粉尘螨变应原Der f 8全长基因克隆及表达

Cloning and expression of the full-length gene of group 8 allergen of Dermatophagoides farinae

目的获得粉尘螨(Dermatophagoides farinae)变应原Der f 8全长基因并构建其原核表达质粒。方法参考Gen Bank登录号为AY283295的Der f 8部分序列设计并合成引物。以粉尘螨总RNA为模板,RT-PCR扩增获得Der f 8部分片段。采用5′cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)获得Der f 8全长序列,连接至pMD19-T载体,热转化至大肠埃希菌(Escherichia coli),挑取阳性菌落,抽提质粒后测序。根据Der f 8全长序列设计并合成引物,以粉尘螨总RNA为模板进行RT-PCR扩增,产物回收后连接pCold TF载体,热转化至E.coli,涂板过夜培养后,挑取阳性菌落,抽提质粒后测序。将pCold TF-Der f 8质粒转化至E.coli BL21,异丙基-β-D-硫代半乳糖苷诱导表达,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物。采用生物信息学软件预测Der f 8的理化特性、结构和功能,并构建系统进化树。结果以Der f 8部分序列设计的引物行RT-PCR扩增获得Der f 8部分片段,长度为600 bp;5′RACE获得Der f 8剩余序列,长度为300 bp;以全长基因序列设计的引物进行RT-PCR扩增获得了Der f 8基因CDS区,长度为696 bp,测序均正确。SDSPAGE结果显示,目的蛋白为可溶性表达,相对分子质量(M_r)为81 000,与预期大小一致。序列经生物信息学分析结果显示,Der f 8全长序列与参考序列(Gen Bank登录号为AY283295)同源性为98.49%,预测其编码的疏水性蛋白具有谷胱甘肽S转移酶活性,二级结构包括α-螺旋(45.45%)、延伸主链(11.26%)和无规卷曲(43.29%)。系统进化树结果显示,粉尘螨与户尘螨(Dermatophagoides pteronyssinus)聚成一簇。结论获得了Der f 8全长基因及其原核表达质粒。

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＇ eDNA ends （RACE）, ligated into pMD19-T vector and transformed into Escherichia coll. 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.