[Construction of expression plasmid for fused idiotypic DNA vaccine of B-cell lymphoma].
The idiotypic determinant of surface immunoglobulin of B-cell lymphoma, as a tumor-specific antigen, has proved to be able to induce immune responses. To analyze whether an idiotypic vaccine fused with cytokine can elicit more effectively protective antitumor immunity, an eukaryotic expression plasmid was constructed, which encoded the fusion gene of single-chain variable fragment as a tumor specific antigen against B-cell lymphoma with monocyte chemotactic protein-3 (MCP3) as immunogen to elicit T-cell-dependent protective antitumor immunity, and EGFP (Enhanced Green Fluorescent Protein) gene as a marker to trace the survival, growth, differentiation and expression of the former exogenetic genes. The cDNAs for immunoglobulin (Ig) VH and IgVL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) connected with a (Gly(4)Ser)(3) linker by recombinant PCR method. Then, the fragments of scFv and MCP3 were ligated with a NDAQAPKS spacer by the same method. The results showed that the fusion genes of scFv and MCP3-scFv were inserted into an eukaryotic expression vector pTARGET, and EGFP was cloned into the downstream of scFv and MCP3-scFv respectively. Finally the constructed plasmids were confirmed by sequencing and restriction analysis. In conclusion, a tumor-derived idiotypic DNA vaccine, encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP3) to elicit a T-cell dependent, antitumor immunity, and the EGFP gene was inserted correctly. The DNA vaccine could be used for further study of DNA vaccine against B cell lymphoma in vivo.
Wang
Fu-Xu
FX
Department of Hematology, The Second Hospital, Hebei Medical University, Shijiazhuang 050000, Hebei Province, China. wangfx@hebmu.edu.cn
Zhang
Yan
Y
Zhao
Bing
B
Pan
Ling
L
Zhang
Xue-Jun
XJ
Luo
Jian-Min
JM
Dong
Zuo-Ren
ZR
D000818:Animals; D019496:Cancer Vaccines / Q000235:genetics* / Q000276:immunology; D045744:Cell Line, Tumor; D054410:Chemokine CCL7 / Q000276:immunology*; D005822:Genetic Vectors; D007135:Immunoglobulin Variable Region / Q000276:immunology; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000276:immunology* / Q000517:prevention & control; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D057127:Single-Chain Antibodies / Q000276:immunology; D019444:Vaccines, DNA / Q000235:genetics* / Q000276:immunology
D019496:Cancer Vaccines; D054410:Chemokine CCL7; D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies; D019444:Vaccines, DNA