虎纹捕鸟蛛毒素-Ⅰ（HWTX-Ⅰ）在巴氏比赤酵母中的表达

【作者】 聂东宋；

【导师】 李敏； 梁宋平； 何宁佳；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 虎纹捕鸟蛛毒素-Ⅰ（Huwentoxin-Ⅰ，简称HWTX-Ⅰ）是湖南师范大学梁宋平教授从中国珍稀毒蜘蛛种虎纹捕鸟蛛（Selenocosmia Huwena）的粗毒中分离纯化并命名的一种肽类神经毒素，其相对分子质量为3750，由33个氨基酸残基组成的含三对二硫键的小分子多肽，对其生物学活性的研究表明：它是一种作用于突触前膜的神经毒素且是一种N-型Ca²⁺通道的阻断剂，最近发现它有明显的镇痛活性，可望开发成一种新型的镇痛药物。 本室李敏教授已在原核表达体系中实现了化学合成的HWTX-Ⅰ基因的融合表达，但因原核表达体系缺乏翻译后加工过程，表达产物需进一步氧化复性才具备生物学活性，且表达产物存在内毒素污染问题，影响了其临床应用。甲醇营养型酵母（Pichia Pastoris）表达体系是一种最近开发出的极具潜力的真核表达体系，该系统具有高表达，高分泌，高稳定的特点，许多蛋白在该系统中都得到了成功的表达。 本文报道了HWTX-Ⅰ全化学合成基因在P. pastoris系统的表达，纯化及生物学活性的鉴定，根据已得到的HWTX-Ⅰ基因的cDNA序列，人工合成了6条寡核苷酸链，经磷酸化，退火连接得到完整的HWTX-Ⅰ基因片段，将该基因插入到去磷酸化的质粒pPIC9K中转化Top10F’菌后，阳性克隆经两次PCR鉴定和DNA序列测定证明插入基因的方向和序列完全正确。将带有HWTX-Ⅰ基因的pPIC9K经BlgII线性化后，转化酵母宿主菌Gs115原生质体后经筛选阳性克隆并经表型鉴定为His⁺Mut^s酵母菌，进一步用遗传毒素G418筛选多拷贝的转化菌株，命名为GH1；将GH1甲醇酵母菌用0.5％的甲醇诱导表达，发酵上清经90％饱和度的（NH₄）₂SO₄沉淀，YW-3（MWC03000）的超滤膜超滤，再经CM阳离子交换，C₁₈反相HPLC纯化得到分子量为4KD左右的组分，其中4289.05的组分经质谱鉴定，氨基酸组成分析和序列测定为正确的表达产物，生物学活性表明其活性为天然毒素活性70%，表达量为80mg/L。另一组分子量为4048的成分经测序分析表明也为正确表达产物。但经c端测序结果表明其C端丢失了二个氨基酸残基。生物学活性分析表明，其活性为天然活性的27%。更多还原

【Abstract】 Huwentoxin- I (HWTX- I )is a neurotoxin purified from the vemon of the Chinese bird spider Selenocosmia huwena which consists of 33 amino acid residues , HWTX- I is a reversible presynaptic inhibitor which can selectively inhibit N-type Ca2+ channel. Recent research study shows that it is a peptide with analgesia activity, so it is promised to be exploited into a new kind of analgesia medicine.The synthetic HWTX- I gene was expressed in a new eukaryotic expression system-Pichia pastoris which has the advantage of high stability ,high secretion and high expression. The HWTX- I gene was chemically synthesized according to its known cDNA sequence,the gene was inserted into vector pPIC9K which contained AOXj promotor and the sequence of a secreting signal peptide- a-factor, the cloning pPIC9K/HWTX- I was constructed and confirmed by two-step PCR and DNA sequence analysis,then it was transformed into host strain GS115, A His+Muts cell line was screened and multicopy transformants were screened by various G418 concentrations, the multicopy transformant was named GH1.GH1 was cultivated in flasks. After 6 days of induction by 0.5% methanol,the supernatant was checked by 16.5% Tricine-SDS PAGE, which showed there was a band in the position of 3.5-6.1KD, then it was isolated and desalted by ultrofiltration followed by ion exchange of CM column, after reverse phase HPLC of Ci8 and vacuum drying,the purified rHWTX-1 was obtained which was proved to be correct recombinant HWTX- I by Tricine SDS-PAGE,MALDI-TOF Mass Spectrometry,amino acid composition analysis, the N-terminal amino acid sequence and its biological activity,the final field of thepurified rHWTX- I was about 80mg/L,accounting for 23.6% of it total secretory proteins.更多还原