关键词: 牛巴贝斯, DXS, 序列分析, 真核表达

Abstract: 【Objective】The apicomplexa parasites, including Plasmodium, Babesia, commonly contain a relic, non-photosynthetic plastid-like organelle that has been named the apicoplast and is vital for their survival. The apicoplast harbors several metabolic pathways that supply essential biosynthetic precursors to the parasite. The synthesis of fatty acids and isoprenoid precursors are its most prominent functions, and the apicoplast has been shown to be essential for the survival of P. falciparum and T. gondii specifically. The isoprenoid mevalonate independent biosynthesis of parasitic protozoa in the apicoplast is a promising chemotherapeutic target, because this pathway is different from the mevalonate pathway in mammals and is essential to such parasites. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) catalyzes the first rate limiting step in the mevalonate independent pathway. To obtain the activive 1-deoxy-D-xylulose-5-phosphate synthase from Babesia bovis Shannxian, dxs gene was cloned and expressed in eukaryotic system. And it is the foundation for further research of the design of anti-babesia agents that can optimally target the active sites of the DXS enzyme.【Method】A full length cDNA of dxs gene from B. bovis Shannxian was obtained using reverse transcription polymerase chain reaction amplification of total RNA extracted. The complete open reading frames (ORF) of dxs was further inserted into T-easy vetctor and the eukaryotic expression vector, then sequenced and bioinformation was analyzed. The positive recombinant plasmids were transfected into the Hela cells by lipofection transfection and detected using inverted fluorescence microscope and Western blot. To obtain the positive monoclonal cells, the limited diluent with G418 was used, and the enzyme activity of the expression product was detected by liquid mass spectrometry in vitro.【Result】Results showed that the full length of DXS gene was 2061bp, encoding 686 amino acid, the similarity with B.bovis T2Bo in GenBank was 98.0%. According to the bioinformation analysis, the enzyme has three domains, namely thiamine pyrophosphate binding region, pyrimidine ring of thiamine pyrophosphate binding region, and transketolase C-terminal region. The enzyme belongs to the common TPP dependent protease family. The positive recombinant plasmids PEGFP-DXS were transfected into Hela cells by lipofection transfection. The inverted fluorescence microscope and Western blot were used to detect the expression of recombinant plasmids. The Hela cells which transfected successfully showed green fluorescence under the inverted fluorescence microscope. And the Western blot results showed a band about 102 kD in accordance with DXS fusion enhanced green fluorescence protein. The positive monoclonal cells were obtained by combining G418 and limited dilution method, the crude cell extracts from the reaction mixture had a product which has the same molecular of product DOXP, the cationic mode m/z=237 by LC-MS/MS, which indicated that the expression product had catalytic activity.【Conclusion】The fusion protein was expressed correctly in Hela cell，and the expression product had enzyme activity. The study is very important for the study of DXS as targets for antiprotozoal drugs.

Key words: Babesia bovis , DXS , sequence analysis , eukaryotic expression