【摘要】 【目的】进行含信号肽ApoC-Ⅰ(signal-ApoC-Ⅰ)和ApoC-Ⅰ成熟肽(mature-ApoC-Ⅰ)的基因克隆、原核表达和纯化。【方法】取肝癌切除手术患者的癌旁组织,提取总RNA后,逆转录成c DNA,并设计带酶切位点的特异引物分别扩增出signal-Apo C-Ⅰ、mature-Apo C-Ⅰ基因;将扩增出的基因通过T克隆入PMDTM19-T载体;T克隆经测序验证后,双酶切出目的基因,连入同样双酶切后的PET28a载体,构建原核表达质粒;再次测序验证后,将质粒转入BL21Star(DE3)中,表达条件优化后进行融合表达;将表达后产物用Ni-NTA树脂亲和层析进行重组蛋白纯化;纯化产物采用Western blot进行验证。【结果】构建了PET28a-signal-ApoC-Ⅰ、PET28a-mature-Apo C-Ⅰ表达质粒,重组质粒都经过测序鉴定正确;PET28a-signalApoC-Ⅰ诱导表达后能抑制宿主菌生长,但经过条件优化2个表达质粒都在BL21Star(DE3)菌中经异丙基-β-D-硫代半乳糖苷(IPTG)诱导大量表达和纯化,Western blot证实了表达的结果。【结论】含信号肽ApoC-Ⅰ、ApoC-Ⅰ成熟肽的基因克隆、原核表达和纯化成功,为进一步研究重组人Apo C-Ⅰ的作用奠定了基础。更多还原

【Abstract】 Objective To clone the genes of ApoC-Ⅰsignal peptide(signal-ApoC-Ⅰ) and ApoC-Ⅰmature peptide(mature-ApoC-Ⅰ) and to induce their prokaryotic expression and protein purification. Methods Peri-tumor tissues were collected from live cancer patients. Total RNA was extracted following by reverse transcription and designing specific primers to amplify the genes of signal-ApoC-Ⅰand mature-ApoC-Ⅰ. Subsequently, the amplified genes were cloned to PMDTM19-T vector by original TA cloning kit, and then the obtained cloning vector was verified by sequencing method. The object genes cut from the obtained vector were linked to PET28 a vector for obtaining a recombinant plasmid with prokaryotic expression. After verification by sequencing method, the recombinant plasmid was transformed into BL21Star(DE3) for fusion expression under the optimized expression conditions. The expressed products were purified by Ni-NTA resin affinity chromatography and then were verified by Western blotting method. Results The plasmids of PET28a-signal-ApoC-Ⅰ and PET28a-matureApoC-Ⅰwere successfully constructed and sequenced exactly. PET28a-signal-ApoC-Ⅰcannot be induced to express in normal ways. After optimizing the induction conditions, the target genes carried by the two plasmids can be induced to express by isopropyl thiogalactoside(IPTG) in BL21Star(DE3) strain. And the purity is verified by Western blotting method. Conclusion The successful cloning, prokaryotic expression and purification of signal-ApoC-Ⅰand mature-ApoC-Ⅰwill lay a foundation for the further study of the effect of recombinant human ApoC.更多还原