【作者】 王宪锋；

【导师】 薛采芳； 甄荣芬；

【作者基本信息】 第四军医大学 ， 病原生物学， 2001， 硕士

【摘要】 早在1897年Ronald Ross就发现疟疾是蚊媒传播的疾病，100余年后的今天，疟疾仍然是人类健康的主要威胁之一。对各种疟原虫基因及其基因产物的功能研究是发现有效疫苗候选抗原和抗疟药物作用靶位的必要前提。基因敲除可为蛋白功能提供重要的信息，但其所反映的信息只是全有或全无的。对疟原虫有重要功能的蛋白基因不能通过基因敲除来研究，否则会导致疟原虫的死亡而无法研究，如MSP1和AMA-1。因此，需要建立一种可诱导性转基因表达系统，以有效控制基因的表达。 目前认为四环素诱导表达系统是最有前途的诱导性系统。GBP130是主要于滋养体期转录的期特异性基因，该基因启动子含有单一的转录起始点，有利于在其附近插入四环素识别调控元件。对GBP130的5’侧翼序列进行分析，发现具有高转录活性的最小调控序列，以及可能的期特异性和非期特异性调控元件，进而还可以建立期特异性和非期特异性诱导表达系统。已有研究认为GBP130的5’侧翼序列的转录起始点上游不存在期特异性调控元件，但转录起始点下游是否具有期特异性调控功能还不清楚。 本研究建立了一种有效的疟原虫瞬时基因转染系统，将GBP130基因5’近端侧翼序列不同片段删除，构建调控序列的系列缺失体，以CAT为报告分子，利用疟原虫基因转染技术对该基因5’近端侧翼序列的调控功能从强度调控和期特异性调控两个方面进行初步研究，为疟原虫诱导性转基因表达系统的构建打下基础。 由于疟原虫特殊的分子生物学特性，其基因转染技术并没有像高等真核细胞基因转染一样成为一项常规技术，在国外仅有个别实验室进行相关研究，而国内还没有实验室开展此类工作。本研究将含有不同长度GBP130启动子的质粒（pGBPCAT、pGBPCATΔ2和pGBPCATΔ3）采用电穿孔法转染入我国云南（YN）株恶性疟原虫（P.f）环状体，以液体闪烁计数法检测各质粒中报告基因CAT的表达。结果发现报告基因CAT在转染虫体中得到表达，各质粒中启动子的表达强度表现出预期的明显差异，说明瞬时转染P.f获得成功，为进一步研究P.f的基因表达调控和基因功能奠定了基础。 第四军医大学硕士学位论文 摘 要 为分析 GBP乃0基因了近端侧翼序列的调控功能，以质粒 pGBPCAT A 2为模板，应用 PCR扩增 GBP130 5’侧翼序列转录起始点上游 615hp序列（含转录起始点），克隆入报告基因CAT上游，即将该基因5’近端片段完全删除，构建该片段缺夫的质粒 p＊BP a 2／615。将 pGBPCAT s 2和 pGBP a 2／615分别转染V环状体，比较两者中报告基因CAT的表达水平，从而初步分析近端序列对基因表达的影响。 为进一步分析近端侧翼序列中可能的特异调控元件，应用PCR分别扩增了侧翼序列近端大小分别为400hp和800hp的2个片段，即F。0和F。…经内切酶消化后分别插入 pGBP A 2／6中相应的限制性位点，即可删除近端序列中部分片段，构建成 2个缺失体，扯泪P A 2／400和 P阳P A 2用00。 用 pGBPCAT凸 2、pGBP凸 2／400和 pGBP凸 2／800分别转染疟原虫，在转染后48h收集虫体制备细胞抽提物，检测报告分子CAT活性，分析近端侧翼序列不同区域的强度调控功能。另将 pGBP A 2／400和 pGBP A 2沿00分别同时转染疟原虫，分别于转染后 sh（sh卜 15hp和 46hp收集虫体，制备细胞抽提物，检测CAT活性，分析近端侧翼序列不同区域的期特异性调控功能。 结果表明，pGBP A 2店 转染的疟原虫表达报告基因 CAT的水平下降，与 pGBPCAT山2转染的疟原虫相比有显著性差异汐功刀5卜说明近端侧翼序列在GBPj30基因的表达调控中具有非常重要的作用，可能含有重要的调控元件，包括强度和期特异性调控元件。 当从转录起始点厂游删除较小的片段一GBP A 2沿00卜 CAT表达水平与pGBPCAT A 2中 CAT水平没有差异（Pt）．05），当删除近端较大片段…GBP A2叶00）时，＊AT表达水平明显下降炉刃．OS）。但序列分析发现，被删除序列中与已知可能的疟原虫调控序列没有同源性，据此推测这种启动子活性强度的差异是因为5’UTR长度的不同，较长的UTR可促进基因的转录表达。同时pGBP凸 2／400的转录活性与对照组也有明显差异厂＜0．05），可能 GBPj30基因近端侧翼序列中2个同聚的…pp：瞩结构在强度调控中也具有作用。 pGBP A 2八00与 pGBP A 2／800相比，在 5 hP时前者转录活性高于后者，但在 15 hpt和 46 hp时，后者转录活性高于前者，提示 2种质粒在疟原虫不同生活阶段具有期特异调控特性。pGBP凸 2／400与 pGBP A 2用00中含有相同的二个同聚…叭吐A：dT）序列，但侧翼序列中SAI－－－－－的长度不同。说明侧翼序列中5”U’mN的长度在GBP130基因的期特异性调控中也具有重要作用，即含有较短了UTIR的启动子主要在环状体期具有增强转录的活性，而较长的5”UTR主要在滋养体期和裂殖体期促进启动子的转录活性。gb火p甘T）结构是否具有期特异调控特性还不清楚。更多还原

【Abstract】 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更多还原