伯氏疟原虫静息巯基氧化酶的生物信息学分析和体外酶活检测

Bioinformatics analysis and enzymatic activity test of quiescin sulfhydryl oxidase from Plasmodium berghei

目的克隆、表达伯氏疟原虫静息巯基氧化酶(PbQSOX)基因,纯化重组Pb QSOX蛋白(rPbQSOX),并对其生物信息学特点和酶活性进行分析。方法分别采用BLAST、SMART、ClustalW对P QSOX蛋白的信号肽、蛋白结构域、活性位点、同源性进行分析;通过BLAST和MAGA5构建PbQSOX的系统进化树;应用SWISS-MODEL对PbQSOX蛋白的三级结构进行预测。BALB/c雌性小鼠经腹腔感染伯氏疟原虫(1×10^7/鼠),于感染后第4天提取疟原虫基因组DNA。PCR扩增PbQSOX基因,经测序正确后,将目的片段连接至原核表达载体pET30a(+),构建重组质粒pET30a(+)-PbQSOX。将鉴定正确的重组质粒转化至大肠埃希菌BL21中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,取菌液进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质免疫印迹(Western blotting)分析;应用镍氨三乙酸琼脂糖柱纯化可溶性的rPbQSOX,并以三(2-羧乙基)膦(TCEP)或二硫苏糖醇(DTT)为酶反应底物分析rPbQSOX的酶活性。P QSOX大部分其他物种的同源基因。多序列蛋白比对与蛋白结构域分析结果显示,PbQSOX蛋白含1个信号肽,存在Trx1、ψErv和Erv/ALR结构域,但完全缺失了Trx2结构域。Pb QSOX蛋白含3个关键的CXXC活化基序(C为半胱氨酸Cys,X为任意氨基酸),即CPAC、CRNC和CNYC;采用MAGA5邻接法构建的系统进化树显示,PbQSOX与约氏疟原虫QSOX(PyQSOX)的亲缘关系最近;SWISS-MODEL同源建模法分析结果显示,用已知的布氏锥虫QSOX(TbQSOX)可预测PbQSOX蛋白的三级结构。PCR扩增PbQSOX基因,获得1486 bp目的条带,经测序为PbQSOX基因序列;pET30a(+)-PbQSOX质粒经NdeⅠ、HindⅢ双酶切与测序鉴定正确。SDS-PAGE和Western bloting分析结果显示,rPbQSOX重组蛋白主要以可溶形式表达,相对分子质量约59000。纯化后的rPbQSOX对TCEP的催化活性为0.84±0.18,DTT的催化活性为0.78±0.14(P<0.01)。结论PbQSOX蛋白为单Trx1结构域的伯氏疟原虫静息巯基氧化酶,构建、表达的rPbQSOX蛋白为可溶性蛋白,具有一定的静息巯基氧化酶催化活性。

Objective To clone and express the gene of Plasmodium berghei quiescin sulfhydryl oxidase(PbQSOX),purify the recombinant protein rPbQSOX,and analyze its bioinformatics characteristics and enzymatic activity.Methods The signal peptides,protein domains,active sites and homology of PbQSOX protein were analyzed by BLAST,SMART and ClustalW softwares.The phylogenetic tree of PbQSOX was constructed by BLAST and MAGA5.The tertiary structure of PbQSOX protein was predicted by SWISS-MODEL.Female BALB/c mice were intraperitoneally injected with 1×10^7 P.berghei protozoan/mouse.On day 4 after infection,P.berghei genomic DNA was extracted.The PbQSOX gene was amplified by PCR.After confirming the product by sequencing,the target product was cloned into pET30 a(+)vector to construct recombinant plasmid pET30 a(+)-PbQSOX.The verified plasmid was transfected into E.coli BL-21 after induction by isopropylβ-D-thiogalactoside,the bacteria culture medium was analysed underwent by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting.The soluble recombinant Pb QSOX(rPbQSOX)was purified by Ni-triacetate agarose column,and the enzyme activity was analyzed using TCEP[Tris(2-carboxyethyl)phosphine]or DL-Dithiothreitol(DTT)as the enzyme reaction substrate.Results BLAST analysis identified homologues of PbQSOX from other species.Multiple-sequence blasting and domain analysis showed that PbQSOX contained a signal peptide,and Trx1,ψErv and Erv/ALR domains,but lacked the Trx2 domain.The PbQSOX protein contained three key CXXC activation motifs(C,cysteine;X,any amino acid):CPAC,CRNC and CNYC.The phylogenetic tree constructed by MAGA5 using the neighbor joining method showed that the PbQSOX was genetically most close to the P.yoelii QSOX(PyQSOX).SWISS-MODEL analysis showed that the tertiary structure of PbQSOX can be predicted using known Trypanosoma brucei QSOX(TbQSOX).PCR amplification of PbQSOX resulted in a specific band of 1486 bp,sequence of which was confirmed by sequencing.The PET30 a(+)-PbQSOX plasmid was verified by sequencing after NdeⅠand HindⅢdigestion.SDA-PAGE and Western blotting analysis showed that r Pb QSOX was expressed in soluble form with a relative molecular weight of about 59000.The enzymatic activities of purified rPbQSOX toward TCEP and DTT were 0.84±0.18 and 0.78±0.14,respectively.Conclusion The expressed PbQSOX protein contains a single Trx1 domain.The rPbQSOX is a soluble protein that has some catalytic activity of the quiescin sulfhydryl oxidase.