驯鹿β-防御素-1(rebd-1)基因原核表达及蛋白活性鉴定-prokaryotic expression and protein activity identification of reindeer β - defensin - 1 ( rebd - 1 ) gene.docxVIP

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驯鹿β-防御素-1(rebd-1)基因原核表达及蛋白活性鉴定-prokaryotic expression and protein activity identification of reindeer β - defensin - 1 ( rebd - 1 ) gene.docx

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驯鹿β-防御素-1(rebd-1)基因原核表达及蛋白活性鉴定-prokaryotic expression and protein activity identification of reindeer β - defensin - 1 ( rebd - 1 ) gene

2.2.5目的片段的回收································································ 112.2.6克隆载体的构建································································ 122.2.7编码 reBD-1 成熟肽基因的扩增 142.2.8原核表达体系的构建·························································· 142.3结果 ················································································ 162.3.1总 RNA 的检测结果 162.3.2RT-PCR 扩增结果 172.3.3reBD-1 重组质粒的筛选和鉴定 182.3.4reBD-1 重组质粒序列分析 192.3.5reBD-1 成熟肽基因的克隆 202.3.6pET-32a（+）- reBD-1 重组质粒的鉴定结果 202.4讨论 ················································································· 212.4.1载体及菌株的选择····························································· 212.4.2关于 reBD-1 成熟肽的特性 222.5小结 ················································································· 223pET-32a（+）/reBD-1 融合蛋白的表达及纯化 223.1材料 ················································································· 223.1.1菌株与质粒······································································ 223.1.2主要试剂 ········································································ 223.1.3主要实验仪器··································································· 233.2实验方法 ··········································································· 233.2.1重组蛋白 pET-32a（+）/reBD-1 的诱导表达233.2.2主要试剂的配制································································ 243.2.3目的蛋白原核表达及诱导表达条件的优化 ······························· 243.2.4SDS－PAGE 电泳253.2.5大量表达靶蛋白································································ 273.2.6目的蛋白的纯化与复性 ······················································· 283.3结果 ················································································· 313.3.1目的蛋白表达条件的优化 ···················································· 313.3.2重组蛋白表达形式的分析 ···················································· 323.3.3目的蛋白的纯化································································ 333.4 讨论··················································································· 343.5 小结···············