The Monoclonal Antibody Of Porcine CR1-like And Its Distribution On Erythrocyte Membrane

Objective:We used swine as the study object and produced mouse anti-porcine CR1-like monoclonal antibody to detect the nature distribution of CRl-like on porcine erythrocyte membrane for further study of the distribution of CR1-like and the its immune regulation. Meanwhile, we expected that we can obtain some theoretical basis and methods that may contribute to the next step study of the mechanism of animal erythrocyte immunological function.Methods:According to the full sequence of cDNA of porcine CR1-like, the peptide fragment was prepared by Beijing Protein Innovation. Mouse anti-porcine CR1-like hybridoma was established and selected using McAb preparative technology. The McAb was purified by affinity chromatographic and AKTA purifier. The characteristics and the purity of McAb were identified by tricine SDS-PAGE and the activity was detected by indirect immunofluorescence. The distribution of CR1-like on porcine erythrocyte membrane was detected using Laser Scanning Confocal Microscopy (LSCM) and the purified McAb. The C3b-beads treated with fresh rabbit serum were incubated with porcine erythrocytes and the erythrocytes were analyzed by Flow Cytometry (FCM).Results:In this study, the mouse anti-porcine CR1-like hybridoma was obtained. SDS-PAGE demonstrated that mouse anti-porcine CR1-like McAb was secreted and purified successfully. Analyzed by indirect immune fluorescence, it showed that the McAb can combine most of the erythrocytes and cells showed green under Fluorescence Microscope. Analyzed by LSCM, the fluorescence points were dispersed on membrane in fixed group, while clustered in unfixed group. The fluorescence points on fresh erythrocyte membrane were obviously less than those in fixed group. FCM data showed that the mean fluorescence intensity of fixed erythrocytes was 302.35,634.44 for unfixed erythrocytes and 4.7 for black control. Fixed group and unfixed group were significantly higher than blank control group (P<0.01) and the unfixed was obviously stronger than the fixed erythrocytes (P<0.01).Conclusion:The prepared hybridoma cell line, CRT-2 CL-5, can secrete mouse anti-porcine CR1-like McAb. The McAb was proved to be bioactive. In summary, CR1-like is dispersed on naive erythrocyte membrane while clustered on membrane of the erythrocytes that showed immune function or combined with immune complexes (ICs). The fluidity of erythrocyte membrane was important physiological basis for the high affinity of porcine erythrocyte CR1-like adhering to immune complexes.