| Male specific antigen is often referred to as male-specific minor histocompati-bility Y antigen, also called H-Y antigen. In earlier studies, H-Y antigen was considered general material caused inbred female mice to reject skin grafts from male syngeneic mice. Serological findings showed that H-Y antigen was located at cell membranes of male animals, and were expressive products from a group of male specific genes, determining the existence of H-Y antigen at cell membrane of embryo is an approach for sexing embryo. H-Y antigen has an important theoretical and practical significance for the study of transplant rejection between different gender donor-recipient groups and sex control. Because H-Y antigen is a weak antigen, specific H-Y sera with high titer were not obtained by the conventional immunolog-ical methods, the titer of H-Y monoclonal antibody prepared by hybridoma technique could not be significantly improved, which limited the feasible application of sex control using H-Y antibody. The phage antibody library technique, also called combinatorial library technique of antibody genes, could express antibody fragments in vitro by using phage antibody library to replace B lymphocytes clones in vivo, which possess enough diversity, short production cycle, technology flexibility, wide screening range and other characteristics. So it is an effective way to prepare a variety of antibodies, and affinity maturation of phage antibody in vitro might break boundary of antibody affinity maturation in vivo, which could make antibody affinity reach supernatural intensity, phage antibody library technique provides a possibility to increase antibody titer.This study based on primary phage antibody library against mouse male specific antigen constructed successfully, aimed to improve affinity of antibody from the phage library by chain shuffling, and was also a preliminary study of sexing mouse preimplantation embryo was also carried out. The main contents of our research are as following:1. Expression and specific identification of Fab antibody against serologically detected H-Y antigenTo study soluble expression and specificity of Fab antibody against serologically detected H-Y antigen, the expressive plasmid was constructed by the ligation reaction system of T4DNA ligase, after the phage gⅢfragment was removed from the recombinant phagemid digested with Spe I and Nhe I. The soluble Fab antibody was expressed in positive XL1-Blue with recombinant phagemid by adding IPTG, the antibody specificity was determined by technique of immunofluorescence staining with FITC\DAPI and ELISA assay. The results showed that a band around 49 kD band appeared in the SDS-PAGE. Based on number of positive cells and mean fluorescence intensity of male or female spleen cells, comparison of cell immunofluorescence staining of Fab antibody and H-Y serum showed that male specificity and binding activity of Fab antibody were higher than those of antiserum. The quantitative analysis of immuno-fluorescence staining of paraffin sections showed that the binding activity of Fab antibody against male mouse liver was obviously higher than that of female mouse, there was high significant difference between them (t=20.73, P=0.0023<0.01), binding activity of H-Y serum against male mouse liver was slightly higher than that of female mouse, there was significant difference between them (t=7.11, P=0.0192<0.05). ELISA assay showed that the soluble Fab antibody had male specific activity with male mouse spleen and testicular cells as antigen, but OD values of Fab antibody were lower than that from antiserum. The results suggest that male specificity of Fab antibody was higher than that of H-Y serum, but its binding activity with male cells was lower than H-Y serum, Fab antibody with male specificity had non-specific binding to a small number of female tissue or cells, affinity maturation of Fab antibody in vitro would be used as follow-up study, in order to screen soluble Fab antibody with high affinity and specificity.2. Improving affinity of Fab antibody against serologically detected male antigen by chain shufflingThe total RNA was isolated from spleen cells of C57BL/7 female mouse, then reverse transcribed into a single strand cDNA. Fab antibody genes with diversity were amplified using cDNA template, mouse combinatorial Fab gene libraries were constructed by inserting both light-chain genes and fd genes into pComb3 vector, respectively. The light-chain genes library and original clone A8 with pComb3+K+fd were digested by Sac I and Xba I, the light-chain shuffling library was constructed by the ligation reaction system of T4DNA ligase, then rescued by the infection with helper phage VCSM 13, the secondary phage antibody library was constructed. The panning cycles include specific absorption, elution and amplication with male or female spleen cells and ELISA were initiated, clones with the higher affinity were obtained after light-chain shuffling. The secondary phage antibody library was constructed again by light-chain gene of this clone and fd genes from high-chain genes library cloned randomly into pComb3 vector. The phage Fab antibody against serologically detected H-Y antigen with high affinity was finally selected by helper phage infection, screening and ELISA. The results showed that the recombination rates of light-chains shuffling library were 80%, the volume of the phage library was 7.6×107, and the recombination rates of high-chains shuffling library were 70%, the volume of the phage library was 6.4×107. The results from panning cycles of phage antibody library showed that the affinity and specificity of phage antibody were gradually improved, and the recovery rate of phage antibody library with light and high chain shuffling increased 35 and 25 times respectively in the fifth screening cycle. After the final panning, the phage antibody in 25 clones screened from final screening cycle were analysed with ELISA,8 (32%) phage antibodies from the library after light- chain shuffling had male specificity,12 (48%) phage antibodies from the library after high-chain shuffling had male specificity. After high chain shuffling, OD values of the best clone B9 increased to 0.961, compared that from the original clone A8 (0.505), which increased 1.90 times. From the point of view, the affinity of phage antibody was improved by chain shuffling.3. The analysis of specificity of Fab antibody with high affinityThe phage Fab antibody gene with high affinity from the library after high-chain shuffling was expressed using pComb3 vector and XL1-Blue strain, the antibody specificity was determined by technique of immunofluorescence staining with FITC\DAPI and ELISA. The sequences of antibody genes were analysed with IMGT online tools. The quantitative analysis of immunofluorescence staining of Kunming mouse spleen cells showed that binding activity of B9 Fab antibody with high affinity against male mouse spleen cells was obviously higher than female mouse spleen cells, and male specificity of B9 Fab antibody (about 3.62 times) with high affinity after high chain shuffling was higher than that of Fab antibody (about 2.07 times) from the original clone. The B9 Fab antibody with high-affinity could specifically bind testis, its specific binding with ovarian was not obvious. The results from comparative analysis of CD3 regions of light or high chain genes between original clone A8 (Fab-Kl, Fab-fdl) and positive clone B9 with high affinity (Fab-K2, Fab-fd2) showed a few site mutations of CD3 regions occurred, which suggested that site mutations and increase of CD3 length may be related to improvement of antibody affinity.4. Sexing mouse preimplantation embryos by Fab antibody with high affinityThe gender of 70 morulas and blastocysts from Kunming mice were identified using indirect immunofluorescence, then were evaluated by PCR amplification system of SRY and 113 genes established.28 morulas and blastocysts with strong fluorescence were determined as male embryos after PCR evaluation of genes from these embryos, the accuracy of embryos sexing (the coincidence rate of two methods) was 82.14%. 36 morulas and blastocysts with gray-green fluorescence or zona pellucida presenting gray-green fluorescence were determined as female embryo by the same PCR evaluation, the accuracy of embryos sexing was 88.57%. The results suggested this B9 Fab antibody had better specificity against serologically detected H-Y antigen, which need animal experiments to further determine antibody specificity.This study developed a practical application of embryo sexing using H-Y Fab antibody, preparation of H-Y Fab antibody with high affinity will be helpful to understand the molecular basis of serologically detected H-Y antigen and antibody, and offer theoretical basis to study and application of serologically detected H-Y antigen with unique mechanism and complex characteristics. |
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