棘阿米巴原虫全长cDNA文库的构建

目的快速构建棘阿米巴滋养体全长cDNA文库,为棘阿米巴滋养体抗原的筛选和基因结构研究奠定基础。方法以棘阿米巴滋养体分离株总RNA为模板,以经修饰的SMART寡聚核苷酸引物逆转录合成单链cDNA,采用长距PCR(long-Distance PCR,LD PCR)方法扩增得到双链cDNA,经蛋白酶K消化和SfiⅠ酶切后通过色谱柱CHROMA SPON-400按分子质量进行分离,进行1%琼脂糖凝胶电泳鉴定,回收纯化0.4~2.5kb分离产物。取1μl PCR产物与λ噬菌体连接,以XL1-BLUE为受体菌扩增得到cDNA文库,分别用梯度法和随机测序法检测文库滴度与重组率。结果成功构建棘阿米巴cDNA文库,文库滴度为3.85×107pfu/ml,插入片段大小在0.4~2.5kb之间,重组率为100%(20/20)。结论棘阿米巴滋养cDNA文库构建成功,为棘阿米巴滋养体抗原的筛选和基因结构研究奠定了基础。

Construction of a full-length cDNA library from Acanthamoeba

Objective To construct a full-length eDNA library from Acanthamoeba trophozoites. Methods Total RNA was extracted from Acantharnoeba healyi trophozoites using Trizol reagent. The single strand （ss） DNA was re verse transcribed from total RNA using the protocol for the super SMART eDNA construction kit. Long-distance PCR （LD-PCR） was used to produce double-stranded （ds） eDNA after proteinase K digestion and SfiI digestion, eDNA of 0.4 -2.5 kb was collected and purified, and then 1 μl of product was ligated to a X phage. The recipient strain XL1 Blue was amplified to construct a cDNA library, and the titer of the eDNA library and recombination efficiency were determined using a gradient method and random sequencing. Results A full-length eDNA library of A. healyi was successfully constructed. The titer of the eDNA library was 3.85× 10^7 pfu/ml. The length of the inserted fragment ranged from 0.4 to 2.5 kb and the recombination efficiency was 100% （20/20）. In total, 20 PCR products from 20 clones were randomly selected and sequenced. The sequences were identified using Blastn in GenBank. There was extensive coverage with fulllength clones （18/20）. Conclusion A full-length eDNA library was successfully constructed from A. healyi trophozoites. This provides a basis for antigen screening and study of the genetic structure of A. healyi.