论文部分内容阅读
【目的】人肠腺病毒Ad41被称为难养腺病毒,其难以培养的特性可能与次要核心蛋白V(Ad41proteinV,pV)表达不充分有关。本研究拟表达纯化Ad41pV抗原,免疫动物制备抗血清,为研究Ad41难养性机理打下基础。【方法】以野生型Ad41基因组DNA为模板,PCR扩增pV,克隆到原核表达载体pET30a(+),测序后,转化大肠杆菌BL21(DE3)菌株,异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的蛋白表达,固化金属亲和层析(IMAC)方法纯化,免疫BALB/c小鼠制备抗血清,将获得的抗血清用于Western blot检测Ad41感染各细胞系后pV的表达。【结果】克隆得到包括完全编码区的pV基因,表达质粒转化BL21(DE3)菌株,使用1mmol/LIPTG37℃诱导4h,pV以包涵体形式表达,或使用0.5mmol/LIPTG25℃诱导8h获得可溶性表达。利用皮下多点注射包涵体的方法免疫小鼠,得到抗pV抗血清;使用纯化的可溶性pV作为抗原对抗血清进行了鉴定,表明该抗血清可用于Western blot检测。等量野生型Ad41感染293或293E12细胞(一株稳定表达Ad41E1B55K基因的293细胞)后,pV在293E12细胞的表达明显高于293细胞。【结论】成功克隆了Ad41pV基因,表达纯化了重组蛋白,获得了可用于Western blot检测的抗血清,为进一步研究Ad41难养性机理打下了基础。
【Objective】 Ad41, a human enteric adenovirus, is known as anaphylactoid adenovirus, and its inability to culture may be related to the insufficient expression of the secondary core protein V (Ad41proteinV, pV). In this study, the purified Ad41pV antigen was expressed and the antisera were prepared from animals for immunization, laying a foundation for studying the mechanism of Ad41 antidepressant. 【Method】 The wild type Ad41 genomic DNA was used as a template to amplify pV by PCR and cloned into prokaryotic expression vector pET30a (+). After sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3) The recombinant protein was purified by immobilized metal affinity chromatography (IMAC) and immunized with IPTG to prepare antiserum. The obtained antiserum was used to detect the expression of pV after Ad41 infection expression. 【Result】 The pV gene was cloned and the expression plasmid was transformed into BL21 (DE3). The recombinant plasmid was transformed into BL21 (DE3) strain. The recombinant plasmid was induced by 1mmol / L IPTG at 37 ℃ for 4h, pV was expressed as inclusion body, or induced by 0.5mmol / L IPTG at 25 ℃ for 8h. Anti-pV antiserum was obtained by immunizing mice with multiple subcutaneous injection of inclusion bodies. Purified soluble pV was used as antigen to identify the antiserum, indicating that the antiserum can be used for Western blot. The expression of pV in 293E12 cells was significantly higher than 293 cells after the same amount of wild-type Ad41 was infected 293 or 293E12 cells (a 293 cell stably expressing the Ad41E1B55K gene). 【Conclusion】 The Ad41pV gene was cloned successfully, and the recombinant protein was expressed and purified. The antiserum that can be used for Western blot detection was obtained, which laid the foundation for further research on the mechanism of Ad41 difficult-to-maintain.