人67KDa层连蛋白受体前体蛋白的基因克隆、表达及其免疫原性

Gene Cloning, Prokaryotic Expression and Immunogenicity of 67kDa Lami-nin Receptor Precursor(LRP)

［摘 要］目的 克隆、表达人67KDa层连蛋白受体前体蛋白（67KDa laminin receptor precursor，LRP）并检测其免疫原性。方法 应用RT－PCR从胃癌细胞9GC7901中扩增编码LRP的cDNA，将其按照T-A克隆法重组入pUCm－T载体并进行DNA序列测定。采用DNA重组技术构建原核表达载体pQE30-LRPP；用M15/pQE系统表达6×组氨酸与 LRP的融合蛋白，并以 SDS－PAGE和Western blot鉴定所获融合蛋白。用含融合蛋白的聚丙烯酸胺凝胶颗粒免疫小鼠，以 Westem blot检测小鼠血清的抗体活性。结果 DNA测序结果证实所扩增的cDNA片段与 LRP的基因序列完全相符。SDS－PAGE检测显示，经IPTG诱导2h后，M15/pQE30－LRP总蛋白中出现一条34KDa的新生蛋白带，Western blot进一步证实该新生蛋白为融合蛋白。其免疫小鼠血清经 Western blot检测仅识别SGC7901细胞中一条大小约为42KDa的蛋白带；其血清即使被稀释2000倍，仍然显示与靶蛋白有较强的结合活性。结论 成功地克隆、表达了人LRP，并具有良好的免疫原性。

[Abstract] Objective To clone, express and study on the immunogenicity of human 67kDa laminin receptor precursor(LRP) .Methods The cDNA encoding LRP was amplified from gastric cancer cell SGC7901 by RT - PCR, then inserted into pUCm - T vector by T - A cloning and sequenced. Prokaryotic expression vector was constructed by recombinant DNA technique. LRP protein fused with histidine tag was expressed in M15/ pQE system and identified by SDS - PAGE and Western blot. Mice were immunized with the polyacrylamide gel particles containing the fusion protein, and the sera of them were detected for antibody activity by Western blot. Results The sequence of amplified cDNA fragment was fully consitent with that of LRP. After M15/pQE -LRP bacterium was induced with IPTG for 2h, a new protein band with a relative molecular weight of 34000 was shown on SDS - PAGE profile, and further proved to be fusion protein by Western blot. The antisera of immunized mice only recognized an unique protein band with a relative molecular weight of 42000 and showed high binding activity to target protein even at a dilution of 1:2000. Conclusion Human LRP with good immunogenicity was successfully expressed.