IN-1重组单链抗体表达载体的构建与融合表达

Construction and Fusion Expression of Recombinant IN-1 Single Chain Antibody Expression Vector

目的 :设计并人工合成IN 1重组单链抗体 (recombinantIN 1single chainantibody)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 genebank中发表的IN 1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 ,该基因双链分 35个小片段合成 ,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后 ,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 :测序结果合成的基因序列与设计的仅差一个碱基 ;SDS PAGE检测显示 ,表达出相对分子量 31kd的融合蛋白。结论 :IN 1重组单链抗体基因表达载体的构建和表达 ,为深入研究其生物活性奠定了基础。

Objective: Our aim was to construct and express a recombinant cloning vector bearing the chemically synthesized cDNA of the recombinant IN 1 single chain antibody. Methods: Thirty five segments with the length ranging from 40 to 50 bp were assembled in only one step by using a method of polymerase chain reaction. The entire gene was cloned into pUC18 plasmid. Determined by auto sequencing, the target gene was cloned into prokaryotic expression vector pET28a(+)in fusion form and transformed into E.coli BL21. Results: Sequence analysis showed that the sequence in the gene was almost correct except one base. SDS PAGE analysis showed that a new protein band with molecule weight of 31kd appeared as the expected size. Conclusion: The successful construction and expression of the recombinant vector pET 28a(+) bearing the cDNA might provide material for the further research about the recombinant IN 1 single chain antibody.