The Cloning Of Sheep Complement Component C3d And The Development Of Recombinant Hydatids Subunit Vaccine

Echinococcosis (Hydatidosis) caused by infection with larval stage of Echinococcus granulosus(Eg), affects both humans and domestic animals, which has been considered as one of the worldwide major zoonoses. This study focus on the cloning of sheep complement component C3d and the development of recombinant hydatids subunit vaccine. At one respect, the subunit vaccine EG95 for the sheep echinococcosis was modified to EG95s to enhance the solubility of the fusion protein, and several copies of Eg95s were connected and expressed to improve the expression of the recombinant protein. At another respect, the gene of sheep C3d was amplified , the plasmid containing the fusion gene EG95s-3C3d was constructed and expressed in order to enhance the immunogenicity of the recombinant protein EG95. The main results are as follows:1. The improvement of the solubility of recombinant protein EG95 and the tandem expression of EG95One segment about 350bp encoding highly hydrophilic dominate epitope sequence was selected according to the analysis of the software, the prokaryotic expression vector was construced, also the prokaryotic expression vector containing two or three copies of EG95s gene was construced, the fusion protein GST-EG95s, GST-2EG95s, GST-3EG95s were expressed and identified by western-blotting.2. cloning and sequence analysis of the complement molecular C3d of sheepC3d gene was amplified by reverse transcription polymerase chain reaction(RT-PCR) using the total RNA extracted from the liver tissue of the sheep and the primer pairs designed according to the published sheep C3d sequence and cloned into pUC-18 vector. Then the recombinant plasmid was sequenced and analysed. Sequencing result reveal the sheep C3d has a similarity of 99.1% with published data, and the amino acids sequences encoded by these two sequence were the same. The similarity between sheep C3d and other mammal animals are nearly 80%, and the bovine C3d has the highest similarity of 95% with sheep C3d, this result revealed sheep and bovine are close in the cladogram.3. the connection and expression of sheep C3d gene and EG95s geneIn order to analysis the immunogenicity of EG95s fused with C3d, the EG95s gene was connected to three tandem copies of sheep C3d, and there was peptide chain adaptor (Linker) between each gene. Then the prokaryotic expression vector pGEX-6P-1-EG95s-3C3d was construced , fusion protein GST-EG95s-3C3d was expressed and identified by SDS-PAGE and western-blotting. The fusion protein expressed as inclusion body was denaturation dissolved and renaturation by dialyse, the fusion protein after intial purification was obtained.