首次对家蚕核型多角体orf25基因进行了描述。扩增Bm25基因,亚克隆到原核表达载体pGEX-4T-2,在大肠杆菌BL21(DE3)中表达含有GST标签的融合蛋白。IPTG诱导后高效表达GST-Bm25融合蛋白。纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体。利用制备的抗GST-Bm25融合蛋白的多克隆抗体进行表达时相分析显示:24h p.i.检测到30 kDa的蛋白条带。RT-PCR方法,在18-72 h p.i检测到Bm25基因的转录本。结论:以上数据表明Bm25基因编码一晚期表达的30kDa蛋白。
Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5′-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mori and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5. 38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee (Apis mellifera). This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.
