结肠癌患者淋巴结构建抗结肠癌噬菌体Fab抗体库

Construction of the natural immune Fab antibody phage display library from patients with colorectal cancer

目的 构建 2个结肠癌患者自然致敏淋巴结抗体Fab段噬菌体呈现库。方法 取 2个结肠癌患者转移淋巴结 ,提取淋巴结总RNA ,逆转录PCR扩增重链Fd和κ轻链cDNA。依次将PCR产物插入载体 pComb3的相应部位 ,噬菌体VCSM13辅助感染。以点印迹检测噬菌体表面Fab的表达。 结果 所选 2种Ig亚类的重链Fd片段、2种κ轻链cDNA得到扩增。Fd片段和κ轻链均插入 pComb3的重组率为 40 %,Fab噬菌体表达库容量达 1.48× 10 6。噬菌体悬液的点印迹免疫染色显示有Fab表达。结论 构建了 2个结肠癌患者自然致敏淋巴结抗体Fab段噬菌体呈现库 ,为筛选结肠癌相关抗体奠定了基础。

Objective To construct the natural immune Fab antibody phage display libraries of colorectal cancer. Methods Extracting total RNA from tissue of local cancer metastasis lymph nodes, which were obtained from 2 patients with colorectal cancer. RT-PCR was used to amplify the heavy chain Fd and light chain κ, and the amplification products were inserted successively into the vector pComb3 followed by phage VCSM13 infecting. Dot immunoblotting was used to monitor Fab phage display. Results The amplified fragments of Fd and κ by RT-PCR were about 650 bp. Fd and κ PCR products were subsequently inserted, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48×10 6. Dot immunoblotting showed that there was Fab expression on the phage libraries. Conclusion Two natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They have been the basis of screening the relative antibodies of colorectal cancer.