人表皮生长因子cDNA的克隆、原核表达和抗体的制备

Cloning and Prokaryotic Expression of Human Epidermal Growth Factor cDNA and Its Antiserum Preparation

根据已发表的人表皮生长因子(hEGF)mRNA序列设计引物,上游引物5＇端引入山羊β-乳球蛋白信号肽序列。PCR从人肝脏混合单链cDNAs中扩增出hEGF cDNA;测序表明,克隆的hEGF cDNA与已发表的序列完全相同;经Signalp V1.1软件分析,山羊β-乳球蛋白信号肽序列与hEGF cDNA能在预计位点切割。将此cDNA克隆到pGEX-6P-1中,经IPTG诱导,在BL21(DE3)大肠杆菌中表达约32 ku的GST-hEGF融合蛋白。将此cDAN插到pcDNA-3中,用此重组质粒免疫青紫蓝兔,经6次免疫后获得抗hEGF的多克隆抗体,该抗体能识别GST-hEGF融合蛋白。

A pair of primers were synthesized according to previously published hEGF mRNA sequence with goat β-lactoglobulin signal peptide sequence fused to the forward primer, a cDNA of correct size was amplified from pooled human liver single-stranded cDNAs by high fidelity PCR. Sequence analysis showed that the cDNA was identical to that previously published and the goat β-lactoglobulin signal peptide was able to be cleaned at expected site. The cDNA was subcloned into the XhoI site of prokaryotic expression vector pGEX-6p-l and the recombinant plasmid was transformed to Escherichia coli BL21 (DE3) for expression. After IPTG induction, an additional band of 32 ku was detected by SDS-PAGE. The cDNA was subcloned into eukaryotic expression vector pcDNA-3 and the recombinant vector was injected into rabbits. After six times of injection, an antiserum to hEGF was obtained, which was able to recognize GST-hEGF fusion protein expressed in E. coli.