新生隐球菌ISC10基因的cDNA克隆及序列分析  

Molecular Cloning and Sequence Analysis of Cryptococcus Neoformans Meiosis-specific Protein Required for Spore Formation(ISC10)

作　　者：赵卓[1] 潘炜华[1] 卫立辛[2] 廖万清[1] 

机构地区：[1]第二军医大学附属长征医院皮肤科,上海200003 [2]第二军医大学东方肝胆外科医院肿瘤免疫与基因治疗研究中心,上海200433

出　　处：《中国皮肤性病学杂志》2008年第6期325-327,共3页The Chinese Journal of Dermatovenereology

基　　金：国家自然科学基金资助项目(编号30771927,No30370082);长征医院“三重三优”学科人才建设计划基金资助

摘　　要：目的克隆新生隐球菌ISC10基因(Meiosis-specific protein required for spore formation)的全长cDNA序列,并进行生物信息学分析。方法从新生隐球菌B3501菌株中分离提取总RNA,逆转录成cDNA,运用逆转录-聚合酶链反应(RT-PCR)技术扩增获得新生隐球菌ISC10基因,构建pGM-T/ISC10重组载体,测序后与GenBank中ISC10基因(DQ332212)进行同源性比较和序列分析。结果所克隆的基因共编码267个氨基酸,分子量为31.65KD,与GenBank中ISC10基因(DQ332212)序列同源性达99.10%,编码的蛋白质在67位氨基酸由Ala(丙氨酸)突变为Pro(脯氨酸),233位氨基酸由Thr(苏氨酸)突变为Ser(丝氨酸)。结论所克隆的基因为新生隐球菌的一个新基因,其相关生物学信息的明确,为应用分子生物学技术进一步深入研究新生隐球菌的感染和致病机制奠定了基础。Objective To clone and analyze the bioinformatics of full-length cDNA sequences of meiosis - specific protein required for spore formation （ ISC10 ） gene from Cryptoeoccus neoformans, Methods Total RNA was isolated from the Cryptococcus neoformans wild strain B3501 and the mRNA was reversly transcribed into cDNA. Cryptococcus neoformans ISC10 gene was ampli- fied by reverse transcription polymerase chain reaction（ RT - PCR） to cloning and inserted into recombinant clone vector of pGM- T/ISC10. Extensive amino acid sequence homology was found when compared with ISC10 of GenBank. Results To clone the full - length cDNA Sequences was meiosis - specific protein required for spore formation （ISC10） gene from Cryptococcus neoformans and to participate in meiosis - specific protein required for spore formation （ ISC10 ） gene from Saccharomyces cerivisiae to compare homology was 99.10%. There were two mutations of amino acid in the catalytic domain and hinge region with this ISC10 gene. The amino acid of Ala （ position 67 ） was mutated into amino acid of Pro, and the amino acid of Thr（ position 233 ） was mutated into a- mino acid of Ser. Conclusion This sequence is a new of Cryptococcus neoformans gene. Identification of the biological characteristics will play important role in the further study of its infection and pathogenic mechanism by molecular biology technique.

关 键 词：新生隐球菌 基因ISC10 逆转录聚合酶链反应 分子克隆 序列分析 

分 类 号：R379.5[医药卫生—病原生物学]