【作者】 张惠中；

【导师】 王文亮； Duan Lingxun；

【作者基本信息】 第四军医大学 ， 病理学， 1997， 博士

【摘要】 单链可变区抗体（Single Chain Antibody Variable Region Fragments sFv）由完整抗体的可变区轻链（L_H）及可变区重链（V_H）组成，中间由一多肽链相连。近年来抗体工程技术的飞速发展使得这一具备完整抗体高特异性和结合性的最小结构单位，可通过适当的表达载体被导入靶细胞内的特定区域，并稳定、持续地表达目的基因，从而起到阻断或开放某缺陷基因的作用，故这一技术在当今肿瘤性疾病及病毒感染性疾病基因治疗中显示出广泛的应用前景。HIV-1为一具有包膜的逆转录病毒，含有2个拷贝的单链RNA（9800bp）。HIV-1借高亲合性的受体（CD₄）进入靶细胞，在宿主细胞酶的作用下脱去包膜并在病毒酶的作用下逆转录成cDNA进而整合于宿主细胞基因组，完成其生命周期。因此整合过程是其生命周期中不可缺少的关键步骤，阻断HIV-1整合酶将遏制HIV-1感染于早期阶段，从而起到治疗目的。据此，我们特构建了抗HIV-1整合酶的sFv（Anti-HIV-1 IN sFv）及带有核内定位信号的抗HIV-1整合酶的sFv（Anti-HIV-1 IN／Nu sFv）。 一、抗HIV-1整合酶sFv的功能鉴定 为探讨IN-sFv在HIV-1感染中的临床应用意义。作者对其进行了功能测试。首先以免疫染色法对该结构的细胞内表达及分布进行了观察。结果显示。转染有IN-sFv及IN／Nu-sFv的NIH3T3细胞分别于胞浆及胞核中有明确的sFv表达。然后对其进行了感染实验，以MOI=0.04-0.06的NL_4-3病毒株同时感染10⁶个转染有IN-sFv的SupT1细胞及转染有CAT、HBVcore-sFv及抗HIV-1 Rev-sFv的SupT1对照组细胞。通过ELISA法测定培养细胞上清中的P²⁴蛋白含量证实IN-sFv阻断病毒复制的作用达更多还原

【Abstract】 Single-chain Fv(sFv) are recombinant antibody fragments consisting of only the variable light chain (V_L) and variable heavy chain (V_H) domains covalently connected to one another by a polypeptide linker. Recent advances in antibody engineering have now allowed the genes encoding the sFv, which is probably the minimal structural domain that retains the specific and high-affinity binding properties of the parent antibody , to be stably expressed in precise intracellular locations inside mammalian cells. As a simple and effective new method to replace defective genes or add functional genes to specific cell types, sFv gene therapy is likely to have widespread impact as potential therapeutic agents on both tumor and infectious diseases.HIV-1 is an enveloped retrovirus containing two copies of a single-stranded RNA genome. After binding to a high-affinity receptor on a cell surface, called the CD4 molecule, the virion penetrates the cell and is uncoated by cellular enzymes. Viral reverse transcriptase catalyzes the synthesis of a double-stranded DNA intermediate from the viral RNA. The DNA is transported to the nucleus by the viral proteins and integrated into the host cell’s genome by viral integrase(IN) to complete its life cycle. As HIV-1 integrase plays a key role in the early stage of its life cycle, successfully blocking the integrase will halt the HIV-1 replication prior to integration. Based on these, we constructed sFv which binds to the HIV-1 integrase C-terminal domain.更多还原