摘要
目的 制备原核表达的OVA12纯化蛋白 ,研究正常人与肿瘤患者血清中抗OVA12抗体水平的差异。方法 克隆OVA12的cDNA至pGEMT载体中 ,经测序确证后 ,将该基因插入原核表达载体pET32a( +)中 ,并转化至E .coli表达菌BL2 1(DE3)中 ,诱导 6×His融合蛋白表达 ,经Ni2 + 亲和层析及胶回收技术纯化蛋白 ,并将蛋白免疫动物制备兔抗OVA12蛋白抗体 ;采用ELISA、Dotblot、Westernblot法比较正常人及肿瘤患者血清中抗体水平的差异。结果 获得纯化OVA12蛋白及高滴度的兔抗血清 ;ELISA法测定显示 ,正常人组OD均值为 0 17± 0 0 7,肿瘤患者组OD均值为0 32± 0 15 ,两组之间有显著性差异 (P <0 0 0 0 1) ,并选择其中样本经Westernblot、Dotblot加以进一步证实。结论 成功克隆OVA12基因 ,并获得纯OVA12蛋白和高滴度的兔抗血清 ;ELISA等方法测定肿瘤患者与正常人血清中OVA12抗体水平有明显差异 ,为临床检测的应用奠定基础。
Objective To obtain OVA12 prokaryotic expression protein and study the difference between human normal and tumor patients in level of serum's anti-OVA12 antibody. Methods OVA12 cDNA was cloned into pGEMT. After the sequence was confirmed, it was inserted into prokaryotic expression vector pET32a(+). The recombinant vector was transfected into BL-21(DE3),which was then expressed as 6×His fusion proteins induced by IPTG. The proteins were flowing through Ni 2+ affinity chromatography and purified by gel extraction. After this, the proteins were injected into rabbits to make its polyclonal antibody. In addition, ELISA, Dot blot and Western blot were used to compare the antibody levels in normal and tumor patient's serum. Results Purification of OVA12 proteins and rabbits' antibody serum of high titer were obtained. ELISA results showed that OD means of normal group was 0 17±0 07, and tumor group's was 0 32±0 15. There difference was significant( P <0 0001). Furthermore, some samples are confirmed by Western blot and Dot blot. Conclusion OVA12 gene was cloned successfully, purified protein and rabbit antibody serum of high titer were prepared. ELISA was established to find that there are significant differences between them. So the basis of clinical application has been set up.
出处
《中国实验诊断学》
2004年第2期135-139,共5页
Chinese Journal of Laboratory Diagnosis
基金
国家自然科学基金资助
No.3 0 3 715 98
