犬新孢子虫巨噬细胞转移抑制因子生物学特性鉴定

Cloning, Expression and Immunoregulatory Activities Identification of Macrophage Migration Inhibitory Factor Derived from Neospora Canine

关键词:  犬新孢子虫  巨噬细胞转移抑制因子  原核表达  免疫调节

Key words:  Neospora Canine  Macrophage Migration Inhibitory Factor  Prokaryotic expression  Immunoregulatory

摘要:

目的 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。方法 通过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 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。结论 实现了NcMIF在大肠杆菌系统中的可溶性表达并对其功能进行了鉴定,为进一步探究NcMIF生物学功能及其MIF在宿主免疫调节中的作用奠定基础。

Abstract:

Prokaryotic expression system was used to express Neospora Canine macrophage migration inhibitory factor (NcMIF) and the enzyme activities of NcMIF was identified. In order to obtain the NcMIF complete open reading frame (ORF) sequence, based on comparison of NcMIF sequences reported in the GenBank, one pair of specific primers were 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. The 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.