弓形虫棒状体蛋白ROP18的优化表达  

Optimized prokaryotic expression of a recombinant ROP18 protein of Toxoplasma gondii

作　　者：程正阳[1] 苏蕊[1] 张贤[1] 李叶林[1] 朱万博[1] 余莉[1] 

机构地区：[1]安徽医科大学病原生物学教研室人兽共患病安徽省重点实验室安徽病原生物学省级实验室,安徽合肥230032

出　　处：《中国病原生物学杂志》2017年第4期345-349,共5页Journal of Pathogen Biology

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。Objectives To construct a vector for prokaryotic expression of Toxoplasma gondii rhoptry protein 18 （ROP18） and to optimize that expression via codon optimization. Methods The ROP18 gene was amplified using RT- PCR and cloned into a pGEX-6P-1 vector to construct the recombinant plasmid pGEX-ROP18. The OptigeneTM platform for codon optimization and analysis was used to optimize the coding sequence of ROP18, and the optimized full-length gene （ROP18u） was synthesized and then subcloned into a pGEX-6P-1 vector to construct the recombinant plasmid pGEX-ROP18u. The optimized ROP18u gene served as a template, and PCR was used to amplify the truncated ROP18u fragment without the N-terminal signal peptide and propeptide （ROP18uc）. The ROP18uc gene was subcloned into a pGEX-6P-1 vector to construct the recombinant plasmid pGEX-ROP18uc. All of the recombinant plasmids were confirmed using PCR, double enzyme digestion, and sequencing, and then transformed into E. coli BL21（DE3）. Expression of the recombinant protein was induced with IPTG and the protein was identified with Western blotting. Results Data indica- ted that pGEX-ROP18 and pGEX-ROP18u produced a band of about 88 ku and that pGEX-ROP18uc produced a band of about 77 ku after incubation at 30℃ for 4-6 h. All of the recombinant proteins were recognized by GST monoclonal anti- bodies and ROP18 polyclonal antibodies. Conclusion The prokaryotic expression vectors pGEX-ROP18, pGEX- ROP18u, and pGEX-ROP18uc were successfully constructed. Codon optimization did not significantly increase the ex- pression of the recombinant ROP18 protein but it did increase the soluble protein content.

关 键 词：弓形虫 ROP18 原核表达 

分 类 号：R382.31[医药卫生—医学寄生虫学]