Plant vector construction and expression of Der f1 allergen of Dermatophagoides pteronyssinu / 中国免疫学杂志

Objective:To construct the plant expression vector of Der f1 allergen of Dermatophagoides pteronyssinu and expression in tobacco lamina.Methods:The Der f1 gene was amplified from the glycerin bacterium which contained pET28a(+)-Der f1 plasmid,cloned into the pMD 19-T plasmid,and then sequenced.The Der f1 gene was digested by ClaⅠand SalⅠ,and cloned into potato virus X (PVX) to construct plant expression vector PVX-Der f1,and then was transformed agrobacterium tumefaciens.The positive one was selected to infect tobacco lamina for expressing target protein.The protein was identified and analysed by SDS-PAGEand Western blot.Results:Digestion and sequence analysis confirmed that the plant expression vector was correct,and the SDS-PAGE and Western blot results showed that the molecular weight of the protein was about 34M_r and it could specific binding with positive serum.Conclusion:The plant expression vector of Der f1 is successfully constructed and the recombinant protein is also produced.

Cloning and characterization of the group 5 allergens from Dermatophagoides farinae / 中华微生物学和免疫学杂志

Objective To obtain the gene coding for the group 5 allergens from Dermatophagoides farinae ( Derf5 ) and predict its molecular characteristics. Methods The total RNA of D. farinae were extracted, and the gene Derf5 was amplified by RT-PCR with the primers designed according to previous sequence published in GenBank. The target gene was linked into pMD19-T Simple plasmid, sequenced and analyzed by bioinformatics software. Results The sequence homology reached to 97.8% between our sequenced result with one complete open reading fragment (ORF) and the reference. The gene encode an extracellular hydrophobic protein with 132 amino acid resides, one signal peptide from 1 to 19 position and one transmembrane domain from 1 to 19 position. The secondary structure was composed of extended strand (1. 52% ), random coil (7.58%) and alpha helix (90.91%). The encoded protein was deduced to have two Casein kinase Ⅱ phosphorylation sites. The similarity of the amino acid sequence of the group 5 allergens were 78% between D. farinae and D. pteronyssinus. Conclusion The gene Derf5 was cloned successfully, and its characteristics was primarily predicted.