Cloning And Expressing Of The Genes Of Surface Antigen SAG2 And SAG7 In Eimeria Tenella

Coccidiosis is an important avian disease which seriously harm to the poutry production. 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 of a complex repertoire by the second generation Merozoite has on host adapted immunity are unknown. We choose the two surface proteins (SAG2 and SAG7) to research:(1)According to the reported sequences of eimeria tenella Houton strain ,we designed the specific primers to amplify the genes of Surface antigen 2 (SAG2)and Surface antigen 7(SAG7)of E.tenella Yangling strain by RT-PCR. The PCR product was purified and cloned into pGEM-T easy vector and the recombinant plasmid was sequenced. The sequences compared with Houton strain, the nucleic acid sequences'homology reach as high as 99%,The the deduced amino acid sequences'homology reach to 99%, These indicated the genes of SAG2 and SAG7 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. We apply the 3 ' RACE (3 ' rapid amplification of cDNA end) to obtain non-translation region of SAG2/SAG7 gene, and the results indicated that the amplified region has the typical characterization of mRNA gene in eukartoyic cell.(2) The gene without the signal peptide was amplified by PCR from the pGEM-SAG2/SAG7 using the specific primers and cloned into the expression vector pET-32a (+), and then they were transformed into E.coli BL21 (DE3). They were conformed by PCR, restrction endonuclease digestion and the genes were sequenced if the coding frames were correct. The recombinant plasmids were induced by IPTG and the expressed proteins...