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.
- Author:
Fu-Xu WANG
1
;
Bing ZHAO
;
Yun-Hui CHENG
;
Ling PAN
;
Jian-Min LUO
;
Xue-Jun ZHANG
;
Zuo-Ren DONG
Author Information
1. Department of Hematology, The Second Hospital, Shijiazhuang 050000, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Animals;
Cancer Vaccines;
biosynthesis;
genetics;
immunology;
Chemokine CCL7;
biosynthesis;
genetics;
immunology;
Genetic Vectors;
Humans;
Immunoglobulin Idiotypes;
immunology;
Immunoglobulin Variable Region;
biosynthesis;
genetics;
immunology;
Lymphoma, B-Cell;
immunology;
Mice;
Mice, Inbred BALB C;
Molecular Sequence Data;
Plasmids;
genetics;
Prokaryotic Cells;
metabolism;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
immunology;
Vaccines, DNA;
biosynthesis;
genetics;
immunology
- From:
Journal of Experimental Hematology
2006;14(6):1151-1155
- CountryChina
- Language:Chinese
-
Abstract:
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.
