大肠杆菌不耐热肠毒素B亚单位在毕赤酵母中的表达及鉴定

Expression and Identification of Recombinant Heat-labile Enterotoxin Subunit B in Pichia pastoris

为了在毕赤酵母中高效表达大肠杆菌不耐热肠毒素B亚单位(LTB)及研制具有粘膜免疫佐剂活性的无毒重组LTB,采用PCR方法从产毒型大肠杆菌H44815菌株扩增获得LTB完整编码区DNA,将其克隆到酵母表达载体pPIC9K的分泌信号肽基因下游,构建真核表达载体pPIC9K-LTB;重组质粒经核苷酸测序确认并线性化后电转化毕赤酵母菌株KM71,PCR筛选转化子,并以小量诱导表达筛选高效表达工程菌株,最后Western blotting分析甲醇诱导上清中的LTB及采用亲和层析和HiTrap Desalting预装柱脱盐纯化蛋白。结果表明,PCR克隆获得的LTB编码DNA序列与GenBank登录DNA序列完全一致,定向插入pPIC9K载体,可获得表达LTB的酵母分泌表达载体pPIC9K-LTB;电转化后的重组酵母菌KM71诱导表达产物经SDS-PAGE和Western blotting分析,发现甲醇诱导的培养基上清中含LTB,且具有较好的免疫原性。

To efficiently express heat-labile enterotoxin subunit B of E.coli in Pichia pastoris and develop a non-toxic recombinant LTB with mucosal immune adjuvant activity,the full length encoding region DNA of LT subunit B was cloned from genome of E.coli strain H44815 by PCR,and inserted into the downstream of the α-factor signal sequence of the Pichia pastoris expression vector pPIC9K.The linearized recombinant plasmid was transformed into P.pastoris strain KM71 by electroporation after sequencing.The recombinant strains were identified by PCR,and LTB in culture supernatant induced by methanol was identified by Western blotting to screen the engineer bacteria with high expressions.The desalting purification of expressed protein was conducted by affinity chromatograph and HiTrap Desalting preloaded column.The results showed that the sequence of LTB DNA obtained was identical with the encoding region of LTB that published on GenBank and the DNA was directly inserted to pPIC9K to obtain a secreted expression vector pPIC9K-LTB.SDS-PAGE and Western blot analysis of induced expression products in invert recombinant strain KM71 showed that there had LTB in the culture supernatant induced by methanol,which had a good antigenicity.