摘要
以山西风陵渡东亚钳蝎（ＢｕｔｈｕｓｍａｒｔｅｎｓｉＫａｒｓｃｈ）的尾腺总ＲＮＡ为模板，根据已知的蝎神经毒素保守氨基酸序列设计引物，利用ＰＣＲ技术，扩增并克隆了两个蝎神经毒素基因．序列分析表明，由两个基因导出的氨基酸序列（ＢｍＫＭｍ１和ＢｍＫＭｍ２）与已知的蝎神经毒素ＢｍＫⅠ、ＢｍＫⅡ、ＢｍＫⅢ、ＢｍＫＭ１、ＢｍＫＭ９有很高的同源性．将ＢｍＫＭｍ２基因重组到大肠杆菌分泌型表达载体ｐＥｘＳｅｃ１中进行表达．ＳＤＳ－ＰＡＧＥ证明表达产物被分泌到细胞周间质及培养液中．
The total RNA was isolated from the venom gland of scorpion Buthus martensii Karsch.According to the reported amino acid sequences of the scorpion neurotoxins BmKⅠ,BmKⅡ,two primers were designed and synthesized.By means of RT PCR one PCR fragment was obtained.The PCR products were cloned into pGEM T vector.Four clones were sequenced.Analysis of the nucleotide sequences showed that they belonged to two genes encoding the anti mammalian neurotoxins (named BmK Mm1 and Bmk Mm2).The deduced amino acid sequences shared a high homology with the known sequences of the anti mammalian neurotoxins BmKⅠ,BmKⅡ,BmKⅢ,BmKM1 and BmKM9.To express the BmKMm2 gene in Escherichia coli a novel expression secretion vector ,pExSec1,had been chosen.This vector contained a strong and tightly regulated T7 promoter.Translation started at the protein A leader sequence followed by the synthetic IgG binding domains (ZZ sequence) of protein A.The BmKMm2 gene was inserted into the multiple cloning site in the correct reading frame.The produced fusion protein ZZ BmK Mm2 was secreted into the culture medium of the host cell.It was isolated by affinity chromatography on IgG Sepharose.The biological activity of the fusion protein was tested on mice.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1999年第2期205-210,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金
山西省自然科学基金
关键词
东亚钳蝎
蝎神经毒素
分泌型
表达
PCR
Buthus martensii Karsch,Scorpion neurotoxin,Expression in Escherichia coli
