中文摘要：

目的利用毕赤酵母表达系统表达BoNT-LH（N）-Elafin融合蛋白,并检测其生物学活性。方法构建pPIC9K-BoNT-LH（N）-Elafin重组真核表达质粒,转化毕赤酵母GS115菌株,甲醇诱导表达,取上清,经阳离子交换层析纯化融合蛋白,并检测其反应原性、抗弹性蛋白酶活性及对SNARE蛋白复合体的裂解作用。结果重组表达质粒pPIC9K-BoNT-LH（N）-Elafin经双酶切及测序证实构建正确;表达的BoNT-LH（N）-Elafin融合蛋白相对分子质量约为110 000,纯化的融合蛋白纯度达92%,浓度为54 mg/L,反应原性良好,具有抗弹性蛋白酶活性及裂解SNARE蛋白复合体的作用。结论已在毕赤酵母GS115中成功表达了重组融合蛋白BoNT-LH（N）-Elafin,该融合蛋白具有良好的生物学活性,为进一步研究其抑制气道黏液高分泌的机制奠定了基础。

英文摘要：

Objective To express BoNT-LH（N）-Elafin fusion protein in Pichia pastoris and determine its bioactivity.Methods Recombinant eukaryotic expression vector pPIC9K-BoNT-LH（N）-Elafin was constructed and transformed to P.pastoris GS115 strain for expression under induction of methanol.The culture supernatant was collected,from which the fusion protein was purified by cation exchange chromatography and determined for reactogenicity,anti-elastase activity and cleaving effect on SNARE protein complex.Results Both restriction analysis and sequencing proved that recombinant plasmid pPIC9K-BoNT-LH（N）-Elafin was constructed correctly.The expressed BoNT-LH（N）-Elafin fusion protein,with a relative molecular mass of about 110 000,reached a purity of 92% and a concentration of 54 mg/L after purification,which showed good reactogenicity,anti-elastase activity as well as cleaving effect on SNARE protein complex.Conclusion Recombinant fusion protein BoNT-LH（N）-Elafin was successfully expressed in P.pastoris GS115 strain and showed high bioactivity,which laid a foundation of further study on the mechanism of its inhibitory effect on airway mucus hypersecretion.