Cloning And Expression Of ADF Gene Of Eimeria Tene Lla Yl Strain And Its Immune Protetc Ion In Chickens

Coccidiosis of poultry, an intestinal disease caused by seven species of apicomplexanprotozoan parasites of the genus Eimeria, is characterized by bloody diarrhea, high morbidityand mortality in young chickens. It is a severe problem for the intensive farming of chickens.E.tenella is one of the most virulent strains of Eimeria in chicken. In the recent years, geneticengineering vaccine as a novel vaccination strategy has gradually drawn more people’sattention. Like all other apicomplexans, the invasive form of E.tenella named zoite, as well asmigration, replication, invasion; takes advantage of the high flexibility of the cytoskeleton.The actin depolymerizing factor (ADF) is a family of actin-binding proteins expressed in alleukaryotic cells. The pathogenicity of E.tenella YL strain, which was passaged for severaltimes, was studied at different dosages. The sequence of ADF gene was analysed after genecloning. The immune protection of ADF recombinant protein was studied after prokaryoticexpression. Four different parts of work have been finished as followings.（1）To study the pathogenicity of multiple passaged E.tenella, seven-day-old chickenswere infected with sporulated oocysts of36passage E.tenella YL strain at different dosages.The pathogenicity was assessed by survival rate, clinical symptom, lesion score, relativeweight gain, and oocysts per gram faeces (OPG). Result showed that E.tenella YL strain hadhigh pathogenicity in chickens although it was passaged for many times.（2）The total RNA was extracted from the prepared sporozoites of E.tenella YL strain.Then the ORF of ADF gene was amplified by RT-PCR. The analysis of the ADF genesequence indicated that the ORF included357nucleotides and encoded118amino acids. Thehomologies of its nucleotide sequence were100%,99%, respectively, compared with thesequence of ADF gene of E. tenella and E.maxima. The homologies of their amino acidsreached100%. The results demonstrated that the ADF gene has very high conservatismamong different geography strains and different species.（3）The ADF gene was cloned into pET-28a(+) vector to proceed prokaryoticexpression in E. coli BL21(DE3) by IPTG induction. The terminal concentration of IPTG was1mmol/L, and IPTG induced for4h in37℃. The results of SDS-PAGE and Western blotshowed that the ADF recombinant protein,18ku as expected, was successfully expressed. The expressed proteins existed mainly in the supernanant of lysate, and were purified usingthe HiTrap Chelating HP.（4）The immune protective effect of ADF recombinant proteins that immunizedchickens was assessed by the parameters, including relative weight gain, OPG, caecal lesionscore, and anti-coccidial index (ACI). The results showed that the ADF recombinant proteinsinjected intramuscularly could reduce the oocyst production and decrease the caecum lesionscores. It indicated that the ADF recombinant proteins induced certain anticoccidial immuneprotection by intramuscular injection, and the ACI value reached159.4.