| 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...
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