抗呋喃它酮代谢物的衍生物噬菌体单链抗体库的构建

Construction of a Phage Single Chain Fv Library against Derivatives of Furaltadone Metabolites

目的:构建抗呋喃它酮代谢物(AMOZ)的衍生物噬菌体单链抗体库。方法:从分泌抗AMOZ的单克隆抗体的杂交瘤细胞系(BC3-E8)中提取总RNA,经RT-PCR反转录成cDNA,设计通用简并引物,PCR扩增抗体重链可变区基因(VH)和轻链可变区基因(VL)。经重叠延伸PCR(SOE-PCR),将VH和VL基因用编码(G1y4Ser)3的linker随机拼接成单链抗体(scFv)基因,然后将其克隆到噬菌粒载体pCANTAB5E中,转化大肠杆菌(Escherichia coli)TG1感受态细胞,经辅助噬菌体M13K07超感染,建立噬菌体单链抗体库。随机挑取10个阳性克隆,经PCR和双酶切鉴定,并测序。登陆DNAMAN软件对序列进行分析、比对。结果:成功扩增VH、VL及scFv基因,并得到库容为1.2×106的噬菌体抗体库,噬菌体的滴度为2.0×1010PFU,PCR鉴定及双酶切鉴定文库重组率较高,软件对序列比对结果显示,scFv基因全长序列之间差异为8.38%,VH序列差异为3.68%,VL序列差异为14.34%,且序列差异多集中在CDR抗原结合区域对应的核酸序列上。结论:已构建抗呋喃它酮代谢物的衍生物噬菌体单链抗体库,为进一步富集筛选并表达抗AMOZ的衍生物的单链抗体提供参考。

Objective：To construct a phage single chain Fv （scFv） library against derivatives of furaltadone metabolites （AMOZ）. Methods：The total RNA extracted from a hybridoma cell line （BC3-E8） secreting monoclonal antibodies against AMOZ was reverse-transcribed into cDNA by RT-PCR. The heavy chain （VH） and the light chain （VL） variable region genes were amplified respectively by PCR with the previously designed degenerate primer. The VH and VL genes were spliced into scFv fragment with a DNA linker encoding （G1y4Ser）3 by splicing overlap extension （SOE）. Then the scFv fragment was cloned into the phagemid pCANTAB5E and after the cloning,the phagemid was transformed into the competent Escherichia coli TG1. With the rescue of helper phage M13K07,a phage scFv library was constructed. Ten positive clones were randomly selected and identified by PCR and double enzymatic digestion. Furthermore,the sequences of these positive clones were sent for sequencing and analyzed by the DNAMAN software. Results：VH,VL and scFv DNA fragments were amplified successfully. The constructed phage scFv library had a capacity of 1.2 × 106 and the titre was about 2.0 × 1010 PFU. PCR and double enzymatic digestion identification showed that the ratio of positive insert was high. Multiple sequence alignment showed that the difference of scFv sequences was 8.38%,and those of VH and VL sequences were 3.68% and 14.34%,respectively,and the discrepancy were mostly concentrated in corresponding nucleic acid sequences of the CDR antigen region. Conclusion：a scFv phage antibody library against derivaties of furaltadone metabolites has been constructed successfully. This will lay a foundation for the further enrichment and expression of scFv.