毒Ad5F11b 抗CD20抗体 IRES 

分 类 号：R73-362[医药卫生—肿瘤]