Cloning And Expression Of Bmbuffy Gene In The Silkworm, Bombyx Mori

In recent years, apoptosis in C. elegans, Drosophila and mammals have made great achievements. As a typical representative of Lepidoptera and a good model organism, the researches on apoptosis of Bombyx mori are still at the primary stage. Bcl-2 gene, as a key regulatory gene in mitochondrial apoptosis pathway, was received extensive attention since Tsujimoto et al have found in the follicular B cell lymphoma in 1985. This gene is a proto-oncogene, which does not trigger the malignant transformation, nor promote cell proliferation, but it can prolong survival times of cells under no neurotrophic factors and growth factors. Bcl-2 is able to inhibit apoptosis induced by variety of Cytotoxins. In this work,the Bmbuffy gene, the Drosophila Buffy Gene Homologous genes in silkworm was cloned and expressed. The main research results are as follows:1 Cloning and sequence analysis of BmbuffyBmbuffy , a homolog of Drosophila buffy from Bombyx mori,was electronic cloned based on the silkworm EST data. The complete cDNA sequence of Bmbuffy was 1632 bp with 879bp ORF (ORF:362-1241). encoded a protein with 292 amino acids with predicting molecular weight 32.4kD and the isoelectric point pH 9.94. The location of 130-241 aa is the conserved domain of Bcl-2 family. Bmbuffy gene was consisting of 6 exons, and its exon/intron boundary was line with GT-AG rule; The first to 5th exons located on on two scaffold of chromosome 7, while the 6th exons located on the 4th chromosome.2 Prokaryotic expression of BmbuffyAccording the complete cDNA sequences of BmBuffy, we designed the primer of the ORFof Bmbuffy gene. The complete ORF of Bmbuffy was subcloned into the pET-28a-c(+) expression vector, and the recombinant plasmid was transformed into Escherichia coli BL21. Then induced Bmbuffy/ pET-28a-c(+)/BL21 to express protein by IPTG We got a recombinant protein with molecular weight about 36.2kD, including a six-His tags protein,which is consistent with prediction. The protein is expressed as inclusion bodies. IPTG concentration, induction temperature and induction time have a little effect on protein expression of Bmbuffy.The optimal condition of protein expression are 1.0mmol/L IPTG and 37℃4 hours.3 Preparation of Bmbuffy antibodyBmbuffy protein was purified by affinity chromatography and then used to produce specific antibody. The antibody titer is 1:32000 detected by indirect ELISA.4 Expression of Bmbuffy in apoptosis cellsWe detected the expression of Bmbuffy\ BmDredd and BmGsk3 genes in normal BmE-S WU1 cells,the apoptosis cells inducted by UVC and actinomycin D. The apoptosis cells inducted by UVC and actinomycin D showed typical features of apoptosis, such as nuclear condensation, chromatin condensation, nuclear cracking, and visible clearly apoptotic bodies.Bmbuffy, BmGsk3 gene expression decreased, but less obvious, BmDredd increased in BmE-SWU1 cell induced by actinomycin D; Bmbuffy and BmDredd gene expression was significantly decreased, BmGsk3 gene expression was significantly increased of BmE-SWU 1 cell induced by UVC.We detected the three sample using antibodies-Bmbuffy by Western blotting. The results consistented with the expression analysis, and suggested Bmbuffy inhibit apoptosis.