【摘要】 目的:构建人泛素样修饰子(hUBL)基因的逆转录病毒载体并转染293T细胞,以探讨其生物学功能。方法:将hUBL克隆到原核表达质粒PET28a中,构建重组质粒PET28a-hUBL,转化BL21菌,以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。用其纯化蛋白免疫小鼠,获得多克隆抗体。重组蛋白用Western blot.鉴定。将hUBL基因克隆到逆转录病毒质粒中并转染293T细胞,用免疫组化染色法鉴定hUBL基因的表达。结果:hUBL在大肠杆菌中获得高效表达,并纯化了hUBL-PET28a的融合蛋白,以此为抗原制备了高效价的抗hUBL蛋白的小鼠多克隆抗体。免疫组化染色表明,hUBL重组逆转录质粒可在293T细胞中高效表达hUBL蛋白。结论:成功地构建了hUBL重组逆转录病毒载体,使得其在293T细胞中表达,获得稳定表达的细胞株。更多还原

【Abstract】 To construct human ubiquitin-like modifier (hUBL) gene recombinant retrovirus vector and transfect into 293T cells for understanding its role further. Methods: Human hUBL gene was inserted into the vector PET28a to construct recombinant vector PET28a-hUBL. The recombinant vectors were transfected into E. coli BL21 to express recombinant protein under isopropy-β-D-thiogalactoside (IPTG) induction. Mouses were immunized with purified proteins to produce polyclonal anti-hUBL antibody. The specificity of the anti-hUBL antisera was tested by Western Blot. In addition, hUBL gene was cloned into retrovirus vector LZRSPBMN and transfected into 293T cell lines by calcium phosphate precipitation. The expression of hUBL gene was examined by immunohistochemical staining. Results: hUBL protein was expressed and purified in E. coli and was used to obtain polyclonal anti-hUBL antibody. The results of immunohistochemistry revealed that hUBL recombinant retrovirus protein was expressed in 293T cells. Conclusion: hUBL recombinant retrovirus vector was constructed and transfected into 293T cells successfully. We obtained polyclonal anti-hUBL antibody, which established the basis for the functional study of hUBL.更多还原