Cloning And Expression Of Caspase-1 Gene From Bombyx Mori And Its Apoptosis Effect

Apoptosis is a phenomenon vital to cells and plays a key role in evolution, stabilization of intemal environment and development of multi-system for organisms. Currently, the mechanism of apoptosis in nematdode, fruit fly and mammalian has been understood to great extent. Caspases are found to be the key executioners of apoptosis. To date, while dozens of caspases have been identified in mammalian, only 2 caspases sequences of Bombyx mori, a model insect, were registered in NCBI with their functions unknown. To further study the function of silkworm caspase-1, we did the following work:The caspase-1 gene was amplified with a pair of primers designed according to the sequence of caspase-1 gene by RT-PCR, and the product was cloned into pMD 18-T vector for sequencing. The result showed that the open reading frame of the gene is 882 base pairs in length and encodes 294 amino acids.To study the expression of caspase-1 gene, the ORF of the gene was inserted into the plasmid pET28a, a prokaryotic expression vector, to construct pET28a-caspase-1 recombinant vector, and the recombinant plasmid was used to be transformed into BL21 cells. Expression of the caspase-1 gene was induced by IPTG, and the protein products were identified by SDS-PAGE and Westem Blotting. The results showed that Caspase-1 product was cleaved after 3 hours following induction by IPTG. It indicated that the expression of caspase-1 gene can be induced in E. coli BL21, and its product can be autoprocessed. Caspase-1 gene was inserted into the baculovirus transfer plasmid pFastBacHTB, as well. The recombinant plasmid pFBH-caspase-1 was obtained and transformed into the competent cells of DH10Bac. Bacmid was extracted, identified by PCR, and the result showed that caspase-1 gene was transposed, thus the recombinant baculovirus Bm/Bacmid-caspase-1 DNA was obtained. Then recombinant virus DNAs were transfected into Bm cells, the recombinant virus Bm/r-caspase-1 was obtained. We performed trypan blue-staining experiment on silkworm cells infected with BmPAK6 virus and the recombinant virus, respectively, and the result showed the expression of the caspase-1 gene advances in the apoptosis process of silkworm cells. Relationships between apoptosis and both ultraviolet overexposure and virus infection were also investigated in this work. Silkworm larvae and silkworm cells were overexposed in ultraviolet and silkworm cells were infected with BmNPV, cellular RNA then extracted and quantitative real-time RT-PCR of caspase-1 gene performed. Silkworm cells were stimulated by ultraviolet for 5 sec, and expression level of Caspase-1 reached its peak at 10 min following stimulation, which suggests that stimulation by ultraviolet for a short time increases the expression level of Caspase-1 rapidly. Silkworms were also stimulated by ultraviolet for different time, and collected at different times after culturing. While 1 hour after culturing following stimulation, the highest expression level was found to be stimulated by a 20-min ultraviolet stimulation, 3 hours after culturing following stimulation, the highest expression level was found to be stimulated by 10-min ultraviolet stimulation. However, compared with the expression level after culturing for 1 hour, the expression level after culturing for 3 hours was relatively low, which indicates that Caspase-1 is an upstream molecule in the cell apoptosis signal pathway induced by ultraviolet. The virus infection of cells reduces the expression level of caspase-1 gene, especially at 0-4 h, indicating Caspase-1 is also an upstream molecule in apoptosis pathway caused by virus. To conclude, Caspase-1 is an upstream molecule in cell apoptosis signal pathway induced by the two factors.