摘要
目的构建鼻咽癌人源抗独特型单链抗体原核表达载体,对其产物做初步鉴定,为鼻咽癌疫苗制备奠定基础。方法采用分子克隆技术,PCR扩增G22ScFv基因,并将其克隆到原核表达载体pET25b(+)上,将重组子转化大肠杆菌BL21(DE3),经IPTG诱导表达后,表达的蛋白经His-tag纯化试剂盒纯化后复性。SDS-PAGE及Western blot分析融合蛋白。结果构建的原核表达载体pET25b(+)-G22经测序检测,序列正确。在大肠杆菌中G22ScFv以包涵体形式获高效表达,SDS-PAGE及Western blot鉴定表达的目的蛋白Mr为25kD,同时证实载体构建及表达均正确。目的蛋白经试剂盒纯化后电泳分析纯度达95%以上。结论利用基因重组技术,在原核细胞中表达出重组G22ScFv并得到纯化,为研究鼻咽癌疫苗打下了基础。
[Objective] To construct a new recombinant expression vector of nasopharyngeal carcinoma anti-idiotypic single chain antibody, and identify the product for the possible clinical use in active immunity therapy of nasopharyngeal carcinoma. [Methods] The G22 gene was emplified by PCR and digest by restrictive enzyme before cloned into pET25b （＋）. It was then transformed into E.Coll DtLSctand the positive recombinant plasmid named pET25b（＋）-G22 was sequenced. The target protein was expressed after transformed into E.Coll BL21 （DE3）, and induced with IPTG. The cell extracts were purified by His-tag fusion protein purification kit under denaturing condition. Western blot analysis was performed after solubility analysis. [Results] The sequence of target gene in recombinant plasmid pET25b（＋）-G22 was the same as G22 and could be efficiently expressed in E.coli. Protein production mainly was the inclusion body,but could be purified by His-tag fusion protein purification kit. The western blot analysis was proved that the protein had immunologic specificity and bioactivity. [Conclusion] The Ab2βScFv G22 is successfully cloned and expressed in this study, The purified product has biological activity. These results pave the way for further studies.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2008年第8期1056-1059,共4页
China Journal of Modern Medicine
基金
国家自然科学基金资助(No:30270512)
关键词
抗独特型抗体
鼻咽癌
原核表达
单链抗体
anti-idiotypic antibody
nasopharyngeal carcinoma
prokaryotic expression
single chain antibody
