马尔尼菲青霉酵母相消减cDNA文库的构建及初步筛选  

Construction of the subtracted cDNA library for the yeast phase of Penicillium marneffei and its preliminary screening

作　　者：刘红芳[1] 席丽艳[1] 李希清[1] 鲁长明[1] 张军民[1] 马黎[1] 

机构地区：[1]中山大学附属第二医院皮肤性病科,广州510120

出　　处：《中国人兽共患病学报》2007年第3期218-221,226,共5页Chinese Journal of Zoonoses

摘　　要：目的构建马尔尼菲青霉酵母相抑制性消减cDNA文库,寻找其在酵母相中的差异表达基因。方法分别提取马尔尼菲青霉菌丝相和酵母相的总RNA并合成cDNA,然后应用抑制性消减杂交技术(SSH),以酵母相为tester(检测子),菌丝相为driver(驱赶子),连接不同的接头,通过两轮杂交和两次抑制性PCR后,将产物与T载体连接并转染大肠杆菌。经PCR鉴定,共得到480条插入片段。序列分析和同源性比较表明一些基因与细胞壁抗原、转运蛋白、氧化还原酶等具有同源性。结果成功构建了一个以酵母相为tester(检测子)的抑制性消减cDNA文库。结论所构建的cDNA消减文库为进一步筛选马尔尼菲青霉致病相关基因奠定了基础。To construct a subtracted cDNA library for the yeast phase of Penicillium marneffei with suppression subtractive hybridization（SSH） in order to look for the genes differentially expressed in yeast form, the total RNA was extracted from the mycelium and yeast phase of P. marneffei and the double-stranded cDNA was synthesized. Then, the SSH technique was carried out using the double-stranded cDNA derived from the yeast phase as tester and the mycelium phase as driver. After adaptor ligation and two rounds of hybridizations and PCR amplifications, the cDNAs were inserted into T vector and transformed to E. coll. Being identified with PCR, 480 insert fragments were obtained, in which homologies existed among some of these genes with genes encoding cell-wall antigens, transport proteins and oxidation-reduction enzymes as demonstrated by sequence analysis and homology comparison. It is evident that the subtracted cDNA library for the yeast phase of P. marneffei was successfully constructed, thus providing solid foundation to screen the related genes involved in the pathogenesis of P. marne f fei infection.

关 键 词：马尔尼菲青零 抑制性消减杂交 聚合酶链反应 

分 类 号：R379[医药卫生—病原生物学]