Constructing And Identification Of Lentivirus -mediated Mouse MIP-1α And B7-1 Gene Vectors

【AIM】The choice of vector is key step for cancer gene therapy. Lentivirus vector reconstructed from human immunodeficiency virus (HIV), not only can infect mitotic phase cells and metabolic stage cells, but also is able to integrate into the host cell genome and get a long-term stable expression. Inaddition, Lentivirus vector could accommodate large-scale exogenous target gene segments and show minor immune response. This study was to construct lentivirus-mediated mouse MIP-1αand B7-1 gene vectors and lay a foundation for double gene therapy with lymphoma in the future.【METHODS】1. The first part, mouse MIP-1αand B7-1 genes were synthesizeed by nest PCR . Lentivirus vector was linearized by Age I enzyme and then was directly connected with objective genes, the production of which were transformed into competent Bacterium coli DH5αcells. The positive recombinant colones were identified by PCR and direct sequencing analysis.2. The second part, the plasmids of MIP-1αand B7-1 genes infected 293T cells, respectively, 48 hours later, green fluorescence protein (GFP) in 293T cells was observed by fluorescence microscope; Western Blot was used to test protein expression of MIP-1αand B7-1 genes.3. The third part, three plasmid system of lentivirus were applied to transfect 293T cells for packing Lentivirus which included pGC-LV restructuring plasmid and packaging plasmid pHelper 1.0 and pHelper 2.0. After 48 hours, supernatant fluid was collected, and then concentrated and purified. Real-time PCR was used to detect the titer of lentivirus. 【RESULTS】1. Lentivirus-mediated mouse MIP-1αand B7-1 gene vectors were successfully constructed; The results of direct sequencing of positive colones were completely coincidence with target genes.2. There were MIP-1αand B7-1 GFP expression confirmed in 293T cells by fluorescence microscope and Western Blot.3. Three plasmid system successfully infected 293T cells and the titer of which was 2.00E+8 TU/ml tested by real-time PCR.【CONCLUSION】Lentivirus-mediated mouse MIP-1αand B7-1 gene vectors were successfully constructed and lay a foundation for double gene therapy with lymphoma in the future.