Expression In Baculovirus And Molecular Evolution Investigation Of Complement Component3d

Complement component3d (C3d) is a fragment from activated C3which iscritically important for both innate immunity and achieved immunity by interactingwith CR2. Current research about C3d is always around its molecular adjuvantactivity, such as the generation of fusion protein binding (C3d)3with antigen protein,which can enhance the immune response by thousands of times. However, research ofthe mechanism of mediating immune system as well as the structure and function ofC3d is not so much. In this work, C3d protein was expressed effectively, then thecorrelation among its molecular evolution, structure and function was analysed, whichwas quite important for other research about C3d.First, C3d genes from16birds were cloned and submitted to Genbank. Aphylogenetic tree about C3d amino acid sequences from both birds and mammals wasestablished through bioinformatics analysis, it was indicated that sequences frombirds are far away from that from mammals in the tree, while there were two partsseparated in avian block, which is not determined by orders of birds. It wasdemonstrated that the evolution of C3d was always under purify selection in spite of8amino acid sites those were suggested to be under positive selection. The tertiarystructure of chicken C3d was generated with the published structure of human C3d asa template and then the potential positive selected sites were positioned into thecorresponding sites. It was demonstrated that those sites were all exposed and mightbe important for sustaining the structure of the antigen binding domain and providingthe energy needed by CR2binding.Secondly, the bac-to-bac baculovirus expression system of chicken C3d wassuccessfully constructed and the recombinant virus named pFB-C3d-BAC wasgenerated, then the C3d protein was expressed effectively. Western and IFA wasemployed for the identification of the biological activities of the expressed protein,which was the foundation for further study, such as the generation of monomerprotein as well as the research of the immune mechanism.Finally, oligonucleotide microarray was conducted to detect the existence of B, E,J subgroup of ALV in16birds, and it was demonstated that there were7birdsinfected with E subgroup while2birds with J subgroup, and there was no significant infection of B subgroup among the birds. Combining with the phylogenetic andpositive selection analysis, it was suggested that birds nearby in the phylogenetic treetended to be infected by the same subgroup of ALV, and the comporison of aminoacid sequences between infected and uninfected birds nearby indicated that someviriation occurred on the positive selected sites which might be important for structureand function, and then the inference that viriation on some special sites in C3dsequences would affect the host immune system was raised.