Preparation And Identification Of Monoclonal Antibodies Gainst Synthesized Peptides C34 And C46 Derived From HIV-1 Envelop Gp41 Core Structure

AbstractPreparation and Identification of a Monoclonal Antibodies against Synthetical Peptides C34 and C46 Derived from HIV-1 Envelop gp4l core structureHIV- 1 envelope transmembrane subunit gp4 1 plays a critical role in the fusion of viral to target cell membranes.The extracellular region of gp4 1 consists of two heptad repeat(HR) regions containing hydrophobic sequences with high a -helical propensity.One is located adjacent to the N-terminal fusion peptide (NHR) ;the other at the C-terminus of gp4 1 ectodomain (CHR) . Biochemical and biophysical studies suggest that in the fusion -active state , the CHR region interacts with the NI-JR region of gp4 1 to form a six elix bundle,consisting of three N-terminal and three C-terminal hel ices packed in the reverse direction and representing the fusion-active gp4l core struture. The peptides derived from the CHR regions of gp4 1 ,designated C- peptides,have potent inhibitory activity on the membrane fusion step of HIV- 1 infection.Previouse studies identified an a -helical complex within the-6-gp4lectodomain consisting of the peptides N-36 and C-34 andshowed that these tWo pePtides associate to form a stable,' Q -helical trimer ofheterodimers. This subdomain displays the salientfeatures of the stable core structure of the isolated gP4l protein.Synthetical pePtides derived from gp4l core structure, C34(628-66l) and C46(628-673 ),contain repeated sequence withSJ2176 and DPl78. Anti-C34 and --C46 monoclonal antibodieswere generated and cloned from mice imrnunized with GST C34and GSTC46. The 3 ofmabs binding C34(lG1,2F8, 2B7)inhibited the bindind of both N46 and C34. The assay for cellproliferation by Mn showed that l Gl can enhance theproliferation of H9anV 1 IIIB cells; but the cell fusion assayshowed that lGl cannot influence the form of syncytiumconfOrmation of HIV l infected cells (H9naVlmB) withuninfected cells(MT2). These results suggested that the epitoperecognized by l Gl may induce a response to enhanceproliferation of HIV1 infected cell.For further identification of above epitoPe, we screened andidentifiy the epitope from l2mer random phage displayed peptide- 7 -libraries with 1G1 mab. After three rounds screening, the positive clones were identified by phage ELISA and binding assay with rabbit anti- gp4 1 polyclonal antibodies. The results showed that 6 positive clone from random 17 clones. In addition these positive clones can bind to 101 and anti-C34 polyclonal antibody, the also can bind to N-36 peptide, which means these clones are mimotopes of C-34 peptide.