Cloning And Expression Of CDNA Coding For Group I And II Allergen Of Dermatophagoides Farinae

Objective To construct and express prokaryotic expression plasmid of the cDNA coding for group 1 and group 2 allergen of Dermatophagoides farinae which collected in Huainan area.Methods Firstly, the Dermatophagoides mites were collected, identified and cultured in our laboratory. Secondly, the total RNA of D. farinae was extracted with the Trizol reagent. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to acquire cDNA. Then PCR was performed in a 50ul reaction mixture in an automatic thermal cycler, and the PCR product was visualized under UV light and photographed. After purified, the target fragments were cloned into the compatible sites of the T-vector pMD-18T, the recombinant plasmid pMD-18T-Der f 1, pMD-18T-Der f 2 were digested with restriction endonuclease and were subcloned into expression vector pET32a (+), respectively. The positive recombinants were transferred into E.coli BL21 and identified by restriction endonuclease digestion and PCR. Finally, the genetically engineered bacteria including pET32a (+)-Der f 1 and pET32a (+)-Der f 2 plasmids were induced by IPTG, and the expression was analyzed with the methods of SDS-PAGE and gel densitometic scanning. Results The results revealed that the cDNA of Der f 1 and Der f 2 were specifically amplified from total RNA of Dermatophagoides farinae by RT-PCR and PCR, recombinant plasmid pMD-18T-Der f 1, pMD-18T-Der f 2 were successfully digested by BamH I /Sac I , Sac I /Not I , and the products of digestion were identical with the predicted one, respectively. Sequence analysis also showed that the cDNA Der f 1 was 99.52% affinity in comparison with DNA sequence published on GenBank (emb | X65196.1), and insertion sequence wasn't be found in cDNA Der f 2, while comparison with other cDNA sequence (locus: AF346905). The prokaryotic expression plasmids pET32a (+)-Der f 1 and pET32a (+)-Der f 2 could express a specific 45kDa and 34 kDa protein, which accoutered for about 15% and 16% of the total protein of recombinant bacterial, in E.coli BL21, respectively. Conclusion The sequence of Der f 1 cDNA and Der f 2 cDNA were different in various fauna, respectively. The prokaryotic expression pl;,smids, which eontair. Der f 1 and Der f 2 cDNA from Huainan area, have been constructed successfully and can express objective protein in E.coli DL21.