Research On Human Genetically Engineered Antibodies To Structural Protein Of Human Immunodificiency Virus Type 1 (HIV-1)

The provention and control of HIV-1 infection is still a confounded problem to humans' health. HIV-1 is a single RNA virus, with elliptic or rounded shape and diameter of 90-140 nm, belongs to lentvirus in retroviridae. HIV-1 has serrated spikes in virion's surface. Virion's outer-membrane is lipoid, its inner has a columned core. The envolope is glycoprotein gp160, one of the structural proteins, which is consisted of gp120, an outer-membrane protein, and gp41, a transmembrane protein. Interaction of gp120 with CD4 induces the conformation change of gp120 and forms high affinity site with CCR5/CXCR4 in gp120, with which gp120 helps HIV-1 entry into host cell. The structural proteins has included p24, a core protein, and p18, a matrix protein,too. In this project, the phage antibody library technology was used to obtain human-derived recombinant anti-HIV-1 gp160 Fab antibody. First, human peripheral blood from asymptomatic HIV-1 infected donors with high titer antibody against HIV-1 was collected and lymphocytes were isolated. Total RNA was extracted from lymphocytes, and cDNA was synthesized with oligo(dT)_12-18 primer. The genes of heavy chains Fd fragment and light chains of antibody were amplified by using PCR with a set of primers specific to human IgG1 Fab. The purified PCR products were cloned into phagmid pComb3X. These phagmids contained Fab genes were transformed into E.coli XL1-Blue by electroporation. A phage Fab library was constructed with 8x10⁶ members under the help of helper phage VCSM13. After panning and screening the phage Fab library against HIV-1 gp160 by three rounds of "absorption-elution-...