| Coccidiosis is an important avian disease which seriously harms the poutry production. Conventional disease control strategies rely heavily on chemoprophylaxis and, to a certain extent, live vaccines. Due to the increasing emergency of drug-resistant strains and the consumers'needing green food, many novel methods have been explored to control the disease. The decisive measure is using the recombinant vaccines and/or DNA vaccines to control the avain coccidiosis. A major hurdle in development of those vaccines is searching for the protective antigens. Many potential coccidial antigens have been characterized, which are surface antigens and secrectory antigens from organelles such as micronemes, rhoptries and refractile bodies. Examining the sequences from the sporozoite and second merozoite of the parasite led to the identification of 37 potential GPI-linked variant surface proteins, some of which are expressed in the sporozoite, majority are expressed in the second merozoite. The biological role(s) of these proteins and the effect(s) of variant surface antigens, that express a complex repertoire by the second generation merozoite, on host adapted immunity are unknown. We chose the two surface proteins (SAG5 and SAG10) to research:(1)According to the reported sequences of Eimeria tenella Houghton strain, we designed the specific primers to amplify the genes of Surface antigen 5 (SAG5) and Surface antigen 10 (SAG10) of E. tenella Yangling strain by RT-PCR(GenBank Locus:EF635426,EF649989). The PCR product was purified and cloned into pGEM-T easy vector and the recombinant plasmid was sequenced. Compared with Houghton strain, the homologies of the sequences of nucleic acid and deduced amino acid in Yangling strain reach as high as 98%. These indicated that the genes of SAG5 and SAG10 have the very high conservative nature between different geography; we have to proof with the further experiments if they have an important role in host cell invasion and immunity. At the same time, we applied the SMART RACE technology to obtain the SAG10 gene full cDNA sequence.(2) The gene without the signal peptide was amplified by PCR from the PGEM-SAG5/SAG10 using the specific primers and cloned into the expression vector, and then they were transformed into E. coli Rosetta. They were conformed by PCR, restrction endonuclease digestion, and the genes were sequenced to confirm the coding frames whether correct or not. The recombinant plasmids were induced and expressed proteins successfully by IPTG. at first, we chose the pET-32a (+) as the expression vector, in which the expressed protein is inclusion body in the mycelium cell. For getting the soluble protein, we turned to the pMAL-c2X expression system and obtained higher concentrated soluble protein by Amylose Resin column purification system.(3) The effect of recombinant proteins immunization was test by ELISA and the protective experiment in E. tenella infected chickens. The parameters including weight gain, oocyte production (OPG), the caecal lesion score and anti-coccidial index (ACI) were observed, to judge the protective rate of the recombined protein. The results showed that the recombinant proteins induced certain degree antibody level and anticoccidial immune protection. |
