Cloning and Subcloning of cDNA Coding for Group Ⅱ Allergen of Dermatophagoides Farinae

摘要

Objective: To clone, sequence and subclone the cDNA coding for group Ⅱ allergen of Dermatophagoides farinae (Der f 2). Methods: The cDNA gene fragment of Der f 2 was amplified by RT-PCR. After being purified, the gene fragment was cloned into a vector pMD-18T. The recombinant plasmid pMD-18T-Der f 2 was transformed into E. coli JM109. Positive clones were screened and identified by PCR and digested with restriction endonuclease, and the sequence of inserted Der f 2 cDNA was also analyzed. Then Der f 2 was sub-cloned into the vector of pET-32a( + ). Results: The Der f 2 cDNA was specifically amplified from RNA by RT-PCR. The recombinant plasmid pMD-18T-Der f 2 and pET-32a( + ) -Der f 2 was constructed and digested by Sac Ⅰ and Not Ⅰ , and the size of gene fragment was 455bp and in accordance with the expected one. Conclusion: The pET-32a( + )-Der f 2 subclone was constructed successfully.