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 |
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引用本文: | 曲光刚,沈志强,王文秀,王金良,唐娜,付石军,唐世云,韩文瑜.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[J].兽医大学学报,2013(11):1679-1684. |
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作者姓名: | 曲光刚 沈志强 王文秀 王金良 唐娜 付石军 唐世云 韩文瑜 |
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作者单位: | [1]吉林大学动物医学学院,吉林长春130062 [2]山东省滨州畜牧兽医研究院,山东滨州256600 |
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摘 要: | 通过原核表达系统表达奥氏奥斯特他线虫(Ostertagiaostertagi)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NematodeV3.0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。
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关 键 词: | 线虫 巨噬细胞转移抑制因子 免疫印迹 氧化还原酶 互变异构酶 |
The clone,expression and enzymatic activity identification of macrophage migra- tion inhibitory factor derived from Ostertagia ostertagi |
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Authors: | QU Guang-gang ' SHEN Zhi-qiang WANG Wen-xiu WANG Jin-liangz TANG Na FU Shi-jun TANG Shi-yun HAN Wen-yu |
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Institution: | 1 (1. College of Animal Science and Veterinary Medicine, Ji lin University, Changchun 130062, China ; 2. Binzhou Animal Science and Veterinary Medicine A cademy , Binzhou, Shandong 256600, China) |
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Abstract: | 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. |
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Keywords: | Nematode MIF Western-blotting oxidoreductase tautomerase |
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