中文摘要：

目的：表达和纯化RpfA融合蛋白．方法：将含有RpfA基因片段的质粒用NdeI与Bam H Ⅰ双酶切，然后将目的基因片段克隆入pcDNA3．1^＋载体构建重组载体pcDNA3．1^＋-RpfA，测序正确后再将目的基因片段亚克隆入pET19b原核表达载体并转化Ecoli DE3，IPTG诱导表达融合蛋白，Westernblot鉴定融合蛋白．在变性条件下Ni^2＋-NTA亲和色谱柱纯化目的融合蛋白,结果：目的基因片段测序与Genbank报道一致（切去了5’端99bp）．SDS—PAGE显示，在M约为80ku处有表达条带，Westernblot鉴定为（His）。融合蛋白，并与小鼠抗Rv1884和抗Rv2389免疫血清有交叉反应．可溶性分析发现融合蛋白主要以包涵体形式存在,经Ni^2＋-NTA亲和色谱柱纯化得到了融合有6个组氨酸残基的RpfA融合蛋白,结论：成功构建了pET19b—RpfA表达载体，并在Ecoli DE3中高效表达，亲和层析后获得了纯化目的蛋白．

英文摘要：

AIM ： To express efficiently Mycobacterium tuberculos/s RpfA in E. coli and purify the fusion proteins. METHODS： RpfA gene segments were digested with NdeI and BamHI and cloned into pcDNA3. 1^＋ vector. After sequencing, RpfA were subcloned into the prokaryotic expression vector pET19b to get pET19b-RpfA. The plasmid pET19b-RpfA were transformed into E. coli DE3 and induced by IPTG for its expression. The RpfA fusion protein expression was analyzed by SDS-PAGE and confirmed by Western blot. Recombinant （His）6 fusion proteins were purified via Ni2^＋ -NTA affinity chromatography. RESULTS： RpfA gene segments were identical to that GenBank reported （lacking 5＇ ends of 99 bp）. The pET19b-RpfA vector expressed RpfA fusion proteins at Mr about 80 ku, which could be caught by anti（His） 6 mAb ,anti-Rv1884 and anti-Rv2389 immune serum. SDSPAGE analysis showed that the fusion proteins mainly existed in inclusion bodies. The expressed proteins could be purified via Ni2^＋ -NTA affinity chromatography in denatured condition. CONCLUSION： The recombinant expression plasmid pET19b-RpfA has been constructed and RpfA fusion proteins been successfully expressed in E. coli and purified, which lay a basis for further study of RpfA functions.