【摘要】 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。更多还原

【Abstract】 The idiotypic determinant of surface immunoglobulin of B-cell lymphoma,as a tumor-specific antigen,has proved to be able to induce immune responses. To analyze whether an idiotypic vaccine fused with cytokine can elicit more effectively protective antitumor immunity,an eukaryotic expression plasmid was constructed,which encoded the fusion gene of single-chain variable fragment as a tumor specific antigen against B-cell lymphoma with monocyte chemotatic protein-3 ( MCP3 ) as immunogen to elicit T-cell-dependent protective antitumor immunity,and EGFP ( Enhanced Green Fluorescent Protein ) gene as a marker to trace the survival,growth,differentiation and expression of the former exogenetic genes. The cDNAs for immunoglobulin (Ig) VH and IgVL were amplified by RT-PCR and assembled into the single-chain variable fragment(scFv)connected with a (Gly4Ser)3 linker by recombinant PCR method. Then,the fragments of scFv and MCP3 were ligated with a NDAQAPKS spacer by the same method. The results showed that the fusion genes of scFv and MCP3-scFv were inserted into an eukaryotic expression vector pTARGET,and EGFP was cloned into the downstream of scFv and MCP3-scFv respectively. Finally the constructed plasmids were confirmed by sequencing and restriction analysis. In conclusion,a tumor-derived idiotypic DNA vaccine,encoding the fusion gene of single-chain variable fragment and monocyte chemotatic protein-3 ( MCP3 ) to elicit a T-cell dependent,antitumor immunity,and the EGFP gene was inserted correctly. The DNA vaccine could be used for further study of DNA vaccine against B cell lymphoma in vivo.更多还原