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Cloning and Expression of Bombyx Mori Apoptosis-Related Gene, ice, and Its Effect on Virus Infection of Bombyx Mori Cells

Author: SongLiNa
Tutor: WangWenBing
School: Jiangsu University
Course: Biochemistry and Molecular Biology
Keywords: Bombyx mori ice clone expression E.coli fluorescence quantify PCR baculovirus expression system
CLC: Q78
Type: Master's thesis
Year: 2006
Downloads: 37
Quote: 0
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Abstract


ICE (interlukin-1β-converting enzyme), the first discovered member of caspases (cys-teinylaspartate specific proteinase) family, plays an important role in programmed cell death of multicellular organisms. To date, caspase members, eleven from human, ten from murine, four from avian, four from fish, eight from amphibian and three from nematode have been identified. However, only one sequence of silkworm similar to ice was found in the gene database with its function still unknown. In this paper, according to the ice gene (GenBank accession number: AY885228) , the special primers were designed. Two new ice genes, named ice-2 and ice-5 (GenBank accession number: DQ360829 and DQ360830) , were obtained from silkworm cells. The open reading frames (ORFs) of the two isoforms contain 852 and 936 base pairs, encoding 284 and 312 amino acids, respectively. Sequence analysis of ice, ice-2 and ice-5 showed that there was something identical and different among these three genes. While both ice and ice-2 contain 7 exons, ice-5 contains 8 exons. However, they shared the same fragments of the N-terminal five exons and the last exon. The difference between ice and ice-2 was that ice-2 has more 30 bases than ice in the middle of the 6th exon.Proteins in ICE family will be cut by themselves in the maturation process. In order to study the splicing pattern of ICE-2 and ICE-5, E.Coli expression system, which can express protein steadily, was used. In this study, ICE-2 and ICE-5 were induced at 37℃and 25℃. At 37℃inducement prophase, ICE-2 and ICE-5 were not cut, thus having obvious bands at 33 kD and 23 kD, respectively. ICE-2 had special bands at both 33 kD and 23 kD 3 hours following inducement, which indicates that excessive ICE-2 can arise to self-splicing partly, while ICE-5 had no change after 3 h following inducement. At 25℃, ICE-5 had only a band at 26 kD induced, while ICE-2 could not be detected by Western, which indicates that the splicing pattern of ICE-2 and ICE-5 were different.In order to study the role of ice-2 and ice-5 obtained in apoptosis pathway, silkworm cells were stimulated by hydrogen peroxide and virus as well as by ultraviolet ray which was also used to stimulate silkworm larvae, and the alteration in the expression level of ice-2 and ice-5 caused by different stimuli was detected by fluorescence quantitative PCR .The results showed that 1) in silkworm larvae and cells that were stimulated by low doses of ultraviolet ray, while the expression level of ice-2 reached a high level at certain time point following stimulation, no significant alteration was observed in that of ice-5, which indicates that ice-2 may be involved in repairing; 2) in silkworm larvae and cells stimulated by high doses of ultraviolet ray, the alteration in the expression level of ice-5 was more pronounced than that of ice-2; 3) in silkworm cells stimulated by hydrogen peroxide and virus, both of the expression level of ice-2 and ice-5 altered with the increase in the doses of stimulation, however, the alteration in the expression level of ice-5 was more pronounced. It is showed that ice-2 and ice-5 were involved in the execution of cell apoptosis in which ice-5 palyed a more important role.The impact of over-expression of ice-2 and ice-5 on virus infection of silkworm cells was studied by baculovirus expression system. Ice-2 and ice-5 were cloned into expression vector HTB, respectively, and then transformed into compETent BmDH10Bac. After screened with antibiotic and X-gal/IPTG, the recombinant baculovirus Bacmid-ICE-2 and Bacmid-ICE-5 were obtained, and they were proved to be positive by PCR with pUC/M13 as primers. The Bacmid-ICE-2 and Bacmid-ICE-5 were used to transfect silkworm cells, and viruses, r-ice-2 and r-ice-5, were obtained. The viruses were further identified by PCR with the primers designed according to the sequences of ice-2 and ice-5, and the result showed that ice-2 and ice-5 were contained in the viruses. The viruses were used to infect silkworm cells, and the change of cells was examined by morphological observation, cells livability measure and DNA Ladder. The results showed that both ice-2 and ice-5 can induce cell apoptosis, and that ice-5 is more effective in inducing apoptosis than ice-2.In this study, two new silkworm ice genes, ice-2 and ice-5, were obtained. It is shown that both ice-2 and ice-5 were involved in cells signaling transduction, and that ice-2 was involved in repairing. In the execution of cell apoptosis, ice-5 may play a more important role in the apoptosis pathways.

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