人源性大肠癌自然致敏噬菌体抗体Fab呈现库的构建与筛选

Construction and screening of natural immune Fab antibody phage library from patients with colorectal cancer

目的 构建大肠癌病人自然致敏淋巴结抗体Ｆab段噬菌体呈现库，初步筛选相关抗大肠癌抗体。方法取大肠癌病 人转移淋巴结，提取淋巴结总ＲＮＡ，逆转录ＰＣＲ扩增重链Ｆｄ和Ｋ轻链ｃＤＮＡ。依次将ＰＣＲ产物插入载体pＣomb３的 相应部位，构建人源性大肠癌噬菌体Ｆab基因库，并应用噬菌体表面呈现技术对该抗体库进行淘选及鉴定。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 体表达库容达１．４８×１０６。经３轮淘选，抗体库得到约１２０倍的富集。３轮抗体库的点印记免疫染色均显示有Ｆab表达；酶 联免疫吸附实验显示其与大肠癌抗原有结合活性。结论成功构建了大肠癌病人自然致敏淋巴结抗体Ｆab段噬菌体呈 现库，利用噬菌体抗体库技术，可筛选相关抗大肠癌抗体。

Objective To construct natural immune Fab fragment phage display library and to screen the antibodies against human colorectal cancer. Methods Total RNA in the metastatic lymph nodes from patients with colorectal cancer was extracted routinely, and reverse transcriptase PCR employed to amplify the heavy chain Fd and light chain κ cDNA. The amplification products were inserted in succession into the vector pComb3 to construct human Fab fragment library, which was screened and identified using phage display technique. Results The heavy chain Fd fragment and light chain κ fragment, (650 hp in length) were amplified, and the products were inserted-into pComb3 vector with a recombination rate of 40%. The constructed phage display Fab library had a capacity of up to 1.48x106, and was enriched approximately 120 times after 3 cycles of panning. Dot immunoblotting found the expression of Fab fragment Which could bind to the antigens of human colorectal cancer. Conclusion The phage display library of the antibody Fab fragment from naturally immunized lymph nodes is successfully established, rendering it possible to screen the antibodies against human colorectal cancer.