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目的通过细胞内表达抗HIV 1整合酶单链抗体(IN sFv)基因 ,阻断病毒基因组与宿主细胞基因组的整合 ,进而抑制病毒复制 ,探讨该基因载体在HIV 1感染基因治疗中应用的意义。方法用带有IN sFv基因的逆转录病毒表达载体 pSLXCMV/IN sFv转染PA317包装细胞 ,并用包装后含有目的基因的逆转录病毒转导SupT1细胞及外周血单个核细胞(PBMC)。以HIV 1NL4 3病毒株感染表达IN sFv的SupT1及PBMC ,用ELISA方法测定病毒感染细胞后不同时间培养上清中HIV 1P24蛋白的含量 ,以监测病毒复制的水平 ;用半定量巢式PCR扩增病毒感染后不同时间点细胞内HIV 1整合前体中的环状病毒DNA ,以明确病毒进入细胞后的整合状态。结果IN sFv在SupT1及PBMC两种细胞中 ,均可明显地抑制病毒的复制。环状病毒DNA在表达IN sFv的SupT1细胞中早于对照细胞8～10h出现。结论细胞内表达的抗HIV 1整合酶单链抗体 ,可显著抑制病毒的整合 ,从而阻断细胞内病毒的复制。该结果为开拓HIV 1基因治疗的新领域奠定了基础。
OBJECTIVE: To detect the integration of HIV-1 integrase (IN sFv) gene into host cell genome by intracellular expression of anti-HIV 1 integrase single-chain antibody (IN sFv) and then to inhibit viral replication. Methods The PA317 packaging cells were transfected with the retroviral vector pSLXCMV / IN sFv with IN sFv gene and the SupT1 cells and peripheral blood mononuclear cells (PBMCs) were transduced with the retrovirus containing the gene of interest. The HIV-1NL4 3 strain was infected with INsFv-expressing SupT1 and PBMC. The HIV 1P24 protein content in the culture supernatant was measured by ELISA at different times to monitor the level of virus replication. The semi-quantitative nested PCR Cytomegalovirus DNA was integrated into HIV-1 cells at different time points after virus infection to clarify the integration of the virus into the cell. Results IN sFv in SupT1 and PBMC two kinds of cells, can significantly inhibit the virus replication. The circular virus DNA appeared in SupT1 cells expressing IN sFv earlier than the control cells for 8-10 hours. Conclusion The intracellular expression of anti-HIV 1 integrase single chain antibody can significantly inhibit the integration of the virus, thereby blocking intracellular viral replication. This result lays the foundation for pioneering new areas of HIV 1 gene therapy.