新生隐球菌荚膜相关基因的实验研究
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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
Background
On the basis of the antigenic composition of its polysaccharicle and biological
differences,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 meth
od.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 of
C.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. Methods
1.The cloning of CAP64 gene of C.neoformans and its importance of detecting C.neoformans
Serval 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
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
Background
On the basis of the antigenic composition of its polysaccharicle and biological
differences,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 meth
od.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 of
C.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. Methods
1.The cloning of CAP64 gene of C.neoformans and its importance of detecting C.neoformans
Serval 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
引文
1． Sakaguchi N,Baba T,Fukuzawam,etal. Ultrastructural study of Cryptococcus neoformans by quick-freezing and deep-etching method.Mycopathologia.1993, 121(1) : 133-141．
2． Spoljar BH,and Tomasic J. A novel ELISA for determination of polysaccharide specific immunoglobulins. Vaccine,2000, 22:19(7-8) :924-30．
3． Buchanan KL and Murphy JW. What makes Cryptococcus neoformans a pathogen? Emerg. Infect. Dis,1998,4(1) :71-83．
4． Feldmesser M, Kress Y, Novikoff P, etal. Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection.Infect Immun,2000, 68(7) :4225-4237．
5． Casadevall A. Characterization of a murine monoclonal antibody to Cryptococcus neoformans polysaccharide that is a candidate for human therapeutic studies. Antimicrob Agents Chemother,1998, 42(6) : 1437-144．
6． Chang YC, and Kwon-Chung KJ. Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol,1994, 14(7) :4912-4919．
7． Chang YC, Penoyer LA and Kwon-Chung KJ.The second capsule gene of Cryptococcus neoformans, CAP64, is essential for virulence.Infect Immun.,1996, 64(6) : 1977-1983．
8． Chang YC and Kwon-Chung KJ. Isolation of the third capsule-associated gene, CAP60, required for virulence in Cryptococcus neoformans. Infect Immun,1998,66(5) : 2230-2236．
9． Chang YC and Kwon-Chung KJ. Isolation, characterization and localization of a capsule-associated gene,CAP10, of Cryptococcus neoformans. J BacteriolJ 999, 181(18) :5636-5643．
10． Yuka N,Rui K,Shinichi W,etal.Molecular analysis of CAP59 gene sequence from five serotypes of Cryptococcus neoformans.J Clin Microbiol,2000,38(3) :992-995．
11． Maurizio DP,Dena LT,Thomas HR,etal.Cryptococcus neoformans differential gene expression detected in vitro and in vivo with green fluorescent protein.Infect
Immun, 1999,67(4) : 1812-1820．
12． 1． Mitchell TG and Perfect JR. Cryptococcosis in the era of AIDS-100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev, 1995, 8(4) : 515-548．
13． Liao WQ,Shao JZ,Wu SX,etal. Cryptococcus Neoformans Var,S8012 caused meningitis.Clin Med J,1983,96(4) :287-290．
14． Sorrell TC, Chen SC,Ruma P,etal.Condordance of clinical and environmental isolates of Cryptococcus neoformans var.gatti by random amplification of polymorphorphic DNA analysis and PCR fingerprinting.J Clin Microbiol, 1996,34(6) : 1253-1260．
15． Wu JH and Liao WQ.Isolation of specific DNA probes for detection of Cryptococcus neoformans.Mycoses, 1997,40(9,10) :385-389．
16． Cherniak R and Sundstrom JB.Polysaccharide antigens of the capsule of Cryptococcus neoformans.Infect Immun,J 994,62(3) : 1507-1512．
