人源抗Rh(D)单链抗体基因的克隆和表达

Cloning and expression of human anti-Rh(D) single-chain Fv fragment

目的:构建人源抗红细胞Rh(D)抗原单链噬菌体抗体库。方法:应用噬菌体展示技术,从分泌抗Rh(D)抗体的细胞中提取总RNA,逆转录合成cDNA第一条链,多次PCR扩增和克隆抗体可变区基因(VH/VLcDNA),经重叠延伸拼接(SOE)用编码连接肽(Gly4Ser)3的互补序列组成scFv基因,经酶切克隆入载体pCANTAB5E,使之呈现于噬菌体表面。使用完整Rh+型红细胞进行四轮淘汰筛选,ELISA对所获抗体进行初步鉴定。结果:构建的噬菌体抗体库库容为1.2×107,噬菌体DNA中全长scFv基因的插入率为0.80,用辅助噬菌体援救后,得到滴度为3×108pfu/ml的初级噬菌体抗体库。以完整Rh+型红细胞进行四轮淘汰筛选,出现特异性富集。经DotELISA实验鉴定,得到1株与Rh+型红细胞特异结合的scFv噬菌体抗体。结论:运用噬菌体抗体库技术构建了人源抗红细胞Rh(D)抗原的噬菌体抗体库,为进一步对所获克隆株进行序列分析、可溶性抗体表达和纯化奠定了基础。

Objective ： To construct a phage display library of human single-chain Fv antibodies against blood group Rh（D） substance. Methods： Combining phage display library techniques, isolated total RNA from B lymphoblastoid cell lines secreting anti-Rh（D） antibodies was used for the synthesis of the first strand of cDNA, VH and VL genes were amplified by 2nd PCR and linked together by splicing overlap extension （SOE） with the use of a （Gly4 Ser）3 linker. The resulted scFv genes were then cloned into pCANTAB5E vectors and displayed on the phage. Phage clones were selected using intact red ceils as a source of antigen. After 4 rounds of ＂binding-elution-enrichment＂ , each clone was assayed for specificity by Dot ELISA. Results： A phage antibody library, with the sink size being 1.2 × 10^7, was obtained. The percentage of full-length scFv gene inserted into phage DNA was 0.80. Rescued by helper phage, a phage scFv library with titer of 3 × 10^8 pfu/ml was established. Specific phages with scFv were acquired after 4 rounds of panning, one clone exhibiting specific binding to Rh^＋ cell was identified by Dot ELISA. Conclusion ： A strategy for construction phage antibody library by means of phage display technique was practicable, which would be useful in screening engineered antibodies against human Rh （D） blood group substances.