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目的:提取健康人外周血淋巴细胞的mRNA,利用反转录PCR方法,建立一个大容量的人抗体Fab段的天然抗体库。方法:收集健康者外周血,提取淋巴细胞,以淋巴细胞mRNA为模板,扩增人抗体Fab段,连入载体pComb3内,利用电转化的方法,构建噬菌体抗体库。结果:最终建立了重链库为1.73×107,轻链库为8.52×104的天然抗体库,并利用酶切鉴定计算出实际库容为重链库1.21×107,轻链库4.26×104,所以理论上所建天然噬菌体抗体库总的库容量可以达到5.15×1011。结论:成功构建了一个大容量的完全人源化的天然抗体库。
OBJECTIVE: To extract the mRNA of peripheral blood lymphocytes from healthy human and establish a large library of natural antibody against human Fab fragment by reverse transcription PCR. Methods: Peripheral blood was collected from healthy volunteers. Lymphocytes were extracted and the Fab fragment of human antibody was amplified with mRNA of lymphocytes. The Fab fragment was inserted into vector pComb3. The phage antibody library was constructed by electroporation. Results: The final library was 1.73 × 107 for heavy chain and 8.52 × 104 for light chain, and the actual library was 1.21 × 107 for heavy chain and 4.26 × 104 for light chain. In theory, the total capacity of the constructed natural phage antibody library can reach 5.15 × 1011. Conclusion: A large library of fully humanized natural antibodies has been successfully constructed.