| Avian coccidiosis is a diseases caused by intracellular protozoa belonging to Eimeria, and has a major economic importance to poultry industry. Nowadays coccidiosis is mainly controlled by the use of chemotherapeutic agents and live vaccines. Although the measures such as shuttle medication, rotation program and combination of drugs are taken to diminish the time for the development of resistance, the drug resistances are still appeared in the field. The live vaccines can provoke an immune protection against coccidiosis, but immunovariant infections within a flock can lead to the emergence of uncovered strain. This may lead to the ingestion of large numbers of oocysts by susceptible animals and increases the risk of disease outbreaks. The subunit recombinant vaccines may be able to resolve these problems. Therefore, cloning of protective antigen gene from coccidian and expression of recombinant proteins have always been a research focus. E. necatrix is an pathogenic, and caused acute small intesnial coccidiosis among eight to eighteen week-old chickens. The number of genes cloned from E. necatrix is very little. In present research, The cDNA library of the second generation merozoites of E. necatrix was constructed by the technology of SMART, and screened using the immunological method. The 3-1E gene were cloned from the second generation merozoites of E. necatrix, and expressed using a prokaryotic expression system. This research established foundation to develop the subunit recombinant vaccines against avian coccidiosis.1. The purification of the second generation merozoites of E. necatrixTwenty-eight-day-old chickens were infected with 20000 sporulated oocysts of E. necatrix. At 150 h postinfection the chickens were killed, and the mucosal tissues in the small intestine was removed. The merozoites were released from the mucosal tissues using the method of grinding and digestion with hyaluronidase. By means of filtration with four layers of cotton cloth,260 mesh nylon mesh and 17 microns PET membrane, respectively, the merozoites were isolated. Finaly after centrifuged at 5 200 rpm for 15 minutes with 30% and 50% percoll, the merozoites were purified. The results showed that the method used here was simple. The purity of merozoites obtained from the tissues were high, and can be used in the molecular biology research.2. The Construction of a cDNA library for the second generation merozoites of E. necatrixThe whole RNA from the second generation merozoites of E. necatrix was extracted using Trizol reagent and the mRNA were purified using the beads with oliogo(dT). Then, the cDNA library was constructed by SMART technique. The results showed that the capacity of the unamplified library was 2.14×106 pfu/mL and the capacity of the amplified library was 4.47×1010 pfu/mL. The amplified library recombination rate was 90%. According to the PCR amplification from 100 monoclonal colonies that were picked randomly, the sizes of amplified fragments were mostly between 400 bp to 1000 bp. The cDNA library constructed in the present study provided a basis for screening, cloning and further studies of new genes of E. necatrix.3. The immunological screening of a cDNA library for the second generation merozoites of E. necatrixFirstly, the positive sera of anti-E. necatrix oocysts and anti-E. necatrix merozoite proteins were prepared from the chickens orally infected with sporulated oocysts of E. necatrix and mice immunized with the proteins isolated from the second generation merozoites of E. necatrix, respectively. The sera titers were detected with ELISA, which showed that sera could be used for immunoscreening cDNA library. Secondly, the cDNA library was screened using both chicken and mouse sera as probes repectively. In the primary screening, eleven positive clones were obtained using chicken serum and seven positive clones using mouse serum. Five clones were positive for both of two sera, which were further screened. Finally, the five positive clones were identified using PCR, and the PCR products were sequenced. The sequences of ESTs were analysesed. The results showed that the five ESTs were 599,723,516, 939 and 899 bp, respectively. The bioinformatics analysis showed that the ESTs had high homologies with E. necatrix hypothetical protein, SJCHGC02770 protein, SJCHGC00839 protein and SJCHGC 09095 protein of Schistosoma japonicum, and ribosomal protein of E. necatrix and E. tenella, and E. maxima cytochrome oxidase I, respectively.4. The cloning and expression of 3-1E gene of E. necatrixFirstly, The 3-1E gene of E. necatrix was amplified by RT-PCR from total RNA of purified the second generation merozoites, and cloned into pGEM-T Easy vector. The recombinant plasmid was identified by PCR, restriction endonuclease analysis and sequencing after blue/white selection. Secondly, the 3-1E gene was subcloned into express vector pET-28a(+). The pET28a-3-1E vector then was transformed into E. coil BL 21, and was induced with IPTG for expression. The expression products were analyzed with SDS-PAGE. Finaly, the recombinant proteins were analyzed by Western-blot using the positive sera of anti-E. necatrix oocysts and anti-E. necatrix merozoite proteins. The results showed that E. necatrix 3-1E gene was consisted of 671 nucleotides and included an complete opening read frame with a length of 513 bp which encoding 170 amino acids. The fusion protein was about 36.47 kDa, and expressed in soluble form. The recombinant proteins can be recognized by the positive sera of anti-E. necatrix oocysts and anti-E. necatrix merozoite proteins, respectively, which suggested that the recombinant protein had good immunogenicity. |
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