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目的 :构建抗木瓜凝乳蛋白酶全套单链抗体 (scFv)噬菌体表面展示文库。方法 :从木瓜凝乳蛋白酶免疫小鼠的淋巴细胞中提取总RNA ,反转录成cDNA后 ,用抗体V区简并引物扩增全套VH 和VL 基因。经重叠延伸反应 ,装配成scFv基因 ,并将其克隆到噬粒载体pFAB5C中 ,构建scFv噬菌体抗体库。对抗体库进行亲和筛选后 ,用ELISA法鉴定抗木瓜凝乳蛋白酶的scFv。结果 :经 4轮“亲和 吸附 洗脱”的富集过程 ,得到ELISA活性较高可与木瓜凝乳蛋白酶结合的克隆。结论 :成功地构建了抗木瓜凝乳蛋白酶的scFv噬菌体展示文库 ,并筛选到与木瓜凝乳蛋白酶具有结合能力的scFv基因
Objective: To construct a full-scale anti-chymopapain scFv phage display library. Methods: Total RNA was extracted from lymphocytes of mice immunized with chymopapain and reverse transcribed into cDNA. Then, a full set of VH and VL genes were amplified by degenerate primers of antibody V region. After overlapping extension reaction, the scFv gene was assembled and cloned into the phagemid vector pFAB5C to construct a scFv phage antibody library. After affinity screening of antibody library, anti-chymopapain scFv was identified by ELISA. Results: After 4 rounds of “affinity adsorption and elution”, the clones with high activity of ELISA and chymopapain were obtained. Conclusion: The anti-chymopapain scFv phage display library was successfully constructed and scFv gene with chymopapain binding ability