摘要
目的 :重新设计并人工合成抗NI 35重组单链抗体 (anti NI 35 scFv)的cDNA克隆 ,构建其表达载体 ,在原核系统中实现初步表达。方法 :根据抗NI 35抗体的轻链重链序列 ,重新设计适于在大肠杆菌中表达的基因片段 ,经退火、复性连接成目的片段后 ,克隆到克隆载体 pUC 18中 ,经全自动序列分析仪测序证实 ,再亚克隆至表达载体 pET 2 8a(+) ,转化大肠杆菌BL2 1,诱导表达。 结果 :合成的基因序列与设计的仅差一个碱基 ,目的基因连接方向及阅读框架正确 ;SDS PAGE检测显示 ,表达出相对分子量 31kDa的融合蛋白 ,并得到了良好的生物活性。结论 :抗NI 35重组单链抗体基因表达载体的构建和表达 。
Objective: Our aim was to construct and express a recombinant cloning vector bearing the chemically synthesized cDNA of the recombinant anti-NI-35 single-chain antibody. Methods: Thirty-five segments of 40 to 50 bp were assembled in only one step by a PCR approach. The entire gene was cloned into pUC18 plasmid. After determined by auto-sequencing, the target gene was cloned into prokaryotic expression vector pET28a(+)in fusion form and transformed into. E.coli BL21. Result: Sequence analysis showed that the splicing order, the direction and the sequence in the gene were correct. SDS-PAGE analysis showed a new protein band with molecule weight of 31kDa, which appeared as expected. Conclusion: The successful construction and expression of the recombinant vector pET-28a(+) bearing the cDNA might provide basis for further research about the anti-NI-35 single-chain antibody.
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2003年第5期398-400,425,共4页
Journal of China Medical University
关键词
抗NI-35重组单链抗体
基因合成
表达
recombinant anti-NI-35 single-chain antibody
gene synthesis
expression
