中国虎纹捕鸟蛛神经毒素肽基因的克隆及在酵母中的表达

从中国珍稀毒蜘蛛种虎纹捕鸟蛛的粗毒中分离纯化的虎纹捕鸟蛛毒素-Ⅰ(HWTX-Ⅰ)是含33个氨基酸的多肽.有关实验已证实,HWTX-Ⅰ是一种作用于突触前膜的神经毒素和高阈值钙通道抑制剂,在电刺激时HWTX-Ⅰ也影响交感神经释放肾上腺素和迷走神经释放乙酰胆碱,这些结果表明,HWTX-Ⅰ具有潜在的镇痛活性.本文报道通过RT-PCR方法克隆、测定了HWTX-Ⅰ的cDNA序列.在此基础上,以pPIC9K为表达载体和GS115为宿主,筛选His Muts克隆,以甲醇为诱导剂,成功地构建并在毕赤酵母系统分泌表达HWTX-Ⅰ基因.进一步分析HWTX-Ⅰ表达产物,生物活性实验观察到酵母表达体系所获得的HWTX-Ⅰ产物具有与天然HWTX-Ⅰ相似的生理活性.图4参10

Clonging and Expression of a Neurotoxin Peptide Gene from Chinese Bird Spider (Selenocosmia huwena) in Yeast

Huwentoxin- Ⅰ ( HWTX- Ⅰ ) is a 33 amino acids peptide purified from the venom of the Chinese bird spider Selenocosmia huwena. HWTX- Ⅰ acts as a presynaptic toxin that blocks the high-voltage activated calcium channel. HWTX- Ⅰ can also affect the release of acetylcholine and adrenalin of sympathetic-vagus nerve terminal by electrical stimulation. The result indicates that HWTX- Ⅰ is a potential analgesic drug candidate. In this report, the sequence of cDNA of HWTX- Ⅰ was identified by RT-PCR and further successfully expressed HWTX-1 peptide in Pichia pastoris expression vector pPIC9K using his4 mutant yeast GS115. The His+ Mut8 clones were screened and recombinant HWTX- Ⅰ was secreted into the culture media using methanol as an inducer. The expressed HWTX-1 product was further analyzed and its biological activities were confirmed by some biological assays. Similar physiological activity was observed in the yeast-expressed HWTX-1 compared with the native HWTX- Ⅰ . Fig 4, Ref 10