恶性疟原虫PfRNase Ⅱ的克隆表达与降解特性的初步分析  

Cloning,prokaryotic expression,and analysis of catalysis by PfRNase Ⅱ of Plasmodiumfalciparum

作　　者：余胜超[1] 常志广[1] 土志伟 黄朋[1] 韩悦[1] 赵帅杰[1] 魏晓艳[1] 周健华[1] 陆慧君[1] 姜宁[1] 陈启军[1] 

机构地区：[1]吉林大学人兽共患病研究所人兽共患病教育部重点实验室,吉林长春130062

出　　处：《中国病原生物学杂志》2016年第3期238-241,共4页Journal of Pathogen Biology

摘　　要：目的利用原核表达系统诱导表达恶性疟原虫融合蛋白pGEX-PfRNaseⅡ,并分析其RNA降解特性。方法分析并选取恶性疟原虫3D7株PfRNaseⅡ的活性区,以cDNA为模板PCR扩增目的片段。将目的片段插入到pGEX-4T-1表达载体中,构建pGEX-4T-1-PfRNaseⅡ重组表达质粒,转化至大肠埃希菌BL21-CodonPlus(DE3),利用IPTG诱导表达目的蛋白pGEX-PfRNaseⅡ。应用Glutathione-SepharoseTM 4B树脂纯化目的蛋白。并用pGEX-PfRNaseⅡ体外分析PfRNaseⅡ的RNA降解特性。结果经PCR、双酶切、测序鉴定,pGEX-4T-1-PfRNaseⅡ重组表达质粒构建正确,转化DE3后在IPTG的作用下表达目的蛋白。纯化的目的蛋白pGEX-PfRNaseⅡ在体外可降解单链RNA,但不能降解双链RNA。pGEX-PfRNaseⅡ在低浓度的Mg2+条件下降解活性较强,高浓度Mg2+条件下活性受到抑制,在5mmol/L EDTA存在下活性降低。结论克隆表达的pGEX-PfRNaseⅡ重组蛋白能水解单链RNA,且该水解活性具有二价金属离子依赖性。Objectives To prepare the fusion protein pGEX-PfRNase II of Plasmodium falciparumin a prokaryotic expression system and analyze its RNA degradation in vitro. Methods The catalytic domain of PfRNase II was determined using bioinformatic analysis.A pair of primers was designed to amplify the selected gene fragment from cDNA of the 3D7 strain of P.falciparum.The specific PCR product was cloned into a pMD18-T vector.The gene fragment was correct according to sequencing;this fragment was inserted into a pGEX-4T-1expression vector.The recombinant plasmid was transformed into E.coli BL21-CodonPlus（DE3）.Expression of the fusion protein pGEX-PfRNase II was induced with100μmol/L IPTG.The protein was then purified with Glutathione-SepharoseTM 4Band used to analyze RNA degradation by PfRNase II in vitro. Results A recombinant expression vector was successfully constructed and identified with PCR,restriction endonuclease digestion,and DNA sequencing.Single-stranded RNA,rather than double-stranded RNA,was cleaved by purified pGEX-PfRNase II in vitro.Activity at a low concentration of Mg2＋（micromole level）was markedly greater than that at a higher concentration（millimole level）,and activity was suppressed by 5mmol/L EDTA. Conclusion The purified fusion protein pGEX-PfRNase II is a hydrolytic ribonuclease that cleaves single-stranded RNA and its catalytic activity is dependent on divalent metal ions.

关 键 词：疟原虫 恶性 PfRNaseⅡ 原核表达 RNA降解 

分 类 号：R382.31[医药卫生—医学寄生虫学]