人促血液血管细胞生成素的原核表达及兔抗体的制备

Expression of recombinant human hemangiopoietin and preparation of its polyclonal antibody

目的:表达人促血液血管细胞生成素(hemangiopoi-etin,HAPO)蛋白并制备兔抗HAPO抗体。方法:利用硫氧还原蛋白TrxA融合表达载体(pET32c),表达可溶性HAPO。表达的HAPO融合蛋白经亲和层析快速纯化,并通过Westernblot进行鉴定。以纯化的人HAPO免疫新西兰大白兔,制备抗体并测定其效价。结果:获得高效表达并纯化的HAPO蛋白。制备的兔抗人HAPO抗体的免疫双扩散的效价为1∶8。结论:建立了HAPO基因的原核表达载体和快速纯化体系并制备出兔抗HAPO的抗体,为研制检测HAPO的ELISA试剂盒奠定了基础。

AIM： To express the recombinant fusion protein of hemangiopoietin （HAPO） and prepare the rabbitanti-human HAPO polyclonal antibody. METHODS： The sequence encoding HAPO was amplified by PCR and cloned into plasmid pET32c to construct recombinant prokaryotic expression system. The recombinant expression vectors were identified by enzyme digestion analysis and transformed into E. coil The HAPO protein was purified by affinity chromatography. Rabbits were immunized with the HAPO protein, and the immune sera of rabbits were collected. Antibodies （IgG） obtained from the immune sera were purified. RESULTS： The purified HAPO protein was successfully obtained. The purified polyclonal antibody of rabbit- anti-human HAPO was also obtained from the immune sera of rabbits, and could response to human HAPO. CONCLUSION： A prokaryotic expression system of human HAPO has been prepared and the polyclonal antibody against HAPO has been prepared, which can be used to determine HAPO protein.