白念珠菌论文：白念珠菌 苹果酸脱氢酶 乳酸脱氢酶抗体 交叉免疫 耐药 蛋白.doc

【关键词】白念珠菌 苹果酸脱氢酶 乳酸脱氢酶抗体 交叉免疫 耐药 蛋白【英文关键词】Candida albicans malate dehydrogenase （MDH） lactatedehydrogenase(LDH) Cross-reactive immune response antifungalresistance protein白念珠菌论文：白念珠菌苹果酸脱氢酶的结构及功能初步研究【中文摘要】随着艾滋病、糖尿病等疾病的发病率不断上升，器官移植及导管技术的推广普及，临床上免疫受损患者日益增多，念珠菌系统感染发病率大幅上升。临床使用抗真菌药物的频率和剂量有明显增加导致真菌耐药情况变得越来越严重，尤其对唑类药物的耐受，给有效控制日益增高的念珠菌系统感染带来严重困难，也使白念珠菌耐药发生影响因素研究具有重大意义。耐药性的研究中发现，白念珠菌对葡萄糖含量极之敏感，培养基中0.001的葡萄糖浓度改变，都会引起菌株调节代谢的核心基因发生改变，对唑类药物的耐药性增加。而白念珠菌在巨噬细胞内以乙醛酸循环功能是该菌引起感染的关键。宿主巨噬细胞是一个葡萄糖，氨基酸等营养缺乏的微环境，微生物无法利用三羧酸循环进行物质和能量代谢。巨噬细胞内白念珠菌调节氧化、乙醛酸循环、糖异生等代谢途径的各个关键酶基因转录水平均明显上调，而糖酵解的关键酶则呈下调趋势。因此我们推测，白念珠菌糖酵解升高，乙醛酸循环下降，这一能量代谢优势途径的转变可能与真菌耐药性存在一定的关系。本研究通过观察MDH在耐伊曲康唑白念珠菌株与非耐药株中表达的差异，探讨白念珠菌MDH11p表达水平与唑类耐药存在关系。通过对白念珠菌MDH交叉免疫反应筛选其高度保守表位结构，为探讨白念珠菌新的药物作用靶点和免疫特异诊断抗体提供理论基础。实验探讨白念珠菌MDH11p表达水平与唑类耐药存在关系；确定LDH/MDH同源序列存在高度保守的表位结构。实验方法：1CaMDH基因的全长cDNA序列生物信息学分析利用NCBI、ExPASy和DNAstar等生物信息学软件包分析CaMDH氨基酸序列，预测蛋白的结构与功能；并与常见病原微生物的同源氨基酸序列进行序列比对，构建进化树。2白念珠菌药物敏感试验及ELISA检测MDH的表达水平初步药敏筛选白念珠菌临床分离株30株，以M27A微量法药敏试验重复鉴定伊曲康唑耐药株、中介株和敏感株各10株，应用ELISA检测比较CaMDH的表达水平，统计采用方差分析,P0.05具有统计学意义。3CaMDH的识别及与同源序列抗血清交叉免疫反应白念珠菌总蛋白与SjLDH多克隆免疫血清反应作Western印迹试验。实验结果：CaMDH与其他物种同源氨基酸序列一致性可达43，比对的同源序列中SjLDH一致性最低18.2%。CaMDH具有乳酸脱氢酶保守区域，8个主要的B细胞表位，苹果酸脱氢酶活化点包含于NAD(P)结合部分中。M27一A微量法药敏试验结果重复鉴定30株临床白念珠菌药敏MIC值，结果同前伊曲康唑耐药株、中介株和敏感株各10株。ELISA检测耐药株与LDH抗体相对结合量为348.714334.3594,中介株与LDH抗体相对结合量为327.000085.5990，敏感株与LDH抗体相对结合量为443.428635.6363.（F=8.516,P0.003）。从氨基酸序列分析CaMDH与SjLDH有三处表位相似，可发生交叉免疫反应，CaMDH与SjLDH多克隆免疫血清Westernblot结果阳性。实验结论：生物信息学分析白念珠菌MDH蛋白的结构与功能与进化较高级生物的LDH蛋白更接近。白念珠菌耐药株MDH蛋白表达量较敏感菌株低，提示白念珠菌MDH表达水平与耐药存在一定关系。白念珠菌MDH与同源序列LDH抗血清发生交叉免疫结合，识别结合位点为LDH/MDH同源序列高度保守的抗原表位决定簇。【英文摘要】BackgroundWith the wide use of antibiotics, antitumor agent and glucocorticoid,especially in transplant patients or AIDS patients,the infections causedby fungus has dramatically increased.Candida albicans is the most commonhuman opportunistic pathogenic fungus and the most significant pathogeneof nosocomial infection.The report of resistance to azole antifungalagents became increased,it was severely challenge to control the fungusinfections effectively.The study of azole antifungal resistancemechanism and the development of new antifungal drug or vaccine areeffective way to solve problem.In a study of antifungal resistance mechanism,the Saccharomycescerevisiae, C.albicans is exquisitely sensitive to glucose,regulatingcentral metabolic genes even in response to0.01%glucose, increased theresistance to an azole antifungal agent.C.albicans is an intracellularpathogen which causes disseminated,progressive and life threateninginfections.During infection,C.albicans in macrophages is considered tobe in a poor environment in nutrients,which cant employed tricarboxylicacid cycle carbon metabolism to maintain survival.In systematic screeningof differentially expressed proteins in an infection model withmacrophages during the infection process in candida albicans, theencoding gene of isocitrate lyase is up-regulated, but those forglycolysis is down-regulation.The