IN-1重组单链抗体基因的克隆和序列分析

Cloning and Sequencing of cDNA of the Recombinant IN-1 Single-chain Antibody

目的 应用基因工程技术获得人工设计的IN - 1重组单链抗体 (IN - 1-scFv)的cDNA克隆。方法 参照 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目的基因片段 ,将该基因双链分成 3 5个小片段合成 ,经退火、复性连接成目的片段后 ,克隆到经过BamHI和HindIII双酶切的克隆载体pUC18中 ,并转化大肠杆菌DH5a,抽提重组子 pUC18/744进行克隆PCR、酶切鉴定及测序分析。结果 测序结果证明获得的基因序列与实验设计仅差一个碱基。结论 正确设计并合成了IN - 1重组单链抗体 (IN - 1-scFv)的cDNA 。

Objective To construct a recombinant cloning vector bearing the chemically synthesized cDNA of the recombinant IN-1 single-chain antibody . Method 35 segments with the length ranging from 40 bp ～50 bp were assembled in only one step by a PCR approach. The entire gene was cloned into cloning plasmid vector pUC18 .The sequence of the cloned cDNA was confirmed by DNA sequencing, clone PCR and restriction enzymes analysis. Results Sequence analysis showed that the splicing order, the direction and the sequence in the gene were almost same as those of experimental design except for one nucleotide acid. Conclusion The successful construction of the recombinant vector pUC18 bearing the cDNA might provide materials for the further research about the IN-1 single-chain antibody. [YM5'BZ?=292,S]