The Immunogenicity Of Eimeria Tenella Surface Antigen SAG21 And SAG23

Protozoa of the Eimeria tenella is intracellular parasite in the caecum of domestic chicken causing coccidiosis, a severe infection particularly of poultry. The infection has to be controlled fundamentally by permant medication with anti-coccidia. In recently, live vaccines have becomed more important. To develop new methods to control the coccidiosis, more should be learned about the pathogen. And now more researchers pay more attendtion to the study of the surface antigen of the genus Eimeria. Fiona Tomley had analysed a great deal of available expressed sequence tags (ESTs) of E.tenella and identified several GPI-linked variant surface antigens (SAGs). SAG21 and SAG23 were two of them. Based on the published sequences of the two genes in GeneBank, the SAG21 and SAG23 genes of Eimeria tenella (Guangdong strain), namely SAG21 and SAG23, were cloned. And then, on one hand, these genes were subcloned into pMAL-c2X vector to express the recombinant protein; on the other hand, the two genes were subcloned into eukaryotic expression vector pcDNA6.0(c) namely pcDNA6(c)-SAG21 and pcDNA6(c)-SAG23. Chickens were immunized with the recombinant protein and recombinant vector, and challenged with sporozoited oocysts to evaluate the protection effect . This experiment includes the followings:Cloning, characterization and sequence analysis of SAG21 and SAG23 genesBased on the published nueleotide sequences of SAG21 and SAG23 genes in GeneBank, paris of gene-special primers were designed using computer software, respectively. Two fragments about 810 bp and 813 bp were amplified by RT-PCR, using the total RNA extracted from the second generation merozoites of Eimieria tenella (Guangdong strain). Then, the two genes were cloned into pEGM-T vector, respectively. It was comformed that the amplified fragments were SAG21 and SAG23 genes via PCR and DNA sequencing. Comparison and analysis indicated that, the SAG21 gene was 810 bp, sharing 99.7% similarity with the reported one (E.tenella Houghton strain), and the SAG23 gene was 813 bp, sharing 99.6% similarity with the reported one. And the amino acids sequences the two genes encoding shared 99.6% and 99.6% with the reported sequences, respectively.The expression of SAG21 and SAG23 in E.coli and purification of recombinant proteinBy the means of genetic engineering technique, the SAG21 and SAG23 genes without signal peptides encoding sequences were subcloned into pMAL-c2X vector, respectively, and transformed into E.coli BL21(DE3). The recombinant protein were expressed by inducing with IPTG . The target protein were detected by western-blot using the antiserum of chicken immunizied with E.tenella as the primary antibody .The result of the SDS-PAGE indicated that the recombinant protein were about 67.8KDa and 67.9KDa, respectively, and both of them were soluble. The two protein were purified using the MBP-tag recombinant protein purification system. The SDS-PAGE indicated that the purified proteins were high concentration and were high purity, indicating that the protein could be used in the following experiments.Construction of recombinant eukaryotic expression vector of SAG21 and SAG23 and the detection of the expression of the recombinant vectors in vivoTaking advantage of DNA recombinant technique, the recombinant eukaryotic expression vectors, pcDNA6(c)-SAG21 and pcDNA6(c)-SAG23 were constructed. After identification by restriction enzymes digestion and DNA sequencing, the recombinant vectors were injected into the breast muscle of 14 day-old chicken, repectively. The chickens were killed 8 days after injection. The target genes' transcription and expression in vivo were detected by RT-PCR and western-blot.Protective effect experiment of the recombinant protein and recombinant eukaryotic expression vector on chicken against E.tenellaAccording to the animal experiment program, the recombinant protein and recombinant eukaryotic expression vector were injected into breast muscle twice at the 14 and 21 day-old chicken, respectively. And challenge with freshly sporulated E.tenella oocysts was carried out 7 days after the second injection. In order to evaluate the protective effects of the immunization, survival rate, relative weight gain rate, reduction of caecum lesion scores, oocysts amount in caecum content and anticoccidial index(ACI) were analysed. The result indicated that, both the recombinant protein and the recombinant vector could reduce the oocyst amount in caecum content and decrease the caecum lsion scores, the effect of the recombinant protein were relative with the dose, and the recombinant vector was better than recombinant protein.