论文部分内容阅读
目的构建靶向EGFR二聚化界面的肿瘤多肽疫苗P64k-EGFR262-328并分析其免疫原性。方法采用重叠延伸PCR来扩增P64k-EGFR262-328融合基因,克隆至pMD18-T,经测序鉴定后与pET-21b构建重组表达载体,并转化大肠杆菌BL21(DE3)。目的蛋白经IPTG诱导表达后用Source Q阴离子层析和Ni-NTA亲和层析纯化,纯化的融合蛋白乳化后免疫BALB/c小鼠分析其免疫原性。结果获得了2 031 bp的融合基因,IPTG诱导后获得了相对分子质量(Mr)70 000大小的目的蛋白,经两步柱层析后,融合蛋白达到了95%以上的纯度,并在小鼠体内产生了高达1∶16 000以上的抗体滴度。结论成功制备了肿瘤多肽疫苗P64k-EGFR262-328。
Objective To construct the tumor peptide vaccine P64k-EGFR262-328 targeting EGFR dimerization interface and analyze its immunogenicity. Methods P64k-EGFR262-328 fusion gene was amplified by overlap extension PCR and cloned into pMD18-T. The recombinant plasmid pET-21b was transformed into E. coli BL21 (DE3) after sequencing. The target protein was induced by IPTG and purified by Source Q anion chromatography and Ni-NTA affinity chromatography. The purified fusion protein was then immunized to immunize BALB / c mice to analyze its immunogenicity. The result showed that a fusion protein of 2 031 bp was obtained. After IPTG induction, the target protein of 70 000 in molecular weight (Mr) was obtained. After two-step chromatography, the fusion protein reached more than 95% purity. Antibody titers of up to 1: 16 000 are produced in vivo. Conclusion The tumor peptide vaccine P64k-EGFR262-328 was successfully prepared.