【作者】 方强；

【导师】 高琪；

【作者基本信息】 苏州大学 ， 病原生物学， 2010， 博士

【摘要】 研究背景:疟疾是一种严重危害人类健康的虫媒传染病,也是一个严重影响我国人民健康的重要问题。间日疟虽然病死率低于恶性疟,但流行范围更为广泛,我国的疟疾流行亦以间日疟为主。而且,间日疟的危害一直被低估,相应的研究也落后于恶性疟,有必要加强间日疟防治的研究。疟疾诊断是疟疾防治工作中的一个重要环节,现有的病原学和分子生物学的诊断方法已不适应疟疾现场防治的需要。以检测疟原虫特异性抗原为诊断靶标的快速免疫诊断方法已在疟疾现场防治中得以应用并取得良好效果。但现有的疟疾快速免疫诊断方法尚不能特异性诊断间日疟,间日疟的现场防治中亟需间日疟特异性快速免疫诊断方法。建立cDNA文库,筛选调取功能性基因是一种成熟的发现功能基因(包括诊断靶标、疫苗候选分子等)的策略,目前尚无高质量的我国间日疟原虫红内期全长cDNA文库,因此,建立高质量的我国间日疟原虫红内期全长cDNA文库对筛选调取包括间日疟特异性诊断靶标在内的功能基因,促进我国疟疾防治工作具有重要意义。谷氨酸脱氢酶(GDH)为疟原虫糖酵解中的关键酶,是一个潜在的诊断靶标,已建立的恶性疟原虫谷氨酸脱氢酶(PfGDH)快速免疫诊断方法已显示了较满意的效果。但间日疟原虫谷氨酸脱氢酶(PvGDH)尚未见报道。因此,克隆表达PvGDH,并制备抗PvGDH特异性单抗,可为进一步研制以PvGDH为诊断靶标的间日疟原虫特异性快速免疫诊断试剂奠定基础,从而为间日疟防治提供有力工具。第一部分红内期间日疟原虫全长cDNA文库的构建与鉴定目的:构建中国红内期间日疟原虫全长cDNA文库。方法:自未经治疗的确诊间日疟患者静脉采血,滤过去除白细胞后浓集含虫红细胞,提取总RNA,RT-PCR法逆转录合成cDNA第一链,LD-PCR合成cDNA第二链及双链cDNA,经蛋白酶K消化、Sfi I酶切、CHROMA SPIN-400柱分离去除小于400 bp的片段后CHROMA SPIN-400柱分离去除小于400 bp的片段后,将cDNA与已经Sfi I酶切的质粒载体连接,转化入DH5α感受态细胞。测定文库的库容和重组率。随机挑选48个菌落,以M13+-引物进行PCR,鉴定插入cDNA片段大小。结果:构建的中国红内期间日疟原虫全长cDNA文库原始库容为1.14×106,重组率为97.2%。重组子插入cDNA片段大小为900~2500bp之间。结论:成功构建了中国红内期间日疟原虫全长cDNA文库。第二部分间日疟原虫谷氨酸脱氢酶基因的克隆表达目的:克隆表达间日疟原虫谷氨酸脱氢酶。方法:根据推测的PvGDH编码基因设计特异性引物,采用PCR自构建的中国红内期间日疟原虫全长cDNA文库扩增PvGDH编码基因,将其克隆至pMD18-T载体,经PCR和双酶切鉴定后进行序列测定后,亚克隆至pCOLDⅡ载体进行表达。通过ELISA和Western-blot检测正常人血清、间日疟患者血清、恶性疟患者血清与rPvGDH的免疫反应性;以表达的rPvGDH免疫小鼠制备抗血清,通过ELISA检测抗血清与rPvGDH的免疫反应性;并以Western-blot检测抗血清与间日疟原虫抗原、恶性疟原虫抗原、正常红红细胞抗原的免疫反应性。结果:PCR产物电泳结果显示所克隆的基因约为1450bp,基因测序结果显示克隆的基因为1442bp,与GenBank间日疟原虫Sal-I株基因组测序预测PvGDH编码基因有3个碱基差异,但编码氨基酸无差异,无内含子。经原核表达获得可溶性的PvGDH重组蛋白(rPvGDH),可为间日疟患者血清和恶性疟患者血清所识别,而不能被正常人血清识别。制备的PvGDH抗血清可以识别rPvGDH、间日疟原虫抗原、恶性疟原虫抗原,而不能识别正常红细胞抗原。结论:成功克隆了PvGDH,并表达了具有免疫学活性的rPvGDH。第三部分间日疟原虫谷氨酸脱氢酶线性B细胞表位的预测与鉴定目的:预测并鉴定PvGDH线性B细胞表位。方法:利用生物信息软件分析PvGDH、PfGDH序列,预测PvGDH、PfGDH线性B细胞表位,并比较分析,预测特异性PvGDH线性B细胞表位;合成预测PvGDH线性B细胞表位肽,免疫小鼠,制备抗血清;通过ELISA和Western-blot检测抗血清与rPvGDH的免疫反应性;通过Western-blot检测抗血清与与间日疟原虫抗原、恶性疟原虫抗原、正常红红细胞抗原的免疫反应性。结果:预测出1个特异性PvGDH线性B细胞表位;该预测表位肽抗血清可以识别rPvGDH和间日疟原虫抗原,而不能识别恶性疟原虫抗原和正常红细胞抗原。结论:预测并鉴定成功1个特异性PvGDH线性B细胞表位。第四部分抗PvGDH单克隆抗体研究目的:分别通过rPvGDH和PvGDH预测表位肽制备抗PvGDH单抗。方法:分别利用rPvGDH和PvGDH预测表位肽作为免疫原免疫小鼠,制备抗PvGDH单克隆抗体,并检测制备的单抗的滴度和抗体亚类;通过ELISA和Western-blot检测制备的单抗与rPvGDH的免疫反应性。结果:通过rPvGDH制备了4株单抗,通过PvGDH预测表位肽制备6株单抗,抗体滴度均大于1:2430;通过PvGDH预测表位肽制备的单抗中有2株抗体亚类为IgG1,其余均为IgG2b。10株单抗均可有效识别rPvGDH。结论:成功地通过rPvGDH和PvGDH预测表位肽2种方法制备得到10株抗PvGDH单抗。更多还原

【Abstract】 Background: Malaria still remain one of most serious vector-born diseases, it’s one of most important disease threat to health in China as well. Plasmodium vivax is the most wide-distributed of human malaria parasites, and contributed as major species of malaria parasite in China, although it’s not as severe as another Plasmodium falciparum. The burden of vivax malaria is always under-estimated, and the research about vivax was neglected comparing with falciparum. Diagnosis is essential for malaria control, however, the malaria diagnostic technique is still lack with the demand of disease control. The rapid diagnosis test based on specific Plasmodium antigen