Construction and solvable expression of the recombinant plasmid pCold TF-Der f 2 of Dermatophagoides farinae.

Objective To construct and express the recombinant plasmid pCold TF-Der f 2 of Dermatophagoides farinae.Methods Using the plasmid pMD19-T-Der f 2 as template,the target gene Der f 2 was amplified by polymerase chain reaction.The gene fragment coding for Der f 2 was inserted into the prokaryotic expression plasmid pCold TF DNA to construct pCold TF-Der f 2 and transformed into E.coli BL21 for protein expression induced by isopropyl β-D-1-thiogalactopyranosid(IPTG).The expression product was analyzed and identified by SDS-PAGE and Western blotting.Results The gene coding for Der f 2 was obtained by PCR and the expression plasmid pCold TF-Der f 2 was successfully constructed.SDS-PAGE showed that the plasmid pCold TF-Der f 2 was successfully expressed in E.coli BL21.Most of the products are soluble and can bind with mouse anti-penta-His antibody according to Western blotting.Conclusion The recombinant plasmid pCold TF-Der f 2 of D.farinae was successfully constructed and highly expressed in E.coli,and the expressed fusion protein is soluble.