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已有研究证实,B细胞淋巴瘤病人自身免疫球蛋白独特型可视为肿瘤特异性抗原用于独特型疫苗。为探究带有细胞因子的融合型独特型肿瘤疫苗是否会提高免疫效果,制备小鼠B细胞型淋巴瘤细胞的独特型单链可变区片段,与免疫佐剂单核细胞趋化因子MCP3融合,同时融合报告基因EGFP,构建融合型独特型淋巴瘤DNA疫苗。用RT-PCR法扩增BALB/c小鼠源B细胞淋巴瘤细胞株A20的IgVH和IgVL基因,重组PCR法将编码(Gly4Ser)3的核苷酸片段连接两基因,制备scFv片段。用相同PCR方法,选用1段编码NDAQAPKS连接肽连接趋化因子MCP3基因与scFv片段,获得MCP3-scFv融合基因片段。将scFv和MCP3-scFv融合基因片段分别插入真核表达质粒pTARGET,并在融合基因的下游插入报告基因EGFP,构建真核表达质粒pTARGET/scFv-EGFP和pTARGET/MCP3-scFv-EGFP。结果表明,成功扩增A20细胞的IgVH和IgVL基因片段及scFv-EGFP、MCP3-scFv-EGFP融合基因片段;酶切鉴定表明,成功制备重组真核表达质粒pTARGET/scFv-EGFP和pTARGET/MCP3-scFv-EGFP。结论:成功构建带有鼠源scFv片段、趋化因子MCP3和EGFP融合的独特型抗B细胞淋巴瘤疫苗表达质粒pTARGET/MCP3-scFv-EGFP和pTARGET/scFv-EGFP。所构建的重组表达质粒为下一步的抗B细胞淋巴瘤基因疫苗的体内动物实验奠定了基础。
Studies have confirmed that patients with B-cell lymphoma idiotype of autoimmune globulin can be considered as tumor-specific antigen for the unique type of vaccine. To explore whether the fusion-type idiomatic tumor vaccine with cytokines enhances the immune response, a unique single-chain variable region fragment of mouse B-cell lymphoma cells was prepared and fused with the immune adjuvant monocyte chemotactic factor MCP3 , At the same time, fusion EGFP reporter gene to construct a fusion type of idiotypic lymphoma DNA vaccine. The IgVH and IgVL genes of BALB / c mouse-derived B cell lymphoma cell line A20 were amplified by RT-PCR, and the two fragments encoding the (Gly4Ser) 3 nucleotide sequence were ligated by the recombinant PCR to prepare the scFv fragment. The MCP3-scFv fusion gene fragment was obtained by the same PCR method using a piece of NDAQAPKS linker peptide linked to the chemokine MCP3 gene and the scFv fragment. The scFv and MCP3-scFv fusion gene fragments were inserted into the eukaryotic expression plasmid pTARGET, and the reporter gene EGFP was inserted downstream of the fusion gene to construct the eukaryotic expression plasmids pTARGET / scFv-EGFP and pTARGET / MCP3-scFv-EGFP. The results showed that the IgVH and IgVL gene fragments and the fusion gene fragments of scFv-EGFP and MCP3-scFv-EGFP in A20 cells were successfully amplified. The restriction endonuclease digestion indicated that the recombinant eukaryotic expression plasmids pTARGET / scFv-EGFP and pTARGET / MCP3- scFv-EGFP. CONCLUSION: The unique anti-B cell lymphoma vaccine expression plasmids pTARGET / MCP3-scFv-EGFP and pTARGET / scFv-EGFP with murine scFv fragment, chemokine MCP3 and EGFP fusion were successfully constructed. The constructed recombinant expression plasmid laid the foundation for the in vivo animal experiment of the next anti-B cell lymphoma gene vaccine.