【摘要】 目的　淡色库蚊抗药性相关基因NYD Ch(糜蛋白酶 )和NYD Tr(胰蛋白酶 )表达与鉴定。　方法　以淡色库蚊抗性品系全长cDNA文库为模板 ,采用PCR方法扩增出抗性品系中高表达的NYD Ch和NYD Tr基因 ,经T/A克隆测序鉴定 ,将扩增产物用NotI、EcoRI双酶切后作为插入片段 ,与具有相同粘性末端的融合表达质粒 pET 2 8a(+ )连接 ,转化入表达菌株E .coliBL2 1中 ,取含重组表达质粒的重组菌株以IPTG进行诱导表达 ,表达产物以SDS PAGE电泳和小鼠抗NYD Ch和NYD Tr抗血清进行Westernblot分析鉴定。　结果　NYD Ch和NYD Tr基因开读框大小分别为816bp和 786bp ,预计可分别编码 2 72个和 2 62个氨基酸。对应分子质量大小分别为 2 9.4ku和 2 8ku ,加上pET 2 8a(+ )载体融合部分氨基酸 ,表达产物条带应在 3 3ku附近 ,与SDS PAGE蛋白电泳胶上蛋白条带一致。Westernblot分析确认诱导后的 pET 2 8a(+ ) /NYD Ch和 pET 2 8a(+ ) /NYD Tr在分子质量约 3 3ku处出现 1条与小鼠抗NYD Ch和NYD Tr抗血清特异反应的条带。　结论　NYD Ch和NYD Tr基因在大肠杆菌中获得表达 ,Westernblot分析表明该蛋白为目的蛋白。更多还原

【Abstract】 Objective To express and identify resistance associated NYD-Ch and NYD-Tr genes of Culex pipiens pallens . Methods The full-length cDNA express library was used as template and 2 full-length genes of NYD-Ch and NYD-Tr were amplified by polymerase chain reaction(PCR). The PCR products were cloned into pGEM-Teasy and the nucleotide sequence were determined. Then the genes were digested by Not I and EcoR I, subcloned into pET-28a(+), expressed in E. coli and induced by IPTG. The fusion product was identified by SDS-PAGE gel electrophoresis and western blot was proceeded with anti-NYD-Ch and NYD-Tr sera from mouse. Results The sizes of NYD-Ch and NYD-Tr genes were 816 bp and 786 bp, encoding 272 and 262 amino acids, respectively. The molecular weight were 29.4 ku and 28 ku respectively. The molecular weight of fusion protein of pET-28a(+) was nearly 4 ku. So molecular weight of the expressed proteins was about 33 ku. On SDS-PAGE gel dyed with Coomassie brilliant blue R250, the protein band was about 33 ku. Western blot proceeded with anti-NYD-Ch and NYD-Tr sera from mouse demonstrated that the specific protein band was about 33 ku. Conclusion The NYD-Ch and NYD-Tr genes from the mosquito, Cx. pipiens pallens were able to express in E. coli and the NYD-Ch and NYD-Tr fusion protein contained specific antigenic epitopes.更多还原