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Expression of CIRP from the BALB/C Mouse in NIH3T3 Cells and Study of Cold-Induced Apoptosis

Author: YinWei
Tutor: LiShiZe
School: Heilongjiang Bayi Agricultural University
Course: Basic Veterinary Science
Keywords: Cold- inducible RNA binding protein Eukaryotic expression Apoptosis
CLC: Q255
Type: Master's thesis
Year: 2009
Downloads: 18
Quote: 0
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Abstract


Cold inducible RNA binding protein (Cold inducible RNA-binding protein, CIRP) is a mammal in the first one to be found in the cold-shock protein, composed of 172 amino acids, the molecular weight of 18 KD, with RNA-binding domain, can be Low large induced expression plays an important role in the cold-induced cell growth inhibition. CIRP may be involved in human and animal neural development and regulation, embryonic development, physiological processes such as tumor, as well as animal hibernation; recent study found, CIRP able to inhibit the process of apoptosis induced by TNF-α. Therefore, CIRP play a wide range of biological effects of the multifunctional protein, to carry out in-depth research and reveal the mechanism into significance on the development of its potential value. In this study, extraction of total RNA from BALB / C mice after cold treatment testicular tissue CIRP The cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) to construct the recombinant cloning plasmid pGEM-T-CIRP by PCR correct restriction endonuclease, send sequencing company sequencing, sequence analysis showed that the BALB / C mice CIRP cDNA clone; cDNA clones, respectively connected to the eukaryotic expression vector pcDNA3 and pECFP-C1, and then transformed into E. coli DH5α competent cells, through the the restriction endonuclease screened positive recombinant bacteria; liposomes recombinant plasmid pcDNA3-CIRP and pECFP-C1-CIRP blank control plasmid were transfected NIH3T3 cells, 36h after inverted fluorescence microscope and RT-PCR method of observation and identification of the CIRP cells showed that the recombinant expression plasmid was constructed correctly and get the expression; pECFP-C1-CIRP transfected group G418 and screening, fluorescence microscopy cells showed strong green fluorescence, indicating that successfully screened to obtain a stable expression cell strains, and the green fluorescence mainly distributed in the cytoplasm and the extracellular described as the pressure screening CIRP fusion protein is secreted out of the cell; Finally, after transfection cells 4 ° C, 14 ° C, under the conditions of 24 ° C cold-induced apoptosis experiments after after Hoechst33258 fluorescence staining, apoptosis fluorescence microscopy test results show that the the CIRP gene transfected cells group the degree of apoptosis is less than the control group, in the protection of cells from cold apoptosis process plays an important role. This study to establish a cold cell apoptosis in experimental models, confirmed the the CIRP to play an active role in the process of anti-apoptotic, laid the foundation to carry out the CIRP functional and mechanism of action.

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