| Human immunodeficiency virus type I (HIV-1) is the etiologic agent of AIDS , the envelope glycoprotein (gp 120) of HIV-l encoded by the env gene , play critical roles in viral entry processes which lead to infection . The first step in the replication of HIV-l is selective binding of gpl2O to CD4+ helper T cells, monocytes macrophages, dendritic cells, langerhans cells, placenta] trophoblasts and neuronal cells , after penetration in these cells , the genome of HIV- 1 is integrated in the human genome . It is believed that eliminating HJV-1 infected cells is crucial in preventing disease progression . The use of immunotoxins to eliminate HJV-1 infected cells is a effective strategies , immunotoxins are chimeric molecules in which cell binding ligands are coupled to toxins and can specifically eliminate undesired cells , HIV-l envelope glycoprotein(gpl2O) is one of the target molecule used for constructed immunotoxins , single chain antibody (ScF v) and CD4 surface antigens are two kinds of gpl2O-binding ligands which could be coupled to toxins to prepare killing HIV-l infected cells immunotoxins. In our experiments A single chain antibody ScFv directed against HIV-l envelope glycoprotein was constructed by using the antibody variable region (V) genes of murine mab against HIV~1 gpl2o , the VH and VL gene were amplified by RT-PCR from a hybridoma cell line 102, producing mouse mab against HIV-l gpl2O protein. The heavy and light chain variable regions were connected by a flexible Linker(Gly4ser)3. The fusion gene (ScFv) were cloned into pGEM-T vector and Sequenced, The nucleotide sequence results showed that the VH and VL genes were homologous with the published mouse antibody variable region gene sequences and were confirmed as functionally rearranged mouse imrnunoglobulin variable region genes. The ScFv gene were 705bp, consisting of VH, Linker and VL genes , VH gene was 336bp,encoding 112 amino acids ,The VL gene was 324bp,encoding 108 amino acids .To express ScFv containing chimeric immunotoxins , we constructed a ScFv expressing plasmids and two immunotoxins expressing plasmids by replacing the receptor binding domain of PE and DT genes with ScFv gene, plasmid pET28-ScFv-PE encodes for a hybrid protein consisting N terminus of ScFv and C terminus of PE , plasmid pET28-DT-ScFv encodes for a hybrid protein composed of DI fragment at the N terminus and ScFv fragment at the C terminus . After transformed these plasmids into Ecoli strain BL2 I (DE3) and induced by IPTG . both immunotoxins (ScFv-PE, DT-ScFv) were expressed successfully. Expressed anti-HIV- I ScFv take more than 51% of the total cell proteins , while the expression level of recombinant immunotoxins ScFv-PE and DT-ScFv were 28% and 26% respectively . After cell lysis and SDS-PAGE analysis , we found all the expression ptoducts exist in forms of inclusion body . To produce active forms of recombinant proteins, after solubilization in the presence of strong denaturants , we achieved refolding of the denatured proteins to an active form by renaturation , by one step of Ni-NTA affinity chromatography the expressed ScFv and recombinant immunotoxin were purified to near homogenity. To prepare antigen used for the detection of antibody activity of recombinant ScFv and immunotoxins , full lengh of subtype B HIV-1 gag and gpl2o gene were fused and cloned into baculoviruses transfer vector pfastbac I and recombinant plasmid pfastGE was constructed . After transforming it into competent Ecoli DH 10 cells , the recombinant... |
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