【作者】 郭秀军；

【导师】 廖万清； 任大明；

【作者基本信息】 第二军医大学 ， 皮肤病与性病学， 2003， 博士

【摘要】 根据新生隐球菌多聚糖荚膜成分和生化方面的差异，人们将新生隐球菌分成3个变种，5个血清型，其中新生变种(血清型A、D和AD)存在世界范围的土壤和鸟粪中，与免疫力低下的患者，尤其是爱滋病患者的隐球菌病有相关性，而格特变种(血清型B和C)和上海变种(血清型B)，严格局限于热带和亚热带地区，通常引发免疫正常人的隐球菌病，因此研究新生隐球菌不同的血清型与致病性之间的关系是重要的流行病学研究课题。临床上主要用免疫学方法检测抗新生隐球菌荚膜多聚糖的抗体，对其进行血清分型，但是该法有时对某些临床分离株不能鉴定。尽管已有一些分子生物学的技术补充了免疫学方法的局限性，但是临床上仍然需要新的、较准确的和简便的方法来对新生隐球菌进行血清型分型。 June Kwon-Chung和她的合作者利用新生隐球菌DNA文库互补了一些荚膜缺陷株，已经克隆了4个与荚膜合成有关的荚膜基因(CAP59，CAP64，CAP60和CAP10)，这些荚膜相关基因是新生隐球菌荚膜合成的必需基因，这些荚膜基因在新生隐球菌荚膜生化合成途径中的作用是不明确的，但是多聚糖荚膜成分是新生隐球菌分型的物质基础，因此它们是找到与分型的物质基础最相关和最可能的地方。 目前，CAP基因与新生隐球菌血清型分型之间的研究并不多，仅发现不同血清型新生隐球菌CAP59基因部分序列有差异性。因此我们根据实验的条件和财力，选择新生隐球菌的CAP59和CAP64基因作为研究对象，来探讨这两个基因在检测新生隐球菌病原体和血清型分型中的意义，以期对临床检测新生隐球菌提供有用的分子生物学工具。 前期的研究发现，无荚膜的突变株和定向的荚膜基因缺失株在动物模型中无致病性，当它们用野生型荚膜基因互补后，菌株形成荚膜，在动物模型中恢复毒性作用，这些研究结果在分子水平证实了荚膜在新生隐球菌毒性方面的重要性，但是人们仍然不清楚CAP基因在荚膜合成途径中的作用。荚膜基因产物的定位研究对探索这一难题是必不可少的步骤，已有的相关研究表明，用常规免疫组织化学的方法定位新生隐球菌CAP基因产物是非常困难的。 绿色荧光蛋白(GFP)在许多生物蛋白质定位研究中已经得到较多的运用，由于不需要固定、洗涤、封闭和染色的步骤，可以直接观察单个活细胞内的基因h 丫K人TWD一沦大 小UN业大达；；1”9年，附；；；；。gw等首次将GH’用于新生隐球菌M！‘m产’物的定位研究，山1技术原山仅仅观察到C川‘则基因产物的细胞位铁t。本课地价内外一十mV姚村深入研究新’＊刘求菌C川‘明和m隅4基因的功能，并将〔TPJ刹。叶为报法0引小爿入以生隐球13i的美）剧变区【产物的定位研究中。实验方祛1．新’＊划求的灭血利1x从wC川‘64的克隆和川临测病原体力八川的忠义：根州调卜I；隐J求菌（咖消）U D）CAP64美映基因的序列设计众多引物则委引物一 如L ＜j。nB。uk进计比对，代才叫恃V州山受好的引物进行研究和验j小。随后将设川小引物）／75个惊彻m．沽W新生踢球菌，已分型和未分型新生隐J求首临床分或采，以及其‘面的致他真菌，以、J找引物的型特异性。2．动广＊组体内划供材1）：某冈（二川。59最大夕卜显厂的序列分析人山。lh测y．如原件人M的思义：分析C川。59 gi肥缺陷株C八＊”基因最大外星于的序列’／（来源村林科厂厂踢球跑卜3以）【相应序列的差别，以期发现该／斗歹小卜一C川‘切从的功能下门工的你从·处分迎过分析5个标准则D．清型12f株C川’59）i引人】的最人外Z．又广J、列‘，j厂’刊11山＄’U新’H辙J求以i（：AI’个）j川I；的最大外显了个歹帅勺差别，来探索利）－I－I（川’19肚I）、I见人外以一厂序列卜＿分5个M人与w新生隐球IYi分于生物学，七MIf卜＞补肝川。隐珠他的小卜系进化大系，升洲小它在】；而风L可行们上 对2（）株新生踢球的叩；此分。幼林！X从几它们而补为进行C八厂59最人爿、显于序歹山沟扩l曾，来研究C川’59的最大外枯广h：斯十踢球的价测病以休人m川勺思义。3＠新生＊田水仙＊E＊1〕59取IC川）64荚＊ 缺；为株＊＊忍＊又变株的筛选河1鉴定三通工 仆夕上人W纸组】支术，川大多数地母都敏感的＊418基区It雨入到新个隐〕求1为CAI。天以州处僻缺陷株1二＊bU＊AS冶】月的特定序列，用电转化将＿上述的。公巨片段V入处刀切决地休，。·Jilt…N人得具有＊418抗上＊勺ur的突变株。考虑到新1隐珠峋认同的卜，］测地八个什、K低，找＊仆们叫月破除二c酯化学诱变分J，对新’＊隐珠的CAI巧9、＊AI‘64川 CAP10灭j份ode除机i为变。4．h把荧光蛋I0］，P从国和（人PJk因融合农达载体的初不构让：以忖化Jc门纠。明们V的如梭J粒pC州旧们三为分寸土日 将＊＊＊64丛囚九Cr〕人二】人！从北1二附 卜人；为纤HKAS．1．引人M则不《厂GAI。7们勾的启动于之卜，构迷了一系列的门】上周U个J则V．实验结果 ．2－“二十队人。了M广沦义 小义伯叱】．（二＊％4从同在检测新生隐球菌病原体更多还原

