Construction of HIV-1 broadly neutralizing antibody gene eukaryotic expression vector and detection of its biological function / 中华实验和临床病毒学杂志

Objective@#To construct a recombinant double gene co-expressing plasmid of HIV-1 broadly neutralizing antibody and to detect its expression in 293T cell line.@*Methods@#HIV-1 neutralizing antibody 2G12 variable region of light chain (VL) and heavy chain (VH) was synthesized, and ligated with vectors containing human IgG constant regions of light and heavy chain to construct a complete 2G12 light and heavy Chain. The VL and VH of 2G12 and IRES were cloned into eukaryotic expression vector pVR by PCR amplification and then the recombinant plasmid was transfected into 293T cells. Expression of the antibody in cell supernatant was detected by ELISA. Binding and neutralizing activity of the cell supernatant were tested by ELISA and micro-neutralization assays.@*Results@#The recombinant double gene eukaryotic expression vector which can express human IgG was constructed successfully. The expression level of the supernatant was 6.43 μg/ml, and the antibody retained the binding and neutralizing activity.@*Conclusions@#The constructed vector can express the antibody with binding and neutralizing activity, this study provides a good platform for the expression of human HIV neutralizing antibody in the eukaryotic expression vector.

Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System / 病毒学报

For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.