摘要
应用抗HIV 1Rev单链抗体细胞内免疫方法 ,研究在人T细胞和周围血淋巴单核细胞内抗病毒复制的效果 ,探讨细胞内免疫抗HIV 1基因治疗的可行性。克隆抗HIV 1Rev单链抗体 (sFv)基因 ,以逆转录病毒为基因载体 ,将包装后的含靶基因的逆转录病毒转导至人CD4 阳性T 细胞株CEM和SupT1,以及HIV 1阴性自愿者的周围血淋巴单核细胞 (PBMC) ,再分别用不同剂量 (MOI)的HIV 1病毒株PNL4 3 和HxB2 进行病毒攻击实验。测定HIV 1p2 4抗原。滴度以了解抗病毒复制的效果 ,检测带报道基因CAT以了解靶基因在细胞内的表达状况。分别应用0 2 4、0 0 6和 0 0 2 4MOI的PNL4 3 或HxB2 病毒株攻击 ,测定p2 4抗原。结果显示 ,抗RevsFv细胞内表达能有效地抑制病毒在细胞内的复制 ,与对照组比较 ,病毒复制被抑制效率达 90 %以上。体外培养细胞 2个月 ,仍可观察到效果 ,病毒攻击后合胞体细胞形成显著减少。CAT实验表明 ,逆转录病毒载体可使靶基因在人T细胞和PBMC内有效表达。抗HIV 1RevsFv细胞内表达可有效地抑制病毒复制 ,有望成为抗HIV
This study was to explore the applicable value of intracellular immunization against human immunodeficiency virus type 1 (HIV 1) infection in human T lymphocytes and peripheral blood mononuclear cells (PBMC). Using gene therapy or intracellular immunization techniques, a single chain variable fragment(sFv), derived from a monoclonal antibody to the HIV 1 regulatory protein Rev, has been constructed into the murine retroviral shuttle vectors. Human T lymphocytic cell lines CEM, SupT1 and normal PBMC were transduced with these anti Rev sFv expressing vector particles from packaging cell line supernatants. The cells transduced by anti Rev sFv were challenged with HIV 1 strains, HxB 2 and PNL 4 3 , at various MOI input values of 0.24、0.06 and 0.024 respectively. The culture supernatants were collected after infection and the levels of HIV 1 p24 antigen were determined by an HIV 1 antigen captured enzyme linked immunosorbent assay. The results showed that HIV 1 infection was dramatically inhibited in anti Rev sFv transduced CEM, SupT1 and PBMC, and inhibition of HIV 1 induced syncytia formation in these cells was also observed. CAT assay revealed that target gene in retroviral vectors pLSXN and pSLXCMV both could effectively express in human T cell lines and PBMC. These data suggest that intracellular expression of anti Rev sFv may be utilized as gene therapy or intracellular immunization for HIV 1 infections in vivo.
出处
《病毒学报》
CAS
CSCD
北大核心
2000年第1期17-23,共7页
Chinese Journal of Virology
关键词
单链抗体
细胞内免疫
人免疫缺陷病毒-1
single chain variable fragment antibody
intracelluler immunization
human immunodeficiency virus type 1
T lymphocytes
gene therapy
