Protective Effects Of Recombinant SO7 Antigen Of Eimeria Tenella

Avian coccidiosis is a serve problem for the poultry industry, caused by intracellular protozoa including several species of the coccidia such as E.tenella, E.maxima, and E.acervulina. Coccidia infection results in extensive destruction of the duodenum epithelium accompanied by severe depression in body weight gain, reduced feed efficiency and intestinal shedding of parasite oocysts. Although coccidiosis is mainly controlled by the use of chemotherapeutic agents, alternative control strategies are needed due to the increasing emergence of drug-resistant parasite strains in commercial settings. Recently, novel vaccinating strategies using recombinant vaccines have been shown to be able to induce protectively cellular and humoral immune responses against coccidia. In order to attempt a new method of controlling coccidiosis in poultry industry of China, the studies had been conducted as the following:1.Clone and sequence analysis of gene SO7 of E.tenella YZ strainSO7 gene of E.tenella YZ strain isolated from Yangzhou city was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of sporulating oocyst at 7 hours. The products of RT-PCR were cloned into pUCm-T vector. Two positive clones were identificated with blue/white selection, restriction enzyme digestion, and PCR method. Nucleotide sequence of the two inserted fragments were determined by dideotide chain-termination method, and showed that SO7 gene of E.tenella YZ strain include 651 bp opening read frame which code for a polypeptide of 216 amino acids. The ORF sequences of the gene showed 99.5 % homologous compared with LS18 strain and BJ strain, but 99.4 % with HB strain, and 99.7 % with GD strain. Comparing its deduced amino acid sequence demonstrated that it showed 99.1% homologous compared with LS18 strain and GD strain, and 98.6% with BJ strain and HB strain. Analysis of secondary structure,flexible regions, antigenic index, and surface probability of SO7 protein of YZ strain indicated that they were coincide with other isolated strains, and might be a suitable gene for study vaccines against Eimeria infection.2.Expression of gene SO7 of E.tenella YZ strain in E.coliSO7 gene of E.tenella YZ strain without signal peptide encoding region was amplified by polymerase chain reaction (PCR) from plasmid named pUCm-T- SO7 which contained the cDNA of SO7 gene. The fragment of SO7 gene was subcloned into express vector pGEX-6P-1, and positive plasmid named pGEX-6P-1- SO7 was identified by restriction enzyme digestion and sequencing. Insoluble 48Ku GST-S07 fusion protein was expressed after induced by IPTG, which was confirmed by SDS-PAGE and Western blot analysis. Fusion protein GST-S07 separated by SDS-PAGE was extracted from the gel and used to immunize the ICR mice once a week for five times. The antiserum was collected from the immunized mice, and its specificity to E.tenella was identified by indirect ELISA. The results suggested that the fusion protein had good immunogenicity, and could be used to study vaccine against Eimeria infection.3.Improvement of the expression condition of recombinant E.coliThe main factors of expression condition including inducing moment, induction time, temperature, and IPTG concentration were examined by SDS-PAGE. To find the optimal inducing moment, the growth curve of recombinant bacteria was also established. The results showed that the optimal incubation time for production of GST-SO7 was 3 h after inoculation, the optimal induction time was 4.5 h after induction, the best temperature was 34-40℃, and the best concentration of IPTG was 0.001 mmol/L. Under the optimal expression condition, the insoluble form fusion protein could accounted for 37.5 % of total bacterial proteins by Tlc-scanning.4.Protective effects of recombinant SO7 antigen against E.tenella infectionsFive-day old chickens were subcutaneously immunized with preliminary purified SO7 antigen (100 microgram per chick) with adjuvant or without adjuvant, and boosted with the same dosage at 12, 19 days of age. Chickens in group 1, 2, and 3 were given antigen mixed with 0.25, 0.5, and 1.0 mg ginsenosides per dose, respectively. Group 4, 5, and 6 were given antigen mixed with 1 000, 5 000, and 10 000 units'chicken recombinant interferon-gamma (chIFN-gamma) per dose, respectively. Group 7, 8, 9, and 10 were given FCA adjuvant, nonadjuvanted antigen (subcutaneously), nonadjuvanted antigen (orally), and 100 sporulated oocysts of E.tenella YZ strain (orally) at 12 days of age, respectively. Group 11, 12 were unimmunized, served as positive and negative control, respectively. Chickens in all groups were orally challenged with 105 homologous sporulated oocysts at 26 days of age, and killed at eight days postchallenge. Mortality, weight gains, lesion scores, oocyst output, and ACI were determined and compared with those of the controls. All groups administered with adjuvant showed higher protective immunity compared to nonadjuvanted antigen (subcutaneously) control and unimmunized negative control, and the protective immunity was affected by the dose of the chIFN-gamma and ginsenosides, in which the group given adjuvanted antigen with 5 000 units chIFN-gamma showed significantly strongest adjuvant effects in terms of mortality, oocyst output, lesion scores, and weight gains (P < 0.05). In nonadjuvanted antigen (subcutaneously and orally) groups, no significant protective immunity was observed. ChIFN-gamma had strong adjuvant effects when mixed with E. tenella recombinant SO7 antigen at a dose of 5 000 units, and would be a potential vaccine adjuvant against avian coccidiosis.5. Cross protective effects of recombinant SO7 antigen against E.maxima infectionsFive-day old chickens were subcutaneously immunized with SO7 antigen (100 microgram per chick), and boosted with the same dosage at 12, 19 days of age. Group 1 were given antigen mixed with 5 000 units'chicken recombinant interferon-gamma (chIFN-gamma) per dose,. group 2 were given nonadjuvanted antigen subcutaneously, group 3 were given 100 sporulated oocysts of E. maxima (orally) at 12 days of age, group 4 were given 100 sporulated oocysts of E.tenella (orally) at 12 days of age. Group5, 6 were unimmunized, served as positive and negative control, respectively. Chickens in all groups were orally challenged with 105 homologous sporulated oocysts at 26 days of age, and killed at 8 days postchallenge. The weight gains and oocyst outputs of all the groups were determined and compared with those of the controls. The results demonstrated that the chickens in group orally immunized with E. maxima oocysts showed best protective effects, while the group immunized with nonadjuvanted antigen subcutaneously was seem to no protective effects. When adjuvanted with chIFN-gamma, recombinant protein showed partial protective effects in terms of weight gain, but no effects on the output of oocysts.