17． Alessandro Bolano,Silvia Stinchi,Roberta Preziosi,etal.Rapid methods to extract DNA and RNA from Cryptococcus neoformans.FEMS Yeast Research.2001,1 (2001) :221-224．
18． Sansinforiano ME, Padilla JA, Hermoso-de-Mendoza J TI,etal. Rapid and easy method to extract and preserve DNA from Cryptococcus neoformans and other pathogenic yeasts. Mycoses. 1998 May-Jun; 41 (5-6) : 195-8
19． Ikeda R,Shinoda T,Fukazawa Y,etal.Antigenic characterization of Cryptococcus neoformans serotypes and its application to serotyping of clinical isolates. J Clin Microbiol, 1982,16(1) :22-29．
20． Liao WQ,Shao JZ,Wu SX,etal. Cryptococcus Neoformans Var,S8012 caused meningitis.Clin Med J,1983,96(4) :287-290．
21． Teresa Barojosep M,Torres R,etal.Serotyping of Cryptococcus neoformans Isolation from Clinical and Enviromental Sources in Spain.J Clin Microbiol, 1999,37(4) : 1170-1172．
22． Polacheck I and Lebens GA.Electrophoretic karyotype of the pathogenic yeast Cryptococcus neoformans.J Gen Microbiol,1989,135(l):67-71．
23． Polacheck I,Lebens GA and Hicks JB. Development of DNA probes for the early diagnosis and epidemiological study of cryptococcosis in AIDS patients.J Clin Microbiol,1992,30(3) :925-930．
24． Currie BP,Freundlich LF and Casadevall A. Restriction fragment length polymorphism analysis of Cryptococcus neoformans isolates from environmental (pigeon excreta) and clinical sources in New York City.J Clin Microbiol,32(4) :1188-1192．
25． Francisco HA,Tamae I,Reiko T,etal.New PCR primer pairs specific for Cryptococcus neoformans serptype A or B prepared on the basis of random amplified polymorphic DNA fingerprint pattern analyses.J Clin Microbiol, 1999,37(2) : 315-320．
26． Dromer F,Mathoulin S,Dupont B etal. Individual and environmental factors associated with infection due to Cryptococcus neoformans serotype D.Clin Infect Dis,1996,23(1) :91-96．
27． Chang YC,Wickes BL and Kwon-Chung KJ.Further analysis of CAP59 locus of Cryptococcus neoformans: structure defined by forced expression and description of a new ribosomal protein encoding gene.Gene,1995,167(l):179-183．
28． Bertout S,Renaud F,Swinne D,etal. Genetic multilocus studies of different strains of Cryptococcus neoformans:Taxonomy and Genetic structure.J Clin Microbiol,1999,37(3) :715-720．
29． Hadfield C,Jordan BE,Mount RC,etal.G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae.Curr Genet,1990,18(l):303-314．
30． A.亚当斯，D.E.戈施林，C.A.凯泽等。酵母遗传学方法实验指南.第1版，刘子铎译，科学出版社，2000,45-60．
31 .Edman J.Isolation of telomerelike sequences from Cryptococcus neoformans and their use in high-efficiency transformatiom.Mol Cell Biol,1992,12(6) :2777-2783．
32． Sambrook J,Fritsch EF,Mamiatis T.et al.分子克隆实验指南.第2版，金冬雁译，北京科学出版社，1998,16-18．
33． Jef D Boeke, Francois La Croute, and Gerald R Fink. A positive selection
for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast:5-fluoro-orotic acid resistance. Mol gen Genet, 1984, 197(2) : 345-346．
34． Edman JC, Kong-Chong KJ, Penoyder LA, etal. Isolation of the ura5 gene from Cryrptococcus neoformans var. neoformans and its use as a selective marker for transformation. Mol Cell Biol, 1990, 10(9) :4538-4544．
35． Mondon P, Chang YC,Varma A,etal.A novel eposomal shuttle vector for transformation of Cryptococcus neoformans with the ccdB gene as a positive seletion marker in bacteria .FEMS Microbiology Letters,2000,187(l):41-45．
36． Toffaletti DL,Rude TH,Johnston SA,etal.Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA.J Bacteriol,1993,175(5) :1405-1411．
37． Chen XJ.Low and high copy number shuttle vector for replication in the budding yeast Kluyeromyces lactis.Gene, 1996,172(1) : 131-136．
38． Heim R, Cubitt AB, Tsien RY, etal.Improved green fluorescence. Nature,1995,373(2) :663-554．