glyoxylate was energy consumptionrequired for persisting in macrophages. Therefore, assessment of in candida albicans carbon metabolism shunt asglycolysis upregulation and glyoxylate down-regulation at the same timemay be related with the occurence of itraconazole resistance.Exploringthe differential of MDH expression between the Itraconazole resistancegroup and sensitive group in Candida albicans,to asses the relationbetween the glyoxylate key enzyme regulation and the occurence of theItraconazole resistance.The bioinformatics analysis predict a potentialdiagnosis and drug target domain. The positiv result with the anti-LDHbody identify the interaction point was highly conserved epitopes of CaMDHhomologous sequence and supply new methods for the vaccine, diagnosis anddrug target study of the CaMDH.：Asses the relation between the glyoxylate key enzyme regulation and theoccurence of the Itraconazole resistance.Identify the interaction pointwas highly conserved epitopes of CaMDH homologous sequence and supply newmethods for the vaccine, diagnosis and drug target study of the CaMDH.Methods:1.Bioinformatics analysis the cDNA sequence of CaMDHThe structure, properties and function about encoding protein of malatedehydrogenase from candida albicans were analyzed and predicted bybioinformatics tool as NCBI、ExPASy and DNAstar in this study.CaMDHsequence with homologous sequence from other species of pathogenicmicroorganism are compared in phylogenetic analysis.2.Screening the Itraconazole-resistant and Itraconazole-sensitiveC.albicans strains and ELISA to measure the MDH expression.Select30strains including ten Itraconazole resistant strains, ten intermediary strains and ten sensitive strains,respectively measure andcompare the expression level of3groups by ELISA. Data was analyzed byanalysis of variance,p0.05was statistically significant for thedifference.3.Identify CaMDH protein and Westernblot analysis with the homologoussequence recombinant protein immunne serum.Extracted the whole intracellular proteins of5strains Candiada albicansby P0013B RIPA lysis buffer,Westernblot analysis with the SjLDHrecombinant protein immunne serum.Results:Analysis show the amino acid sequence of schistosoma japomicum lacticdehydrogenase (SjLDH) had lowest identiy(18.2%) with CaMDH than that ofother pathogenic microorganism species(43%).CaMDH contain LDH conserveddomain and7main B cell epitopes. The MDH active site located at the NAD(P)binding domain.Determination of the MIC values of30candida albicans strains by the brothmicrodilution assay as NCCLS document M27-A described.The result of ELISA show combination with anti-LDH antibody was348.714334.3594in resistant Candiada albicans isolates, significantly lowerthan that in sensitive isolates(443.428635.6363) and the mediumisolates was327.000085.5990（F=8.516,P0.003）。CaMDH contain3epitopes display high levels of similarity with that ofSjLDH, and the Westernblot analysis with the SjLDH recombinant proteinimmunne serum show positive result.Conclusions