detection was applied in field and showing promising results. But most of them can not specific detect Plasmodium vivax, the vivax specific RDT is urgent needed. To address it, a full length cDNA library of erythrocytic stage Plasmodium vivax was constructed for vivax specific diagnostic antigen screening.Glutamate dehydrogenase (GDH) is an essential enzyme for glycolysis in Plasmodium, is a potential diagnostic target as well, the established immunology diagnosis test based on Plasmodium falciparum glutamate dehydrogenase (PfGDH) shows satisfactory results, however, no report revealed about Plasmodium vivax glutamate dehydrogenase (PvGDH) so far. Therefore, the PvGDH was cloned and expressed, and monoclonal antibody against recombinant PvGDH was raised for further Plasmodium vivax specific diagnosis test development.PART 1 Construction and Identification of Full-length cDNA Library of Erythrocytic Stage Plasmodium vivaxObjective: To construct a full-length cDNA library of erythrocytic stage Plasmodium vivax of China isolation.Methods: The blood was collected from confirmed vivax malaria patients before treatment, and WBCs were removed using Plasmodipur filter, and Plasmodium vivax infected RBCs were enriched by Porcoll gradient purification. Total RNA was extracted using TRIZOL reagent,first-stranded cDNA was synthesize by reverse transcription polymerase chain reaction ( RT-PCR ) method. Then, the second-stranded cDNA and double-stranded cDNA was synthesized using long distance polymerase chain reaction (LD-PCR) method. The ds-cDNAs were digested by proteinase K and Sfi I restriction enzyme, to ensure the length of inserts, fragments less than 400 bp were separated by CHROMA SPIN-400 column, meanwhile the cDNA was ligated to the Sfi I digested vector and transformed into E. Coli DH5αcompetent cells. Then the titration and the recombinant rate of the cDNA library were evaluated, and 48 colonies were choose randomly and identified the size of the inserted cDNA using PCR with M13+-primers.Results: The full-length cDNA library was constructed, with 1.14×106 independent clones, a recombinant rate of 97.2%. The lengths of the inserted cDNA fragments ranged from 900 to 2500bp.Conclusion: A high-quality full-length cDNA library of erythrocytic stage Plasmodium vivax of China isolation was successfully constructed.PART 2 Cloning and Expression of Plasmodium vivax Glutamate DehydrogenaseObjective: To clone and express Plasmodium vivax Glutamate Dehydrogenase (PvGDH)Methods: The primers were designed based on putative Plasmodium vivax Glutamate Dehydrogenase gene, then PvGDH coding sequence was amplified from emplicationcon was the full-length cDNA library of erythrocytic stage Plasmodium vivax, and inserted into pMD18-T vector. The sequence was confirmed by sequencing after identification using PCR and restriction enzyme digestion, then sub-cloned into pCOLD II vector for expression. The immunoreactivity of the serum from