39． Ormo M,Cubitt AB,Kallio K,etal.Crystal structure of the Aequorea Victoria green fluorescent protein.Science,1996,273(5280) :1392-1395．
40． Wickes BL and Edman JC.The Cryptococcus neoformans GAL7 gene and its use as an inducible promoter.Mol Microbiol, 1995,16(60) : 1099-1109．
41． Chang YC,Cherniak R,Kozel TR ,etal.Structure and Biological activities of acapsular Cryptococcus neoformans 602 complemented with the CAP64 gene.Infect Immun,1997,65(5) :1584-1592．
42． Chalfiem T,Tu Y,Euskirchen G,etal.Green fluorescent protein as a marker for gene expression.Science,1994,263(5148) :802-805．
2． Spoljar BH,and Tomasic J. A novel ELISA for determination of polysaccharide specific immunoglobulins. Vaccine,2000, 22:19(7-8) :924-30．
3． Buchanan KL and Murphy JW. What makes Cryptococcus neoformans a pathogen? Emerg. Infect. Dis,1998,4(1) :71-83．
4． Feldmesser M, Kress Y, Novikoff P, etal. Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection.Infect Immun,2000, 68(7) :4225-4237．
5． Casadevall A. Characterization of a murine monoclonal antibody to Cryptococcus neoformans polysaccharide that is a candidate for human therapeutic studies. Antimicrob Agents Chemother,1998, 42(6) : 1437-144．
6． Chang YC, and Kwon-Chung KJ. Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol,1994, 14(7) :4912-4919．
7． Chang YC, Penoyer LA and Kwon-Chung KJ.The second capsule gene of Cryptococcus neoformans, CAP64, is essential for virulence.Infect Immun.,1996, 64(6) : 1977-1983．
8． Chang YC and Kwon-Chung KJ. Isolation of the third capsule-associated gene, CAP60, required for virulence in Cryptococcus neoformans. Infect Immun,1998,66(5) : 2230-2236．
9． Chang YC and Kwon-Chung KJ. Isolation, characterization and localization of a capsule-associated gene,CAP10, of Cryptococcus neoformans. J BacteriolJ 999, 181(18) :5636-5643．
10． Yuka N,Rui K,Shinichi W,etal.Molecular analysis of CAP59 gene sequence from five serotypes of Cryptococcus neoformans.J Clin Microbiol,2000,38(3) :992-995．
11． Maurizio DP,Dena LT,Thomas HR,etal.Cryptococcus neoformans differential gene expression detected in vitro and in vivo with green fluorescent protein.Infect
Immun, 1999,67(4) : 1812-1820．
12． 1． Mitchell TG and Perfect JR. Cryptococcosis in the era of AIDS-100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev, 1995, 8(4) : 515-548．
13． Liao WQ,Shao JZ,Wu SX,etal. Cryptococcus Neoformans Var,S8012 caused meningitis.Clin Med J,1983,96(4) :287-290．
14． Sorrell TC, Chen SC,Ruma P,etal.Condordance of clinical and environmental isolates of Cryptococcus neoformans var.gatti by random amplification of polymorphorphic DNA analysis and PCR fingerprinting.J Clin Microbiol, 1996,34(6) : 1253-1260．
15． Wu JH and Liao WQ.Isolation of specific DNA probes for detection of Cryptococcus neoformans.Mycoses, 1997,40(9,10) :385-389．
16． Cherniak R and Sundstrom JB.Polysaccharide antigens of the capsule of Cryptococcus neoformans.Infect Immun,J 994,62(3) : 1507-1512．
17． Alessandro Bolano,Silvia Stinchi,Roberta Preziosi,etal.Rapid methods to extract DNA and RNA from Cryptococcus neoformans.FEMS Yeast Research.2001,1 (2001) :221-224．
18． Sansinforiano ME, Padilla JA, Hermoso-de-Mendoza J TI,etal. Rapid and easy method to extract and preserve DNA from Cryptococcus neoformans and other pathogenic yeasts. Mycoses. 1998 May-Jun; 41 (5-6) : 195-8
19． Ikeda R,Shinoda T,Fukazawa Y,etal.Antigenic characterization of Cryptococcus neoformans serotypes and its application to serotyping of clinical isolates. J Clin Microbiol, 1982,16(1) :22-29．
20． Liao WQ,Shao JZ,Wu SX,etal. Cryptococcus Neoformans Var,S8012 caused meningitis.Clin Med J,1983,96(4) :287-290．
