论文部分内容阅读
目的:构建包含甲胎蛋白强抗原决定簇hAFP542-550、EGFP和GPI锚定蛋白GPC3三者组成的融合蛋白GPC3-hAFP542-550-EGFP表达质粒pGPC3-EGFP,确定其定位表达在真核细胞膜上,以强化靶细胞的免疫原性。方法:①采用RT-PCR方法从人胎盘组织总RNA中调取GPC3基因,化学合成基因片段-KOZAK-GPCN+afp542-550-,并从pEG-FP-N1质粒中扩增EGFP基因,三者嵌合构建融合蛋白的表达质粒pcDNA3.1(+)/GPCN+afp542-550-EGFP-GPCC(pG-PC3-EGFP);②以Lipofectamine 2000转染质粒pGPC3-EGFP入肝癌细胞株HepG2(GPC3+AFP+),转染后24h和48h引物GPCN-F和EGFP-rRT-PCR及Western blot检测融合蛋白GPC3-hAFP542-550-EGFP表达;③对照质粒pEGFP-N1和pG-PC3-EGFP转染HepG2,荧光显微镜观察比较两种质粒EGFP蛋白表达的部位;pGPC3-EGFP转染人胚肾HEK293细胞(GPC3-AFP-),提取细胞膜蛋白和可溶蛋白Westernblot检测融合蛋白表达。结果:①成功构建pGPC3-EGFP质粒;②融合蛋白可在HepG2中表达;③与pEGFP-N1不同,融合蛋白主要在胞膜上出现荧光反应,胞质内仅见散在零星荧光;转染后HEK293细胞的膜蛋白中检测到融合蛋白表达,而可溶蛋白中未检出。结论:pGPC3-EGFP可以在真核细胞中表达,表达的融合蛋白GPC3-hAFP542-550-EGFP仍然定位表达在细胞膜,是一种新型GPI再锚定蛋白。
OBJECTIVE: To construct a fusion protein GPC3-hAFP542-550-EGFP expression plasmid pGPC3-EGFP containing the strong epitopes of alpha-fetoprotein hAFP542-550, EGFP and GPI anchoring protein GPC3, and to determine its localization on eukaryotic cell membrane To enhance the immunogenicity of the target cells. Methods: ①The GPC3 gene was extracted from the total RNA of human placenta by RT-PCR and the gene fragment-KOZAK-GPCN + afp542-550- was chemically synthesized. The EGFP gene was amplified from pEG-FP-N1 plasmid The recombinant plasmid pcDNA3.1 (+) / GPCN + afp542-550-EGFP-GPCC (pG-PC3-EGFP) was transfected into HepG2 cells by Lipofectamine 2000. The recombinant plasmid pGPC3- AFP +). The expression of GPC3-hAFP542-550-EGFP was detected by GPCR-F, EGFP-rRT-PCR and Western blot at 24h and 48h after transfection; ③The control plasmid pEGFP-N1 and pG-PC3-EGFP were transfected into HepG2, Fluorescence microscopy was used to compare the expression sites of the two plasmids EGFP protein; pGPC3-EGFP was transfected into human embryonic kidney HEK293 cells (GPC3-AFP-), and the expression of the fusion protein was detected by Western blot. Results: ① The plasmid pGPC3-EGFP was successfully constructed; ② The fusion protein was expressed in HepG2; ③ Unlike pEGFP-N1, the fusion protein mainly reflected fluorescence on the cell membrane, scattered sporadic fluorescence appeared in the cytoplasm; The expression of the fusion protein was detected in the membrane protein of the soluble protein but not in the soluble protein. Conclusion: pGPC3-EGFP can be expressed in eukaryotic cells. The expressed fusion protein GPC3-hAFP542-550-EGFP is still located in the cell membrane and is a novel GPI re-anchor protein.