猪囊尾蚴排泄分泌抗原M13h基因的克隆及原核表达  

Cloning and prokaryotic expression of M13h ES antigen gene from Cysticercus cellulosae

作　　者：赵光辉[1] 王秋霞[1] 鲁琨[1] 张改平[2] 宁长申[1] 王选年[2] 张龙现[1] 菅复春[1] 宋海涛[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省农业科学院河南免疫学重点实验室,河南郑州450002

出　　处：《中国病原生物学杂志》2008年第10期749-753,共5页Journal of Pathogen Biology

基　　金：国家“863”计划项目(No.2003AA249030)

摘　　要：目的研究猪囊尾蚴排泄分泌抗原M13h基因的克隆及原核表达。方法利用RT-PCR技术从猪囊尾蚴中扩增排泄分泌抗原M13h蛋白去除信号肽序列的基因,对其进行序列分析,并亚克隆至原核表达载体pET43a上,转化至大肠埃希菌BL21(DE3),用IPTG诱导重组菌株表达融合蛋白,并用Western blot对纯化后的融合蛋白进行鉴定。结果从猪囊尾蚴中克隆到去除信号肽序列的M13h的基因,其序列长度为204 bp,包含198 bp的编码区,与GenBank中西班牙分离株的核苷酸和氨基酸序列的同源性均为100%,与韩国的M13h蛋白的核苷酸与氨基酸序列同源性分别为95.7%和95.3%;经SDS-PAGE电泳鉴定,重组质粒表达产物分子质量单位约74 ku,其中M13h基因表达蛋白的分子质量单位为7.87 ku,与预期结果一致;经Western blot检测,纯化后的复性蛋白质可与兔抗全囊囊尾蚴血清发生特异性反应。结论pET43M13h重组质粒表达产物具有较高的保守性和特异性,可用于猪囊尾蚴病的免疫诊断。Objective To study the cloning and prokaryotic expression of M13h ES antigen gene from Cysticercus cellu losae. Methods The M13h gene sequence was analyzed and subclone with 198 GenBank t signal peptide sequence was amplified from C. cellulosae by RT-PCR, and its pET43a vector and transformed into E. coli BL21 （DE3）. The recombinant were induced with lPTG and purified fusion protein was identified by Western blot. Results M13hgene hp of coding region was 204 bp in length and the was the same as that from Spain strain of but 95.7% in nucleotide acid and 95.3% in amino acid to thai of Korea strain. TbeSI）S-PAGEelectrophoresis indicated that molecular weight of the expressed fusion protein was approximately 74 ku, and the molecular weight of the M13h antigen within the fusion protein was about 7.87 ku. which was the same as the expected results. The Western blot showed that the purified reannealing fusion protein could be recognized by the positive sera of rabbit against the whole C. cellulosae. Conclusion The expression product of recombinant plasmid has higher conservatism and specificity and can be used for immunodiagnosis of eysticercosis.

关 键 词：猪囊尾蚴 M13h 克隆 原核表达 

分 类 号：R383.34[医药卫生—医学寄生虫学]