粉尘螨Ⅰ类变应原的cDNA克隆测序及亚克隆

目的　获得粉尘螨I类变应原(Derf1)cDNA克隆及亚克隆,并进行测序。　方法　设计合成引物,从粉尘螨体内提取RNA,经逆转录聚合酶链反应(RTPCR)获得cDNA,PCR扩增目的片段经纯化回收后克隆至pMD18T,转化大肠埃希菌(E.coli)JM109,经PCR初筛挑选阳性克隆并测序,将PCR筛检阳性重组子及pET32a(+)表达载体分别用BamHⅠ和SacⅠ双酶切,连接转化至E.coli感受态细胞JM10 9中过夜培养,挑选菌落进行酶切鉴定。　结果　从粉尘螨基因组RNA中扩增出Derf1基因,获得pET32a(+)Derf1亚克隆,酶切产物的大小与预期相符。　结论　对粉尘螨Derf1基因进行体外扩增并获得pET32a(+)Derf1亚克隆。

Cloning, Sequencing and Subcloning of cDNA Coding for Group Ⅰ Allergen of Dermatophagoides farinae

OBJECTIVE: To clone, sequence and subclone the cDNA coding for group 1 allergen of Dermatophagoides farinae (Der f 1). METHODS: The cDNA of Der f 1 was amplified by RT-PCR and PCR. After purified, the gene fragment was cloned into a vector pMD-18T. The recombinant plasmid pMD-18T-Der f 1 was transformed into E. coli JM109. Positive clones were screened and identified by PCR and digestion with restriction enzyme. The sequence of inserted Der f 1 gene fragment was also detected. Der f 1 was then subcloned into the vector of pET-32a(+). RESULTS: The Der f 1 gene fragment of Dermatophagoides farinae was specifically amplified from RNA by RT-PCR and PCR. The recombinant plasmid pMD-18T-Der f 1 and pET-32a(+)-Der f 1 was constructed and digested by Bam H I and Sac I, the size of gene fragment was 646 bp and in accordance with the expected one. CONCLUSION: The pET-32a(+)-Der f 1 subcloning has been constructed successfully.