Masri Saad A, Rast Heidi, Hu Wei-Gang, Nagata Les P, Chau Damon, Jager Scott, Mah David
Canadian Food Inspection Agency, Centre for Plant Health, 8801, East Saanich Road, Sidney, British Columbia, Canada.
Mol Immunol. 2007 Mar;44(8):2101-6. doi: 10.1016/j.molimm.2006.09.007. Epub 2006 Oct 12.
Human lymphocytes derived from the blood of a donor immunized with anthrax vaccine were isolated and enriched for B-cells by Nycoprep density centrifugation. Individual anti-anthrax protective antigen (PA) B-cells were isolated by fluorescence activated cell sorting with fluorescence-labeled recombinant PA (rPA). The RNA from sorted single B-cells was extracted using plant total RNA as the carrier prior to purification by Nanoprep RNA isolation columns and then cDNA was prepared. Donor specific human Fab primer sets were developed based on rapid amplification of 5'-complementary DNA end results. Heavy chain and light chain of human Fab were amplified from the donor single B-cells by PCR. The amplified heavy and light chain were then cloned into the expression vector pASK-IBA2 and expressed in Escherichia coli (E. coli). The chains combined in vivo to form a functional Fab which was then purified as one protein. The human Fab antibodies produced by this technique were functional when tested in Western blots where the rPA was the target as well as in ELISA. This approach allowed us to obtain human Fab that retained the natural heavy and light chain pairing, which is supposed to have a high antigen-binding affinity.
从接种炭疽疫苗的供体血液中分离出人类淋巴细胞，并通过Nycoprep密度离心法富集B细胞。使用荧光标记的重组保护性抗原（rPA），通过荧光激活细胞分选技术分离出单个抗炭疽保护性抗原（PA）的B细胞。在通过Nanoprep RNA分离柱纯化之前，先以植物总RNA为载体，从分选的单个B细胞中提取RNA，然后制备cDNA。基于5'-互补DNA末端结果的快速扩增，开发了供体特异性人Fab引物组。通过PCR从供体单个B细胞中扩增出人Fab的重链和轻链。然后将扩增的重链和轻链克隆到表达载体pASK-IBA2中，并在大肠杆菌（E. coli）中表达。这些链在体内结合形成功能性Fab，然后作为一种蛋白质进行纯化。当在以rPA为靶标的蛋白质免疫印迹和酶联免疫吸附测定中进行测试时，通过该技术产生的人Fab抗体具有功能。这种方法使我们能够获得保留天然重链和轻链配对的人Fab，这应该具有高抗原结合亲和力。
