Cloning And Identification Of Two Thioredoxin Genes From

Babesiosis is a kind of parasitic diseases caused by Babesia that is a blood parasite in the host red blood cells, mainly transmitted by ticks. Babesia microti infects in animals and human with the main symptoms of fever, headache, fatigue,anemia, and specially to immune deficiencies and immunocompromised people with fatal.Babesiosis become an emerging tick-borne diseases in the world.Thioredoxin(Trx), involved in the thioredoxin system with thioredoxin reductase(Trx R) and NADPH, is a small molecule protein and widely presents in prokaryotes and eukaryotes.Research on malaria and other blood parasites found that their survival depends on redox system to geting rid of oxides.Thioredoxin system plays key functions in redox regulation and antioxidant defense.Currently, Babesia thioredoxin system remains to research.This study will identify Babesia microti(B.Microti) Trx molecular, to understand the molecular mechanism of Babesia thioredoxin system and to found drug targetsIn the present study,there are the following four contents:1 By transcriptome sequence, bioinformatics analysis, B.microti thioredoxin(Trx)gene sequence frangments were screened. The full length c DNA of B.microtia Trx-2and Trx-3 genes were obtained by 3 ’and 5’ RACE. bioinformatics analysis showed that B.Microtia Trx-2 and Trx-3 both have typical Trx domain and WCGPC active site.2 recombinant expression plasmid using p ET-30 a as a carrier were constructed and transformed into BL21 competent cells. In 1m M IPTG induction, recombinant Trx-2 and Trx-3 proteins were expressed,and purified by affinity chromatography.3 Both recombinant Trx-2 and Trx-3 protein showed reducing activity under insulin reduction assays.With the reaction time extending,the Trx-2 activity increased slowly,and reached the peak after 4 minutes; however, the Trx-3 reduction activity was quickly and instantly achieve a smooth peak, similar to the positive control group.4 The Trx-2 and Trx-3 native protein are 15.5k Da and 21.7k Da respectively by western blotting, and abundantly expressed in the peak of the parasitmia in red blood cells.The Indirect immunofluorescence test(IFAT) proved that the Trx-2 and Trx-3 are mainly present in the cytoplasm.In a word, this study cloned the full-length c DNA of the B.Microti Trx-2 andTrx-3 genes,completed expression and activity analysis of the two recombinant proteins, determined the size and initial positioning of the two molecules.Results Provide a basis for understanding the molecular mechanisms of Babesia thioredoxin antioxidant system and screening of a new drug targets.