人源性单纯疱疹病毒II型抗gD蛋白噬菌体单链抗体库的构建

Construction of a phage display library of human single-chain Fv antibodies against HSV-2 gD protein

目的:利用HSV-2病毒IgG、IgM抗体阳性的生殖器疱疹患者外周血淋巴细胞,构建人源单链抗体库。方法:取患者外周血淋巴细胞提取总RNA,逆转录成cDNA,以其为模板,分别扩增抗体重链及轻链可变区基因,将重链、轻链可变区基因拼接成单链抗体(scFv)基因,酶切后连接到噬菌粒载体pFAB5c中,将重组噬菌粒载体电转化大肠杆菌XL-1Blue,构建噬菌体抗体库并测定其库容量。结果:成功构建人源性单纯疱疹病毒抗gD蛋白噬菌体抗体库,库容达1.86×106。结论:利用RT-PCR和噬菌体表面展示技术成功构建了HSV-2抗gD蛋白人源性单链抗体库,为其进一步的研究打下了基础。

Objective： Tn construct the phage antibody library, of human single chain Fv antibodies from peripheral blood lymphoeytes of genital herpes patient with positive serum antibody against HSV - 2 gD proteins, Methods： Total RNA was extracted from peripheral blood lymphocytes of patient and was transeribed reversely into cDNA. Using the cDNA as template, the heavy chain and light chain variable region gene of human immunoglobulin were amplified individually. Combined through a DNA linker encoding the peptide to construct single - chain variable fragments （scFv） gene and cloned into phagemid pFAB5c. Then the recombinant phagemid was electroporated into E. coli XL- 1 Blue. The recombinant rate was detected by enzyme digestion analysis, and library repertoire was detected. Results： The phage antibody library of human single - chain Fv antibodies against HSV - 2 gD protein has been constructed successfully. The library size was 1.86 × 10^6. Conclusion： A typical phage display library of human single- chain Fv antibodies has been constructed, by RT- PCR and phage display technique, and human single- chain Fv antibodies can be screened from this library.