| Babesia orientalis is an intraerythrocytic protozoan parasite of the buffalo Bubalus bubalis transmitted by the tick Rhipicephalus haemaphysaloides haemaphysaloides. It is the cause of babesiosis, one of the most important diseases of buffalo in central and southern China and is characterized by apathy, fever, anemia, icterus, haemoglobinuria, and in some cases death. It is a new Babesia species which was isolated and named by our laborary.In this study, the Babesia orientalis cDNA library been build the last two years had been used to sequence and analysis the ESTs sequences, and to e-clone each of the sequences to gain the longer ones to get more informations about the Babesia orientalis gene. And then the mitochlndrial DNA had been chosen for the further study according to the results of the ESTs analysis and e-clone study. Further more the cytochrome b gene was been cloned by PCR and sequenced for the complete gene which was based on to build the phylogenetic tree.1) The sequencing and analysis of the B. orientalis ESTs sequencesNearly there were 1514 B.orientalis ESTs sequences which were almost over 300bp had been sequenced from the B.orientalis cDNA library randomly from negative colony to E.coli Bm25.8 by the PCR identification. And then 1171 unigenes with high quality were clustered and analysised by the phrap and BLAST software, encluding the mitochlndrial DNA, RNA binding protein, papain family cysteine protease and the hypothetical protein et cetera, and so on. According to the results of the analysis ,maybe there were 5 potential metabolic pathways conclude the oxidative phosphorylation pathway, ribosome pathway ,cytokine receptor interaction pathway,apoptosis pathway and antigen processing and presentation pathway and so on.2) The elongation and analysis of the e-cloned B. orientalis ESTs sequencesEach of the 1514 ESTs had been e-cloned and analysis one by one from theaverage 593.7bp to 819bp which was nearly 1.4 times than before. There may be 83 sequences could be complete the new ones and 15 of which had great homology with the protozoa, meanwhile 69 ESTs were associated with the protozoa genes and had 51 of them completely extended.3) The clone and bioinformatics analysis of B. orientalis mitochlndrial DNAConsidering the analysis results of the ESTs and e-clone the Contig18 had beenchosen to continue the study of B.orientalis mitochlndrial DNA which had high expression during the lifetime of this kind of parasite. Several primers had been designed to try to clone this gene and the sequencing results were almostly exactly the same with the e-clone datas while only few parts of the mitochondria gene could not be cloned due to their own characters, which proved the effectiveness of the e-clone technology. The genes on mitochondria had been predicted by bioinformatics analysis software and the cytochrome b gene of the mitochondria had been analysis using the NCBI's databases, forether more, it was used to make Babesia orientalis more exactly localized on the phylogenetic tree.This was a three-part-research including The sequencing and analysis of the B.orientalis ESTs sequences, The elongation and analysis of the e-cloned B.orientalis ESTs sequences and The clone and bioinformatics analysis of B.orientalis mitochlndrial DNA. Babesia orientalis was still a kind of unkown parasite to most of the researchers , so it will have great importance in further more studies about the genes on mitochondrial DNA of B.orientalis for the further research .
|
PDF Full Text Request