α-银环蛇毒素基因的克隆及其非融合型原核表达  

Cloning of Alpha-Bungarotoxin Gene and Its Prokaryotic Expression as a Non-fusion Protein

作　　者：胡延春[1] 张乃生[2] 邓俊良[1] 左之才[1] 廖玉辉[1] 欧阳红生[2] 

机构地区：[1]四川农业大学动物科技学院,雅安625014 [2]吉林大学畜牧兽医学院,长春130062

出　　处：《遗传》2006年第4期463-469,共7页Hereditas（Beijing)

摘　　要：根据文献报道α-银环蛇毒素的氨基酸序列推导出其DNA序列,设计并人工合成两两互补的14条寡核苷酸片段。经片段延伸、PCR、克隆,成功构建α-银环蛇毒素基因克隆质粒;质粒经XbaⅠ和EcoRⅠ双酶切回收后连接于表达载体pET28a(+)中,分别转化BL21(DE3)、BL21(DE3)Codon plus、BL21(DE3)plysS进行诱导表达,表达产物经Tris/tricine系统进行SDS-PAGE分析。结果表明:该基因已在大肠杆菌BL21(DE3)宿主菌中进行了非融合表达,其表达量占细菌总蛋白的11.98%,主要以包涵体形式存在;同时对表达条件进行了优化,其表达量可达16.28%。经Western blot分析,在大约8 kDa处出现明显的目的带,与预计蛋白分子量大小一致,说明表达产物与天然α-银环蛇毒素具有相似的免疫原性。表达产物纯化、复性后经动物毒性试验表明:表达的α-银环蛇毒素蛋白具有生物学活性,小鼠腹腔注射其LD50约为1.28μg/g。On the basis of the reported amino acid sequence of alpha-bungarotoxin （α-BGT）, DNA sequence of α-BGT was deduced and fourteen partially complementary oligonucleotides were designed and synthesized. A plasmid carrying the coding region of α-BGT was obtained by primer extension, PCR and ligation with pMD-18-T. The target fragment was digested with Xba Ⅰ and EcoR Ⅰ , recovered and ligated with pET28a（ ＋ ）. The resultant expression vector was transformed into BL21 （DE3）, BL21 （DE3） Codon plus, and BL21 （DE3） plysS, respectively. Recombinant α-BGT was expressed in BL21（DE3） and was analyzed by 15% Tris/tricine SDS-PAGE. The result showed that the recombinant protein, mostly found in inclusion bodies, accounted for 11.98 % of the total bacterial lysate. The expression capacity could be increased to 16.28% by optimizing expression conditions. Western blotting results showed that the expressed protein had similar immunogenicity with the natural α-BGT protein purified from the venom of Krait Bungarus spp. In vivo toxicity assay of purified and renatured proteins in mice showed that LD50 was about 1.28μg/g.

关 键 词：Α-银环蛇毒素 基因克隆 非融合原核表达 免疫原性 生物学活性 

分 类 号：Q78[生物学—分子生物学] S852[农业科学—基础兽医学]