Cloning And Expession Profiling Of EtppGalNAC-T2 And EtGPT Genes During The Development Stages Of Eimeria Tenella

Coocidiosis is a parasitic protozoal disease,causing considerable economic loss in poultry industry.At present,the prevention and treatment of chicken coocidiosis was mainly depended on chemical drugs.However,there was severe drug resistance for chicken coocidium against the drugs used.So it is urgent to search new drug targets and develop new drugs.Studies indicated that the glycosylation modification affected not only the spatial conformation,biological activity,transport and location of protein,but also played an important role in molecular recognition and signal transduction.Besides,it was also very important in the process of parasite invasion into the host.And it was reported that there were glycosylation modification in the apicomplexan protozoa such as Toxoplasma gondii,Cryptosporidium and Plasmodium,while it is rare reported in coocidium.We found there were glycosylation modification in coocidium by qualitative protein mass spectrometry.It was reported in the papers that UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase T2(EtppGal NAc-T2)and UDP-N-acetylglucosamine-1-phosphate transferase(Et GPT)were key enzymes and played important role in the glycosylation modification.In this study,we cloned and prokaryoticly expressed EtppGalNAc-T2 and EtGPT,and then analyzed their mRNA transcriptional levels during the developmental process for Eimeria tenella.First,we tested the proteins of E.tenella from the five developmental stages which are the second generation merozoites,unsporulated oocycts,sporulated 7 h oocycts,sporulated oocycts and sporozoites by mass-spectrometric technique and identified 15,9,9,9,10 glycosylated proteins respectively.Then we found all the proteins participated in the functions of response to external stimuli,regulation of biological process,catalytic activity,cell and cell component by GO analysis.And the main pathways are ribosomes,carbon metabolism and glycolysis / gluconeogenesis by KEGG pathway mapping.The above results indicate that glycosylation do exist in coocidium and may play a role in its developmental stages.Furthermore,we cloned the full-length ORF for Etpp GalNAc-T2 and EtGPT and analyzed them primarily by bioinformatics tools.The lengths of ORF for genes EtppGalNAc-T2 and EtGPT are 1983 bp and 1089 bp,respectively and the sizes of their coding proteins are 75.14 ku and 39.25 ku.There are not signal peptides indicating that they are intracellular localized and biological activated and the corresponding theoretical isoelectric points are 8.66 and 7.92.Next,we adopted the real-time PCR with relative quantitative method to calculate the mRNA transcriptional levels for tppGalNAc-T2 and EtGPT during the developmental process for E.tenella in which the E.tenella 18 S rRNA was chosen as the reference gene.The results indicated that there were significant differences in mRNA level for the two genes and they had the highest expression level in the stage of sporozoite and lowest expression level in the stage of sporulated oocysts.Finally,we constructed the prokaryotic expression vector for pGEX-6p-1-EtppGalNAc-T2 and pET30a-EtGPT and transferred the recombinant plasmid to Transetta(DE3)and expressed the fusion proteins by IPTG with 1 mmol/L.The above experiments showed that EtppGalNAc-T2 and EtGPT had potential as the target for anti-coccidiosis.Our study established a theoretical foundation for discussion of their functions in glycosylation modification and the development of anti-coccidiosis drugs.