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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。
OBJECTIVE: To express and express pGEX-PfRNaseⅡ in Plasmodium falciparum by using prokaryotic expression system and to analyze its RNA degradation characteristics. Methods The active region of PfRNase Ⅱ of Plasmodium falciparum 3D7 strain was analyzed and selected. The cDNA fragment was amplified by PCR. The recombinant plasmid pGEX-4T-1-PfRNaseⅡ was constructed and transformed into Escherichia coli BL21-CodonPlus (DE3). The target protein pGEX-PfRNaseⅡ was induced by IPTG. Glutathione-Sepharose 4B resin was used to purify the target protein. The RNA degradation properties of PfRNaseII were analyzed in vitro with pGEX-PfRNase II. Results The recombinant plasmid pGEX-4T-1-PfRNaseⅡ was identified by PCR, double enzyme digestion and sequencing. The recombinant plasmid pGEX-4T-1-PfRNaseⅡ was constructed and expressed under the action of IPTG. The purified target protein pGEX-PfRNaseⅡ can degrade single-stranded RNA in vitro, but can not degrade double-stranded RNA. pGEX-PfRNaseⅡhad a strong degradation activity under the condition of low concentration of Mg2 +. The activity of pGEX-PfRNaseⅡwas inhibited under the condition of high concentration of Mg2 + and decreased in the presence of 5mmol / L EDTA. Conclusion The cloned pGEX-PfRNaseⅡ recombinant protein can hydrolyze single-stranded RNA, and its hydrolytic activity is divalent metal ion-dependent.