抗鹅α-干扰素单链抗体基因扩增与表达

Amplification and Expression of the Single-chain Fv Gene Specific for Goose IFN-α

为制备抗鹅α-干扰素的单链抗体,以抗鹅α-干扰素的杂交瘤细胞株总RNA为模板,RT-PCR法扩增鹅α-干扰素的抗体轻、重链基因,再采用SOE-PCR法,以编码柔性多肽(Gly4Ser)3为接头,组装出完整的鹅α-干扰素的单链抗体可变区片段(Sc Fv)基因,并将Sc Fv基因克隆到p GEMT-Easy载体中进行测序分析,将测序正确的Sc Fv基因片段克隆入p ET-30a载体中进行诱导表达。结果显示,成功组装了Sc Fv基因,其全长为726 bp,为VH-Linker-VL结构,其中VH长357 bp、VL长324 bp,成功表达了Sc Fv蛋白,大小为27 ku。

In order to obtain the single chain variable fragment( Sc Fv) gene specific for goose IFN-α,total light and heavy domains of the immunoglobulin were amplified from the RNA isolated from anti goose IFN-α monoclonal hybridoma cells by RT-PCR method. Full length of Sc Fv was assembled by overlap extension PCR with a linker containing flexible polypeptide( Gly4Ser)3. The Sc Fv fragment was cloned into the vector p GEMT-Easy and sequenced. And then it was cloned into p ET-30 a for expression. As a result,the Sc Fv gene related to goose IFN-α,consisting of 726 bp with the structure of VH-Linker-VL was successfully amplified. The VH gene was 357 bp,while the VL gene was 324 bp. Sc Fv could be expressed in p ET-30a(27 ku).