【Abstract】 BackgroundOn the basis of the antigenic composition of its polysaccharicle and biologicaldifferences,Cryptococcus neoformans has been subdivided into three varieties and five serotypes. C. neoformans var.neoformans(serotype A ,serbtype D and serotype AD) is prevalent worldwide in association with soil and avian excreta,and generally responsible for cryptococcosis in the setting of immune compromise,particularly in AIDS patients.By contrast,C.neoformans var.gattii(serotype B and C), and C. neoformans Var,S8012(serotype B) are geographically restricted to tropical or sub-tropical climates,associated with eucalyptus trees and usually cause infection in immunocompetent individiuals.The association of various cryptococcal serotypes with palhogenesis of cryptococcosis has been identified as an important epidemiological research. The serotyping of C.neoformans has been performed, immunologically with antisera against the mucopolysaccharide capsule component of this yeast.However,there are some limitions in serotyping,since noncapsulated mutants and nontypeable isolates were not idetntified by this method.Although some molecular serotyping methods haved been used to compensate for the limitions of the imnumological method, some new,easier and more reliable approaches are still needed in clinical serotyping study of C.neoformans isolates.By complementing serval noncapsulated mutants with cryptococcal genomic DNA libraries, June Kwon-Chung and her co-workers have cloned four capsule-related gcnes(CAP59, CAP64, CAP60 and CAP 10 gene) required for normal capsule synthesis.Although the role of CAPgenes in the capsular polysaccharide biosynthetic pathway remains unknown,but we have known that the major capsular polysaccharide,gluciironoxylomannan(GXM),is the basis for defining the serotypes of C.neoformans,so CAP genes are the most related and possible places where we can find the material basis of serotyping.The relationship of CAPgenes and serotypes of C.neoformans has been relatively neglected.The previous study showed that there are serospecific differences amomg the nucleotide sequences of CAP59 genes from five serotypes ofC.neofonnans.According to our laboratorial condtions and financial support,we chose C’AP59 gene and CAP64 gene to investigate their important roles in detecting and serotyping of C.neoformans as a useful molecular tool.The previous studies demonstrated that acapsular isolates and mutants created by particular CAP gene deletion were avirulent in animal models.But when the capsule-deficient phenotype was complementde by CAPgene of C.neoformans wild-type isolate,they restored the capsule and became virulent.These studies provided molecular evidences that capsule is a virulence factor. The role of CAPgenes in the capsular polysaccharide biosynthetic pathway is not clear.The study on the location CAP genes products is an essential approach to resolve the problem. Difficulities in using immunological histochemical methods to locate CAP genes products in C.neoformans have been encountered.The green -fluorescent protein(GFP) from jellyfish Aequorea victoriae has been emerged as a useful marker in studying protein location in a variety of organisms.Direct visualization of gene expression in individual cells is therefore possible without distortion cauded by fixation,washing,sectioning and staining.In 1999,Chang YC firstly used GFP as a reporter in stud ing location of CA10 gene products.Because of technical problem,they only observed the place where CAP 10 gene produts located.The another objective of our research is to further study I he functions of CAP59 gene and CAP64 gene,and still use GFP as a reporter in studing the location of the two CAP genes’ products. Methods1.The cloning of CAP64 gene of C.neoformans and its importance of detecting C.neoformansServal primers were designed according to CAP64 gene of serotype D C.neoformans.These primers were analysed one by one in GenBank and the most specific primers were selected.Then the primers were synthesized to amply fragment to更多还原