| The Rhoptry, as well as the miconeme and conoid, formed a conserved structural feature in the Apicomplexan protists for their phylogeny and their interaction with host cell. As the results from Plasmodium spp and Toxoplasma goddi, the complex formed by proteins secreted from rhoptry neck (RONs) and microneme (Apical Membrane antigen, AMA), which is named as Moving Juction (MJ), serves as the mediation to cell invasion of these parasites. However, no any knowledge about the rhoptry proteins (ROPs) and RONs in Eimeria parasites was revealed up to date.In this paper, the sequence encoding Eimeria tenella RON2 (EtRON2) protein were predicted from the crow date of Eimeria tenella gene sequences using the bio informatics tool, amplified by RT-PCR from Eimeria tenella (Guangdong strain) total RNA template, using gene specific primer based on the predicted sequence. This ORF is 4020bp in full lenth, and coding a peptide of 1339 aa in lenth with a signal peptide sequence.The genome DNA sequence transcripted this ORF was obtained by PCR from E.tenella (Guangdong strain) DNA template, using the gene specific primer based on the cloned EtRON2 ORF. This gene including 11 introns, and is 5197bp in lenth. Amino Acid sequences alignment of EtRON2 with those from other several apicomplexan parasites by Clustal W showed EtRON2 shared 15-41% amino acid identeis with other RON2s.A fragment encoding 76 amino acid residues (EtRON2Ag) was hitted based on the antigenicity prediction by Expasy online tools, and Blastp alignment against GenBank, which will be subcloned into the pMAL-c2x and transformed into E.coli Rosseta (DE3) for expressing the antigenic peptide. The BALB/c mice were immunized with this purified recombinant peptide three times to produce polyckonal antibodies against EtRON2. High resolution image-capture and processing were performed using a confocal scanning laser microscope, and revealed the EtRON2 was located in the apex of the E.tenella sporozoites. This recombinant antigenic peptide also used as a probe for detection. The serum of chicken chanllenged with Eimeria tenella at 5,7,10,15,20 days post infection, both a result that the specific polyclonal antibodies against EtRON2 who produced at 10 days post infection.The parasites at different developmental stages were harvested and their total RNA were substracted by real-time PCR using EtActin gene as a reference for establishing standard curves to evaluate the copy number of EtRON2 and EtAMA genes. The results showed that all of quantative analyses were reliable because the melting curve showed a single peak. Etron2 gene transcription reached a peak during the unsporulated oosysts stage, then merozoites, sporulated oosysts and the lowest during the sporozoites stage. So it is likely that the transcription messages were stored during the unsporulated oosysts stage, and then the EtRON2 protein can be translated and screted into the rhoptry soon when the oosysts were sporulated. And it may play an important role in the process of invasion. Etama gene transcription reached a peak during the sporozoites stage, but it was very low during all other stages. So it demonstrated the Etama gene we got may be the ama2 gene, and the EtAMA2 protein may play a key role in the process of invasion like EtRON2.The no signal peptide of EtRON2 fragments was subcloned into the pET-32a(+). pMAL-c2x,pCold-TF and pCold-SUMO vectors, and transformed into E.coli Rosseta(DE3) and E.coli BL21 respectively with IPTG induction at different temperature. Comparison of these expression indicates that onlypET-32a(+)-EtRON2 was expressed as inclusion bodies in the E.coli Rosseta(DE3) at 16℃. |
