T载体的构建及在恶性疟原虫RAP2基因克隆中的应用研究

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

CONTRUCTING T VECTOR AND USING IT TO CLONE RAP2 GENE OF PLASMODIUM FALCIPARUM

Aim To construct high effective clone vector for PCR product and use it to clone Plasmodium falciparum isolate FCC1/HN RAP2 gene. Methods Plasmid PUC18 was digested by small and purified, added dTTP into it, incubated at 70℃ for 3h to construct T vector. Using PCR technique, RAP2 gene was amplified from genomic DNA of Plasmodium falciparum isolate FCC1/HN of southem China. The PCR product was purified and cloned into T vector, then transformed into E.coli strain DH5α. The recombinant plasmids were screened and idetified by using EcoR/XhoI and PCR method. Results RAP2 gene was amplified from the genomic DNA of Plasmodium falciparum by PCR, it is about 1215bp. The recombint plasmid pT-RAP2 was constructed.Conclusion Successful constructed T vector and got recombint plasmid pT-RAP2, which provided the condition for sequencing RAP2 gene and study on its structure and function.