奥氏奥斯特他线虫巨噬细胞转移抑制因子克隆表达与酶功能鉴定

The clone,expression and enzymatic activity identification of macrophage migration inhibitory factor derived from Ostertagia ostertagi

通过原核表达系统表达奥氏奥斯特他线虫(Ostertagia ostertagi)巨噬细胞转移抑制因子(OoMIF),并对该蛋白的酶功能进行鉴定。通过GenBank和Nematode V 3.0数据库发表的序列,利用分子生物学软件设计了1对特异的引物,通过RT-PCR扩增OoMIF全基因,经测序分析后,将OoMIF亚克隆到pET28a(+),然后将鉴定为阳性的重组质粒转化到BL21中用IPTG进行诱导表达。利用HPLC对可溶性表达的重组OoMIF蛋白进行纯化,同时通过免疫印迹对线虫自身的MIF及纯化后的OoMIF进行鉴定,最后对OoMIF的互变异构酶和氧化还原酶活性进行鉴定。结果表明OoMIF具有互变异构酶活性,但没有氧化还原酶活性。为进一步探究OoMIF生物学功能及其MIF在宿主免疫调节中的作用提供依据。

Prokaryotic expression system was used to express Ostertagia ostertagi macrophage mi- gration inhibitory factor （OoMIF） and the enzymic activities of OoMIF was identified. In order to obtain the OoMIF complete open reading frame sequence, based on comparison of OoMIF se- quences reported in the GenBank and Nematode V 3.0 database,one pair of specific primers was designed to amplify the OoMIF gene from cDNA by using polymerase chain reaction （PCR） and the OoMIF ORF was sequenced and subcloned into pET28a（＋）. The recombinant plasmid was transformed into Ecoli BL21 and induced by IPTG. The recombinant protein was expressed in sol- uble way and purified by HPLC. Western-blotting was used for testing the recombinant protein and native proteins. The results of enzymic activities showed that OoMIF has tautomerase activity but is lack of oxidoreductase activity.