Construction Of Phage Display Library Of Fab Antibodies Repertoies And Screening Of Anti-Mouse Male Specific Antigen

Male-specific antigen is usually defined as a male minor histocmpatibility Y antigen, which was confirmed by Eichwald in 1955. Male-specific antigen is a group of products of male specific genes. It will provide assistance to study the rejection of male tissue graft by female recipients and sex determination. Because polyclonal antibodies are short of specificity, while the process of preparation of monoclonal antibodies by hybridoma technology is so complicated that the period from immune mice to specific monoclonal antibodies to be screened is several months. Moreover, these technologies are laborious, additionally, difficult to develop and produce a high-affinity antibody against H-Y antigen. These limit their application in mammal sex selection. Fortunately, in recent years the development of phage display technology has made it possible to solve these above problems.First, H-Y antisera can be raised in isogenic C57BL/7 female mouse by inoculation with male spleen cells from the same strain, the antibodies of sera of two groups of immune female mice were assayed by sperm Cytotoxicity test or ELISA technique. RNA was isolated from immunized spleen cells. The mixture of Fd fragment of the H chain and the mixture of 3 full-length light chain(κ)were amplified using degenerate oligonucleotide primers from total RNA with RT-PCR. A mouse combinatorial Fab library was constructed by inserting both light chain gene and Fd gene fragments into phagemid vector pComb3, and followed by the help phage VCSM 13 infection. The recombination rates of light chains and Fd fragments were 80%. The volume of the phage library was 5.2×l0⁶, and the titre was 3.2×l011 pfu/mL. The presence of mouse Fab on the phage sruface was determined by ELISA. Three cycles of affinity enrichment of the mouse phage antibody library with male spleen cells and two cycles of nonspecific absorption with female spleen cells were performed. Recombination rate of the phage antibody library clones is 60%. 9 of 15 strains can bind to male spleen cells with the specific activity. Sequence analysis of the PCR products of plasmid DNA of E5 clones show V_h and Vk had common characteristics shared by other known variable region of antibodies. We successfully constructed the Fab phage antibody library of mouse male specific antigen, these new molecules are potentially valuable tools in the study of H-Y antibody and the application of sex embryos.