vivax malaria patients , falciparum malaria patients and health volunteers to recombinant PvGDH (rPvGDH) was tested using ELISA and Western-blot. The serum was collect from mice after immunization using rPvGDH, and its immunoreactivity to rPvGDH was tested using ELISA, as well as the immunoreactivity to antigen of Plasmodium vivax, Plasmodium falciparum, and normal human RBC was tested using Western-blot.Results: A 1442 bp gene was amplified by PCR, and cloned into vector, the sequence has 3 bp difference with the coding sequence of PvGDH from sal-I strain genomic database, but without encoding amino acid changing. The recombinant PvGDH was expressed as soluble form, and could be recognized by the serum from vivax malaria patients or falciparum malaria patients , but not be recognized by the serum from health volunteers. The serum was collect from mice after immunization using rPvGDH could recognize rPvGDH, both antigen of Plasmodium vivax and Plasmodium falciparum, but not normal human RBC.Conclusion: PvGDH was successful cloned and expressed with immunogenicity.PART 3 Prediction and Identification of B Cell Epitope of Plasmodium vivax Glutamate DehydrogenaseObjective: To predict and identify B cell epitope of Plasmodium vivax Glutamate Dehydrogenase (PvGDH)Methods: The B cell epitope of PvGDH was predicted through the sequences analysis and comparing of PvGDH and PfGDH using bioinformatics software. The peptide was synthesized according to the predicted linear epitope, and for raising anti-serum by mice immunization. The immunoreactivity of serum to rPvGDH was tested using ELISA and Western-blot, as well as the immunoreactivity to antigen of Plasmodium vivax, Plasmodium falciparum, and normal human RBC.Results: A specific linear B cell epitope of PvGDH was predicted, and the serum against the peptide could recognize rPvGDG and antigen of Plasmodium vivax, but either Plasmodium falciparum or normal human RBC.Conclusion: A specific linear B cell epitope of PvGDH was successful predicted and confirmed.PART 4 Study of Monoclonal Antibody against Plasmodium vivax Glutamate DehydrogenaseObjective: To produce monoclonal antibodies against PvGDH using recombinant PvGDH and predicted PvGDH B cell epitope.Methods: Monoclonal antibodies against PvGDH were produced using recombinant PvGDH and predicted PvGDH B cell epitope for mice immunization. The titer and subtype of monoclonal antibodies were tested, the immunoreactivity of monoclonal antibodies to rPvGDH was tested using ELISA and Western-blot.Results: 4 and 6 monoclonal antibodies were produced respectively using rPvGDH, and predicted PvGDH B cell epitope. All of them can specifically recognized rPvGDH with titer higher than 1: 2430 Out of total 10, 2 monoclonal antibodies rose using predicted PvGDH B cell epitope are IgG1, others all are IgG2b.Conclusion: 10 monoclonal antibodies against PvGDH were successfully produced using rPvGDH and predicted PvGDH B cell epitope.更多还原