【作者】 刘君梁；

【导师】 梁宋平； 谢锦云；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 根据已知的HWTX-Ⅳ的一级结构和酵母的偏爱密码子，人工合成了4条寡聚核苷酸链，经磷酸化、退火连接得到完整的HWTX-Ⅳ基因。HWTX-Ⅳ基因被克隆进表达载体pPIC9K，重组载体被命名为pPIC9K／HWTX-Ⅳ。pPIC9K／HWTX-Ⅳ由BglⅡ线性化后被导入宿主菌GS115，整合HWTX-Ⅳ基因的宿主菌经0.5％甲醇诱导表达72h后，16.5％Trcine SDS-PAGE鉴定，表达HWTX-Ⅳ的重组菌株被命名为HWTX-Ⅳ／GS115。重组菌株扩大培养，发酵上清经硫酸铵沉淀、CM阳离子交换柱层析、C18反相柱层析分离纯化，MALDI-TOF鉴定，表明HWTX-Ⅳ被正确表达，表达产物为N-端多4个氨基酸残基（FEVY）的HWTX-Ⅳ，且有严重的C-端不均一现象。不均一的混合物理化性质接近，因而未能得到均一的rHWTX-Ⅳ进行生理活性分析。 由pPIC9K／HWTX-Ⅳ克隆得到HWTX-Ⅳ的基因在5`端加上编码thrombin识别序列后，被克隆到原核分泌表达载体pMAL-p2X中，重组载体被命名为pMAL-p2X／HWTX-Ⅳ。pMAL-p2X／HWTX-Ⅳ转化DH5α感受态细胞，0.3mmol／L IPTG诱导表达4h后，16.5％TrcineSDS-PAGE鉴定，电泳结果表明HWTX-Ⅳ在E．coli DH5 α中以融合蛋白的形式被分泌表达。扩大培养，培养液离心，取沉淀。沉淀经冷渗透休克，渗透液上清透析脱盐、冻干。融合蛋白的产率为20mg／L，融合蛋白用凝血酶酶切后，Superdex^TM75分子筛分离，目的蛋白再经C18反相柱脱盐纯化、冻干，rHWTX-Ⅳ的产率为0.4mg／L，膜片钳定性实验表明重组HWTX-Ⅳ与天然HWTX-Ⅳ有一致的生物活性。 HWTX-Ⅺ是一种Kunitz型丝氨酸蛋白酶抑制剂，从其cDNA中克隆获得的虎纹捕鸟蛛毒素Ⅺ基因在大肠杆菌中被分泌表达。采用pMAL-p2X载体，表达产物N端为麦芽糖结合蛋白的融合蛋白被分泌到大肠杆菌的胞间质。经冷渗透休克后，透析脱盐，用凝血酶切割融合蛋白，再经Superdex^TM75分子筛柱层析、高效液相色谱反相C18柱纯化，得到重组虎纹捕鸟蛛毒素Ⅺ，质谱分析表明，rHWTX-Ⅺ系正中文摘要确表达产物。重组HWTX一xI表现出与天然HWTX一xI一致的生物学活性· 以重组载体pVT 1 OZU/H WTX一XI为模板，采用PCR定点突变的重叠延伸法获得突变基因，克隆到pVTIOZU中，六种突变体均在酿酒酵母S一78中被分泌表达，表达量从4.2一34.smg/L不等。胰蛋白酶抑制活性表明突变体的活性与天然毒素都有差异，其中K14A活性下降了3107.5倍，说明K14是HwTX一xI胰蛋白酶抑制活性的关键残基。更多还原

【Abstract】 The spider Selenocosmia huwena Wang[=Ornithoctonus huwena (Wang)] is distributed in the hilly areas of Guangxi and Yunnan in the south of China. In previous work, many components with different structures and functions were purified and characterized. The expression of spider toxins by genetic engineering often encountered difficulties for the pairs of disulfide bonds, short-chain peptide and other unknown reasons. In this study, we tried to express toxins from the spider of S. huwena Wang in different paths such as prokaryotic secretion expression and eukaryotic secretion expression. First, the synthetic HWTX-IV gene was expressed in the eukaryotic secretion expression vector of pPIC9K, but unfortunately, we can’ t purify the recombinant HWTX-IV. Then the gene of HWTX-IV has been subcloned into the prokaryotic vector pMAL-p2X. The fusion protein, whose N terminus encodes the Maltose Binding Protein, was expressed by IPTG induction. After cold osmotic shock, the fusion protein was cleaved with thrombin. And the recombinant HWTX-IV was purified by size exclusion chromatography and reversed phase HPLC. The purified rHWTX-IV was proved to be correctly expressed by mass spectrum. The recombinant HWTX-IV was proved to have the same biological activity as the native one.Secondly, HWTX-XI, the first serine proteinase inhibitor isolated from spider venoms which belongs to the BPTI/Kunitz type serine proteinase inhibitor group, was obtained using the vector of pMAL-p2X, whose yield is 0.8mg/L, was proved to be correctly expressed by massspectrum. The recombinant HWTX-XI was proved to have the same inhibition activity as the native one. Then we got a new genetic engineering pathway to express spider toxins and this will benefit the whole spider toxin research.Finally, for the sake of the structure and function of HWTX-XI, some mutants of HWTX-XI were expressed and purified using the eukaryotic secretion expression vector pVT102U. The mutants were analyzed using photospectrometry method and surface plasmon resonance assay. The analysis shows that the mutant K14A’ s inhibition level has declined 3107. 5 times. So the main active site of HWTX-XI as a trypsin inhibitor was K14. and the effects of some other side position mutations were also analyzed using photospectrometry method and surface plasmon resonance assay.更多还原