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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。
Objective To construct a full-length cDNA library of trophozoites of Amomum acuminatum and lay a foundation for the screening and gene structure study of Acanthamoeba trophozoite antigen. Methods The total RNA of trophozoites of Acanthamoeba mellitus was used as a template to reverse-transcribe single-stranded cDNA with modified SMART oligonucleotide primers and amplified by using long-distance PCR (LD-PCR) The double-stranded cDNA was digested with proteinase K and digested with SfiI and separated by molecular mass with CHROMA SPON-400 column. The DNA was identified by 1% agarose gel electrophoresis and the 0.4 ~ 2.5kb isolated product was recovered. Take 1 μl of PCR product and λ phage were connected to XL1-BLUE as recipient bacteria amplified cDNA library, respectively, using a gradient method and random sequencing library titers and recombination rate. Results The Acanthamoeba mellitus cDNA library was successfully constructed. The titer of the library was 3.85 × 107pfu / ml. The insert size ranged from 0.4 to 2.5kb and the recombination rate was 100% (20/20). Conclusion The cDNA library of Acanthamoeba nopal is successfully constructed, which lays the foundation for the screening and gene structure study of acanthamoeba trophozoite antigen.