| Specific cellular immune response, particularly CD8~+ CTL response, plays an important role in the control of the tumor growth and metastasis. CD8~+ CTLs can recognize the antigenic peptide displayed by class I major histocompatibility complex (MHC) molecules on tumor cells, and kill tumor cells. It was reported that class I MHC molecules-antigenic peptide single chain trimer (SCT) consisting of an antigenic peptide-linker-p2-microglobulin (|32m)-linker-H chain could efficiently maintain their covalent structure, and was unusually stable at the cell surface. Immunization of mice with SCT could efficiently induce the generation of antigenic peptide-specific CD8~+CTL response, hereby efficiently eradicating viral infection and tumor cells in animal models.Angiogenesis plays a critical role in the growth, invasion and metastasis of tumors. It has been shown that tumor growth is generally limited to 1 to 2 mm3 in the absence of a vascularized blood supply and inhibition of tumor angiogenesis is associated with suppression of tumor progression. Thus, antiangiogenic immunotherapy represents a novel modality for cancer treatment. Vascular endothelial growth factor (VEGF) receptor2 (VEGFR-2, also known as KDR) mediated VEGF signaling is the key rate-limiting step in angiogenesis. The approach to block VEGFR-2 signaling seems to be the most economy, most effective method of antiangiogenic therapy. In addition, three H-2D~b candidate epitopes were predicted and synthesized. Three peptides, designated as KDR1, KDR2, and KDR3, respectively, were found to contain motifs with high binding potential to the H-2D~b molecules by two computer programs: Bimas and SYFPEITH. Two of them, KDR2 and KDR3, were from the extracellular domain; KDR1 was from the intracellularpart of the protein. Furthermore, experiments suggest that KDR2 is more efficiently processed by proteasomes and/or transported to the endoplasmic reticulum by the TAP transporter rather than KDR3, offsetting the higher affinity of the latter.This study aims to construct recombinant plasmid that encodes MHC class I-VEGFR-2 antigenic peptide single-chain trimer, to pave a way for further research and development. ObjectiveConstruction of recombinant plasmid encoding MHC class I -VEGFR-2 antigenic peptide single-chain trimer. Method1. DNA fragments encoding Linker-1, Linker-2 and KDR2 with P2m signal peptide respectively were synthesized by Sangong Biothechnology Company.2. cDNA of H-2D~b and β2m was cloned from splenocytes of C57BL/6 mice by RT-PCR.3. DNA fragments encoding β2m signal peptide-KDR2 antigenic peptide and β2m were linked by Linker-1, and then cloned into pcDNA3.1(+) to form pcDNA3.1(+)/VEGFR-2-Linker-1-β2m recombinant plasmid.4. DNA fragment encoding H-2D~b was ligated with Linker-2, and then cloned into pcDNA3.1(+) to form pcDNA3.1(+)/Linker-2-H-2D~b recombinant plasmid.5. DNA fragment encoding Linker-2-H-2D~b gained by double enzymatic digestion, and then cloned into pcDNA3.1(+)/VEGFR-2-Linker-l-p2m to form pcDNA3.1(+)/VEGFR-2-β2m-H-2D~b (pcDNA3.1(+)/SCT).ResultConstruction of pcDNA3.1/β2m and pcDNA3.1/antigenic peptide with P2m signal peptide-Linker-1-β2m recombinant plasmids were verified by double enzymatic digestion and DNA sequencing. The results were consistent with those expected, showing successfully construction of recombinant plasmids mentionedabove. Construction of pcDNA3.1/Linker-2 recombinant plasmid was verified by double enzymatic digestion, and the result matched with which we have expected, proved that the recombinant plasmid was successfully constructed. H-2D~b sequence amplified by PCR was verified by agarose electrophoresis and DNA sequencing, and the results were consistent with those expected. ConclusionSuccessfully constructed three recombinant plasmids pcDNA3.1/β2m, pcDNA3.1/Linker-2 and pcDNA3.1/antigenic peptide with β2m signal peptide-Linker-1-β2m, and successfully amplified H-2D~b sequence.
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