Bovine CR4 Is Involved In Mannheimia Haemolytica Leukotoxin-induced Cytotoxicity

Bovine respiratory disease complex is a kind of important bacterial pathogeny in Bovine respiratory disease complex（BRDC） which jeopardize the upper respiratory tracts of flocks and herds and lead to bronchopneumonia and fibrinous pneumonia. Leukotoxin（LKT） is the foremost virulence factor of Mannheimia haemolytica, and play a conclusive role in the generating process of BRDC. Recent evidence indicates that LKT can recognize cell membrane specific receptor of leukocyte, and finally cause the apoptosis and cytolysis, such as β2 integrin LFA-1（CD11a/CD18） and Mac-1（CD11b/CD18）. Whether CR4 can serve as a cell membrane specific receptor for LKT ? Our research was on the basis of biological characteristics research, PCR amplification method was used to obtain the gene sequence of bovine monocytes CD11 c and CD18.We also carried out the study of mutual effect of LKT and CR4. Our work may provide a new theoretical and practical basis for the pathogenic mechanism of Mannheimia haemolytica. The research contents of this study were showed as follows:Firstly, MTT assay comparative analysis of crude extracts and purified LKT to bovine neutrophil proliferation. In this study,three pairs of primers were designed to amplify CD18, sheep and bubalus CD11 c gene fragments according to the published sequence of M81233.1, FJ601824.1 and XM₀06070380.1 in GenBank. To extract the monocytes total RNA as a template, PCR amplification of Holstein cow and native cattle CD18 and CD11 c genes. CD18 gene was cloned to T vector and the production was double enzyme-cut（EcoRI and XbaI） and link up pCI-neo toeukaryotic expression vector. The products were subsequently introduced into pcDNA3.1 eukaryotic expression vectors and eukaryotic expression plasmid pcDNA3.1-CD11 c were constructed. Bovine CD18, CD11 c and CR4（CD18/CD11c） were built in 293 T cell line through transient expression assay. Indirect immunofluorescence, RT-PCR and Western Blot technique were used to identify the protein expression status of bovine CD18, CD11 c and CR4.And eventually, purified LKT were used to infect 293 T cells expressed CD18, CD11 c and CR4 to test MTT dye reduction cytotoxicity assay,293 T cells as a negative control.The results showed that molecular weight of purified LKT was approximately 100 kDa and the recombinant eukaryotic expression plasmid pCI-neo-CD18 was expressed correctly in pCI-neo. The overall length of CD18 sequence was 2310 bp, encoded 758 amino acids. Eukaryotic expression plasmid pc DNA3.1-CD11 c was successfully constructed. The overall length of CD11 c sequence was 3477 bp, encoded 1158 amino acids. The homology of dairy cow and cattle compared with buffalo and goat in Gene bank was 97.5%, 95.5% and 97.2%, 95.2% respectively. On the 19 amino acid residue front of N- terminal of dairy cow CD11 c was predicted the signal peptide and one-time transmembrane protein.The indirect immunofluorescence, RT-PCR and Western Blot test showed that CD18, CD11 c and CR4（CD11c/CD18） expression in 293 T cell line. MTT test showed that the strongest cytotoxicity of LKT was observed in CR4 transfected 293 T cells.CR4 can serve as a cell membrane specific receptor for LKT.In conclusion, we demonstrated that CR4（CD11c/CD18） receptor located on the surface of peripheral blood mononuclear cells in Holstein cow and native cattle. In this research, we obtained the nucleotide sequence of CD11 c for the first time, the overall length of the sequence was 3477 bp and encoded 1159 amino acid. In this study we also had a preliminary discussion on cytotoxic effect receptor induced by Holstein cow mononuclear leucocyte CR4 mediated leukotoxin LKT in Mannheimia heamolytica.