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目的 构建PCR产物高效克隆载体T载体 ,并应用于克隆恶性疟原虫RAP2基因。方法 PUC18用SmaI酶切纯化后 ,与dTTP在 70℃孵育 3h ,构建T载体。另设计一对特异引物 ,采用PCR方法从恶性疟原虫FCC1/HN株基因组DNA中特异扩增RAP2基因 ,并将其克隆入T载体 ,重组克隆经蓝白斑初筛后 ,再用双酶切及PCR法进行鉴定。结果 从恶性疟原虫基因组DNA中特异扩增出大小为 12 15bp基因片段 ,与预期长度相符。克隆入T载体后的重组克隆经双酶切及PCR鉴定均正确无误。结论 成功构建T载体 ,并获得阳性重组克隆T -RAP2 ,为进行该RAP2基因的序列测定及研究该基因的结构与功能奠定基础
Objective To construct efficient cloning vector T vector of PCR product and to clone RAP2 gene of Plasmodium falciparum. Methods PUC18 was digested with SmaI and incubated with dTTP for 3h at 70 ℃ to construct T vector. In addition, a pair of specific primers was designed to amplify the RAP2 gene from the genomic DNA of Plasmodium falciparum FCC1 / HN strain by PCR and cloned into T vector. The recombinant clones were identified by blue-white staining and double digestion PCR method for identification. Results The 12 15bp gene fragment was amplified specifically from genomic DNA of P. falciparum and was consistent with the expected length. The recombinant clones cloned into T vector were identified by double enzyme digestion and PCR. Conclusion The T vector was successfully constructed and a positive recombinant clone T -RAP2 was obtained, which laid the foundation for sequencing the RAP2 gene and studying its structure and function