Cloning And Expression Of RB1-a Gene Of Eimeria Tenella YL Strain And Immune Protection In Chickens

Coccidiosis is a global epidemic intestinal parasites protozoosis which caused by 7 species of the Eimeria, and it harms the poultry industry seriously. When chickens are infected with coccidia, a large number of intestinal epithelial cells are damaged, causing hemorrhage, even death. Presently, the control of coccidiosis chiefly depends upon anticoccidial drugs. But with the drug resistance of anti-coccidia drug continuous strengthening and the high cost of new drug development, coupled with that drug residue may harm human health, people have to find a new method to prevent and cure the chicken coccidiosis. In recent years, genetic engineering vaccine as a new measure to prevent poultry coccidiosis draws people's attention increasingly. Coccidiosis vaccines used to prevent coccidiosis become a primary mean of the prevention and treatment of coccidiosis in the future, so it gets more and more important to find and identify the antigen gene of coccidian. This paper studied on the cloning, sequence analysis and prokaryotic expression of the antigen gene RB1-a of E. tenella YL strain and finished the immune protection in chinkens. The results were as followings.(1)RB1-a gene of E. tenella YL strain isolated from Yangling city was amplified by two-step reverse transcription polymerase chain reaction(RT-PCR) from total RNA of sporulated oocysts. The sequence analysis of the RB1-a gene suggests that the ORF of the RB1-a gene consists of 618 nucleotides and encodes 205 amino acids, and contains rich AGC and CAG repeat sequences. The sequence homologies of nucleotides and deduced amino acids between the RB1-a gene and the E. acervulina PAPa46 strain were 99.84% and 100%, respectively. The result shows that there is high conservatism in different Eimeria species about the RB1-a gene.(2)The RB1-a gene of E. tenella YL strain without signal peptide encoding region were amplified by PCR from the pMD18-T-RB1-a using specific primers and cloned into the expressive vector pET-32a(+), and then expressed in BL21(DE3) strain of E. coli. after induced by IPTG. SDS-PAGE showed that the fusion protein was successfully expressed and the molecular weight of the protein was 42ku. There was a lot of soluble fusion protein expressed in the triple-checked condition that IPTG was 0.4mmol/L, induced by 4~6h in 28℃. It will engage to the correct protein folding, in the meanwhile, it will provide an underlying platform to the analysis of the immunological function in the future.(3)The recombinant proteins was used to immunize chickens infected with E. tenella YL strain to decide the preventive effect. The parameters, including the caecal lesion score, OPG, weight gain and anti-coccidial index (ACI), were observed and recorded to judge the protection rate of the recombined proteins. The results showed that the recombinant proteins induced certain immunologic potency of anti-coccidia infections.