| Avian coccidiosis is an economically important disease for the poultry industry throughout the world. Many methods besides chemotheraphy have been explored to eliminate its damage due to the increasing emergence of drug-resistant strains and the consumers' appeal for green food. Recently, novel vaccinating strategies using antigen-encoding DNA plasmids have been shown to be able to induce protectively cellular and humoral immune responses against many kinds of infectious diseases and cancer. In order to attempt a new method of controlling coccidiosis in poultry industry of China, we have conducted the following studies:1. Clone of the Gene EtlA. The total RNA of Eimeria tenella BJ strain sporulated oocysts was extracted and used as the template for RT-PCR. The primers were designed with the software Oligo according to the published data , which involved an EcoR I or Hindlll cleavage site respectively at its upper or lower primer in order to be easily ligated to the plasmid later. After RT-PCR, the amplified EtlA was ligated to the vector pGEM-T and transformated into the JM109 competent cell. Identified by PCR and cleaved with endonucleases, the positive clone was used to determine its nucleotide sequence. Results indicated that, the full-length of EtlA was 1978bp in E. tenella BJ strain, which had an ORF of 1944bp. Compared with the EtlA sequence from GenBank, mutations occurred at 7 bases of 6 regions and their homology were 99.5%. EtlA had a 79.8% identity with the gene EalA, which was also a gene encoding the refractile body protein cloned out from E.acervulina. The derived protein of EtlA is of functional similarity to nucleotide transhydrogenase. It is a speculated energy support during the invasion of sporozoites into host cells.2. Expression and Identification of the Gene TA4. The antigen TA4 is a 25kDa protein composed of 2 polypeptides of 17kDa and SkDa respectively, linked by a disulflde bond. It was located on the surface of sporozoites and synthesized throughout the latter half period of sporulation. The TA4 gene of Eimeria tenella BJ strain has a 99% homology compared with the data of GenBank. In this study, aimed to utilize the expressed fusion antigen to induce the antibody production for the later identifying the expression of pCT and pCTE in vitro and in vivo, the gene TA4 of E. tenella BJ strain was ligated to the prokaryotic expression vector pGEX-KG and induced with IPTG to express in E.coli BL21 (DE3). The results indicated that, the fusion GST-TA4 protein was about 43kDa instead of SlkDa shown from the western-blot result after SDS-PAGE. This means that the disulflde bond linking the 17kDa and SkDa polypeptides will be broken during the boiling treatment of samples beforeSDS-PAGE, and only the 17kDa polypeptide is linked to the 26kDa GST protein. In the large-scale purification of the fusion protein, the cells were lysed supersonically; the inclusion bodies were denatured and dissolved in 8M urine solution containing reduced glutathione. SDS-PAGE with the recombinant protein purified with glutathione Sepharose 4B showed 2 bands of 51kDa (primary) and 43kDa (secondary) respectively. This means that the disulfide bond wouldn't break completely in our purifying conditions. In the further investigation, when the purified protein was treated boilingly, the percent of 51kDa decreased while that of 43kDa protein increased simultaneously. This means the purifying conditions will affect the structure of GST-TA4 protein. Further investigation should be done to compare the immune-protective effects of the different kinds of fusion proteins.3. Construction of the DNA Vaccine pCT and pCTE, Identification of Their Expression in vitro and Their Efficacy against Experimental Coccidiosis in Chickens. In order to construct the DNA vaccine, the gene TA4 was ligated to the mammalian expression vector pcDNA3.1 (Zeo+), and then the gene Etl A was ligated to its upper stream, aiming to express them in a fusion protein form. The IFAT was established to examine the in vitro expression of the c...
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