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.
出处
《遗传》
CAS
CSCD
北大核心
2006年第4期463-469,共7页
Hereditas（Beijing)
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
alpha-bungarotoxin
gene cloning
non-fusion prokaryotic expressions immunogenicity
biological activity
