Construction And Identification Inducible Transgenic Expression System Of Plasmodium Falciparum Based On Tetracycline

Malaria is infamous as a tropical scourge responsible (largely infection with Plasmodium falciparum) for as many as 2 million deaths per year. Despite intensive research efforts, an effective global vaccine is still a distant possibility. In the absence of success with the more traditional approaches of vaccination and drug development, it is becoming clear that greater biological information about the parasite would be helpful in developing knowledge-based strategies to combat the Plasmodium parasites. In the recent years the development of stable, drug-selectable, genetic transformation has offered the possibility of investigating and exploiting the biology of Plasmodium parasites, ranging from the means to obtain specific information at the structure-function level of proteins to the functional expression of transgenes.However, the expression of target genes is unregulated using this system, which is hence not suitable for studying genes that are essential or potentially growth-inhibiting or toxic to the cells when over-expressed For the study of such genes, a controllable system that allows regulated induction of gene expression in P.falciaprum is required. The regulatory elements of the tetracycline (Tet) resistance operon of Eschenchia coli, Tet repressor (TetR) and Tet operator (TetO), have served as the basis for the establishment of heterologous repression-based inducible system in many eukaryotes including yeast, plants, insects, mammals and parasitic protozoa, such as Trypanosomes, Entamoeba,Giardia, Toxoplasma, Leishmania and Trichomonas.In this paper, we tried to construct an inducible transgenic expression system based on tetracycline for falciparum parasites. But the inducible promoters available now are not functional in malarial parasites, and no inducible promoter derived from this parasite was found. To construct a recombinant promoter bearing the inducible response to tetracycline or its analogues in malarial parasites, 7-copy of TetO (7cot) was inserted into the vicinity of single transcriptional initiation site (TIS) of GBP130 promoter. To this end, a BamHi restriction site was introduced into different points relative to the TIS, namely -5, -2, +2 and +5, respectively, by overlap extension PCR, because no appropriate restriction site near the TIS could be applied in cloning. PCR products of 7cot amplified from plasmid pTL-8 having a BgtII site at both ends were ligated into the BamHI site produced in GBP 130 promoter as above, respectively, yielding 4 responsive plasmids: pG/7T(-5), pG/7T(-2), pG/7T(+2) and pG/7T(+5), as identified by PCR amplifications, restrictions and confirmed by DNA sequencing.To test the insertion effects of 7cot on the activity of GBP 130 promoter, 4 plasmids constructed above and plasmid pGBPCATA2 were electroporated into ring-stage falciparum parasites and the expression level of reporter gene CAT was detected by CAT ELISA. CAT detection in samples indicated that CAT expression level in each derivative of pGBPCATA2 plasmid [pG/7T(-5), pG/7T(-2), pG/7T(+2), pG/7T(+5)] was higher than that of pGBPCATA2 plasmid, suggesting that modified GBP130 promoter in each derivative plasmid had much higher activity than the primitive promoter, and location of 7cot downstream of the promoter showed more significant promoter activity than those located upstream. And of the derivative ones, the promoter in plasmid pG/7T(+5), in which 7cot was inserted into +5 point of GBP130 promoter, showed the highest activity. Then plasmid pG/7T(+5) could be chosen as the responsive plasmid in establishing tetracycline-controlled transgenic expression system of malarial parasites.To produce a regulatory plasmid, TetR coding sequence was amplified and cloned into BamHi, the single cloning site in plasmid pD, and plasmid pDT was constructed, in which TetR expression was controlled by the 5f and 3' flanking sequence of dihydrofolate reductase-thymidylate synthase gene of P. berghei.Since both responsive and regulatory plasmids were constructed successfully, co-transfection of pG/7T(+5) and p...