四种蜱源MIF基因的原核表达及表达产物的反应原性分析  

Prokaryotic expression and reactionogenicity analysis of MIF gene from four ticks

作　　者：袁小松[1,2] 罗金[2] 谢俊仁[2] 田占成[2] 田美媛[2] 郑进峰[2] 王芳芳[2] 张以芳[1] 刘光远[2] 

机构地区：[1]云南农业大学动物科学技术学院,云南昆明650201 [2]中国农业科学院兰州兽医研究所家畜疫原病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2013年第10期1028-1034,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31201899);甘肃省创新研究群体计划项目(1210RJIA006)

摘　　要：为研究不同蜱源巨噬细胞移动抑制因子(MIF)基因的生物学特性,本研究设计了1对特异性引物,采用PCR从亚洲璃眼蜱、青海血蜱、血红扇头蜱、麻点璃眼蜱中扩增MIF基因的完整阅读框,并对该序列一级结构特征进行分析。PCR产物经BamHⅠ+EcoRⅠ双酶切后亚克隆到表达载体pGEX-4T-1上,重组质粒转入感受态细胞BL21(DE3),37℃下用1mmol/L IPTG诱导表达,通过SDS-PAGE分析MIF基因体外的表达特征;接着用亚洲璃眼蜱全蜱血清与四种不同蜱种MIF重组蛋白进行Western-blot分析。结果显示,MIF基因完整阅读框全长351bp,编码116个氨基酸。氨基酸比对结果显示,亚洲璃眼蜱、青海血蜱、血红扇头蜱、麻点璃眼蜱MIF基因推导的氨基酸序列与已知的长角血蜱MIF基因推导的氨基酸序列相似性分别为98%、93%、93%、92%。SDS-PAGE结果显示,诱导的MIF重组蛋白的分子质量约为38.5ku。亚洲璃眼蜱全蜱血清与亚洲璃眼蜱MIF重组蛋白有较强的反应原性,且该血清与青海血蜱、血红扇头蜱、麻点璃眼蜱MIF重组蛋白存在交叉反应。结果表明,蜱MIF是一种良好的抗原分子。To study the biological characters of macrophage migration inhibitory factor（MIF） gene in ticks,the complete open reading frames（ORF） of MIF were amplified by PCR using specific primers from H yalornma asiaticum , Haerna ph ysalis qinghaiensis , Rhi picephalus sanguineus and H yalomma ru f i pes , and the primary structure was analyzed. The PCR products were digested by BarnH I and EcoR I and cloned into pGEX-4T-1 vector, and then the recombinant plasmid were transformed into H. coli BL21 （DE3）. For induction of MIF expression at 37 ~C, IPTG was added to a final concentration of 1ram. The characteristic of MIF from four ticks were analyzed using SDS-PAGE. The reactionogenicity of recombinant proteins was analyzed by Western-blotting. The result showed that the MIF gene was 351 bp which encodes 116 amino acids. Compared with the Haemaphysalis longicornis, the homology of the amino acid MIFsequences from four ticks were 98% ,93% ,93 % and 92% ,respectively. The recombinant proteins were approximately 38.5 ku in size by SDS-PAGE. The rabbit antiserum against the adults of Hyalomma asiaticum ,which had cross reaction with the MIF recombinant proteins of Haemaphysalis qinghaiensis ,Rhipicephalus sanguineus and Hyalomma rufipes, showed stronger reactionogenicity with the recombinant MIF of Hyalomrna asiaticum. The above-result showed that MIF was a good antigen in ticks.

关 键 词：蜱 巨噬细胞移动抑制因子 原核表达 

分 类 号：S852.746[农业科学—基础兽医学]