21． Teresa Barojosep M,Torres R,etal.Serotyping of Cryptococcus neoformans Isolation from Clinical and Enviromental Sources in Spain.J Clin Microbiol, 1999,37(4) : 1170-1172．
22． Polacheck I and Lebens GA.Electrophoretic karyotype of the pathogenic yeast Cryptococcus neoformans.J Gen Microbiol,1989,135(l):67-71．
23． Polacheck I,Lebens GA and Hicks JB. Development of DNA probes for the early diagnosis and epidemiological study of cryptococcosis in AIDS patients.J Clin Microbiol,1992,30(3) :925-930．
24． Currie BP,Freundlich LF and Casadevall A. Restriction fragment length polymorphism analysis of Cryptococcus neoformans isolates from environmental (pigeon excreta) and clinical sources in New York City.J Clin Microbiol,32(4) :1188-1192．
25． Francisco HA,Tamae I,Reiko T,etal.New PCR primer pairs specific for Cryptococcus neoformans serptype A or B prepared on the basis of random amplified polymorphic DNA fingerprint pattern analyses.J Clin Microbiol, 1999,37(2) : 315-320．
26． Dromer F,Mathoulin S,Dupont B etal. Individual and environmental factors associated with infection due to Cryptococcus neoformans serotype D.Clin Infect Dis,1996,23(1) :91-96．
27． Chang YC,Wickes BL and Kwon-Chung KJ.Further analysis of CAP59 locus of Cryptococcus neoformans: structure defined by forced expression and description of a new ribosomal protein encoding gene.Gene,1995,167(l):179-183．
28． Bertout S,Renaud F,Swinne D,etal. Genetic multilocus studies of different strains of Cryptococcus neoformans:Taxonomy and Genetic structure.J Clin Microbiol,1999,37(3) :715-720．
29． Hadfield C,Jordan BE,Mount RC,etal.G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae.Curr Genet,1990,18(l):303-314．
30． A.亚当斯，D.E.戈施林，C.A.凯泽等。酵母遗传学方法实验指南.第1版，刘子铎译，科学出版社，2000,45-60．
31 .Edman J.Isolation of telomerelike sequences from Cryptococcus neoformans and their use in high-efficiency transformatiom.Mol Cell Biol,1992,12(6) :2777-2783．
32． Sambrook J,Fritsch EF,Mamiatis T.et al.分子克隆实验指南.第2版，金冬雁译，北京科学出版社，1998,16-18．
33． Jef D Boeke, Francois La Croute, and Gerald R Fink. A positive selection
for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast:5-fluoro-orotic acid resistance. Mol gen Genet, 1984, 197(2) : 345-346．
34． Edman JC, Kong-Chong KJ, Penoyder LA, etal. Isolation of the ura5 gene from Cryrptococcus neoformans var. neoformans and its use as a selective marker for transformation. Mol Cell Biol, 1990, 10(9) :4538-4544．
35． Mondon P, Chang YC,Varma A,etal.A novel eposomal shuttle vector for transformation of Cryptococcus neoformans with the ccdB gene as a positive seletion marker in bacteria .FEMS Microbiology Letters,2000,187(l):41-45．
36． Toffaletti DL,Rude TH,Johnston SA,etal.Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA.J Bacteriol,1993,175(5) :1405-1411．
37． Chen XJ.Low and high copy number shuttle vector for replication in the budding yeast Kluyeromyces lactis.Gene, 1996,172(1) : 131-136．
38． Heim R, Cubitt AB, Tsien RY, etal.Improved green fluorescence. Nature,1995,373(2) :663-554．
39． Ormo M,Cubitt AB,Kallio K,etal.Crystal structure of the Aequorea Victoria green fluorescent protein.Science,1996,273(5280) :1392-1395．
40． Wickes BL and Edman JC.The Cryptococcus neoformans GAL7 gene and its use as an inducible promoter.Mol Microbiol, 1995,16(60) : 1099-1109．
41． Chang YC,Cherniak R,Kozel TR ,etal.Structure and Biological activities of acapsular Cryptococcus neoformans 602 complemented with the CAP64 gene.Infect Immun,1997,65(5) :1584-1592．
42． Chalfiem T,Tu Y,Euskirchen G,etal.Green fluorescent protein as a marker for gene expression.Science,1994,263(5148) :802-805．