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[Cloning, sequencing and subcloning of cDNA coding for group I allergen of Dermatophagoides farinae]
1School of Medicine, Anhui University of Science & Technology, Huainan 232001, China.
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.
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