[Construction and expression of a prokaryotic expression plasmid of idiotypic vaccine against B cell lymphoma: encoding the fusion genes of single-chain variable fragment and MCP-3].
The aim was to construct a prokaryotic expression plasmid encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP-3). The cDNAs of immunoglobulin (Ig) VH and Ig VL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) by recombinant PCR method. The cDNAs of Ig VH and Ig VL were connected by a (Gly4Ser)3 linker. Then, the fragments of scFv and MCP-3 were connected with a NDAQAPKS spacer, using recombinant PCR method again. The results indicated that the fusion gene of scFv-MCP-3 were constructed correctly and cloned into the prokaryotic expression plasmid successfully identified by sequencing and restriction endonucleases examination. Finally, the fusion protein was expressed in E coli DH5alpha under induction by arabinose. And the fusion protein was 65 kD and account for 30% of the total protein of the bacteria. In conclusion, a prokaryotic plasmid, encoding the fusion gene of single-chain variable fragment with MCP-3 and expressing idiotype protein vaccination against B cell lymphoma, was constructed correctly.
Wang
Fu-Xu
FX
Department of Hematology, The Second Hospital, Shijiazhuang 050000, China.
Zhao
Bing
B
Cheng
Yun-Hui
YH
Pan
Ling
L
Luo
Jian-Min
JM
Zhang
Xue-Jun
XJ
Dong
Zuo-Ren
ZR
D000595:Amino Acid Sequence; D000818:Animals; D019496:Cancer Vaccines / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D054410:Chemokine CCL7 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D005822:Genetic Vectors; D006801:Humans; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D016393:Lymphoma, B-Cell / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D019444:Vaccines, DNA / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
C515450:CCL7 protein, human; D019496:Cancer Vaccines; D054410:Chemokine CCL7; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA