Effects of Diclazuril on G3PDH in Second-generation Merozoites of Eimeria Tenella
|School||Nanjing Agricultural College|
|Course||Basic Veterinary Science|
|Keywords||Diclazuril Second-generation merozoites GAPDH Cloning and expression Apoptosis Anzymatic activity|
Coccidiosis, one of the poultry industry’s most common and expensive diseases, is caused by infection with Eimeria species parasites with severe economic loss worldwide. The important species involved include Eimeria tenella, Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria praecox, and Eimeria necatrix. Among them, Eimeria tenella, is the most pathogenic species for chickens. E. tenella develops in the caeca but can colonise the terminal ileum and the rectum in severe infections.The parasitic cycle of E. tenella includes two steps:one is an endogenous phase in the chicken intestine, and the other is exogenous phase in the environment. At the first developmental stage, the sporozoites invade the epithelium of the tip of the villi and cross the epithelial cells, then the sporozoites penetrate the epithelial cells from the base and develop into first-generation meronts under the cell nucleus. After released into the lumen of the gland, the merozoites I develop rapidly into large second-generation meronts which emigrate into the submucousal layer. During the parasitic cycle of Eimeria necatrix, when the merontsⅡburst, they cause severe tissue haemorrhages, and result in considerable mortality.Despite the advances in novel vaccine, to date, coccidiosis is mainly controlled by the chemotherapy. Diclazuril, one of effective triazine-based agents against multiple types of coccidia in numerous hosts, has been used in poultry for a long time. In our previous study, we found that after the treatment with diclazuril, in the infected/treatment group, merozoites showed obvious apoptotic features, including reduced amounts of chromatin, which was condensed and compressed against the nuclear envelope and aggregated into large dark, compact masses. However, the mechanism of E.tenella apoptosis has not been clear.In the present work, a glyceraldehydes-3-phosphate dehydrogenase(GAPDH) of E.tenella, named mz-GAPDH was sequenced and analyzed.1 Sequencing and analysis of mz-GAPDH from second-generation merozoites of E.tenellaThe flanking region of mz-GAPDH was gotten by reverse transcription PCR (RT-PCR), and then sequenced. The result revealed the presence of a 1020 bp open reading frame (ORF), and its gene was named mz-gapdh. This ORF, mz-gapdh, encodes a polypeptide of 339 amino acids with a calculated molecular mass 36.38 KDa. The result of BLASTP and FASTA algorithms showed that mz-GAPDH was significant homologous to the GAPDH of Toxoplasma gonhe. Sequence of this gene was submitted to GenBank/DDBJ/EMBL database. Accession number assigned to this sequence is HQ317455.2 The expression of GAPDH and specific enzyme assaysThe mz-gapdh gene was ligated to the prokaryotic expression vector pET-28a. Then, the recombinant plasmid pET-28a-mz-GAPDH was transformed to host strain E.coli BL21 (DE3). Trying the different condition, I found the best condition for fusion protein production was pH 7.0 LB medium,0.1 mmol/L IPTG,120 rpm/min of rotation speed, and at 20℃temperature. After purified by His-Bind (?)Kit, the specific enzymatic activity of the fusion protein was tested by UV spectrophotometry, and the titer was calculated through the method ELISA. The result showed the titer could acquire 1:20000.3 Effects of diclazuril treatment on mz-GAPDH gene and protein expression in second generation merozoites of E.tenellaThe effects of diclazuril treatment on mz-gapdh mRNA and protein expression were examined by Real Time PCR (RT-PCR), Reverse Transcription PCR, western blot, respectively. These results revealed that after the diclazuril treatment, the mz-GAPDH protein was extremely upregulated, however, no significant change was observed on the quantity of mz-gapdh mRNA.4 ImmunofIuorescence localization and enzymatic activity of GAPDH in second-generation merozoites of E. tenellaIn this chapter, by the method of transmission electron microscopy(TEM), the Ultrastructural changes of second-generation merozoites of E. tenella was monitored, in the diclazuril group, merozoites that were characterized by obvious apoptotic features including reduced amounts of chromatin, which was condensed and compressed against the nuclear envelope and aggregated into large dark, compact masses. Furthermore, the mz-GAPDH was observed aggregated abundantly in the cell nucleus by fluorescence microscope. In accordance with the result of western-blot, the specific enzymatic activity of mz-GAPDH was very higher than the negative control group. These findings indicated that mz-GAPDH involved the apoptosis of merozoites caused by diclazuril, which was possibly one of mechanisms of diclazuril against coccidian.