A Preliminary Study On Pro-apoptotic Gene BAD Of Schistosoma Japonicum

Schistosomiasis is a widely distributed and cause infection in humans and mammals by schistosome cercariae of serious harm zoonotic parasitic diseases.The disease is prevalent in Africa,Latin America,Southeast Asia and China.The major epidemic strain is Schistosoma japonicum Chinese strain in southern China.Currently,Using praziquantel is still the major method to cure schistosomiasis japonicium which inducing drug resistance.It is urgent finding new drug targets to treat schistosomiasis japonicium.Apotosis is also named programmed cell death which is strictly controlled by multiple genes.It can prevent the variation of passing to offspring by DNA damage.It also play a very important role in maintaining a normal growth environment as well as the organism homeostasis.Our previous works showed that the morphology,growth and apoptosis in worms is significant different in different sources of host Japan schistosomulum.It suggestted that apoatosis may be an important factor in the growth and development of the parasite Schistosoma impact.BAD is the bcl-2 associated death promoter.It belongs to the Bcl-2 protein family which containing BH3-only.We refered the SjBAD gene sequence(SjD AY814710.1)which was published on the European Molecular Biology Laboratory(EMBL,http://www.ebi.ac.uk/).We desgined primers and SjBAD was amplified by PCR which is a gene with 594 bp,encoded 197 amino acids.The gene SjBAD was subcloned into a pET28a(+)vector and the recombinant plasmid was transformed into competent E.coil/BL21 for producing recombinant protein.The recombinant plasmid was successfully expressed in E.coil/BL21 and purified recombinant protein pET28a(+)-SjBAD with molecular weight of 27 kDa.SjBAD was transcribed at various developmental stages of Schistosoma japonicum.It expressed highest in 14-day-old schistosomula while the expression level in 42-day male worms was higher than that in female worms.Its antigenicity was confirmen by western blotting analysis.The recombinant protein SjBAD rejected in the mouse could produce high levels of IgG by ELISA analysis.Two independent animal trials showed that 27.50%and 33.57%worm reduction,as well as 39.80%and 42.06%liver eggs reduction were obtained in rSjBAD vaccinated group compared with those of the blank control group.Here,we constructed eukaryotic recombinant plasmid PcDNA3.1(+)-SjBAD and transfected it into 293T cells.We detected the pro-apoptotic function of SjBAD by flow cytometry and the results showed that SjBAD could promote early apoptotic in 293T cells.The SjBAD was transfected into 293T cells,respectively,12h,24h,36h,48h,the cells were collected.Then we extrated the RNA,and reverse transcribed into cDNA.We analysis the level of SjBAD transcriptional by real-time quantitative PCR.This article studied the biological function of SjBAD and it will provide the basis for screening the new drug targets to prevent of schistosomiasis.