犬新孢子虫巨噬细胞转移抑制因子(NcMIF)克隆表达与其免疫调节作用鉴定

摘要

通过原核系统表达犬新孢子虫巨噬细胞转移抑制因子(NcMIF),并对该蛋白的免疫调节作用进行分析.根据GenBank发表的序列,利用分子生物学软件设计了一对特异性引物,通过RT-PCR方法扩增出NcMIF全基因,经测序分析后,将NcMIF亚克隆到原核表达载体pET28a(+),然后将鉴定为阳性的重组质粒转化到E.coli BL21(DE3)中用IPTG进行诱导表达.利用HPLC对可溶性表达的重组NcMIF蛋白进行纯化,去除内毒素,最后对NcMIF的免疫调节作用进行鉴定.结果显示,NcMIF没有明显的抗糖皮质激素的免疫抑制作用,也没有上调巨噬细胞TNF-α和NO表达量的作用,为进一步探究NcMIF的生物学功能及MIF在宿主免疫调节中的作用奠定了基础.%To express macrophage migration inhibitory factor ( NcMIF ) of Neospora canium by prokaryotic expression system and identify the enzyme activities of NcMIF, the NcMIF complete open reading frame ( ORF) sequence was obtained based on sequences reported in the GenBank. And one pair of specific primers was designed to amplify the NcMIF gene from cDNA by using polymerase chain reaction ( PCR) and the NcMIF ORF was sequenced and subcloned into pET28a(+) . The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. The recombinant protein was expressed in soluble way and purified by HPLC and endotoxin was removed from the recombinant protein and native proteins. Results showed that NcMIF had no effect to counter-regulate glucocorticoid activity in macrophages or to regulate TNF-α and nitric oxide production by macrophages.It will establish the foundation to explore the biological function of NcMIF and its role in host immune regulation of MIF.