The idiotype (Id) of immunoglobulin expressed by B cell lymphoma can serve as the only widely accepted tumor associated antigen. But the Id vaccines have failed to elicit anti-tumor immunity for its weak immunogenic. Monocyte chemoattractant protein-3 (MCP3) can recruit various subsets of immune cells, such as DCs, which would uptake and properly process and present Id, activating both arms of the immune system, humoral and cellular. So the Id-MCP3 fusion proteins are potential vaccines for immunotherapy of B cell lymphoma. In this study, two vaccine candidates were constructed by fusing allogeneous MCP3 to the amino-(MCP3-scFv) or carboxyl-(scFv-MCP3) terminus of the A20 (BABL/c murine B-lymphocyte) Id scFv with a flexible polypeptide spacer encoding NDAQAPKS to prevent dissociation and keep their respective natural construction and function. And VH and VL domains were linked with a current linker encoding (Gly4Ser)3. Firstly, the cDNAs of Ig VH and Ig VL were amplified by RT-PCR from A20 mRNA, and then assembled into scFv by recombinant PCR method. Secondly the fusion genes of scFv/MCP3 were formed using the same method. After sequencing, MCP3/scFv fusion genes were cloned into pET-39b vector. Lastly MCP3/scFv fusion proteins were expressed in E.coli BL21. And the fusion protein is about 62 kD. We found that, under the same condition, MCP3-scFv fusion protein was expressed successfully and accounted for 40% of the total protein of the bacteria but not scFv-MCP3. Our result indicated that fusing MCP3 to carboxyl-terminus of scFv protein may have cytotoxicity to the host cells or maybe not stable inside the host cells. Next we will determine the activity of the fusion protein MCP3-scFv with cell-chemotatic-experiment in vitro and bearing-tumor mice experiment in vivo. Once the results would suggest that there may be an anti-tumor effect, we can make individual vaccines to lead to a better survival.
Disclosure: No relevant conflicts of interest to declare.