抗AFP重链可变区单域抗体融合蛋白的构建、表达及初步鉴定

Construction,expression and identification of anti-human AFP V_H single domain antibody fusion protein gene

目的构建抗甲胎蛋白(AFP)重链可变区(VH)单域抗体融合蛋白基因在大肠杆菌中的表达,并初步鉴定表达产物的活性。方法从已构建的抗AFP单链抗体(ScFv)的载体中,经PCR扩增出VH单域抗体基因,再克隆到融合蛋白表达载体pET32a(+)中进行表达;用SDS-PAGE及Western-blot鉴定表达产物;并通过竞争抑制ELISA分析TrxA-VH融合蛋白的结合活性;细胞免疫组化染色分析其内化及结合情况。结果VH单域抗体基因全长339 bp,将其克隆到pET32a(+)中,转化大肠杆菌可获得高效表达,Western-blot证实在相应分子质量处,有TrxA-VH融合蛋白的显色条带。经初步纯化和复性后,获得抗AFP的VH单域抗体融合蛋白。竞争抑制ELISA及细胞免疫组化证明,表达产物具有与AFP特异结合的活性。结论成功构建了抗AFP的V单域抗体融合蛋白,为临床的应用研究奠定了基础。

OBJECTIVE To construct anti - human AFP Vn single domain antibody fusion protein gene, transform it into E. coli BL- 21 （DE3） for expression and identify its bioactivity. METHODS The VH single domain antibody gene was amplified by PCR from the vector constructed, and cloned into the prokaryotic fusion protein expression vector pET32a（ ＋ ）and expressed in E. coli BL - 21 （ DE3 ）. The expression product was identified by SDS - PAGE ,Western - blot ,competitive inhibition ELISA and immunocytochemistry. RESULTS The VH single domain antibody gene consisted of 339 bp,which was cloned into an expression vector pET32a（ ＋ ）and then transformed into E. coli BL - 21 （DE3）. The pET32 - VH vector producted a 33 kD fusion protein in E. coli BL - 21 （ DE3 ） at a high level of total cellular protein, which was in the form of cytorrhyctes. After preliminary purification and renaturation, the VH single domain antibody fusion protein was obtained. Competitive inhibition ELISA and immunocytochemistry proved that the expression product has the special affinity with AFP. CONCLUSION Anti - AFP single domain antibody fusion protein has been successfully constructed and expressed for the use in clinical studies in future.