摘要
目的：克隆和表达人源化小鼠抗人交联纤维蛋白单链抗体。方法：首先依照人源化单链抗体的蛋白序列设计并优化人源化单链抗体的核苷酸序列，然后在化学合成 １４ 条人源化单链抗体基因片段的基础上，利用二次 Ｐ Ｃ Ｒ 方法构建完整的人源化单链抗体基因，并把构建的全长人源化单链抗体基因直接克隆到表达载体上，利用表达筛选和测序验证相结合的方法挑选正确的基因，并在大肠杆菌中进行了人源化单链抗体基因的 Ｉ Ｐ Ｔ Ｇ 诱导表达。结果：构建并挑选到序列正确的全长人源化单链抗体基因，而且在大肠杆菌中实现了目的基因的高表达。结论：基因的部分化学合成， Ｐ Ｃ Ｒ构建及表达筛选是克隆基因的一种有效方法。
Objective: To clone and express humanized mouse ScFv against human cross linked fibrin(HScFv). Methods: Based on the amino acid sequence of HScFv, its nucleotide sequence was designed and optimized. Using the method of PCR, the HScFv gene was constructed from fourteen chemically synthesized oligonucleotides. The constructed HScFv gene was cloned into the expression vector, and the correct HScFv gene was obtained by screening the clones with inducing expression strategy and verified by sequencing. By inducing with IPTG, the cloned HScFv gene was expressed in E.coli. Results: The correct HScFv gene was constructed, cloned and overexpressed in E.coli. Conclusion: It is an effective way to use combined methods of partial chemical synthesis, PCR construction and expression screening for cloning a gene.
出处
《军事医学科学院院刊》
CSCD
北大核心
1999年第3期175-178,共4页
Bulletin of the Academy of Military Medical Sciences
基金
国家"八六三"高技术基金
关键词
交联纤维蛋白
单链抗体
大肠杆菌
表达
克隆
humanization
cross linked fibrin
ScFv
Escherichia coli
expression
