【作者】 杨利敏；

【导师】 扈廷茂； 王景林；

【作者基本信息】 内蒙古大学 ， 微生物学， 2005， 硕士

【摘要】 为实现肉毒毒素A重链C-端结合区（BoNT/AHc-C）在原核表达系统中高效表达,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/A Hc-C基因片段,插入表达载体pET-His,再转化大肠杆菌BL21（DE3）plysS中进行诱导表达,重组蛋白约占细胞总蛋白的65%,以金属螯合亲和层析法纯化重组蛋白,然后对其进行了稀释复性,获得纯度大于95%的可溶性重组蛋白,Western印迹和间接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。 用纯化的rBoNT/A-Hc-C免疫BALB/c小鼠,取其脾细胞与骨髓瘤Sp2/O细胞融合,经间接ELISA筛选和克隆化制备杂交瘤细胞系,获得3株杂交瘤细胞株,命名为2A8、4F7和2F2,可稳定分泌单克隆抗体,IgG亚类鉴定均为IgG1,2A8可保护小鼠抵抗10 LD₅₀ BoNT/A的攻击,以蛋白G亲和层析纯化腹水抗体,获得纯度大于95%的抗体蛋白。 为获得核糖体展示所必需的全部组件,采用重叠PCR技术合成pT7PD质粒,它可以有效的进行体外转录和体外翻译。然后,通过酶切连接引入编码随机12肽的核苷酸序列,即得到核糖体展示随机12肽库的核酸文库。对该文库进行了体外转录和体外翻译鉴定,证明其可以有效地进行体外转更多还原

【Abstract】 In this experiment, to express, purify and analyze the antigenicity of the heavy chain C-terminal binding domain of botulinum neurotoxin type A(BoNT/AHc-C), the gene encoding BoNT/AHc-C was optimized by replacing rare codons with high-frequency ones and adjusting AT contents, then synthesized using overlapping PCR. The synthetic gene was subcloned into the pET-His vector for expression in Escherichia coli, and recombinant protein was one-step purified by using Ni-NTA affinity agarose chromatography column, then refolded. The antigenicity of recombinant BoNT/AHc-C was identified by western blot and indirect ELISA. The rBoNT/AHc-C, which obtained a 65 % overexpression in the genetically engineering strain BL21/pET-BoNT/AHc-C, was one-step purified and refolded to 95% purity.To prepare the monoclonal antibodies (mAbs) against rBoNT/AHc-C, BALB/c mice were intraperitoneally immunized with purified rBoNT/AHc-C, and splenocytes of immunized mice were fused with myeloma cells Sp2/0. Hybridoma cells were screened by indirect ELISA and limited dilution was used to obtain mAbs. Three hybridomas cell lines producing the mAbs against BoNT/A, named 2A8 4F7 and 2F2, were successfully established. Identification of subclass showed that all mAbs belong to IgGl These mAbs showed high specificity to recombinant BoNT/A-Hc and nativeBoNT/A by the indirect ELISA. The mAb 2A8 showed preferable neutralizing activity.To obtain all elements used for ribosome display, plasmid pT7PD was constructed by Overlapping PCR, which can be transcribed and translated in vitro efficiently. Then, a high-complexity DNA library which encodes random 12-mers peptide used for prokaryotic ribosome display was generated by PCR and DNA ligation. The DNA library was identified by sequencing and in vitro transcription/translation. Ribosome display was applied to select the immobilized mAb 2A8 and positive result was obtained. This paves the way for epitope mapping for monoclonal antibodies by ribosome display technology.更多还原