弓形虫棒状体蛋白ROP18的优化表达

发布时间：2018-03-06 11:35

  本文选题：弓形虫　切入点：ROP　出处：《中国病原生物学杂志》2017年04期 　论文类型：期刊论文

【摘要】：目的构建弓形虫棒状体蛋白ROP18原核表达载体,并通过密码子优化等方法优化其表达。方法运用RT-PCR扩增ROP18基因,将其克隆入pGEX-6P-1原核表达载体,构建pGEX-ROP18重组质粒;运用OptigeneTM密码子优化分析平台对弓形虫ROP18编码基因进行优化,合成优化后的全长基因(ROP18u)并将其克隆入pGEX-6P-1原核表达载体,构建pGEX-ROP18u重组质粒;以优化后的ROP18u基因为模板,PCR扩增去除N端信号肽和前功能区序列的截短ROP18片段(ROP18uc),将其克隆入pGEX-6P-1原核表达载体,构建pGEX-ROP18uc重组质粒。所有重组质粒经PCR、双酶切和测序鉴定正确后,转化大肠埃希菌BL21(DE3),用IPTG诱导表达重组蛋白,并进行Western blot鉴定。结果 pGEX-ROP18、pGEX-ROP18u和pGEX-ROP18uc于30℃诱导培养4h-6h,分别检测到分子质量约为88ku和77ku的重组蛋白,该蛋白能被GST单克隆抗体和ROP18多克隆抗体识别。结论成功构建了pGEX-ROP18、pGEX-ROP18u和pGEX-ROP18uc原核表达载体,密码子优化未能显著提高ROP18重组蛋白的表达量,但提高了可溶性蛋白的含量。
[Abstract]:Objective to construct the prokaryotic expression vector of ROP18 of Toxoplasma gondii and optimize its expression by codon optimization. Methods ROP18 gene was amplified by RT-PCR and cloned into pGEX-6P-1 prokaryotic expression vector to construct pGEX-ROP18 recombinant plasmid. The ROP18 encoding gene of Toxoplasma gondii was optimized by using OptigeneTM codon optimization analysis platform. The optimized full-length gene was synthesized and cloned into pGEX-6P-1 prokaryotic expression vector to construct pGEX-ROP18u recombinant plasmid. Using the optimized ROP18u base as template, the truncated ROP18 fragment of N terminal signal peptide and prefunctional region sequence was amplified and cloned into pGEX-6P-1 prokaryotic expression vector to construct pGEX-ROP18uc recombinant plasmid. All the recombinant plasmids were confirmed by PCR, double enzyme digestion and sequencing. The recombinant protein was induced by IPTG and identified by Western blot. Results pGEX-ROP18, pGEX-ROP18u and pGEX-ROP18uc were cultured at 30 鈩，

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