东亚钳蝎毒素BmαTX14的基因组克隆、真核表达纯化及功能的初步研究

Genomic DNA Sequences and Functional Expression, Purification of BmαTX14 Neurotoxin from Scorpion Buthus martensii Karsch

以东亚钳蝎(Buthus martensii Karsch,BmK)BmαTX14全长cDNA序列设计引物,克隆BmαTX14成熟肽,插入毕赤酵母表达载体pPIC9K中,转化酵母,诱导表达。Tricine-SDS-PAGE和Westernblot分析显示,表达产物以可溶性分子形式存在于培养上清中,诱导84h时的表达量达到高峰,约为120mgL。通过Ni金属螯合层析柱亲和层析、聚乙二醇沉淀、凝胶分子筛层析获得纯化的rBmαTX14融合蛋白,经毒性实验表明,具有明显的抗昆虫活性。同时以蝎基因组总DNA为模板进行PCR扩增,得到BmαTX14基因克隆,序列分析显示:BmαTX14在编码信号肽的基因序列中有1个长408bp的内含子,其基因组织结构具有α型Na+通道毒素的特征。从功能活性和基因组织结构两个角度证实BmαTX14为α型Na+通道毒素。

Based on the full-length cDNA of BmαTX14 from Chinese scorpion Buthus martensii Karsch （BmK） , gene of the mature peptide of BmαTX14 was cloned into the yeast expression vector pPIC9K. After transforming, screening and inducing, tricine-SDS-PAGE and Western blot proved that rBmαTX14 protein was expressed in the medium for up to 84 hours, getting nearly 120mg/L. Recombinant BmαTX14 was purified rapidly and efficiently through Ni-NTA-agarose, polyethylene glycol precipitation and gel filtration chromatography. The purified rBmαTX14 proved to have the anti-insect activity by toxicity assay. Meanwhile, genomic gene of BmαTX14 was cloned and sequenced by PCR method, sequence analysis of this gene showed that BmαTX14 had an intron of 408 base pairs located at the signal peptide encoding region, which was similar with the characteristic of othera-type sodium ion-channel toxin. Considering both the genomic organization and the peptide function, BmαTX14 proved to be a membership belonging to a-type sodium ion-channel toxin.