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［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 and express 67KDa laminin receptor precursor (LRP) and test its immunogenicity. Methods cDNA encoding LRP was amplified from gastric cancer cell line 9GC7901 by RT-PCR and cloned into pUCm-T vector according to T-A cloning method. The DNA sequence was determined. The prokaryotic expression vector pQE30-LRPP was constructed by DNA recombination technique. The fusion protein of 6 × histidine and LRP was expressed by M15 / pQE system and the fusion protein was identified by SDS-PAGE and Western blot. Mice were immunized with fusion protein-containing polyacrylamide gel particles and mouse serum antibody activity was detected by Western blot. Results The results of DNA sequencing confirmed that the amplified cDNA fragment exactly matches the gene sequence of LRP. SDS-PAGE analysis showed that a 34 kDa neo-protein band appeared in M15 / pQE30-LRP total protein after induced by IPTG for 2 h. Western blot further confirmed that the neo-protein was a fusion protein. Western blot analysis of the immunized mouse sera only identified a protein band of about 42 kDa in SGC7901 cells. The serum of the immunized mice still showed strong binding activity with the target protein even after being diluted 2000-fold. Conclusion Human LRP was successfully cloned and expressed, and had good immunogenicity.