Construction And Screening Of A Phage Display Library Of Mouse Single-Chain FV Antibodies Against HSV-2gB Protein

Herpes simplex virus type 2(HSV-2)is a linear, double stranded DNA virus, HSV-2 is the primary cause of genital herpes. Following the primary infection, the virus migrates to sensory ganglia and establishes latency in dorsal root ganglia. In pregnant women, the infection causes unplanned abortion, premature delivery and congenital neonatal herpes. It has been is also associated with an increased risk of HIV infection with a significant increase in the disease severity.gB,gD, which plays an important part in virion reproduction and antibody production, are the manly component of the surface of virions, and the key of HSV inefetion. not only can induce humoral immunity and cell-mediated immunity, but also can give birth to DTH reaction and T-cell multiplication.AIM:To construct and screen specific mouse ScFv phage antibody library against HSV2-gB. METHODS:Using a recombinant phage-display antibody technique, BALB/c mice were immunized with herpes simplex virus typeⅡenvelope glycoprotein B of the N-terminal epitope fusion protein (Trx-gBN), and splenic lymphocytes were isolated for extraction of total RNA from which antibody heavy chain variable region gene (VH) and light chain variable region gene (VL) were amplified by RT-PCR, genes encoding ScFv fragments were constructed by random linkage of VH gene and VL gene by SOE-PCR, and then the constructed ScFv gene were cloned into phagemid vector pCANTAB-5E and transfected into E.coli TG1, by helper phage M13K07 rescue by phage single chain antibody library. To herpes simplex virus typeⅡenvelope glycoprotein B of the N-terminal epitope fusion protein(Trx-gBN)target site for the screening, panning positive recombinant phage, was identified for its sequence analysis, non-competitive ELISA, the initial detection of ScFv specificity of recombinant antigen binding activity and neutralizing activity. RESULTS:Recombinant ScFv phage antibody library was successfully constructed, its size was 4×109 and the recombination rate was 93% as analysis by colonies PCR. After three rounds of screening, we screened the specific ScFv of virus neutralizing activity. CONCLUSION:The specific mouse ScFv phage display antibody library against HSV2-gB was successfully constructed, screened the specific ScFv, which laid a foundation of preparation of therapeutic humanized antibody against HSV2-gB.