| Malaria still remains a major threat to mankind nowadays. The efforts for gene functions will facilitate the development of effective malarial vaccines and the discovery of drug targets. Gene targeting is one of the important methods which provide vital information about genes and proteins, but the information is none or all. Moreover, malaria parasites will die when some key genes such as merozoite surface protein 1 (MSP1) and apical membrane antigen1(AMA-1} are disrupted, which would prevent further investigations. To control the expression of the gene of interest effectively, an inducible transgenic expression system is in need.Now inducible transgenic expression system based on tetracycline is considered the best one. For it's convenient to insert the tetracycline regulation element into the vicinity of the single transcription start site of glycophorin binding protein 130(GBP130),a stage-specific gene transcribed in trophozoite parasites, GBP130 promoter is the best candidate to be used in the inducible transgenic expression system of malaria parasite. The findings of minimal regulation sequence with high transcription activity and of elements with and without stage-specificity, based on deletion analysis of 5' flanking sequence of GBP130, will lead to the establishment of an inducible system with stage-specificity. Recently, investigations suggested that no stage-specific element existed in the sequence upstream of GBP130 transcription start site, but the regulation role of the proximal fragment of GBP130 5' flanking sequence remains unknown.Here a malaria transient transfection system was established firstly, using a chloramphenicol acetyltransferase (CAT)-based reporter. The regulation functions of the proximal fragment of GBP130 5' flanking sequence were studied by the introductions of a few plasmids bearing deletions of different fragments in the proximal sequence into ring-stage malaria parasites and the detections of theexpression level of reporter gene CAT.Malaria transfection, both transient and stable, marked a significant breakthrough in the investigation at molecular level, and opened a new chapter in the molecular analysis of malaria. However, this new tool is still limited in one or two foreign labs, and none of home lab does some work about it.Plasmids pGBPCAT, pGBPCAT A 2 and pGBPCAT A 3 containing different deletions of upstream of the GBP130 promoter were electroporated into ring-stage Plasmodiwn falciparum YN strain, respectively, and the expression level of CAT in each plasmid was detected by liquid scintillation counts(LSC). Consistent with previous results, the reporter gene CAT expressed in the parasites and the promoter activities were significantly different, showing that transient transfection of'falciparum malaria was successfully performed.To analysis the functions ofGBP130 5' proximal sequence, several deletions were constructed. In plasmid pGBPCAT A 2, the reporter gene CAT was flanked by the truncated 5' flanking sequence of GBP130 gene (Genebank:Z11832), the first 292 bp removed, and 3' flanking sequence of histidine rich protein (HRPII). A distal fragment, 615bp (2487bp~3101bp), of 5' noncoding sequence of gene GBP130 in pGBPCAT A 2 was PCR amplified from pGBPCAT A 2 and cloned into pGBPCAT A 2 excised with KpnllNsil to yield pGBP A 2/615.Based on pGBP A 2/615, pGBP A 2/400 and pGBP A 2/800 were constructed. To this end, F4oo(3788bp~4200bp) and F800 (3329 bp~4200bp) were amplified from 5' proximal flanking fragment ofGBPJSO taking the plasmid pGBPCAT A 2 as a template. The PCR products purified in 2% agrose gel were restricted by Nsil and inserted into the Nsil site of pGBP A 2/615. Individual clones containing the restricted PCR products were isolated. Positive clones bearing single insert with correct direction were identified by enzyme restrictions. For the identification of pGBP A 2/400, the plasmid was restricted with Nsil, KpnUNcol, respectively. And for pGBP A 2/800, restricted with Nsil, Kpnl/Spel, respectively.All the plasmids, maxiprepared w...
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