关键词: CR1-like, C3b, 酵母双杂交, 免疫粘附

Abstract:

【Objective】In order to provide scientific data for elucidating the molecular mechanism of porcine erythrocyte immune adhesion function, it was investigated whether CR1-like (Complement receptor 1-like, CR1-like) of porcine erythrocyte could bind to the C3b or not.【Method】In this study, the recombinant plasmids of CR1-like_(3-6) and CR1-like_(8-11) functional domain fragments were constructed first, which were used to establish a yeast two-hybrid detection system. The bait plasmid (recombinant pGBKT7-CR1-like) and capture plasmid (recombinant pGADT7-C3b) were co-transformed into Y2HGold yeast cells. The single deficient SD/-Leu, SD/-Trp and double-deficient SD/-Leu/-Trp (DDO) media were used to strictly screen the co-transformed yeast cells. Then, according to the expression of report factor, the growth of transformants were identified on the double-deficient medium SD/-Leu/-Trp/X-α-Gal (DDO/X) or SD/-Leu/-Trp/X-α-Gal/Aba (DDO/X/A) combined with the color change phenomenon of the colony to comprehensively determine whether CR1-like active fragments and complement C3b bind to each other in yeast cells or not. The CR1-like-C3b binding complex in yeast cells was then separated by immunoprecipitation, and the specificity of the complex was identified by Western blot. 【Result】The co-transformed yeast clones showed normal growth on SD/-Leu, SD/-Trp, DDO and DDO/X, DDO/X/A media with blue color colonies, and this indicated that positive yeast colonies were successfully obtained. The results of PCR reverse identification showed that the co-transformed yeast contained the target genes CR1-like_(3-6) and CR1-like_(8-11). The C3b gene fragment appeared after the plasmid was digested, indicating that the recombinant plasmid pGBKT7-CR1-like and pGADT7-C3b were successfully co-transformed into yeast cells. In the immunoprecipitation test, the tag antibody c-Myc of the pGBKT7 vector was used to precipitate the fusion protein in yeast cells. Western blot detection with c-Myc as the primary antibody revealed that the fusion protein transformed pGBKT7-CR1-like_(3-6) and pGBKT7-CR1-like_(8-11) separately showed a specific band at 50 kDa; the yeast fusion protein co-transformed with pGBKT7-CR1-like_(3-6) + pGADT7-C3b and pGBKT7-CR1-like_(8-11) + pGADT7-C3b showed a specific band at 83 kDa; when the HA monoclonal antibody was used as the primary antibody for Western blot detection, no specific bands appeared in the pGBKT7-CR1-like_(3-6) and pGBKT7-CR1-like_(8-11) fusion proteins, and only the yeast fusion protein co-transformed in lane 3 and 4 showed a specific band at 83 kD. It showed that there was a complex of CR1-like and C3b in Y2HGold yeast cells. Using CR1-like monoclonal antibody to precipitate the fusion protein in yeast cells, Western blot detection with CR1-like as the primary antibody revealed that the fusion protein transformed with pGBKT7-CR1-like_(3-6) and pGBKT7-CR1-like_(8-11) separately showed a specific band at 50 kD; the yeast fusion protein co-transformed with pGBKT7-CR1-like_(3-6) + pGADT7-C3b and pGBKT7-CR1-like_(8-11) + pGADT7-C3b showed a specific band at 83 kD; when the C3 monoclonal antibody was used as the primary antibody for Western blot detection, no specific bands appeared in the pGBKT7-CR1-like_(3-6) and pGBKT7-CR1-like_(8-11) fusion proteins, lanes 3 and 4 showed that only the co-transformed yeast fusion protein had a specific band at 83 kD. This indicated that there was a biologically active CR1-like and C3b binding complex in Y2HGold yeast cells. The bait plasmid expression products CR1-like_(3-6), CR1-like_(8-11) fragments and capture plasmid expression products C3b fragment could be combined in yeast cells.【Conclusion】In summary, the recognition ligand for porcine erythrocyte CR1-like to exert immune adhesion function was C3b, which provided an important data basis for the further analysis of the molecular structure of CR1-like functional domain.

Key words: CR1-like, C3b, yeast two-hybrid, immune adherence