白念珠菌菌丝相全长cDNA文库的构建和鉴定

Construction and Identification of Full-Length cDNA Library from Candida albicans in the Hyphal Phase

目的利用SMART（Switching Mechanism At5′end of RNA Transcript Method）技术,构建高质量的白念珠菌菌丝相（芽管）全长cDNA文库。方法不完全1640培养基诱导高于95%的白念珠菌带芽管孢子,提取总RAN并纯化poly A＋RNA作为模板,应用SMART技术利用SMARTScribeTMMMLV反转录酶和经修饰的oligo（dT）引物（CDSⅢ/3′PCR引物）合成第一链cDNA,SMARTⅣTM寡核苷酸作为mRNA5′端延伸出去的模板,采用LD-PCR合成双链cDNA,经蛋白酶K消化、SfiⅠ酶切及过柱分级分离后,与λTriplE×2载体连接经噬菌体包装后转导大肠杆菌,构建全长cDNA文库。结果原始菌丝相白念珠菌cDNA噬菌体表达文库滴度为1.87×10^7PFU/mL,重组率达100%,库容量9.35×10^6;文库扩增后,滴度达5×10^9PFU/mL,插入cDNA长度多在0.4-2.5kb,平均长度在1.5kb左右。文库的代表性和重组片段的序列完整性均达建库要求。结论成功构建了菌丝相白念珠菌cDNA噬菌体表达文库,为进一步筛选和克隆白念珠菌芽管特异性抗原表达基因及致病性相关基因奠定了基础。

Objective To construct a full-length cDNA library of Candida albicans in the hyphal phase by SMART（Switching Mechanism At 5＇ end of RNA Transcript Method）technique.Methods RMPI 1640 culture was used to induce the germ tube formation of C.albicans with the formation ratio of more than 95%.The total RNA was extracted from the hyphal cells and the mRNA was purified.The first-strand cDNA was synthesized through reverse transcription with a modified oligo（dT）primer（CDS Ⅲ/3＇PCR Primer）while the SMART Ⅳ oligonucleotide was utilized as a template at the 5＇end of mRNA.The double-strand cDNA was amplified by Long-Distance PCR and then digested by Sfi Ⅰ（ⅠA ⅠB）restriction enzyme.After cDNA was purified through CHROMA SPIN-400 Column,the ds cDNA was ligated to λTriplE×2 vector and then the ligation products were packaged in vitro.Results The titer of the primary cDNA library was 1.87×107PFU/mL,the library capacity was 9.35×10^6 independent clones and the percentage of recombinant clones was about 100%.The titer of the amplified cDNA library was 5×109 PFU/mL.The range of the exogenous inserts length of the recombinants was between 0.3～2.5kb,with an average of 1.5kb.Conclusion The cDNA phage expression library of Candida albicans in the hyphal phase has been constructed successfully,which meets the requirement for screening and cloning C.albicans germ tube specific antigen genes and pathogenicity-associated genes.