摘要
目的 构建大肠癌患者转移淋巴结Fab段噬菌体抗体库并结合蛋白质组学相关技术进行初步筛选大肠癌特异性抗原蛋白及相应的噬菌体抗体。方法 取大肠癌患者系膜转移淋巴结 ,提取总RNA ,RT PCR扩增重链Fd和轻链cDNA ,将扩增产物依次插入载体pComb3,电转感受态大肠杆菌XLI Blu ,加噬菌体VCSM13超感染。以二维凝胶电泳分离大肠癌细胞和正常大肠粘膜蛋白组并转至PVDF膜 ,将银染胶图用Melanie 112 DE分析软件进行分析 ,以大肠癌组织匀浆为靶抗原对初级抗体库进行三轮淘洗 ,再用富集抗体库和PVDF膜进行Western Blot印迹实验。结果 成功建立了大肠癌Fab段噬菌体抗体库。建立了平均 877个斑点的大肠癌平均胶图和 84 7个斑点的正常大肠粘膜平均胶图。经Western Blot印记实验我们发现了至少八个大肠癌表达而正常大肠粘膜不表达的有噬菌体抗体特异性结合活性的抗原蛋白。结论 成功建立了双盲、双向、高通量筛选大肠癌特异性抗原及其相应噬菌体抗体的方法。
Objective Construction of bacteriophage antibody library large intestine cancer, and combination of protomics technology to screen human of cancer. Methods Prepare total RNA of patient of large intestine cancer serum, RT-PCR of weight chain Fd and light chain kcDNA, the PCR product were inserted into carrier pComb3, electransterred into E. coli XL1-Blu, added bacteriophage VCSM13. Separates cancer cell's proteins with the 2D gel electrophoresis and transfers to PVDF membranes. 2D gels were analysis by Melanie 11 2-DE software, the specific antigen were selected by bacteriophage antibody library throughout 3 rounds. Results Weight chain Fd and light chain mRNA were amplified, ELISA test revealed that the 8 specific antigen were detected in tumor tissue and not in normal tissue. Conclusion Successfully construct the human antibody library, and the Fab of lymph gland, and used the proteomics technology to screen and determine specific antigen.
出处
《中国实验诊断学》
2004年第3期219-221,共3页
Chinese Journal of Laboratory Diagnosis
关键词
大肠癌
噬菌体抗体库
蛋白质组
2-DE
large intestine cancer
bacteriophage antibody library
proteomics
2-DE
