家蚕Bax inhibitor-1基因的原核表达及功能分析

When screening Silkworm cDNA library, we found a gene which has a higher homology with Bax inhibator-1 and named BmBI-1 (Bombyx mori Bax inhibator-1). In order to research the function of BmBI-1, the ORF of this gene was cloned into the pET-28a(+) vector, and constructed the procaryotic expression plasmid pET-28a(+)-BmBI-1. The recombinant plasmid was transformed into E.coli BL21 (DE3). After inducing with IPTG , the analysis of SDS-PAGE showed that the recombinant protein was expressed compared to control. The recombinant protein was soluble and was purified with metal-chelating affinity chromatography. Immunization of New Zealand rabbit with recombinant protein generated high titer polyclonal antibodies (1:12800). Subcellular localization study with polyclonal antibodies in silkworm cell Bm5 suggest that BmBI-1 is mainly distributed in the cytoplasm, may be positions in the endoplasmic reticulum.At the same time, The gene expression features in different organizations and differences stages were analysis using fluorescence quantitative PCR and western blot method. The results showed that in all tissues BmBI-1 are expressed as following from the most to the lest : Malpighian tubes, the testicles, head, intestines, valves, skin, fat body, silk gland; In the four Development stages, moth the is the highest followed by larva, and the next is pupa ,but in eggs , few expression was detected. From the above results we can have a conclusion that BmBI-1 may be related to development and maintenance of normal physiological functions in silkworm as other similar proteins.