發(fā)布時間：2018-03-06 11:35

  本文選題：弓形蟲　切入點：ROP　出處：《中國病原生物學(xué)雜志》2017年04期 　論文類型：期刊論文

【摘要】：目的構(gòu)建弓形蟲棒狀體蛋白ROP18原核表達載體,并通過密碼子優(yōu)化等方法優(yōu)化其表達。方法運用RT-PCR擴增ROP18基因,將其克隆入pGEX-6P-1原核表達載體,構(gòu)建pGEX-ROP18重組質(zhì)粒;運用OptigeneTM密碼子優(yōu)化分析平臺對弓形蟲ROP18編碼基因進行優(yōu)化,合成優(yōu)化后的全長基因(ROP18u)并將其克隆入pGEX-6P-1原核表達載體,構(gòu)建pGEX-ROP18u重組質(zhì)粒;以優(yōu)化后的ROP18u基因為模板,PCR擴增去除N端信號肽和前功能區(qū)序列的截短ROP18片段(ROP18uc),將其克隆入pGEX-6P-1原核表達載體,構(gòu)建pGEX-ROP18uc重組質(zhì)粒。所有重組質(zhì)粒經(jīng)PCR、雙酶切和測序鑒定正確后,轉(zhuǎn)化大腸埃希菌BL21(DE3),用IPTG誘導(dǎo)表達重組蛋白,并進行Western blot鑒定。結(jié)果 pGEX-ROP18、pGEX-ROP18u和pGEX-ROP18uc于30℃誘導(dǎo)培養(yǎng)4h-6h,分別檢測到分子質(zhì)量約為88ku和77ku的重組蛋白,該蛋白能被GST單克隆抗體和ROP18多克隆抗體識別。結(jié)論成功構(gòu)建了pGEX-ROP18、pGEX-ROP18u和pGEX-ROP18uc原核表達載體,密碼子優(yōu)化未能顯著提高ROP18重組蛋白的表達量,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，

本文編號：1574673