【摘要】 目的表达和纯化伯氏疟原虫类巨噬细胞移动抑制因子(PbMIF)。方法根据GenBank中PbMIF mRNA的预测序列,设计特异引物,采用RT-PCR方法从伯氏疟原虫ANKA株红内期RNA扩增获得PbMIF基因。将PbMIF基因与T载体连接并测序,利用NCBI中Blast程序分析测序结果。阳性T/A克隆质粒经BamHⅠ和XhoⅠ双酶切后,将目的片段克隆至原核表达载体pET28a,并转化大肠埃希菌BL21(DE3),收集经IPTG诱导表达的细菌进行SDS-PAGE分析,并对表达产物进行亲和纯化。结果从伯氏疟原虫RNA中扩增到PbMIF cDNA,长度为351 bp,编码116个氨基酸。测序结果显示,其核苷酸序列与GenBank中PbMIF cDNA的预测序列完全一致,编码的氨基酸在一级结构上与恶性疟原虫类巨噬细胞移动抑制因子(PfbbMIF)的同源性为76%,与人类和小鼠MIF的同源性为31%。表达产物为可溶性的PbMIF蛋白,亲和纯化后的PbMIF蛋白纯度为71%。结论表达并纯化出可溶性PbMIF蛋白,为进一步研究疟原虫的生物学特性奠定了物质基础。更多还原

【Abstract】 Objective To express and purify the macrophage migration inhibitory factor homologue of Plasmodium berghei(PbMIF).Methods The PCR primers were designed according to the nucleotide sequence of putative PbMIF in GenBank.Blood-stage RNA sequence from P.berghei ANKA strain was reverse transcripted and amplified.The amplified cDNA was cloned into T vector and sequenced.The sequencing result was searched for homology identity with NCBI Blast program.After positive plasmids of T/A clone were hydrolyzed by BamHⅠ and XhoⅠ,the fragments of products were cloned into prokaryotic expression vector pET28a.Recombinant plasmids were transformed into BL21(DE3).Expression of PbMIF was induced with IPTG and analyzed by SDS-PAGE.The expression products were purified through affinity column.Results The cloned PbMIF cDNA was composed of 351 nucleotides and encoded 116 amino acids.The sequence of cloned PbMIF cDNA was identical to that of putative PbMIF in GenBank.The predicted amino acid sequence has an identity of 76% with MIF-like protein of P.falciparum,and 31% with MIF protein of human or mouse. Soluble PbMIF protein was expressed in BL21(DE3).The purity of affinity purification products was 71%.Conclusion PbMIF protein was successfully expressed and purified,which provided the basis for further study.更多还原