Cloning, Sequencing and Subcloning of cDNA Coding for GroupⅠAllergen of Dermatophagoides farinae / 中国寄生虫学与寄生虫病杂志

ABSTRACT

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 HⅠand SacⅠ, 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.