EBV转化B型健康人B淋巴细胞构建噬菌体抗体库

Construction of a phage antibody library for blood group A substance by using EBV-transformed B-lymphocytes of B blood type

目的构建人源抗红细胞A抗原单链噬菌体抗体库。方法应用EB病毒转化方法和噬菌体抗体库技术,从EB病毒转化成功的B型个体淋巴细胞中提取总RNA,逆转录合成cDNA第一条链,PCR分别扩增VH和Vκ基因,经重叠延伸拼接法(SOE)组成ScFv基因并将其克隆入pCANTAB5E,转化E.coliTG1,加入辅助噬菌体M13KO7援救,构建人源抗红细胞A抗原单链噬菌体抗体库,并测定文库的库容、滴度及ScFv基因的插入率。使用完整A型红细胞对该库进行4轮“吸附-洗脱-扩增”的筛选,细胞ELISA、红细胞血凝实验对所获抗体进行初步鉴定。结果所构建的噬菌体抗体库,其库容为1.2×106,噬菌体DNA中全长ScFv基因的插入率为0.90,用辅助噬菌体援救后,得到滴度为2.52×1010PFU/mL的初级噬菌体抗体库。以完整A型红细胞进行四轮淘筛,出现特异性富集。经细胞ELISA、红细胞血凝实验鉴定,得到2株与A型红细胞特异结合的ScFv噬菌体抗体。结论EB病毒转化方法联合噬菌体展示技术构建噬菌体抗体库切实可行,可进行亲和筛选以得到人源抗红细胞A抗原的基因工程抗体。

Objective To construct a phage display library of human single-chain Fv antibodies against blood group A substance. Methods Total RNA was extracted from EB-transfected lymphoblastoid cell lines of B blood group by using EB virus transformation technique and phage display hbrary technique. The first strand of cDNA was synthesized by reverse trauscription assay. With amplification of VH and VK genes by PCR, the single-chain fragment variable （ScFv） gene was constructed by splicing overlap extension （SOE）, and then cloned into vector pCANTABST. The pCANTAB5E was transformed into E. coli TGl cells, and then rescued with M13KO7 helper phage. The size, titre, and insertion rate the constructed library were detected. Phages from the library were selected using intact red cells as a source of antigens. After 4 rounds of ＂absorption-elution-enfichment＂, individual clones were assayed for specificity by cell ELISA and agglutination. Results Phage antibody library with size of 1.2 × 10^6 was obtained. The percentage of full-length scFv gene inserted into phage DNA was 90%. The titer of the phage scFv library was 2.52 × 10^10 PFU/mL after rescuing by helper phage. Specific phage scFv was acquired after 4 rounds of panning and 2 clones exhibiting specific binding to red blood cells of type A were identified by cell ELISA and agglutination. Conclusion A strategy for constructing phage antibody library by means of phage display technique and EB virus transformation technique is practicable, which would be useful in screening human engineering antibody against A blood group substances.