关键词: 粉尘螨, 变应原, 基因克隆, 基因表达, 生物信息学

Abstract:

Objective To obtain the full-length gene of group 8 allergen of Dermatophagoides farinae, Der f 8, and construct a prokaryotic expression vector to express this gene. Methods Primers were designed according to the previously published partial sequence of Der f 8 (GenBank Accession No.AY283295) and synthesized. The Der f 8 fragment was amplified by RT-PCR using the total RNA of Dermatophagoides farinae as template. The full-length Der f 8 was obtained by rapid amplification of 5′ cDNA ends (RACE), ligated into pMD19-T vector and transformed into Escherichia coli. Positive colonies were selected for plasmid extraction followed by sequencing. Primers were designed based on the full sequence of Der f 8, and RT-PCR was performed using total RNA as template. The products were recycled from gel and incorporated into the pCold TF plasmid to construct the pCold TF-Der f 8 recombinant plasmid, which was transformed into E. coli, and incubated overnight. The positive colonies were used for plasmid extraction and sequencing. The pCold TF-Der f 8 plasmid was again transformed into E. coli BL21 for expression under the induction of IPTG. The expressed product was validated by SDS-PAGE. The physical and chemical properties, structure and function of Der f 8 were predicted by bioinformatics software. Phylogenetic tree was constructed. Results The Der f 8 fragment was amplified by RT-PCR with the product size of 600 bp. Sequencing result was as expected. The left part of full-length Der f 8 was obtained by 5′ RACE with a length of 300 bp, and confirmed by sequencing. The Der f 8 CDS region was amplified by RT-PCR using the primers designed based on the full-length Der f 8, with a length of 696 bp, and was confirmed by sequencing. SDS-PAGE showed that the target protein was expressed in a soluble form, with a relative molecular weight of 81 000. Bioinformatics analysis revealed a 98.49% homology between full-length Der f 8 and the reference sequence (GenBank No.AY283295). Functional analyses through ScanProsite, InterProScan and MotifScan identified glutathione S transferase activity of its protein, with its secondary structure comprising of alpha helixes (45.45%), an extended main strand(11.3%), and random coils (43.3%). The phylogenetic tree showed that Dermatophagoides farinae and Dermatophagoides pteronyssinus were clustered together. Conclusions The full-length Der f 8 cDNA has been obtained, and the prokaryotic expression vector has been constructed to express this gene.

Key words: Dermatophagoides farinae, Allergen, Gene cloning, Gene expression, Bioinformatics