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Expression and Functional Analysis of TRAPα Gene from Silkworm

Author: ZhuLiMing
Tutor: TongFuDan；ZhangYaoZhou
School: Zhejiang University of Technology
Course: Biochemistry and Molecular Biology
Keywords: Silkworm TRAPα Fluorescence quantitative Tissue localization Subcellular localization
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 11
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Abstract

The mammalian TRAPα a glycosylated protein size of 34 kD by Ca ^{2 regulation and distribution in the endoplasmic reticulum and the nuclear membrane. MRNA transcription by TRAPα the positive role of granulocyte macrophage colony-stimulating factor in human bone marrow cells. The study found that in mouse skeletal muscle and heart cells after birth TRAPα express some slightly different systems in the body for the first time determined TRAPα protein may muscle and in the mouse heart endothelial cell maturation process of the substrate from the specific role. Screened cDNA library from home silkworm chrysalis one containing cDNA TRAPα conserved domains, and named BmTRAPα (Bombyx the translocon associated protein alpha), The GenBank accession landing No. DQ311413. The length of the gene sequence for the 1389 bp ORF of is 840 bp, encoding 279 amino acid residues, predict a molecular weight of 30.8 kD. Will the BmTRAPα amplified gene product was digested recovered, cloned into fusion expression vector pET-28a appropriate restriction enzyme sites, the recombinant plasmid pET-28a-BmTRAPα proved correct recombinants by PCR and restriction enzyme digestion, The recombinant was transformed into E. coli BL21 (DE3) to a final concentration of 1 mM IPTG induction, cells were harvested and sample preparation by SDS-PAGE analysis showed a concentration-specific protein bands in the the Marker Size 35 kD near, and consistent with the expected value (fusion tag of 3.56 kD, BmTRAPα is 30.8 kD). Fusion protein exists in the form of inclusion bodies, by processing the inclusion bodies and nickel affinity chromatography purified fusion protein, and the fusion protein was used to immunize New Zealand white rabbits polyclonal antibody ELISA detection titers greater than 1:12800. Gel column using Protein A purified polyclonal antibody. Extracted each period of the silkworm and fifth instar larvae of the organizations of the protein for Western blot and ELISA half quantitative determination BmTRAPα of each period and fifth instar larvae of the content of the organizations. Western blot analysis showed the highest expression levels BmTRAPα protein in silkworm pupal stage; gonads of fifth instar larvae of the silk gland, Malpighian tubules and fat body expression, the expression of different higher expression levels in the silk gland and fat body , while the clear signal is not detected in the hemolymph. The basic ELISA semi-quantitative test results with the experimental results of the Western blot match. Quantitative PCR method to compare the amount of target mRNA in the developmental stages of silkworm eggs, fifth instar larvae, pupae and moth, and the organization of the fifth instar larvae, there are some differences in the level of translation and transcription level. The BmN cells immunocytochemistry experiments, the results show that throughout the cell cycle, BmTRAPα proteins are mainly distributed in the cytoplasm.}

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