粉尘螨变应原第10组分基因克隆表达及生物信息学分析

目的:克隆粉尘螨变应原第10组分(Der f 10)基因并建立其原核表达体系。方法:提取粉尘螨总RNA,反转录后经套式PCR扩增出目的基因并克隆至pMD19-T载体测序,将测序正确的目的基因插入表达质粒pET28a,转化E.coliBL21(DE3)用IPTG诱导表达,用SDS-PAGE和Western blot鉴定重组蛋白。结果:RT-PCR扩增获得Der f 10,琼脂糖凝胶电泳见一条约888 bp的条带,与参考序列(GenBank No.EU 106617)同源性高达99.8%。将pET28a(+)-Der f 10质粒转化宿主菌E.coli BL21,SDS-PAGE电泳时出现特异性条带,Western blot表明重组蛋白可与抗His-Tag Mab抗体结合。生物信息学软件预测获得的Der f 10重组体由295个氨基酸组成的疏水性蛋白,相对分子质量为34 233.1 Da,二级结构包括α-螺旋(91.86%)、延伸主链(1.36%)和无规卷曲(6.78%),含有5个原肌球蛋白模序分别位于84～101、120～140、145～173、175～198和231～256氨基酸处。结论:获得了Der f 10编码基因,成功建立其原核表达体系,为生产重组变应原用于临床诊断与治疗奠定基础。

Cloning,expression,and bioinformatics of the group 10 allergen of Dermatophagoides farinae

Objective:To clone the gene encoding of group 10 allergen of Dermatophagoides farinae(Der f 10),and to develop its prokaryotic expression system.Methods:The total RNA of Dermatophagoides farinae was extracted,and the gene encoding Der f 10 was amplified and inserted into pMD19-T simple vector.After sequencing,the target gene was then inserted into the expression plasmid pET28a,transformed into E.coli BL21(DE3),induced by IPTG,and identified by SDS-PAGE and Western blot.Results:The gene encoding Der f 10 was amplified by RT-PCR,which had 99.8% similarity to reference sequence.The expression plasmid pET28a(+)-Der f 10 was transformed into E.coli BL21,and a specific band was observed on SDS-PAGE and Western blot with antibody His-Tag Mab.By analysis of bioinformatics software,the recombinant protein rDer f 10 was composed with 295 hydrophobic residues.The secondary structure of Der f 10 appears to comprise alpha helix(91.86%),extended strand(1.36%),and random coils(6.78%).Additionally,five tropomyosin motifs were predicted at 84-101,120-140,145-173,175-198 and 231-256 amino acid position.Conclusion:The gene encoding Der f 10 was obtained,and a prokaryotic expression system was developed,which lay the groundwork for future studies,including large-scale production of recombinant Der f 10 allergen for diagnostic and therapeutic agents.