Gene Cloning And Expression Analysis Of Bmcaspase-9 In The Silkworm, Bombyx Mori

Apoptosis is a normal physiology phenomenon that the cell can finish its own vital process according to own molecular genetic regulation procedure under certain physiological or pathological conditions. Caspase protease family is key elements of apoptotic regulation network. Through the method of bioinformatics and molecular biology, this article has cloned the Bombyx mori caspase-9 homologous gene, and carried out prokaryotic expression and polyclonal antibody preparation. Using Western Blotting, we have analysed the expression quantity difference of Bmcaspase-9 in the normal cells, UV-induced apoptotic cells and ActD-induced apoptotic cells. The results are as follows.(1)The gene cloning and sequence analysis of Bmcaspase-9Two homologene of caspase-9 in Bombyx mori have been obtained by cloning in this research, and named Bmcaspase-9L and Bmcaspase-9S. The length of Bmcaspase-9L is 1454bp, and ORF length is 1245 bp. Bmcaspase-9L contains nine exons and encodes 415 amino acids, formed by a caspase recruitment structural domain CARD, a large catalytic subunit P20 and a small catalytic subunit P10. The protein molecular weight and isoelectric point separately is 47.8kD and 7.66 by forecasted.The length of Bmcaspase-9S is 830bp, and ORF length is 549 bp. Bmcaspase-9S contains five exons and encodes 183 amino acid, is only composed of CARD and P10, lacking of P20 and QACR(orG/Q)G active site. The protein molecular weight and isoelectric point separately is 21.4kD and 9.35 by forecasted. We extrapolated that Bmcaspase-9S may be Bmcaspase-9's variant and the negative regulation molecule of the endogenous Bmcaspase-9, having the function of inhibit apoptosis.We carried on the cluster analysis using musle.exe and the MEGA4.0 software. The result indicates that Caspase-9 homolog of silkworm and Caspase-9 homolog of insect are clustered together,the Caspase-9 homolog congener evolutionary distance of Drosophila melanogaster is nearer Bombyx mori than Echinoidea, Pisces, Ammphibia, Aves and mammalia. The higher structure conservatism of Caspase-9 homolog suggests the function conservatism and importance of Caspase-9. The data analysis result of Bombyx mori genome array showed that Bmcaspase-9 is non-organizational and non-sexual specific, and universally expressed in different developmental stages from ripe silkworm to silk moth of thely-silkworm and arrheno-silkworm, and in 10 different tissues (or sample) from 5th instar larva of Bombyx mori, such as the body wall, blood, ovaries, and so on.but the expression levelsis lower. These spatio-temporal differential expressions may be connected to the development, differentiation and apoptosis of bombyx mori, et al.(2) The prokaryotic expression and polyclonal antibody preparation of Bmcaspase-9The Bmcaspase-9L gene fragment, coding the protein sequence from 180th to 415th amino acid residue, was choosed and inserted to the prokaryotic expression vector pET-28a-c(+).The recombinant vector was transformed into BL21(DE3) and induced the expression of the inserted protein coding sequence. The target protein was purified by Ni2+-based immobilized metal ion affinity chromatograph. Taking this purified target protein as the antigen, we have prepared the polyclonal rabbit anti-Bmcaspase-9 antibody. The Western blotting analysis of prokaryotic expressed target protein, using this prepared antibody, indicated that the fusion protein has the antigenicity of Caspase-9 in Bombyx mori, and this antibody has high immunologic specificity.(3) The expression of Bmcaspase-9 in silkworm BmE-SWU1 apoptotic cellsBmE-SWU1 cells had happened apoptosis by 24h normal cell culture after being treated by UV and actinomycin D, the final concentration is 200ng/mL. This may be due to that Bmcaspase-9 protein itself exists in the form of inactive zymogen in the cytoplasm, and can cause apoptosis by autoactivation after receiving apoptotic signal stimulus, but its expression quantity don't obviously raised in the whole event.