摘要
目的寻找高效克隆马尔尼菲青霉新基因的方法。方法从生物信息库中找出已知的酿酒酵母、白念珠菌、新生隐球菌、烟曲霉、构巢曲霉SKN7氨基酸保守序列,设计简并引物,PCR扩增获得部分马尔尼菲青霉SKN7 cDNA片段,随后应用RACE技术分别扩增其5′端和3′端未知序列。结果简并PCR扩增可产生多个条带,以预期大小1100bp处条带最清晰。5-′RACE得一约900bp大小产物,无非特异性扩增。3-′RACE扩增产物为多条片段,其中以700bp,400bp和200bp条带相对较清晰,纯化、克隆、测序、比对分析后证实700bp大小产物为目的片段。将上述产物序列进行对位拼接,获得一全长约为2.5kb的序列,它编码的蛋白与其他物种Skn7蛋白高度同源,为马尔尼菲青霉SKN7 cDNA。结论简并PCR结合RACE技术是一种高效、简单、有效的克隆马尔尼菲青霉新基因的方法。
Objective To find out an effective method in the cloning of unknown gene of Penicillium marneffei. Methods Degenerate oligonucleotide primers were designed according to the conserved amino acids sequences information of Skn7 proteins of several fungi. Partial cDNA sequences of SKN7 were amplified by degenerate PCR from Penicillium marneffei. Special primers of RACE method were designed based on these partial sequences to amplify the 3′ cDNA and 5′ cDNA. Results A full length （2.5kb） cDNA fragment was acquired by degenerate PCR,3′ RACE and 5′ RACE. Sequence analysis showed this cDNA fragment had an open reading frame （ORF） of 1776bp, encoding 591 amino acid residues. The deduced amino acid sequence of the ORF was similar to the Skn7 protein in others fungi. Conclusion Degenerate PCR combined with RACE is an effective tool for search- ing and discovering new genes of Penicillium marneffei.
出处
《中国皮肤性病学杂志》
CAS
北大核心
2007年第11期660-662,共3页
The Chinese Journal of Dermatovenereology
基金
教育部高校博士点专项基金资助课题(项目编号20050001103)
