人B细胞淋巴瘤细胞系Namalwa CDR3基因片段的克隆及其DNA疫苗制备

Construction of the DNA vaccine plasmid against CDR3 region of immunoglobulin heavy chain of the human B-cell lymphoma cell line Namalwa

目的 :为探讨B细胞淋巴瘤细胞膜表面免疫球蛋白可变区的互补决定区 3能否在动物体内激发特异性抗独特型抗体 ,以人B细胞淋巴瘤细胞系Namalwa为模型 ,克隆其膜表面免疫球蛋白重链可变区互补决定区 3(CDR3)基因片段作为抗原基因 ,构建DNA疫苗质粒。方法 :以RT PCR的方法获得互补决定区 3(CDR3)基因片段 ,进而克隆了小鼠单核细胞趋化因子 (MCP 3)基因作为佐剂分子 ,以重组PCR的方法获得CDR3和MCP 3基因的融合基因片段 ,克隆在真核表达载体pcDNA3 1中构建DNA疫苗质粒 ,然后通过脂质体转染的方法验证该质粒在真核细胞中的表达情况。结果 :应用分子生物学的方法获得了以Namalwa细胞mIgCDR3作为抗原基因的DNA疫苗质粒。结论 :人B细胞淋巴瘤细胞系NamalwaCDR3基因的DNA疫苗构建正确 ,体外瞬时转染实验证明该质粒能够在真核细胞中正确表达。

Objective:To analyze whether the DNA vaccine against the CDR3 regio n of immunoglobulin heavy chain of the human B-cell lymphoma could elicit the s p ecific idiotypic antibody,the authors established a model with the human B-cell lymphoma c ell line Namalwa.The CDR3 gene fragment of Namalwa membranous immunoglobulin hea vy chain was amplified.The sequenced CDR3 fragment was used as the antigen gene to construct the DNA vaccine plasmid. Methods:The authors acquired the CDR3 gene fragment using Ig superfamily primers by means of RT-PCR and the murine monocyte chemot ic protein(MCP-3) as the adjuvant molecular.The fused gene fragment of CDR3 and MCP-3 was obtained by recombinant PCR and then cloned into the eukaryonic plas mid vector pcDNA3.1 to construct the DNA vaccine plamid.Then the vaccine plasmid was transiently expressed in the eukaryonic cell COS-7. Results:The authors acquired th e DNA vaccine plasmid in which the mIgCDR3 of the Namalwa cell was used as the a ntigen gene by the molecular biology. Conclusion:The transient transfection assa y proved that the recombinant plasmid could express in eukaryonic cells in right way.They have constructed an expression plasmid containing fused MCP3-CDR3 seq uen ce which could be used in further study of DNA vaccine against B-cell lymphoma in vivo. [