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目的:构建羧基末端缺失核蛋白的真核表达载体并在小鼠肥大细胞瘤细胞株P815细胞中表达.方法:用RT-PCR方法从西安地区丙型肝炎患者血清中扩增HCVC区及部分E1区基因并进行克隆、测序,以此为模板扩增羧基末端缺失突变核蛋白基因片段(C507),将其定向克隆入真核表达载体pCI-neo中并转染P815细胞,经间接免疫荧光染色、激光共聚焦显微镜检测细胞中突变核蛋白(P169)的表达.结果:从患者血清中成功克隆出HCVCcDNA,构建了编码羧基末端缺失核蛋白的重组质粒pCI-507,并经酶切鉴定和测序证实;间接免疫荧光染色表明P169主要在P815细胞胞质中表达,少数可见于细胞核内.结论:重组体pCI-507构建正确,其表达产物P169在P815中得到有效表达,为在此基础上的DNA免疫研究提供了实验依据.
OBJECTIVE: To construct an eukaryotic expression vector carrying the carboxyl terminal deletion nucleoprotein and express it in the mouse mastocytoma cell line P815.Methods: The HCVC region and some E1s were amplified from the serum of patients with hepatitis C by RT-PCR (C507) was amplified by PCR and cloned into the eukaryotic expression vector pCI-neo and transfected into P815 cells. After indirect immunofluorescence staining (P169) were detected by laser scanning confocal microscope.Results: The recombinant plasmid pCI-507 encoding the carboxyl terminal deletion nucleoprotein was successfully cloned from the patient’s serum and identified by restriction enzyme digestion and sequencing Confirmed that indirect immunofluorescence staining showed that P169 was mainly expressed in the cytoplasm of P815 cells and a few could be found in the nucleus.Conclusion: Recombinant pCI-507 is constructed correctly, and its expression product P169 is effectively expressed in P815, on the basis of which DNA immunization provides experimental evidence.