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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 a Fab phage display library of two naturally sensitized lymph node antibodies in patients with colon cancer. Methods Lymph nodes were dissected from 2 colon cancer patients, total RNA was extracted from lymph nodes, and heavy chain Fd and κ light chain cDNAs were amplified by reverse transcription PCR. The PCR product was inserted into the corresponding sites of the vector pComb3 in turn, and phage VCSM13 assisted infection. Dot blotting was used to detect phage surface Fab expression. Results The heavy chain Fd fragments of the two selected Ig subclasses were amplified and the two kappa light chain cDNAs were amplified. The recombination rate of Fd fragment and kappa light chain inserted into pComb3 was 40%, and the Fab phage expression library capacity reached 1.48 × 10 6. Dot blot immunostaining of phage suspensions showed Fab expression. Conclusions The Fab phage display library of two naturally sensitized lymph node antibodies in colon cancer patients was established, which laid the foundation for the screening of colon cancer related antibodies.