PCR合成的A型肉毒毒素重链C端结合区基因的高效表达与免疫原性分析  

Immunogenicity and overexpression of PCR-synthesizing gene of heavy chain from C-terminal binding domain of botulinum neurotoxin type A

作　　者：杨利敏[1] 王景林[2] 扈庭茂[1] 

机构地区：[1]内蒙古大学生命科学学院,呼和浩特010021 [2]军事医学科学院微生物流行病学研究所病原微生物与生物安全国家重点实验室,北京100071

出　　处：《中华微生物学和免疫学杂志》2006年第1期27-31,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目(30370081)

摘　　要：目的A型肉毒毒素重链C-端结合区(BoNT/A Hc-C)的PCR拼接合成、重组表达与免疫原性分析。方法经密码子优化软件对编码基因序列重新优化设计,以16条长片段寡核苷酸引物经重叠PCR合成BoNT/AHc-C基因片段,插入表达载体pET-His,再转化大肠杆菌BL21(DE3)plys S中进行诱导表达,以金属螯合亲和层析法纯化重组蛋白,并进行鉴定和抗原性分析。结果表达工程菌裂解液经SDS-PAGE分析,重组蛋白约占细胞总蛋白的65%,为包涵体形式,经亲和层析一步纯化和柱上复性获得纯度大于95%的可溶性重组蛋白,Western blot和ELISA均证明,该重组蛋白与抗天然BoNT/A马血清抗毒素有特异性结合反应,是BoNT/AHc-C抗原,且免疫小鼠可耐受10个LD50BoNT/A的攻击。结论所获重组BoNT/AHc-C蛋白具有良好的免疫原性,为研制基因工程疫苗奠定了基础。Objective To express, purify and analyze the anfigenieity of the heavy chain from C-terminal binding domain of botulinum neurotoxin type A（BoNT/AHc-C）. Methods The gene encoding BoNT/AHc-C was optimized by replacing rare cedons with high-frequency ones and adjusting AT contents, then synthesized with overlapping PCR. The synthetic gene was subcloned into the pET-His vector for expression in Escherichia coli, and recombinant protein was purified and refolded by Ni-NTA affinity agarese chromatography column. The antigenieity of recombinant BoNT/AHc-C was identified by Western blot and indirect ELISA. Results The recombinant BoNT/ AHc-C protein was one-step purified and refolded with 95% purity and yields up to 20 mg/L of induced culture. A good antigenicity was proved with Western blot and indirect ELISA. Mice that received three injections of 5 μg of purified rBoNTAHc - C were completely protected against i . p. challenge with LD50 of BoNT / A toxin. Conclusion of recombinant BoNT/AHc-C was obtained in Escherichis coli, and the results laid a solid base for applying BoNT/AHc-C as a candidate of new vaccine.

关 键 词：A型肉毒毒素 亲和层析 柱上复性 抗原性 

分 类 号：R392[医药卫生—免疫学]